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.
The intact human growth hormone (hGH) gene is negatively regulated by triiodothyronine (T3) treatment in transfected rat pituitary tumor cells. We now demonstrate that this responsiveness is mediated by a negative thyroid hormone response element (nTRE) localized to the proximal 3'-untranslated/3'-flanking region (3'-UT/FR). This region binds thyroid hormone receptors specifically and with high affinity. nTRE function was promoter-dependent, since it suppressed the activity of a positive TRE in the human chorionic somatomammotropin promoter, partially repressed activity of the herpes simplex virus TK promoter, but did not function with the human actin or Rous sarcoma virus promoters. T3 treatment did not alter transcript termination sites nor selectively affect the stability of transcripts containing the hGH 3'-UT/FR when transcription was blocked by actinomycin D treatment. The function of the nTRE depended on its location in the 3'-UT/FR; it was inactive when positioned down-stream of the simian virus 40 (SV40) 3'-UT/FR, and it acted as a positive TRE when placed upstream of the hGH promoter. These results demonstrate a novel localization of a TRE with unique properties which suggests expanded mechanisms by which thyroid hormone receptors can affect gene expression.
Triiodothyronine (T3) induces the transcription of the human chorionic somatomammotropin (hCS) promoter transfected into rat pituitary (GC) cells, but does not stimulate the homologous human growth hormone (hGH) promoter. As demonstrated by forward and reverse mutagenesis, this differential T3 responsiveness is due to subtle structural differences in a T3 response element located between nucleotides -64 and -44 of the 5'-flanking DNA of the hGH and hCS promoters. Synthetic hCS(-70/-40) DNA binds thyroid hormone receptors with a 4-fold higher affinity than the corresponding hGH T3 response element, indicating that small differences in receptor binding properties are reflected by major differences in T3 responsiveness. Analysis of circular permutation fragments containing the native hGH and hCS or mutated hCS(-70/-40) sequences demonstrates that the thyroid hormone receptor induces DNA bending. The extent of bending shows a possible correlation with the function of these sequences, suggesting that the receptor-induced changes in DNA conformation may be required for thyroid hormone receptor action.
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)
While steroid response is generally restricted by the availability of steroid receptors, the theoretical limits of the response are not known. We have constructed a series of cell lines that stably express the estrogen receptor (ER) at levels up to 5,000,000 ERs per cell and employed these cells to explore the limits of the estrogen response. Several reporter genes with estrogen response elements upstream of the herpes thymidine kinase promoter showed hyperbolic saturation kinetics with increasing ER. Maximum response was 10 times that seen in cell lines with receptor titers comparable to physiological levels. Half-maximal responses required 500,000 receptors per cell, and cells with 5,000,000 ERs showed greater than 90% maximum induction. Estradiol dose-response studies indicated that the receptors are limiting below 500,000 ERs per cell, but at higher ER titers there are spare receptors. In contrast to most reporters, the widely used reporter pA2-CAT, which has 200 base pairs of Xenopus vitellogenin DNA between the response element and the promoter, showed squelching at ER levels beyond 500,000 per cell. Cell lines that expressed ER above this level activated pA2-CAT with a distorted hormone dependence, where saturating ligand concentrations were inhibitory. All reporters displayed squelching when the ER was provided by transient transfection at a level that we judge is 20,000,000 per cell by extrapolation from the behavior of stable cell lines. These findings suggest that saturation of the cellular capacity to mediate an estrogen response and ER-dependent squelching occur at receptor titers well above those encountered in nature. If current models of steroid hormone action are correct, the findings also imply that estrogen response elements are occupied to very small extents under normal conditions.
The rat GH (rGH) gene is expressed in the pituitary in a highly tissue-specific manner. A pituitary-specific transcription factor, Pit-1 (or GHF-1), and other, more tissue-general factors, including the thyroid hormone receptor (T3R), are important for regulating rGH promoter activity. The relative roles of Pit-1, T3R, and protein kinases in the activation of the rGH promoter were studied. Each component was supplied individually or in combination with the others to human monocyte U937 cells. The transfected rGH promoter was inactive in these cells even when it was cotransfected with either Pit-1 or T3R expression vectors. The rGH promoter carried in a truncated pUC vector could be activated by expression of the T3R if the cells were cultured with inducers of protein kinase-A (forskolin) and protein kinase-C [phorbol 12-myristate 13-acetate (PMA)] activity. By contrast, the PMA- and forskolin-dependent activation of the rGH promoter by Pit-1 expression was comparatively insignificant unless 1) the sequences deleted from the pUC vector (including a putative site for the transcription factor AP1) were restored to the plasmid carrying the rGH promoter; or 2) the T3R was coexpressed, which led to a marked synergistic response. These results indicate the relative inactivity of Pit-1 in isolation from other factors. Activation by forskolin and PMA did not require de novo protein synthesis. The synergistic activation by Pit-1 and the T3R was enhanced, but was not dependent upon, thyroid hormone (T3). The T3-dependent effect operated predominately through a thyroid hormone response element located up-stream of the two Pit-1-binding sites within the rGH promoter, whereas the T3-independent effect did not require any of the known T3R-binding sites on the rGH promoter. These results suggest a role for the more tissue-general T3R and protein kinases in the activation of the rGH promoter. They demonstrate the synergistic interplay between the T3R and Pit-1, underscore the dependence of Pit-1 action on other transcription factors, and implicate Pit-1 as a cofactor, rather than the dominant factor, influencing the tissue-specific expression of the rGH promoter.
