Biomedical subjects
Publications and source records attributed to J D Baxter.
The therapeutic benefit of glucocorticoids appears to derive chiefly from their suppression of immunologic and inflammatory responses, but beyond these generalized phenomena, there are a number of tissue-specific effects. Increasing knowledge of these actions is shedding light on such issues as steroid resistance and steroid-induced adrenocortical insufficiency.
Coinfection with the human immunodeficiency virus (HIV) and Treponema pallidum may predispose to accelerated neurosyphilis. The mechanism underlying this interaction is undefined, but usually presumed to result from HIV-induced suppression of cell-mediated immunity as reflected in the CD4 T-lymphocyte count. We report a patient infected with HIV who developed aggressive neurosyphilis despite a CD4 count of 1000/mm3. The CD4 cells constituted 17% of his total lymphocytes. Our case suggests that while severe neurosyphilis can occur in HIV-infected persons with normal absolute CD4 counts, the percentage of CD4 cells may be a better indicator of the risk of neurosyphilis. These observations are relevant to designing treatment strategies for patients coinfected with HIV and T pallidum based on measures of their immunocompetence.
Activators of protein kinase C, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), are known to regulate the expression of many genes, including the tumor necrosis factor alpha (TNF) gene, by affecting the level or activity of upstream transcription factors. To investigate the mechanism whereby TPA activates the TNF promoter, a series of 5'-deletion mutants of the human TNF promoter linked to chloramphenicol acetyltransferase was transfected into U937 human promonocytic cells. TPA produced a 7- to 11-fold activation of all TNF promoters tested, even those promoters truncated to contain only the core promoter with no upstream enhancer elements. The proximal TNF promoter containing only 28 nucleotides upstream and 10 nucleotides downstream of the RNA start site confers TPA activation to a variety of unrelated upstream enhancer elements and transcription factors, including Sp1, CTF/NF1, cyclic AMP-response element, GAL-E1a, and GAL-VP16. The level of activation by TPA depends on the TATA box structure, since the TPA response is greater in promoters containing the sequence TATAAA than in those containing TATTAA or TATTTA. These findings suggest that the core promoter region is a target for gene regulation by second-messenger pathways.
Transgenic mice harboring a chimeric gene linking mouse protamine 1 5'-flanking sequence to the coding sequence of the simian virus 40 T-antigen develop spontaneous rhabdomyosarcomas of the right atria. The presence of the tumors is accompanied by dramatic elevations in plasma atrial natriuretic peptide (ANP) immunoreactivity (1,698 +/- 993 vs. 60 +/- 18 fmol/ml for controls) and hematocrit (56 +/- 8 vs. 51 +/- 2 for controls). The immunoreactive ANP (irANP) present in the tumors is similar in size to irANP found in normal mouse atria. ANP mRNA transcripts present in the tumors also appear to be very similar in overall size and 5'-termini to those produced in normal cardiac tissue. Microscopically, the tumors are composed of a disorganized array of densely packed abnormal-appearing cells. Immunocytochemistry and in situ hybridization analysis reveal considerable heterogeneity in ANP gene expression. ANP peptide and mRNA are detectable throughout the parenchyma of the tumors, but absolute levels of expression vary widely among different cells in the population. These tumors represent a potentially valuable model for the study of inappropriate ANP secretion and may provide a tissue source for the development of an ANP-producing atrial cell line.
