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beta2-glycoprotein I (beta2GPI) and annexin V (AV) have been implicated in the pathophysiology of the antiphospholipid syndrome (APS). We investigated their placental expression in normal villous tissues throughout gestation; first trimester n = 10, early second trimester; n = 4, preterm; n = 5) and term; n = 7 and in APS (2 first trimester, 1 preterm and 8 term deliveries). beta2GPI and AV were both expressed by the placenta from as early as seven weeks gestation and were colocalised to the syncytiotrophoblast. beta2GPI staining was also observed in stromal cells, being present in phagocytic Hofbauer cells and surrounding newly formed fetal vessels in a perivascular pattern, from seven to seventeen weeks gestation. An abnormal morphological distribution of AV was noted in one first trimester APS placenta, and for beta2GPI in a further first trimester placenta. When placental proteins were extracted from villous tissue, the concentration of AV/mg protein in term APS placentas (median, interquartile range) (aPS; 8.16, 7.879.72 microg/mg) was significantly higher (p <0.005) than normal term levels (normal; 2.47, 2.28-2.54 microg/mg). beta2GPI increased with advancing gestation (first trimester; 0.93, 0.64-1.26 microg/mg, term; 3.67, 2.58-4.48 microg/mg) in normal pregnancy. Term APS placentas had a reduced beta2GPI content (2.31, 1.87-2.49 microg/mg), p <0.05. The placental role of these proteins remains to be identified.
The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[3H]ethylcarboxamidoadenosine [( 3H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [3H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.
Atrial natriuretic factor (ANF), a 28 amino acid peptide, is primarily produced and secreted by cardiac atrial myocytes to modulate cardiovascular and renal functions. Although ANF is also produced in tissues other than the heart, it remains uncertain whether the peptide is synthesised by human placentae. We provide here evidence from in vitro studies suggesting de novo production of ANF by the human placenta. Placental tissues were collected from normal term pregnancies by elective Cesarean section and acid extracted for ANF radioimmunoassay. The level of placental immunoreactive (ir) ANF was 186 +/- 33 pg/gm wet tissues (mean +/- SE, n = 6). Perfusates from in vitro perfusion of the fetoplacental compartment of placental lobules yielded 26 +/- 6 pg/min/gm wet tissue (n = 3) of irANF. Furthermore, pro-ANF mRNA signals were localised by colorimetric in situ hybridization in a subpopulation of placental cytotrophoblast-like cells, but not in syncytiotrophoblasts nor in chorionic cells of placental sections. Northern blot analysis of placental tissue extracts showed a single band of pro-ANF mRNA signals (-0.85 Kb) similar in size to that found in the rat heart. Our findings suggest that ANF is expressed and produced by a small population of human placental cytotrophoblast-like cells. The possibility that placental ANF may be secreted locally or into the fetoplacental circulation to exert paracrine, autocrine or both effects now needs to be considered.
New artificial biomaterials were tested for support of gastro-intestinal tract wound healing in the rat. Double layered collagenic matrices were prepared with purified collagens extracted from human placental tissues. Two types of patches were tested, the first constituted from a collagen type I + III layer covered by collagen IV in a liquid phase (patch I + III/IV) and the second from a collagen IV layer covered by liquid collagen IV (patch IV/IV). The matrices were applied with fibrin sealant to the edges of a 1 cm diameter colonic wall defect in the rat. Healing evolution was determined by macroscopic, microscopic and immunostaining studies. The reconstitution of the three colonic wall layers was achieved within 45 days without retraction or inflammatory reaction, while the biomaterial was resorbed. Human collagen I and III antibodies failed to stain extracellular matrix. This failure may be a consequence of outdated antibodies or more likely epitope alteration during extraction and preparation of the collagens. A human collagen type IV antibody staining of the scar zone showed the basement membranes of newly developed vessels within 10 days, and newly formed colonic mucosa within 20 days. The collagen reconstituted matrix was able to assist healing of normal digestive tract defects as shown by the labelling of the new synthesized extracellular matrix by collagen type IV antibody. These findings support the use of collagen biomaterial in gastro-intestinal anastomosis. This new surgical approach allowing healing of colonic wall defects could reduce occurrence of anastomotic leakage in human.
