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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human placentae obtained early in pregnancy or at full term were examined for RNA content per cell, RNA polymerase types and activities, and chromatin template availability. The RNA:DNA ratio fell from 0.7 at 15 to 20 weeks to 0.4 at 40 weeks of pregnancy. Since RNase activities were similar at both times, the reduction in RNA content was attributed not to increased degradation, but to reduced synthesis. At both stages of pregnancy, about 55 to 60 per cent of the RNA polymerase activity in isolated placental nuclei was accounted for by RNA polymerase II, as judged by suppression of activity with alpha-amanitin and by separation of the extracted polymerases on DEAE-Sephadex. The relative roles of changes in polymerase activity and template availability were measured in nuclei from 20- and 40-week placentae. Nuclei showed 20 per cent greater polymerase activity in full-term than in early placentae, but the template availability of isolated chromatin for transcription by RNA polymerase II was 70 per cent less at full term. We conclude that the reduced amount of RNA per cell in the full-term placenta is due to reduced template availability that more than offsets the slight increase in polymerase activity.
Placenta
PMID:Control of RNA content of developing human placenta. 616 May 73

A simple method for the purification of human placental nuclei is described. Nuclei were isolated by homogenizing tissue in standard saline citrate solution in the presence of zinc chloride to stabilize the nuclear membranes, NP40 as non-ionic detergent and sodium bisulphite for inhibition of proteolytic activity. Nuclei purification was achieved by low-speed centrifugation through a discontinuous sucrose gradient. The purified nuclei were evaluated by morphological criteria using phase contrast and electron microscopy. The extent of contamination by cytoplasmic debris was estimated by Papanicolaou's staining technique. Biochemical criteria include measurements of alkaline phosphatase activity as a plasma membrane enzyme marker and DNA-dependent RNA polymerase activity for the functional integrity of nuclear components. Transcriptionally active nuclei were obtained but the yield of nuclei was low; however, this low yield is compensated by the high degree of purity, the simplicity of the method and the functional and morphological integrity of the purified nuclei.
Placenta
PMID:Low-speed purification of human placental nuclei. 652 84

Many previous studies in both mouse and human placenta have implicated a role for colony stimulating factor-1 (CSF-1) in the regulation of placental development. In this study we have examined CSF-1 production by an immortalized cell line (TCL-1) derived from the choriodecidua, transfected with a retrovirus gene coding for the large-T antigen. TCL-1 cells were uniformly positive by immunocytochemistry for the composite sub-units of human chorionic g gonadotrophin (hCG) but were negative for markers of other cell types localized at the fetal-maternal interface. Gelatinase enzymes were secreted by TCL-1 cells cultured on extracellular matrix in a manner indicative of extra-villous trophoblast. Dot-blot immunoassays and ELISA indicated that CSF-1 was secreted by TCL-1 cells, at levels comparable to primary trophoblast cells and BeWo choriocarcinoma (trophoblast tumour) cells. Reverse transcriptase-polymerase chain reaction analysis confirmed the presence in TCL-1 cells of CSF-1 receptor mRNA (c-fms gene product), indicating that the components of a potential autocrine loop were present in these cells. Proliferation of TCL-1 cells was not affected by the addition of exogenous CSF-1 but was elevated in response to treatment with a CSF-1 neutralizing antibody. The immortalized cell line, TCL-1, provides a potential model in which to investigate regulation of growth and differentiation of trophoblast cells in vitro.
Placenta
PMID:Partial characterization of an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1 autocrine loop. 917 33

In order to characterize the expression of adrenomedullin during pregnancy, we measured the mature and total concentrations in maternal plasma and amniotic fluid, and examined its expression in fetoplacental tissues. Plasma samples were obtained from 13 normal normotensive non-pregnant women and 14 normal normotensive post partum women. Maternal plasma and amniotic fluid samples were obtained from 37 normal pregnant women (10 in the first trimester, 13 in the second trimester and 14 in the third trimester). Fetoplacental tissues were obtained from first and third-trimester pregnancies. Mature and total adrenomedullin concentrations in plasma and amniotic fluid were determined by using specific radioimmunoassay. The distribution and expression of adrenomedullin were determined using immunohistochemistry, reverse-transcriptase polymerase chain reaction, and in situ hybridization. Plasma total adrenomedullin concentrations were increasing with advancing gestation. The mature/total adrenomedullin ratio in the second trimester was the highest during pregnancy. Mature and total adrenomedullin concentrations in the amniotic fluid were significantly higher than those in the maternal plasma throughout gestation (P< 0.05). Mature adrenomedullin concentrations and the mature/total adrenomedullin ratio in the amniotic fluid increased with advancing gestation. There was a significant linear correlation between amniotic fluid and maternal plasma mature/total adrenomedullin ratio in the first or second trimester of pregnancy. Adrenomedullin mRNA was identified in the amniotic membrane and chorionic villi, and within the endothelial layers of villous blood vessels. These results suggest that the mature/total adrenomedullin ratio is modified in maternal plasma and amniotic fluid with advancing gestation.
Placenta
PMID:Change of adrenomedullin concentrations in plasma and amniotic fluid, and human placental adrenomedullin expression with advancing gestation. 1117 Aug 30