In humans, active renin is generated by the removal of a 43-amino acid prosegment from the zymogen prorenin. This cleavage event is highly specific, occurring at only one of the seven pairs of basic amino acids in the body of preprorenin. This cleavage site selectivity is also displayed by a number of other proteases in vitro and in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector, suggesting that specificity of cleavage is directed in part by the primary sequence, the higher order structure, or both of prorenin itself. To test this hypothesis, single amino acid mutations were introduced in the region of human preprorenin surrounding the natural cleavage site, and the resultant recombinant proteins were expressed in cultured Chinese hamster ovary and AtT-20 cells. The results suggest that amino acids in addition to the pair of basic amino acids surrounding the cleavage site affect the ability of both trypsin and the endogenous AtT-20 processing enzyme to cleave prorenin. Notably, although a proline at position -4 is essential for processing of prorenin in AtT-20 cells and is correlated with predicted formation of a beta-turn at this position, site-directed mutations suggest that this structural feature in addition to a pair of basic amino acids is not sufficient to lead to proteolytic activation of prorenin. Displacement of sequences surrounding the cleavage site to a position 10 amino acids toward the amino terminus led to partial processing of a mutated prorenin.(ABSTRACT TRUNCATED AT 250 WORDS)
T3 binds to intranuclear thyroid hormone receptors (TRs) on target DNA elements and exerts profound influences on gene expression by mechanisms not yet characterized. We used gel shift assays and cross-linking experiments to demonstrate that T3 greatly induced the monomeric binding of the hTR beta produced in Escherichia coli to DNA. T3 also increased the gel mobility of these monomer-DNA complexes suggesting they undergo a ligand-induced conformational change. This effect did not depend on the orientation and spacing of the half-site motifs within the DNA structure. In contrast, T3 had diverse effects on the dimeric interaction. T3 increased the dimeric interaction to the palindrome GGTCA.TGACC (an effect lost by spacing the half-sites with 3 base pairs) and decreased the dimeric interaction to the inverted palindrome containing the TGACC.GGTCA motif. Scatchard analyses indicated that the T3 enhancement on binding was due to an increase in the number of TR with high affinity DNA-binding activity and not by increasing the affinity of TR that could bind to DNA. The effects of various T3 analogs were directly related to their affinities for the TR. These ligand effects on in vitro TR-DNA binding may reflect mechanisms by which T3 regulates transcription in vivo.
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The effect of media conditions on the glucocorticoid response has been examined in three types of cultured cells. In rat pituitary tumor cells (GC cells) growth hormone production was stimulated by glucocorticoids provided fresh culture media was present (enriched media conditions). In contrast, dexamethasone either failed to induce or deinduce growth hormone synthesis if added to cultures which had not received fresh media for 3 days (depleted media condition). With human skin fibroblasts, cortisol stimulated [3H]thymidine incorporation in the enriched condition but inhibited this response in the depleted condition. In mouse lymphoma (S49) cells the enriched media conditions significantly delayed the killing response to glucocorticoids (20% killing after 24 h versus 90% killing after 24 h for the depleted condition). Thus, the magnitude and in some cases, the direction of the glucocorticoid response are sensitive to the conditions to which the cells are exposed. In all three cell types the steroid also rapidly (detectable by 15 min, maximal by 2 h) altered chromatin structure as detected by a change in the number of initiation sites for Escherichia coli RNA polymerase assayed under cell-free conditions. This early nuclear response could be in a positive or negative direction and was also affected by the culture conditions; enriched media favored a positive or less negative effect on the initiation sites by the steroid, while depleted media favored a steroid-induced inhibition of this chromatin function. In S49 and GC cells the kinetics and magnitude of the change in chromatin closely followed receptor . glucocorticoid complex binding to nuclei while removal of dexamethasone from the culture media resulted in a rapid (t 1/2 = approximately 20 min) disappearance of the effect which paralleled loss of bound hormone from the nucleus. The glucocorticoid effect on chromatin was not observed in two lines of glucocorticoid-resistant mutant S49 cells. One line (R-) lacks detectable glucocorticoid receptors; the other line (Nti) has receptors that bind the hormone normally, but the receptor . glucocorticoid complexes bind more avidly to the nucleus. These results suggest that the receptor is involved in both the stimulatory and the inhibitory effects on chromatin. The findings in the Nti cells and of a slight lag between nuclear binding of receptors and initiation site alteration implies that some receptor property, in addition to nuclear binding per se, is responsible for the influence on chromatin. These results are discussed in terms of a model in which steroid hormones initiate their actions by influencing a reaction that modifies chromatin structure. The direction and magnitude of the reaction, and its effect on the expression of specific genes, are dictated by the metabolic state and differentiation of the cell.
