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[Relation between quality of dietary protein, plasma corticosteroid concentration and various enzyme activities in liver and mucosa of small intestine].

Comprehensive studies were performed in Wistar rats on the relations between the dietary protein quality and the function of the adrenocortical system. The authors determined: the increase in body mass, the urea and corticosteroid concentrations in the blood plasma, the GOT (EC 2.6.I.I.) and GPT (EC 2.6.I.2.) activities in the liver, the reactivity of the adrenocortical system in vitro, the ACTH content in the hypophyses and the content of corticosteroid-binding globulin in the blood plasma. In adrenalectomized and normal rats as well as in rats treated with corticosterone or cortisol, the authors determined the GPT, GOT, amino-acid aryl amidase (EC 3.4.I.2.) and tripeptidase (EC 3.4.I.3.) activities in the liver and the mucosa of the small intestine, and the total, alpha and beta-amylase activities in the small intestine.

Adrenal Cortex Hormones↗

3H-estradiol and 3H-R5020 binding in cytosols of normal and neoplastic human ovarian tissue.

High-affinity cytoplasmic estrogen and progesterone receptors in normal and abnormal ovarian tissues were studied. Estradiol receptor was detectable in 65% and progesterone receptor in 36% of the malignant tumors; 39% of all malignant ovarian tissues were estradiol- as well as progesterone-receptor-positive. Tumors were said to be receptor-positive when the receptors bound greater than 5 fM steroid/mg cytosol protein. No correlations were found between receptor status and histopathological diagnosis. In normal ovarian tissues collected at various phases of the menstrual cycle no changes in [3H]-estradiol and [3H]-R5020 binding could be detected. Analysis of the receptor concentration for both steroid hormones with regard to the menopausal status demonstrated highest levels in postmenopausal women. No significant difference could be found when two groups of patients with advanced ovarian carcinoma associated with the cytosol estrogen receptor status were compared in terms of two different therapeutic schemes (cytosolic chemotherapy with and without tamoxifen).

Cytosol↗

Biochemical and immunological characterization of the acrosome reaction-inducing substance (ARIS) of hFF.

Gel filtration experiments with 3H-progesterone labeled hFF demonstrated that the AR-inducing activity of hFF might be mediated by a progesterone-binding protein. Further immunological investigations added evidence in support to ARIS being identical with the SERPIN CBG. Moreover, only the protein-progesterone-complex was able to induce AR. It is suggested that the CBG-progesterone-complex is proteolytically cleaved at the plasma membrane of spermatozoa, releasing a high local concentration of progesterone which leads to induction of the AR.

Acrosome↗

A linkage disequilibrium between genes at the serine protease inhibitor gene cluster on chromosome 14q32.1 is associated with Wegener's granulomatosis.

The frequency of carriers of the alpha1-antitrypsin (alpha1-AT) deficiency allele PI*Z is increased in patients with Wegener's granulomatosis (WG). The polymorphic protease inhibitor (PI) gene is part of a cluster of serine protease inhibitor (serpin) genes (AACT; alpha1-antichymotrypsin, PCI; protein C inhibitor, CBG; corticosteroid binding globulin, PIL; PI-like pseudogene) at chromosome 14q32.1. In this study we investigated whether the serpin gene cluster contributes to the background of Wegener's granulomatosis. Therefore, phenotyping of alpha1-AT was performed and simple tandem repeat polymorphisms (STRP) in the genes for CBG, PI, and PCI as well as two STRP (D14S55 and D14S48) flanking the centromeric and one (D14S51) flanking the telomeric region of the gene cluster were examined in a population of 79 patients with WG and 128 unrelated healthy controls. In WG patients an increased frequency of the PI*Z defective allele is demonstrated as well as a linkage disequilibrium between all members of this gene cluster plus the centromeric and telomeric STRP. These results indicate an involvement of the serpin genes in the pathogenesis of Wegener's granulomatosis and it is possible that other genes located in the vicinity of D14S55 or D14S51 contribute to the genetic background of the disease.

