Expression of cyclooxygenase-1 and -2 in the baboon endometrium during the menstrual cycle and pregnancy.

Cyclooxygenase (COX) is the rate-limiting enzyme in the biosynthesis of PGs. PGs together with ovarian steroids play important regulatory roles in the establishment and maintenance of pregnancy in a number of different species. In the primate, little is known about the role of PGs in these processes. In this study, the uterine expression of COX-1 and COX-2 throughout the menstrual cycle [late follicular, day 5 postovulation (PO), day 10 PO, and day 14 PO] and pregnancy (days 12-18, day 39, day 51, and near term) was analyzed using semiquantitative RT-PCR, in situ hybridization, and immunocytochemistry. During the menstrual cycle, the highest expression of COX-1 occurred in luteal phase endometrium and was localized to the surface and glandular epithelium. The stromal cells did not express detectable levels of COX-1 at any time. COX-2 messenger RNA (mRNA) expression, as measured by RT-PCR, was evident at all stages of the menstrual cycle, and in situ hybridization showed specific localization for this mRNA in the epithelial cells during the cycle. Treatment of animals with the antiprogestin (ZK 137.316) for 9 days (beginning on the day of the LH surge) inhibited COX-1 expression in the epithelium when the tissue was analyzed on day 10 PO, whereas COX-2 expression disappeared in the epithelium and increased in the stroma. With the onset of pregnancy, COX-1 expression in epithelial cells decreased dramatically. In contrast, COX-2 continued to be detected on the surface epithelium and was also strongly expressed specifically in the stromal cells at the site of implantation. Immunocytochemical staining for COX-2 showed the same pattern of expression for the protein as the message. Finally, near-term decidua expressed very little COX-1 or COX-2 mRNA. These studies suggest that in the baboon endometrium, COX-1 expression is regulated primarily by progesterone, whereas regulation of COX-2 expression may involve additional mediators of embryonic origin at the site of implantation.     

Interpreting the clinical significance of the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2.

The International Consensus Meeting on the Mode of Action of COX-2 Inhibition (ICMMAC) brought together 17 international experts in arthritis, gastroenterology and pharmacology on 5 6 December 1997. The meeting was convened to provide a definition of COX-2 specificity and to consider the clinical relevance of COX-2-specific agents. These compounds are a new class of drugs that specifically inhibit the enzyme COX-2 while having no effect on COX-1 across the whole therapeutic dose range. The objectives of the meeting were to review the currently available data regarding the roles and biology of COX-1 and COX-2, and to foster a consensus definition on COX-2 specificity. At the present time, no guidelines exist for the in vitro and in vivo assessment of COX specificity, and it was felt that consensus discussion might clarify some of these issues. The meeting also reviewed recent clinical data on COX-2-specific inhibitors. The following article reflects discussion at this meeting and provides a consensus definition of COX-2-specific inhibitors.     

Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy

Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.     

Construction, expression and characterization of human interferon α2b-（G4S）n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNc(2b and To(1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris.METHODS: Using PCR and molecular clone techniques,the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1(n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex^TM 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex^TM 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins‘ function in vivo.     

Tissue-specific modulation of cyclooxygenase-2 (Cox-2) expression in the uterus and the v. cava by estrogens and phytoestrogens

Cyclooxygenase 2 (Cox-2), an enzyme involved in prostaglandin production, is a key player in the development of pathologic changes, such as colorectal cancer, arteriosclerosis and thrombosis. In this study, we investigated the effects of estrogens, selective estrogen receptor modulators (SERMs), pure antiestrogens and phytoestrogens on the tissue-specific expression of Cox-2 in the uterus and the v. cava of ovariectomized female rats. Cox-2 expression could be detected in the uterine epithelium and in the endothelium of the v. cava. Cox-2 expression was time-dependently stimulated after administration of 17beta estradiol (E2) in the uterus. In the v. cava, E2 treatment resulted in a stimulated expression of the progesterone receptor (PR), a gene known to be regulated by E2, whereas Cox-2 was simultaneously down-regulated. Administration of the pure antiestrogen faslodex (Fas) had no effect on Cox-2 expression. In contrast, administration of tamoxifen (Tam) resulted in a decrease of Cox-2 expression in the v. cava but does not stimulate Cox-2 expression in the uterus. Interestingly, the same expression pattern of Cox-2 could be detected after dose-dependent administration of genistein (Gen). Here, down-regulation of Cox-2 could already be detected after administration of merely 0.5 mg/(kgBW) Gen, a dose where no effects on uterine weight were observed. In summary, our results demonstrate a reverse tissue-specific regulation of Cox-2 expression by estrogens in the v. cava and uterus indicating the existence of complex molecular mechanisms which have to be characterized in future studies. Remarkably, Tam and the phytoestrogen Gen, both share the ability to decrease the expression of Cox-2 in the v. cava without effecting its uterine expression. These observations may be of great importance with respect to potential beneficial or adverse effects of estrogens, SERMs and phytoestrogens on the cardiovascular tissue.     

