1984449 Induction of NF-KB during monocyte differentiation by HIV type 1 infection. The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression. 1986254 Positive and negative regulation of immunoglobulin gene expression by a novel B-cell-specific enhancer element. A new B-cell-specific enhancer element has been identified 3' of E4 and the octamerlike motifs in the human immunoglobulin heavy-chain gene enhancer. Tandem copies of this 67-bp MnlI-AluI fragment, when fused to the chloramphenicol acetyltransferase gene driven by the conalbumin promoter, stimulated transcription in B cells but not in Jurkat T cells or HeLa cells. Footprinting analysis revealed that the identical sequence CCGAAACTGAAAAGG, designated E6, was protected by nuclear extracts from B cells, T cells, or HeLa cells. Gel mobility shift assays using a synthetic E6 motif detected a B-cell-specific complex in addition to a ubiquitous band found also in T cells and HeLa cells. In agreement with the results of gel retardation assays, tandem copies of the E6 motif stimulated transcription in ARH77 and Raji cells but not in Jurkat or HeLa cells. Furthermore, a mutant E6 motif lost both in vitro binding activity and in vivo enhancer activity. In striking contrast to the mouse Ig heavy-chain enhancer, in which the octamer motif acts as a B-cell-specific enhancer element, the human enhancer contains an octamerlike sequence with one base substitution which bound octamer-binding proteins with only very low affinity and showed no enhancer activity of its own. Interestingly, the MnlI-AluI fragment could suppress the basal-level activity of the conalbumin promoter in both Jurkat and HeLa cells. Moreover, simian virus 40 enhancer activity was blocked by the MnlI-AluI fragment in HeLa cells but not in B cells. Thus, the novel enhancer element identified in this study is probably a target site for both positive and negative factors. 1987353 The NF kappa B independent cis-acting sequences in HIV-1 LTR responsive to T-cell activation. The rate of transcription initiation directed by the long terminal repeat (LTR) of HIV-1 increases in response to mitogenic stimuli of T cells. Here we show that the response of the HIV-1 LTR may be governed by two independent sequences located 5' to the site of transcription initiation sequences that bind either NFAT-1 or NF kappa B. The rate of LTR-directed gene expression increased in response to treatment with either a phorbol ester or tumor necrosis factor alpha if either the NFAT-1 or NF kappa B binding sites were deleted, but failed to respond to these mitogenic stimuli if both sequences were absent. The HIV-1 mutant virus containing both NF kappa B and NFAT-1 deletion was able to replicate although at a much decreased growth rate, while the deletion of NFAT-1 alone increased the viral growth rate in Jurkat cells. Neither deletion of NF kappa B nor deletion of NFAT-1 decreased activation of viral replication by phorbol ester. 1988951 Specific depletion of the B-cell population induced by aberrant expression of human interferon regulatory factor 1 gene in transgenic mice. Interferons (IFNs) are well known both as antiviral proteins and as potent regulators of cell growth and differentiation. In fact, IFNs inhibit growth of various normal and transformed cell types. Previously, a nuclear factor, IRF-1 (interferon regulatory factor 1), which binds to type I IFN and some IFN-inducible gene promoters, was identified and cloned. Since the IRF-1 gene is both virus and IFN inducible, an intriguing issue is raised as to whether the IRF-1 gene is functioning in IFN-mediated regulation of cell growth and differentiation. In this study, we generated transgenic mice carrying the human IRF-1 gene linked to the human immunoglobulin heavy-chain enhancer. In the transgenic mice, all the lymphoid tissues examined showed a dramatic reduction in the number of B lymphocytes (B cells). Preparation and analysis of bone marrow cells from the chimeric mice indicated that the bone marrow is the effective site for specific depletion of the B-cell population. In fact, transgenic bone marrow cells cocultured with a bone marrow-derived stromal cell line revealed an altered B-cell maturation pattern. 1989880 Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. CD3-epsilon expression is controlled by a downstream T lymphocyte-specific enhancer element. We report the identification of a T cell-specific transcription factor, TCF-1, binding to this element. The multimerized recognition motif of TCF-1 constituted a T cell-specific enhancer. Subsequent cloning of TCF-1 identified three splice alternatives. TCF-1 contained a single DNA-binding HMG box most closely related to similar boxes in the putative mammalian sex-determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene. TCF-1 mRNA was expressed uniquely in T lymphocytes. Upon cotransfection into non-T cells, TCF-1 could transactivate through its cognate motif. These results identify TCF-1 as a T cell-specific transcription factor, which might play a role in the establishment of the mature T cell phenotype. 1990263 Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes. Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered. 1899335 Expression of c-jun, jun B and jun D proto-oncogenes in human peripheral-blood granulocytes. We have found that purified human peripheral-blood granulocytes express constitutively significant levels of proto-oncogenes c-jun, jun B and jun D mRNA. Upon functional activation of granulocytes by 4 beta-phorbol 12-myristate 13-acetate (PMA), the levels of c-jun, jun B and jun D transcripts were increased. The three jun genes showed a similar time course in their induction by PMA, maximal mRNA levels being reached after 60 min of induction. These results suggest that expression of c-jun, jun B and jun D genes might be involved in terminal granulocyte differentiation or in regulating granulocyte functionality. 1847170 Platelet-activating factor induces phospholipid turnover, calcium flux, arachidonic acid liberation, eicosanoid generation, and oncogene expression in a human B cell line. Platelet-activating factor is a potent mediator of the inflammatory response. Studies of the actions of platelet-activating factor have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of platelet-activating factor on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that platelet-activating factor significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, lyso-platelet-activating factor, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that platelet-activating factor from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas lyso-platelet-activating factor was again ineffective. We further demonstrate the impact of platelet-activating factor binding to B cells by measuring platelet-activating factor induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, platelet-activating factor was capable of inducing transcription of the nuclear proto-oncogenes c-fos and c-jun. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates platelet-activating factor induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, platelet-activating factor is shown here to have a direct and profound effect on a pure B cell line. 1994572 A novel HIV-1 isolate containing alterations affecting the NF-kappa B element. Three molecular clones of HIV-1, derived from a single isolate (AL1), exhibited distinct replicative and cytopathic properties during propagation in a human T cell line. The phenotypic differences observed were attributable, in large part, to changes affecting the viral LTR. Nucleotide sequence and PCR analyses demonstrated the presence of novel duplications or deletions involving the NF-kappa B motif. These changes in the enhancer element were identified in the original AL1 virus stock. Subcloning of the variant NF-kappa B segments into LTR-driven CAT expression vectors confirmed a correlation between promoter activity and replicative/cytopathic capacity. 1705836 1,25-Dihydroxyvitamin D3 receptor RNA: expression in hematopoietic cells. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however ; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes. 2005404 Kappa B binding proteins are constitutively expressed in an IL-2 autocrine human T cell line. The IL-2 and the IL-2-R alpha genes are both expressed transiently in normal T lymphocytes after Ag or mitogen activation. In contrast, the human T cell line, IARC 301, expresses these two genes constitutively and we have previously demonstrated that its growth depends on the autocrine production of this T cell growth factor and high affinity IL-2R. To dissect the molecular basis for the unusual persistent expression of the IL-2 and IL-2-R alpha genes in these IARC 301 T cells, we have analyzed the interactions of constitutively expressed nuclear proteins with the 5' flanking regions of the IL-2 and IL-2-R alpha genes using both DNase I footprinting and gel retardation techniques. We have found that a region in both genes (-276 to -250 for IL-2-R alpha and -203 to -183 for IL-2), which corresponds to a kappa B enhancer element, is specifically protected by nuclear proteins from IARC 301. In agreement with this finding, both the IL-2 and IL-2-R alpha promoters are active in transient transfection assays in IARC 301 cells. In contrast, mutation of the kappa B enhancer results in markedly attenuated activities of both promoters. Two proteins binding the kappa B sequence, NF-kappa B and KBF1, are constitutively expressed in IARC 301 nuclei and induced by PMA and PHA in Jurkat. They bind to the kappa B motifs with different relative affinities that may reflect their different contribution in the expression of various promoters. 1672442 The functional domains of the murine Thy-1 gene promoter. The Thy-1 gene promoter resembles a " housekeeping " promoter in that it is located within a methylation-free island, lacks a canonical TATA box, and displays heterogeneity in the 5'-end termini of the mRNA. Using transgenic mice, we show that this promoter does not confer any tissue specificity and is active only in a position-dependent manner. It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site. We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1, an inverted CCAAT box, and sequences proximal to the transcription start site. DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements, including Sp1 and CP1. Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences. 2006151 Comparison of constitutive and inducible transcriptional enhancement mediated by kappa B-related sequences: modulation of activity in B cells by human T-cell leukemia virus type I tax gene. The kappa B sequence (GGGACTTTCC) binds a factor, NF-kappa B, that is constitutively found in its functional, DNA binding form only in B lymphocytes. A factor with apparently indistinguishable sequence specificity can be induced in many other cell types, where it is used to regulate inducible gene expression. For example, kappa B-related sequences have been shown to be important for the transcription of a few inducible genes, such as the interleukin 2 receptor alpha-chain gene and the beta-interferon gene. However, these genes are not constitutively active in B lymphocytes, suggesting that other regulatory mechanisms must play a role in determining the patterns of expression. We have investigated the constitutive and inducible transcriptional activity mediated by five kappa B-related sequence elements in two different cell types. We show that in S194 plasma cells the activity of each element correlates well with the relative affinity of B-cell-derived NF-kappa B for that element. This leads to significantly lower transcription enhancement by sites derived from the interleukin 2 receptor or T-cell receptor genes in S194 cells. However, in either EL-4 ( T ) cells or S194 cells, both lower-affinity sites can be significantly induced by the tax gene product of human T-cell leukemia virus type I, showing that NF-kappa B activity can be modulated even in a B-cell line that constitutively expresses this factor. 2006423 Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B [published erratum appears in Science 1991 Oct 4 ; 254 ( 5028 ) : 11] A DNA probe that spanned a domain conserved among the proto-oncogene c-rel, the Drosophila morphogen dorsal, and the p50 DNA binding subunit of NF-kappa B was generated from Jurkat T cell complementary DNA with the polymerase chain reaction (PCR) and degenerate oligonucleotides. This probe was used to identify a rel-related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes. In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons (kD). The translated protein showed weak DNA binding with a specificity for the kappa B binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex. Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex. 2006697 Lymphocyte glucocorticoid receptor number in posttraumatic stress disorder. OBJECTIVE: The authors ' objective was to investigate the possibility that glucocorticoid receptor changes may be involved in the dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in posttraumatic stress disorder (PTSD). METHOD: They measured the number of lymphocyte cytosolic glucocorticoid receptors and plasma cortisol concentrations in 15 consecutively admitted male combat Vietnam veterans with PTSD and in a normal comparison group of 11 subjects. RESULTS: Both the patients and the normal comparison subjects showed a morning-to-afternoon decline in glucocorticoid receptor concentrations, paralleling the normal diurnal decline in cortisol levels. The number of glucocorticoid receptors was 63 % greater in the morning and 26 % greater in the afternoon in the patients with PTSD than in the normal subjects. No group differences in cortisol levels were observed, nor were glucocorticoid receptor number and cortisol levels correlated. The number of morning glucocorticoid receptors was positively correlated with symptoms of PTSD and anxiety. CONCLUSIONS: These results provide further evidence for a dysregulation of the HPA axis in PTSD. The finding that patients with PTSD had a substantially greater number of lymphocyte glucocorticoid receptors than normal comparison subjects is consistent with the authors ' previous observations of low 24-hour urinary cortisol excretion in subjects with PTSD. Furthermore, the receptor changes observed are opposite of those reported in major depressive disorder. The present data, along with other findings of HPA abnormalities in PTSD, support the possibility of a greater negative feedback sensitivity at one or more levels of the HPA axis. 2010090 A thymus-specific member of the HMG protein family regulates the human T cell receptor C alpha enhancer. The human T cell-specific transcription factor TCF-1 alpha plays a key role in the tissue-specific activation of the T cell receptor ( TCR ) C alpha enhancer and binds to pyrimidine-rich elements (5'-PyCTTTG-3') present in a variety of other T cell-specific control regions. Using amino acid sequence information derived from the DNA affinity-purified protein, we have now isolated cDNA clones encoding TCF-1 alpha. The TCF-1 alpha cDNA contains a single 68-amino-acid domain that is homologous to a region conserved among high-mobility group (HMG) and nonhistone chromosomal proteins. Expression of full-length and mutant cDNA clones in bacteria reveal that the single HMG motif, which is predicted to contain two extended alpha-helical segments, is sufficient to direct the sequence-specific binding of TCF-1 alpha to DNA. Northern blot experiments demonstrate further that TCF-1 alpha mRNA is highly tissue specific, found primarily in the thymus or T cell lines. The immature CEM T cell line expresses relatively low levels of TCF-1 alpha mRNA, which are increased upon activation of these cells by phorbol esters. Interestingly, the cloned TCF-1 alpha protein is a potent transcriptional activator of the human TCR alpha enhancer in nonlymphoid cell lines, whereas the activity of the endogenous protein in T cell lines is strongly dependent on an additional T cell-specific protein that interacts with the core enhancer. TCF-1 alpha is currently unique among the newly emerging family of DNA-binding regulatory proteins that share the HMG motif in that it is a highly tissue-specific RNA polymerase II transcription factor. 1707027 Immune response of peripheral blood mononuclear cells to HBx-antigen of hepatitis B virus. The hepatitis B virus genome encodes a transcriptional transactivator protein designated HBxAg. We have investigated whether this antigen is a target structure for human T-lymphocytes. Using recombinant HBxAg protein, we found HBxAg-specific stimulation of peripheral blood mononuclear cells in patients with acute hepatitis B virus infection (6 of 6) and chronic hepatitis B virus infection (6 of 17) but not in healthy individuals. With HBxAg-specific synthetic polypeptides, several T-cell epitopes were identified. Most were located in the carboxyterminal half of the HBxAg protein. Five T-cell clones specific for a T-cell epitope located at the carboxyterminal region of HBxAg were established and found to belong to the CD2/CD4-positive, CD8-negative subtype. These data establish for the first time HBxAg as an antigen in the cellular immune response. 1901389 A study on the circadian rhythm of glucocorticoid receptor. Circadian rhythm in glucocorticoid receptor (GR) was studied in the rat liver and human peripheral leukocytes. For rats exposed to a natural environmental photic cycle or a 12L : 12D artificial light regime, peak values of hepatic GR were detected between 23:00 and 02:00 h. Except for a 4-hour advancement of the peak, a similar circadian rhythm of hepatic GR was detected in rats reared under a reversed lighting regimen (12D : 12L; lights on between 18:30 and 06:30 h). In human leukocytes, the peak value of GR was found to parallel that of plasma cortisol with high and low values detected at 04:00-08:00 h and 23:00-24:00 h, respectively. In patients suffering from Cushing 's syndrome, the circadian rhythm of plasma cortisol either disappeared or was inverted while that of GR did not significantly deviate from the normal subjects. For apoplexic patients with lesions localized to the base of the brain as indicated by computerized tomography, the diurnal variation of GR was abolished. Conversely, diurnal rhythmicity persisted in apoplexy patients whose lesions were in the cerebral cortex. Thus, we postulated that the circadian modification of GR was independent of the diurnal fluctuations in plasma cortisol level or the circadian variations in environmental lighting and that the rhythmicity might be regulated by the ' circadian pacemaker ' located in the human basal brain. These diurnal variations in GR might serve to coordinate the reactivity of the target cells to cortisol because the diurnal rhythms of a GR-mediated response, the fractional inhibition of chemotactic migration rate of polymorphonuclear leukocytes by cortisol, were found to be synchronous with those of GR. 2011512 Multiple Oct2 isoforms are generated by alternative splicing. The interaction of the Oct2 transcription factor with the cognate octamer motif ATGCAAAT is a critical determinant of the lymphoid-specific expression of immunoglobulin genes. Ectopic expression of cloned Oct2 cDNA was shown to be sufficient to reconstitute at least some aspects of this regulation in non-lymphoid cells. We describe the isolation and characterization of multiple cDNAs encoding mouse Oct2 from a mature B-cell line and we show that a variety of isoforms of this transcription factor is generated from a single gene by an alternative splicing mechanism. All the isoforms retain the previously characterized POU-domain and are therefore able to bind to the octamer motif. Different amounts of the various isoforms are present within the same B-cell regardless of the developmental stage of B-cell differentiation and at least some of the isoforms are conserved between mouse and humans. In cotransfection experiments we show that all the isoforms are able to activate an octamer containing promoter element in fibroblasts revealing an unexpected functional redundancy. Finally, we show that one of the isoforms encodes the previously described lymphoid-specific Oct2B protein which has been suggested to be involved in the function of the octamer motif in the context of the immunoglobulin heavy-chain (IgH) enhancer. 2017177 Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer. A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation. 2017258 Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection. Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is regulated in part by cellular factors and is stimulated by activation of latently infected T cells. T-cell activation also correlates with the induction of the factor NF-kappa B which binds to two adjacent sites in the HIV-1 long terminal repeat. This factor consists of two DNA-binding subunits of relative molecular mass 50,000 (50K) associated with two 65K subunits. It is located in the nucleus in mature B cells, but is present in other cell types as an inactive cytoplasmic complex. External stimuli, including those that activate T cells, result in nuclear translocation of active NF-kappa B. The cloning of the complementary DNA for the 50K subunit helped to identify an exclusively cytoplasmic 105K precursor (p105) (V.B., P.K. and A.I., manuscript submitted). The expression of active NF-kappa B might therefore also be regulated by the extent of processing of p105. Because HIV-1 requires active NF-kappa B for efficient transcription, we tested the effect of HIV-1 infection on the processing of the human 105K precursor. We show here that the HIV-1 protease can process p105 and increases levels of active nuclear NF-kappa B complex. 1850412 Vitamin D receptor expression in human lymphocytes. Signal requirements and characterization by western blots and DNA sequencing. The signals controlling the expression of the receptor protein for 1 alpha,25-dihydroxyvitamin D3 in normal human lymphocytes and the relationship of this protein to the classical vitamin D receptor were examined. Lymphocytes activated with the OKT3 antibody to the T-cell antigen receptor expressed fewer binding sites as compared to lymphocytes that were activated by the polyclonal activator phytohemagglutinin (PHA). However, combination of OKT3 and phorbol myristate acetate produced a concentration of binding sites similar to the PHA-activated cells. The receptor from OKT3 and OKT3 + phorbol myristate acetate-activated lymphocytes exhibited decreased binding to DNA-cellulose compared to PHA-activated lymphocytes. In lymphocytes activated either by PHA or OKT3 ( but not in resting cells), a 50-kDa species cross-reacting with a monoclonal antibody against the intestinal vitamin D receptor was detected. Finally, RNA from activated lymphocytes was amplified by polymerase chain reaction using oligonucleotide primers flanking the 196 base pair long region encoding the DNA-binding domain of the human intestinal receptor. The amplified product showed an identical nucleotide sequence to the DNA-binding domain of the human intestinal receptor. These findings suggest that expression of the 1,25-(OH)2D3 receptor in lymphocytes is triggered by distinct and contingent signals, and that the protein and the mRNA encoding it are identical to the classical vitamin D receptor. 1827138 Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers. CD3-epsilon gene expression is confined to the T cell lineage. We have recently identified and cloned a human transcription factor, TCF-1, that binds to a functional element in the T lymphocyte-specific enhancer of CD3-epsilon. In a panel of human cell lines, TCF-1 expression was restricted to T lineage cells. TCF-1 belonged to a novel family of genes that contain the so-called high mobility group 1 ( HMG ) box. Here we report the cloning of murine TCF-1. Two splice alternatives were identified that were not previously observed in human TCF-1. Murine and human TCF-1 displayed a 95.5 % overall amino acid homology. Recombinant murine and human TCF-1 recognized the same sequence motif in the CD3-epsilon enhancer as judged by gel retardation and methylation interference assays. With the murine cDNA clones several aspects of TCF-1 were analyzed. First, deletion analysis revealed that a region of TCF-1 containing the HMG box was sufficient for sequence-specific binding. Second, by high stringency Northern blotting and in situ hybridization, TCF-1 expression was shown to be confined to the thymus and to the T cell areas of the spleen. Third, TCF-1 bound specifically to a functional T cell-specific element in the T cell receptor alpha (TCR-alpha) enhancer. The T lineage-specific expression and the affinity for functional motifs in the TCR-alpha and CD3-epsilon enhancers imply an important role for TCF-1 in the establishment of the mature T cell phenotype. 2023633 HIV enhancer activity perpetuated by NF-kappa B induction on infection of monocytes [see comments] Permissiveness to replication of human immunodeficiency virus (HIV) differs in T lymphocytes and macrophages. In T cells, HIV transcription is poorly detected in vivo. Cloned, normal T lymphocytes show very little, if any, basal activity of the HIV enhancer and low nuclear expression of NF-kappa B, a potent transcriptional activator of the HIV enhancer. In contrast, fixed tissue macrophages express detectable HIV proteins, indicating permanent virus transcription. One explanation for the perpetuation of virus infection in macrophages could be sustained nuclear NF-kappa B expression. However, the U937 monocytic cell line, which is fully permissive to HIV replication, is known to express only low levels of nuclear NF-kappa B. We show here that chronic HIV infection results in both induction of a nuclear factor with antigenic properties indistinguishable from those of NF-kappa B and permanently increased HIV enhancer activity. This phenomenon, which is independent of tumour necrosis factor, is associated with HIV replication, and is thus likely to explain at least in part the perpetuation of HIV infection in monocytes. 1850841 Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection. During latent Epstein-Barr virus (EBV) infection of human B lymphocytes, six viral nuclear antigen (EBNAs) are expressed from long primary transcripts by means of alternative splicing and alternative polyadenylylation sites. These transcripts initiate from one of two promoters, Cp or Wp, that function in a mutually exclusive fashion. Wp is exclusively utilized during the initial stages of infection of primary B lymphocytes, followed by a switch to Cp usage. These studies have been extended to show that ( i ) a mutant EBV strain lacking the gene encoding EBNA 2 fails to switch from Wp to Cp usage in primary B lymphocytes, although the virus contains a functional Cp; ( ii ) a region from -429 to -245 base pairs upstream of Cp is essential for Cp activity in B lymphocytes, but only in the context of upstream and downstream sequences; ( iii ) this region contains an EBNA 2-dependent enhancer; and ( iv ) DNase I protection employing nuclear extracts from B and T lymphocytes revealed a B-cell-specific footprint in the region of the EBNA 2-dependent enhancer. These results support a model for viral promoter switching during the initial stages of infection in which Wp activity leads to the expression of EBNA 2, followed by activation of Cp through the EBNA 2-dependent enhancer. 2024810 Glucocorticoid receptor characteristics in monocytes of patients with corticosteroid-resistant bronchial asthma. The mechanism of corticosteroid resistance in bronchial asthma has been studied by determining the rank order of potency for different corticosteroids in inhibiting the generation of a 3 kD molecule from peripheral blood monocytes isolated from corticosteroid-sensitive (CS) and corticosteroid-resistant (CR) asthmatic subjects, which augments leukotriene B4 (LTB4) generation by human neutrophils (PMN) stimulated by calcium ionophore. In addition, binding studies with ( 3H ) dexamethasone have been performed to determine the dissociation constant (Kd) and receptor numbers (Ro) in the monocytes of these two groups of subjects. The concentration of corticosteroid producing 50 % inhibition (IC50) was 600 nM, 70 nM, and 0.5 nM for hydrocortisone, methylprednisolone, and dexamethasone, respectively, in monocytes from CS individuals. There was only weak inhibition of the generation of enhancing activity by the corticosteroids in the CR asthmatic individuals. The dexamethasone Kd was 2.45 ± 0.58 nM (mean ± SEM, n = 6) in the CS group and 1.6 ± 0.35 nM (mean ± SEM, n = 6) in the CR group of patients (p = 0.14). The Ro in the CS group was 3,605 ± 984 binding sites per nucleus (mean ± SEM, n = 6) and 4,757 ± 692 binding sites per nucleus (mean ± SEM, n = 6) in the CR group (p = 0.23). These findings indicate that corticosteroid resistance in bronchial asthma can not be explained by abnormalities in corticosteroid receptor characteristics. 2026605 Tissue-specific expression of the platelet GPIIb gene. One of the major objectives in the study of thrombogenesis is to determine the mechanisms by which a hematopoietic progenitor is activated and committed to the megakaryocytic lineage. Recent development of primary cultures of human megakaryocytes and the molecular cloning of genes that are specific to this lineage offer the possibility of getting some insights into the genetic mechanisms that control megakaryocytopoiesis. One gene of interest is the glycoprotein IIb (GPIIb) gene; GPIIb, the alpha subunit of the platelet cytoadhesin GPIIb-IIIa, is produced in megakaryocytes at an early stage of the differentiation, whereas the other subunit of this complex, GPIIIa, is expressed in other cells. For these reasons, the 5'-flanking region of the GPIIb gene was used to identify the regions that interact with DNA-binding nuclear factors. A fragment extending from -643 to +33 is capable of controlling the tissue-specific expression of the CAT gene in transfection experiments. Within this region, we have identified several sequences that are implicated in DNA protein interactions as shown in DNAse I footprints and gel mobility shift assays. One region, centered at -54, is similar to a nuclear factor E1-binding site, and a region located at position -233 contains a CCAAT motif. Two domains centered at positions -345 and -540, respectively, bind proteins that are present in megakaryocytic cells and nonrelated cells as well. Finally, two other domains, located at positions -460 and -510, interact with proteins that are only present in megakaryocytic cells. In addition, deletion of the region containing these two domains results in a significant decrease of the promoter activity. It is very likely that these domains bind megakaryocyte-specific nuclear proteins acting as positive transcription factors. 1851743 Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells. To examine the role of protein phosphatases in T cell activation, Jurkat cells were treated with okadaic acid, an inhibitor of type 1 and 2A phosphatases, and nuclear extracts were examined for the presence of AP1 as a measure of early T cell activation. Okadaic acid was found to be a potent inducer of AP1. In contrast to phorbol esters such as phorbol myristate acetate (PMA), the induction of AP1 by okadaic acid occurs predominantly by transcriptional activation of the jun and fos family of proto-oncogenes. Surprisingly, while the addition of phytohemagglutinin further enhanced the induction of AP1, the addition of PMA inhibited it. Okadaic acid treatment was found to dramatically increase mRNA transcripts of the jun family of proto-oncogenes including c-jun, junD, and junB and to a lesser extent the fos family including c-fos and fra-1. By comparison, PMA is a very inefficient inducer of the jun gene family in Jurkat cells. Similar to its effect on the induction of AP1 by okadaic acid, PMA inhibits the induction of c-jun mRNA by okadaic acid. Transfection of c-jun promoter constructs confirmed the marked difference between PMA and okadaic acid in inducing c-jun transcription. The induction of AP1 by okadaic acid suggests that protein phosphatases 1 and 2A (PP1 and PP2A) may be involved in T cell activation as important negative regulators of the transcription factor AP1. 2033090 Demonstration of estrogen and progesterone receptors as well as Ki-67 and p-145 antigens in single tumor cells from blood and pleural effusions using a slide assay. We describe a slide assay that allows the demonstration of antigens localized in the nucleus from isolated white blood cells as well as from single tumor cells derived from malignant effusions. With the antibodies Ki-67 and anti-p-145 an increased rate of nuclear and nucleolar staining resulted in cells from highly malignant lymphomas. An almost identical reaction was obtained when tumor cells from malignant effusions were tested. Cells isolated from the blood of patients with leukemic spread of lymphomas of low malignancy yielded a weak staining comparable to that of normal mesothelial cells from non-tumorous cavity fluids. The detection of estrogen and progesterone receptors (ER and PR) localized in the cell nucleus can be achieved by the same assay. The reaction is enhanced by incubation of the tumor cells for 30 min at 37 degrees C prior to fixation. Pleural effusions from 20 patients with breast cancer were tested. ER was positive in 13 and PR was positive in 12 of the 20 samples. In 5 cases there was a divergent reaction with ER and PR antibody. The hormone receptors of the primary tumor were known in 15 (ER) and 14 (PR) patients, respectively. In each cohort there was only one case with a negative reaction of the primary tumor and a positive reaction with the isolated tumor cells from the pleural effusions. These results indicate that the demonstration of hormone receptor proteins in cells from malignant effusions is possible and that there is a correlation with the status of the primary site of cancer. 