Peptide-activator of cyclic nucleotide phosphodiesterases in the rat and human uterus

The presence of the cyclic nucleotide phosphodiesterase peptide-activator previously isolated from the calf myometrium was studied in uterine tissue of rats at different functional states (in puberty, estrus and diestrus stages, pregnancy) and in human myometrium and myoma. The peptide isolated from calf myometrium, immature rat myometrium and uterine tissue in myoma caused a 3-4-fold stimulation of the phosphodiesterase activity.     

Hypertrophy of the myometrial cells in women with myoma

A rapid growth of the uterus myoma results from the cell hypertrophy, which involves essential ultrastructural changes in the cytoplasm and the nucleus, thus suggesting the intensification cell functions. The increase in the cell volume and intensification of secretion is followed by the enlargement of the interface between the nucleus and the cytoplasm resulting in formation of numerous invaginations of the nuclear membrane. The autoradiographical data demonstrate a considerable DNA synthesis in the peripheral zone of the myoma, unlike the situation seen in the centre of the myoma and the unchanged myometrium, where DNA synthesis is not considerable. The cytospectrophotometric analysis confirms the above data showing the highest content of DNA in the peripheral zone of the myoma.     

Ultrastructure and relations between cells in the myometrium and in myomas of the human uterus

The state of myocytes and their intercellular connections in an unaltered myometrium in the peripheral zone and in the central part of the myoma node has been studied electron microscopically. When analysing the peripheral zone of the uterine myoma, myocytes have resemblence with myometrial cells in the folliculine phase of the menstrual cycle. Simultaneously, certain changes are observed both in the intracellular components and in intercellular interrelations: myocytic nuclei swell, most part of chromatin is decondenced, cell cytoplasmic volume increases, in the prenuclear zone numerous agregates of ribosomes are revealed. The myocytes draw nearer and form specialized junctions, that ensure their cooperation.     

The TRAF2 and TRAF6 expression in myomas and myometrium of women in reproduction and perimenopausal age

Uterine myomas represent one of the most common female diseases. Uterine myomas or fibromas are benign, hormone-responding tumours of, respectively, smooth muscles and fibroblasts and their aetiology induces a significant interest. In myomas the presence of aromatase was detected and, in addition, oestrogen was found to be synthesized in myoma cells. The studies were performed on myoma patients of generative age and those in peri-menopausal age. Expression of TRAF2 and TRAF6 proteins was examined using immunohistochemistry and Western blot approach in small and large uterine myomas isolated from women of various age. In addition, the evaluation was conducted at the periphery of every myoma. We indicated that the level of both tested proteins in myomas is higher than in control. TRAF2 level in myometrium was lower than in myomas but higher than in control. In the case of TRAF6 those changes were ambiguous. Age didn't have influence the level of expression in both tested TRAF in studied structures.     

Ghrelin in the human myometrium

Background  

Ghrelin is a 28-amino acid octanolyated peptide, synthesised primarily in the stomach. It stimulates growth hormone release, food intake and exhibits many other diverse effects. Our group have previously determined that ghrelin inhibited human contractility in vitro. The aim of this study therefore, was to investigate the expression of ghrelin, its receptor, the growth hormone secretagogue receptor type 1 (GHS-R1), ghrelin O-acyltransferase (GOAT) which catalyses ghrelin octanoylation, prohormone convertase 1/3 (PC1/3) responsible for pro-ghrelin processing, in human myometrium, during pregnancy prior to labour, during labour and in the non-pregnant state. Modulation of ghrelin and ghrelin receptor expression in cultured myometrial cells was also investigated.     

Processes of apoptosis and cell proliferation in uterine myomas originating from reproductive and perimenopausal women

We studied uterine myomas originating from females of reproductive age and from females of perimenopausal age. Uterine myomas represent benign tumors of the myometrium, and they develop frequently in women of reproductive age. The frequency of uterine myomas increases with age until women reach the menopause. The study included patients with a myomatous uterus, in the reproductive age or peri-menopausal age, independently evaluating small and large myomas. Myometrial alterations in their direct vicinity were evaluated independently of the myomas. The study included evaluation of immunolocalization of two index proteins which participate in myoma cells growth control: Ki-67 nuclear antigen and caspase 3. In women of reproductive age, both in small and large myomas, elevated immunostaining of Ki-67 was noted in parallel to low levels of caspase 3 staining, which indicated the ongoing process of proliferation. In women of peri-menopausal age with small or large myomas, no Ki-67 immunostaining was detected, while staining of caspase 3 manifested low levels. Proliferation in reproductive age women myomas is higher than in the peri-menopausal age.     

