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PubMed1981

Translation of vesicular stomatitis and Sindbis virus mRNAs in cell-free extracts of Aedes albopictus cells.

We have developed a cell-free system from Aedes albopictus (mosquito) cells which is able to carry out endogenous protein synthesis and is stable to freezing and thawing. Successful preparation of extracts was found to depend on the presence of purified placental RNase inhibitor during cell breakage. Micrococcal nuclease-treated extracts translated exogenously added Sindbis 26S or vesicular stomatitis virus mRNA with a high degree of fidelity, demonstrating that initiation of protein synthesis had occurred. Evidence is presented showing that when cell fractions containing intracellular membranes were used to translate vesicular stomatitis virus mRNA, the G protein was glycosylated and inserted into microsomal vesicles. Additional studies indicate that initiation of protein synthesis in this system is dependent on a capped and methylated 5'-terminal structure in the mRNA.

Aedes↗

PubMedDate not supplied

A high molecular weight species of placental alkaline phosphatase in human syncytiotrophoblast microvilli.

A high molecular weight form of human placental alkaline phosphatase has been detected in extracts of placenta at term by electrophoresis in starch gels containing 0.5 per cent Triton X-100. The enzyme has a mobility intermediate between the previously described A and B forms of the enzyme and has been called the 'M' form of placental alkaline phosphatase. The M form is the major form of the enzyme found in microvilli extracted from syncytiotrophoblast, though trace amounts of membrane-associated M form can be found in extracts of placentae which had previously been experimentally depleted of microvilli. The M form is present in both of the two recently described subfractions of placental microvilli (see Davies, Parry and Sutcliffe, 1981; Truman, Wakefield and Ford, 1981). A variety of experiments show that the M form is not an artefact of extraction. The characteristic mobility of the M form in starch/Triton gels is the same, whether the microvilli are extracted in butanol, chloroform/methanol, Nonidet P40, Triton X-100 or Na deoxycholate. Serological, heat-stability and genetic studies showed that the A and M forms contain the same enzymatic polypeptide. Gel filtration of butanol/H2O and butanol/saline extracts of microvilli provided an estimated molecular weight of the A form of 127000 and of the M form of 725000; these values were unaffected by the presence of Triton in the medium.

Alkaline Phosphatase↗

PubMed1986

Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives.

Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.

Antibodies, Monoclonal↗

PubMed1983

Comparative measurement of the lactogenic activity of ovine placental lactogen in rabbit and ewe mammary gland.

Ovine placental lactogen is known to bind to prolactin receptors and to initiate milk synthesis in the rabbit mammary gland. However, this hormone exhibited a very low capacity of competing with 125I-labeled human growth hormone for the binding to membranes extracted from ewe mammary gland. Ovine placental lactogen was very efficient in provoking the accumulation of beta-casein mRNA in rabbit mammary explants but was much less active on ewe mammary explants. These data indicate that the placental hormone is not a potent lactogen in the homologous species and that its role in the control of mammary gland development and activity may have been previously overestimated.

Animals↗

PubMed1986

Tissue origin of serum placental-like alkaline phosphatase in cigarette smokers.

A variety of solubilised tissue extracts from cigarette smokers and non-smokers have been screened quantitatively for both placental alkaline phosphatase (PLAP) and placental-like alkaline phosphatase (PLAP-like AP) in order to identify the possible tissue origins of the circulating PLAP-like AP found in most smokers. Lung alveolar tissue, and to a lesser extent colonic tissues, contained both PLAP-like AP and PLAP. Tissues from smokers and non-smokers contained comparable proportions and amounts of PLAP and PLAP-like AP. No other tissue source of PLAP-like AP was found other than those previously reported for testicular, endometrial and thymic tissue. Selective release of PLAP-like AP from the lung in cigarette smokers seems likely to be a major source for this isoenzyme in peripheral circulation.

Alkaline Phosphatase↗

PubMed1982

A cell spreading factor, abundant in human placenta, contains fibronectin and fibrinogen.

A salt extract from human normal-term placental extracellular matrix contains material that rapidly adsorbs to glass or tissue-culture plastic and promotes the spreading of fibroblastic and epithelioid cell lines. The cellular response to placental spreading factor (PSF) is characterized and compared with the response to fibronectin. The active component is shown to contain antigenic determinants that cross-react with antibodies to fibronectin and fibrinogen. The activity is not retained on gelatin-Sepharose but another component of the extract (approximately 185 000 molecular weight) is retained. Partial purification is reported, and the possible involvement of PSF in trophoblastic invasion of the uterine wall is discussed.