1903417 Transforming growth factor-beta suppresses human B lymphocyte Ig production by inhibiting synthesis and the switch from the membrane form to the secreted form of Ig mRNA. Transforming growth factor-beta (TGF-beta) inhibits B cell Ig secretion and reduces B cell membrane Ig expression. The addition of TGF-beta to human B lymphocyte cultures stimulated with Staphylococcus aureus Cowan strain I and IL-2 completely inhibited B cell Ig secretion (greater than 90 %) and decreased B cell surface IgM, IgD, kappa L chain, and lambda L chain expression. In contrast, TGF-beta had only minimal effects on two other B cell membrane proteins, HLA-DR and CD20. Internal labeling with [35S]methionine and immunoprecipitation with anti-IgM, anti-kappa, and anti-lambda antibodies revealed a striking reduction in kappa L chain in the presence of TGF-beta. A less pronounced reduction in lambda L chain and microH chain was also noted. Northern blot analysis of RNA purified from B cells treated with TGF-beta for varying time intervals revealed a significant decrease in steady state kappa and lambda L chain mRNA levels. Furthermore, a significant decrease in the switch from the membrane forms of mu and gamma to their respective secreted forms was noted in the presence of TGF-beta. Nuclear run-on experiments demonstrated decreased transcription of kappa L chain. The effects of TGF-beta on two transcriptional regulatory factors, Oct-2 and nuclear factor (NF) kappa B, known to be important in Ig gene transcription were examined. Oct-2 mRNA levels and both Oct-2 and NF-kappa B proteins in nuclear extracts were not altered by treatment with TGF-beta. In contrast, levels of the transcriptional factor AP-1, which is not known to be important in B cell Ig production, were reduced by TGF-beta. These findings demonstrate that TGF-beta decreases B lymphocyte Ig secretion by inhibiting the synthesis of Ig mRNA and inhibiting the switch from the membrane form to the secreted forms of mu and gamma mRNA. The mechanism by which TGF-beta inhibits Ig chain synthesis is unclear although it does not involve inhibition of the binding of NF-kappa B or Oct-2 to their respective target sequences. 1851858 Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia. The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells. We have generated a monoclonal antibody, BZ1, to BZLF1 which reacts in immunohistology, immunoblotting, and immunoprecipitation and which recognizes both the active, dimeric form and the inactive, monomeric form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-associated lesion characterized by high-level EBV replication, were examined by immunohistochemistry using the BZ1 monoclonal antibody. A differentiation-associated pattern of BZLF1 expression was observed, BZ1 reacting with nuclei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication. The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium. 1851861 Transactivation of the human immunodeficiency virus promoter by human herpesvirus 6 (HHV-6) strains GS and Z-29 in primary human T lymphocytes and identification of transactivating HHV-6 ( GS ) gene fragments. Human herpesvirus 6 (HHV-6) can activate the human immunodeficiency virus (HIV) promoter and accelerate cytopathic effects in HIV-infected human T cells. This study examines the regions of the HIV promoter required for HHV-6 transactivation in a heterogeneous population of primary human T lymphocytes with or without antigenic stimulation. Two different strains of HHV-6, GS and Z29, transactivated the HIV promoter. The GS strain transactivated the promoter in both stimulated and resting T cells, while the Z29 strain increased HIV promoter activity only in stimulated T cells. Three DNA clones containing HHV-6 ( GS ) genomic fragments transactivated the HIV promoter in cotransfected T cells. A 21.4-kb DNA clone, pZVB70, showed the highest transactivating ability, while two other DNA fragments, pZVB10 (6.2 kb) and pZVH14 (8.7 kb), showed lower activity. One of these clones, pZVH14, activated the HIV promoter construct containing a mutation in the NF kappa B site. However, this mutated NF kappa B promoter was not transactivated during HHV-6 ( GS ) infection or after cotransfection with pZVB70 or pZVB10. These data indicate that the NF kappa B sites of the HIV promoter are essential for its transactivation during HHV-6 ( GS ) infection. By increasing HIV promoter activity in primary T lymphocytes, HHV-6 may consequently increase HIV replication, leading to an increase in the cytopathic effect on coinfected human T cells. 2034676 The rhombotin family of cysteine-rich LIM-domain oncogenes: distinct members are involved in T-cell translocations to human chromosomes 11p15 and 11p13. A chromosomal translocation in a T-cell leukemia involving the short arm of human chromosome 11 at band 11p15 disrupts the rhombotin gene. This gene encodes a protein with duplicated cysteine-rich regions called LIM domains, which show homology to zinc-binding proteins and to iron-sulfur centers of ferredoxins. Two homologues of the rhombotin gene have now been isolated. One of these, designated Rhom-2, is located on human chromosome 11 at band 11p13, where a cluster of T-cell leukemia-specific translocations occur; all translocation breakpoints at 11p13 are upstream of the Rhom-2 gene. Human and mouse Rhom-2 are highly conserved and , like rhombotin, encode two tandem cysteine-rich LIM domains. Rhom-2 mRNA is expressed in early mouse development in central nervous system, lung, kidney, liver, and spleen but only very low levels occur in thymus. The other gene, designated Rhom-3, is not on chromosome 11 but also retains homology to the LIM domain of rhombotin. Since the Rhom-2 gene is such a common site of chromosomal damage in T-cell tumors, the consistency of translocations near the rhombotin gene was further examined. A second translocation adjacent to rhombotin was found and at the same position as in the previous example. Therefore, chromosome bands 11p15 (rhombotin) and 11p13 (Rhom-2) are consistent sites of chromosome translocation in T-cell leukemia, with the 11p15 target more rarely involved. The results define the rhombotin gene family as a class of T-cell oncogenes with duplicated cysteine-rich LIM domains. 1645452 The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for : 1 ) the presence of functional 1,25-(OH)2D3 receptors; 2 ) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3 ) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200 % the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o 2039752 Cortivazol mediated induction of glucocorticoid receptor messenger ribonucleic acid in wild-type and dexamethasone-resistant human leukemic ( CEM ) cells. Cortivazol is a phenylpyrazolo glucocorticoid of high potency and unusual structure. In both wild-type and highly dexamethasone(dex)-resistant clones of the human leukemic cell line CEM, exposure to cortivazol leads to cell death. It has been shown recently that in wild-type CEM cells but not in a dex-resistant, glucocorticoid receptor ( GR ) -defective clone ICR-27 TK-3, dex induces GR mRNA. To test the hypothesis that cortivazol acts in dex-resistant cells by making use of the residual GR found there, wild-type and dex-resistant clones were treated with various concentrations of cortivazol and induction of GR mRNA was studied. Cortivazol significantly induced GR mRNA in the normal CEM-C7 as well as in two classes of dex-resistant clones, although the dex-resistant clones needed at least 10 times more cortivazol than the normal cells for significant GR mRNA induction. Increased levels of GR mRNA were noticed as early as 3 h after treatment. A general correlation between induction of GR mRNA and lysis of the normal and dex-resistant cells was found. Positive induction of GR mRNA might be one of the earliest crucial steps in the lysis of normal and dex-resistant CEM cells, or might serve as a marker for the process. However, the lysis pathway in the dex-resistant cells is defective in that dex-resistant clones needed significantly more cortivazol than the normal cells for lysis of the cells. 1646583 Expression of 1,25(OH)2D3 receptors on alveolar lymphocytes from patients with pulmonary granulomatous diseases. 1,25(OH)2D3 is known to be produced at sites of granulomatous reactions. In order to characterize the cell types that are targets for this immunoregulatory hormone, we have evaluated the expression of 1,25(OH)2D3 receptors on peripheral blood T-lymphocytes and those recovered from the lung by bronchoalveolar lavage from patients with pulmonary granulomatous diseases (tuberculosis and sarcoidosis) and from normal control subjects using combined autoradiographic and immunohistochemical techniques. Lavage T-lymphocytes from patients with tuberculosis or with sarcoidosis, but not those from normal control subjects, expressed 1,25(OH)2D3 receptors as demonstrated by binding of [3H]1,25(OH)2D3, which was inhibited by the presence of excess unlabeled 1,25(OH)2D3, but not by the presence of unlabeled 25(OH)D3 (receptor-positive lymphocytes: sarcoidosis, 20 ± 12%; tuberculosis, 31 ± 17%). In contrast, blood lymphocytes from patients with granulomatous diseases did not express detectable 1,25(OH)2D3 receptors. The percentage of lavage T-lymphocytes expressing 1,25(OH)2D3 receptors was significantly greater for patients with tuberculosis presenting with isolated hilar adenopathy than for patients with pulmonary infiltrates and/or cavities. 1,25(OH)2D3 receptors were expressed to a greater extent on CD8+ T-lymphocytes than on CD4+ T-lymphocytes in sarcoidosis, whereas a greater proportion of CD4+ than of CD8+ T-lymphocytes from patients with tuberculosis were receptor-positive. These findings support the conclusion that the interaction of 1,25(OH)2D3 with its receptor on T-lymphocytes may play an important role in the regulation of granulomatous reactions, but because these receptors are expressed on different lymphocyte populations, the net effect of this potent immunoregulatory molecule is likely different in sarcoidosis and tuberculosis. 1675604 Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant. The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel ( proto ) oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 ( delta SP ), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family. 2050125 Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA. 5-Aminolevulinate synthase (ALAS) catalyzes the first step of the heme biosynthetic pathway. cDNA clones for the human erythroid ALAS isozyme were isolated from a fetal liver library. It can be deduced that the erythroid ALAS precursor protein has a molecular weight of 64.6 kd, and is similar in size to the previously isolated human housekeeping ALAS precursor of molecular weight 70.6 kd. The mature mitochondrial forms of the erythroid and housekeeping ALAS isozymes are predicted to have molecular weights of 59.5 kd and 64.6 kd, respectively. The two isozymes show little amino acid identity in their N-terminal signal sequences but have considerable sequence identity in the C-terminal two-thirds of their proteins. An analysis of the immediate promoter of the human erythroid ALAS gene revealed several putative erythroid-specific cis-acting elements including both a GATA-1 and an NF-E2 binding site. An iron-responsive element (IRE) motif has been identified in the 5'-untranslated region of the human erythroid ALAS mRNA, but is not present in the housekeeping ALAS mRNA. Gel retardation experiments established that this IRE motif formed a protein-RNA complex with cytosolic extracts from human K562 cells and this binding was strongly competed with IRE transcripts from ferritin or transferrin receptor mRNAs. A transcript of the ALAS IRE, mutated in the conserved loop of the IRE, did not readily form this protein-RNA complex. These results suggest that the IRE motif in the ALAS mRNA is functional and imply that translation of the mRNA is controlled by cellular iron availability during erythropoiesis. 2056282 Human tumor necrosis factor alpha gene regulation in phorbol ester stimulated T and B cell lines. The minimal region of the human tumor necrosis factor alpha ( TNF-alpha ) gene promoter necessary for its transcriptional induction by phorbol esters (PMA) in human T and B lymphocyte cell lines has been localized between -52 and +89 nucleotides (nt) relative to the gene 's transcriptional start site. Comparison of these sequences to those required to mediate virus or lipopolysaccharide (LPS) induction of the gene reveal significant differences, and thus , the sequence requirements for PMA induction are distinct from those that mediate induction by virus or LPS. Although three sites in the TNF-alpha promoter (kappa 1, kappa 2, and kappa 3) specifically bind the transcription factor NF-kappa B in lymphoid nuclear extracts, TNF-alpha mRNA induction by PMA does not correlate with NF-kappa B binding activities displayed by different T and B cell lines. Moreover, kappa 1-kappa 3 can each be deleted from the TNF-alpha promoter with little effect on the gene 's inducibility by PMA. Therefore, TNF-alpha mRNA induction by PMA, like its induction by virus and LPS, is not primarily mediated by NF-kappa B, but rather is mediated through other sequences and protein factors. Surprisingly, multimers of kappa 1-kappa 3 can confer PMA inducibility on a heterologous promoter in a B (Raji), but not a T (HUT78) cell line. However they are not functional on a truncated TNF-alpha promoter, indicating that promoter context and cell type specificity influence the PMA inducible function of these NF-kappa B binding sites. 1676267 Towards a molecular understanding of T-cell differentiation. Lymphoid differentiation is one of the best studied examples of mammalian development. Here Hans Clevers and Michael Owen describe how the cloning of the genes that encode T-cell-specific membrane proteins allows the identification of transcription factors that control the expression of these T-cell genes. Such transcription factors play a key role in the development of the mature T-cell phenotype by functioning as 'master regulators of T-cell differentiation'. 1712226 Regulation of jun and fos gene expression in human monocytes by the macrophage colony-stimulating factor. The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. However, the signaling events responsible for these effects remain unclear. The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes. Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes. Treatment of these cells with 10(3) units/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels. Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts. M-CSF treatment was also associated with a rapid induction of the jun-B gene, although expression of this gene was prolonged compared to that of c-jun. We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels. Maximal induction of the c-fos gene was followed by that for the fos-B gene. Moreover, M-CSF-induced expression of the fos-related gene, fra-1, was delayed compared to that for both c-fos and fos-B. Taken together, the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation. 1676597 Cloning of a human homeobox gene that resembles a diverged Drosophila homeobox gene and is expressed in activated lymphocytes. A new homeobox gene, HB24, has been isolated from a human B-lymphocyte cDNA library. Northern blot analysis of polyadenylated RNA purified from activated human B cells revealed a single mRNA transcript of approximately 2.3 kb. Two cDNA clones were sequenced and provided 2,250 nucleotides (nt) of DNA sequence information. There is a single methionine codon-initiated open reading frame of 1,458 nt in frame with a homeobox and a CAX repeat, and the open reading frame is predicted to encode a protein of 51,659 daltons. When the homeodomain from HB24 was compared to known mammalian and Drosophila homeodomains it was found to be only moderately conserved, but when it was compared to a highly diverged Drosophila homeodomain, H2.0, it was found to be 80 % identical. The HB24 mRNA was absent or present at low levels in normal B and T lymphocytes; however, with the appropriate activation signal HB24 mRNA was induced within several hours even in the presence of cycloheximide. Characterization of HB24 expression in lymphoid and select developing tissues was performed by in situ hybridization. Positive hybridization was found in thymus, tonsil, bone marrow, developing vessels, and in fetal brain. HB24 is likely to have an important role in lymphocytes as well as in certain developing tissues. 1648430 Severe 5-fluorouracil toxicity secondary to dihydropyrimidine dehydrogenase deficiency. A potentially more common pharmacogenetic syndrome. This study describes the inheritance of a defect in pyrimidine catabolism and its association with drug-induced toxicity in a patient receiving 5-fluorouracil (FUra) as adjuvant chemotherapy for breast carcinoma. The study population included the affected patient (proband), nine of her blood relatives, and seven healthy volunteers. The activity of dihydropyrimidine dehydrogenase (DPD), the initial enzyme of pyrimidine ( and FUra) catabolism, in peripheral blood mononuclear cells was measured in each subject by a specific radiometric assay using FUra as the substrate. The proband had no detectable DPD activity. When enzyme levels in the proband and relatives were compared with that in controls, an autosomal recessive pattern of inheritance was demonstrated. This is the third patient with severe FUra toxicity secondary to an alteration in pyrimidine catabolism and the second from our clinic population suggesting that the frequency of this genetic defect may be greater than previously thought. Monitoring DPD activity may be important in the management of patients experiencing severe toxicity secondary to FUra chemotherapy. 1829648 Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity. We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60 % similar (46 % identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but not that of the p50/p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation. 2065663 Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes. In this study, we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line. The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form. N-acetyl-L-cysteine (NAC), a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates (ROI) in living cells, prevented the activation of NF-kappa B by H2O2. NAC and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide, double-stranded RNA, calcium ionophore, TNF-alpha, active phorbol ester, interleukin-1, lipopolysaccharide and lectin. This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI. ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B. 1906155 HTLV-1 Tax induces expression of various immediate early serum responsive genes. Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of adult T-cell leukemia (ATL). We showed here by mobility-shift assay that T-cell lines transformed with the virus contained high levels of AP-1 activities. Consistent with this result, these cell lines expressed increased levels of mRNAs encoding the AP-1 proteins, c-Fos, Fra-1, c-Jun, JunB, and JunD. Previously, transcription of the c-fos gene has been reported to be transactivated by the viral transcription factor, Tax1. By using the human T-cell line (JPX-9), in which expression of the Tax1 is inducible, we showed that expression of mRNAs for Fra-1, c-Jun, and JunD was also transactivated by Tax1. Moreover, Tax1 activated expression of two other transcription factors having zinc finger motifs, Egr-1 and Egr-2, in the same cells. The Tax1-inducible transcription factors identified here are encoded by the members of immediate early genes under the control of growth signals. Thus , Tax1 was suggested to replace growth signals, at least in part, by this mechanism. 2072454 Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types. Starting with a replication-incompetent molecular clone of human immunodeficiency virus type 1, lacking all the NF-kappa B and Sp1 binding sites present in the native long terminal repeat (LTR), proviruses containing reconstructed LTRs with individual or combinations of NF-kappa B and Sp1 elements were generated and evaluated for their capacity to produce virus progeny following transfection-cocultivation. Virus stocks obtained from these experiments exhibited a continuum of replicative capacities in different human T-cell types depending on which element ( s ) was present in the LTR. For example, in experiments involving proviral clones with LTRs containing one or two NF-kappa B elements ( and no Sp1 binding sites ), a hierarchy of cellular permissivity to virus replication (peripheral blood lymphocytes = MT4 greater than H9 greater than CEM greater than Jurkat) was observed. Of note was the associated emergence of second-site LTR revertants which involved an alteration of the TATA box. These results suggest that the human immunodeficiency virus type 1 LTR possesses functional redundancy which ensures virus replication in different T-cell types and is capable of changing depending on the particular combination of transcriptional factors present. 1650477 Enhancement of human immunodeficiency virus 1 replication in monocytes by 1,25-dihydroxycholecalciferol. Human immunodeficiency virus (HIV) expression and replication are under tight regulatory control. We demonstrate that 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances the replication of monocyte- and lymphocyte-tropic strains of HIV-1 up to 10,000-fold in monocyte cell lines, peripheral blood monocytes, and unfractionated peripheral blood mononuclear cells. 1,25(OH)2D3 is therefore one of the most potent regulators of HIV-1 replication described to date. Precursors of 1,25(OH)2D3 enhance HIV-1 replication in proportion to their affinity for the 1,25(OH)2D3 intracellular receptor, suggesting that 1,25(OH)2D3 influences HIV-1 replication by mechanisms involving this receptor. These studies may have important implications for the design of effective therapy of HIV-1 infection. 1907460 Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives. HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B. HIV-1-infected individuals have , on average, abnormally high levels of tumour necrosis factor alpha (TNF alpha) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione ( GSH ) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-kappa B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals. 1865413 Glucocorticoid receptors in systemic lupus erythematosus. Glucocorticosteroids remain the major treatment modality for systemic lupus erythematosus (SLE), but their mechanism of action is unclear. Over the past decade it has become clear that glucocorticosteroid receptors play a significant role in the mechanism of glucocorticosteroid action. We studied glucocorticosteroid receptor density and affinity on peripheral blood mononuclear cells by the glucocorticosteroid binding assay in 33 patients with SLE who had taken no glucocorticosteroid for the previous 6 months and in 32 healthy controls. Patients ' disease activity was measured by the SLE Disease Activity Index (SLEDAI). Glucocorticosteroid receptors on leukocytes of patients with SLE were significantly higher than in healthy controls (4419 ± 306 vs 3369 ± 196, p less than 0.005). The binding affinity was not different between patients and controls. There was no correlation between glucocorticosteroid receptor number and SLE disease activity. 1874085 [Changes in leucocytic estrogen receptor levels in patients with gynecomastia] The number of estrogen receptor (ER) in human peripheral leucocytes in 13 men with gynecomastia were measured by radioligand binding method. The results were compared with those of 13 sex- and age-matched healthy subjects. It was found that the number of ER in leucocytes was significantly increased in gynecomastia (Rs of leucocytes were 1054 ± 254 sites/cell). It suggested that increase of ER levels play an important role in the pathogenesis of gynecomastia. 1879243 [Changes in levels of leucocytic estrogen receptor in patients with menopausal type II diabetes and its significance] The number of estrogen receptors (ER) in human peripheral leucocytes in 12 women with menopausal type II diabetes was measured with radio-ligand binding method. The results were compared with those of 12 menopausal women without diabetes and 12 normal women of childbearing age. It was found that the number of ER in the patients was significantly decreased. Our data indicate that decrease of ER level in leukocytes may be related to the pathogenesis of type II diabetes in menopausal period. 1652603 Lymphocyte glucocorticoid receptor binding during depression and after clinical recovery. Lymphocyte glucocorticoid receptor binding parameters were studied in 15 severely depressed patients during depression and after clinical recovery, and in 15 healthy controls. There was no difference in glucocorticoid receptor number or affinity between depressed patients and recovered or control subjects. Afternoon ACTH and cortisol concentrations did not differ significantly between the three groups. No relationship could be established between glucocorticoid receptor binding and antidepressant medication. These data support the view of an impaired ligand-induced plasticity of glucocorticoid receptor regulation rather than the hypothesis of decreased glucocorticoid receptor numbers during depression. 1715516 Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A [see comments] Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit. 1883525 Regulation of M-CSF expression by M-CSF: role of protein kinase C and transcription factor NF kappa B. Macrophage-colony-stimulating factor (M-CSF), also referred to as CSF-1, regulates the survival, growth, differentiation and functional activity of monocytes by binding to a single class of high-affinity cell surface receptors, known to be the product of the c-fms protooncogene. The detection of both M-CSF and c-fms expression by cells of the monocyte lineage has suggested that M-CSF may act by an autocrine mechanism. Interestingly, it has been shown that M-CSF can induce the expression of its own gene. Although sensitivity to M-CSF can be modulated by regulation of receptor expression and function, M-CSF responsiveness is largely determined at a postreceptor level. To date, little is known about the intracellular pathway of M-CSF signal transduction. We have therefore investigated the changes in protein kinase C (PKC) activity upon exposure of monocytes to M-CSF. We show that M-CSF activates and translocates PKC. Inhibition of PKC by the isoquinoline derivative H7 abolishes induction of M-CSF by M-CSF. Furthermore, activation of PKC was pertussis-toxin-sensitive and was associated with the detection of an NF kappa B protein in nuclear extracts of M-CSF-induced blood monocytes but not in monocytes exposed to medium treatment only. The results suggest that M-CSF induction of M-CSF involves G proteins, PKC and NF kappa B. 1653056 NF-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca(2+)-regulated kinases. NF-kappa B is a DNA-binding regulatory factor able to control transcription of a number of genes, including human immunodeficiency virus (HIV) genes. In T cells, NF-kappa B is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor alpha (TNF alpha). In the present work, we investigated the molecular events leading to NF-kappa B activation by TNF alpha in a human T cell line (Jurkat) and its subclone JCT6, which presents a deficiency in the PKA transduction pathway. We found that in both cell lines, both phorbol ester and TNF alpha were able to activate NF-kappa B. Phorbol activation was positively modulated by Ca2+ influx while TNF alpha activation was not. Furthermore, while PMA activation was inhibited by the PKC inhibitor staurosporin, the TNF alpha effect was unchanged. TNF alpha did not activate cAMP production and its signal was not modulated by cAMP activators. Moreover, cAMP activators did not activate NF-kappa B in Jurkat cells. Thus, TNF alpha-induced NF-kappa B activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C (PKC), protein kinase A, or Ca(2+)-regulated kinases. Furthermore, we found that cytoplasmic acidification facilitated NF-kappa B activation by both TNF alpha and PKC, by a mechanism that increases NF-kappa B/I kappa B dissociation without affecting the NF-kappa B translocation step. 1679576 NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I. The effect of constitutive Tax expression on the interaction of NF-kappa B with its recognition sequence and on NF-kappa B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-kappa B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-kappa B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85 , 75 , and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters. 1888683 Regulation of glucocorticoid receptors in human mononuclear cells: effects of glucocorticoid treatment, Cushing 's disease and ketoconazole. Glucocorticoid receptors (GcR) were determined by a whole cell assay in human mononulear leukocytes (hMNL) from control subjects, patients receiving glucocorticoid therapy for systemic diseases and Cushing 's disease patients with or without ketoconazole therapy. Prolonged corticosteroid treatment resulted in down-regulation of GcR, while the mean level of GcR in Cushing 's disease was normal. In this group, however, receptor levels and morning plasma cortisol values showed a negative correlation, indicating a subtle down-regulatory effect. Furthermore, GcR were unaltered after these patients received ketoconazole, in spite of a marked reduction in morning plasma cortisol and urinary free cortisol. We also observed that ketoconazole was a weak competitor of GcR in intact cells, although it significantly inhibited [3H] dexamethasone binding in cytosolic preparations from rat tissues. The results suggested that GcR in hMNL are down-regulated by synthetic steroids given in vivo, but they showed very mild down-regulation in hypercortisolemic patients suffering from Cushing 's disease. Finally, we did not observed either up-regulation or antagonism of GcR by ketoconazole treatment, at the time that cortisol levels of patients with Cushing 's disease were reduced. This indicates that the beneficial effects of ketoconazole in Cushing 's disease are due to adrenal cortisol suppression and not to interaction with GcR of target cells, and that the process of GcR regulation in hMNL is a complex phenomenon awaiting further elucidation. 1889142 Glucocorticoid receptors in lymphocytes in anorexia nervosa. OBJECTIVE : The aim was to explore the down-regulation of the glucocorticoid receptors during hypercortisolaemia in anorexia nervosa. DESIGN : Urine and plasma samples were obtained for cortisol determination and blood lymphocytes were isolated for receptor binding studies. PATIENTS : Sixteen anorexic patients, aged 16-27 years, with a mean ± SEM body mass index of 14.2 ± 2.0 (ranging from 11.1 to 17.4), and 15 normal women were studied. Six patients were reinvestigated after a significant weight gain. MEASUREMENTS : The binding capacity and affinity of the glucocorticoid receptors were measured with dexamethasone as ligand on lymphocytes. RESULTS : In patients, both total and free plasma cortisol concentrations were higher than in the normal women, as was their urinary free cortisol; the number of glucocorticoid receptors per cell (Ro) and the binding affinity (Kd) for dexamethasone were , however, not significantly different (Ro: 7687 ± 1750 vs 7347 ± 1285 sites/cell; Kd: 7.7 ± 2.4 vs 7.4 ± 1.7 nM at 24 degrees C). After weight gain (14 ± 2 to 16 ± 2 kg/m2), receptor numbers were 8421 ± 2126 (pre) and 9011 ± 500 (post) sites/cell, which are not significantly different (P greater than 0.2); the Kd was unchanged (9.3 ± 2.6 vs 9.2 ± 2.4 nM). CONCLUSIONS Hypercortisolaemia does not down-regulate the lymphocyte glucocorticoid receptors in anorexia nervosa and a post-receptor defect might be involved in peripheral tissue resistance to the effects of glucocorticoid hormones in undernutrition. 