Estrogen regulation of aquaporins in the mouse uterus: potential roles in uterine water movement

Estrogen stimulates water imbibition in the uterine endometrium. This water then crosses the epithelial cells into the lumen, leading to a decrease in viscosity of uterine luminal fluid. To gain insight into the mechanisms underlying this estrogen-stimulated water transport, we have explored the expression profile and functionality of water channels termed aquaporins (AQPs) in the ovariectomized mouse uterus treated with ovarian steroid hormones. Using immunocytochemical analysis and immunoprecipitation techniques, we have found that AQP-1, -3, and -8 were constitutively expressed. AQP-1 expression was restricted to the myometrium and may be slightly regulated by ovarian steroid hormones. AQP-3 was expressed at low levels in the epithelial cells and myometrium, whereas AQP-8 was found in both the stromal cells and myometrium. AQP-2 was absent in vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus. This localization implicates all four isotypes in movement of water during uterine imbibition and, based on their localization to the luminal epithelial cells, AQP-2 and -3 in facilitating water movement into the lumen of the uterus. The analysis of the plasma membrane permeability of luminal epithelial cells by two separate cell swelling assays confirmed a highly increased water permeability of these cells in response to estrogen treatment. This finding suggests that estrogen decreases the luminal fluid viscosity, in part, by enhancing the water permeability of the epithelial layer, most likely by increasing the expression of AQP-2 and/or the availability of AQP-3. Together these results provide novel information concerning the mechanism by which estrogen controls water imbibition and luminal fluid viscosity in the mouse uterus.     

Fibroblast growth factors (FGF) in human myometrium and uterine leiomyomas in various stages of tumour growth

We previously described that the growth of human uterine leiomyomas was associated with a significant remodelling of the extracellular matrix of these tumours. Significant weight-related increase of collagen and heparan sulphate contents was detected. The latter was known as a component, which bound some peptide growth factors, mainly FGFs, therefore it was decided to evaluate the amounts of acidic FGF (aFGF) and basic FGF (bFGF) in human myometrium and in leiomyomas of various weight and FGF-binding to tissue components. It was found that myometrium and uterine leiomyomas contain picogram amount of aFGF and nanogram amounts of bFGF. No free aFGF was found. Slight amounts of free bFGF were detected both in myometrium and in the tumours. The aFGF and most of bFGF existed in a form of complex with a high molecular component(s). These complexes were very stable and they did not dissociate in denaturation conditions. In comparison to myometrium the tumours contained several times more FGFs and their amounts distinctly increased during the tumour growth. The expression of FGF-receptor I (FGF RI) in the tumours was more distinct in comparison to myometrium. The extracts from myometrium did not bind exogenous 125I-bFGF. In contrast to that the tumours of different weights contained at least two high molecular weight FGF-binding components. One of them (150 kDa) corresponded to FGF-receptor. The other one (190-200 kDa) might be a heparan sulphate-proteoglycan. It seems that aFGF and bFGF play an important role in transformation of normal myometrium into leiomyoma and further growth of this tumour. The action of FGFs on tumour cells enhances biosynthesis of collagen and sulphated glycosaminoglycans, especially heparan sulphate which binds FGFs in the vicinity of cells and facilitates their interaction with membrane receptors. The effect of these processes may be further stimulation of tumour growth and remodelling of tumour extracellular matrix.     

Platelet-activating factor acetylhydrolase isoforms I and II in human uterus. Comparisons with pregnant uterus and myoma

The concentrations of platelet-activating factor (PAF) that possesses the ability to stimulate myometrial contraction are partially regulated by intracellular type of platelet-activating factor acetylhydrolase (PAF-AH) in many tissues. Tissue cytosol contains at least two intracellular PAF-AH, isoforms I and II. To examine the relationship between the activity and isoforms of intracellular PAF-AH in human uterine myometrium and myoma, we assayed the PAF-AH activity and identified the PAF-AH isoforms I and II by Western blot analysis. The intense bands of the alpha2 and ss subunits of PAF-AH isoform I were detected in nonpregnant uterus; however, the specific bands of the alpha1 subunit of PAF-AH isoform I and the PAF-AH isoform II were extremely weak. The levels of the alpha2 and ss subunits and PAF-AH activity in pregnant uterus (37-39 wk gestation) were significantly lower than those in nonpregnant uterus. On the other hand, the level of ss subunit and the PAF-AH activity in myoma were significantly higher than those in nonpregnant uterus. No significant difference was found in the expression of the PAF-AH isoform II among three tissues. These results indicate that the change in the PAF-AH activity observed in pregnant uterus and myoma are due to the lower or higher protein expression of the PAF-AH isoform I, especially the alpha2 and/or ss subunits. The decrease of the uterine PAF-AH activity in the late stage of pregnancy may facilitate the action of PAF to stimulate myometrial contraction.     

