Search

Experiments to determine Cyprinus carpio iridovirus (CCIV) virulence to fish of different ages and species were performed; viz, carp, Cyprinus carpio L, goldfish, Carassius auratus (L.). and rainbow trout, Oncorhynchus mykiss (Walbaum). Neither signs of disease nor mortality occurred in any of the fish species tested. Five passages of the virus in carp resulted in its loss and, not surprisingly, there was no enhancement of virulence. In carp inoculated intraperitoneally, the virus persisted for more than 6 weeks at 18-20°C, more than 12 weeks at a water temperature increasing from JO to 20°C and more than 27 weeks at 6'5-8°C. The fish cleared itself of the virus after more than 6 weeks maintenance at 18-20°C. The viral infectivity via water is very low. It was impossible to infect the fish with the cell culture grown virus either by water immersion or by prolonged cohabitation with experimentally infected fish. From infectivity data, the authors conclude that the investigated iridovirus is not the aetiological agent of carp gill necrosis. [ABSTRACT FROM AUTHOR]

Progesterone is a hormone indispensable for pregnancy maintenance that acts through two pathways: via nuclear receptors it regulates gene expression while via membrane receptors it exerts quick nongenomic effects. Activation of spermatozoa and oocytes are among such nongenomic effects. Human membrane progesterone receptors (hmPRs) have been cloned, however, their hormone binding features are still weakly characterized. The knowledge of these features is a prerequisite for design of selective progesterone analogs that might be applied in obstetrics, gynecology, and also in oncology. In order to develop a system for the analysis of hmPRs ligand-binding properties, we constructed expression plasmid vectors containing nucleotide sequence insert encoding for the isoform alpha of hmPR under the yeast GAL1 promoter. The transfection of yeast strains with the constructs thus obtained gave an appearance of hmPRalpha mRNA in the cells, while cellular extracts containing membrane fractions acquired an ability for saturable binding of 3H-progesterone. Analogous vectors were constructed for expression of isoforms beta and gamma of hmPR. The results create a methodical base for selection of agents (agonists and antagonists) targeted preferably or exclusively to membrane progesterone receptors. [ABSTRACT FROM AUTHOR]

Progesterone (P4) and its analogues regulate various reproductive processes, such as ovulation, implantation, pregnancy maintenance and delivery. In these processes, an important role is played by the immune cells recruited to the female reproductive organs and tissues, where they are exposed to the action of P4. Progestins regulate cellular processes, acting through nuclear steroid receptors (nSRs), membrane P4 receptors (mPRs), and through the sensors. It remains unclear, what type of receptors is used by P4 and its derivatives to exert their effect on the immune cells and how similar their effects are in different types of these cells. We have previously synthesized new progesterone derivatives, among which two selective mPRs ligands, not interacting with nSRs were identified. The objective of this study was to examine the effects of P4 and new selective mPRs ligands on the expression of pro- and anti-inflammatory cytokines in activated human peripheral blood mononuclear cells (PBMCs), THP-1 monocyte cells, and Jurkat T cells. It was demonstrated that the action of P4 and selective ligands was unidirectional, but in different types of the immune cells, their effects were different, and sometimes even opposite. In PBMCs, exposure to these steroids resulted in the increase of mRNA and secreted protein levels of IL-1β, TNFα, and IL-6 cytokines, as well as in the increase of INFγ mRNA level, decrease of IL-2 mRNA level, increase of TGFβ mRNA level, and decrease of IL-4 mRNA and IL-10 secreted protein levels. In monocytes, similarly to PBMCs, expression of IL-1β and TNFα mRNA was increased, but expression of IL-10 was also increased, and the TGFβ expression statistically significantly remained the same. In Jurkat T cells, expression of IL-2 and TNFα mRNA decreased, while expression of IL-10 increased, and expression of TGFβ did not change. Thus, progestins act on the immune cells through mPRs and have both pro- and anti-inflammatory effects, depending on the phenotypes of these cells. The data obtained are important for understanding the complexity of the immune system regulation by progestins, which depends on the type of the immune cells and individual characteristics of the immune system., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The review considers the effect of progestins on the function, proliferation, and apoptosis of cells of various organs in health and noncancerous disorders. Data are summarized to describe the mechanism of progestin action through various progesterone receptors and sensors and the regulation of their levels. The effects of progestins depend on the cell phenotype, including the composition and proportion of different receptors, activity of signaling pathways, and expression of transcription factor coregulators and steroid metabolism enzymes. The role paracrine regulation plays in the progestin effect is described. Particular attention is paid to the progestin effect on the tissues where progestins are thought or known to affect carcinogenesis or to stimulate or suppress the tumor growth, that is, to modulate cell proliferation, apoptosis, and the epithelial-mesenchymal transition.

