The effects of digestive enzymes on characteristics of placental insulin receptor. Comparison of particulate and soluble receptor preparations.
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The role of the surrounding membrane structure on the binding characteristics of the insulin receptor was studied by using several digestive enzymes. The effects observed with particulate membrane preparations are compared with those from soluble receptor preparations. beta-Galactosidase and neuraminidase had no effect on insulin binding to either particulate or soluble receptors from human placentae. Exposure to 2 units of phospholipase C/ml increased insulin binding to particulate membranes, but was without effect on the soluble receptor preparation. The increase in binding to particulate membranes was shown to be due to an increase in apparent receptor number. After 5 min exposure to 500 microgram of trypsin/ml there was an increase in insulin binding to the particulate membrane fraction, owing to an increase in receptor affinity. After 15 min exposure to this amount of trypsin, binding decreased, owing to a progressive decrease in receptor availability. In contrast, this concentration of trypsin had no effect on the solubilized receptor preparation. Because of the differential effects of phospholipase C and trypsin on the particulate compared with the solubilized receptor preparations, it is concluded that the effects of these enzymes were due to an effect on the surrounding membrane structure. Changes in receptor configuration due to alterations within the adjoining membrane provide a potential mechanism for mediating short-term alterations in receptor function. (+info)
Mrj encodes a DnaJ-related co-chaperone that is essential for murine placental development.
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We have identified a novel gene in a gene trap screen that encodes a protein related to the DnaJ co-chaperone in E. coli. The gene, named Mrj (mammalian relative of DnaJ) was expressed throughout development in both the embryo and placenta. Within the placenta, expression was particularly high in trophoblast giant cells but moderate levels were also observed in trophoblast cells of the chorion at embryonic day 8.5, and later in the labyrinth which arises from the attachment of the chorion to the allantois (a process called chorioallantoic fusion). Insertion of the ROSAbetageo gene trap vector into the Mrj gene created a null allele. Homozygous Mrj mutants died at mid-gestation due to a failure of chorioallantoic fusion at embryonic day 8.5, which precluded formation of the mature placenta. At embryonic day 8.5, the chorion in mutants was morphologically normal and expressed the cell adhesion molecule beta4 integrin that is known to be required for chorioallantoic fusion. However, expression of the chorionic trophoblast-specific transcription factor genes Err2 and Gcm1 was significantly reduced. The mutants showed no abnormal phenotypes in other trophoblast cell types or in the embryo proper. This study indicates a previously unsuspected role for chaperone proteins in placental development and represents the first genetic analysis of DnaJ-related protein function in higher eukaryotes. Based on a survey of EST databases representing different mouse tissues and embryonic stages, there are 40 or more DnaJ-related genes in mammals. In addition to Mrj, at least two of these genes are also expressed in the developing mouse placenta. The specificity of the developmental defect in Mrj mutants suggests that each of these genes may have unique tissue and cellular activities. (+info)
JunB is essential for mammalian placentation.
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Lack of JunB, an immediate early gene product and member of the AP-1 transcription factor family causes embryonic lethality between E8.5 and E10.0. Although mutant embryos are severely retarded in growth and development, cellular proliferation is apparently not impaired. Retardation and embryonic death are caused by the inability of JunB-deficient embryos to establish proper vascular interactions with the maternal circulation due to multiple defects in extra-embryonic tissues. The onset of the phenotypic defects correlates well with high expression of junB in wild-type extra-embryonic tissues. In trophoblasts, the lack of JunB causes a deregulation of proliferin, matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) gene expression, resulting in a defective neovascularization of the decidua. As a result of downregulation of the VEGF-receptor 1 (flt-1), blood vessels in the yolk sac mesoderm appeared dilated. Mutant embryos which escape these initial defects finally die from a non-vascularized placental labyrinth. Injection of junB-/- embryonic stem (ES) cells into tetraploid wild-type blastocysts resulted in a partial rescue, in which the ES cell-derived fetuses were no longer growth retarded and displayed a normal placental labyrinth. Therefore, JunB appears to be involved in multiple signaling pathways regulating genes involved in the establishment of a proper feto-maternal circulatory system. (+info)
Canine preprorelaxin: nucleic acid sequence and localization within the canine placenta.
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Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E-->D and L-->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta. (+info)
Production of prostaglandin f2alpha and its metabolite by endometrium and yolk sac placenta in late gestation in the tammar wallaby, Macropus Eugenii.
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In this study, we investigated production of prostaglandin (PG) F2alpha and its metabolite, PGFM, by uterine tissues from tammar wallabies in late pregnancy. Endometrial explants were prepared from gravid and nongravid uteri of tammars between Day 18 of gestation (primitive streak) and Day 26.5 (term) and were incubated in Ham's F-10 medium supplemented with glutamine and antibiotics for 20 h. PGF2alpha and PGFM in the medium were assayed by specific, validated RIAs. Control tissues (leg muscle) did not produce detectable amounts of either PG. Both gravid and nongravid endometria secreted PGF2alpha, and production increased significantly in both gravid and nongravid uteri towards term. PGFM was produced in small amounts by both gravid and nongravid uteri, and the rate of production did not increase. Neither oxytocin nor dexamethasone stimulated PG production in vitro in any tissue at any stage. Thus, the surge in peripheral plasma PGFM levels seen at parturition may arise from increased uterine PG production, but further study is needed to define what triggers this release. (+info)
Expression of trophinin, tastin, and bystin by trophoblast and endometrial cells in human placenta.
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Trophinin, tastin, and bystin comprise a complex mediating a unique homophilic cell adhesion between trophoblast and endometrial epithelial cells at their respective apical cell surfaces. In this study, we prepared mouse monoclonal antibodies specific to each of these molecules. The expression of these molecules in the human placenta was examined immunohistochemically using the antibodies. In placenta from the 6th week of pregnancy, trophinin and bystin were found in the cytoplasm of the syncytiotrophoblast in the chorionic villi, and in endometrial decidual cells at the utero placental interface. Tastin was exclusively present on the apical side of the syncytiotrophoblast. Tissue sections were also examined by in situ hybridization using RNA probes specific to each of these molecules. This analysis showed that trophoblast and endometrial epithelial cells at the utero placental interface express trophinin, tastin, and bystin. In wk 10 placenta, trophinin and bystin were found in the intravillous cytotrophoblast, while tastin was not found in the villi. After wk 10, levels of all three proteins decreased and then disappeared from placental villi. (+info)
Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network.
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SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells. (+info)
The CTLA-4 gene is expressed in placental fibroblasts.
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In order to elucidate the mechanisms that ensure survival of the allogeneic fetus, we are investigating the expression pattern of genes that are involved in peripheral self-tolerance in tissues at the maternal-fetal interface. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T cell activation and may modulate peripheral self-tolerance. Previously, we reported the preferential transmission of maternally-inherited shorter alleles at a 3'-UTR microsatellite locus to liveborn children, but random transmission of paternally-inherited alleles, suggesting that CTLA-4 may be involved in the maintenance of tolerance at the maternal-fetal interface. In this report, we demonstrate that CTLA-4 mRNA and protein are indeed expressed in fetal tissues at the maternal-fetal interface throughout gestation. (+info)