1909740 Tumor necrosis factor-alpha mRNA accumulation in human myelomonocytic cell lines. Role of transcriptional regulation by DNA sequence motifs and mRNA stabilization. The cytokine TNF mediates many of the pathologic signs of cachexia, inflammation, and sepsis. The current work describes the regulation of TNF in human myelomonocytic cell lines after PMA stimulation. The cell lines exhibit a low level of constitutive TNF mRNA expression. Within 2 to 4 h of PMA exposure, steady state levels of TNF mRNA are markedly elevated in all myelomonocytic cell lines studied. This rise is due to increased mRNA stability, which increased by almost twofold, and to an overall increase in transcription, which rises by more than sixfold. At the level of the genomic TNF gene, a DNase I hypersensitive site is detected within the TNF promoter between -200 to -100 bp relative to the transcription initiation site. Although absent in nonexpressing erythroleukemia cell lines, the DNase I site is present in uninduced myelomonocytic cell lines and is not changed after PMA induction. The PMA induction of c-fos mRNA correlated well with TNF gene induction; expression of genes encoding other proteins in the AP-1 complex (junB and junD) were also induced by PMA. The nuclear extracts from resting and induced ML-1 cells contain proteins binding specifically to the AP-1, AP-2, and NF kappa B sequence located within the TNF promoter. PMA induction increases the level of a number of specific binding complexes relative to the resting cells. The regulatory mechanisms of the human and murine TNF genes are discussed. 1653950 USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1. The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60 % decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1. 1832547 The effect of toremifene therapy on serum immunoglobulin levels in breast cancer. Estrogens and anti-estrogens enhance the number of immunoglobulin ( Ig ) -secreting cells in pokeweed mitogen ( PWM ) -stimulated lymphocyte cultures. Lymphocytes from patients who have received anti-estrogen therapy show similar enhancement of Ig-secreting cells after PWM stimulation. In this study the effect of anti-estrogen ( toremifene ) therapy on serum immunoglobulin (IgA, IgM, IgG) levels in breast cancer patients was investigated. Serum Ig levels were followed up to two years after or during the therapy. An unexpected finding was that the Ig levels decreased during the follow-up period. This decrease was seen in patients who responded to the therapy as well as in those who did not. 1896644 HIV1 infection of human monocytes and macrophages promotes induction or translocation of NF-KB-related factors. In 1991, we demonstrated , using electrophoretic mobility shift assays, that 3 different factors (termed B1, B2 and B3) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages. The B2 factor was induced in the nuclei of these cells only upon HIV1 infection. The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes. Its expression remained very low in nuclei of HIV1-infected macrophages. In this paper, we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein, indicating that it is not associated with an inhibitor (IKB). This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection. The B3 factor is detected in the cytosol only when cells are HIV1-infected. The role of HIV1 infection in the expression and the translocation of these factors is discussed. 1896645 Induction of NF-kappa B during monocyte differentiation is associated with activation of HIV-gene expression. Cells of the monocyte-macrophage lineage are important targets of HIV infection. We report here that the phenotypic differentiation of monocyte cell lines induced by phorbol esters or tumour necrosis factor alpha (TNF alpha) is associated with expression of nuclear factor kappa B (NF-kappa B). In parallel with such differentiation, HIV transcription, monitored using an HIV long terminal repeat reporter gene construct, is activated in such cells under the influence of enhanced NF-kappa B expression. Also, in a promonocyte cell line chronically infected with HIV, NF-kappa B expression and HIV transcription were enhanced on stimulation with phorbol ester or TNF alpha. Thus, stimulation of monocyte cell lines by phorbol esters or TNF alpha induces cell differentiation and activates HIV transcription. Such a process may have fundamental implications in AIDS pathogenesis in vivo and may be important in disease progression induced by opportunistic infections directly or indirectly involving macrophages. 1911548 A nuclear factor NF-GM2 that interacts with a regulatory region of the GM-CSF gene essential for its induction in responses to T-cell activation: purification from human T-cell leukemia line Jurkat cells and similarity to NF-kappa B. Activation of T cells by antigen, lectin, or a combination of phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) leads to the induction of genes for a set of lymphokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA/A23187. This region contains two DNA-binding motifs, GM2 and GC-box. The GM2 sequence (GGTAGTTCCC) is recognized by an inducible factor NF-GM2; the other (CCGCCC) by constitutive factors A1, A2, and B. To elucidate the mechanism of GM-CSF gene activation, we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity. The purified NF-GM2 consists of 50 (p50) and 65 kDa (p65) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa (GGAAAGTCCC) enhancers. Electrophoretically purified p50 alone can form a protein-DNA complex, but in the mixture, p50 associates preferentially with p65 to form the NF-GM2 complex. In addition, p65 gave per se, with low affinity, a protein-DNA complex that migrated more slowly than native NF-GM2 complex. Furthermore, an antiserum against KBF1 (identical to 50 kDa NF-kappa B protein) reacted with the p50 of NF-GM2, indicating that the NF-GM2 polypeptide can not be immunologically differentiated from the 50 kDa subunit of NF-kappa B. The purified NF-GM2 activated in vitro transcription from the kappa B enhancer, while it failed to stimulate transcription from the GM-CSF promoter harboring the GM2 sequence. This suggests that the activation mechanism of the GM-CSF gene through the GM2/GC-box sequence is different from that of genes carrying the kappa B enhancer alone. 1914170 Glucocorticoid receptors in normal leukocytes: effects of age, gender, season, and plasma cortisol concentrations. We measured glucocorticoid receptors (GR) in mononuclear leukocytes (MNL) isolated from peripheral blood of 145 apparently healthy volunteers (86 men and 59 women). An age-related decrease in the number of GR was suggested between subjects younger than 20 years and elderly subjects; there was no apparent seasonal variation in GR. Gender difference in the number of GR was not significant, although women showed slightly fewer GR. Eight patients with dermatomyositis/polymyositis were examined to determine whether the number of GR in MNL could be down-regulated by their cognate ligands. The number of GR in MNL from these patients was significantly decreased one month after the initiation of prednisolone therapy. However, in normal subjects, the GR in MNL did not demonstrate circadian variation, in contrast to concentrations of plasma cortisol. 1680914 Anti-CD2 receptor antibodies activate the HIV long terminal repeat in T lymphocytes. The CD2 T lymphocyte glycoprotein surface molecule mediates both cell to cell adhesion and T cell activation, two processes that are involved in the spread of HIV infection. Treatment of chronically HIV-infected PBMC with anti-CD2 mAb has been shown to induce the expression of infectious virus from these cultures. In this study we investigated the mechanisms whereby anti-CD2 antibodies stimulate viral production. We demonstrate that treatment of transiently transfected T lymphocytes with anti-CD2 antibodies results in activation of the HIV long terminal repeat. Furthermore, CAT assays using mutated HIV long terminal repeat-CAT constructs and gel shift assays demonstrate that this activation is dependent on the NF-kappa B enhancer. These studies suggest that interaction of CD2 with its natural ligand, LFA-3, may play a role in regulation of HIV expression. 1655897 Nuclear transcription factors that bind to elements of the IL-2 promoter. Induction requirements in primary human T cells. Prior studies have identified several elements that contribute to the activity of the IL-2 promoter in the stimulated T cell line, Jurkat. The sites and their corresponding nuclear binding factors include : NF-kappa B, AP-1, AP-3, OCT-1, and NF-AT. The latter "nuclear factor for activated T cells" likely contributes to the tissue specificity of IL-2 gene expression. Using electrophoretic mobility shift assays, we have studied these transcription factors in primary T cells from human blood to verify their presence in a physiologic setting and to identify the signals that stimulate factor activity. All factors are induced in the nuclei of T cells upon activation with mitogens but not with exogenous IL-2 growth factor. However, the signaling requirements and sensitivity to protein synthesis inhibitors differ considerably. Only the activities for NF-AT and AP-1 sites require two signals for optimal induction, i.e., PMA plus either lectin or antibody to the CD3 or CD28 surface molecules. Other factors are induced by lectin, antibody, and/or PMA alone. After appropriate stimulation, both NF-AT and AP-1 are peculiarly sensitive to the protein synthesis inhibitor anisomycin. Our data correlate the activity of NF-AT and AP-1 in gel shift assays with the two signals requirements for IL-2 gene expression. 1656391 An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1. The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site. 1656441 Demonstration of a 1,25-dihydroxyvitamin D3-responsive protein in human lymphocytes: immunologic crossreactivity and inverse regulation with the vitamin D receptor. Using Western blot analysis with a monoclonal antibody recognizing a 17-amino acid epitope of the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]receptor, we have detected two crossreacting proteins in activated normal human lymphocytes. The smaller of the two proteins (50 kDa) was indistinguishable from the classical 1,25(OH)2D3 receptor and , similar to the classical 1,25(OH)2D3 receptor, was upregulated in a dose-dependent fashion by 1,25(OH)2D3. The larger crossreacting protein exhibited an electrophoretic mobility of 80 kDa, was localized in the cell cytosol, and appeared to be specific for activated lymphocytes since it was not detected in several other human cells including monocytes. More strikingly, the 80-kDa protein was downregulated in a dose-dependent fashion by 1,25(OH)2D3; this effect was independent of the mode of lymphocyte activation and specific for the 1,25(OH)2D3 metabolite of vitamin D3. However, two potent immunosuppressive agents, glucocorticoids and cyclosporin A, also inhibited the 80-kDa protein. 1718025 T-helper-cell determinants in protein antigens are preferentially located in cysteine-rich antigen segments resistant to proteolytic cleavage by cathepsin B, L, and D. We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may , therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described. 1930693 A human putative lymphocyte G0/G1 switch gene containing a CpG-rich island encodes a small basic protein with the potential to be phosphorylated. Genes actively involved in the G0/G1 switch (G0S genes) may be differentially expressed during the lectin-induced switch of lymphocytes from the G0 to the G1 phases of the cell cycle. This paper presents studies of G0S2, a member of a set of putative G0S genes, for which cDNAs were cloned and selected on the basis of differential cDNA hybridization. G0S2 mRNA increases transiently within 1-2 hr of the addition of lectin or cycloheximide to cultured blood mononuclear cells. Comparison of a nearly full-length cDNA sequence with the corresponding genomic sequence reveals one small intron and an open reading frame in the second exon. The derived 103-amino-acid basic protein has two potential alpha-helical domains separated by a hydrophobic region with the potential to generate turns and assume a beta-sheet conformation. Consistent with involvement in the G0/G1 switch, the protein contains potential sites for phosphorylation by protein kinase C and casein kinase II. The gene contains a CpG-rich island suggesting expression in the germ line. An upstream segment contains tandem dinucleotide repeats (CT)19/(CA)16. There is a suitably located TATA box, but potential sites for CCAAT-box binding factors are far upstream, embedded in a 42-nucleotide repeat element. Potential sites for transcription factors AP1, AP2, and AP3 are consistent with rapid transcriptional activation in response to inducing agents. 1931834 Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I. We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I (HTLV-I) using the human T-cell lines Jurkat and MOLT 4, which are negative for HTLV-I, and MT-2 and TL-Mor, which carry the proviral genome of HTLV-I. Two distinct elements have been implicated in function of the HTLV-I enhancer. One is the 21-base-pair (bp) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor ( CREB ) -like factor ( s ). The other is a region interposed between the 21-bp elements. In this study we demonstrate that a subfragment (C26) in the region between the 21-bp elements is involved in trans-activation by p40tax, possibly through binding to an NF-kappa B-like nuclear factor or factors. Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax. The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that, by itself, showed little activation in response to p40tax. However, the C26 element alone, even when repeated, could not be activated by p40tax, unlike other NF-kappa B-binding elements. In contrast, the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate. These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements, including binding sites for at least CREB- and NF-kappa B-like factors, which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax. Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element. 1682217 v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII. The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone ( T3/T4 ) receptor c-erbA ( type alpha ). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein. 1939341 Inhibition of phorbol ester-induced monocytic differentiation by dexamethasone is associated with down-regulation of c-fos and c-jun (AP-1). Previous studies have shown that treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation and expression of the c-jun and c-fos early response genes. The present work demonstrates that the glucocorticoid dexamethasone inhibits TPA-induced increases in c-jun and c-fos mRNA levels in U-937 leukemia cells. These findings were associated with a block in appearance of the monocytic phenotype, including inhibition of TPA-induced increases in lamin A, lamin C, and vimentin transcripts. Other studies have demonstrated that TPA-induced monocytic differentiation and expression of the c-jun and c-fos genes in myeloid leukemia cells are regulated by protein kinase C (PKC). The finding that dexamethasone has no effect on TPA-induced activation of PKC suggests that this glucocorticoid inhibits signals downstream or parallel to this enzyme. Nuclear run-on assays demonstrate that : ( 1 ) induction of c-jun and c-fos expression by TPA is regulated by transcriptional mechanisms, ( 2 ) TPA-induced expression of c-jun and c-fos does not require protein synthesis, and ( 3 ) TPA-induced expression of both genes is inhibited at the transcriptional level by dexamethasone. To further define the effects of dexamethasone at the molecular level, we prepared a series of deleted c-jun promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene. Increases in CAT activity during transient expression of these constructs in TPA-treated U-937 cells could be assigned to the region (-97 to -20) of the promoter that contains the AP-1 binding site. This induction of CAT activity was sensitive to dexamethasone. These findings suggest that dexamethasone down-regulates TPA-induced transcription of the c-jun gene during monocytic differentiation by inhibiting activation of the AP-1 site. 1719077 Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation. The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis. 1944294 Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor. We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element ( GRE ) -containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding. 1945879 One base pair change abolishes the T cell-restricted activity of a kB-like proto-enhancer element from the interleukin 2 promoter. The inducible, T cell-specific enhancers of murine and human Interleukin 2 (Il-2) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif. When cloned in multiple copies this so-called TCEd (distal T cell element) acts as an inducible proto-enhancer element in E14 T lymphoma cells, but not in HeLa cells. In extracts of induced, Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA. The binding of the most prominent factor, named TCF-1 (T cell factor 1), is correlated with the proto-enhancer activity of TCEd. TCF-1 consists of two polypeptides of about 50 kD and 105 kD; the former seems to be related to the 50 kD polypeptide of NF-kB. Purified NF-kB is also able to bind to the TCEd, but TCF-1 binds stronger than NF-kB to TCEd DNA. The conversion of the TCEd to a ' perfect ' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and , as a functional consequence, to the activity of the ' converted ' TCEd motifs in HeLa cells. Thus, the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells. These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene. 1946356 Negative regulation of human immunodeficiency virus type 1 expression in monocytes: role of the 65-kDa plus 50-kDa NF-kappa B dimer. Although monocytic cells can provide a reservoir for viral production in vivo, their regulation of human immunodeficiency virus type 1 (HIV-1) transcription can be either latent, restricted, or productive. These differences in gene expression have not been molecularly defined. In THP-1 cells with restricted HIV expression, there is an absence of DNA-protein binding complex formation with the HIV-1 promoter-enhancer associated with markedly less viral RNA production. This absence of binding was localized to the NF-kappa B region of the HIV-1 enhancer; the 65-kDa plus 50-kDa NF-kappa B heterodimer was preferentially lost. Adding purified NF-kappa B protein to nuclear extracts from cells with restricted expression overcomes this lack of binding. In addition, treatment of these nuclear extracts with sodium deoxycholate restored their ability to form the heterodimer, suggesting the presence of an inhibitor of NF-kappa B activity. Furthermore, treatment of nuclear extracts from these cells that had restricted expression with lipopolysaccharide increased viral production and NF-kappa B activity. Antiserum specific for NF-kappa B binding proteins, but not c-rel-specific antiserum, disrupted heterodimer complex formation. Thus, both NF-kappa B-binding complexes are needed for optimal viral transcription. Binding of the 65-kDa plus 50-kDa heterodimer to the HIV-1 enhancer can be negatively regulated in monocytes, providing one mechanism restricting HIV-1 gene expression. 1946405 Isolation of a candidate repressor/activator, NF-E1 (YY-1 , delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site. We have determined that the developmental control of immunoglobulin kappa 3' enhancer ( kappa E3' ) activity is the result of the combined influence of positive- and negative-acting elements. We show that a central core in the kappa E3' enhancer is active at the pre-B-cell stage but is repressed by flanking negative-acting elements. The negative-acting sequences repress enhancer activity in a position- and orientation-independent manner at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to the negative-acting segment of the kappa E3' enhancer. This protein also binds to the immunoglobulin heavy-chain enhancer mu E1 site. NF-E1 is encoded by the same gene as the YY-1 protein, which binds to the adeno-associated virus P5 promoter. NF-E1 is also the human homologue of the mouse delta protein, which binds to ribosomal protein gene promoters. The predicted amino acid sequence of this protein contains features characteristic of transcriptional activators as well as transcriptional repressors. Cotransfection studies with this cDNA indicate that it can repress basal promoter activity. The apparent dual function of this protein is discussed. 1658795 Clone pAT 133 identifies a gene that encodes another human member of a class of growth factor-induced genes with almost identical zinc-finger domains. We report the structure and regulation of a gene represented by clone pAT 133, which is induced upon transition from a resting state (G0) through the early phase of the cell cycle (G1). The pAT 133 gene is immediately induced, with FOS-like kinetics, in human T cells and in fibroblasts. Primary structure analysis showed that the encoded protein contains three tandem zinc-finger sequences of the type Cys2-Xaa12-His2. This zinc-finger region, which is thought to bind DNA in a sequence-specific manner, is similar (greater than 80% on the amino acid level) to two previously described transcription factors pAT 225/EGR1 and pAT 591/EGR2. Except for the conserved zinc-finger domains, the amino acid sequences of the three proteins are distinct. This structural similarity suggests that the pAT 133 gene encodes a transcription factor with a specific biological function. Comparing the regulation of these related zinc-finger-encoding genes showed coordinate induction upon mitogenic stimulation of resting T lymphocytes and of resting fibroblasts. However, upon transition from a proliferating (G1) to a resting state of the cell cycle the three genes were differently regulated. In human histiocytic U937 cells mRNA of clone pAT 133 was constitutively expressed, whereas mRNA of pAT 225/EGR1 was induced upon induction of terminal differentiation. In contrast mRNA representing pAT 591/EGR2 was not expressed in these cells. This difference in gene regulation suggests distinct biological roles in the control of cell proliferation for the respective proteins. 1719551 cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins. We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester- , and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways. 1952426 Glucocorticoid resistance in chronic asthma. Glucocorticoid pharmacokinetics, glucocorticoid receptor characteristics, and inhibition of peripheral blood T cell proliferation by glucocorticoids in vitro. A total of 37 chronic, severe, nonsmoking asthmatic patients with documented reversible airways obstruction were classified as glucocorticoid-sensitive or -resistant on the basis of changes in FEV1, FVC, and peak expiratory flow (PEF) after oral prednisolone. The resistant patients showed no significant improvements in airflow limitation. Phytohemagglutinin ( PHA ) -induced proliferation of peripheral blood T lymphocytes from the sensitive but not the resistant asthmatic patients was significantly (p less than 0.01) inhibited by dexamethasone (10(-7) mol/L), reflecting a shift of the dose-response curve. When all the asthmatic patients were analyzed together, there was a significant correlation between the degree of sensitivity of T cells to dexamethasone and the clinical responsiveness to prednisolone (p less than 0.01). No differences were observed between six of the sensitive and resistant patients in the clearance of plasma prednisolone derived from orally administered prednisone. Peripheral blood mononuclear cell glucocorticoid receptors were also characterized in five sensitive and seven resistant patients. The numbers and binding affinities of these receptors could not account for the observed difference in the susceptibility of these cells to functional inhibition by dexamethasone in vitro. These results suggest that clinical glucocorticoid resistance in chronic asthma does not reflect abnormal glucocorticoid clearance but may be due at least partly to a relative insensitivity of T lymphocytes to glucocorticoids. This lack of sensitivity is unexplained but is not attributable to abnormalities of cellular glucocorticoid receptors. 1953785 Regulation of interleukin-1 beta production by glucocorticoids in human monocytes: the mechanism of action depends on the activation signal. Glucocorticoids are known to downregulate interleukin-1 beta production in monocytic cells by two different mechanims: direct inhibition of the gene transcription and destabilization of the preformed interleukin-1 beta mRNA. Now we have examined the effect of the nature of the monocyte activating signal on these two inhibitory mechanims. When human monocytes were preincubated with dexamethasone for 1 hour and then stimulated either with bacterial lipopolysaccharide or phorbol myristate, it was found that dexamethasone inhibited the lipopolysaccharide-induced interleukin-1 beta protein production, but the phorbol myristate-induced production was increased 3-10 fold. This difference was also seen at the mRNA level. When dexamethasone was added to the cultures 3 hours after the stimulators, it clearly decreased the interleukin-1 beta mRNA levels regardless of the stimulator used (although the effect was clearly weaker on the PMA-induced mRNA). Thus these data suggest that the phorbol myristate-induced signal (prolonged protein kinase C activation?) can not be inhibited by prior incubation with dexamethasone and it also protects the induced mRNA for the degradative action of dexamethasone. 1956769 Identification of transcriptional suppressor proteins that bind to the negative regulatory element of the human immunodeficiency virus type 1. Two different proteins which independently bound to neighboring sequences within the negative regulatory element (NRE) of human immunodeficiency virus type 1 (HIV-1) were detected in the nuclear extract of a virus-infected human T cell line. One of the factors bound to a novel dyad symmetrical sequence. This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene. Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay. The other factor bound to a putative AP-1 recognition sequence predicted for the HIV-1 NRE. However, this factor did not bind to a typical AP-1 site. The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third, respectively. Thus, each factor may function as a novel negative regulator of transcription. 1958222 Constitutive activation of NF-kB in human thymocytes. NF-kB is a eukaryotic transcription regulatory factor. In T cells and T cell lines, NF-kB is bound to a cytoplasmic proteic inhibitor, the IkB. Treatment of T cells with mitogens (phorbol esters) or cytokines (TNF alpha) induces NF-kB nuclear translocation and the subsequent expression of NF-kB dependent T cell genes. Here we examined the activation of NF-kB in human T cell thymic progenitors. We report differences in (Ca2+)i requirement for NF-kB activation in thymocytes as compared to mature T cells. Furthermore, our results indicated that thymocytes have a constitutively active form of NF-kB, suggesting that they are activated in vivo. 1751404 The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation. Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process. 1836958 TCF-1, a T cell-specific transcription factor of the HMG box family, interacts with sequence motifs in the TCR beta and TCR delta enhancers. We have recently identified and cloned TCF-1, a T cell-specific transcription factor with specificity for the AACAAAG motif in the CD3 epsilon enhancer and for the TTCAAAG motif in the TCR alpha enhancer. TCF-1 belongs to the family of transcription-regulating proteins which share a region of homology termed the HMG-box. Here, we show by gel retardation analysis that TCF-1 specifically recognizes the T beta 5 element of the TCR beta enhancer and the T delta 7 element of the TCR delta enhancer. Comparison of the sequences of all elements recognized by TCF-1 defines a consensus motif A/T A/T C A A/G A G. These observations imply that TCF-1 is involved in the control of several T cell-specific genes and might thus play an important role in the establishment and maintenance of the mature T cell phenotype. 1309587 The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells. Regulation of replicative functions in the Epstein-Barr virus (EBV) genome is mediated through activation of a virally encoded transcription factor, Z (BZLF1). We have shown that the Z gene product, which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos, can efficiently activate the EBV early promoter, BMRF1, in certain cell types (i.e. , HeLa cells) but not others (i.e. , Jurkat cells). Here we demonstrate that the c-myb proto-oncogene product, which is itself a DNA-binding protein and transcriptional transactivator, can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells (a T-cell line) or Raji cells (an EBV-positive B-cell), whereas the c-myb gene product by itself has little effect. The simian virus 40 early promoter is also synergistically activated by the Z/c-myb combination. Synergistic transactivation of the BMRF1 promoter by the Z/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein. A 30-bp sequence in the BMRF1 promoter which contains a Z binding site (a consensus AP-1 site) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z/c-myb combination to a heterologous promoter. That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests c-myb is likely to engage in similar interactions with cellularly encoded transcription factors. 1763035 The 29-kDa proteins phosphorylated in thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein. Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. We have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, we isolated and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the " estrogen receptor-related protein " is HSP27, and the three major 29-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation. 1763325 Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but , rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex. 1662496 High affinity aldosterone binding to plasma membrane rich fractions from mononuclear leukocytes: is there a membrane receptor for mineralocorticoids? In vitro effects of aldosterone on the intracellular concentrations of sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in intact human mononuclear leukocytes (HML). In the present paper, the binding of a [125I]-labeled aldosterone derivative to plasma membrane rich fractions of HML was studied. High affinity binding of the radioligand with an apparent Kd of approximately 0.1 nM was found. Aldosterone displaced the tracer at a similar Kd. Both canrenone and cortisol were inactive as ligands up to concentrations of 0.1 microM. The findings are the first to demonstrate membrane binding sites with a high affinity for aldosterone, but not for cortisol. These data are perfectly compatible with major properties of steroidal effects on the sodium-proton-antiport in HML and thus very likely represent membrane receptors for aldosterone. 1765275 Transcription factor requirements for U2 snRNA-encoding gene activation in B lymphoid cells. Transcription of a human U2 small nuclear RNA ( snRNA ) -encoding gene in HeLa cells requires a distal enhancer element, which is composed of one octamer motif (Oct) and three Sp 1-binding sites. To study the transcription factor requirement in B-cells, different U2 enhancer constructions were transfected into the lymphoid cell line, BJA-B. The results showed that the activation of U2 snRNA transcription in B-cells also requires an enhancer comprising both the Oct and at least one Sp 1-binding site. Deletion of all the Sp 1-binding sites from the enhancer reduces transcription by 80-90% in HeLa, as well as in BJA-B cells, whereas the removal of the octamer-binding site reduces transcription to levels below detection in both cell types. Enhancers containing a single Oct have , nevertheless, the capacity to partially activate U2 snRNA transcription in both HeLa cells, in which only OTF-1 is expressed, and in BJA-B cells in which OTF-2 is the predominantly expressed octamer-binding factor. The most likely interpretation of our results is that both the ubiquitous transcription factor, OTF-1, and the B-cell-specific transcription factor, OTF-2, can activate U2 snRNA transcription. The results also revealed a similar functional cooperation between the transcription factors which bind to the Oct and the adjacent Sp 1-binding site in BJA-B cells, as has been observed in HeLa cells, since a template which contains a weak binding site for OTFs expresses wild-type levels of U2 snRNA in both cell types when the weak octamer-binding site is combined with a Sp 1-binding site. 1309833 Cortisol receptor resistance: the variability of its clinical presentation and response to treatment. Primary ( partial ) cortisol receptor resistance was previously reported in a total of 7 patients and 14 asymptomatic family members. Its occurrence is considered to be extremely rare. In the present study we report on 6 patients (2 males and 4 females) with the syndrome. The first male patient presented with mild hypertension. Hydrochlorothiazide therapy resulted in life-threatening hypokalemia. The second male patient had slight hypertension without hypokalemia. All four female patients presented between the age of 20-30 yr with acne, hirsutism, and irregular menstruations. Low dose dexamethasone therapy (1-1.5 mg/day) was of clinical benefit in these patients. All patients showed insufficient suppression of serum cortisol concentrations in the overnight 1-mg dexamethasone test. The diurnal rhythm of ACTH and cortisol was intact, albeit at an elevated level. There was a normal increase in ACTH, cortisol, and GH (except in one obese patient) in response to insulin-induced hypoglycemia, while cortisol production was elevated in three patients. Circulating adrenal androgen levels were increased in all patients. Glucocorticoid receptors were investigated in a whole cell dexamethasone binding assay in mononuclear leukocytes. In the first male patient, the number of receptors was very low, while the affinity was lower than that in controls. A lowered affinity to dexamethasone was found in one female patient, while a lowered number of receptors was found in three patients. In the second male patient, no abnormalities were found. As a bioassay for glucocorticoid action we also measured dexamethasone suppressibility of mitogen-stimulated incorporation of [3H]thymidine in mononuclear leukocytes. In the male patient with normal receptor status, dexamethasone suppressibility of [3H]thymidine incorporation was significantly lower than that in healthy controls with respect to both maximal suppression and IC50. Partial cortisol receptor resistance might be less rare than previously thought. In the six patients presented, at least three different forms can be recognized. Therapy with dexamethasone was successful in female patients with acne and hirsutism, as the secondary increase in the production of adrenal androgens was effectively controlled. 