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Objective: To elucidate the mode of action of chlormadinone acetate (CMA) in reducing dysmenorrheic pain by studying the effects of CMA and dexamethasone (DEX) on messenger RNA (mRNA) abundance of cyclo-oxygenase-2 (COX-2), annexin-1 (ANXA1), glucocorticoid receptor (GR), progesterone receptor (PR), and concentrations of prostaglandin F(2α) (PGF(2α)) and leukotrienes B(4) (LTB(4)) and C(4) (LTC(4)) in human endometrial explants., Design: Ex vivo study., Setting: University hospital., Patient(s): Fifteen premenopausal patients undergoing surgery for benign gynecologic disorders., Intervention(s): Endometrial explants were obtained by aspiration curettage and stimulated ex vivo with interleukin-1β before exposure to CMA or DEX; mRNA levels were determined via reverse transcription-quantitative real-time polymerase chain reaction, and concentrations of arachidonic acid metabolites by enzyme immunoassays., Main Outcome Measure(s): Messenger RNA levels of COX-2, ANXA1, PR, and GR; concentrations of PGF(2α), LTB(4), and LTC(4) in endometrial explants treated with CMA or DEX., Result(s): In IL-1β-treated explants COX-2 mRNA and PGF(2α), concentrations were significantly down-regulated by CMA but not by DEX. Chlormadinone acetate did not affect mRNA abundance of ANXA1, PR, and GR., Conclusion(s): Our data suggest that CMA is a suppressor of COX-2 expression. Comparison with DEX revealed that progestin-specific activity of CMA may mainly be responsible for suppression of prostaglandin biosynthesis in human endometrium., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17β-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The human endometrium is unique among adult tissues. Its functions are modulated by numerous hormones and mediators. The aim of this study was to evaluate the suitability of human endometrial explants for studying functional effects of chemicals and drugs on gene expression biomarkers. Endometrial tissues were obtained by aspiration curettage and cultivated for up to 24 h. Relative mRNA concentrations were determined by reverse transcription quantitative real-time PCR. Viability was assessed by light microscopy, lactate dehydrogenase assay and scanning electron microscopy. It was acceptable after 6 h of culture but reduced after 24 h. Culture-induced alterations of mRNA levels were found for progesterone receptor, estrogen receptor(α), leukemia inhibitory factor and cyclooxygenase-2 in tissues from all cycle stages. The suitability of the model to detect chemical effects was demonstrated by the down-regulation of cyclooxygenase-2 mRNA by chlormadinone acetate in proliferative and secretory endometrium. The model is mainly restricted by interindividual variations and varying tissue quality. An advantage is the preservation of tissue composition. We conclude that human endometrial explants are a complex model due to limited viability, difficult standardization and intrinsic alterations during culture. Experiments with this model should be performed over a limited time period under strictly controlled conditions.

The human endometrium is a fertility-determining factor. Its receptivity during the implantation window may be altered by chemicals. Since human embryo implantation is unique chemical risk assessment cannot be based solely on animal studies. We established a tissue-specific in vitro test based on human endometrial adenocarcinoma (Ishikawa) cells. Progesterone receptor (PR) was selected as primary target gene for estrogenic effects. Changes of mRNA levels were investigated by reverse transcription quantitative real-time PCR. Sigmoidal dose-response curves for up-regulation of PR mRNA and EC(50) values were established for 17beta-estradiol, diethylstilbestrol and the weak xenoestrogen bisphenol A. Nonylphenol also had a clear PR mRNA up-regulating effect. Several other chemicals were characterized as negative compounds. Among them was methoxyacetic acid which may produce false positive results in reporter gene assays. Up-regulation of PR protein by 17beta-estradiol, diethylstilbestrol, bisphenol A and nonylphenol was confirmed by Western Blotting., (Copyright 2010 Elsevier Inc. All rights reserved.)

In this article real-time quantitative RT-PCR strategies for investigation of mRNA distribution in stress-related corticotropin-releasing hormone (CRH), CRH-binding protein (CRH-BP), and CRH receptors (CRH-R1, CRH-R2) in human gestational tissues are described. The effect of sample and RNA preparation, reverse transcription, and the quantitation strategy were investigated. Both thawing of the sample before homogenization and the RT reaction were identified as critical steps. In contrast, the time-lag from the biopsy until snap-freezing and the homogenization procedure had little effect. The "housekeeping" gene cyclophilin was found to be differently expressed in gestational tissues, compromising its use as internal reference. For relative quantitation of mRNA levels by TaqMan PCR the standard curve method and the comparative C (T) method (DeltaDeltaC (T) or DeltaC (T)' method) were compared. Both calculation strategies delivered similar relative mRNA amounts in identifying the placenta as the main source of CRH and CRH-BP mRNA compared with myometrium and decidua. Consistent results were also obtained for CRH-R1 and CRH-R2 by both methods of calculation. We conclude that the simpler comparative C (T) method is adequate for assessing the mRNA levels of CRH, CRH-BP, and CRH receptors in human gestational tissues.