Alleles↗

Polarized secretion of human corticosteroid binding globulin by MDCK and BeWo cells.

Polarized epithelial cells are able to faithfully direct certain secretory protein components to either their apical or basolateral environments. The mechanism by which these cells accomplish this is still not entirely understood. It is hypothesized that a membrane-associated "sorting receptor" recognizes an intrinsic signal contained within the sorted protein. This interaction directs the secretory protein into the appropriate domain-specific vesicle for transport to either the apical or basolateral face. The nature of this sorting signal and the recognition receptor have not been established. In an effort to understand this phenomenon, a study was undertaken to ascertain whether human corticosteroid binding globulin (hCBG) contains intrinsic signals capable of directing its secretion to a particular side of polarized epithelial cells. The results of these studies have revealed that hCBG is selectively secreted into the apical environment by both MDCK and BeWo cells. Furthermore, this polarized secretion is unaffected by either (1) agents that inhibit N-linked oligosaccharide processing or (2) lysomotrophic drugs, which alter the intravesicular pH. It is concluded that hCBG possesses an intrinsic signal for apical secretion, which can be recognized by two polarized cell types of differing origins. This signal does not appear to be present in the N-linked oligosaccharide moieties of hCBG nor is it affected by an elevation of the intravascular pH within the trans-Golgi network. The use of hCBG-transfected MDCK and BeWo cells constitute a useful model system for the investigation of the signals involved in the sorting of secreted proteins.

1-Deoxynojirimycin↗

Effects of experimentally elevated testosterone on plasma corticosterone and corticosteroid-binding globulin in dark-eyed juncos (Junco hyemalis).

An earlier study of free-living male dark-eyed juncos found an increase in plasma corticosterone (B) in response to experimental elevation of plasma testosterone (T) (E. D. Ketterson et al., 1991, Horm. Behav. 25, 489-503). To investigate whether the increase was caused by enhanced secretion of corticosterone or by a slower clearance rate, or both, we exposed 52 captive yearling male dark-eyed juncos (Junco hyemalis) to day lengths corresponding to those of spring and implanted them with one or two testosterone-filled or sham implants (10 T-I, 22 T-II, and 20 C-males). We then examined the effect of experimentally elevated testosterone on plasma corticosterone and on corticosteroid-binding globulin (CBG), as measured by the ability of steroid-stripped plasma to bind labeled corticosterone. Plasma samples were taken five times, 2 weeks before experimental prolongation of day length and approximately every 3 weeks thereafter. Treatment with testosterone increased both plasma testosterone and plasma corticosterone two to three times above control levels, and the degree of elevation was dose-dependent. Only when all treatment groups were pooled, however, were plasma testosterone and corticosterone significantly correlated. The relationship between plasma corticosterone and time required to bleed the birds was similar for all three treatment groups, suggesting that there was no effect of treatment on the stress response. Testosterone significantly increased the capacity of the plasma to bind corticosterone, presumably because it contained more CBG, when compared to the plasma of controls. However, treatment with testosterone did not affect the affinity of the plasma for corticosterone. It seems likely that exogenous testosterone elevated corticosterone by slowing the corticosterone clearance rate via an increase in CBG. It is not clear what the net effect of chronic elevation of testosterone would be on the availability of corticosterone to target tissues.

Animals↗

Distinct specificity for corticosteroid binding sites in amphibian cytosol, neuronal membranes, and plasma.