Effect of caffeine and cyclosporin A on the transmembrane exchange of Ca ions in smooth muscle mitochondria

The effects of cyclosporin A and caffeine on the active and passive transport of Ca2+ in mitochondria isolated from adult rat myometrium were studied by fluorescent technique using Ca2+-sensitive probe tetracycline (TC). It was shown that 5 microM cyclosporin increases Ca2+ accumulation by the mitochondria matrix. But it fails to exhibit such effect when 20 mM caffeine was also present in the incubation medium, while the inhibitory action of caffeine on the accumulation of Ca2+ reveals nevertheless in the absence or presence of cyclosporin A. In case of the preliminary incubation of mitochondria with 10 mM caffeine before the initiation of transport process one could also observe the inhibition of kinetic parameters of the active accumulation of Ca2+ by the mitochondria. It was also shown, that caffeine stimulates passive efflux of Ca2+ from the myometrium mitochondria. Thus we conclude, that the stimulating effect of cyclosporin on Ca2+ accumulation by the myometrium mitochondria is sensitive to caffeine, while caffeine has no direct effect on Ca2+-uniporter, but it evidently disturbs the barrier function of the inner mitochondria membrane in such way, that stimulating effect of cyclosporin A cannot develop.     

Arachidonic acid metabolites in pregnant rat uterus

Production of prostaglandin E2 (PGE2), F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by pregnant rat uterus were measured in vitro. At mid-pregnancy, myometrium incubated with decidua attached released more prostanoids into the culture medium than when incubated without. As pregnancy progressed to 21 days more prostanoids were detected in the culture medium. However, no significantly increased conversion of exogenous arachidonic acid (AA) by myometrium was found.     

An accumulation of insulin-like growth factor I (IGF-I) in human myometrium and uterine leiomyomas in various stages of tumour growth

It is commonly thought that uterine leiomyomas result from hyperstimulation of myometrium by ovarian hormones. Some observations suggest that cytokines and growth factors are intermediate elements through which the ovarian hormones may exert their growth-stimulatory effects on leiomyomas. Human myometrium and uterine leiomyomas of various weights were homogenised and extracted with 1 M acetic acid or with 0.05 M Tris/HCl, pH 7.6. The extracts were assayed for IGF-I using the ELISA technique. It was found that 0.05 M Tris/HCl extracts contained several times more IGF-I than the 1 M acetic acid extracts. Nanogram amounts of IGF-I were found in both control myometrium and in leiomyomas. It was found that the amounts of IGF-I extracted from leiomyomas were distinctly higher in comparison to control myometrium and they increased as a function of tumour growth. Polyacrylamide gel electrophoresis, followed by Western immunoblotting, demonstrated that IGF-I in acidic and alkaline extracts exists as stable complexes, probably with extracellular matrix components. No free IGF-I was detected. Furthermore, it was found that some components of both the acidic and alkaline extracts were able to bind exogenous (125)I-labeled IGF-I. It is suggested that IGF-I plays an important role both in myometrium biology and in the growth of uterine leiomyomas.     

Analysis of fibroblast growth factor influence on growth and developmental potential of rat foetuses in the in vitro culture model

The fibroblast growth factor's (FGF) influence on the growth and differentiation of 8- and 9- day-old rat foetus has been studied, whereas foetuses were grown in an in vitro culture model. Proliferation was analysed by the expression of proliferating cell nuclear antigen (PCNA). It was established that the usage of FGF in the first period of the culture lowers the growth no matter the foetus age at the moment of culturing and no matter whether it is a medium with or without a serum. If FGF is applied in the second culture period, it also lowers the growth, however younger foetuses in the in vitro culture model are more sensible to FGF negative influence. When FGF was applied in a lower concentration the growth of whole foetuses was improved in the in vitro culture model, which shows that the FGF influence on growth depends on the concentration. Stereological analyses have been done and showed that, in the in vitro culture model, FGF has no influence on proliferating cartilage tissue, but it stimulates the survival of nervel tissue cells. It has been shown that the quantitative research of growth processes in cultivated foetuses can precisely be done by combining classic methods of measuring whole foetus diameters and analysing the expression of proliferating antigen.     

Extracellular matrix components in uterine leiomyoma and their alteration during the tumour growth

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.     

Regulation of proliferation of embryonic heart mesenchyme: role of transforming growth factor-beta 1 and the interstitial matrix

Proliferation of atrioventricular cushion mesenchyme of the embryonic avian heart maintained in three-dimensional aggregate culture is stimulated by interaction with the interstitial matrix. Chicken serum or transforming growth factor-beta 1, which stimulates proliferation, induces matrix deposition in regions of the aggregate showing high labeling indices with tritiated thymidine. Dispersed heart mesenchyme interstitial matrix introduced into serum-free culture is incorporated into the aggregate and stimulates cellular proliferation similar to serum or transforming growth factor-beta 1. Proliferation is reversibly inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro. It is suggested that transforming growth factor-beta 1 stimulates the production of interstitial matrix and that a sufficient stimulus for proliferation in this system is the presence of the matrix, which acts as the adhesive support for cellular anchorage.     