The candidate tumor-specific soluble mammary tumor glycoprotein with a molecular weight of approximately 20,000 (MTGP20) has been isolated from human breast carcinomas and characterized biochemically. Although with the use of xenoantisera this glycoprotein has previously been demonstrated only in breast carcinomas, analyses of body fluids and conclusions regarding putative tumor specificity have been limited by the sensitivity of assays. In the present study, a specific radioimmunoassay has been developed. With appropriate selection of buffer, the assay has a sensitivity threshold of less than 0.1 unit of MTGP20 per ml, equivalent to less than 250 pg type I MTGP20 per ml or less than 530 pg type II MTGP20 per ml, which is more than 200-fold more sensitive than previously described assays. MTGP20 type I, which contains tyrosine, was labeled with 125I and used as the immunochemical ligand in a double antibody competitive inhibition assay. A partial weak cross-reaction was observed with a mixture of placental glycoprotein (perchloric acid extract), but this reaction was abolished by absorption of antisera with placental glycoprotein. Normal tissue and tumors of other than breast origin were devoid of MTGP20-related antigens. MTGP20-related antigens were not detectable in sera or concentrated urine specimens from normal individuals or patients with metastatic breast carcinomas. The present studies further support the probable tumor specificity of this glycoprotein but indicate that it does not represent a circulating secretory product of the cancer cell and does not provide a serum marker for breast carcinoma.
Colony-stimulating factor-1 (CSF-1), a growth factor produced by monocytes, macrophages, fibroblasts, and endothelial cells, has been implicated in the functional regulation and growth of the murine placenta through the presence of the CSF-1 receptor, c-fms, found in this tissue. In this study we examined the tissue levels of CSF-1 by RIA and the relative expression of CSF-1 and c-fms mRNA by Northern blot analysis in human endometrial, decidual, and placental tissues during the normal menstrual cycle and early pregnancy. All endometrial, decidual, and placental tissues demonstrated extractable immunoreactive CSF-1 and expressed the 4.0-kilobase CSF-1 mRNA species. First trimester decidual tissue expressed higher levels of CSF-1 mRNA than proliferative (3.2-fold higher; P less than 0.01) or secretory (2.4-fold higher; P less than 0.01) endometrial tissues, whereas proliferative and secretory endometrial tissues expressed similar levels of CSF-1 mRNA. Tissue extractable levels of immunoreactive CSF-1 were 3.2-fold (P less than 0.05) higher in first trimester decidual tissue and 2.9-fold (P less than 0.05) higher in secretory endometrial tissue compared to levels in proliferative endometrial tissue, whereas first trimester decidua and secretory endometrial tissues had similar levels of immunoreactive CSF-1. There was expression of c-fms mRNA in all endometrial and first trimester decidual tissue samples, with little change during the menstrual cycle and early pregnancy. In placenta, there was a positive correlation of increasing CSF-1 and c-fms mRNA expression with increasing gestational age. These results suggest that there is increased local production of CSF-1 in tissues found at the maternal-fetal interface during the time of implantation and early pregnancy. This increased production of CSF-1 may play a role in decidual function and placental growth through the presence of c-fms in these tissues.
OBJECTIVE: Our purpose was to examine regulatory linkages between fetal oxygenation and fetal and placental growth. We determined umbilical cord PO (2) and oxygen saturation, fractional oxygen extraction, and birth to placental weight ratio values in relation to size at birth for a large tertiary hospital population delivering at term. STUDY DESIGN: The computerized perinatal database of St Joseph's Health Care London, London, Ontario, was used to obtain the umbilical cord gases, pH, birth weight, placental weight, and other selected information for all term, singleton, liveborn infants between January 1990 and December 1999 (N = 27,043). Oxygen saturation values were calculated from the umbilical cord PO(2) and pH data with a previously derived empirical equation; fractional oxygen extraction values were calculated from the umbilical cord oxygen saturation data. Size at birth was divided into the following 5 birth weight categories using neonatal growth standards: fetal growth restriction, <3%; borderline fetal growth restriction, >or=3% and <10%; appropriate for gestational age, >or=10% and 90% and 97%. RESULTS: Infants in the borderline fetal growth restriction and fetal growth restriction groups had umbilical vein and artery PO(2) and oxygen saturation values that were stepwise lower than respective values for infants in the appropriate for gestational age group. Conversely, infants in the borderline large for gestational age and large for gestational age groups had umbilical vein PO(2) and oxygen saturation values that were stepwise higher than respective appropriate for gestational age group values; infants in these groups showed no change in arterial PO (2) and oxygen saturation values. Therefore infants in the borderline fetal growth restriction and fetal growth restriction groups had fractional oxygen extraction values that were stepwise higher than the appropriate for gestational age group value, whereas values for infants in the borderline large for gestational age and large for gestational age groups remained unchanged. Birth weight was disproportional to placental weight for infants in the borderline fetal growth restriction and fetal growth restriction groups when compared with that of the infants in the appropriate for gestational age group, with the birth to placental weight ratio values stepwise decreased. Conversely, birth weight was proportional to placental weight for infants in the borderline large for gestational age and large for gestational age groups with the birth to placental weight ratio values thus unchanged when compared with that of the infants in the appropriate for gestational age group. CONCLUSION: We conclude that fetal oxygenation is related to size at birth across the entire range of birth weights as studied at term from macrosomic to growth-restricted infants; this conclusion supports oxygen as a primary determinant of fetal growth. However, there are differences in the linkage between fetal oxygenation and metabolic rate or growth for these cohort groups that may relate to underlying etiologic processes.