Cell Adhesion↗

PubMed2001

Cytokine patterns in maternal blood after premature rupture of membranes.

OBJECTIVE: To compare two types of cytokines, type 1, which activate cell-mediated reactions and are important in cytotoxic and delayed-type hypersensitivity reactions, and type 2, which encourage vigorous antibody production and are commonly found in association with humoral immune responses, in blood of women with premature rupture of membranes (PROM). METHODS: Forty-four women with histories of at least three successful pregnancies and who currently delivered normally served as controls. The PROM group consisted of 30 women with spontaneous rupture of fetal membranes at term. Peripheral blood mononuclear cells were stimulated separately with a mitogen, placental cells, and a trophoblast antigen extract, and the supernatants examined for type 1 and type 2 cytokines. RESULTS: Mitogen-stimulated blood cells produced significantly higher levels of type 1 cytokines in PROM women than in normal controls. Higher levels of the type 1 cytokine interferon-gamma were produced by PROM samples stimulated with autologous placental cells and with trophoblast antigens. Ratios of type 1 to type 2 cytokines were higher in PROM compared with normal pregnancy, and in some cases as much as 25-fold higher. CONCLUSION: Women in the PROM group had a stronger type 1 reactivity whereas normal women were more predisposed to type 2 immunity; thus, PROM appears to be associated with a maternal type 1 bias.

Adult↗

PubMed1976

Properties of biologically active messenger RNA from human placenta. Cell-free synthesis of two immunoreactive forms of placental lactogen.

In order to understand better the regulation of human placental proteins the activity of placental lactogen messenger RNA has been examined. Total RNA was extracted from normal term placentas and purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in wheat germ cell-free extracts, and immunoprecipitation of the translation products with antiserum directed against human placental lactogen (hPL) suggests that about 2% of the peptides contain hPL determinants. Analysis of the material precipitated with hPL antiserum by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed two major species, one co-migrating with hPL and the other migrating slightly slower than hPL. On DEAE-cellulose chromatography the former material eluted close to authentic hPL while the latter material eluted at higher ionic strength than hPL, indicating a difference in net charge of these two species. Tryptic peptide analysis of the large material and authentic hPL shows marked similarities in the primary structure of these two proteins. The slower migrating peptide has an apparent molecular weight about 3000 larger than hPL and thus may represent a precursor molecule. Both cell-free products could be competed out of immunoprecipitates by a large excess of authentic hPL, confirming their immunologic similarities. Centrifugation of the placental poly(A)-containing RNA through aqueous glycerol gradients indicates that the hPL mRNA sediments at about 14 S.

Aminoacylation↗

PubMed1975

Identification of "big" human placental lactogen in placenta and serum.

Because of increasing evidence for the heterogeneity of polypeptide hormones, studies of the molecular species of human placental lactogen (hPL) were initiated. When extracts of freshly delivered human placentas were passed over Sephadex G-100 in 0.05M ammonium carbonate, three immunoreactive peaks were detected. In addition to a peak corresponding to native hPL (Kav = 0.39) and one in the void volume, a consistent peak which eluted before hPL (Kav = 0.20) was present. The latter represented 2-25% of total hormonal activity and could be rerun without significant conversion to hPL. In 8M urea, the peak continued to behave as a large molecular weight form on both Sephadex chromatography and on polyacrylamide disc gel electrophoresis. Extraction procedures at both neutral and alkaline pH produced similar quantities of the larger material. [125I]iodo-hPL was not converted to the larger form by the conditions of extraction or analysis. These properties are consistent with a larger molecular weight, non-aggregated form of hPL. In comparison with the native hormone, the idsplacement curves for the larger form were parallel in radioimmunoassay studies. Sera obtained from pregnant women during various stages of gestation also showed consistent evidence for a large molecular weight form of the hormone. These observations provide direct evidence, both in placental tissue and in serum for "big" hPL.

Female↗

PubMed1997

Prenatal diagnosis of beta-thalassaemia by coelocentesis.

Coelomic fluid and placental tissue were obtained from four women undergoing termination of pregnancy at 7-9 weeks gestation for psychological reasons. All four women and their partners were known carriers of beta-thalassaemia and DNA analysis in their blood identified the mutation carried by each of them. Allelespecific polymerase chain reaction and denaturing gradient gel electrophoresis techniques were used to detect and identify the mutations in the DNA extracted from the coelomic cells and placental tissue. Three fetuses were found to be carriers of either the paternal or maternal mutation, while one was found to be affected by beta-thalassaemia. There was concordance in the results obtained from the chorionic villi and coelomic cells. Amplification of the apolipoprotein B gene variable number tandem repeats (VNTR), in the DNA of the coelomic cells showed normal sagregation of alleles in the fetuses, thus excluding maternal contamination. The results of this study demonstrate that coelocentesis may be a reliable alternative technique for the diagnosis of beta-thalassaemia from as early as 7 weeks gestation.