To address mechanisms of corticosteroid action, one needs tools for distinguishing between the major classes of corticosteroid binding sites: neuronal membrane-associated receptors, intracellular ligand-activated transcription factors, and corticosteroid binding globulins (CBG) in plasma. We characterized the binding parameters for three classes of binding sites in an amphibian, Ambystoma tigrinum, and found that each class had a distinct pharmacological specificity. Equilibrium saturation and kinetic experiments indicated that [3H]corticosterone binds to neuronal membranes with high affinity (Kd approximately 0.37 nM). Aldosterone and two synthetic ligands for mammalian intracellular receptors, dexamethasone and RU486, displayed low affinity for brain membrane sites. In cytosol prepared from brain and liver, [3H]corticosterone bound to a single class of receptors with high affinity (Kd approximately 0.75 and 4.69 nM, respectively) and the rank order potencies for steroid inhibition of [3H]corticosterone binding was RU486 > dexamethasone approximately = corticosterone > aldosterone. In kidney and skin cytosol, [3H]corticosterone binding was best fit with a model having a high-affinity and a lower-affinity site; these sites are not consistent with the pharmacology of mammalian Type I (MR) and Type II (GR) receptors. [3H]Corticosterone also bound to presumed CBG in plasma with high affinity (Kd approximately 2.7 nM), but dexamethasone and androgens bound to plasma CBG with equivalently high affinity. These data demonstrate that pharmacological specificity can be a useful tool for distinguishing corticosteroid binding to different classes of binding sites. These data also indicate that there may be marked species differences in the specificity of corticosteroid binding sites.

Adrenal Cortex Hormones↗

Testosterone, corticosterone, and photoperiod interact to regulate plasma levels of binding globulin and free steroid hormone in dark-eyed juncos, Junco hyemalis.

The pharmacology and regulation of corticosteroid binding globulins (CBG) in Dark-eyed Juncos, Junco hyemalis, was investigated. The equilibrium dissociation constant for [(3)H]corticosterone (CORT) binding to plasma was <5 nM. This binding site had a similar high affinity for progesterone, approximately fivefold lower affinity for androgens, and negligible affinity for estradiol. The following data suggested that plasma CBG levels are regulated by both testosterone and day length: (1) CBG binding capacity in free-living adult males was greater in early than in late breeding season and greater in males than in females and (2) CBG levels were higher in testosterone-treated, castrated males than in castrated males receiving no testosterone and still higher in testosterone-treated males exposed to long days than in similar males exposed to short days. Birds apparently lack a sex steroid-specific binding globulin, but it was estimated that more than 90% of testosterone in junco plasma should bind to CBG. An increase in plasma CORT, such as occurs during a stress response, was judged to acutely increase free testosterone levels as much as fivefold. Corticosterone and testosterone may thus interact in a complex manner in species that lack sex hormone binding proteins.

Animals↗

A 370-kb cosmid contig of the serpin gene cluster on human chromosome 14q32.1: molecular linkage of the genes encoding alpha 1-antichymotrypsin, protein C inhibitor, kallistatin, alpha 1-antitrypsin, and corticosteroid-binding globulin.

The human genes encoding alpha 1-antitrypsin (alpha 1AT, gene symbol PI), corticosteroid-binding globulin (CBG), alpha 1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an approximately 370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen-CBG-ATR-alpha 1 AT-KAL-PCI-AACT-tel. The genes occupy approximately 320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by approximately 170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies approximately 63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is approximately 12 kb of intergenic DNA between KAL and PCI and approximately 19 kb between PCI and AACT. The proximal subcluster includes alpha 1AT, ATR, and CBG; it occupies approximately 90 kb of genomic DNA, with approximately 12 kb of DNA between alpha 1AT and ATR and approximately 40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.

Carrier Proteins↗

Long-range chromatin reorganization of the human serpin gene cluster at 14q32.1 accompanies gene activation and extinction in microcell hybrids.