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.
New IMI of cows milked with high and low slip rate milking machine liners were compared. High slip liners used in this study averaged 6.1 initial slips and 7.6 major vacuum fluctuations per cow milking, and low slip liners averaged 3.6 initial slips and 3.1 major vacuum fluctuations per cow milking. Least squares means for new IMI rates per 100 cow days were .49 in the high slip group and .27 in the low slip group. Rates of clinical cases per 100 cow days were .35 in the high slip group and .20 in the low slip group. New IMI rates for cows entering the trial with an existing infection were .81 in the high slip group and .39 in the low slip group. More new IMI tended to occur with cows milked with the high slip liners than with low slip liners.
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Longitudinal observations made during the past two decades on the prevalence of chronic ear disease in Inuit Elementary School Children in the Eastern Canadian Arctic reveal that as the current segment of this population enters into the 1990's a significant decrease in the prevalence is occurring. While we can speculate ad infinitum why this is happening we really do not know the answer. The usual cliché is adaptation to contact, improved medical/social services, housing, diet, etc.
Prorenin, the inactive biosynthetic precursor of renin, is proteolytically cleaved in the renal juxtaglomerular cells to renin. The activity of renin is rate-limiting for generation of angiotensin II in the circulation. We identified a renal thiol protease which activates and accurately cleaves the 43-amino acid prosegment of human recombinant prorenin. In the current studies, 6.5 mg of this protease was purified from human renal cortex using a three-step procedure dependent upon Leu-Leu-arginyl affinity chromatography. This represented an overall 766-fold purification and resulted in three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of molecular weights 30,000, 25,000, and 24,000. All three bands cross-reacted with an anti-human liver cathepsin B antibody upon immunoblot analysis; electrolution of each band and amino-terminal sequence analysis confirmed that the Mr 30,000 protein was mature cathepsin B and the Mr 25,000 and 24,000 bands were cathepsin B subunits. The pH optimum for the hydrolysis of pure human recombinant prorenin by pure renal cathepsin B was 6, and the Michaelis-Menten constant, Km, of the reaction was 1.4 x 10(-9) M. Immunostaining of human kidney using a sheep anti-human cathepsin B antibody demonstrated the presence of cathepsin B in the juxtaglomerular areas of the kidney, as well as in the renal proximal tubules. Electron microscopic immunohistochemistry using the same antibody demonstrated cathepsin B in dense secretory granules of the juxtaglomerular cells. Renin was also shown to be present in these granules. This study provides both biochemical and morphological evidence that renal cathepsin B is a human prorenin-processing enzyme.
The regulation by tumor necrosis factor alpha (TNF) of its own promoter has been investigated by transient transfection and nuclear protein binding assays. In human K652 erythroleukemia cells TNF produced an 8-10-fold activation of the human TNF promoter linked to the chloramphenicol acetyltransferase gene. The TNF-responsive element was localized to the -125 to -82 region by examining the TNF activation in 5'-deletion or site-directed mutants of the TNF promoter and by demonstrating that the -125 to -82 fragment confers TNF responsiveness to the thymidine kinase promoter. This region contains a palindrome, 5' TGAGCTCA 3', that resembles the consensus binding sequences for the transcription factors, activator protein-1 (AP-1), cyclic AMP-responsive element binding protein (CREB), and activation transcription factor (ATF). An internal deletion in the palindrome abolished the TNF responsiveness, whereas known AP-1 and CREB/ATF elements were unresponsive to TNF. In band shift analyses a nuclear factor from U937 cells specifically bound to the -125 to -82 TNF-responsive fragment in or near the palindromic sequence. Oligonucleotides containing AP-1 or CREB/ATF sites did not effectively compete for the binding, indicating that the U937 cell factor is different from these factors. Anti-c-fos antiserum did not affect binding of the U937 cell factor, whereas anti-c-jun antiserum did block its binding, indicating that either c-jun or a protein antigenically related to c-jun is a component of the factor. These results suggest that the TNF-responsive element is not activated by AP-1 or CREB in U937 cells and that a novel DNA binding factor is important for constitutive and inducible TNF gene expression.
