Partial characterization of human amniotic membrane interferon

The molecular weight and the electrofocusing profile of human amniotic membrane interferon (IFN-AM) were determined. When submitted to gel filtration, IFN-AM showed a single 26-28 kDa component; in polyacrylamide gel electrophoresis one component of 19,500. In electrofocusing, IFN-AM displayed a terogeneity was reduced by previous treatment of IFN-AM with neuraminidase. IFN-AMis a siaglycoprotein similarto human beta IFN in terms of antigenicity but different from it in electrofocusing profile

La placenta humana: estudio bibliográfico de su composición química y sus extractos / Human placenta: bibliographic study of its chemical composition and extracts

Se presentan los resultados de la revisión bibliográfica realizada hasta el año 1987 sobre la composición química de la placenta humana, así como de los diferentes extractos reportados que tienen aplicación en la terapéutica o en la elaboración de cosméticos. Una gran parte de la literatura consultada contempla solamente publicaciones referativas como el Chemical Abstracts. Debido a lo amplio del tema a tratar, sólo se profundiza en algunos tópicos que se han considerado los más importantes, mientras que en otros se han mencionado los trabajos sin detallar los mismos. La placenta es un órgano que contiene gran variedad de sustancias que forman un conjunto biológico bastante complejo. En este trabajo se exponen aquellas de mayor importancia de las tantas reportadas en la literatura

Polycyclic aromatic hydrocarbon-DNA adducts in spontaneously aborted fetal tissue.

Fetal tissue and placentas from 15 human spontaneous abortions were evaluated for DNA adducts of polycyclic aromatic hydrocarbons (PAHs), using a competitive enzyme-linked immunosorbent assay (ELISA) with fluorescent end-point detection. PAH-derived adducts were found in 43% of placentas, 27% of fetal liver samples and 42% of fetal lung specimens, thus confirming that the human fetus is a target for DNA damage. As there was only 60% concordance between placenta and fetal lung or liver on the presence or absence of detectable PAH adducts, the placenta was not a good surrogate for adduct formation in other fetal organs. PAH-derived adducts in fetal liver and lung presumably form as a result of transplacental exposure to environmental stimuli. Since none of the positive fetal samples were from women who reported smoking during pregnancy, cigarette smoke is, in this case, an unlikely candidate and the adducts detected must be due to some other common source(s) of hydrocarbon exposure. The high frequency of positive samples in our small series casts some doubt on whether fetal PAH-DNA adducts identify a population at increased risk for transplacental carcinogenesis.

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins.

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.

Vascular angiotensin II and atrial natriuretic peptide receptors in normal and growth-retarded human placentae.

Receptors for angiotensin II (AII) and atrial natriuretic peptide (ANP) were characterized in a membrane fraction from resistance-type artery from human placentae. Placentae from normal pregnancies and pregnancies complicated by intrauterine growth retardation (IUGR) were studied. High- and low-affinity receptors for AII (dissociation equilibrium constant (Kd) 1.7 and 15.7 nmol/l respectively) and ANP (Kd 0.2 and 55.5 nmol/l respectively) were identified; these parameters were unchanged in IUGR, but there was a reduction in high-affinity receptor number by approximately 50% for AII and 80% for ANP in this condition. Both peptides may have a role in the regulation of fetoplacental blood flow. The alterations in IUGR are consistent with sustained activation of the fetal renin-angiotensin system and suggest altered vascular responsiveness to ANP.

Molecular characterization of a t(11;14)(q23;q32) chromosome translocation in a B-cell lymphoma.

We have analyzed the molecular features of a t(11;14)(q23;q32) chromosome translocation of a cell line established from a B-cell lymphoma. Somatic hybrid cells carrying the 11q- and/or 14q+ chromosome(s) were produced in order to map the breakpoints. Southern blot analyses of DNAs from these hybrid cell lines together with various probes from the IGH locus on chromosome 14 and the ETS-1 and CD3 genes on chromosome 11 showed that the breakpoints of the translocation occurred between the constant regions of the C phi gamma and C gamma 2 genes on chromosome 14 and between the CD3 and ETS-1 genes on chromosome 11. The t(11;14)(q23;q32) translocation does not seem to involve the same mechanism that is responsible for translocations occurring at the immunoglobulin heavy chain joining segment (JH).