1768652 Kappa B-specific DNA binding proteins are differentially inhibited by enhancer mutations and biological oxidation. Kappa B (kappa B) enhancer binding proteins isolated from the nuclei of activated human T cells produce two distinct nucleoprotein complexes when incubated with the kappa B element from the interleukin-2 receptor-alpha (IL-2R alpha) gene. These two DNA-protein complexes are composed of at least four host proteins (p50, p55, p75, p85), each of which shares structural similarity with the v-rel oncogene product. Nuclear expression of these proteins is induced with distinctly biphasic kinetics following phorbol ester activation of T cells (p55/p75 early and p50/p85 late). DNA-protein crosslinking studies have revealed that the more rapidly migrating B2 complex contains both p50 and p55 while the more slowly migrating B1 complex is composed of p50, p55, p75, and p85. Site-directed mutagenesis of the wild-type IL-2R alpha kappa B enhancer (GGGGAATCTCCC) has revealed that the binding of p50 and p55 (B2 complex) is particularly sensitive to alteration of the 5' triplet of deoxyguanosine residues. In contrast, formation of the B1 complex, reflecting the binding of p75 and p85, critically depends upon the more 3' sequences of this enhancer element. DNA binding by all four of these Rel-related factors is blocked by selective chemical modification of lysine and arginine residues, suggesting that both of these basic amino acids are required for binding to the kappa B element. Similarly, covalent modification of free sulfhydryl groups with diamide (reversible) or N-ethylmaleimide (irreversible) results in a complete loss of DNA binding activity. In contrast, mild oxidation with glucose oxidase selectively inhibits p75 and p85 binding while not blocking p50 and p55 interactions. These findings suggest that reduced cysteine thiols play an important role in the DNA binding activity of this family of Rel-related transcription factors. 1531086 A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site. A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors. 1773463 [Changes in leucocytic estrogen receptor levels in patients with climacteric syndrome and therapeutic effect of liuwei dihuang pills] The numbers of estrogen receptor (ER) in human peripheral leucocytes in 22 women with climacteric syndrome were measured by radioligand method. The results were compared with those of 12 normal child-bearing-age women. It wat found that the contents of leucocytic ER in climacteric syndrome patients were significantly lower than normal child-bearing-age women. The authors used a Chinese prescription--Liuwei Dihuang Pills (LDP) to treat the patients for 2 months. The numbers of leucocytic ER were significantly increased after treatment. The data indicate that decrease of ER levels in cell may involve in the pathogenesis of climacteric syndrome. LDP not only increases plasma estradiol levels, but also increases the leucocytic ER levels. This may be the basis of the therapeutic effect on the disease. 1734865 A novel primer extension method to detect the number of CAG repeats in the androgen receptor gene in families with X-linked spinal and bulbar muscular atrophy. X-linked spinal and bulbar muscular atrophy (SBMA), an adult-onset form of motor neuron disease, was recently reported to be caused by amplification of the CAG repeats in the androgen receptor gene. We report here a simple and rapid strategy to detect the precise number of the CAGs. After the DNA fragment containing the CAG repeats is amplified by the polymerase chain reaction, a primer extension is carried out; the extension of the end-labelled reverse primer adjacent to 3' end of CAG repeats stops at the first T after CAG repeats with the incorporation of dideoxy ATP in the reaction mixture. The resultant primer products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. This method could be quite useful to detect not only CAG repeats in SBMA but also other polymorphic dinucleotide and trinucleotide repeats. 1777483 Activity of the kappa B enhancer of the interleukin-2 receptor alpha chain in somatic cell hybrids is accompanied by the nuclear localization of NF-kappa B. The two nuclear proteins NF-kappa B (consisting of subunits p50 andp65) and the DNA-binding subunit of NF-kappa B (p50) by itself, also called KBF1, are constitutively expressed and localized in the nucleus of the human T-cell line IARC 301.5. In order to define the roles of these two factors, which bind to the same kappa B enhancers, in transcription activation we have prepared somatic cell hybrids between IARC 301.5 and a murine myeloma. Most hybrids express both KBF1 and NF-kappa B in their nuclei, but one hybrid expresses only KBF1. The kappa B enhancer of the gene encoding the interleukin-2 (IL-2) receptor alpha chain (IL-2R alpha) is functional only in the hybrids expressing nuclear NF-kappa B. These findings show that nuclear NF-kappa B is necessary to activate the kappa B enhancer, while KBF1 by itself is not sufficient. We propose that KBF1 is a competitive inhibitor of NF-kappa B and discuss how these factors may be involved in the transient expression of IL-2 and IL-2R alpha genes during the immune response. 1734946 Influence of estradiol and tamoxifen on susceptibility of human breast cancer cell lines to lysis by lymphokine-activated killer cells. The design of combination hormonal and immunotherapeutic protocols for breast cancer patients may be facilitated by analysis of preclinical in vitro model systems. Estrogen receptor positive (ER+: MCF-7) and negative (ER-: MDA-MB-231) human breast cancer cell lines were utilized to evaluate the effects of tamoxifen (TAM) and estradiol (E2) on modulation of breast cancer target susceptibility to lysis by lymphokine-activated killer (LAK) cells. E2-stimulated ER+ cells were more susceptible to lysis by LAK cells than corresponding TAM-treated or control cells, while treatment of ER- cells with either E2 or TAM alone did not alter from control their susceptibility to this immune-mediated lysis. All ER+ and ER- cells tested remained sensitive after treatment with TAM to lysis by LAK cells. In addition, an adenocarcinoma reactive human-mouse chimeric monoclonal antibody (ING-1) was able to significantly boost in vivo generated LAK cell-mediated lysis of control, E2-treated, and TAM-treated ER+ and ER- cells. These in vitro results provide a preclinical rationale for in vivo testing of TAM, interleukin-2 (IL-2), and breast cancer reactive antibody-dependent cellular cytotoxicity facilitating antibody in patients with refractory or high risk breast cancer. 1736549 Glucocorticoid receptor and inhibition of 3-O-methyl-D-glucose uptake by glucocorticoids in peripheral blood leukocytes from normal humans: correlation between receptor level and hormone effect in vitro. We have measured the glucocorticoid receptor concentration in mononuclear and polymorphonuclear leukocytes, both of which were isolated from peripheral blood from ten healthy male volunteers. In parallel, the inhibitory effect of dexamethasone on 3-O-methyl-D-glucose uptake was assayed in the corresponding mononuclear leukocytes. The glucocorticoid receptor levels in mononuclear leukocytes correlated with those in polymorphonuclear leukocytes, and there was a linear relationship between the cellular glucocorticoid receptor levels and glucocorticoid-mediated inhibition of the uptake of 3-O-methyl-D-glucose in mononuclear leukocytes. When mononuclear leukocytes were incubated in the presence of 8-bromo-cAMP, cellular glucocorticoid receptor levels increased and a more pronounced inhibitory effect of dexamethasone was observed on the transport of 3-O-methyl-D-glucose. We conclude that the cellular glucocorticoid receptor levels in peripheral blood leukocytes reflect in vitro responsiveness to glucocorticoids in mononuclear leukocytes from healthy males, and that the individual responsiveness may alter upon changes in the cellular levels of glucocorticoid receptor. 1782151 Stimulation of interferon beta gene transcription in vitro by purified NF-kappa B and a novel TH protein. The human interferon beta (IFN-beta) regulatory element consists of multiple enhanson domains which are targets for transcription factors involved in inducible expression of the promoter. To further characterize the protein-DNA interactions mediating IFN-beta induction, positive regulatory domain (PRD) II binding proteins were purified from phorbol ester induced Jurkat T-cells and from IFN primed, cycloheximide/polyinosinic-polycytidylic acid treated HeLa S3 cells. From HeLa cells, two major proteins of 52 and 45 kilodaltons (kD) copurified with DNA binding activity, whereas from T-cells, four proteins--a major protein of 52 kD and three minor proteins of 82 , 67 , and 43-47 kD--were purified. Also, an induction specific DNA binding protein was purified from HeLa cells that interacted with the ( AAGTGA ) 4 tetrahexamer sequence and the PRDI domain. This protein is immunologically distinct from IRF-1/ISGF2. Uninduced or Sendai virus induced HeLa extracts were used to examine transcription in vitro using a series of IFN beta promoter deletions. Deletions upstream of the PRDII element increased transcription in the uninduced extract, indicating predominantly negative regulation of the promoter. A 2-4-fold increase in IFN-beta promoter transcription was observed in Sendai virus induced extracts, and deletion of PRDI and PRDII elements decreased this induced level of transcription. When purified PRDII and tetrahexamer binding proteins were added to the induced extract, a 4-fold increase in transcription was observed. These experiments demonstrate that it is possible to modulate IFN-beta transcription in vitro but indicate that additional proteins may be required to fully activate IFN-beta transcription. 1310680 Structure function analysis of vitamin D analogs with C-ring modifications. Analogs of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH) 2D3) with substitutions on C-11 were synthesized. Small apolar substitutions (11 alpha-methyl, 11 alpha-fluoromethyl) did not markedly decrease the affinity for the vitamin D receptor, but larger (11 alpha-chloromethyl or 11 alpha- or 11 beta-phenyl) or more polar substitutions (11 alpha-hydroxymethyl, 11 alpha-(2-hydroxyethyl] decreased the affinity to less than 5% of that of 1 alpha,25-OH)2D3. Their affinity for the vitamin D-binding protein, however, increased up to 4-fold. The biological activity of 11 alpha-methyl-1 alpha,25-(OH)2D3 closely resembled that of the natural hormone on normal and leukemic cell proliferation and bone resorption, whereas its in vivo effect on calcium metabolism of the rachitic chick was about 50% of that of 1 alpha,25-(OH)2D3. The 11 beta-methyl analog had a greater than 10-fold lower activity. The differentiating effects of the other C-11 analogs on human promyeloid leukemia cells (HL-60) agreed well with their bone-resorbing activity and receptor affinity, but they demonstrated lower calcemic effects in vivo. Large or polar substitutions on C-11 of 1 alpha,25-(OH)2D3 thus impair the binding of the vitamin D receptor but increase the affinity to vitamin D-binding protein. The effects of many C-11-substituted 1 alpha,25-(OH)2D3 analogs on HL-60 cell differentiation exceeded their activity on calcium metabolism. 1531412 Transcriptional regulation during T-cell development: the alpha TCR gene as a molecular model. The regulation of gene expression during lymphocyte differentiation is a complex process involving interactions between multiple positive and negative transcriptional regulatory elements. In this article, transcriptional regulation of the archetypal T-cell-specific gene, alpha TCR, is discussed. Major recent developments, including the identification of novel families of transcription factors that regulate multiple T-cell genes during thymocyte ontogeny and T-cell activation, are described. 1740494 Cortisol resistance in acquired immunodeficiency syndrome. This study concerns 9 iv drug abusers with acquired immunodeficiency syndrome (AIDS) who developed hypercortisolism without the clinical signs or metabolic consequences of hypercortisolism. All patients were characterized by an Addisonian picture (weakness, weight loss, hypotension, hyponatremia, and intense mucocutaneous melanosis). An acquired form of peripheral resistance to glucocorticoids was suspected. We, therefore, examined glucocorticoid receptor characteristics on mononuclear leukocytes by measuring [3H]dexamethasone binding and the effect of dexamethasone on [3H]thymidine incorporation, which is one of the effects of glucocorticoid receptor activation. Glucocorticoid receptor density was increased in AIDS patients with an Addisonian picture (group 1; 16.2 ± 9.4 fmol/million cells) compared to values in 12 AIDS patients without an Addisonian picture (group 2; 6.05 ± 2.6 fmol/million cells; P less than 0.01) and sex- and age-matched controls (3.15 ± 2.3 fmol/million cells; P less than 0.01). The affinity of glucocorticoid receptors (Kd) was strikingly decreased (9.36 ± 3.44 nM in group 1; 3.2 ± 1.5 nM in group 2; 2.0 ± 0.8 nM in controls; P less than 0.01). [3H]Thymidine incorporation was decreased dose-dependently by dexamethasone in controls and patients; the effect was significantly blunted (P less than 0.05) in group 1 patients, which suggests that activation of glucocorticoid receptor is impaired as a result of the glucocorticoid receptor abnormality. In conclusion, AIDS patients with hypercortisolism and clinical features of peripheral resistance to glucocorticoids are characterized by abnormal glucocorticoid receptors on lymphocytes. Resistance to glucocorticoids implies a complex change in immune-endocrine function, which may be important in the course of immunodeficiency syndrome. 1740663 Induction of monocytic differentiation and NF-kappa B-like activities by human immunodeficiency virus 1 infection of myelomonoblastic cells. The effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression. 1740667 The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter. Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter. 1537389 Interleukin 6-induced differentiation of a human B cell line into IgM-secreting plasma cells is mediated by c-fos. The role of the protooncogene c-fos in interleukin ( IL ) 6-induced B cell differentiation was assessed. Treatment of SKW 6.4 cells with IL 6 induced a transient and early stimulation of c-fos sense mRNA expression. The effect appeared within 30 min and returned to basal levels after 2 h. The addition of antisense oligonucleotides to c-fos significantly inhibited IL 6-induced IgM production by SKW 6.4 cells (p less than 0.001), whereas control oligonucleotides had no inhibitory effect. These results indicate that activation of c-fos is involved in IL 6-induced differentiation of SKW 6.4 cells into IgM-secreting cells. 1537556 Binding of erythroid and non-erythroid nuclear proteins to the silencer of the human epsilon-globin-encoding gene. To clarify the molecular mechanisms involved in the developmental control of hemoglobin-encoding genes we have been studying the expression of these genes in human cells in continuous culture. We have previously reported the presence of a transcriptional control element with the properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin-encoding gene [Cao et al., Proc.Natl.Acad.Sci.USA 86 ( 1989 ) 5306-5309]. We also showed that this silencer has stronger inhibitory activity in HeLa cells, as compared to K562 human erythroleukemia cells. Using deletion mutants and cis-cloned synthetic oligodeoxyribonucleotides in transient expression assays, nucleotide sequences responsible for this effect have now been further delimited to 44 bp located from -294 to -251 bp. Gel electrophoresis mobility shift assays and DNaseI footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. Two binding proteins are detected in K562 nuclear extracts, while only one is found in extracts from HeLa cells. Possible mechanisms by which these proteins may regulate transcription of the epsilon-globin-encoding gene in erythroid and non-erythroid cells are discussed. 1371788 Gangliosides suppress tumor necrosis factor production in human monocytes. Both normal and malignant cells contain gangliosides as important cell membrane constituents that, after being shed, may influence cells of the immune system. We have studied the impact of gangliosides on the expression of TNF in blood monocytes and in the monocytic cell line Mono Mac 6. Although under standard culture conditions, bovine brain gangliosides (100 micrograms/ml) suppressed LPS-stimulated TNF production 5-fold in PBMC and 10-fold in Mono Mac 6 cells, suppression was more efficient under serum-free conditions. Looking at highly purified gangliosides, GD3, GD1a, GM3, GM2, and GM1 were all effective in reducing TNF production in PBMC, and in Mono Mac 6 by factor 10 to 50. The suppressive activity was lost in molecules, lacking the sugar moiety or the lipid moiety. Gangliosides appear to act at an early step of activation in that TNF transcripts were reduced and the mobilization of the nuclear factor kappa B was blocked. Furthermore, in time kinetics, gangliosides were effective for up to 30 min after addition of LPS, but not thereafter. However, the expression of the CD14 Ag, a receptor molecule for LPS-LPS binding protein complexes, was unaffected by gangliosides. Finally, when using Staphylococcus aureus or platelet activating factor as a stimulus, gangliosides were able to suppress TNF production in Mono Mac 6 cells by factor 5 to 10, as well. On the other hand, phorbol ester-induced production of O2- was similar in cells treated with and without gangliosides. Taken together, our data demonstrate that TNF gene expression in monocytes induced by different types of stimuli can be blocked by gangliosides at an early step of signal transduction. 1541828 T cell-specific negative regulation of transcription of the human cytokine IL-4. IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells, B cells, and mast cells. We investigated the upstream regulatory elements of the human IL-4 promoter. A novel T cell-specific negative regulatory element (NRE) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene: -311CTCCCTTCT-303 (NRE-I) and -288CTTTTTGCTT-TGC-300 (NRE-II). A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II, respectively, were identified. Furthermore, a positive regulatory element was found 45 bp downstream of the NRE. The enhancer activity of the PRE was completely suppressed when the NRE was present. These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element. These data may have implications for the stringent control of IL-4 expression in T cells. 1796314 [Regulatory effect of insulin on glucocorticoid receptor in human peripheral leukocytes] The regulatory effect of insulin on the specific binding power of glucocorticoid receptor (GR) of human leukocytes was assessed by the unoccupied receptor sites capable of combining with [3H] labelled dexamethasone measured at 3 and 24 h after incubation with various concentrations of insulin added to the medium. After 3 h incubation the specific binding power with [3H] Dex was decreased by 23.3 ± 10.0, 32.2 ± 13.2 and 54.3 ± 9.2% (P greater than 0.05, P greater than 0.05 and P less than 0.01 as compared with the control value of 100 in the absence of insulin) respectively in the presence of 20 mU/L (physiological testing concentration), 200 mU/L (physiological upper limit) and 2,000 mU/L (pharmacological concentration) insulin in the incubation medium. After 24 h incubation the decrease of these values increased respectively to 43.5 ± 19.0, 56.1 ± 20.7 and 80.2 ± 15.5 (P less than 0.05, P less than 0.01 and P less than 0.01 compared with control). Thus the inhibitory effect of insulin on the GR binding power is both dose- and time-dependent, which strongly suggests that GR is tonically controlled by insulin concentration change under physiological conditions. 1545132 Human T cell activation through the activation-inducer molecule/CD69 enhances the activity of transcription factor AP-1. The induction of the AP-1 transcription factor has been ascribed to the early events leading to T cell differentiation and activation. We have studied the regulation of AP-1 activity in human peripheral blood T lymphocytes stimulated through the activation inducer molecule ( AIM ) /CD69 activation pathway. Phorbol esters are required to induce AIM/CD69 cell-surface expression as well as for triggering the proliferation of T cells in conjunction with anti-AIM mAb. Mobility shift assays showed that addition of anti-AIM mAb to PMA-treated T lymphocytes markedly enhanced the binding activity of AP-1 to its cognate sequence, the phorbol ester response element. In contrast, anti-AIM mAb did not induce any change in the binding activity of NF-kappa B, a transcription factor whose activity is also regulated by protein kinase C. The increase in AP-1-binding activity was accompanied by the marked stimulation of the transcription of c-fos but not that of c-jun. Blockade of the DNA-binding complexes with an anti-Fos mAb demonstrated a direct participation of c-Fos in the AP-1 complexes induced by anti-AIM mAb. Most of the AP-1 activity could be eliminated when the anti-AIM mAb was added to the culture medium in the presence of cycloheximide, suggesting that de novo protein synthesis is crucial for the induction of AP-1-binding activity. These data provide the evidence that activation of human peripheral blood T cells through the AIM activation pathway regulate the activity of AP-1. Therefore, this pathway appears as a crucial step in the initiation of early T cell activation events. 1545787 cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1. The recent definition of a consensus DNA binding sequence for the Ets family of transcription factors has allowed the identification of potential Ets binding sites in the promoters and enhancers of many inducible T-cell genes. In the studies described in this report, we have identified two potential Ets binding sites, EBS1 and EBS2, which are conserved in both the human and murine interleukin-2 enhancers. Within the human enhancer, these two sites are located within the previously defined DNase I footprints, NFAT-1 and NFIL-2B, respectively. Electrophoretic mobility shift and methylation interference analyses demonstrated that EBS1 and EBS2 are essential for the formation of the NFAT-1 and NFIL-2B nuclear protein complexes. Furthermore, in vitro mutagenesis experiments demonstrated that inducible interleukin-2 enhancer function requires the presence of either EBS1 or EBS2. Two well-characterized Ets family members, Ets-1 and Ets-2, are reciprocally expressed during T-cell activation. Surprisingly, however, neither of these proteins bound in vitro to EBS1 or EBS2. We therefore screened a T-cell cDNA library under low-stringency conditions with a probe from the DNA binding domain of Ets-1 and isolated a novel Ets family member, Elf-1. Elf-1 contains a DNA binding domain that is nearly identical to that of E74, the ecdysone-inducible Drosophila transcription factor required for metamorphosis (hence the name Elf-1, for E74-like factor 1). Elf-1 bound specifically to both EBS1 and EBS2 in electrophoretic mobility shift assays. It also bound to the purine-rich CD3R element from the human immunodeficiency virus type 2 long terminal repeat, which is required for inducible virus expression in response to signalling through the T-cell receptor. Taken together, these results demonstrate that multiple Ets family members with apparently distinct DNA binding specificities regulate differential gene expression in resting and activated T cells. 1312541 Mineralocorticoids and mineralocorticoid receptors in mononuclear leukocytes in patients with pregnancy-induced hypertension. To examine the role of mineralocorticoids in the pathophysiology of pregnancy-induced hypertension (PIH), we studied plasma aldosterone and 18-hydroxycorticosterone levels in 25 women with PIH and 25 normal pregnant women, as controls. Furthermore, we evaluated the mineralocorticoid receptor (MR) status in mononuclear leukocytes in the 2 groups. MR count was significantly (P less than 0.0005) decreased in the PIH group (148 ± 9 binding sites/cell) compared with the control group (300 ± 17 binding sites/cell; mean ± SEM). Plasma aldosterone in women with PIH was 281 ± 61 pmol/L; in normal pregnant women it was 697 ± 172 pmol/L (P less than 0.025). Plasma 18-hydroxycorticosterone was also significantly (P less than 0.025) lower (PIH, 1071 ± 149 pmol/L; controls, 1907 ± 318 pmol/L). These values were determined at the onset of clinical symptoms of PIH. These results can not be explained by receptor down-regulation due to higher levels of mineralocorticoids in PIH; a hitherto unknown mineralocorticoid may , thus, be responsible for the hypertension and altered MR status. 1312543 Mineralocorticoid effector mechanism in preeclampsia. Mineralocorticoid effector mechanisms were evaluated in 29 patients with preeclampsia and in 25 uncomplicated pregnancies by measurement of plasma aldosterone, levels of mineralocorticoid receptor (MR) in mononuclear leucocytes, and subtraction potential difference (SPD; rectal minus oral values). Mean values for plasma aldosterone were not different between the two groups, but significant differences were observed for MR (preeclampsia, 81 ± 44 receptors/cell; controls, 306 ± 168) and SPD (preeclampsia, 65 ± 7 mV; controls, 12 ± 5 mV). In six cases we determined MR, plasma aldosterone, and SPD in patients with preeclampsia before and 3 months after delivery. MR were reduced before delivery (96 ± 27 receptors/cell), and SPD increased (64 ± 8 mV), with both parameters normalizing after delivery (MR, 242 ± 79; SPD, 14.0 ± 4 mV). Aldosterone levels returned to normal nonpregnant values after delivery. These data suggest an important role for abnormalities in mineralocorticoid effector mechanisms in the etiology of preeclampsia and could be an useful marker for diagnosis. 1372388 A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells. The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family, which includes the p50 and p65 subunits of nuclear factor kappa B. We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs. In particular, the c-Rel homodimer has a high affinity for interleukin-6 (IL-6) and beta interferon kappa B sites. In spite of its association with p50 in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes; this factor, termed IL-6 kappa B binding factor II, appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs. Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1/tumor necrosis factor-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells. 1347914 Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for v-erbA oncogene action. The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene c-erbA in erythropoiesis. Here we demonstrate that the endogenous thyroid hormone receptor alpha (c-erbA/TR-alpha) and the closely related retinoic acid receptor alpha (RAR-alpha) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human RAR-alpha in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the RAR-alpha is essential for the RA effect. Likewise, enhanced expression of exogenous c-erbA/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors. 1313226 Leukotriene B4 stimulates c-fos and c-jun gene transcription and AP-1 binding activity in human monocytes. We have examined the effect of leukotriene B4 (LTB4), a potent lipid proinflammatory mediator, on the expression of the proto-oncogenes c-jun and c-fos. In addition, we looked at the modulation of nuclear factors binding specifically to the AP-1 element after LTB4 stimulation. LTB4 increased the expression of the c-fos gene in a time- and concentration-dependent manner. The c-jun mRNA, which is constitutively expressed in human peripheral-blood monocytes at relatively high levels, was also slightly augmented by LTB4, although to a much lower extent than c-fos. The kinetics of expression of the two genes were also slightly different, with c-fos mRNA reaching a peak at 15 min after stimulation and c-jun at 30 min. Both messages rapidly declined thereafter. Stability of the c-fos and c-jun mRNA was not affected by LTB4, as assessed after actinomycin D treatment. Nuclear transcription studies in vitro showed that LTB4 increased the transcription of the c-fos gene 7-fold and the c-jun gene 1.4-fold. Resting monocytes contained nuclear factors binding to the AP-1 element, but stimulation of monocytes with LTB4 induced greater AP-1-binding activity of nuclear proteins. These results indicate that LTB4 may regulate the production of different cytokines by modulating the yield and/or the function of transcription factors such as AP-1-binding proto-oncogene products. 1532661 An 11-base-pair DNA sequence motif apparently unique to the human interleukin 4 gene confers responsiveness to T-cell activation signals. We have identified a DNA segment that confers responsiveness to antigen stimulation signals on the human interleukin (IL) 4 gene in Jurkat cells. The human IL-4 gene, of 10 kilobases, is composed of four exons and three introns. A cis-acting element (P sequence) resides in the 5' upstream region; no additional DNA segments with enhancer activity were identified in the human IL-4 gene. For further mapping purposes, a fusion promoter was constructed with the granulocyte/macrophage colony-stimulating factor basic promoter containing 60 base pairs of sequence upstream from the cap site of the mouse granulocyte/macrophage colony-stimulating factor gene and various lengths of the 5' upstream sequence of the IL-4 gene. The P sequence was located between positions -79 and -69 relative to the transcription start site of the human IL-4 gene, and this location was confirmed by base-substitution mutations. The plasmids carrying multiple copies of the P sequence showed higher responsiveness to the stimulation. The binding protein ( s ) that recognize the P sequence of the IL-4 gene were identified by DNA-mobility-shift assays. The binding of NF(P) (a DNA binding protein that specifically recognizes the P sequence) to the P sequence was abolished when oligonucleotides carrying base substitutions were used, indicating that the NF(P) interaction is sequence-specific and that binding specificity of the protein paralleled the sequence requirements for IL-4 expression in vivo. The P sequence does not share homology with the 5' upstream sequence of the IL-2 gene, even though surrounding sequences of the IL-4 gene share high homology with the IL-2 gene. We conclude that a different set of proteins recognize IL-2 and IL-4 genes. 1557508 Glucocorticoid receptor binding in three different cell types in major depressive disorder: lack of evidence of receptor binding defect. 1 . In order to further understand the apparent glucocorticoid resistance in major depressive disorder, circadian variation in cortisol concentration, dexamethasone suppression and glucocorticoid receptor binding in mononuclear leukocytes, polymorphonuclear leukocytes and cultured skin fibroblasts were measured in rigidly defined major depressive disorder patients and non-depressed psychiatric controls. 