Enhanced renal immunocytochemical expression of ANG-(1-7) and ACE2 during pregnancy

Previously we demonstrated that kidney concentration and urinary excretion of angiotensin-(1-7) are increased during normal pregnancy in rats. Since this finding may reflect local kidney production of angiotensin-(1-7), we determined the immunocytochemical distribution of angiotensin-(1-7) and its newly described processing enzyme, ACE2, in kidneys of virgin and 19-day-pregnant Sprague-Dawley rats. Sprague-Dawley rats were killed at the 19th day of pregnancy, and tissues were prepared for immunocytochemical by using a polyclonal antibody to angiotensin- (1-7) or a monoclonal antibody to ACE2. Angiotensin-(1-7) immunostaining was predominantly localized to the renal tubules traversing both the inner cortex and outer medulla. ACE2 immunostaining was localized throughout the cortex and outer medulla and was visualized in the renal tubules of both virgin and pregnant rats. The quantification of angiotensin-(1-7) and ACE2 immunocytochemical staining showed that in pregnant animals, the intensity of the staining increased by 56% and 117%, respectively (P<0.05). This first demonstration of the immunocytochemical distribution of angiotensin-(1-7) and ACE2 in kidneys of pregnant rats shows that pregnancy increases angiotensin-(1-7) immunocytochemical expression in association with increased ACE2 intensity of staining. The findings suggest that ACE2 may contribute to the local production and overexpression of angiotensin-(1-7) in the kidney during pregnancy.     

Soluble proteins in the human atherosclerotic plaque. With spectral reference to immunoglobulins, C3-complement component, alpha 1-antitrypsin and alpha 2-macroglobulin.

A number of soluble proteins contained in human aortic intimal tissue was extracted into buffered saline (pH 7.4) and identified and quantitated by immunoelectrophoresis and immunodiffusion. The proteins included IgA, IgG, IgM, B1C (C3), alpha 1-antitrypsin, alpha 2-macroglobulin, fibrinogen, albumin, LDL, HDL, alpha 1-acid glycoprotein, beta 2-glycoprotein, transferrin and ceruloplasmin. The concentration of soluble proteins was significantly higher in the atherosclerotic intima than in the normal intima. The diseased intima also contained a small amount of tissue-bound IgG, IgA and B1C which was extractable with citrate buffer at pH 3.2. The vascular band IgG, and B1C were shown by enzymatic and immunohistochemical studies to be closely associated with the collagenous tissue of the plaque. The Ig contained in the atherosclerotic plaque may be derived in part from the biosynthesis of Ig by the artery, since the incorporation of 14C-labeled leucine into IgG by the atheromatous plaque was demonstrable by radioimmunoelectrophoresis. In contrast to the diseased artery, the normal artery did not synthesize IgG and did not contain vascular bound IgG or complement. However, the normal artery was capable of fixing IgG and B1C eluted from the diseased artery. The present studies suggested that the IgG contained and synthesized by the plaque might represent an immune response to an endogenous or exogenous antigen closely associated with plaque collagen. IgG and B1C either alone or in the form of an immune complex also may play an important role in phagocytosis in the plaque and thereby influence the course of atherosclerosis. The proteolytic inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin, found in relatively high concentrations in the plaque, could enhance fibrosis of the lesion because of thier known inhibitory effects on collagenase and elastase.     

Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy

Uteroplacental prostaglandins (PGs) play pivotal roles in maintenance and /or termination of pregnancy in mammals. Regulation of PG biosynthetic and signaling mechanisms in uteroplacental tissues during maintenance of pregnancy is largely unknown. In the present study, we have characterized the expression of PGE2 receptors (EP2, EP3, EP4), PGF2alpha receptor (FP), and cyclooxygenase (COX) types 1 and 2 in placentome caruncle (CAR), intercaruncle, and fetal membrane tissues during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. The levels of expression of EP2, EP3, and FP mRNAs differ depending on tissues and days of gestation (days <50 to >250). EP4 mRNA was undetectable in all the tissues studied. The expression levels of PG receptor mRNAs were as follows: placentome CAR FP>EP2>P3, intercaruncle EP2>EP3> or =FP, and fetal membranes EP3> or =EP2 >FP. EP2 and EP3 expressions were modulated in uteroplacental tissues, depending on days of pregnancy, whereas FP was uniformly expressed. COX-1 mRNA and protein were constitutively expressed, whereas COX-2 was highly modulated in uteroplacental tissues throughout pregnancy. Immunohistochemistry showed that EP2 and COX-2 proteins were colocalized in most cell types of placentome CAR, endometrium, and myometrium. Our study indicates that EP2 is the primary cAMP-generating PGE2 receptor expressed in uteroplacental tissues during bovine pregnancy. Temporal and tissue-specific expression of PGE2 and PGF2alpha receptors and COX-1 and -2 at the maternal-fetal interface suggests a selective and distinctive role for PGE2 and PGF2alpha in uterine activities during pregnancy in bovine.     

Antagonism by PGF2alpha of the vasodilation induced by PGE1, PGE2 and PGA1 in the canine uterus.

Prostaglandins appear to play an important role in a number of reproductive processes. The present study was designed to determine if the vasodilator action of prostaglandins of the A and E series on uterine vascular smooth muscle was mediated via a receptor and if PGF2alpha could compete for this same receptor. The data demonstrate that PGF2alpha, which by itself has no uterine vascular effect, can produce a parallel shift in the dose-response curves for certain vasodilator prostaglandins. This action, which suggests competitive antagonism, may well play a role in regulating blood flow in the nonpregnant uterus.     

The effects of labor on differential gene expression in parturient women, placentas, and fetuses at term pregnancy

Labor and its associated pain are thought to have unique impacts on parturient women. The goal of this study was to investigate the effects of labor and associated pain on differential gene expression profiles in the maternal, fetal, and placental compartments. We used microarrays to analyze maternal blood (MB), fetal cord blood (CB), and placental tissue samples in pregnant women after term vaginal deliveries (laboring group) and in term pregnant women after scheduled Ceasarean sections (nonlaboring group). The upregulated genes in the MB of the laboring group are involved in cytokine and nuclear factor-kappa B signaling pathways, regulation of the networks of toll-like receptor 4, and suppressor of cytokine signaling 3. Upregulated genes in the CB of the laboring group are involved in responding to stress and stimuli by regulating the network genes of the T-cell receptor beta locus and the FK506 binding protein 8. Differentially expressed genes in the placenta of the laboring group are involved in nitric oxide transport, gas transport, response to hydrostatic pressure, oxygen transport, acute phase responses, and the tumor necrosis factor-mediated signaling pathway, which are important during the transient hypoxemia and hypoperfusion that occur in the placenta during uterine contractions. Interestingly, few of the genes exhibited simultaneous changes in all three compartments, indicating that different pathways and complex interactions may be involved in human labor. In conclusion, human labor and its associated pain elicit unique gene regulatory changes in MB, placenta, and CB.     

Differential regulation of estrogen receptor alpha and beta mRNAs in the rat uterus during pregnancy and labor: possible involvement of estrogen receptors in oxytocin receptor regulation

Oxytocin receptor (OTR) mRNA levels in the uterus dramatically increase in the near term human and rat. Estrogen is believed to be a potent stimulator of OTR mRNA expression. However, estrogen does not stimulate rat OTR mRNA expression on day 18 of pregnancy or in progesterone-treated rats. Thus, the regulation of uterine responsiveness to estrogen in the near term rat appears to be an important mediator of estrogen action. To determine the effect of altering uterine responsiveness to estrogen on OTR induction, uterine ERalpha and ER beta mRNA levels were examined by competitive RT-PCR in pregnant and parturient rats, progesterone-treated ovariectomized (OVX) virgin rats and OVX pregnant rats. In pregnant and parturient rats, OTR mRNA levels were highest at 2200-2230 h on day 21 of pregnancy (P21pm) and during labor when compared with other groups. ERalpha mRNA levels significantly increased during labor compared with days 15-21 of pregnancy. Compared with control animals, ERalpha mRNA levels decreased significantly in OVX virgin rats implanted with tubes containing progesterone for one week; 24 h following the removal of the progesterone tubes, ERalpha mRNA levels were found to be similar to control levels. Estrogen treatment following OVX on day 18 of pregnancy caused increased OTR mRNA levels, whereas ovariectomy alone increased ERbeta mRNA but not ERalpha mRNA. Results from the present study suggest that ERalpha and ERbeta mRNA expressions are differentially regulated in the rat uterus. Moreover, during spontaneous labor our findings appear to suggest that ERalpha plays a more prominent role than ERbeta in mediating estrogen action in the induction of uterine OTR mRNA before labor.     