The clinical significance of absent subchorionic fibrin in the placenta.

Patchy fibrin is normally present beneath the chorionic plate of the placenta. The present study attempted to determine whether it has any clinical significance. In 31,622 placentas the amount of this fibrin was related to markers of fetal activity. Fibrin was often absent when the markers indicated hypoactivity. The markers of fetal hypoactivity were neonatal hypotonia, neonatal lethargy, the presence of Down's syndrome, and a short umbilical cord. Umbilical cord length is strongly influenced by tension applied to it by fetal movements--the fewer the movements, the shorter the cord. Children with no subchorionic fibrin subsequently had increased frequencies of cerebral palsy and low intelligence quotient values. Children who were hyperactive at one year of age had increased levels of subchorionic fibrin, which suggests that they were hyperactive before birth. All of these findings raise the possibility that normal fetal movements sometimes traumatize the placenta, which leads to fibrin deposits beneath its surface.

Localization of renin substrate in fetoplacental tissue.

Specific polyclonal rabbit anti-human renin substrate-antibodies were used in order to study the distribution of renin substrate immunoreactivity in human fetal and placental tissue. Renin substrate was immunohistochemically detected in human decidua and placenta, as well as in 19 weeks old human fetal liver and kidney. The presence of renin substrate in fetoplacental tissue supports the concept of a locally functioning renin-angiotensin system.

Structural requirements for signal transduction of the insulin receptor.

Structural requirements for signal processing by human placental insulin receptors have been examined. Insulin binding has been found to change the physico-chemical properties of (alpha beta)2 receptors solubilized with Triton X-100, indicating a marked alteration of the form, i.e. size and shape, of the molecular complex. (a) The Stokes radius decreases from about 9.5 nm to 7.9 nm, as determined by PAGE with Triton X-100 in the buffer (Triton X-100/PAGE), and from 9.1 nm to 8.7 nm, as assessed by gel filtration. (b) The sedimentation coefficient s20,w rises from 10.1 S to 11.4 S. Upon dissociation of the receptor-hormone complex, the alterations are reversed. After autophosphorylation of hormone-bound (alpha beta)2-insulin receptors, phosphate incorporation was found for 7.9-nm receptor forms when receptor-insulin complexes were crosslinked with disuccinimide suberate prior to Triton X-100/PAGE. However, phosphate incorporation was demonstrated for the 9.5-nm receptor forms when receptor-insulin complexes were not prevented from dissociation. This strongly indicates that the (alpha beta)2 receptor is autophosphorylated after assuming its 7.9-nm form upon insulin binding. Moreover, the insulin-dependent structural alterations are not affected by autophosphorylation. In contrast to (alpha beta)2 receptors, the diffusion and the sedimentation behaviour of alpha beta receptors, which carry a dormant tyrosine kinase even in the hormone-laden state, has been found to be insensitive to insulin binding. Different molecular properties of alpha beta and (alpha beta)2 receptors have also been detected by hormone binding studies. Insulin binding to (alpha beta)2 and alpha beta receptors differs markedly with respect to pH, ionic strength, and temperature. This might indicate that the structure of the hormone binding domain of alpha beta receptor changes on association into the (alpha beta)2 species. Alternatively, distinct hormone-induced conformational alterations at the molecular level of alpha beta and (alpha beta)2 receptor species may lead to the different binding properties. Our data demonstrate that the (alpha beta)2-insulin receptor undergoes extended conformational alterations upon insulin binding. This capacity for structural changes coincides with the hormone-inducable enhancement of tyrosine autophosphorylation of the 7.9-nm insulin-bound receptor form. In contrast, alpha beta receptors appear to be locked in an inactive nonconvertable state. Thus, interaction between two alpha beta receptor units is required to allow extended conformational alterations, which are assumed to be the triggering event for augmented auto-phosphorylation.

High concentrations of plasma immunoreactive inhibin during normal pregnancy in women.