Alleles↗

PubMed1984

Determination of placental alkaline phosphatase phenotypes by starch-gel and acrylamide gel electrophoresis.

The polymorphism of placental alkaline phosphatase (ALP) is usually studied by butanol extraction followed by starch gel electrophoretic separation at two different pH's, 8.6 and 6.0. Acrylamide gel electrophoresis (7.5%) in the presence of Triton X-100 is shown to be preferable in speed, reliability and reproducibility of results. All the placental ALP phenotypes detected had the same molecular mass by gradient acrylamide electrophoresis. The enzyme butanol extract is not sufficiently pure to enable the different placental ALP's to be distinguished by isoelectric focusing on polyacrylamide gels.

Alkaline Phosphatase↗

PubMed1978

Properties of receptors for IgG on human placental cell membranes.

The amount of 125I-IgG which bound to membranes isolated from the human placenta was competitively inhibited by the presence of increasing amounts of unlabeled IgG but not by unlabeled albumin. The relationship between membrane-bound and free IgG indicated the presence of membrane receptors with an appreciable affinity for IgG. Incubation of membranes with collagenase or neuraminidase did not results in appreciable reduction of IgG-membrane binding, indicating that neither intact collagen nor sialic acid play an important role in the binding. Placental surface membranes isolated by salt extraction bound 3.79+/- 1.78 (SD) pmol IgG/microgram membrane protein, whereas membranes isolated by differential centrifugation bound only 1.61 +/- 0.24 pmol/microgram (p less than 0.02). The fraction of a preparation of solubilized membranes which bound to an IgG affinity column yielded on polyacrylamide gel electrophoresis three prominent protein bands which had molecular weights of 3.7 X 10(4), 4.5 X 10(4) and 6.0 X 10(4) daltons. These findings are consistent with the existence of a limited number of receptors for IgG on placental membranes, including IgG receptors on the microvillus membrane of the syncytial trophoblast. The latter, in accordance with Brambell's hypothesis, could be of importance in the transplacental transport of maternal IgG.

Binding Sites↗

PubMed1986

Characterization of a gonadotropin-releasing hormone receptor site in term placenta and chorionic villi.

The properties of GnRH receptor sites in the human placenta were analyzed by binding studies performed in particulate and solubilized receptor preparations. The binding affinities (Ka) of the membrane receptor of term placenta for the GnRH superagonists [D-Lys6]- and [D-Ala6]des-Gly10- GnRH-N-ethylamide were 1.6 X 10(6) and 5.4 X 10(5) M-1, respectively. The binding affinities of native GnRH and a potent GnRH antagonist were similar to those of the superagonists (1.1 and 2.0 X 10(6) M-1, respectively). The placental sites could be solubilized by extraction with the detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane sulfonate, with retention of 40-45% of the specific binding activity, though with a significant decrease in binding affinity. The sites were also solubilized with sodium dodecyl sulfate after covalent labeling with a photoreactive 125I-labeled [D-Lys6] GnRH agonist derivative. Analysis of the solubilized hormone-receptor complex on polyacrylamide gradient gels showed a single band of 53,700 mol wt, similar to the mol wt of the pituitary GnRH receptor in other species. It is clear that the placental receptor differs markedly from the GnRH receptor of the pituitary gland in its low binding affinity and lack of selectivity for GnRH analogs. However, it is possible that the human placental receptor for GnRH could serve as a low affinity regulatory site for locally formed GnRH or related low affinity regulatory site for locally formed GnRH or related peptides within the placenta, and that the placental GnRH system has a significant role in the maintenance of pregnancy.

Binding, Competitive↗

PubMed1995

Isolation and characterisation of a hyaluronan binding protein, hyaluronectin, from human placenta and its colocalisation with hyaluronan.