The apoptosis cascade that plays a central role in normal and pathological processes is strictly controlled, in part by FLIP (Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein), an inhibitor of caspase-8. Here, we report the expression of long and short isoforms of FLIP mRNAs and proteins in early and late gestation human placentas, term cytotrophoblast cells and two choriocarcinoma cell lines, JEG-3 and Jar. Reverse transcriptase polymerase chain reaction identified mRNAs derived from the FLIP gene in all samples. Analysis by immunoblotting revealed that both long and short forms of FLIP proteins are present in early and late gestation human placentas with increasing levels over gestation and that FLIP proteins are present in normal and transformed trophoblast cells. Immunohistochemical experiments performed on paraformaldehyde-fixed tissue sections taken from early and late stages of pregnancy demonstrated that FLIP proteins are present in caspase-8-expressing cells and that expression patterns of FLIP differed according to cell lineage and stage of cell differentiation. The results of this study are consistent with the postulate that FLIP proteins have critical roles in placental cell survival and suggest that FLIP may protect normal and transformed trophoblast cells from cell death.
Placenta
PMID:FLICE-inhibitory protein: expression in early and late gestation human placentas. 1617 30

Angiogenesis is fundamental to normal placental development. Aberrant angiogenesis within the placental terminal villi is a characteristic of significant placental pathologies and includes structural and vascular abnormalities as well as altered endothelial cell function, which substantially impacts on maternal-fetal exchange. Homeobox gene transcription factors regulate vascular development in embryonic and adult tissues, but their role in the placental microvasculature is not well known. In this study, we isolated and enriched human placental microvascular endothelial cells (PLEC) by a perfusion-based method and compared homeobox gene expression between PLEC and macrovascular human umbilical vein endothelial cells (HUVEC). Reverse transcriptase PCR detected mRNA expression of homeobox genes DLX3, DLX4, MSX2, GAX and HLX1 in both PLEC and HUVEC. DLX4 and HLX1 have not been previously detected in PLEC and with the exception of GAX, none of these homeobox genes have been previously identified in HUVEC. There was lower expression of HLX1 mRNA in HUVEC compared with PLEC. Using real-time PCR analysis PLEC HLX1 mRNA expression relative to housekeeping gene GAPDH was 0.9+/-0.06 fold of the calibrator (n=6) versus 0.2+/-0.06 (n=6) for HUVEC, p<0.001. These data provided evidence of heterogeneity in homeobox gene expression between microvascular PLEC and macrovascular HUVEC that most likely reflects significant differences in endothelial cell function in the two different cellular environments.
Placenta
PMID:Homeobox genes are differentially expressed in macrovascular human umbilical vein endothelial cells and microvascular placental endothelial cells. 1664 16

Angiogenesis is fundamental to normal placental development and aberrant angiogenesis contributes substantially to placental pathologies. The complex process of angiogenesis is regulated by transcription factors leading to the formation of endothelial cells that line the microvasculature. Homeobox genes are important transcription factors that regulate vascular development in embryonic and adult tissues. We have recently shown that placental homeobox genes HLX, DLX3, DLX4, MSX2 and GAX are expressed in placental endothelial cells. Hence, the novel homeobox genes TLX1, TLX2, TGIF, HEX, PHOX1, MEIS2, HOXB7, and LIM6 were detected that have not been reported in endothelial cells previously. Importantly, these homeobox genes have not been previously reported in placental endothelial cells and, with the exception of HEX, PHOX1 and HOXB7, have not been described in any other endothelial cell type. Reverse transcriptase PCR was performed on cDNA from freshly isolated placental microvascular endothelial cells (PLEC), and the human placental microvascular endothelial cell line HPEC. cDNAs prepared from control term placentae, human microvascular endothelial cells (HMVEC) and human umbilical vein macrovascular endothelial cells (HUVEC) were used as controls. PCR analyses showed that all novel homeobox genes tested were expressed by all endothelial cells types. Furthermore, real-time PCR analyses revealed that homeobox genes TLX1, TLX2 and PHOX1 relative mRNA expression levels were significantly decreased in HUVEC compared with microvascular endothelial cells, while the relative mRNA expression levels of MEIS2 and TGIF were significantly increased in macrovascular cells compared with microvascular endothelial cells. Thus we have identified novel homeobox genes in microvascular endothelial cells and have shown that homeobox genes are differentially expressed between micro- and macrovascular endothelial cells.
Placenta 2008 Jul
PMID:Novel homeobox genes are differentially expressed in placental microvascular endothelial cells compared with macrovascular cells. 1851 8