The plasma inhibin concentrations in 190 normal pregnant women at 5-40 weeks gestation and in 4 puerperal women were measured by a specific RIA for human inhibin. The average plasma inhibin concentrations in pregnant women throughout pregnancy (minimum, 2.25 +/- 0.48 IU/mL at 17 weeks gestation; maximum, 24.15 +/- 6.99 IU/mL at 39 weeks gestation) were much higher than those in nonpregnant women with a normal menstrual cycle (0.46 +/- 0.04 IU/mL in the midfollicular phase and 2.02 +/- 0.47 IU/mL in the midluteal phase). The inhibin concentrations were already high at 5 weeks gestation (7.54 +/- 1.10 IU/mL) and rose to peak at 8-10 weeks gestation. The concentrations then decreased and remained relatively low during 14-30 weeks gestation, but rose again during the third trimester. The inhibin concentrations decreased to undetectable levels after delivery. Immunoreactive inhibin was demonstrated in the corpus luteum and term placental extracts, and the dose-response curves were parallel to an inhibin preparation from human follicular fluid. Immunoreactive inhibin concentrations were also high in both the umbilical vein and artery (7.77 +/- 0.80 and 7.84 +/- 0.78 IU/mL, respectively). These observations suggest that both the corpus luteum and placenta are likely sources of inhibin.

Purification and functional characterization of integrin alpha v beta 5. An adhesion receptor for vitronectin.

We have purified a novel member of the integrin gene family from placenta that serves as a vitronectin receptor. This integrin is composed of the alpha v subunit and a beta subunit that we designate beta 5. Purification was accomplished by immunodepleting a placental extract of integrin alpha v beta 3, allowing us to purify alpha v beta 5 from the remaining extract by monoclonal antibody affinity chromatography on LM 142-Sepharose, which binds to the alpha v subunit. Purification to homogeneity was subsequently achieved by affinity chromatography on wheat germ lectin-Sepharose. Western blot analysis with antibodies raised against alpha v beta 5 and alpha v beta 3 demonstrated that beta 3 and beta 5 were distinct but confirmed that the alpha subunit of the two integrins were immunologically identical. Similarly, antibodies that bind beta 3 proximal to the ligand-binding site failed to react with beta 5, indicating an architectural difference at the ligand-binding site of these related integrins. This structural difference apparently results in a functional distinction, since purified alpha v beta 3 bound to vitronectin, fibrinogen, von Willebrand factor, and fibronectin, whereas integrin alpha v beta 5 bound preferentially to vitronectin. Finally, we demonstrate by three criteria that beta 5 and beta x, the latter of which was identified in lung carcinoma cells (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), are identical. First, peptide maps of beta x and beta 5 are identical. Secondly, polyclonal antibodies raised against alpha v beta 5 immunoprecipitate both beta 5 and beta x, and finally, the amino-terminal amino acid sequences of beta x and beta 5 are identical.

Synthesis and secretion of ovine placental lactogen and its biochemical properties.

The biochemical properties of ovine placental lactogen (oPL) have been previously determined following purification, which has yielded various results. To clarify the properties of oPL prior to purification, oPL was examined in solubilized fetal cotyledonary tissue (d 100 of gestation) or conditioned culture medium by electrophoretic, immunoblotting and immunoprecipitation techniques. In cotyledonary tissue or conditioned culture medium, oPL has an apparent molecular weight (Mr) of 22,000 with an isoelectric point (pI) of 9.2. Incorporation of [3H]-glucosamine or [3H]-mannose into immunoreactive oPL could not be detected, nor did the presence of tunicamycin in explant culture medium alter the apparent Mr of oPL. In vitro translation of d 100 fetal cotyledonary mRNA, followed by immunoprecipitation, provided evidence that pre-oPL has an apparent Mr of 25,000. The size of oPL mRNA was determined to be approximately 1,350 base pairs by Northern hybridization procedures using an oligonucleotide probe which was generated from oPL amino acid sequence data. These experiments suggest that the only intracellular processing oPL undergoes is removal of a amino-terminal signal sequence. We conclude that oPL is synthesized and secreted as a single nonglycosylated-basic protein, at a time during gestation when circulating oPL is elevated.