Hyaluronan (HA) is a major component of the extracellular matrix and is known to influence cell behaviour and to play a role in angiogenesis, morphogenesis and tissue remodelling, although little is known concerning the regulation of these effects. Until now its detection in the placenta has been by indirect methods, which has led to conflicting conclusions as to its distribution and hence its role. Hyaluronectin (HN) is one of a group of proteins with HA binding ability which may regulate the effects of HA. Although nervous tissue HN has been partly characterised with regard to its distribution, structure and biochemistry, little is known about the mesenchymal isoform and its distribution in placenta has not previously been reported. Using specific probes we have characterised the distribution of HA and HN in human placental tissue. At all stages of development studied (8, 10, 12, 30 and 38 wk gestation) HA and HN were unequivocally colocalised, being distributed in the extracellular matrix of stromal tissue of placental villi, chorioallantoic membranes and umbilical cord. Particularly strong immunoreactivity was observed in the villous stroma immediately adjacent to fibrinoid depositions at sites of denudation of the trophoblast layer. Extraction and characterisation of the HN from placental villi have revealed 4 major glycoproteins of 47, 52, 57 and 67 kDa, this being a different pattern and smaller molecular range than observed for the nervous tissue form. This is the first direct demonstration of the presence of HA and HN in the placenta and identifies an abundant new source of mesenchymal HN. The functions of mesenchymal HN are unknown but may include ion exchange, immunosuppression and regulation of the effects of HA in such roles as maintenance of tissue architecture, cell migration and angiogenesis.

Blotting, Western↗

PubMed1997

Expression of the apoptosis-inducing Fas ligand (FasL) in human first and third trimester placenta and choriocarcinoma cells.

The Fas (Apo-1/CD95) ligand (FasL) belongs to the tumor necrosis factor family and acts through its receptor (FasR/ Apo-1/CD95) to induce apoptosis in target cells. FasL is expressed in several immunologically privileged sites. Induction of apoptosis by FasL in invading lymphocytes acts as a mechanism of immune privilege and is important in preventing graft rejection. Furthermore, FasL is expressed in certain malignancies and it has been implicated as a possible key mechanism in immune privilege of these tumors. Since the invading placental trophoblast is another very important site with a privileged immune status, we investigated whether FasL is expressed in the normal and tumoral human placenta. For this purpose, mRNA was extracted from first and third trimester human placental samples as well as from JEG3 choriocarcinoma cells and reverse transcribed to obtain cDNAs. These were used as templates for PCR analysis of FasL expression, in which specific primers were employed to amplify an 853 bp fragment spanning the whole FasL coding region. A product of the appropriate length was amplified from normal placenta as well as from the choriocarcinoma cells. Expression of FasL protein was confirmed by Western Blot and was localized to trophoblast by immunohistochemistry using a FasL-specific antibody. Expression of FasL in the human placenta indicates that induction of apoptosis in lymphocytes by the invading trophoblast could be an important mechanism implicated in the immune tolerance of the fetal semi-allograft.

Apoptosis↗

PubMed1983

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease.

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.

Female↗

PubMed1987

The corticotropin releasing hormone gene is expressed in human placenta.

Maternal plasma immunoreactive corticotropin-releasing hormone (IR-CRH) increases progressively with pregnancy. This elevated plasma IR-CRH is presumably secreted by the placenta. To investigate further this hypothesis, we searched for the CRH mRNA and its peptide product in full term human placentae. Using a radiolabelled 48-mer oligonucleotide probe complementary to a portion of human CRH mRNA, we identified a 1300 nucleotide RNA from human placenta and rat hypothalami. We next examined the chromatographic characteristics of the placental IR-CRH. The bulk of the IR-CRH extracted from placenta and the IR-CRH secreted in vitro by placental fragments had the same chromatographic profiles as synthetic CRH. These findings indicate that the CRH gene is expressed in human placenta and imply that this organ is a site of CRH biosynthesis during pregnancy.

Chromatography, Gel↗

PubMed1990

Characterization of receptors for recombinant human tumor necrosis factor-alpha from human placental membranes.

High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-alpha) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-alpha iodinated by the iodogen method was found to bind placental membranes in a displaceable manner with an approximate dissociation constant (KD) of 1.9 nM. The membrane bound TNF-alpha receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-alpha to the solubilized receptor was found to be time and temperature dependent, yielding maximum binding within 1 h, 24 h and 48 h at 37 degrees C, 24 degrees C and 4 degrees C, respectively. However, the maximum binding obtainable at 4 degrees C was only 40% of that at 37 degrees C. The binding 125I-rhTNF-alpha to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-alpha, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-beta; previously called lymphotoxin). This is similar to the behavior of TNF-alpha receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-alpha and rhTNF-beta bind with equal affinity to TNF receptors. The Scatchard analysis of the binding data of the solubilized receptor revealed high affinity binding sites with a KD of approximately 0.5 nM and a receptor concentration of about 1 pmole/mg protein. Gel filtration of the solubilized receptor-ligand complexes on Sephacryl S-300 revealed two different peaks of radioactivity at approximate molecular masses of 50,000 Da and 400,000 Da. The 400,000 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-alpha are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-beta.

Binding Sites↗