This article describes investigations of several aspects of the molecular biology of the human renin gene and the three-dimensional structure of renin and its precursor, prorenin. Because of the importance of the RAS in hypertension, heart failure, renal failure, and possibly other disorders such as atherosclerosis, it is critical to understand the detailed control of this system. This control involves regulation at the transcriptional level, folding of prorenin, sorting of prorenin to a regulated pathway where it is proteolytically cleaved to renin and released in response to secretogogues, constitutive release of uncleaved prorenin, and nonproteolytic activation of prorenin. Currently there is great interest not only in the control of renin in the kidney, the sole source of circulating renin, but also at extrarenal sites where RAS activity may regulate cardiovascular functions. The renin gene was found to be expressed significantly in the renal juxtaglomerular cells and several other cell types. Most tissue culture cells did not express the gene; exceptions were cultured SK-LMS-1 cells and cAMP-stimulated human lung fibroblasts. Cultured human uterine-placental cells expressed the human renin gene at levels higher than in other cell types assessed. Renin mRNA had the same start site in the placental cells as the kidney and was regulated by calcium ionophores and cAMP. Thus, these cells provide primary nontransformed human cells to study the homologous human promoter. Transfected renin promoters showed cell type-specific expression and cAMP responsiveness in these cells in constructs containing as few as 102 bp of 5'-flanking DNA. DNA upstream from this appears to contain an inhibitory element(s) that may have some tissue specificity in its distribution. The cAMP response is not due to cAMP induction of a transcription factor that secondarily affects the renin promoter. A novel element may be involved, since the promoter does not contain a CRE element that mediates many cAMP responses, and the cells do not appear to respond to another known cAMP-responsive transcription factor, AP-2. Studies with transfected vectors expressing a mutant cAMP-responsive protein kinase A regulatory subunit suggest that cAMP is not responsible for basal renin promoter activity in the placental cells. By contrast, cAMP induces in essence gene activation in WI26VA4 transformed human lung fibroblasts in which renin mRNA levels increase by up to 150-fold in response to forskolin. Thus, cAMP may activate renin gene expression under certain circumstances and tissue-specific renin gene expression may be directed by more than one mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
Advances in molecular biology research continue to have a major impact on clinical medicine. These advances have provided a means to produce proteins previously available in limited supply and allow for the production of novel proteins that are improved agonists or else antagonists with greater specificity for therapeutic targets. Newer drug delivery systems should facilitate delivery of these proteins. By combining the capabilities to produce drug targets in acceptable quantities with improved methods for determining the three-dimensional structures of these targets, novel organic therapeutic molecules that act on these targets will be designed. Gene transfer therapy using genes that express important proteins or that encode "antisense" RNAs that inhibit the translation of specific mRNAs will soon become a reality. The use of RFLPs and PCR methodologies promises increased means to diagnose specific genetic diseases and infections. Most importantly, molecular biology is helping to understand the mechanisms of disease such that novel diagnostic and therapeutic approaches can be described. These advances are providing an understanding of the mechanisms involved in cancers of the thyroid gland. They have already led to an enhanced understanding of (1) the growth factors that control proliferation of the thyroid gland, (2) the potential steps in thyroid nodule and neoplasia development, (3) particular mutations that may occur as thyroid cancers develop, (4) oncogenes that are expressed in thyroid cancers, and (5) the genetic defects that are responsible for thyroid gland malignancies in the multiple endocrine neoplasia (MEN) syndromes. With the latter, the RFLP technology has already provided an enhanced means to diagnose the disorder. With further progress, enhanced means for diagnosis and treatment should emerge. Molecular biology techniques are contributing to an increased understanding of the mechanisms of development of autoimmune thyroid disease as with Graves' disease and Hashimoto's thyroiditis. The potential role of infections, histocompatibility antigens, thyroid gland and extraglandular antigens, immune modulators, subpopulations of T-cells such as suppressor and helper cells, other cells involved in immune responses, and a combination of influences of several different functions on the thyroid gland are being defined. This knowledge should soon suggest improved means for diagnosis and treatment. Understanding of the function of the thyroid hormone receptors should have clinical importance. This knowledge suggests a means to develop thyroid hormone antagonists that may be used to more rapidly ameliorate the effects of hyperthyroidism and could be useful in nonthyroidal disorders such as cardiac arrhythmias.(ABSTRACT TRUNCATED AT 400 WORDS)
Human prorenin is the enzymatically inactive biosynthetic precursor of renin. Recent interest has focused on the posttranslational sorting and processing of prorenin to renin since markedly increased levels of circulating prorenin have been associated with both physiological and pathological changes. These observations raise the question of whether prorenin processing may be a regulatory event in renin production in the kidney. In the juxtaglomerular cells of the kidney, prorenin can be sorted to either of two pathways: 1) the regulated pathway, which is mediated by secretory granules, where a thiol protease resembling cathepsin B processes prorenin to renin by cleavage of the amino terminal 43-amino acid prosegment, which allows exposure of the active site of renin, or 2) the constitutive pathway, which is not regulated and does not involve conversion of prorenin to renin. Studies in which segments of prorenin are modified by site-directed mutagenesis suggest that the prosegment and glycosylation are not required for sorting, although they may influence or participate in sorting, or both. Certain areas in the prosegment are important determinants of enzyme activity and ability to cleave the prosegment. Further structural analysis of prorenin will be useful to assess details of its sorting and processing. In addition, a number of extrarenal tissues such as uterine lining, ovarian theca, corpus luteum, pituitary, and adrenal, express the renin gene. These tissues have different capabilities to sort and process prorenin compared with kidney, and some tissues secrete only prorenin. Whether prorenin-to-renin conversion is necessary to activate these local renin-angiotensin systems is a key issue.(ABSTRACT TRUNCATED AT 250 WORDS)