The genes encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of six serine protease inhibitor (serpin) genes located on human chromosome 14q32.1. Both genes are actively transcribed in the liver and in human hepatoma cells, but they are not expressed in most other cell types. In this study we mapped DNase I-hypersensitive sites (DHSs) in an approximately 130-kb region of 14q32.1 that includes both genes. The distributions of DHSs in expressing (HepG2) vs nonexpressing (HeLa S3) cells were very different: HepG2 cells displayed 29 DHSs in this interval, but only 7 of those sites were present in HeLa cells. To determine the chromatin organization of activated or extinguished serpin alleles, we transferred human chromosome 14 into rat hepatoma cells or fibroblasts, respectively. Human alpha1AT and CBG gene expression was activated in rat hepatoma microcell hybrids containing human chromosome 14, but extinguished in rat fibroblast hybrids with the same genotype. DHS mapping in these microcell hybrids demonstrated that the chromatin structure of the entire 130-kb region was reorganized in microcell hybrids, and the distributions of DHSs in activated and extinguished alleles recapitulated those of expressing and nonexpressing cells, respectively. Thus, microcell hybrids provide a system in which reproducible changes in gene activity and long-range chromatin organization can be induced experimentally. This provides a basis for studying the effects of targeted modifications of the alpha1AT and CBG loci on the regulation of gene activity and chromatin structure.

Animals↗

Persistent effects of prenatal, neonatal, or adult treatment with flutamide on the hypothalamic-pituitary-adrenal stress response of adult male rats.

To explore the role of androgens in early development on adult hypothalamic-pituitary-adrenal (HPA) function in males, we administered flutamide or vehicle injections: (1) to pregnant dams on embryonic days 15-20; (2) to neonatal pups on days 0-5; or (3) to adults on days 55-60. At approximately 70 days of age, trunk blood was collected to determine corticosterone levels (1) upon removal from the home cage, (2) immediately after 30 min of restraint stress, or (3) 60 min after return to home cage following the stressor. Flutamide treatment resulted in higher basal levels of testosterone and stress levels of corticosterone compared to vehicle treatment, and there was no interaction of treatment with age at time of treatment. This suggests that testosterone is less effective at inhibiting HPA function in flutamide-treated males. In addition, prenatally treated males had higher stress levels of corticosterone than neonatally and adult-treated males, regardless of the type of treatment. There were no differences in CBG levels among the groups. The results suggest that, in males, flutamide treatment has a long-lasting effect on HPA function. These results are consistent with our previous research on neonatally gonadectomized males and the hypothesis of organizational effects of sex hormones on HPA function.

Age Factors↗

Decidual cell function: evidence for a role in the regulation of serum CBG and a 60 kDa protein during early pregnancy in the hamster.

Several serum proteins increase in titer during pregnancy. We tested the hypothesis that decidual cells may signal the production of certain serum proteins in the hamster. Measurement of serum CBG by equilibrium binding using either [3H]-progesterone or [3H]-cortisol in conjection with ion exchange chromatography showed that decidualization increased CBG levels. Two-dimensional gel electrophoresis revealed that a 60 kDa++ protein increases markedly in the serum of the hormonally pseudopregnant (PSP) animal soon after artificial induction of decidualization on PSP day 4. The 60 kDa serum protein remains low in the nondecidualized PSP animal, but it increases in the pregnant animal. A photoaffinity labeling procedure was used to covalently bind [3H]-androstadienolone to CBG. Fluorography of 2D gels run under denaturing conditions established that the 60 kDa protein did not bind steroid as did CBG (69 kDa). To determine whether decidual cells could induce the 60 kDa and CBG proteins, different numbers of decidual cells were injected IP into PSP recipients. A single injection of 50 x 10(6) decidual cells induced both serum proteins within 48h, whereas the same number of hamster fetal cells was ineffective. Thus, these results demonstrate that hamster decidual cells induce a 60 kDa protein of unknown function and serum CBG. Since the decidual cell itself does not appear to be the source of either protein, it follows that the decidual cell signals the synthesis and secretion of these proteins elsewhere in the body, most likely in the liver. To our knowledge, this is the first demonstration that the decidual cell regulates serum CBG and other proteins in this manner.

Animals↗