Structural analysis of the regulatory region of the human corticotropin releasing hormone gene.

A DNA fragment containing the human corticotropin releasing hormone (CRH) gene, along with 9 kb of upstream and 4 kb of downstream sequences, was isolated from a human genomic DNA library. Nucleotide sequence analysis of the proximal 918 nucleotides 5' flanking the putative major mRNA start site of the human gene and comparison to the 866 nucleotide long homologous ovine sequence, revealed that this region of the CRH gene consists of two distinct areas with different degrees of homology, varying from 72% to 94%. The putative functional features of the human sequence were identified. Many, but not all, features were conserved in the ovine sequence. The highly conserved nature of the regulatory region of this gene makes it a good candidate for tracing possible related genetic defects of the hypothalamic-pituitary-adrenal (HPA) axis.

Analysis of cultured chorionic villi in a case of osteogenesis imperfecta type II: implications for prenatal diagnosis.

We examined collagens produced by cultured cells from skin, chorionic villi, and placental membranes of a 32 week fetus with osteogenesis imperfecta (OI) type II. We observed that skin fibroblasts synthesized two populations of pro alpha 1(I) chains of type I procollagen; one population was normal, while the other population had excessive post-translational modification. The thermal stability of helices containing the overmodified chains was reduced 1-2 degrees C. Most significantly, the cells cultured from chorionic villi produced type I collagen chains with the same electrophoretic abnormalities as the skin collagen. This suggests that chorionic villus sampling (CVS) is a means of prenatal diagnosis for families with a previous type II or type IV OI infant.

A compositional map of human chromosome 21.

GC-poor and GC-rich isochores, the long (greater than 300 kb) compositionally homogeneous DNA segments that form the genome of warm-blooded vertebrates, are located in G- and R-bands respectively of metaphase chromosomes. The precise correspondence between GC-rich isochores and R-band structure is still, however, an open problem, because GC-rich isochores are compositionally heterogeneous and only represent one-third of the genome, with the GC-richest family (which is by far the highest in gene concentration) corresponding to less than 5% of the genome. In order to clarify this issue and, more generally, to correlate DNA composition and chromosomal structure in an unequivocal way, we have developed a new approach, compositional mapping. This consists of assessing the base composition over 0.2-0.3 Mb (megabase) regions surrounding landmarks that were previously localized on the physical map. Compositional mapping was applied here to the long arm of human chromosome 21, using 53 probes that had already been used in physical mapping. The results obtained provide a direct demonstration that the DNA stretches of G-bands essentially correspond to GC-poor isochores, and that R-band DNA is characterized by a compositional heterogeneity that is much more striking than expected, in that it comprises isochores covering the full spectrum of GC levels. GC-poor isochores of R-bands may, however, correspond to 'thin' G-bands, as visualized at high resolution, leaving GC-rich and very GC-rich isochores as the real components of (high-resolution) R-band DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioligand binding assay of epidermal growth factor receptor: causes of variability and standardization of the assay.

Although experimental evidence indicates a probable role of epidermal growth factor receptor (EGFr) in clinical oncology, no standardized method for its determination has been yet described, and discrepant results have been reported in clinical studies. In standardizing a radioligand binding assay for EGFr, we evaluated the causes of variability in each step of the assay. Entrapment of EGFr in the nuclear fraction and contamination of the crude membrane fraction by cytosol protein were eliminated through preliminary purification steps. Both Scatchard and Rosenthal analysis of the saturation reaction of the membrane fraction with a wide range of concentrations of 125I-labeled EGF revealed a double class of binding sites. Study of the saturation reaction showed a partial exchange of 125I-labeled EGF with endogenous EGF within 20 h. The present method--incubation of partly purified membrane fraction with 125I-labeled EGF, 0.5 nmol/L, with and without 100-fold excess of cold EGF, for 20 h at 26 degrees C, followed by centrifugation at 5000 x g for 30 min to separate membrane-bound 125I-labeled EGF--shows good sensitivity, precision, and accuracy; is reasonably simple; and may be suitable for routine clinical use.