Study of the multiple endocrine neoplasia type 1, growth hormone-releasing hormone receptor, Gs alpha, and Gi2 alpha genes in isolated familial acromegaly

Familial acromegaly may occur as an isolated pituitary disorder or as a feature of hereditary syndromes, such as multiple endocrine neoplasia type 1 (MEN1) or the Carney complex. Herein, we characterized a newly identified kindred with isolated acromegaly and searched for germline mutation in genes that have been associated with endocrine tumors [i.e. MEN1, Gs alpha (GNAS1), and Gi2 alpha (GNAI2)], as well as the GHRH receptor (GHRH-R) gene. Genomic DNA was used to amplify exons 2-10 of MEN1, followed by dideoxy fingerprinting mutation analysis and direct sequencing. The GHRH-R gene was analyzed via direct sequencing of PCR-amplified fragments representing the coding exons and intron-exon junctions. To exclude mutation at hot spot areas of GNAS1 and GNAI2, exons 8 and 9 of GNAS1 and exons 5 and 6 of GNAI2 were amplified and screened for mutation via denaturing gradient gel electrophoresis. No mutations were detected in any of the four genes. The present data extend prior reports of the absence of mutation in MEN1, GHRH-R, and GNAS1 and describe the first family with isolated acromegaly in which germline mutation in GNAI2 has been searched.     

Regulation of insulin-like growth factor-binding proteins in the baboon (Papio anubis) uterus during early pregnancy.

The baboon uterus begins to synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) in the deep glands of the late secretory endometrium, and this protein then becomes the major secretory product of the term decidua. We hypothesized that the placenta and/or conceptus may regulate the synthesis and secretion of IGFBP-1 by decidualized stromal cells during pregnancy. To test this hypothesis, tissue was obtained from pregnant baboons on days 18, 25, and 32 postovulation. The uterus was separated into three regions: RI (directly below the implantation site), RII (adjacent to the implantation site), and RIII (opposite the implantation site). Portions of the tissue were fixed in Bouin's solution for immunocytochemistry, and the remainder was subdivided into functionalis, basalis, and myometrium and subjected to organ explant culture. The placenta was fixed or cultured separately. Ligand blot analysis of functionalis medium showed that the major IGFBP had a mol wt (Mr) of 29,000-31,000; however, a doublet of 37,000-43,000 Mr and a band at 24,000 Mr were also present. The functionalis from all regions expressed the majority of the IGFBPs, but basalis from RI tissue also secreted the same array of IGFBPs on days 25 and 32. Ligand blot analysis of placental medium proteins revealed a doublet at Mr 37,000-43,000 on days 25 and 32, but not on day 18. Immunoprecipitation followed by ligand blot analysis of medium proteins using polyclonal antibodies to IGFBP-1 and IGFBP-2 and -3 confirmed that IGFBP-1 and -2 were the predominant products of the endometrium and decidua, while IGFBP-3 was synthesized by the placenta. Immunocytochemistry with a monoclonal antibody to IGFBP-1 demonstrated intense glandular epithelial staining in all regions on days 18, 25, and 32. Stromal staining for IGFBP-1 was first evident on day 25 and was only present in stromal cells in intimate contact with the trophoblastic tissue. By day 32, IGFBP-1 expression was not limited to the endometrial-trophoblastic junction, but extended to the deeper stromal cells and included the perivascular regions. IGFBP-1 staining was most intense in RI, but stromal cells at the luminal surface and those surrounding the spiral arteries also showed some staining in RII and RIII on day 32. These studies suggest that the baboon placenta and/or conceptus regulate IGFBP expression in the uterine endometrium during the initial stages of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)