2 . Mononuclear leukocytes binding to glucocorticoid correlated significantly with polymorphonuclear leukocytes binding to glucocorticoid, but both determinations failed to differentiate major depressive disorder and control subjects. 3 . Initial and post-dexamethasone in vitro fibroblast binding to glucocorticoid was not different between major depressive disorder and non-depressed control subjects. 4 . The phenomenon of glucocorticoid resistance in major depressive disorder remains unexplained. 1313693 Kinetics of nuclear translocation and turnover of the vitamin D receptor in human HL60 leukemia cells and peripheral blood lymphocytes-- coincident rise of DNA-relaxing activity in nuclear extracts. High affinity receptors (VDR) for 1,25-dihydroxycholecalciferol (calcitriol) are expressed in HL60 human leukemia cells and in low numbers in peripheral blood lymphocytes (PBL). HL60 cells, expressing some characteristics of promyelocytes, can be induced to monocytoid differentiation by calcitriol. Specific nuclear translocation of [3H]calcitriol/VDR was examined after exposure of whole cells to 10(-9) M/l calcitriol in the presence and absence of a 500-fold excess of unlabeled ligand and subsequent isolation of nuclei. Specific nuclear translocation of [3H]calcitriol/VDR was found to be time dependent reaching a maximum of approximately 2100 binding sites/nucleus after 3 h of incubation in HL60 cells, whereas a maximum of approximately 310 binding sites/nucleus was found after 3 h in PBL. Pulse exposure of HL60 to radiolabeled hormone for 3 h followed by culture in medium without serum and calcitriol lead to nuclear retention of approximately 1600 radiolabeled VDR by 8 h and approximately 1000 VDR by 24 h. Radiolabeled VDR disappeared from the nuclear compartment with a halflife of approximately 30 min if cells were cultured with identical concentrations of unlabeled hormone after the pulse (pulse/chase-experiments). No difference of VDR retention in pulse and pulse/chase-experiments was seen in PBL, where VDR halflife was approximately 30 min. No specific translocation into the nuclear compartment was seen when isolated nuclei were incubated in [3H]calcitriol. Radiolabeled hormone/receptor complexes of nuclei isolated from cells exposed for 3 h to radiolabeled hormone--in contrast to identical experiments with intact cells--did not disappear from the nuclear compartment upon incubation of nuclei with identical concentrations of the unlabeled compound. The activity of DNA relaxing enzymes (e.g. topoisomerases I and II) in nuclear extracts was measured using a PBR 322-relaxation-assay. Enhanced overall enzyme activity was found in nuclear extracts by 1 h after incubation with calcitriol (final ethanol concentration 0.0001 % v/v) in HL60 and PBL. The enhanced activity disappeared after 2 h in PBL, whereas it was still enhanced by 4 h in HL60. No effect was seen in ethanol treated controls. We conclude that a specific nuclear translocation mechanism exists for calcitriol in both cell types examined, most likely due to translocation of receptor proteins after hormone binding. Translocated hormone/receptor complexes compete for a limited number of specific nuclear binding sites. Enhanced activity of topoisomerases in nuclear extracts upon translocation of VDR might reflect interaction of both within the nuclear compartment, thus initiating DNA-unwinding, a prerequisite of transcription initiation. 1560002 The B cell-specific nuclear factor OTF-2 positively regulates transcription of the human class II transplantation gene, DRA. The promoter of the major histocompatibility class II gene DRA contains an octamer element (ATTTGCAT) that is required for efficient DRA expression in B cells. Several DNA-binding proteins are known to bind this sequence. The best characterized are the B cell-specific OTF-2 and the ubiquitous OTF-1. This report directly demonstrates that OTF-2 but not OTF-1 regulates the DRA gene. In vitro transcription analysis using protein fractions enriched for the octamer-binding protein OTF-2 demonstrate a positive functional role for OTF-2 in DRA gene transcription. In contrast, OTF-1-enriched protein fractions did not affect DRA gene transcription although it functionally enhanced the transcription of another gene. Recombinant OTF-2 protein produced by in vitro transcription/translation could also enhance DRA gene transcription in vitro. In vivo transient transfection studies utilizing an OTF-2 expression vector resulted in similar findings : that OTF-2 protein enhanced DRA gene transcription, and that this effect requires an intact octamer element. Together these results constitute the first direct evidence of a positive role for the lymphoid-specific octamer-binding factor in DRA gene transcription. 1807404 [Regulation of intracellular cholesterol synthesis in hypercholesterolemia by glucocorticoids] The rate of endogenous cholesterol synthesis in blood lymphocytes and skin fibroblasts from patients with type IIa hyperlipidemia was found to be increased in comparison with healthy donors. The cells of hyperlipidemic patients had lowered levels of glucocorticoid receptors concomitantly with a partial loss of their sensitivity to glucocorticoids. In fibroblasts from patients with hereditary hypercholesteremia of homozygous type the number of glucocorticoid receptors did not exceed 10 % of their content in normal cells. The decrease of the number of glucocorticoid receptors in patients with type IIa hyperlipidemia seems to be a compensatory response of cells culminating in activation of endogenous cholesterol synthesis. 1314139 Transcription factor activation and functional stimulation of human monocytes. Activation of expression of genes encoding transcription factors: c-fos and c-jun and formation of AP1 transcriptional complex in human monocytes was investigated. It was found that lipopolysaccharide induced strongly both c-fos and c-jun expression as well as AP1 formation. Interferon gamma activated strongly c-fos and weakly c-jun and AP1. Tumor necrosis factor induced slightly c-fos and had almost no effect on c-jun and AP1. The data suggest that differences in functional responses elicited in monocytes by all three factors may be dependent on different routes on nuclear signalling employed by the factors. 1566834 Corticosteroid receptors and lymphocyte subsets in mononuclear leukocytes in aging. Plasma cortisol and aldosterone levels and number of related receptors in mononuclear leukocytes were measured in 49 healthy aged subjects (62-97 yr) and in 21 adult controls (21-50 yr). In all subjects, in addition, lymphocyte subsets were determined as an index of corticosteroid action. The mean number of type I and type II receptors was significantly lower in aged subjects than in controls (respectively, 198 ± 96 and 272 ± 97 receptors/cell for type I, and 1,794 ± 803 and 3,339 ± 918 for type II receptors). Plasma aldosterone and cortisol and lymphocyte subsets were not different in the two groups. All of the parameters were also tested for correlation, and a significant inverse correlation was found between age and type I and type II receptors when all subjects were plotted and between aged and CD4 and age and CD4/CD8 in the aged group. These data show that aged subjects have reductions of corticosteroid receptors that are not associated with increase of related steroids and that this situation probably represents a concomitant of the normal aging process. 1349016 Stable expression of HB24, a diverged human homeobox gene, in T lymphocytes induces genes involved in T cell activation and growth. A diverged homeobox gene, HB24, which is known to be induced following lymphocyte activation, was introduced into Jurkat T cells under the control of a constitutive promoter. Stable transfectants of HB24 were established that expressed high levels of HB24 mRNA and possessed an altered phenotype suggestive of activated T cells. A number of genes known to be induced following T cell activation and associated with cell growth were increased in the transfectants, including c-fos, c-myc, c-myb, HLA-DR, lck, NF-kappa B, interleukin-2 and interleukin-2 receptor alpha (IL-2R alpha). Analysis of IL-2R alpha expression by transient transfection of IL-2R alpha promoter constructs into the HB24 transfectants revealed constitutive expression (about 60% of phytohemagglutinin- and phorbol ester-activated Jurkat cells) that was dependent on the kappa B site in the IL-2R alpha promoter. Furthermore, as a consequence of the increased HB24 mRNA levels, the Jurkat HB24 transfectants proliferated more rapidly than control cell lines. Thus, stable expression of HB24 confers an activation phenotype on a human T cell line, implicating this gene as an important transcriptional factor during T cell activation and growth. 1533227 Studies on the biological activity of triiodothyronine sulfate. Hepatic microsomes and isolated hepatocytes in short term culture desulfate T3 sulfate (T3SO4). We, therefore, wished to determine whether T3SO4 could mimic the action of thyroid hormone in vitro. T3SO4 had no thyromimetic effect on the activity of Ca(2+)-ATPase in human erythrocyte membranes at doses up to 10,000 times the maximally effective dose of T3 (10(-10) mol/L). In GH4C1 pituitary cells, T3SO4 failed to displace [125I]T3 from nuclear receptors in intact cells or soluble preparations. Thus, T3SO4 was not directly thyromimetic in either an isolated human membrane system or a pituitary cell system in which nuclear receptor occupancy correlates with GH synthesis. Thyroid hormones inhibit [3H]glycosaminoglycan synthesis by cultured human dermal fibroblasts, and T3SO4 displayed about 0.5% the activity of T3 at 72 h. Human fibroblasts contained roughly the same level of microsomal p-nitrophenyl sulfatase activity as that previously observed in hepatic microsomes. Propylthiouracil (50 mumol/L) did not affect the action of T3SO4, suggesting that deiodination was not important for this activity of T3SO4. Thus, it appears T3SO4 has no intrinsic biological activity, but , under certain circumstances, may be reactivated by desulfation. 1533441 Nuclear factor of activated T cells contains Fos and Jun. The nuclear factor NF-AT (ref. 1) is induced in T cells stimulated through the T-cell receptor/CD3 complex, and is required for interleukin-2 (IL-2) gene induction. Although NF-AT has not been cloned or purified, there is evidence that it is a major target for immunosuppression by cyclosporin A (CsA) and FK506 (refs 2-7). NF-AT induction may require two activation-dependent events: the CsA-sensitive translocation of a pre-existing component and the CsA-resistant synthesis of a nuclear component. Here we report that the newly synthesized nuclear component of NF-AT is the transcription factor AP-1. We show that the inducible nuclear form of NF-AT contains Fos and Jun proteins. Furthermore, we identify a pre-existing NF-AT-binding factor that is present in hypotonic extracts of unstimulated T cells. On the basis of binding, reconstitution and cotransfection experiments, we propose that activation of NF-AT occurs in at least two stages: a CsA-sensitive stage involving modification and/or translocation of the pre-existing NF-AT complex, and a CsA-insensitive stage involving the addition of newly synthesized Fos or Fos/Jun proteins to the pre-existing complex. 1315834 Interferon-gamma potentiates the antiviral activity and the expression of interferon-stimulated genes induced by interferon-alpha in U937 cells. Binding of type I interferon (IFN-alpha/beta) to specific receptors results in the rapid transcriptional activation, independent of protein synthesis, of IFN-alpha-stimulated genes (ISGs) in human fibroblasts and HeLa and Daudi cell lines. The binding of ISGF3 (IFN-stimulated gene factor 3) to the conserved IFN-stimulated response element (ISRE) results in transcriptional activation. This factor is composed of a DNA-binding protein (ISGF3 gamma), which normally is present in the cytoplasm, and other IFN-alpha-activated proteins which preexist as latent cytoplasmic precursors (ISGF3 alpha). We have found that ISG expression in the monocytic U937 cell line differs from most cell lines previously examined. U937 cells express both type I and type II IFN receptors, but only IFN-alpha is capable of inducing antiviral protection in these cells. Pretreatment with IFN-gamma potentiates the IFN-alpha-induced protection, but IFN-gamma alone does not have any antiviral activity. ISG15 mRNA accumulation in U937 cells is not detectable before 6 h of IFN-alpha treatment, peaks at 24 h, and requires protein synthesis. Although IFN-gamma alone does not induce ISG expression, IFN-gamma pretreatment markedly increases and hastens ISG expression and transcriptional induction. Nuclear extracts assayed for the presence of ISRE binding factors by electrophoretic mobility shift assays show that ISGF3 is induced by IFN-alpha within 6 h from undetectable basal levels in untreated U937 cells. Activation of ISGF3 alpha, the latent component of ISGF3, occurs rapidly. However, the increase in ISGF3 activity ultimately correlates with the accumulation of ISGF3 gamma induced by IFN-alpha or IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS) 1374612 The mechanism of action of cyclosporin A and FK506. CsA and FK506 are powerful suppressors of the immune system, most notably of T cells. They act at a point in activation that lies between receptor ligation and the transcription of early genes. Here, Stuart Schreiber and Gerald Crabtree review recent findings that indicate CsA and FK506 operate as prodrugs: they bind endogenous intracellular receptors, the immunophilins, and the resulting complex targets the protein phosphatase, calcineurin, to exert the immunosuppressive effect. 1813169 [Plasma cortisol concentration and blood leukocyte content of glucocorticoid receptors in patients with deficiency-cold vs deficiency-heat syndromes] Plasma cortisol concentration and blood leukocyte content of glucocorticoid receptors (GCR) were assayed in 20 patients with deficiency syndromes, 10 cold in property (deficiency-cold), the other 10 hot in property (deficiency-heat), and also in 10 healthy individuals as normal control for the purpose of investigating the nature of cold and heat syndromes. As a result, the cases of deficiency-cold syndrome (DCS) had a normal concentration of plasma cortisol but a lowered content of GCR in leukocytes when compared with the normal control (P less than 0.05); the cases of deficiency-heat syndrome (DHS) had a higher concentration of plasma cortisol than the normal control (P less than 0.05) and a slightly higher content of GCR in leukocytes. It was concluded that the DCS is characterized by diminished biological effects of adrenocortical activity, while the DHS, by augmented biological effects of adrenocortical activity. 1583734 Specific NF-kappa B subunits act in concert with Tat to stimulate human immunodeficiency virus type 1 transcription. NF-kappa B is a protein complex which functions in concert with the tat-I gene product to stimulate human immunodeficiency virus (HIV) transcription. To determine whether specific members of the NF-kappa B family contribute to this effect, we have examined the abilities of different NF-kappa B subunits to act with Tat-I to stimulate transcription of HIV in Jurkat T-leukemia cells. We have found that the p49 ( 100 ) DNA binding subunit, together with p65, can act in concert with Tat-I to stimulate the expression of HIV-CAT plasmid. Little effect was observed with 50-kDa forms of p105 NF-kappa B or rel, in combination with p65 or full-length c-rel, which do not stimulate the HIV enhancer in these cells. These findings suggest that the combination of p49 ( 100 ) and p65 NF-kappa B can act in concert with the tat-I gene product to stimulate the synthesis of HIV RNA. 1533884 Activation of the human immunodeficiency virus type 1 enhancer is not dependent on NFAT-1. The function of a putative NFAT-1 site in the human immunodeficiency virus type 1 enhancer has been analyzed. Activation by the T-cell antigen receptor is minimal in Jurkat cells and is mediated by the kappa B sites. The putative NFAT-1 region is not required for the response to anti-CD3 or to mitogens in T-cell, B-cell, or monocyte/macrophage leukemia lines, nor is it a cis-acting negative regulatory element. 1814434 Protein kinase C activation and protooncogene expression in differentiation/retrodifferentiation of human U-937 leukemia cells. Human U-937 leukemia cells differentiate along the monocytic lineage following 3-day exposures to 12-O-tetradecanoylphorbol-13-acetate (TPA). This induction of differentiation is accompanied by adherence and loss of proliferation, as well as expression/repression of differentiation-associated genes. Long term culture of TPA-differentiated U-937 cells in the absence of phorbol ester for 32-36 days resulted in a process of retrodifferentiation. The retrodifferentiated cells detached from the substrate and reinitiated proliferation. Other cellular parameters, such as glycosidase activities, cytokine release, and filament expression, returned to levels similar to that observed in uninduced cells. Treatment of U-937 cells with TPA resulted in a rapid translocation of protein kinase C (PKC) from the cytosol to cell membrane fractions within 2-8 min. Increased levels of membrane-associated PKC activity persisted until 17-29 days. However, longer periods of incubation were associated with a return to the distribution of PKC in control cells. Activation of PKC has been implicated in the regulation of certain immediate early response genes, and in the present studies, TPA rapidly induced c-fos and c-jun gene expression. Levels of c-fos and c-jun transcripts remained elevated during periods of PKC activation and also returned to levels observed in control cells by 30-36 days, when the cells entered retrodifferentiation. Staurosporine, a nonspecific inhibitor of PKC, partially blocked TPA-induced adherence and growth inhibition and concomitantly prevented TPA-induced c-fos and c-jun gene expression.(ABSTRACT TRUNCATED AT 250 WORDS) 1375324 The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene. 1351090 Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway. We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 ( -139 ) long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection. 1603822 Eicosanoids in breast cancer patients before and after mastectomy. In 19 patients with a malignant breast tumor, tumor tissue and blood were taken to determine the eicosanoid profile and platelet aggregation. Values were compared with those of patients with benign tumors (n = 4), or undergoing a mammary reduction (n = 7). Postoperatively, blood was taken as well in order to compare pre- and postoperative values. Eicosanoids were measured in peripheral blood monocytes and mammary tissue by means of HPLC; furthermore, TXA2, 6-keto-PGF1 alpha, and PGE2 were determined by RIA. Differences in pre- and postoperative values of cancer patients were seen in plasma RIA values: PGE2 and 6-k-PGF1 alpha were significantly higher preoperatively when compared with postoperatively, however , such differences were seen in the control groups as well. Compared to benign tumor or mammary reduction test material the eicosanoid profile of tissue obtained from malignant mammary tumors showed important differences. Except for PGF2 alpha, HHT and 15-HETE no detectable quantities of eicosanoids were found in the non-tumor material, whereas in the malignant tumor material substantial quantities of a number of eicosanoid metabolites were present. Statistically significant correlations could be established between patient/histopathology data and the results of the platelet aggregation assays, e.g. between menopausal status and ADP aggregation; oestrogen receptor (+/-) and collagen and arachidonic acid aggregation, inflammatory cell infiltration score and arachidonic acid aggregation and fibrosis score and ADP aggregation. The results show that eicosanoid synthesis in material from mammary cancer patients is different from that in benign mammary tissue. The implications, in particular, in relation to future prognosis of the patient, remain obscure. 1613697 c-myc mRNA expression in minor salivary glands of patients with Sjogren 's syndrome. c-myc protooncogene is implicated in the pathogenesis of B cell lymphoid malignancies and high levels of c-myc mRNA expression are observed in activated blood mononuclear cells. Sjogren 's syndrome (SS) is characterized by lymphocytic infiltrates of exocrine glands, remarkable B cell hyperreactivity and a strong predisposition to B cell neoplasia. In this study, c-myc protooncogene mRNA expression in 29 labial minor salivary gland biopsies from patients with primary SS and 15 controls was examined using in situ hybridization histochemistry. Two 40mer oligonucleotides from the 1st and the 2nd exon of the c-myc gene, labeled with 35S, were used as probes. To detect the origin of the cell hybridized with a c-myc probe, a combined immunochemistry in situ hybridization histochemistry technique was used. High c-myc mRNA expression was detected on acinar epithelial cells. c-myc did not correlate with c-fos and c-jun protein expression. Stronger c-myc mRNA expression was detected in labial salivary glands of patients with longer disease duration (p less than or equal to 0.002) and more intense T lymphocyte infiltrates (p less than 0.05) although these patients revealed no hypergammaglobulinemia. No correlation was observed between c-myc mRNA and B lymphocyte monoclonicity or lymphoma. In conclusion, strong c-myc mRNA expression was observed on epithelial cells of labial salivary glands from patients with primary SS. Our findings may indicate the presence of a reactivated virus hosted in these cells. 1535455 Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. B lymphocytes and macrophages express closely related immunoglobulin G (IgG) Fc receptors (Fc gamma RII) that differ only in the structures of their cytoplasmic domains. Because of cell type-specific alternative messenger RNA splicing, B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells. Transfection of an Fc gamma RII-negative B-cell line with complementary DNA 's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation. The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation. Instead, regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms. In contrast, the insertion did contribute to the formation of caps in response to receptor cross-linking, consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton. 1668145 Every enhancer works with every promoter for all the combinations tested: could new regulatory pathways evolve by enhancer shuffling? The promoters and enhancers of cell type-specific genes are often conserved in evolution, and hence one might expect that a given enhancer has evolved to work best with its own promoter. While this expectation may be realized in some cases, we have not found evidence for it. A total of 27 combinations of different promoters and enhancers were tested by transfection into cultured cells. We found that the relative efficiency of the enhancers is approximately the same, irrespective of the type of promoter used, i.e. , there was no strong preference for any given enhancer/promoter combination. Notably, we do not see particularly strong transcription when the immunoglobulin kappa enhancer ( or the immunoglobulin heavy chain enhancer) is used to activate a kappa gene promoter. We propose that a generally permissive enhancer/promoter interaction is of evolutionary benefit for higher eukaryotes: by enhancer shuffling, genes could be easily brought under a new type of inducibility/cell type specificity. 1618911 Heterodimerization and transcriptional activation in vitro by NF-kappa B proteins. The NF-kappa B family of transcription proteins represents multiple DNA binding, rel related polypeptides that contribute to regulation of genes involved in immune responsiveness and inflammation, as well as activation of the HIV long terminal repeat. In this study multiple NF-kappa B related polypeptides ranging from 85 to 45 kDa were examined for their capacity to interact with the PRDII regulatory element of interferon beta and were shown to possess distinct intrinsic DNA binding affinities for this NF-kappa B site and form multiple DNA binding homo- and heterodimer complexes in co-renaturation experiments. Furthermore, using DNA templates containing two copies of the PRDII domain linked to the rabbit beta globin gene, the purified polypeptides specifically stimulated NF-kappa B dependent transcription in an in vitro reconstitution assay as heterodimers but not as p50 homodimers. These experiments emphasize the role of NF-kappa B dimerization as a distinct level of transcriptional control that may permit functional diversification of a limited number of regulatory proteins. 1620119 Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity. Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position. 1620122 Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate antigen. Oct-2 is a transcription factor that binds specifically to octamer DNA motifs in the promoters of immunoglobulin and interleukin-2 genes. All tumor cell lines from the B-cell lineage and a few from the T-cell lineage express Oct-2. To address the role of Oct-2 in the T-cell lineage, we studied the expression of Oct-2 mRNA and protein in nontransformed human and mouse T cells. Oct-2 was found in CD4+ and CD8+ T cells prepared from human peripheral blood and in mouse lymph node T cells. In a T-cell clone specific for pigeon cytochrome c in the context of I-Ek, Oct-2 was induced by antigen stimulation, with the increase in Oct-2 protein seen first at 3 h after activation and continuing for at least 24 h. Oct-2 mRNA induction during antigen-driven T-cell activation was blocked by cyclosporin A, as well as by protein synthesis inhibitors. These results suggest that Oct-2 participates in transcriptional regulation during T-cell activation. The relatively delayed kinetics of Oct-2 induction suggests that Oct-2 mediates the changes in gene expression which occur many hours or days following antigen stimulation of T lymphocytes. 1621110 [Changes in plasma interleukin-1 and their possible relationship with the changes in glucocorticoid receptor in aged long-distance runner] For the study of the changes in plasma interleukin-1 (IL-1) and their possible relationship with the changes in glucocorticoid receptor (GR), plasma IL-1 and GR in peripheral blood leukocytes in aged long-distance runner were measured simultaneously. The activity of IL-1 was expressed as its ability to stimulate 3H-TdR incorporation in the thymocytes of C57 mice. GR was determined by whole cell assay with 3H-Dex. The results showed that the activity of plasma IL-1 in aged long-distance runner was 209 %, 223 % and 145 % of the control at 14.7-18.7, 3.8-7.0 and 1.5-2.6 KD fractions. The GR in peripheral blood leukocytes in aged runner was 65 % of the control. Possible relationship between the changes in IL-1 and GR in aged long-distance runner and its physiological significance are discussed. 1628621 Transcription factor AP-2 activates gene expression of HTLV-I. The HTLV-I LTR contains three conserved regulatory elements known as 21 base pair repeats which are required for stimulation of gene expression by the transactivator protein tax. Mutagenesis indicates that the 21 bp repeats can be subdivided into three motifs, A, B and C, each of which influences the level of tax activation. The A site in the 21 bp repeat has strong homology with previously described binding sites for the transcription factor AP-2. We demonstrated that AP-2 mRNA was present in T-lymphocytes and that cellular factors from both non-transformed and transformed T-lymphocytes specifically bound to the consensus motif for AP-2 in each 21 bp. To determine the role of AP-2 in the regulation of the HTLV-I LTR gene expression, we used an AP-2 cDNA in DNA binding and transient expression assays. Gel retardation and methylation interference studies revealed that bacterially produced AP-2 bound specifically and with high affinity to all three 21 bp repeats, and that it required the core sequence AGGC for specific binding. Binding of AP-2 prevented the subsequent binding of members of the CREB/ATF family to an adjacent regulatory motif in the 21 bp repeat. Transfection of an AP-2 expression construct into T-lymphocytes activated gene expression from the HTLV-I LTR. At least two 21 bp repeats were required for high levels of AP-2 activation and mutagenesis of the AP-2 consensus binding sequences in the 21 bp repeats eliminate this activation.(ABSTRACT TRUNCATED AT 250 WORDS) 1631130 Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors. The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which " pure " early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocyte-macrophage pathway by differential treatment with hematopoietic growth factors. The GATA-1 gene, though virtually silent in quiescent progenitors, is activated after entrance into the cell cycle upon stimulation with hematopoietic growth factors. Subsequently, increasing expression along the erythroid pathway contrasts with an abrupt downregulation in the granulocyte-macrophage lineage. These results suggest a microenvironment-directed, two-step model for GATA-1 expression in differentiating hematopoietic progenitors that involves ( i ) cycle-dependent initiation and ( ii ) lineage-dependent maintenance or suppression. Hypothetically, on/off switches of lineage-restricted transactivators may underlie the binary fate decisions of hematopoietic progenitors. 1386811 [Age-related changes in glucocorticoid and mineralocorticoid receptors in lymphocytes of healthy persons and patients with hypertension] It has been found that the number of glucocorticoid receptors in lymphocytes of the peripheral blood of healthy elderly subjects increases , while the number of mineralocorticoid receptors decreases. The mechanisms of hormone-receptor interactions in hypertension are activated: the number of glucocorticoid and mineralocorticoid binding sites grows in hypertensive patients. Still a more essential rise in the number of receptors is observed in mid-age hypertensive patients than in elderly ones. 1502171 In vivo footprint analysis of the HLA-DRA gene promoter: cell-specific interaction at the octamer site and up-regulation of X box binding by interferon gamma. Analysis of the major histocompatibility complex class II gene promoter DRA has previously identified at least five cis-acting regions required for maximal expression. We have examined the DRA promoter for protein-DNA interactions in the intact cell, which may mediate transcriptional activation. Using in vivo genomic footprinting we identified interactions in B-cell lines at the octamer site and the Y, X1, and X2 boxes. Class II antigen expressing T-cell lines maintained contacts identical to B-cell lines, while class II-negative T-cell lines exhibited no interactions. In lymphoid cell lines, the octamer site is occupied and required for maximal expression. This is most likely due to the presence of the lymphoid-specific OTF-2 factor. In contrast, the class II-positive nonlymphoid glioblastoma cell line does not exhibit interactions at the octamer site despite the presence of the ubiquitous OTF-1 factor and an open binding site. Thus, the DRA promoter discriminates against OTF-1 activation at the level of DNA binding in the glioblastoma line. Interferon gamma induces class II expression in this glioblastoma cell line and , in parallel, up-regulates X1 and X2 box protein-DNA interactions, while all other interactions remain unchanged. These results suggest that interferon gamma functions on a poised promoter by altering weak , nonproductive interactions at the X boxes to strong interactions. These findings provide direct in vivo evidence to strongly suggest that the modulation of X1 and X2 interactions is an important constituent of the interferon gamma induction pathway. 1502179 Simple derivation of TFIID-dependent RNA polymerase II transcription systems from Schizosaccharomyces pombe and other organisms, and factors required for transcriptional activation. Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters. This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae, human lymphocytes, and Schizosaccharomyces pombe. TFIIDs from all three organisms are interchangeable among all three systems. The S. cerevisiae and Sch. pombe systems support effects of acidic activator proteins, provided a further protein fraction from S. cerevisiae is supplied. This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators. 