The aim was to test for evidence of transcriptional activity within the nuclei of the syncytiotrophoblast of the human placenta. The syncytiotrophoblast forms the epithelial covering of the villous tree, and is a multinucleated, terminally-differentiated syncytium generated through fusion of the underlying progenitor cytotrophoblast cells. Its nuclei are heterogeneous with respect to chromatin condensation, and previous functional studies of 3H-uridine uptake in vitro have indicated that they are transcriptionally inactive. This observation is surprising given the key roles this tissue plays in active transport, hormone synthesis and metabolic regulation, and has widespread implications for trophoblast physiology and pathophysiology. We used three different approaches to look for evidence of transcriptional activity. First, immunofluorescence staining was performed on paraffin-embedded early pregnancy and term placental villi, using an antibody directed specifically against the actively transcribing form of RNA polymerase II. Second, a nucleoside incorporation assay was applied to placental villi maintained in short-term culture, with and without the transcription blocker alpha-amanitin. Third, histone modifications associated with active chromatin were identified by immunohistochemistry and immunofluorescence. Each of these methods showed transcription to be occurring in a proportion of syncytiotrophoblast nuclei, with qualitative evidence for transcription being more abundant in the first trimester than at term. These findings correlated with electron microscopical observations of prominent nucleoli within the nuclei, particularly during early pregnancy, signifying transcription of ribosomal RNA. Contrary to previous findings, these results confirm that a proportion of syncytiotrophoblast nuclei actively produce mRNA transcripts.
Placenta 2009 Apr
PMID:Evidence for transcriptional activity in the syncytiotrophoblast of the human placenta. 1921 81

Regulatory T cells (Tregs) support pregnancy maintenance by suppressing placental inflammation, while diminished Treg function may accompany reproductive failure. Experimental FIV infection frequently results in vertical transmission and increased pregnancy failure in the cat. The mechanism of reproductive compromise is unknown. We hypothesized that FIV infection alters endometrial Treg population dynamics and function, potentiating vertical transmission and reproductive failure. RNA collected from early and late gestation reproductive tissue and fetuses from FIV infected and control cats was probed for expression of FIV gag and Treg markers CD25, FOXP3, and CTLA4, using real time reverse-transcriptase (RT)-PCR. Frequent placental and fetal infection and reproductive failure were detected at early and late pregnancy. Expression of FOXP3 and CTLA4 was higher in early gestation tissues from control cats. FIV infection significantly reduced expression of FOXP3 and CTLA4 at early, but not late pregnancy. At late pregnancy, CTLA4 was expressed to higher levels in infected tissues. The number of tissues with decreased co-expression of FOXP3 and CTLA4 was significant in infected cats at early pregnancy. No significant changes in CD25 expression occurred between FIV-infected and control animals at early or late pregnancy. Differences in Treg marker expression were not significant between viable and non-viable pregnancies in infected cats. The detection of Treg markers in these feline tissues provides the first evidence of feline endometrial Tregs and suggests that such cells diminish as pregnancy progresses. These cells may be depleted or rendered less functional by viral infection, but understanding their role in pregnancy requires further study.
Placenta 2010 Sep
PMID:Expression of regulatory T cell (Treg) activation markers in endometrial tissues from early and late pregnancy in the feline immunodeficiency virus (FIV)-infected cat. 2067 72

The placenta is an essential organ for embryo development in the uterus of eutherian mammals. Large contributions in unveiling molecular mechanisms and physiological functions underlying placental formation were made by analyzing mutant and transgenic animals. However, it had been difficult to elucidate whether the placental defects observed in such animals originate from the placenta itself or from the fetus, as both placental and fetal genomes are modified. Therefore strategies to modify the placental genome without affecting the "fetal genome" had been needed. Through the ingenious use of lentiviral (LV) vectors, placenta-specific modification is now possible. Lentivirus is a genus of retroviruses that use reverse-transcriptase to convert its single-strand RNA genome to double-strand DNA and integrate into the host genome. Previous studies showed that when LV vectors were used to transduce embryos at the 2-cell stage, the viral genome is systemically introduced into host genome. Interestingly, by delaying the timing of transduction to the blastocyst stage, the transgene is expressed specifically in the placenta as a consequence of trophectoderm-specific viral transduction. This review summarizes the development of the LV vector-mediated placenta-specific gene manipulation technology and its application in placental research over the past decade. A perspective for future application of LV vectors to further placenta research, especially in combination with next generation genome editing technologies, is also presented.
Placenta 2017 Nov
PMID:Placenta-specific gene manipulation using lentiviral vector and its application. 2898 26


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