The nucleotide sequence of a DNA complementary to human growth hormone messenger RNA was cloned; it contains 29 nucleotides in its 5' untranslated region, the 651 nucleotides coding for the prehormone, and the entire 3' untranslated region (108 nucleotides). The data reported predict the previously unknown sequence of the signal peptide of human growth hormone and, by comparison with the previously determined sequences of rat growth hormone and human chorionic somatomammotropin, strengthens the hypothesis that these genes evolved by gene duplication from a common ancestral sequence. The human growth hormone gene sequences have been linked in phase to a fragment of the trp D gene of Escherichia coli in a plasmid vehicle, and a fusion protein is synthesized at high level (approximately 3 percent of bacterial protein) under the control of the regulatory region of the trp operon. This fusion protein (70 percent of whose amino acids are coded for by the human growth hormone gene) reacts specifically with antibodies to human growth hormone and is stable in E. coli.
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An overview of glucocorticoid action is presented including aspects of historic, clinical, and physiologic interest. Despite the diversity of effects, glucocorticoid hormone action at the cellular level simplifies to a rather universal model that involves binding to a soluble receptor, followed by interaction of the complex with the chromatin and modification of gene expression. This unitary concept has important implications in pathology, pharmacology, and therapeutics. Finally, a definition of a glucocorticoid in terms of its receptor is presented, which we feel is better than the traditional terminology based on specific biological effects. It is hoped that this review will also provide the reader with a framework in which the various contributions in the Monograph can be integrated.
In cultured rat pituitary cells, glucocorticoids regulate growth hormone production by modulating the number of growth hormone messenger RNA molecules. The effect is quite specific, since only a few other mRNAs are affected by the hormones. This response is demonstrated by assays involving cell-free mRNA translation and cDNA-RNA hybridization. Furthermore, the inducibility by the glucocorticoids is regulated by at least one other class of hormones, thyroid hormone. Thus, this system serves as a model for studying not only the glucocorticoid regulation of specific mRNA, but also the control of this regulation by other factors in the target tissue.
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The spectrum of physiological, pathological, and genetic variations in sensitivity to glucocorticoids is reviewed. The receptor for these hormones is common to most mammalian tissues, and yet the responses are widely divergent. Although there may be differences in the receptors to account for some of this diversity, it is likely that it is largely due to cellular programming not involving the receptors. In addition to the intertissue differences in sensitivity, it is also clear that intra-tissue differences occur. The greatest amount of information has been accumulated with lymphoid cell systems and there are sensitivity differences to specific responses such as cell killing or effects on immunological functions. In these systems, there can be major variations in either the extent of the response (e. g., from mild growth inhibition to cellular killing) or whether any effect is observed. Further, dose requirements for certain responses can vary by several orders of magnitude. Within a given tissue there may be developmental changes in sensitivity that are not due to obvious changes in the receptor, and decreased sensitivity with aging that in some cases has been associated with changes in receptor binding activity. Finally, the cellular sensitivity can either be influenced by hormones and other factors that affect the ability of the glucocorticoid to elicit a particular response (in a synergistic or antagonistic manner), or the same function regulated by the glucocorticoid can be inducible by the steroid, appearing some time after administration of the steroid and disappearing after steroid removal. Genetic variations in sensitivity to glucocorticoids also occur. In humans these may be generalized, affecting glucocorticoid action in all responsive tissues, and could be important in the pathogenesis of certain diseases. Perhaps the most striking genetic alterations, however, are observed in cultured lymphoid and fibroblastic cells and in acute lymphoblastic leukaemia cells ordinarily growth inhibited or killed by the glucocorticoid. Mutant cell lines arise that are highly resistant and most of these have abnormalities in the glucocorticoid receptor. In some cases binding activity is totally lost, easily expalining the resistance. In other cases, there is a more modest reduction in binding or a change in receptor properties that give it increased or decreased nuclear and DNA binding activity. An analysis of these cell lines suggests that many of the defects are in some receptor property presently not understood that makes the receptor ineffective rather than the defect being due to the quantitative changes in receptor levels detected. The frequency of emergence of steroid-resistant cells can vary widely from about 10(-5) in S49 cells to less than 10(-8) in certain thymic cell lines...