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Isolation and characterization of two different molecular forms of basic fibroblast growth factor extracted from human placental tissue.

Basic fibroblast growth factor (bFGF) was purified to homogeneity from human placental tissue on a semi-large scale. Placental bFGF consists of two proteins of apparent molecular masses 16,000 and 18,000 dalton, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions. Microsequence analysis showed that both proteins have the same N-terminal sequence Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe..., which is identical with that of (1-146) bFGF extracted from human brain. After reduction by dithiothreitol or mercaptoethanol, placental bFGF appears as a single protein of 16,000 dalton. The reduced protein displays the same ability to stimulate the proliferation of CCL39 fibroblasts as the non-reduced doublet. These data indicate that bFGF extracted from placental tissue consists of two proteins with different apparent molecular masses which do not differ in their N-terminal sequence but in their oxidation state.

Amino Acid Sequence↗

[Human placental tissue: a source of natural hematopoietic regulators].

In this paper, it has been shown that human placental tissular extracts are a potent source of natural haemopoietic growth factors. The colony-stimulating activities (CSA) recovered by extraction from washed placental pulp were active both on human and murine haemopoietic progenitors, from monocytic and granulocytic lineages. Crude tissular extracts contained CSA titers at least ten fold the titers usually found in placenta culture media. Placenta is the only human tissue easily available for the study of natural tissue-bound haemopoietic regulators. Extraction on an industrial scale, as proposed for the first time in this paper, should also benefit the identification and purification of new minor molecular classes of growth and maturation factors or inhibitors involved in human haemopoiesis.

Animals↗

Characterization of parathyroid hormone-related protein in the human term placenta.

To characterize parathyroid hormone-related protein (PTHrP) in the human placenta, we measured PTHrP-like immunoreactivity (PRP-LI) in the term placenta and studied the elution profiles of placental tissue extracts on Sephadex G-75 chromatography with a specific RIA. We also examined the gene expression of PTHrP mRNA by Northern blot analysis and the localization of PRP-LI in the placenta by immunohistochemistry. The amount of PRP-LI in placental extracts (n = 7) was 20.9 +/- 2.2 pg/g wet tissue (mean +/- SE). Dilution curves of placental tissue ran parallel to those of synthetic PTHrP (1-34) standards. Sephadex G-75 gel chromatography demonstrated two major PRP-LI peaks; the first peak was eluted around the molecular size between 10 kilodaltons (Kda) and 20 Kda and the other around 5 Kda. Northern blot analysis of PTHrP mRNA extracted from placental tissues showed a major hybridization signal around 18S. PTHrP immunohistochemistry showed PRP-LI staining in the cytoplasm of syncytiotrophoblasts and stroma cells (Hofbauer cells) in the term placenta. These results suggest that syncytiotrophoblasts and stroma cells in the term placenta synthesize PTHrP in two major molecular forms, 10 Kda-20 Kda and around 5 Kda.

Adult↗

Minoxidil: inhibition of proliferation of keratinocytes in vitro.

Previous studies have suggested that minoxidil stimulates growth of keratinocytes, possibly in a manner similar to the action of epidermal growth factor. Using both a short-term assay, thymidine incorporation, and a longer term assay, cell counting, to assess proliferative growth, we tested the activity of minoxidil in human keratinocyte cultures grown in 0.1 mM Ca(++). Minoxidil failed to stimulate growth in these assays. At concentrations of 5-10 micrograms per ml, minoxidil showed half-maximal inhibition of both EGF- and placental extract-stimulated thymidine incorporation. Minoxidil also inhibited proliferative growth in the presence or absence of placental extract. Direct measurement of the ability of minoxidil to compete for binding to the EGF receptor indicated that minoxidil probably does not bind to the EGF receptor. Minoxidil was not toxic, as keratinocytes continued to survive and grow, although at a slower rate, in the presence of minoxidil.

Cell Division↗

Distribution of cells bearing the HNK-1 epitope in the human placenta.