1502202 NF-kappa B-dependent induction of the NF-kappa B p50 subunit gene promoter underlies self-perpetuation of human immunodeficiency virus transcription in monocytic cells. The molecular mechanisms underlying the sustained nuclear translocation of NF-kappa B observed in U937 monocytic cells chronically infected with human immunodeficiency virus (HIV) were studied. The activity of the promoter regulating the synthesis of the p105 precursor of the NF-kappa B p50 subunit was enhanced in these cells. Deletions in this promoter indicated that this upregulation was mediated through the NF-kappa B- but not the AP-1-binding motif, by bona fide p50/p65 heterodimers. Analysis of cytosolic extracts indicated that NF-kappa B levels were increased in HIV-infected cells. In contrast to the transient NF-kappa B activation induced by phorbol ester, the permanent NF-kappa B translocation induced by HIV infection was not dependent on PKC isoenzymes alpha and beta as shown by the use of a specific inhibitor (GF 109203X). These observations indicate that during chronic HIV infection of U937 cells, continuous NF-kappa B (p50/p65) translocation results in p105 promoter upregulation with subsequent cytosolic NF-kappa B accumulation, ready for further translocation. This HIV-mediated mechanism results in a self-perpetuating loop of NF-kappa B production. 1386962 The development of functionally responsive T cells. The work reviewed in this article separates T cell development into four phases. First is an expansion phase prior to TCR rearrangement, which appears to be correlated with programming of at least some response genes for inducibility. This phase can occur to some extent outside of the thymus. However, the profound T cell deficit of nude mice indicates that the thymus is by far the most potent site for inducing the expansion per se, even if other sites can induce some response acquisition. Second is a controlled phase of TCR gene rearrangement. The details of the regulatory mechanism that selects particular loci for rearrangement are still not known. It seems that the rearrangement of the TCR gamma loci in the gamma delta lineage may not always take place at a developmental stage strictly equivalent to the rearrangement of TCR beta in the alpha beta lineage, and it is not clear just how early the two lineages diverge. In the TCR alpha beta lineage, however, the final gene rearrangement events are accompanied by rapid proliferation and an interruption in cellular response gene inducibility. The loss of conventional responsiveness is probably caused by alterations at the level of signaling, and may be a manifestation of the physiological state that is a precondition for selection. Third is the complex process of selection. Whereas peripheral T cells can undergo forms of positive selection (by antigen-driven clonal expansion) and negative selection (by abortive stimulation leading to anergy or death), neither is exactly the same phenomenon that occurs in the thymic cortex. Negative selection in the cortex appears to be a suicidal inversion of antigen responsiveness:instead of turning on IL-2 expression, the activated cell destroys its own chromatin. The genes that need to be induced for this response are not yet identified, but it is unquestionably a form of activation. It is interesting that in humans and rats, cortical thymocytes undergoing negative selection can still induce IL-2R alpha expression and even be rescued in vitro, if exogenous IL-2 is provided. Perhaps murine thymocytes are denied this form of rescue because they shut off IL-2R beta chain expression at an earlier stage or because they may be uncommonly Bcl-2 deficient (cf. Sentman et al. , 1991; Strasser et al. , 1991). Even so, medullary thymocytes remain at least partially susceptible to negative selection even as they continue to mature. 1380242 SRC-related proto-oncogenes and transcription factors in primary human T cells: modulation by cyclosporin A and FK506. Activation of T lymphocytes induces transcription of genes encoding for lymphokines. Interleukin-2 (IL-2) gene expression is controlled transcriptionally by the cooperative activity of specific trans-activating factors that bind to the IL-2 enhancer. Cyclosporin A (CsA) and FK506 inhibit the production of IL-2 in T lymphocytes at the level of gene transcription. A member of the src gene family, the lymphocyte-specific protein tyrosine kinase, p56lck, has been implicated in IL-2 production. CsA was found not to inhibit lck gene expression, nor the activity of the lck gene product. However, CsA and FK506 inhibit the appearance of DNA binding activity of factors that bind to the NF-AT and AP-1 sites in the IL-2 enhancer. Since the induction of NF-AT and AP-1 is induced by the same stimuli that stimulate IL-2 production, these results indicate that the immunosuppressant action of CsA and FK506 is exerted at the level of these trans-activating factors. 1505523 TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation. The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen.(ABSTRACT TRUNCATED AT 250 WORDS) 1510811 Bcl-2: a repressor of lymphocyte death. The genes and mechanisms that control programmed cell death are currently the subject of intense study. The bcl-2 gene, a repressor of lymphocyte death, is perhaps the best understood of the programmed cell death associated genes. Here, Stanley Korsmeyer provides a brief overview of bcl-2, concentrating on its roles in B- and T-cell development and in oncogenesis. 1510878 Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity. We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC. 1355356 Functional interaction between the two zinc finger domains of the v-erb A oncoprotein. The v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor. The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor. The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A, one of which, at serine 61, is known to be important for v-erb A function in the neoplastic cell. We report here that the second alteration, at threonine 78, also plays an important, although more indirect, role: alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61. Threonine 78 lies within the D-box of the v-erb A protein, a region thought to mediate receptor-receptor dimerizations, and is not in physical proximity to the serine at position 61. It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function. 1516825 Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis. BSAP has been identified previously as a transcription factor that is expressed at early, but not late, stages of B-cell differentiation. Biochemical purification and cDNA cloning has now revealed that BSAP belongs to the family of paired domain proteins. BSAP is encoded by the Pax-5 gene and has been highly conserved between human and mouse. An intact paired domain was shown to be both necessary and sufficient for DNA binding of BSAP. Binding studies with several BSAP recognition sequences demonstrated that the sequence specificity of BSAP differs from that of the distantly related paired domain protein Pax-1. During embryogenesis, the BSAP gene is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other Pax genes in the developing central nervous system (CNS). Later, the expression of the BSAP gene shifts to the fetal liver where it correlates with the onset of B lymphopoiesis. BSAP expression persists in B lymphocytes and is also seen in the testis of the adult mouse. All of this evidence indicates that the transcription factor BSAP may not only play an important role in B-cell differentiation but also in neural development and spermatogenesis. 1520341 Okadaic acid is a potent inducer of AP-1, NF-kappa B, and tumor necrosis factor-alpha in human B lymphocytes. Treatment of human B lymphocytes with an optimal concentration of okadaic acid, an inhibitor of phosphatases 1 and 2A, resulted in the induction of the transcription factor, AP-1 and a marked increase in NF-kappa B levels. In contrast, no effect on the levels of the octamer binding proteins, Oct-1 or Oct-2, were found. Since both AP-1 and NF-kappa B have been reported to be important in the induction of the tumor necrosis factor-alpha (TNF-alpha) gene we examined the effects of okadaic acid on TNF-alpha mRNA levels. Treatment with okadaic acid resulted in a striking increase in TNF-alpha mRNA transcripts within 1 h of stimulation and large amounts of TNF-alpha were released into the culture media. Although okadaic acid provides a potent inductive signal for AP-1 and NF-kappa B it did not induce either B cell proliferation or immunoglobulin secretion. 1527846 A novel Ets-related transcription factor, Elf-1, binds to human immunodeficiency virus type 2 regulatory elements that are required for inducible trans activation in T cells. Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are structurally related retroviruses which both cause AIDS in humans. Although both viruses establish latency in quiescent human-peripheral-blood T cells, the asymptomatic phase of HIV-2 infection may be more prolonged than that of HIV-1. The latent phases of both HIV-1 and HIV-2 infection have been shown to be disrupted by T-cell activation, a process that requires host cell transcription factors. In the case of HIV-1, the transcription factor NF-kappa B is sufficient for inducible transcriptional activation. In contrast, factors in addition to NF-kappa B are required to activate HIV-2 transcription in infected T cells. In this report, we demonstrate that a novel Ets-related transcription factor, Elf-1, binds specifically to two purine-rich motifs in the HIV-2 enhancer. Mutagenesis experiments demonstrated that these Elf-1 binding sites are required for induction of HIV-2 transcription following T-cell-receptor-mediated T-cell activation. Moreover, Elf-1 is the only factor present in activated T-cell nuclear extracts that binds to these sites in electrophoretic mobility shift assays. Thus, Elf-1 is a novel transcription factor that appears to be required for the T-cell-receptor-mediated trans activation of HIV-2 gene expression. These results may explain differences in the clinical spectra of diseases caused by HIV-1 and HIV-2 and may also have implications for the design of therapeutic approaches to HIV-2 infection. 1527859 Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells. Human immunodeficiency virus type 1 (HIV-1) can establish a persistent and latent infection in CD4+ T lymphocytes (W.C.Greene, N.Engl.J. Med.324 : 308-317, 1991; S.M.Schnittman, M.C.Psallidopoulos, H.C. Lane, L.Thompson, M.Baseler, F.Massari, C.H.Fox, N.P.Salzman, and A.S.Fauci, Science 245 : 305-308, 1989). Production of HIV-1 from latently infected cells requires host cell activation by T-cell mitogens (T.Folks, D.M.Powell, M.M.Lightfoote, S.Benn, M.A. Martin, and A.S.Fauci, Science 231 : 600-602, 1986; D.Zagury, J. Bernard, R.Leonard, R.Cheynier, M.Feldman, P.S.Sarin, and R.C. Gallo, Science 231 : 850-853, 1986). This activation is mediated by the host transcription factor NF-kappa B [G.Nabel and D.Baltimore, Nature ( London ) 326 : 711-717, 1987]. We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-kappa B DNA-binding activity by T-cell mitogens. However, Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation, including SP-1, USF, URS, and NF-AT. Additionally, Nef inhibits the induction of HIV-1- and interleukin 2-directed gene expression, and the effect on HIV-1 transcription depends on an intact NF-kappa B-binding site. These results indicate that defective recruitment of NF-kappa B may underlie Nef 's negative transcriptional effects on the HIV-1 and interleukin 2 promoters. Further evidence suggests that Nef inhibits NF-kappa B induction by interfering with a signal derived from the T-cell receptor complex. 1326675 [Effect of antihypertensive therapy with captopril on gluco- and mineralocorticoid receptors of peripheral blood lymphocytes in hypertensive patients of various age] Binding of 3H-dexamethasone and 3H-aldosterone by peripheral lymphocyte receptors was investigated in healthy persons and hypertensive patients before and after 2-week captopril treatment. The number of glucocorticoid and mineralocorticoid binding sites was increased in hypertensives vs normotensives. The treatment with the ACE inhibitor captopril led to activation of hormone-receptor interactions. There was a more marked rise of the number of receptors in middle-aged (44-55 years) hypertensives vs elderly (61-80 years) subjects after captopril treatment. 1327803 Leukotriene B4 transcriptionally activates interleukin-6 expression involving NK-chi B and NF-IL6. Leukotriene B4 (LTB4) is a notable participant in inflammation and chemotaxis. It is , however, still unclear whether LTB4 acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines. Here we report that LTB4 induces synthesis of interleukin (IL)-6 by human blood monocytes through transcriptional activation of the IL-6 gene. We furthermore demonstrate that this process involves activation of the transcription factor NF-chi B and, to a lesser extent, of NF-IL6, while the activity of the transcription factor AP-1, shown to otherwise confer IL-6 inducibility, appeared to be unaffected by LTB4. Involvement of NF-chi B and NF-IL6 in induction of IL-6 transcription by monocytes was demonstrated using deleted forms of the IL-6 promoter. Activation of the IL-6 promoter by LTB4 was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional IL-6 protein as well. In addition, LTB4 mediated transactivation of a heterologous promoter construct containing the NF-chi B or the NF-IL6 enhancer, but not the AP-1 enhancer. The signaling events mediating this effect appeared to involve the release of H2O2, since LTB4 failed to induce NF-chi B or NF-IL6 in the presence of the scavenger of H2O2, N-acetyl-L-cysteine. 1397976 Estrogen binding sites in peripheral blood monocytes and effects of danazol on their sites in vitro. 1 . This study was designed to investigate the presence of estrogen type I (high affinity, low capacity) and type II (low affinity, high capacity) binding sites in human peripheral blood monocytes and the effects of danazol on these sites. 2 . These two types of estrogen binding sites existed in human peripheral blood monocytes. 3 . Danazol bound to these sites in high concentration (10(-6) M, clinical serum concentration during danazol therapy) and decreased the number of both sites. 4 . It is suggested that danazol has an anti-estrogenic action to the monocytes through the competition and suppression of estrogen binding sites as seen in the estrogen target organ. 1398205 [Mechanism of action of steroid hormones . I . Estrogens] The steroid hormone are very versatile molecules: although they are related among them by their chemical structure, they have very diverse functions and including antagonic. Their action mechanism is not completely cleared. The estrogens participate in the regulation of practically all the reproductive and sexual events of the female, although the intracellular actions by which they take place are not well known and the proposed models do not adequately satisfy the questions. Currently it is accepted the existence of a cytoplasmic and/or nuclear receptor, without explaining satisfactorily how the hormones come to the nucleus. The endocrine events that are rapidly expressed (seconds) are due to a possible interaction with cellular membrane. The purpose of this review is to analyze and concilliate the reported data on the mechanism of action of estrogens. 1402661 Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication. The human immunodeficiency virus (HIV) Rev protein is essential for viral structural protein expression (Gag, Pol, and Env) and , hence , for viral replication. In transient transfection assays, mutant forms of Rev have been identified that inhibit wild-type Rev activity and therefore suppress viral replication. To determine whether such transdominant Rev proteins could provide long-term protection against HIV infection without affecting T cell function, T leukemia cell lines were stably transduced with a retroviral vector encoding a transdominant mutant of the Rev protein, M10. While all the M10-expressing cell lines remained infectable by HIV-1, these same cells failed to support a productive replication cycle when infected with a cloned isolate of HIV-1. In addition, two out of three M10-expressing CEM clones were also resistant to highly productive infection by a heterogeneous HIV-1 pool. Expression of M10 did not affect induction of HIV transcription mediated by the kappa B regulatory element or Tat. Importantly, constitutive expression of Rev M10 did not alter the secretion of interleukin 2 in response to mitogen stimulation of EL-4 and Jurkat cells. The inhibition of HIV infection in cells stably expressing a transdominant Rev protein, in the absence of any deleterious effect on T cell function, suggests that such a strategy could provide a therapeutic effect in the T lymphocytes of acquired immunodeficiency syndrome patients. 1404124 Glucocorticoid receptor in patients with lupus nephritis: relationship between receptor levels in mononuclear leukocytes and effect of glucocorticoid therapy. We investigated the clinical significance of glucocorticoid receptor determination in 20 patients with systemic lupus erythematosus (SLE) who afterwards developed nephrotic syndrome. Glucocorticoid receptor concentrations in mononuclear leukocytes (MNL) in these patients were comparable with those in both other patients with SLE and healthy persons. Improvement in urinary protein excretion and in disease activity, which was scored according to the SLE Disease Activity Index system of the University of Toronto, closely related to the glucocorticoid receptor concentrations in MNL isolated from the corresponding patients. In summary, glucocorticoid receptor determination in patients with lupus nephritis may be a predictive clue for assessing responsiveness to glucocorticoid therapy. 1406630 Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation. Analysis of the p50 and p65 subunits of the NF-kappa B transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known kappa B DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50- and c-Rel-selected sequences. Using either a p50- or p65-selected kappa B motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappa B complex. Similarly, in transfection experiments in which the selective kappa B binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65- and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-kappa B activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappa B complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific kappa B motifs present. 1328873 Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II ( NF-kappa B ) element. We have biochemically and functionally characterized a new transcription factor, NP-TCII, which is present in nuclei from unstimulated T and B lymphocytes but is not found in nonhematopoietic cells. This factor has a DNA-binding specificity similar to that of NF-kappa B but is unrelated to this or other Rel proteins by functional and biochemical criteria. It can also be distinguished from other previously described lymphocyte-specific DNA-binding proteins. 1406939 The candidate oncoprotein Bcl-3 is an antagonist of p50/NF-kappa B-mediated inhibition. The candidate oncogene bcl-3 was discovered as a translocation into the immunoglobulin alpha-locus in some cases of B-cell chronic lymphocytic leukaemias. The protein Bcl-3 contains seven so-called ankyrin repeats. Similar repeat motifs are found in a number of diverse regulatory proteins but the motifs of Bcl-3 are most closely related to those found in I kappa B proteins in which the ankyrin repeat domain is thought to be directly involved in inhibition of NF-kappa B activity. No biological function has yet been described for Bcl-3, but it was noted recently that Bcl-3 interferes with DNA-binding of the p50 subunit of NF-kappa B in vitro. Here we demonstrate that Bcl-3 can aid kappa B site-dependent transcription in vivo by counteracting the inhibitory effects of p50/NF-kappa B homodimers. Bcl-3 may therefore aid activation of select NF-kappa B-regulated genes, including those of the human immunodeficiency virus. 1411249 A microtitre assay system for glucocorticoid receptors: decreased receptor concentration in myocardial infarction. A major difficulty in determination of glucocorticoid receptor sites is the very complicated assay procedure. Therefore, we describe a microtitre assay system for glucocorticoid receptors which is a whole-cell competitive binding radioassay using [3H]-dexamethasone as radioligand. This modification of a previously described protocol simplifies and reduces laboratory work and allows assay reproducibility to be controlled more reliably. Thus enabled to perform the test on multiple blood samples in parallel, we investigated cardiac infarction patients over a 12-day period to test if glucocorticoid receptor binding is altered in this ' stressful ' disease. On the first day of the disease, glucocorticoid receptor capacity was significantly decreased without alteration of the receptor-ligand affinity, whereas on days 4 and 12 the number of receptor sites was normal again. This result fits well into the general observation of stress-induced down-regulation of immune responses. 1419903 Regulation of c-jun expression during induction of monocytic differentiation by okadaic acid. The present work has examined the effects of okadaic acid, an inhibitor of type 1 and 2A protein phosphatases, on the regulation of c-jun expression during monocytic differentiation of U-937 leukemia cells. The results demonstrate that okadaic acid treatment is associated with induction of a differentiated monocyte phenotype characterized by : ( a ) growth arrest; ( b ) increases in Mac-1 cell surface antigen expression; ( c ) down-regulation of c-myc transcripts; and ( d ) induction of tumor necrosis factor gene expression. This induction of monocytic differentiation was associated with transient increases in c-jun mRNA levels, which were maximal at 6 h. Similar effects were obtained for the c-fos gene. Run-on analysis demonstrated detectable levels of c-jun transcription in U-937 cells and that this rate is increased approximately 40-fold following okadaic acid exposure. c-jun mRNA levels were superinduced in cells treated with both okadaic acid and cycloheximide, whereas inhibition of protein synthesis had little, if any, effect on okadaic acid-induced c-jun transcription. The half-life of c-jun mRNA was similar (45-50 min) in both untreated and okadaic acid-induced cells. In contrast, treatment with both okadaic acid and cycloheximide was associated with stabilization (t 1/2 = 90 min) of c-jun transcripts. Taken together, these findings indicate that the induction of c-jun transcription by okadaic acid is controlled primarily by a transcriptional mechanism. Since previous studies have demonstrated that the c-jun gene is autoinduced by Jun/AP-1, we also studied transcription of c-jun promoter (positions -132/+170)-reporter gene constructs with and without a mutated AP-1 element.(ABSTRACT TRUNCATED AT 250 WORDS) 1419905 Activation of NF-kappa B by interleukin 2 in human blood monocytes. We report here that interleukin 2 (IL-2) acts on human blood monocytes by enhancing binding activity of the transcription factor NF-kappa B to its consensus sequence in the 5' regulatory enhancer region of the IL-2 receptor alpha chain (p55). Similarly, IL-2 activates NF-kappa B in the human monocytic cell line U 937, but not in resting human T-cells. This effect is detectable within 15 min and peaks 1 h after exposure to IL-2. Enhanced NF-kappa B binding activity is followed by functional activation in that inducibility of the IL-2 receptor alpha chain is mediated by enhanced NF-kappa B binding and that a heterologous promoter containing the NF-kappa B consensus sequence (-291 to -245) of the IL-2 receptor alpha chain gene is activated. In addition, IL-2 is capable of increasing transcript levels of the p50 gene coding for the p50 subunit of the NF-kappa B transcription factor, whereas mRNA levels of the p65 NF-kappa B gene remained unchanged. 1421207 Single point estimation of glucocorticoid receptors in lymphocytes of normal subjects and of children under long term glucocorticoid treatment. A single point assay of glucocorticoid receptors (GR) in human lymphocytes based on the measurement of specific dexamethasone binding has been developed and compared with a common multi-point Scatchard analysis. The assay conditions-concentration of the ligand 20 nmol/l, incubation time 2 h and the cell count 2-6 mil. cells/tube in the assay volume 0.25 ml were found to be optimal. An attempt was also undertaken to use a cell harvester for the separation of cells from unbound ligand. Though specifically bound dexamethasone measured by whole-cell assay and that using cell harvester correlated well, almost by one order lower values obtained with the latter method render it non-applicable for receptor quantitation. The results from 9 healthy volunteers (average GR concentration 7131 ± 1256 sites/cell) correlated excellently with those obtained by the Scatchard analysis. The single point assay has been also applied for determination of GH in 10 children treated with large doses of prednisone. The average values from healthy volunteers did not differ significantly from those found in these children, though much broader range was found in patients. 1423591 A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors. A novel B cell-restricted activity, required for high levels of octamer/Oct-dependent transcription from an immunoglobulin heavy chain (IgH) promoter, was detected in an in vitro system consisting of HeLa cell-derived extracts complemented with fractionated B cell nuclear proteins. The factor responsible for this activity was designated Oct coactivator from B cells (OCA-B). OCA-B stimulates the transcription from an IgH promoter in conjunction with either Oct-1 or Oct-2 but shows no significant effect on the octamer/Oct-dependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USF- and Sp1-regulated promoters. Taken together, our results suggest that OCA-B is a tissue-, promoter-, and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters. In light of the evidence showing physical and functional interactions between Oct factors and OCA-B, we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters. 1429562 The regulation of the human tumor necrosis factor alpha promoter region in macrophage, T cell, and B cell lines. The 1311-base pair human tumor necrosis factor ( TNF ) alpha promoter region was fused to the luciferase (Luc) reporter gene and studied in a transient transfection system in three TNF producing cell lines, the U937 macrophage cell line, the MLA 144 T cell line, and the 729-6 B cell line. This full length promoter construct can be induced by phorbol 13-myristate acetate (PMA) in each of these cell types. Analysis of a series of 5'-truncations showed several peaks of basal and PMA induced activity suggesting the presence of several positive and negative regulatory elements. A PMA responsive element was localized to a region between -95 and -36 bp relative to the transcription start site. Within this region, single AP-2- and AP-1-like consensus sequences were noted. These AP-2 and AP-1 sites were each modified with a double point mutation. A modest (20-50%) reduction in TNF promoter activity was observed with the AP-2 site mutation. However, mutation of the AP-1 site markedly diminished both the basal and PMA-activated promoter activity. Also co-transfections of the wild-type promoter construct with an AP-1/c-jun expression vector resulted in augmented basal and PMA-induced promoter activity. 1431113 Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells. We used phenolic, lipid-soluble, chain-breaking antioxidants (butylated hydroxyanisole (BHA), nordihydroquairetic acid, or alpha-tocopherol (vitamin E) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation. BHA was found to suppress not only PMA- or TNF-induced, but also constitutive, HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. This was also true for KBF (p50 homodimer) binding activity in U937 cells. Secretion of TNF, the product of another NF-kappa B-dependent gene, was abolished by BHA in PMA-stimulated U937 cells. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cell, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation. These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert their effect only if cells are in an appropriate redox status, because prior modification toward reduction with BHA treatment prevents this activation. It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes. 1434944 Expression of c-fos, c-jun and jun B in peripheral blood lymphocytes from young and elderly adults. The expression of c-fos, c-jun and jun B proto-oncogenes was studied in phytohemagglutinin (PHA) activated peripheral blood lymphocytes (PBL) from young and aged humans. Specific mRNAs for c-fos and c-jun were detectable within 30 min after cell activation and reached maximal levels within 2 h. Both c-fos and jun B mRNAs decreased to pre-activation levels within 6 h, while c-jun mRNA remained elevated. In PHA-activated PBL, no age-related differences were observed in c-fos or jun B mRNA expression. However, c-jun mRNA levels decreased significantly (1.73 ± 0.08 vs. 1.16 ± 0.09 arbitrary units, P < 0.01, young vs. old) in PBL from elderly individuals activated with PHA. Because previous work has demonstrated that T cells from elderly individuals may display normal proliferative responses when activated via the anti-CD2 pathway, c-jun and jun B mRNA expression was also studied in anti-CD2-activated purified T cells. No age-related differences were found in the expression of either of these two proto-oncogenes by anti-CD2 activated T cells. These results suggest that the decreased IL-2 production and proliferative response displayed by PHA-activated PBL from elderly adults may be related to age-related changes in c-jun mRNA expression and in the ratio of c-fos to c-jun mRNA. 1437560 Characterization of a novel T lymphocyte protein which binds to a site related to steroid/thyroid hormone receptor response elements in the negative regulatory sequence of the human immunodeficiency virus long terminal repeat. We have previously identified a T lymphocyte protein which binds to a site within the LTR of the human immunodeficiency virus type 1 (HIV-1) and exerts an inhibitory effect on virus gene expression. The palindromic site (site B) recognized by this protein is related to the palindromic binding sites of members of the steroid/thyroid hormone receptor family. Here we characterize the T cell protein binding to this site as a 100 kD protein which is most abundant in T cells and which binds to site B as a 200 kD complex. This protein is distinct from other members of the steroid/thyroid hormone receptor family including the COUP protein which has a closely related DNA binding specificity. 1279685 A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1. Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g. , endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4)9. In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription. 1443130 Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action. Rapid nongenomic in vitro effects of aldosterone on intracellular electrolytes, cell volume, and Na(+)-H+ antiport have been found in human mononuclear leukocytes (HML). Binding of 125I-labeled aldosterone to plasma membranes of HML shares important features with these functional data. This includes a very low apparent dissociation constant (Kd) of 0.1 nM for both aldosterone and the effect on the Na(+)-H(+)-antiport, a high turnover rate, and the almost exclusive binding selectivity for aldosterone. Dexamethasone, RU 26988, corticosterone, ouabain, amiloride, and 18-hydroxyprogesterone were inactive as ligands. Deoxycorticosterone acetate had an intermediate activity with an apparent Kd of 100 nM. These findings are the first to demonstrate membrane binding of aldosterone being compatible with major aspects of its nongenomic effects. 1332912 Reticuloendotheliosis virus long terminal repeat elements are efficient promoters in cells of various species and tissue origin, including human lymphoid cells. Promiscuous transcriptional activity of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) was detected in transient expression assays using LTR-chloramphenicol acetyltransferase-encoding gene chimeras, and cells of diverse species and tissue type; levels of expression from two different REV LTRs correlate with reports of pathogenicity of the respective viruses in vivo. REVs do not encode a transactivator targeted to the viral LTR, and cells infected with Marek 's disease virus, a herpesvirus with an overlapping host range, do not express factors that preferentially enhance expression from REV or avian sarcoma/leukemia virus LTRs. REV LTRs work efficiently in human lymphoid cells, and are viable alternatives to promoters commonly used for expression of cloned genes. They may also prove useful in the identification of new, ubiquitous cellular transcription factors. 