Electrophoretic mobility shift assay was used to determine whether pregna-D'-pentaranes allow progesterone receptor (PR) from rat uterine cytosol to bind hormone response element (HRE)-containing oligonucleotide duplexes and to measure the affinity of this interaction. The formation of DNA-protein complexes in low salt medium was progesterone-related ligand-, temperature-, and PR-dependent, and specific for HRE. The highest affinity of PR to DNA (equilibrium K(a) = 0.420 +/- 0.185 nM(-1)) was found in the presence of the partial agonist/antagonist RU486, while the lowest affinity (K(a) = 0.074 +/- 0.013 nM(-1)) was demonstrated with the full agonist 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone. With the exception of the strong full agonist R5020, there was a tendency toward correlation between the induced lower affinity of PR for DNA in the context of tyrosine aminotransferase HRE and the full agonistic activity of tested compounds.

Radioligand and immunohistochemical analysis was used for studying the expression of an unusual estrogen-binding protein (UEBP) in the regenerated liver of male rats poisoned with CCl4. As a result of CCl4 poisoning, most hepatocytes intensely producing UEBP and located in the central part of the liver lobules are destroyed (40 to 90% of the liver parenchymal cells). Dead cells are substituted by new hepatocytes formed due to proliferation of periportal hepatocytes in which UEBP is expressed at a low level. In regenerated liver, however, a high level of UEBP expression characteristic of the control animals is restored, as well as a gradient mode of distribution of UEBP-containing cells with the maximum UEBP concentration in hepatocytes surrounding the central vein. The results obtained with animals with either intact or removed testes were similar, except that the dynamics of liver regeneration was slower in the castrated rats. These data suggest the existence in all hepatocytes of the complete androgen-related program for UEBP expression. This program is inherited by daughter cells in the absence of an inducing hormone and is similar in all cells, independently of the extent of its phenotypic expression in hepatocytes with different localization within the liver lobule.

The localization of an unusual estrogen-binding protein (UEBP) was studied in the rat liver using indirect immunoperoxidase reaction in intact rats and after different hormonal influences. The distribution of the UEBR in normal males displays a form of a gradient with the maximum near the central veins. The gradient is absent in normal females. Castration or hypophysectomy of males, or their injection with estradiol or triiodyronine leads to a decrease in the UEBP concentration, though the gradient character of its distribution is persisting. There is a stable increase in the UEBP concentration in the first two layers of cells adjacent to the central veins in the female rat liver after ovariectomy and especially in combination with androgen injections. The revealed changes in the intensity of immunoperoxidase reaction after different hormonal influences correlate with the UEBP concentration in liver studies performed by the radioligand assay.

Estrophilic forms of rabbit liver cytosolic hydroxysteroid-dehydrogenase (HSD) were obtained as a highly purified preparations by means of fractionation with ammonium sulfate, gel-filtration, ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on estradiol-Sepharose and ion-exchange chromatography on DEAE-Toyopearl 650M. The protein express 4 different kinds of NADP-dependent activities: 3 alpha, 3 beta- and 17 beta-HSD activities with androgens and 20 alpha-HSD with progesterone as substrates. Revealed multiplicity of HSD enzymatic activity is demonstrated here for the first time. 17 beta-HSD activity of the protein preparations with estradiol is extremely low. Absence of a real metabolic activity of the protein with a ligand interacting with it rather intensively suggests that the isolated HSD forms can act not only as an enzyme, but also as a buffer-reserving mechanism for some steroids.

The accumulation of [3H]estradiol-receptor complexes by liver nuclei after preliminary incubation of the hormone with rat liver cytosol was studied. It was demonstrated that addition to female rat liver cytosol of a purified preparation of the unusual estrogen-binding protein (UEBP) from male rat liver causes a dose-dependent inhibition of subsequent accumulation of specifically bound [3H]estradiol in the nuclei. Addition to male rat liver cytosol of 1.5 microM 2 alpha-hydroxytestosterone, testosterone, 1-dehydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-dihydrotestosterone, i. e. compounds possessing marked affinity for UEBP, resulted in a 2-5-fold increase of the subsequent nuclear accumulation of estrogen-receptor complexes. The use of UEBP-deficient female rat liver cytosol revealed that the afore-mentioned steroids are ineffective with respect to estrogen reception. It is concluded that UEBP of male rat liver is capable of modulating estrogen reception.

The structure and properties of an unusual estrogen-binding protein (UEBP) from male rat liver that was purified by affinity adsorption chromatography was studied. A high degree of purity of UEBP (greater than 99%) associated with appreciable microheterogeneity was demonstrated. The latter seems to be due to partial proteolysis of the protein at the N-end in the course of the isolation procedure. The purified UEBP molecules have the following characteristics: Mr = 31 000 (data from SDS-PAAG electrophoresis), sedimentation coefficient 3.75 S, Stockes'radius 25.6 A, friction coefficient ratio 1.11. The protein absorbance maximum in the UV region lies at 276 nm; extinction coefficient--26, alpha-helix content is 25-30%. The value of equilibrium association constant for estradiol is 5 X 10(7) M-1. Estriol (greater than 100%) and, in a weaker degree, estrone and testosterone (approximately 10%) compete for the binding sites with [3H]estradiol; androsterone has no competitive effect. The amino acid composition of UEBP was determined. The protein was shown to possess a great number of residues carrying hydrophobic side groups (34.4%) and more acidic amino acids over basic ones as well as a low content of cystein, threonine and histidine.