Cytochrome P450 metabolism of arachidonic acid (AA) was investigated in human peritoneal macrophages which play a central role in chronic pelvic diseases in women (for example in endometriosis). The formation of eicosanoids other than prostaglandins (PGs) by these cells is still unknown. In non-activated macrophages obtained from women in the reproductive age, the main [(3)H]-AA metabolites coeluted with epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids (DHETs) and hydroxyeicosatetraenoic acids (HETEs) in reverse-phase HPLC. After zymosan activation a shift to PGs pathway was observed. Treatment with low doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin increased the formation of a metabolite coeluting with 5,6-DHET. By gas chromatography/mass spectrometry 5,6-DHET (after beta-naphthoflavone induction), and 14,15-DHET as well as 11,12-DHET (after AA stimulation) were identified as major epoxygenase metabolites, respectively. The enantioselective formation of 12(S)-HETE was demonstrated by chiral-phase HPLC. Our findings demonstrate that non-activated peritoneal macrophages produce substantial amounts of bioactive cytochrome P450 metabolites of AA.

Human term myometrium is poorly characterized as a source of proinflammatory mediators involved in parturition. We have investigated the basal expression of cytokines in myometrium, as well as the effects of CRH and lipopolysaccharide (LPS) on cytokine release. Explants from term myometrium were challenged with CRH or LPS (1 microg/mL each) in short-term tissue culture. Interleukin (IL)-1beta++, IL-6, IL-8, and tumor necrosis factor (TNF)alpha concentrations in the medium were quantified by enzyme immunoassay. The major cytokines released after 24 h were IL-6 and IL-8. All cytokines investigated were stimulated significantly by LPS (P: < 0. 05) but not by CRH. Messenger RNA levels of these cytokines were investigated by RT-PCR. IL-1beta+ and IL-6 messenger RNA were present in preterm and term myometrium before and during labor, whereas IL-8 and TNFalpha were expressed only by myometrium in active labor. Furthermore, myometrial CRH receptors and macrophages were characterized immunohistochemically. We conclude that human term myometrium is a site of production of proinflammatory cytokines and is involved in the inflammation-like reactions mediating the birth process. Cytokine release in term myometrium seems not to be under control of CRH.

Objective: To determine concentrations of environmental estrogens, antiandrogens, and organochlorine compounds in human endometrium and body fat., Design: Cross-sectional, population-based study., Setting: Patient recruitment was done at a university hospital; chemical analysis was performed in a specialized private laboratory., Patient(s): Premenopausal, unexposed women undergoing hysterectomy for uterine myoma., Intervention(s): None., Main Outcome Measure(s): Concentrations of environmental modulators in human endometrium and body fat were quantified by high-resolution gas chromatography/high-resolution mass spectrometry., Result(s): Among known endocrine modulators, the antiandrogenic p, p'-dichlorodiphenyl-dichloroethylene was found in the highest concentrations in endometrium (median 4.7 microg/kg wet weight) and body fat (median 446 microg/kg wet weight). Only trace amounts of the identified environmental estrogens beta-hexachlorocyclohexane, o, p'-dichlorodiphenyl-trichloroethane, bisphenol A, hydroxylated polychlorinated biphenyls, and genistein were found in the endometrium (median <1 microg/kg wet weight). As major organochlorine contaminants without endocrine activities, polychlorinated biphenyls and hexachlorobenzene were found., Conclusion(s): Our data demonstrate that nonchlorinated environmental estrogens do not build up cumulative tissue concentrations in the endometrium. The risk of reduced fertility because of ambient levels of environmental estrogens in the endometrium is negligible.

This paper describes the sequence of adenylate kinase (Mg-ATP+AMP<-->Mg-ADP+ADP) from maize chloroplasts. This light-inducible enzyme is important for efficient CO2 fixation in the C4 cycle, by removing and recycling AMP produced in the reversible pyruvate phosphate dikinase reaction. The complete sequence was determined by analyzing peptides from cleavages with trypsin, AspN protease and CNBr and subcleavage of a major CNBr peptide with chymotrypsin. N-terminal Edman degradation and carboxypeptidase digestion established the terminal residues. Electrospray mass spectrometry confirmed the final sequence of 222 residues (M(r) = 24867) including one cysteine and one tryptophan. The sequence shows this enzyme to be a long-variant-type adenylate kinase, the nearest relatives being adenylate kinases from Enterobacteriaceae. Alignment of the sequence with the adenylate kinase from Escherichia coli reveals 44% identical residues. Since the E. coli structure has been published recently at 0.19-nm resolution with the inhibitor adenosine(5')pentaphospho(5')adenosine (Ap5A) [Müller, C. W. & Schulz, G. E. (1992) J. Mol. Biol. 224, 159-177], catalytically essential residues could be compared and were found to be mostly conserved. Surprisingly, in the nucleotide-binding Gly-rich loop Gly-Xaa-Pro-Gly-Xaa-Gly-Lys the middle Gly is replaced by Ala. This is, however, compensated by an Ile-->Val exchange in the nearest spatial neighborhood. A Thr-->Ala exchange explains the unusual tolerance of the enzyme for pyrimidine nucleotides in the acceptor site.

Adenylate kinases modulate the three adenine nucleotide pools and were found to be localized as isoenzymes in different tissues and organelles in animals and plants. For investigations of adenylate kinase isoenzymes from plant tissues different plant extracts were examined by anion-exchange chromatography. During investigations with the strong anion exchanger Mono Q, adenylate kinase activity eluted in the void volume. This void volume activity did not always occur, but depended on the age of the plants and light treatment. The nature of the factors affecting void volume activity could only be partially resolved. It could be shown that RNase treatment at the beginning of extraction led to the disappearance of void volume activity, whereas an untreated extract still showed this activity.