1448931 Natural variants of the HIV-1 long terminal repeat: analysis of promoters with duplicated DNA regulatory motifs. Sequence variation in the long terminal repeat (LTR) region of HIV-1 was analyzed in viral isolates of 17 infected individuals. Two classes of LTR size variants were found. One HIV-1 variant was detected containing an additional binding site for the transcription factor Sp1. Another LTR size variation was observed in four patients in a region just upstream of the NF-kappa B enhancer. This variation was the result of a duplication of a short DNA sequence (CTG-motif). Cell culture experiments demonstrated that the natural variant with four Sp1 sites had a slightly higher promoter activity and viral replication rate than the isogenic control LTR with three Sp1 sites. No positive effect of the duplicated CTG-motif could be detected. In order to measure small differences in virus production more accurately, equal amounts of a size variant and the wild-type plasmid were cotransfected into T-cells. The virus with four Sp1 sites did outgrow the three Sp1 virus in 35 days of culture and CTG-monomer virus outcompeted the CTG-dimer virus in 42 days. Based on these results we estimate a 5-10% difference in virus production of the LTR variants when compared to that of wild-type. 1453013 SCL and related hemopoietic helix-loop-helix transcription factors. The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus implying a role for SCL in erythroid differentiation events. However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL suggest it is also important in proliferation and self-renewal events in erythroid cells. 1454801 Transcription of the hypersensitive site HS2 enhancer in erythroid cells. In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream beta-like globin genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem Ap1 sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may ( i ) open up the chromatin structure of a gene domain and ( ii ) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site. 1464736 Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells. NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias. There is little information available on the response of NF kappa B to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF kappa B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF kappa B in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SP1 was unaffected by addition of TPA. The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NF kappa B species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NF kappa B. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes. 1299224 Ablation of transplanted HTLV-I Tax-transformed tumors in mice by antisense inhibition of NF-kappa B [published erratum appears in Science 1993 Mar 12 ; 259 ( 5101 ) : 1523] Mice transgenic for the human T cell leukemia virus (HTLV-I) Tax gene develop fibroblastic tumors that express NF-kappa B-inducible early genes. In vitro inhibition of NF-kappa B expression by antisense oligodeoxynucleotides (ODNs) inhibited growth of these culture-adapted Tax-transformed fibroblasts as well as an HTLV-I-transformed human lymphocyte line. In contrast, antisense inhibition of Tax itself had no apparent effect on cell growth. Mice treated with antisense to NF-kappa B ODNs showed rapid regression of transplanted fibrosarcomas. This suggests that NF-kappa B expression may be necessary for the maintenance of the malignant phenotype and provides a therapeutic approach for HTLV-I-associated disease. 1334806 Inhibition of anti-CD3 monoclonal antibody-induced T-cell proliferation by dexamethasone, isoproterenol, or prostaglandin E2 either alone or in combination. 1 . The purpose of these studies was to investigate the modulation of the proliferation of human T cells obtained from peripheral blood by dexamethasone (DEX), isoproterenol (ISO), and prostaglandin E2 (PGE2). The former two substances interact with T cells via the glucocorticoid and beta-adrenergic receptors respectively. When occupied by their natural ligands, glucocorticosteroids and catecholamines, these receptors have a role in modulating T-cell function during stress. During the inflammatory response increased levels of PGE2 bind to their receptors on T cells and thus alter responsiveness. Proliferation of T cells was induced by immobilized anti-CD3 monoclonal antibody (mAb) in the presence or absence of an additional costimulatory signal delivered by anti-CD28 mAb. 2 . Various physiologic concentrations of DEX, ISO, or PGE2 were added at the time of initiation of the cultures and subsequent proliferation of the unstimulated T cells was determined. The results demonstrate that physiologic concentrations of all three of these agents inhibit the anti-CD3 mAb-induced proliferation of T cells. 3 . Although DEX and PGE2 were equipotent in suppressing T-cell proliferation, ISO was much less effective. 4 . Because concomitant elevations in the peripheral levels of these substances may occur, experiments were performed to determine the T-cell inhibitory effects of DEX together with either PGE2 or ISO. Synergistic suppression of T-cell proliferation was observed when various concentrations of DEX and PGE2, but not DEX and ISO, were added to cultures. This synergistic suppression could not be explained by an increase in cAMP accumulation in T cells stimulated with DEX and PGE2. 5 . Finally, the addition of anti-CD28 mAb to anti-CD3 mAb-stimulated T cells overcame much of the suppression of proliferation induced by PGE2 or ISO but less so than that induced by DEX. 1470918 Targeted degradation of c-Fos, but not v-Fos, by a phosphorylation-dependent signal on c-Jun. The proto-oncogene products c-Fos and c-Jun heterodimerize through their leucine zippers to form the AP-1 transcription factor. The transcriptional activity of the heterodimer is regulated by signal-dependent phosphorylation and dephosphorylation events. The stability of c-Fos was found to also be controlled by intracellular signal transduction. In transient expression and in vitro degradation experiments, the stability of c-Fos was decreased when the protein was dimerized with phosphorylated c-Jun. c-Jun protein isolated from phorbol ester-induced cells did not target c-Fos for degradation, which suggests that c-Fos is transiently stabilized after stimulation of cell growth. v-Fos protein, the retroviral counterpart of c-Fos, was not susceptible to degradation targeted by c-Jun. 1472057 Mutations in the Pit-1 gene in children with combined pituitary hormone deficiency. Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone and prolactin genes. In three unrelated Japanese children with combined pituitary hormone deficiency, we identified three point mutations in the Pit-1 gene, Pro24Leu, Arg143Gln, and Arg271Trp, located on the major transactivation region, POU-specific domain, and POU-homeodomain, respectively. 1335357 Calcitriol : a hematolymphopoietrope? [editorial] A MEDLINE search of the English-language literature was conducted using the indexing terms 'immunology, calcitriol and vitamin D' to identify studies indicating a role for calcitriol as a primary immunomodulator. Sixty-six papers published between January 1956 and June 1991 were identified. Forty-five of these reports are cited in this review. The data strongly suggest an endocrine, autocrine and/or paracrine role for calcitriol in immune regulation. No unifying hypothesis has yet emerged explaining this collection of data. This paper provides a brief review of immune properties currently attributed to calcitriol. 1335418 Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells. We have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription. 1478011 The use of interferon-gamma-treated U937 cells in chemiluminescence assays to detect red cell, platelet and granulocyte antibodies of potential clinical significance. The chemiluminescent (CL) response of interferon-gamma-treated U937 (IFN-U937) cells to sensitized target cells has been used to detect red cell, platelet and granulocyte antibodies. A clone of U937 cells was selected which expressed Fc receptor I (Fc gamma RI) and which, after incubation with IFN-gamma for 72 h, was capable of generating high levels of lucigenin-enhanced CL. The CL responses of IFN-U937 cells and peripheral blood human monocytes to sensitized red cells, platelets or granulocytes were then compared. Assays using monocytes or IFN-U937 cells were of comparable sensitivity for detection of antibodies against all three types of target cell. In addition, the use of IFN-U937 cells reduced interassay variation and simplified assay performance. The potential clinical usefulness of these CL assays was suggested by the ability of both monocytes and IFN-U937 cells to respond to red cells, platelets or granulocytes sensitized with sera from pregnant women whose babies had either haemolytic disease of the newborn (HDN), alloimmune thrombocytopenia or alloimmune neutropenia respectively. In addition, monocytes and IFN-U937 cells both responded to red cells sensitized with antibodies against a variety of specificities of assumed ( although not documented) clinical significance for blood transfusion recipients. In contrast, monocytes and IFN-U937 cells responded only weakly to red cells sensitized with either anti-D in sera from mothers of babies unaffected by HDN, or with antisera containing high titre antibodies with specificities not normally associated with significantly reduced red cell survival. 8419337 Involvement of Alu sequences in the cell-specific regulation of transcription of the gamma chain of Fc and T cell receptors. The Fc epsilon RI-gamma chains are expressed in a variety of hematopoietic cells where they play a critical role in signal transduction. They are part of the high affinity IgE receptor in mast cells, basophils, Langerhans cells, and possibly other cells; a component of the low affinity receptor for IgG (Fc gamma RIIIA or CD16) in natural killer cells and macrophages; and part of the T cell antigen receptor in subsets of T cells. Here we have investigated the transcriptional regulation of the gamma chain gene by analyzing the 2.5-kilobase sequence upstream of the transcription start site. This sequence contains a promoter specific to cells of hematopoietic lineage. However, the tissue specificity of this promoter is only partial because it is active in all of the hematopoietic cells tested here, regardless of whether they constitutively express Fc epsilon RI-gamma chain transcripts. We have identified two adjacent cis-acting regulatory elements, both of which are part of an Alu repeat. The first (-445/-366) is a positive element active in both basophils and T cells. The second (-365/-264) binds to nuclear factors, which appear to be different in basophils and T cells, and acts as a negative element in basophils and as a positive one in T cells. Thus, this Alu repeat (90% identical to Alu consensus sequences) has evolved to become both a positive and negative regulator. 8419644 Human immunodeficiency viruses containing heterologous enhancer/promoters are replication competent and exhibit different lymphocyte tropisms. The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) contains binding sites for nuclear factor kappa B (NF-kappa B) and the constitutively expressed transcription factor Sp1, both of which are highly conserved in HIV and simian immunodeficiency virus isolates. To delineate the effects of these motifs on the replicative capacity of HIV and to explore the possibility of extending the virus host range, known heterologous enhancer/promoters were inserted into the HIV-1 LTR in place of the NF-kappa B and Sp1 binding sites. The effects of these substitutions on viral replication in transfected HeLa cells and on HIV infection of human peripheral blood lymphocytes or continuous T-leukemia cell lines were evaluated. HIVs in which the NF- kappa B/Sp1 enhancer plus the downstream TATA element were replaced with heterologous enhancer/promoters were also constructed. Viruses containing the human cytomegalovirus immediate-early enhancer exhibited infectious kinetics similar to that of wild-type HIV in activated human peripheral blood lymphocytes and AA2 cells but replicated less efficiently in H9 and CEM cells. These studies indicate that heterologous enhancer elements are capable of restoring Tat responsiveness to the HIV LTR in the context of directing reporter gene expression as well as in the production of infectious progeny virions. 8419931 Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B- lymphocyte precursors [ published erratum appears in Proc Natl Acad Sci U S A 1993 Apr 15;90 ( 8 ) : 3775 ] Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to ( i ) stimulation of phosphatidylinositol turnover; ( ii ) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and ( iii ) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. 1482357 Photoaffinity labeling of plasma membrane receptors for aldosterone from human mononuclear leukocytes. Non-genomic effects of aldosterone on the sodium-proton-antiport have been shown in human mononuclear leukocytes which could be related to a new aldosterone membrane receptor. In the present paper plasma membranes from human mononuclear leukocytes were covalently photolabeled with a [125I]-aldosterone derivative. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed significant aldosterone binding at a molecular weight of approximately 50000 Dalton which was absent with 1 microM cold aldosterone, but not cortisol in the binding media. The presence of the sulfhydryl agent dithiothreitol did not affect results suggesting the absence of disulfide bridges in the steroid binding domain of the receptor. These data are the first to define the molecular weight of the membrane receptor for aldosterone. 1482376 Alpha-lipoic acid is a potent inhibitor of NF-kappa B activation in human T cells. Acquired immunodeficiency syndrome (AIDS) results from infection with a human immunodeficiency virus (HIV). The long terminal repeat (LTR) region of HIV proviral DNA contains binding sites for nuclear factor kappa B (NF-kappa B), and this transcriptional activator appears to regulate HIV activation. Recent findings suggest an involvement of reactive oxygen species (ROS) in signal transduction pathways leading to NF-kappa B activation. The present study was based on reports that antioxidants which eliminate ROS should block the activation of NF-kappa B and subsequently HIV transcription, and thus antioxidants can be used as therapeutic agents for AIDS. Incubation of Jurkat T cells (1 x 10(6) cells/ml) with a natural thiol antioxidant, alpha-lipoic acid, prior to the stimulation of cells was found to inhibit NF-kappa B activation induced by tumor necrosis factor-alpha (25 ng/ml) or by phorbol 12-myristate 13-acetate (50 ng/ml). The inhibitory action of alpha-lipoic acid was found to be very potent as only 4 mM was needed for a complete inhibition, whereas 20 mM was required for N-acetylcysteine. These results indicate that alpha-lipoic acid may be effective in AIDS therapeutics. 8422274 A human putative lymphocyte G0/G1 switch gene homologous to a rodent gene encoding a zinc-binding potential transcription factor. G0S24 is a member of a set of genes (putative G0/G1 switch regulatory genes) that are expressed transiently within 1-2 hr of the addition of lectin or cycloheximide to human blood mononuclear cells. Comparison of a full-length cDNA sequence with the corresponding genomic sequence reveals an open reading frame of 326 amino acids, distributed across two exons. Potential phosphorylation sites include the sequence PSPTSPT, which resembles an RNA polymerase II repeat reported to be a target of the cell cycle control kinase cdc2. Comparison of the derived protein sequence with those of rodent homologs allows classification into three groups. Group 1 contains G0S24 and the rat and mouse TIS11 genes (also known as TTP, Nup475, and Zfp36). Members of this group have three tetraproline repeats. Groups 1 and 2 have a serine-rich region and an " arginine element " (RRLPIF) at the carboxyl terminus. All groups contain cysteine- and histidine-rich putative zinc finger domains and a serine-phenylalanine " SFS " domain similar to part of the large subunit of eukaryotic RNA polymerase II. Comparison of group 1 human and mouse genomic sequences shows high conservation in the 5' flank and exons. A CpG island suggests expression in the germ line. G0S24 has potential sites for transcription factors in the 5' flank and intron; these include a serum response element. Protein and genomic sequences show similarities with those of a variety of proteins involved in transcription, suggesting that the G0S24 product has a similar role. 8422459 Interleukin-4 inhibits the lipopolysaccharide-induced expression of c-jun and c-fos messenger RNA and activator protein-1 binding activity in human monocytes. We studied the effect of interleukin-4 (IL-4) on the lipopolysaccharide (LPS) induction of two immediate early genes c-fos and c-jun. These genes encode proteins that form the dimeric complex activator protein-1 (AP-1), which is active as a transcriptional factor. Maximal accumulation of either c-fos and c-jun messenger RNA (mRNA) occurred 30 minutes after LPS addition. When cells were treated with IL-4 for 5 hours before LPS activation, both the c-fos and the c-jun mRNA expression was decreased. The inhibition of c-fos and c-jun expression by IL-4 in LPS-treated cells was shown to be due to a lower transcription rate of the c-fos and c-jun genes. IL-4 did not affect the stability of the c-fos and c-jun transcripts. Finally, using electrophoretic mobility shift assays, evidence was obtained that IL-4 inhibits LPS-induced expression of AP-1 protein. These data indicate that IL-4 suppresses the induction of transcription factors in human activated monocytes. 8423993 Ras oncogene transformation of human B lymphoblasts is associated with lymphocyte activation and with a block of differentiation. The p21ras small GTP binding proteins participate in signal transduction from cell surface receptors and affect neoplastic transformation and development in many different cell types. In the present study, we examined the relationship between ras transformation and differentiation of human B lymphocytes. We show that the constitutive expression of the T24 Ha-ras oncogene in EBV-immortalized B lymphoblasts was associated with the induction of the interleukin 2 receptor alpha subunit, with an impaired immunoglobulin gene expression, altered adhesion properties and increased survival in serum-free medium. Since induction of the IL-2 receptor alpha subunit is a hallmark of lymphocyte activation, we suggest that p21ras naturally triggers B cell activation. The ras-transformed lymphocytes displayed a fully functional IL-2r, as assessed by c-fos induction following treatment with IL-2; nevertheless, they were not growth stimulated by this lymphokine. The decreased expression of immunoglobulin genes indicates that the ras oncogene blocks terminal differentiation to plasma cells, possibly by inhibiting the activity of lymphocyte-specific transcription factors. Somewhat unexpectedly, the constitutive p21ras activity did not cause an increased DNA binding of transcription factors PEA1 (AP1), PEA3, Oct-2 or NF-kB. 8424101 Transcription factor GATA-1 and erythroid development. In summary, we derived an experimental system that allows us to dissect the function of GATA-1 in red cell development at a genetic level. We have established the essential nature of GATA-1 during both primitive and definitive erythropoiesis. By ablating the expression of the endogenous GATA-1 gene, we are in a position to introduce a variety of constructs that harbor subtle modifications in flanking or protein-coding sequences. We can now study regulatory regions and functional domains of the protein in the context of a true erythroid environment, experiments that have not been possible heretofore. Although the assay involves the dramatic loss of red cell production, it should be possible to define important regulatory domains that can then be assayed using less stringent systems, such as cell-free extracts for in vitro transcription. The ideal situation would be analyses conducted in GATA-1- erythroid cells. However, these cells have been impossible to generate given the requirement of GATA-1 for Epo receptor expression and red cell viability (C. Simon and S. Orkin, unpublished observations). It may be possible to produce such cells by first expressing the Epo receptor under the influence of a constitutive promoter and then targeting the GATA-1 gene. If GATA-1- red cells were available, the analyses would involve the actual transcription of or chromatin structure surrounding the globin genes. Structure-function studies of the GATA-1 protein could be greatly simplified and a larger number of mutants studied. However, the ES cell system can be used as an alternative until targeted erythroleukemia cells become available. Other applications involve the introduction of other GATA-binding protein family members to determine whether they rescue the mutation. If they can not, chimeric proteins can be tested to identify which amino acids distinguish the different family members. We feel that these experiments are vital to understanding the function of GATA-1 during erythroid ontogeny. How does GATA-1 regulate red cell genes like globin or the Epo receptor? Once we identify the functional domains of the GATA-binding proteins, we hope to learn what proteins GATA-1 binds to in the basic transcription machinery or in chromatin. Is GATA-1 necessary for globin gene switching? GATA-1 may be modified differently during development so that the locus control region can interact with different globin promoters. We may find that one region of the protein is required for embryonic expression and another for adult globin gene expression. 7678779 The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage. We have isolated cDNA clones of myeloid differentiation primary response (MyD) genes, activated in the absence of de novo protein synthesis following induction for differentiation along either the macrophage or granulocyte lineage in human myeloblastic leukemia HL-60 cells. One cDNA clone of a primary response gene, expressed upon macrophage differentiation, encoded for Egr-1, a zinc finger transcription factor. The Egr-1 gene was observed to be transcriptionally silent in HL-60 cells, but active in U-937 and M1 cells, the latter two being predetermined for macrophage differentiation. Egr-1 antisense oligomers in the culture media blocked macrophage differentiation in both myeloid leukemia cell lines and normal myeloblasts. HL-60 cells constitutively expressing an Egr-1 transgene (HL-60Egr-1) could be induced for macrophage, but not granulocyte, differentiation. These observations indicate that expression of Egr-1 is essential for and restricts differentiation of myeloblasts along the macrophage lineage. 1492121 Activation of lymphokine genes in T cells: role of cis-acting DNA elements that respond to T cell activation signals. Activation of T cells is initiated by the recognition of antigen on antigen presenting cells to exert the effector functions in immune and inflammatory responses. Two types of helper T cell (Th) clones (Th1 and Th2) are defined on the basis of different patterns of cytokine (lymphokine) secretion. They determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Although lymphokine genes are coordinately regulated upon antigen stimulation, they are regulated by the mechanisms common to all as well as those which are unique to each gene. For most lymphokine genes, a combination of phorbol esters (phorbol 12-myristate 13 acetate, PMA) and calcium ionophores (A23187) is required for their maximal induction. Yet phorbol ester alone or calcium ionophore alone produce several lymphokines. The production of the granulocyte-macrophage colony stimulating factor (GM-CSF) is completely dependent on the two signals. We have previously found a cis-acting region spanning the GM-CSF promoter region (positions -95 to +27) that confers inducibility to reporter genes in transient transfection assays. Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and conserved lymphokine element (CLE0). GM2 defines a binding site for protein ( s ) whose binding is inducible by PMA. One protein, NF-GM2 is similar to the transcription factor NF-kB. GC-box is a binding site for constitutively bound proteins. CLEO defines a binding site for protein ( s ) whose optimum binding is stimulated by PMA and A23187. Viral trans-activators such as Tax (human T cell leukemia virus-1, HTLV-1) and E2 (bovine papilloma virus, BPV) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor (TCR) mediated signaling. The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them. The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Sp1 to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation. 7678994 Expression of tal-1 and GATA-binding proteins during human hematopoiesis. Tal-1 rearrangements are associated with nearly 30% of human T acute lymphoblastic leukemia. Tal-1 gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells. We investigated the pattern of tal-1 expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis. Both methods demonstrated that the tal-1 gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes. In addition, our results indicate that the tal-1 1A promoter, which contains two consensus GATA-binding sites, is active mainly in these lineages. Because the GATA-1 gene is known to transactivate several genes specific for the erythroid, megakaryocytic, and mastocytic/basophilic lineages, we studied GATA-1 expression in these purified hematopoietic cells. We found that GATA-1 and tal-1 genes are coexpressed in these three lineages. Remarkably, the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation. In immature hematopoietic cells, tal-1 and GATA-1 genes are coexpressed in committed progenitors cells (CD34+/CD38(2+)), whereas they are not detectable in the most primitive cells (CD34(2+)/CD38-). In contrast, GATA-2 is strongly expressed in both most primitive and committed progenitors cells, whereas GATA-3 is mostly detected in most primitive ones. Altogether our results strongly suggest that GATA-1 modulates the transcription of tal-1 during the differentiation of the erythroid, megakaryocytic, and basosophilic lineages. 8427983 Cell-type-specific transactivation of the parathyroid hormone-related protein gene promoter by the human T-cell leukemia virus type I (HTLV-I) tax and HTLV-II tax proteins. The human T-cell leukemia virus type I (HTLV-I) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression. Using deletion mutants, the downstream parathyroid hormone-related protein (PTHrP) promoter is shown to be responsive to both HTLV-I and HTLV-II Tax as well as the AP1/c-jun proto-oncogene. Transactivation of PTHrP by Tax was seen in T cells but not in B-cell lines or fibroblasts. A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but not the HTLV-I long terminal repeat (LTR). Exogenous provision of NFkB rescued IL2R alpha expression but not the PTHrP promoter. Thus, HTLV-I Tax, HTLV-II Tax, and c-jun transactivate PTHrP and may contribute to the pathogenesis of hypercalcemia in adult T-cell leukemia. 8428000 Interleukin-3 expression by activated T cells involves an inducible, T-cell-specific factor and an octamer binding protein. Interleukin-3 (IL-3) is exclusively expressed by activated T and natural killer cells, a function that is tightly controlled both in a lineage-specific and in a stimulation-dependent manner. We have investigated the protein binding characteristics and functional importance of the ACT-1-activating region of the IL-3 promoter. This region binds an inducible, T-cell-specific factor over its 5' end, a site that is necessary for the expression of IL-3 in the absence of other upstream elements. Over its 3' end, it binds a factor that is ubiquitously and constitutively expressed. This factor is Oct-1 or an immunologically related octamer-binding protein, and it plays a role in coordinating the activity of several regulatory elements. These characteristics make the ACT-1 site analogous to the activating ARRE-1 site in the IL-2 promoter. Furthermore, and despite a lack of sequence homology, the promoters of IL-3 and IL-2 share an organizational pattern of regulatory elements that is likely to be important for the T-cell-specific expression of these genes. 1493333 I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding. The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits, NF-kappa B p65 and NF-kappa B p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B, termed I kappa B. In contrast, NF-kappa B p50 alone fails to stimulate kappa B-directed transcription, and based on prior in vitro studies, is not directly regulated by I kappa B. To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B/MAD-3, a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding, I kappa B-mediated inhibition, and I kappa B-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following: 1 ) I kappa B/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes, 2 ) the binding of I kappa B/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm, 3 ) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B/MAD-3, and 4 ) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B/MAD-3. Unexpectedly, our in vivo studies also demonstrate that I kappa B/MAD-3 binds directly to NF-kappa B p50. This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65. 1493370 Surrogate thyroglobulin receptors and T cell proliferation in Hashimoto 's thyroiditis. Immunoglobulin molecules on the surface of a B lymphocyte are the endogenous " receptors " to which specific antigens bind. Studies in mice have shown that a monoclonal antibody, conjugated with palmitate to provide a lipid tail, can be inserted into the cell membrane to provide a " surrogate " antigen receptor. We have investigated whether a palmitate conjugate of a human monoclonal antibody specific for thyroglobulin (TG) could function as a surrogate TG receptor on blood mononuclear cells separated into fractions enriched for T cells or depleted of T cells (non-T cells). Using flow cytometry, we detected surrogate TG receptors on non-T ( but not on T) cells from 11 of 11 individuals studied (5 Hashimoto patients and 6 control donors). In contrast, endogenous TG receptors could only be detected on non-T cells from 1 of 3 Hashimoto patients and from 0 of 4 control donors. Because of the efficient binding of TG by surrogate receptors on non-T cells, we assessed the ability of such cells to present TG to T cells. Proliferation in response to TG was observed in T cells from only 1 of 5 Hashimoto patients. This low frequency of response was no different from that previously detected using cultures of T cells and autologous dendritic cells. Therefore, the successful generation of surrogate receptors on non-T cells is not associated with more efficient TG presentation of T cells. Furthermore, the significance of the present study is that the T cells, not the antigen-presenting cells, are likely to be the limiting element in the T cell proliferative response to TG and other thyroid autoantigens. 8381349 The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter. The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis-acting element, various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein-DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter. 8428943 ras protein activity is essential for T-cell antigen receptor signal transduction. In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC. 8428966 Characterization of the nuclear and cytoplasmic components of the lymphoid-specific nuclear factor of activated T cells (NF-AT) complex. The lymphoid-specific transcription complex, NF-AT, is involved in early gene activation in T cells and is assembled from a pre-existing, T cell restricted cytoplasmic factor and an inducible ubiquitous nuclear component within 30 min after activation through the antigen receptor. Recent studies have implicated the family of AP1 factors as components of the murine NF-AT complex. Evidence is provided here that the nuclear component of human NF-AT contains the phorbol ester-inducible transcription factor AP1 (Jun/Fos). We further characterize which AP1 family members can assume this role. Antisera to Fos inhibits NF-AT DNA binding as does an oligonucleotide containing a binding site for AP1. Constitutive expression in vivo of Fos, and to a lesser extent Fra-1, eliminates the requirement for phorbol 12-myristate 13-acetate (PMA) stimulation, leaving NF-AT-directed transcription responsive to calcium ionophore alone. Overexpression of cJun or JunD, but not JunB, also eliminates the requirement for PMA, indicating that many but not all Jun- and Fos-related proteins functionally activate NF-AT-dependent transcription in the presence of the cytoplasmic component. NF-AT DNA binding can be reconstituted in vitro using semi-purified AP1 proteins mixed with cytosol from T lymphocytes. Fos proteins are not needed for this reconstitution, and although JunB is not functional, it can participate in the NF-AT DNA binding complex. Finally, we have partially purified the cytoplasmic component of NF-AT and show by elution and renaturation from SDS-polyacrylamide gel electrophoresis gels that it has a molecular mass between 94 and 116 kDa and may have multiple differentially modified forms. 