A procedure has been developed for purification of a special rat liver estrogen-binding protein. It includes protein precipitation by ammonium sulfate, gel filtration, ion exchange chromatography, and affinity chromatography on estradiol sepharose. The protein is purified 2260-fold with a 27% yield. Upon electrophoresis in 10% PAG in the presence of sodium dodecylsulfate the protein gives one polypeptide strip with a molecular weight of 31.200 +/- 400 dalton.

The effects of rabbit polyclonal antibodies (AB) against an unusual estrogen-binding protein (UEBP) isolated from rat liver by immunoadsorption on the interaction of [3H]estradiol with UEBP, estrogen receptors of uterus and other tissues, of [3H]dihydroxytestosterone with prostate androgen receptors, of [3H]progesterone with uterine progesterone receptors and of [3H]dexamethazone with rat thymus glucocorticoid receptors were investigated. It was shown that preincubation of cytosol of the above tissues with AB decreases the ability of UEBP, estrogen and androgen receptors to bind the corresponding ligands. The hormone-binding activity of progesterone and glucocorticoid receptors in the presence of AB remains thereby unchanged. The binding activity of UEBP in the presence of ABUEBP diminishes due to the decrease in the concentration of protein binding sites, whereas that of estrogen and androgen receptors decreases due to the diminution of affinity for their ligands which, in turn, is the result of reduction of the association rate constant. A cross influence of AB on the activity of uterine estrogen receptors of rabbit, guinea pig and mouse was observed. It was assumed that the structure of UEBP and sex steroid receptors has some similar elements.

A new modification of specific estrogen-binding protein (SEBP) assay in the liver was used to measure the content of this protein at different stages of ontogenesis in male and female rats after administration of testosterone propionate (TP). It was shown that liver capability to form SEBP could be initially programmed by androgens. The crucial period of the SEBP-determining action of androgens in females is not restricted, since potential capability of androgens of primary induction of SEBP may be realized both neonatally and in the course of pubertation. The emergence of SEBP in the liver of males may be the result of natural pre(neo)natal androgenization. Androgens do not play any decisive role in the control of the level of induced SEBP, inasmuch the content of SEBP in the liver of non-pubertal and pubertal males does not appreciably change after TP administration but grows with pubescence. SEBP is capable of long-term existence in the absence of androgens. The presence of the ovaries produces an inhibitory effect on the level of androgen-induced SEBP and the time of its existence after TP discontinuation. Hypophysectomy does not provoke SEBP emergence in pubertal intact and ovariectomized females but prevents its induction with androgens. Adrenal- and thyroectomy produce no influence on the SEBP-programming effect of TP or promote its spontaneous appearance in the same groups of female rats.

Tissue distribution in male and female rats of unusual immunoreactive material specific for the hepatic estrogen-binding protein (UEBP) was studied by means of immunoblotting analysis. From 28 organs and tissues studied only the liver of animals of both sexes was found to contain polypeptide with Mr, close to Mr of purified UEBP (approximately 31,000), which reacts with anti-UEBP specific antiserum. Detection of the immunoreactive material in female liver, which does not contain any hormone-binding activity of UEBP, suggests pre- or posttranslational modifications of the protein. It is concluded that the regulatory action of UEBP on steroid biodynamics bears a narrow-directed, organ-specific character.

The authors compared the properties of nonreceptor estrogen-binding protein (NREBP) of the ovaries of immature rats, specific estrogen-binding protein (SEBP) of the liver and alpha-fetoprotein (alpha-FP) of the rat blood serum. It was shown that all 3 proteins with moderate affinity bound 3H-estradiol (Ka approximately 10(8)M-1) and were characterized by a high rate of interaction with ligand (K+1 approximately 10(6)M-1c-1) and dissociation of hormone-protein complexes (K-1 approximately 10(-2)c-1). NREBP and alpha-FP manifested a very close hormonal specificity of affinity differing from SEBP specificity: both proteins bound intensively estrone and estradiol, to a lesser degree, estriol, and did not bind androgens and hexestrol. Stokes' radii of the molecules of 3 proteins were 4.4 and 2.5 nm, respectively. Rabbit antiserum to alpha-FP suppressed the hormone-binding activity of blood alpha-FP and ovarian NREBP but not liver SEBP. With growth of animals a study of the time course of the levels of ovarian NREBP and blood alpha-FP has shown that the presence of NREBP in the ovarian cytosol does not probably result from blood proteins mechanical contamination of the sample in homogenization. It has been assumed that intracellular alpha-FP or its close analog may be involved in the regulation of ovarian endocrine function, reversibly binding estrogens.