Comparative analysis of the cardiac glycosides action on the growth of the cardiac tissue explants

The method of organotypical cell culture was used. The long-term cell culture of cardiac embryonic tissue of 10-12-days old chicken was investigated. The effects of ouabaine, strophantin K and digoxin on the growth of cardiac tissue explant were measured. The ouabain concentration which stimulates activity of Na+, K+-ATPase as the signal transducer, was determined. It was equal to 10(-10) M. Strophantin K and digoxin stimulate growth of cardiac tissue in concentration equal to 10(-16) M and 10(-18) M, resp. The data obtained show that application of cardiac glycosides led to control of cardiac tissue growth in dose-dependent manner. We hypothesize that alpha3 isoform of Na+, K+-ATPase is a signal transducer that controls the cardiac cell metabolism and growth.     

Expression of estrogen receptor alpha and beta in myometrium of premenopausal and postmenopausal women

Although a clear role for estrogen receptor (ER) alpha has been established, the contribution of ERbeta in estrogen-dependent development, growth and functions of the myometrium is not understood. As a first step towards understanding the role of ERbeta, we have examined the expression of ERalpha and ERbeta in the human myometrium. With competitive RT-PCR assays, the level of ERbeta mRNA was 10-200 times lower than that of ERalpha mRNA in both premenopausal and postmenopausal myometrium. In premenopausal myometrium, the expression pattern of ERbeta mRNA during the menstrual cycle was similar to that of ERalpha mRNA, with highest levels in peri-ovulatory phase. In postmenopausal myometrium, ERbeta mRNA was significantly higher than it was in premenopausal myometrium, while the level of ERalpha mRNA was lower. The net result was a change in the ratio of ERbeta to ERalpha mRNA expression. The ratio changed from 0.6-1.5 in premenopausal to 2.5-7.6 in postmenopausal myometrium. In premenopausal women, the gonadotropin releasing hormone analogue, leuprorelin acetate, elicited a decrease in ERalpha and an increase in ERbeta mRNA expression to cause a postmenopausal receptor phenotype. Estradiol, on the other hand, reversed ERalpha and ERbeta mRNA expression and their ratio in postmenopausal myometrium to those of premenopausal myometrium. Immunohistochemical staining and Western blot analysis of ERalpha and ERbeta with semiquantitative analysis showed good agreement between mRNA and protein levels. The data indicate that coordinated expression of ERalpha and ERbeta might be necessary for normal estrogen action in myometrium. Furthermore, estrogen appears a dominant regulator of both receptors in the myometrium.     

The synergistic action of the copper chelator bathocuproine sulphonate and cysteine in enhancing growth of L1210 cells in vitro

The growth stimulating effect of a copper-specific chelator, 2,9-dimethyl-4,7-diphenyl-1,10-phenonthroline-sulfonic acid on mouse lymphoma L1210 cells in vitro has been studied. Since they are defective in cystine transport, these cells require cysteine for their growth in vitro. However, addition of cysteine does not greatly enhance cell growth because it is rapidly oxidized to cystine. We have observed that the copper chelator potently inhibited oxidation of cysteine in culture medium and that simultaneous addition of cysteine and the chelator greatly enhanced cell growth. The chelator alone stimulated cell growth slightly by stabilizing a small amount of cysteine effluxed from the cells to the medium. The chelator also enhanced the growth promoting activity of 2-mercaptoethanol by stabilizing cysteine produced in the medium during culture. These results suggest that the chelator stimulates cell growth by inhibiting copper mediated oxidation of cysteine in culture medium.     

Receptor-level interrelationships of amino acids and the adequate amino acid type hormones in Tetrahymena: a receptor evolution model

Histidine stimulates the phagocytosis of Tetrahymena to the same extent as histamine, and also stimulates its division, which histamine does not. Tyrosine and diiodotyrosine equally stimulate the growth of the Tetrahymena. Both amino acids inhibit the characteristic influence of the adequate amino acid hormone when added to Tetrahymena culture 72 h in advance of it. Primary interaction with diiodotyrosine and tyrosine notably increases the cellular growth rate. Histamine has a similar, although less notable effect than histidine. In the light of these experimental observations there is reason to postulate that the receptors of the amino acid hormones have developed from amino acid receptors.     

Isolation,Purification, and Identification of the Secretion Compound Pantoea brenneri AS3 with Fungicidal Activity

Applied Biochemistry and Microbiology - The pantoea brenneri AS3 strain stimulates plant growth and is active against pathogens, releasing secondary metabolites into the culture medium that inhibit...