8430069 The c-rel protooncogene product represses NF-kappa B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus. The long terminal repeat (LTR) of the type 1 human immunodeficiency virus (HIV-1) and the 5' regulatory region of the gene encoding the interleukin 2 receptor alpha subunit (IL-2R alpha) share functional kappa B enhancer elements involved in the regulation of these inducible transcription units during T-cell activation. These kappa B enhancer elements are recognized by a structurally related family of interactive proteins that includes p50, p65, and the product of the c-rel protooncogene (c-Rel). Recent biochemical studies have shown that p65 and p50 form the prototypical NF-kappa B complex, which is rapidly translocated from the cytoplasm to the nucleus during T-cell activation. This intracellular signaling complex potently stimulates kappa B-directed transcription from either the HIV-1 LTR or the IL-2R alpha promoter via the strong transactivation domain present in p65. We now demonstrate that nuclear expression of human c-Rel, which is induced by either phorbol ester or tumor necrosis factor alpha with delayed kinetics relative to p65, markedly represses p65-mediated activation of these transcription units. These inhibitory effects of c-Rel correlate with its DNA-binding activity but not with its ability to heterodimerize with p50, suggesting that c-Rel inhibition involves competition with p50/p65 for occupancy of the kappa B enhancer element. Together, these findings suggest that one function of c-Rel is as a physiologic repressor of the HIV-1 LTR and IL-2R alpha promoters, serving to efficiently counter the strong transcriptional activating effects of p65. 8436816 Protease treatment of nuclear extracts distinguishes between class II MHC X1 box DNA-binding proteins in wild-type and class II-deficient B cells. The X box region is critical for directing the expression of class II major histocompatibility complex genes in B lymphocytes. Although several class II promoter-specific DNA binding factors have been described, only the X box region factor, RFX, shows a genetic correlation with class II expression, being deficient in some B cell lines derived from patients with class II-deficient congenital immunodeficiency. To further evaluate the role of X box DNA-binding proteins in class II gene expression, the role of the X box region was examined in both class II-positive and -negative lymphoid cells. In addition to the wild-type B cell line Raji, two class II transcriptional mutant cell lines, SJO and RJ2.2.5, and Jurkat, a class II negative T cell line, were examined. In contrast to wild-type B cells, neither of the class II mutant cell lines could use the X box region to direct the expression of a transiently transfected reporter gene, indicating that the X box-dependent transcriptional pathway is defective in these cells. The binding activity of the X1 box DNA-binding protein RFX was examined and found to be present in wild-type B cells and the mutant RJ2.2.5 but was absent in SJO and Jurkat. However, other X1 box-specific activities were detected in all these cell lines. To determine whether these different X1 box activities represented distinct DNA binding proteins or multimeric forms of the same factor ( s ), protease treatment of the crude nuclear extracts followed by DNA-binding assays were carried out and demonstrated that B cell extracts contain at least two X1-specific factors. One of these cleaved products (band 1 pk) correlates with RFX activity. A similar comparison with protease-treated extracts prepared from Jurkat cells demonstrated the presence of the band 1pk activity despite an absence of the native RFX activity. In contrast, protease treatment and analysis of SJO extracts showed no detectable levels of the band 1pk activity. These results demonstrate that multiple X1 box-specific DNA-binding activities exist in all lymphoid cells, but the presence of an actively binding RFX species correlates with class II transcription. 8437235 Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the -201 to -130 region of the long terminal repeat. In previous transfection analyses using the chloramphenicol acetyltransferase reporter gene system, we determined that linker substitution (LS) mutations between -201 and -130 (relative to the transcription start site) of the human immunodeficiency virus type 1 long terminal repeat (LTR) caused moderate decreases in LTR transcriptional activity in a T-cell line (S.L.Zeichner, J.Y.H. Kim, and J.C.Alwine, J.Virol.65:2436-2444, 1991). In order to confirm the significance of this region in the context of viral replication, we constructed several of these LS mutations (-201 to -184, -183 to -166, -165 to -148, and -148 to -130) in proviruses and prepared viral stocks by cocultivation of transfected RD cells with CEMx174 cells. In addition, two mutations between -93 and -76 and between -75 and -58 were utilized, since they affect the nuclear factor kappa B (NF-kappa B)- and Sp1-binding sites and were expected to diminish viral replication. Our results suggest that while transfection analyses offer an adequate approximation of the effects of the LS mutations, the analysis of viral replication using a mutant viral stock presents a more accurate picture, which is sometimes at variance with the transfection results. Three mutants (-201/-184 NXS, -165/-148 NXS, and -147/-130 NXS) had effects on viral replication that were much more severe than the effects predicted from their performance in transfection analyses, and the effects of two LS mutations (-201/-184 NXS and -183/-166 NXS) were not predicted by their effects in transfection. In addition, we observed cell type-specific permissiveness to replication of some mutant viruses. In the cell types tested, the LS mutations indicated an apparent requirement not only for the intact NF-kappa B and SP1-binding sites but also for several regions between -201 and -130 not previously associated with viral infectivity. 8441377 Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3). Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. (ABSTRACT TRUNCATED AT 250 WORDS) 8441379 The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction. In these studies, we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 1 beta (proIL-1 beta) gene. A cell-type-independent lipopolysaccharide ( LPS ) -responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site (cap site) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes. The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts. Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences, two of which appeared to be important for gene induction. One of the essential proteins which bound to the enhancer was similar or identical to members of the C/EBP family of transcription factors required for both IL-1- and LPS-specific induction of the IL-6 gene (i.e., the NF-IL6 proteins). When ligated to the proIL-1 beta cap site-proximal region (located between -131 to +12), both the proIL-1 beta and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells, which are not normally competent for IL-1 beta expression. When ligated to the murine c-fos promoter, however, the proIL-1 beta enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells, suggesting the existence of a proIL-1 beta promoter-proximal requirement for tissue specificity. 8443122 Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells. Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA). We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression. Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA. The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133. In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA. This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts. This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it. 8095167 Human CD4 lymphocytes specifically recognize a peptide representing the fusion region of the hybrid protein pml/RAR alpha present in acute promyelocytic leukemia cells. Fusion proteins present in leukemic cells frequently contain a new amino acid at the fusion point. We tested whether a peptide (BCR1/25) encompassing the fusion region of the hybrid molecule pml/RAR alpha, which is selectively expressed by acute promyelocytic leukemia (APL) cells, can be recognized by human T lymphocytes in vitro. CD4+ lymphocytes, at both polyclonal and clonal level, recognized peptide BCR1/25 in an HLA-DR--restricted fashion on presentation by autologous antigen-presenting cell (APC) or by APC expressing the HLA-DR11 restricting molecule. Control peptides corresponding to the normal pml and RAR alpha proteins were not recognized. One clone (DEG5) also exerted a high and specific cytotoxicity against autologous cells pulsed with BCR1/25. The autologous DE LCL containing a transduced pml/RAR alpha fusion gene and expressing a bcr1 type of the pml/RAR alpha hybrid protein induced the proliferation of DE anti-BCR1/25 T cell clones. It is concluded that the bcr1 type-pml/RAR alpha fusion protein of APL contains an antigenic site, absent from the normal parent molecules and recognized by human CD4+ lymphocytes. 8444876 A serum response element and a binding site for NF-Y mediate the serum response of the human thrombospondin 1 gene. The expression of thrombospondin 1 (TSP 1), a member of the TSP gene family, is rapidly induced by growth factors. We tested the ability of human TSP 1-chloramphenicol acetyltransferase constructs to respond to serum in stably transfected NIH-3T3 cells. Two transcriptional elements in the TSP 1 promoter, a distal element at -1280 and a proximal element at -65, were required for the response of the human TSP 1 gene to serum. The distal element contains the 5'-CC(A+T)6GG-3' consensus sequence characteristic of a serum-response element (SRE). Deletions or mutations in this element reduced the serum response of the TSP 1 gene by 80-90%. In gel-shift assays, the -1280 element and the c-fos SRE cross-competed, whereas their functional and binding mutants did not. The proximal element contains the sequence 5'-GGCCAATGGG-3', which closely resembles the consensus binding motif for the CCAAT-binding factor NF-Y (CBF, CP1, alpha CP1). Deletions or mutations in this element also reduced the serum response by 80-90%. Methylation interference analysis of the -65 region identified a pattern of contacts with nuclear factors resembling that for NF-Y, and an NF-Y-binding site and the proximal TSP 1 element cross-competed in gel-shift assays, whereas their binding mutants did not. Finally, an abbreviated TSP 1 promoter/5'-flank, containing the SRE- and NF-Y-binding sites, mediated a serum response that was close in magnitude to that of the parent promoter. We conclude that the serum response of the human TSP 1 gene requires the coordinated function of an SRE- and NF-Y-binding site. 8444885 Suppression of a cellular differentiation program by phorbol esters coincides with inhibition of binding of a cell-specific transcription factor (NF-E2) to an enhancer element required for expression of an erythroid-specific gene. Induction by hemin increases, while induction with 12-O-tetradecanoylphorbol-13-acetate (TPA) represses, erythroid-specific gene expression in the human cell line K562. We analyzed the effects of hemin or TPA induction on the binding and activity of transcription factors at a regulatory element found within the transcriptional regulatory sequences of many erythroid-specific genes. TPA induction increases the binding of ubiquitous AP-1 factors to this element. TPA induction inhibits the binding of the lineage limited transcription factor NF-E2 to this transcriptional control element. Hemin induction of K562 cells does not facilitate the binding of NF-E2 to its recognition site. Hemin induction appears to nonspecifically increase the expression of transiently transfected genes in K562 cells. Beyond this nonspecific increase in gene expression, hemin induction acts to increase the activity of the lineage limited transcription factor NF-E2. The divergent effects of hemin and TPA on gene expression in K562 cells are mediated, in part, by their contrasting effects on the transcription factor NF-E2. 8383323 The interleukin 2 CD28-responsive complex contains at least three members of the NF kappa B family: c-Rel, p50, and p65. Optimal activation of T cells requires at least two signals. One signal can be delivered by the antigen-specific T-cell receptor, and the second signal is provided by the costimulatory molecule ( s ) delivered by the antigen-presenting cell. CD28 is a T-cell surface molecule and stimulation through this protein plays an important role in delivering the second activation signal. In this report, we show that in human peripheral blood T cells, CD28-mediated signal transduction involves the rel family proteins--c-Rel, p50, and p65. Treatment of peripheral blood T cells with phorbol 12-myristate 13-acetate (PMA) and anti-CD28 monoclonal antibody (mAb) results in augmentation of nuclear c-Rel, p50, and p65, and this augmentation can occur in the presence of the immunosuppressant cyclosporin A. It is also shown in this report that , in response to PMA/anti-CD28 mAb or anti-CD3/anti-CD28 mAb, c-Rel, p50, and p65 are associated with CD28-responsive element present in the promoter of the human interleukin 2 gene. The functional significance of c-Rel involvement in the CD28-responsive complex is demonstrated by transient transfection analysis, where cotransfection of c-Rel augments the level of expression of a chloramphenicol acetyltransferase reporter gene linked to the CD28-responsive element. 8446774 Expression of the Tat protein of HIV1 in human promonocytic U937 cells. Numerous studies have shown that , upon HIV1 infection, human promonocytic U937 cells were induced to differentiate , as indicated, for example, by increased expression of adhesion molecules. One of the viral proteins involved in this process might be the Tat protein. Indeed, this viral protein, which is essential for productive infection, has also been shown to display growth-stimulating properties and immunomodulatory activities. In order to apprehend the role of the HIV1 tat gene in inducing the differentiation of HIV1-infected U937 cells, we have successfully introduced this gene into U937 cells by infecting them with retroviral particles transducing tat. The effect of the Tat protein constitutively expressed by these cells upon their differentiation was then evaluated by looking for the expression of the c-fos and of the c-fms proto-oncogenes which are linked to the differentiation of myelomonoblastic cells. Northern blot analysis revealed in these cells, an increase in the transcription of these two proto-oncogenes, and this increase was amplified after treatment with phorbol myristate acetate. No such increase was observed in control U937 cells. These results indicate that , among HIV1 gene products, the Tat protein appears to trigger monocytic differentiation, and suggests that this viral protein directs progenitors of the monocyte/macrophage lineage towards a differentiation stage in which production of viral antigens and virions might be more efficient. 8449904 Transcriptional regulation of the pyruvate kinase erythroid-specific promoter. Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does not bind TFIID. Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites indicate that only the association of CCACC/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter. Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells. 8383677 Stimulation of interleukin-1 alpha and interleukin-1 beta production in human monocytes by protein phosphatase 1 and 2A inhibitors. Protein phosphatases 1 and 2A are important in regulating cellular functions by controlling the phosphorylation state of their substrates. In human monocytes, the inhibitors of these phosphatases, okadaic acid and calyculin A, were found to increase the mRNA accumulation and cytokine production of interleukin-1 beta and interleukin-1 alpha. The increased mRNA accumulation was found to be primarily because of the increase in the transcription rate of the interleukin-1 genes. Stimulation of interleukin-1 gene transcription may be caused by the stimulation of transcription factor activities, including those of AP-1, by these protein phosphatase inhibitors. Okadaic acid increased the synthesis of the interleukin-1 beta precursor and mature forms and their secretion. This increased processing and secretion correlated with the stimulation of IL-1 beta convertase mRNA accumulation. The stimulation of interleukin-1 alpha production by okadaic acid was more modest than that of interleukin-1 beta. However, the phosphorylation of the precursor interleukin-1 alpha cytokine was increased. These results show that protein phosphatase 1 and 2A inhibitors exert multiple effects on cytokine production in human monocytes and suggest that these two phosphatases play important roles in regulating interleukin-1 production. 7680653 Cell type- and stage-specific expression of the CD20/B1 antigen correlates with the activity of a diverged octamer DNA motif present in its promoter. The CD20 ( B1 ) gene encodes a B cell-specific protein involved in the regulation of human B cell proliferation and differentiation. Studies with 5' deletion CD20 promoter-CAT constructs have previously revealed two regions of the promoter between bases -186 and -280 and between bases -280 and -454 which contained positive regulatory elements. In this study we identified a sequence element present in the most proximal region located between bases -214 and -201, TTCTTCTAATTAA, which is important in the high constitutive expression of CD20 in mature B cells and the induction of CD20 in pre-B cells. This sequence element was referred to as the BAT box and its deletion significantly reduced the activity of a CD20 promoter-CAT construct in B cells. Mobility shift assays with various mutant probes and B cell nuclear extracts demonstrated that the core sequence TAAT was essential for binding to this site. Cross competition experiments with an octamer sequence from the Ig heavy chain promoter, the BAT box, and a TA-rich sequence present in the CD21 promoter revealed that all three sequences bound the same nuclear proteins suggesting that the BAT box binding proteins were Oct-1 and Oct-2. Southwestern blotting and UV cross-linking studies confirmed that the BAT box binding proteins were Oct-1 and Oct-2. The affinity of the BAT box binding proteins for the BAT box was approximately 25-fold less than for the octamer sequence and the BAT box binding proteins dissociated from the BAT box 10-fold more rapidly than from the octamer sequence. Despite this lower affinity, a trimer of the BAT box sequence was as efficiently transactivated by an Oct-2 expression vector as was a trimer of the octamer sequence in HeLa cells. The BAT box and Oct-2 were also implicated in the induction of CD20 in the pre-B cell line, PB-697, via phorbol esters. The induction of CD20 mRNA was temporally associated with induction of Oct-2 mRNA and a BAT box-deleted CD20-CAT construct, in contrast to the wild type, was poorly induced by phorbol esters. Together these results suggest that the BAT box binding proteins are important in the B cell specific expression of CD20 and perhaps CD21. 8454114 Transcription factor jun-B is target of autoreactive T-cells in IDDM. Target antigens defined by autoantibodies in IDDM include insulin, a putative glycolipid that reacts with islet cell antibodies, and a 64,000-M(r) protein recently identified as glutamic acid decarboxylase. In addition, some IDDM sera that contain antibodies to glutamic acid decarboxylase also coprecipitate a 38,000-M(r) protein from islets. This study used a high titer anti-38,000-M(r) serum to screen bacteriophage lambda cDNA expression libraries and identified human islet and placental clones encoding jun-B, the nuclear transcription protein, of predicted 38,000 M(r). Peripheral blood T-cells exhibited significant proliferation in response to a recombinant fragment of jun-B (amino acids 1-180) in 12 of 17 (71 %) recent-onset IDDM subjects, 8 of 16 (50 %) ICA-positive first-degree relatives of IDDM subjects who were at risk, 3 of 12 (25 %) other autoimmune disease subjects, and 0 of 10 healthy control subjects. Proliferation to tetanus toxoid did not differ significantly between the groups. Responses to jun-B were not related to age, sex, or human leukocyte antigen status. Thus, autoreactive T-cells identify a novel antigen, p38 jun-B, in IDDM and appear to indicate subjects at risk for the development of clinical disease. 8454603 Transcriptional regulation of interleukin 3 (IL3) in primary human T lymphocytes. Role of AP-1- and octamer-binding proteins in control of IL3 gene expression. We have investigated the molecular and biochemical basis for activation of interleukin 3 (IL3) gene expression in primary human T lymphocytes following CD3 and CD2 receptor stimulation or activation by phytohemagglutinin plus phorbol 12-myristate 13-acetate. Using transfection and reporter gene assays specifically designed for primary T lymphocytes in conjunction with gel retardation assays, Western blot analyses and UV cross-linking studies, we found that c-Jun, c-Fos, and octamer-binding proteins play a major role in transcriptional activation of the IL3 gene via their interaction with two specific regions contained within the IL3 5'-flanking sequence. Additionally, the region between bases -107 and -59 of the IL3 promoter containing putative AP-2 and Sp1 binding motifs appears necessary for basal level expression of the IL3 gene. The data also indicate that CD2 receptor activation and phytohemagglutinin plus phorbol 12-myristate 13-acetate stimulation augment T cell IL3 gene expression through the same cis- and trans-activating signals. These results should contribute to a better understanding of the regulation of IL3 gene expression in human T lymphocytes. 8455611 Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes. We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor ( s ) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters. 8455627 Interaction between NF-kappa B- and serum response factor-binding elements activates an interleukin-2 receptor alpha-chain enhancer specifically in T lymphocytes. We find that a short enhancer element containing the NF-kappa B binding site from the interleukin-2 receptor alpha-chain gene (IL-2R alpha) is preferentially activated in T cells. The IL-2R alpha enhancer binds NF-kappa B poorly and is only weakly activated by the NF-kappa B site alone. Serum response factor (SRF) binds to a site adjacent to the NF-kappa B site in the IL-2R enhancer, and both sites together have strong transcriptional activity specifically in T cells. Surprisingly, the levels of SRF constitutively expressed in T cells are consistently higher than in other cell types. Overexpression of SRF in B cells causes the IL-2R enhancer to function as well as it does in T cells, suggesting that the high level of SRF binding in T cells is functionally important. 8455941 The transcriptionally active factors mediating the effect of the HTLV-I Tax transactivator on the IL-2R alpha kappa B enhancer include the product of the c-rel proto-oncogene. The transactivator HTLV-I Tax activates the promoter of the gene coding for the interleukin 2 alpha-chain receptor (IL-2R alpha) via a kappa B site that can bind several protein species of the rel family. Tax1 strongly activates the enhancer activity of this motif, in both epithelial HeLa and lymphoid Jurkat cells. This activation was not observed in undifferentiated embryocarcinoma F9 cells. Overexpression of the p50, p65 and Rel proteins in these cells showed that significant activation of the IL-2R alpha kappa B site was observed only with Rel and Rel plus p65. Moreover, whereas both Tax and phorbol 12-myristate 13-acetate (PMA) are able to efficiently induce the binding of NF-kappa B to the IL-2R alpha kappa B site, PMA is functionally inactive. Using the DNA affinity precipitation assay, we observed that Tax1 is able to efficiently induce the binding of Rel, whereas PMA is not. This established a clear difference between both stimuli, indicating that Rel is the functionally active factor. We conclude from these results that the functional activity of members of the rel family is regulated by their interaction with DNA and that Rel can be a potent transcriptional activator on specific kappa B sites. 8096091 NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. The eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression. Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B. Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway. 8458581 p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction. The Rel/NF-kappa B family of transcription factors is composed of two distinct subgroups, proteins that undergo proteolytic processing and contain SWI6/ankyrin repeats in their carboxyl termini (p105, p98), and those without such repeats that do not require processing (p65, c-Rel, RelB, and Dorsal). We demonstrate that the p105 and p98 precursors share functional properties with the I kappa B proteins, which also contain SWI6/ankyrin repeats. Both p105 and p98 were found to form stable complexes with other Rel/NF-kappa B family members, including p65 and c-Rel. Association with the precursors is sufficient for cytoplasmic retention of either p65 or c-Rel, both of which are otherwise nuclear. These complexes undergo stimulus-responsive processing to produce active p50/c-Rel and p55/c-Rel complexes. These observations suggest a second pathway leading to NF-kappa B induction, in which processing of the precursors rather than phosphorylation of I kappa B plays a major role. 8460169 Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. The NK-kappa B transcription factor complex is sequestered in the cytoplasm by the inhibitory protein I kappa B-alpha (MAD-3). Various cellular stimuli relieve this inhibition by mechanisms largely unknown, leading to NF-kappa B nuclear localization and transactivation of its target genes. It is demonstrated here with human T lymphocytes and monocytes that different stimuli, including tumor necrosis factor alpha and phorbol 12-myristate 13-acetate, cause rapid degradation of I kappa B-alpha, with concomitant activation of NF-kappa B, followed by a dramatic increase in I kappa B-alpha mRNA and protein synthesis . Transfection studies reveal that the I kappa B-alpha mRNA and the encoded protein are potently induced by NF-kappa B and by homodimers of p65 and of c-Rel. We propose a model in which NF-kappa B and I kappa B-alpha mutually regulate each other in a cycle: saturating amounts of the inhibitory I kappa B-alpha protein are destroyed upon stimulation, allowing rapid activation of NF-kappa B. Subsequently, I kappa B-alpha mRNA and protein levels are quickly induced by the activated NF-kappa B. This resurgence of I kappa B-alpha protein acts to restore an equilibrium in which NF-kappa B is again inhibited. 8462662 Induced myeloid differentiation of K562 cells with downregulation of erythroid and megakaryocytic transcription factors: a novel experimental model for hemopoietic lineage restriction. The human erythroleukemia cell line K562 can be induced to differentiate along the erythroid and megakaryocytic lineages. Here we demonstrate that hexamethylene bisacetamide (HMBA) induced K562 cells to differentiate along a third pathway. This was accompanied by downregulation of two transcription factors normally expressed in erythroid, mast and megakaryocyte lineages. Northern analysis demonstrated coordinate downregulation of alpha globin and gamma globin in addition to the two lineage-restricted transcription factors, SCL and GATA-1. Proliferation of the K562 cells was also suppressed. Clonal assay showed that the suppression was irreversible and appeared analogous to the commitment of murine erythroleukemia (MEL) cells to terminal differentiation. In contrast to MEL cells, however, K562 cells acquired a macrophage-like morphology and exhibited a complete failure to generate benzidine-positive cells. Electron microscopy revealed a marked increase in granules resembling those specific for eosinophils. Surface marker analysis showed that HMBA-induced cells expressed reduced levels of glycophorin A, CD5, CD7 and CD11b. No upregulation of megakaryocyte or lymphoid markers occurred. Thus the response of K562 cells to HMBA may provide a useful experimental system for studying the molecular mechanisms responsible for downmodulation of lineage-restricted transcription factors during hemopoietic lineage commitment. 8096519 The Sp1 transcription factor binds the CD11b promoter specifically in myeloid cells in vivo and is essential for myeloid-specific promoter activity. The myeloid integrin CD11b is expressed selectively on the surface of mature macrophages, monocytes, neutrophils, and natural killer cells. Lineage-specific expression is controlled at the level of mRNA transcription. Recent isolation of the CD11b promoter shows that 92 base pairs (bp) of 5'-flanking DNA are sufficient to direct myeloid-specific expression of a reporter gene. To characterize regulatory sequences important for promoter activity, we performed linker scanning analysis of the 92-bp CD11b promoter and demonstrate that a sequence at bp -60 is essential for CD11b promoter activity. We show that this sequence binds the transcription factor Sp1 in vitro and in vivo. In vivo the Sp1 site is bound only in myeloid (U937) cells, not in cervical carcinoma (HeLa) cells. In addition, the macrophage transcription factor PU.1 binds the CD11b promoter in vitro and in vivo close to the Sp1 site. We propose a model in which binding of a myeloid-specific factor (PU.1) allows a general factor (Sp1) to bind in a tissue-specific fashion thereby contributing to the myeloid-specific expression of CD11b. 8468462 Costimulation of peripheral blood T cell activation by human endothelial cells. Enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1. Endothelial cells (EC) act as APC for resting PBL in vitro, and may have important roles in vivo in the pathogenesis of allograft rejection and delayed hypersensitivity. We previously reported that human umbilical vein EC provide costimulatory signals to PHA-stimulated PBL via CD2:lymphocyte function-associated Ag-3 and an unidentified ligand pair, resulting in a three- to eight-fold enhancement of IL-2 production. The physiologic relevance of this increase was demonstrated by the proliferative advantage provided by EC to PBL suboptimally stimulated with mAb OKT3. We now report that EC costimulation causes increased levels of IL-2 mRNA as a result of increased IL-2 transcription in PBL. We therefore examined the effects of EC on T cell nuclear factors known to regulate IL-2 transcription, including c-jun and c-fos-two components of the transcription factor AP-1, NFAT, and others. PBL constitutively express c-jun transcripts, and the level of c-jun mRNA is not altered by PHA activation in the absence or presence of EC. In contrast, c-fos mRNA is absent from resting T cells and is induced on PHA activation. EC alone do not induce c-fos mRNA but augment the level of c-fos mRNA in PHA-activated T cells by 3- to 10-fold. This effect is largely independent of the CD2:lymphocyte function-associated Ag-3 pathway. Gel-shift analysis reveals the constitutive presence of nuclear factors in resting PBL that bind to the proximal AP-1 site of the IL-2 promoter and that contain immunoreactive c-Jun but not c-Fos protein. In contrast, AP-1 from PHA-activated cells contains c-Jun and low levels of c-Fos. Strikingly, costimulation with EC results in a dramatic increase (up to 15-fold) in the c-Fos content of AP-1. Levels of other nuclear factors involved in IL-2 regulation were not altered by EC, although NFAT-DNA complexes migrated at a slightly different mobility. In summary, our data suggest that changes in the composition of transcription factor AP-1 is a key molecular mechanism for increasing IL-2 transcription and may underlie the phenomenon of costimulation by EC. 8468470 A protein of the AP-1 family is a component of nuclear factor of activated T cells. Nuclear factor of activated T cells (NF-AT) is a transcriptional activator involved in the induction of IL-2 gene expression. The response element for NF-AT is a sequence localized between -285/-254 in the IL-2 regulatory region. The composition of NF-AT protein is still not fully elucidated. We demonstrate that , in normal human T cells, an AP-1 protein is a component of the NF-AT protein complex. This was evidenced by the ability of the AP-1 site to compete with the NF-AT site for binding to NF-AT and by the capacity of immobilized anti-Jun and anti-Fos antibodies to deplete NF-AT-binding activity from nuclear extracts of activated T cells. There was no detectable binding of in vitro translated Jun/Fos heterodimer (AP-1) to the NF-AT sequence, and the NF-AT sequence was unable to inhibit the binding of Jun/Fos to the AP-1 sequence. The presence of an AP-1 protein in the NF-AT protein complex may regulate NF-AT-binding activity through protein-protein interaction. 7682243 Functional antagonism between vitamin D3 and retinoic acid in the regulation of CD14 and CD23 expression during monocytic differentiation of U-937 cells. 1,25 alpha-Dihydroxicholecalciferol (VitD3) and retinoic acid (RA) are important regulators of the proliferation and differentia