Characterization of recombinant factor XIIIa produced in Saccharomyces cerevisiae.

Recombinant factor XIIIa (FXIIIa), produced in Saccharomyces cerevisiae, was recovered as a fully active cytosolic component and rigorously compared to natural F XIIIa from human placenta with respect to physicochemical and functional properties. Identical parameters were found in SDS polyacrylamide gel electrophoresis, analytical ultracentrifugation and HPLC gel filtration, and all spectral characteristics including derivative UV absorbance, fluorescence and circular dichroism were identical. Similarly, the interaction of both proteins with polyclonal antibodies directed against the entire FXIIIa or its N-terminal 4 kD activation peptide were identical. Furthermore, thrombin cleavage and fibrin cross-linking showed indistinguishable patterns. The only difference we observed was with respect to endgroup analysis. The recombinant protein is homogeneous, whereas placental FXIIIa shows multiple electrophoretic bands caused by microheterogeneity in the C-terminal part of the protein.

Estudio morfométrico de la placenta a término en las grandes alturas / Morphometry of the at term placenta at high altitudes

Este trabajo presenta el estudio morfométrico de 10 placentas de La Paz, Bolivia (altitud 3600 metros) y 14 de Santa Cruz de la Sierra, Bolivia (altitud 400 metros). En cada grupo fueron analizados peso, volumen y número de cotiledones de las placentas, área de superficie de las vellocidades coriónicas, de los vasos capilares y de las membranas vásculo-sinciciales, y el volumen de los diferentes componentes macro y microscópicos. Esta investigación permitió el reconocimiento de un patroón morfométrico de las placentas de grandes altitudes. Las mayores diferencias fueron observadas en las estructuras relacionadas con el intercambio de gases entre las circulaciones fetal y materna, ésto es en los volúmenes, áreas de superficie y cantidad de vasos fetales y de las membranas vásculo-sinciales. Los resultados son comparados con otros trabajos realizados en diferentes altitudes .

cDNA and gene analyses imply a novel structure for a rat carcinoembryonic antigen-related protein.

The gene encoding the human tumor marker carcinoembryonic antigen (CEA) belongs to a gene family which can be subdivided into the CEA and the pregnancy-specific glycoprotein subgroups. The corresponding proteins are members of the immunoglobulin superfamily, characterized through the presence of one IgV-like domain and a varying number of IgC-like domains. Since the function of the CEA family is not well understood, we decided to establish an animal model in the rat to study its tissue-specific and developmental stage-dependent expression. To this end, we have screened an 18-day rat placenta cDNA library with a recently isolated fragment of a rat CEA-related gene. Two overlapping clones containing the complete coding region for a putative 709 amino acid protein (rnCGM1; Mr = 78,310) have been characterized. In contrast to all members of the human CEA family, this rat CEA-related protein consists of five IgV-like domains and only one IgC-like domain. This novel structure, which has been confirmed at the genomic level might have important functional implications. Due to the rapid evolutionary divergence of the rat and human CEA gene families it is not possible to assign rnCGM1 to its human counterpart. However, the predominant expression of the rnCGM1 gene in the placenta suggests that it could be analogous to one of the human pregnancy-specific glycoprotein genes.

Purification and characterization of six annexins from human placenta.

Isolation of six calcium-binding proteins from human placenta is described by means of hydrophobic chromatography, calcium-dependent adsorption to heparin-Sepharose and ion-exchange chromatography. These proteins were characterized and identified as PP4, PP4-X, PAP III, p68 and lipocortins I and II belonging to the family of annexins. Antibodies raised against PP4, PAP III and p68 revealed to be highly specific, while those raised against PP4-X reacted with all investigated annexins, except PP4. Cross-reactivity was also observed between lipocortins I and II. All annexins inhibited in a concentration-dependent manner blood coagulation but with different potencies as was determined by means of a modified thromboplastin time test. The most potent inhibitors turned out to be PP4 and PAP III, followed by PP4-X, lipocortin I, p68 and lipocortin II.