HNK-1 (Leu-7 antigen or CD57) is a unique carbohydrate moiety found in certain glycosphingolipids and several cell adhesion glycoproteins on the cell membrane. Previous studies have suggested that HNK-1 carbohydrates act as adhesive ligands in cell-cell interactions. Using a monoclonal antibody reactive to the HNK-1 moiety and an immunoperoxidase method on formalin-fixed paraffin-embedded tissue, the expression of the HNK-1 epitope in human placentae was confined to the intermediate trophoblast (IT) in trophoblastic columns. The number of HNK-1 immunoreactive IT cells increased from the proximal to the midportion of the trophoblastic column, and then disappeared at the junction of the column with the basal plate where IT infiltrates the endomyometrium and becomes extravillous IT. Neither cytotrophoblast nor syncytiotrophoblast reacted with the HNK-1 antibody. In hydatidiform moles, HNK-1 immunoreactivity was localized to areas that structurally resembled trophoblastic columns. In contrast, placental site trophoblastic tumours which do not contain structures analogous to trophoblastic columns did not express HNK-1 epitope. Expression of HNK-1 was only rarely observed in choriocarcinomas, being present in less than 5 per cent of trophoblastic cells in two of 13 cases. The murine placenta, which lacks trophoblastic columns, was negative for HNK-1. Thin-layer chromatography immunostaining demonstrated the HNK-1 reactive glycosphingolipids in placental lipid extracts, whereas Western blot analysis from placental protein extract failed to reveal detectable glycoproteins that demonstrated HNK-1 immunoreactivity. In conclusion, the specific localization of HNK-1 reactive glycosphingolipids in trophoblastic columns of the human placenta suggests that the HNK-1 moiety may play an important role in maintaining cohesion between intermediate trophoblastic cells in the trophoblastic columns thereby contributing to the structural integrity of the villi that anchor the placenta to the basal plate.

Animals↗

Determination of 2',3'-dideoxycytidine in maternal plasma, amniotic fluid, placental and fetal tissues by high-performance liquid chromatography.

2',3'-Dideoxycytidine (DDC) is a nucleoside reverse transcriptase inhibitor that has been shown to inhibit the human immunodeficiency virus (HIV). DDC is a candidate for treatment of pregnant women to prevent prenatal transmission of HIV/AIDS to their unborn children. A quick and simple high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DDC concentrations in samples collected from a pregnant rat model (maternal plasma, amniotic fluid, placental and fetal tissues). Extraction of DDC and its internal standard 2',3'-dideoxy-3'-thiacytidine (3TC) in plasma and amniotic fluid was carried out by protein precipitation. Extraction from placental and fetal homogenates was achieved by solid phase extraction using Waters Oasis HLB solid phase extraction cartridges. Chromatographic separation was achieved on a Waters Spherisorb S3W silica column (4.6 mm x 100 mm) equipped with a Phenomenex guard column. The mobile phase used was 10% methanol in water with 22 mM formic acid. The flow rate was 0.5 ml/min, and the detection wavelength was optimized at 275 nm. Under these chromatographic conditions, DDC eluted around 12 min, and 3TC eluted around 10 min. The calibration curves for each day of validation and analysis showed good linear response through the range of 0.15-75.0 microg/ml in each of the four matrices. The relative recovery for DDC in each of the matrices ranged from 87.8% to 103.0%. Acceptable intra- and inter-day assay precision (<15% R.S.D.) and accuracy (<15% error) were observed over 0.15-75.0 microg/ml for all four matrices.

Amniotic Fluid↗

Analysis of FA contents in individual lipid fractions from human placental tissue.

Data on FA contents in the human placenta are limited. Different methods have been used for the FA analysis, and only percentage results have been presented. We developed and evaluated a method for the determination of FA concentrations in placental tissue. Lipids were extracted from placental tissue with a chloroform/methanol mixture; and phospholipids (PL), nonesterified FA (NEFA), TG, and cholesterol esters (CE) were isolated by TLC. Individual lipid fractions were derivatized with methanolic hydrochloric acid, and the FAME were quantified by GC with FID. The CV of intra-assay (n = 8) of absolute concentrations were evaluated for FA showing a tissue content > 0.01 mg/g. CV ranges were 4.6-11.0% for PL, 6.4-9.3% for NEFA, 6.1-8.9% for TG, and 11.4-16.3% for CE. The relative FA composition across a term placenta indicated no differences between samples of central and peripheral locations of maternal and fetal site (CV 0.5-9.9%), whereas the absolute FA concentrations were only reproducible in the PL fraction (CV 7.0-12.8%). The method shows a reasonably high precision that is well suited for physiological and nutritional studies.

Butylated Hydroxytoluene↗

Identification of a placental enhancer for the human leptin gene.

Leptin is a hormone that regulates metabolic efficiency, energy expenditure, and food intake. Leptin is produced chiefly in adipose cells, but in humans, mRNA encoding leptin is also present in the placenta. Here we elucidate the basis for placental leptin production. The same promoter is used for adipose and placental transcription. An upstream enhancer functions in the JEG-3 and JAR choriocarcinoma cell lines but not in adipocytes or HeLa cells. The minimal positive acting region is 60 base pairs in length. This region is within a MER11 repetitive element, suggesting that human placental expression of leptin is the result of insertion of this element. Binding analyses demonstrated three protein binding sites, designated placental leptin enhancer elements (PLE)1, PLE2, and PLE3. PLE2 binds Sp1. Enhancer activity was reduced by mutation of the PLE1 or PLE3 sites but was unaffected by alteration of PLE2. Proteins binding to PLE3 were present in JEG-3 and human placental nuclear extracts but not in extracts from non-placental sources. Upon triplication, the PLE3 element was a strong enhancer in choriocarcinoma cells but not in HeLa cells. The protein binding to the PLE3 motif appears to be a novel, placenta-specific transcription factor.

Base Sequence↗

Gastrin-releasing peptide-like immunoreactivity is present in human maternal and fetal placental membranes.

Extracts of human term amnion, placenta, and chorion/decidual tissue (n = 5) contained gastrin-releasing peptide-like immunoreactivity (GRPLI) in amounts of 4.7 +/- 2.9 (pmol/g wet wt; mean +/- SEM), 3.6 +/- 1.1 and 2.9 +/- 1.5, respectively. Using C-terminally directed antisera and gel filtration chromatography and reverse-phase high-performance liquid chromatography (HPLC), each tissue contained molecular forms consistent with the presence of GRP1-27 and GRP18-27 but also contained larger amounts of two GRPLI peaks, which apparently are novel GRP-like peptides. In contrast, tissue extracts of human fetal lung contained only GRP1-27, GRP14-27, and GRP18-27. Using RT-PCR and specific GRP primers and probes, messenger RNA encoding for GRP was readily demonstrable from 6-weeks gestation throughout pregnancy to term in full-thickness membranes, placental villi, and decidua. Positive immunohistochemical staining for GRP occurred in extravillous trophoblasts in decidual septa and fetal membranes, cytotrophoblasts, syncytiotrophoblast, and certain stromal cells in placental villi and amniotic epithelium. GRPLI and GRP messenger RNA were present from the earliest dates examined (6-9 weeks) throughout pregnancy to term. Given the proven trophic nature of GRP and related peptides, these peptides may play important roles in maternal, placental, and fetal development during human pregnancy.

Amnion↗

Peroxidase activity in the mouse uterus, placenta and fetus during pregnancy.

Changes in peroxidase activity during pregnancy were examined in CD-1 mice. Peroxidase activity was measured with guaiacol as the substrate in uterine extracts of nonpregnant mice and in uterine, placental, and fetal extracts of pregnant mice on days 9, 12, 14, 16, and 18 of gestation. Uterine peroxidase activity in nonpregnant mice was high, but declined logarithmically to only 0.2% by day 18 of pregnancy. In contrast to this decline, a concomitant 50-fold logarithmic increase in fetal peroxidase activity was observed between day 12 and 18. Activity in placental extracts did not change significantly throughout the gestational period examined. These results suggest that membrane bound peroxidase in mouse uterus and fetus undergoes major shifts during pregnancy.

Animals↗

[Measuring the concentration of various plasma and placenta extract proteolytic and vascular factors in pregnant patients with HELLP syndrome, pre-/eclampsia and highly pathologic Doppler flow values].

OBJECTIVE: Impaired trophoblast invasion plays a major role in the development of preeclampsia. Therefore various factors that are involved in invasion were investigated in gestational disease. METHODS: In pregnant women with HELLP-syndrome (n = 18), pre-/eclampsia (n = 21) and highly pathological Doppler flow measurements (hpD) (n = 13), plasma and placental tissue extract concentrations of uPA, uPA-receptor, tPA, PAI-1, MMP-8, MMP-9, TIMP-1, thrombomodulin, and angiogenin were measured using ELISA. RESULTS: In all three collectives, PAI-1 plasma concentrations were significantly higher (p < 0,05) than in normal pregnancies, in patients with HELLP-syndrome, tPA and TIMP-1 plasma levels were also elevated. MMP-9 concentrations in placental tissue extracts were lower in pre-/eclampsia than in normal pregnancies. CONCLUSIONS: Impaired placental implantation and remodelling in gestational disease is reflected by changes in plasma and placental tissue extract concentrations of various factors that are involved in these processes.

Adult↗

The effect of active immunity against placental proteins on pregnancy in monkeys.

Squirrel monkeys were actively immunized against human placental lactogen (HPL) and/or an extract of human placental tissue, or squirrel monkey placenta. Only half of the monkeys immunized with HPL developed a detectable antibody titer to HPL prior to the subsequent mating season. During the subsequent breeding season, the pregnancy rate was cut in half. The titer developed against HPL did not seem to be related to their ability to become pregnant. When the group of animals which had previously been immunized with HPL were challenged with extracts of human placenta, all animals developed detectable circulating antibody. There was no increase in the effectiveness of this immunization as compared to HPL in pregnancy prevention, however. A third group of monkeys were immunized with extracts from their own placental extracts, and all developed some degree of antibody. The titer obtained did not seem to be related to whether or not they became pregnant. In this group, however, the pregnancy rate was decreased to one fourth of what it had been before immunization.

Animals↗