Lipids, particularly sphingolipids, are emerging as novel regulators of cellular activity. A placental total lipid fraction (PTLF), the total lipid prepared from an hydroalcoholic extract of fresh term human placenta, was previously shown to have a pigment-inducing activity in an animal model. The PTLF contains sphingolipids which stimulate DNA synthesis and melanin formation with marked morphological changes in B16F10 melanoma cells. In order to identify the mechanism underlying the increased melanin synthesis, B16F10 cells were treated with PTLF to assess the catalytic activities of tyrosinase (i.e. tyrosine hydroxylase and DOPA oxidase), the key regulatory enzyme of melanin synthesis. Tyrosine hydroxylase (estimated by the release of (3)H(2)O) as well as DOPA oxidase (measured spectrophotometrically and also in non-denaturing gels), was stimulated significantly by PTLF. Western blot analysis demonstrated an increase in the expression of tyrosinase, tyrosinase related proteins 1 and 2 (TRP1 and TRP2) at the protein level and RT-PCR analysis revealed stimulated transcription of tyrosinase, TRP1 and TRP2 mRNAs in PTLF-treated B16F10 cells. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PTLF-induction of tyrosinase activity with a corresponding decrease in melanogenesis. In all cases, the response to PTLF was similar to that induced by alpha-melanocyte stimulating hormone, a well-known stimulator of melanogenesis. Thus, these results provide the basis of action of PTLF stimulated melanogenesis in B16F10 cells showing that this placental extract is a strong inducer of pigmentation at the transcriptional and translational levels.
The high plasma cortisol and ACTH levels present in pregnant women as well as the non-parrallelism of their plasma extract dilution curves in comparison with the standard curve in the ACTH radioimmunoassay, are evidence for the presence of an ACTH-like substance during pregnancy which would interfere with the assay. Placental extracts were obtained by acid-acetone extraction, followed by partial purification with oxycellulose and by extraction with porous glass powder. A substance was detected which partially cross-reacted with synthetic human ACTH in the radioimmunoassay and which showed biological activity using the assay procedure described by Liscomb & Nelson. The data sustain the existence of an ACTH-like placental hormone: human chorionic corticotrophin (HCC).
Intact human laminin and laminin type IV collagen complexes were extracted from term placental membranes and their structures were examined by electroimmunoblot and by rotary shadowing electron microscopy. Rotary shadowing electron microscopy revealed a structure of human laminin which is essentially similar to the cruciform structure of the mouse tumor (EHS) laminin, but with some notable differences. The observed lengths of the short arms in the human laminin are different. One short arm has an average length of 34 nm and another short arm a length of 42 nm. In the mouse laminin all three short arms are of equal length. The average length of the long arm is 97 nm, which is longer than that of mouse tumor laminin (77 nm). The distal portion of the long arm has two clearly separated globular domains instead of the single one observed with tumor laminin. Human laminin was found in various states of aggregation including dimers, trimers and higher aggregates. Laminin molecules appeared to attach to a point in type IV collagen located 87 nm from C-terminus, or at 174 nm from C-terminus and one with less frequency, at 251 nm from C-terminus. A small number of molecules appeared to bind at the N-terminus of the collagen. These laminin-collagen interactions occurred via the distal globular domains of both the short and long arms of laminin. The data suggest that human laminin extracted from placenta is structurally different from that isolated from the mouse EHS tumor.
Recombinant human IFN alpha 2b coupled to a silica support was used for the purification of the IFN alpha-binding proteins from placental cell membrane extracts. The 100-kDa (p100) and 64-kDa (p64) proteins, which bind preferentially to an IFN alpha 2b-silica matrix, were identified. Using a monoclonal antibody (A6) against IFN-gammaR1, it was able to isolate p100 and p70, but only if IFN alpha 2b was present during chromatography. Similar interactions were observed using polyclonal antibody anti-IFN gamma binding proteins, as assayed in Western blot. These interactions were identified as conformation dependent. We speculate that IFN alpha 2b receptor complex shares an IFN gamma receptor complex epitope.
Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT29 cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g(o)) is caused by inhibition of small non-resolvable channels, which are impermeable to Na+ and SO4(2-), but permeable to Cl-. The regulation of these small Cl(-)-conducting channels (g(o)) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (Po) of the ICOR or on the g(o)) were observed. The Po of the ICOR was reduced by 22% in a Ca(2+)-free solution. g(o) was insensitive to changes in the Ca2+ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT29 cells contain ICOR and small non-resolvable Cl(-)-conducting channels which are similarly inhibited by C. I.(ABSTRACT TRUNCATED AT 250 WORDS)
Administration of Benzo(a)pyrene (BP, 50 mg/kg/d) to pregnant rats significantly increased Glutathione S-transferase (GST) activity in placental tissue-extract (Vmax = 40 nmol/min/mg protein and 69 nmol/min/mg protein in controls versus treated animals respectively; P less than 0.01) and total fetal tissue-extract (Vmax = 51 nmol/min/mg protein and 82 nmol/min/mg protein in controls versus treated animals respectively; P less than 0.01) indicating an induction effect of BP on the GST system. An increase in the Km values was also observed: 1.61 x 10(-3) M and 2.84 x 10(-3) M in control versus treated placentae; 1.38 x 10(-3) M and 2.05 x 10(-3) M in control versus treated fetuses. A competitive effect on the enzyme by the BP present in the sample may also be involved. The glutathione content in both tissues did not show any changes after the treatment with BP. This increase in the GST system was not sufficient to protect the fetus. BP affected the reproductive performance of pregnant rats by significantly increasing the number of resorptions and fetal wastage, and, also, by decreasing the fetal weight.
The peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT) was extracted from human placental membranes using CHAPS as a detergent in the presence of 1 M KCl. Prior to assay dipalmitoylphosphatidylcholine was added to the sample as eluted from the various columns in order to stabilize the protein for subsequent enzyme activity measurements at 37 degrees C. The enzyme was purified from the placental membrane using ocytl-Sepharose CL-4B chromatography, Hydroxyapatite HTP chromatography, CM-Sepharose CL-6B, PBE 94 chromatofocusing and TSK G3000 SW size exclusion chromatography. A final purification of more than 8000-fold with respect to the placental membranes was achieved with a final yield of about 5%. Upon chromatofocusing the peak of activity eluted at a pH of 5.1-5.3 indicating a low isoelectric point. A native M(r) of 60-80 kDa was calculated from HPLC size exclusion chromatography. SDS-PAGE of the final purified fraction showed one major band with a M(r) of 65 kDa. These results suggest that DHAPAT is a monomeric protein. A polyclonal antiserum raised against the purified fraction was prepared in rabbits. Immunoprecipitation experiments showed complete precipitation of DHAPAT activity in fractions prepared from human placenta, liver and skin fibroblasts. Immunoprecipitation was also used to determine the residual amount of DHAPAT protein in liver from a patient with the Zellweger syndrome. A value of about 10% was found, which closely corresponds to the residual amount of enzyme activity.
The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 x 10(5) pmol dG, while antibody 1F11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6- and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/10(5) dG using 100 micrograms DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.
We describe a case of a complete hydatidiform mole with a coexistent embryo. A 19-year-old Hispanic woman presented with an intrauterine pregnancy at 7 weeks gestational age. An ultrasound evaluation at that time showed a gestational sac and fetal heart activity was observed. Four weeks later, subsequent ultrasound study revealed no evidence of an embryo, and a pattern consistent with a hydatidiform mole. Dilation and curettage was subsequently performed that showed a classic hydatidiform mole on histological examination. Chromosome analysis revealed a normal 46,XX karyotype. DNA was extracted from the placental tissue, as well as maternal and paternal blood. Molecular genetic analysis was; performed with four variable number of tandem repeats (VNTR) probes and showed the placental tissue to consist of only paternal DNA with two genomic copies of each allele studied. These findings are consistent with the diagnosis of complete hydatidiform mole and its origin from an empty ovum fertilized by a single sperm. This is the first reported case of a living embryo coexistent with a complete hydatidiform mole documented by genetic analysis.
Mitochondrial transcription factor A (TFAM), a member of the high mobility group proteins, is essential for maintenance of mitochondrial DNA (mtDNA). Most TFAM and mtDNA (both of which are normally soluble) was recovered from the particulate fraction of human placental mitochondria when extracted with the non-ionic detergent Nonidet P-40. mtDNA and TFAM were co-immunoprecipitated by anti-TFAM antibodies. TFAM was released into the supernatant by DNase I digestion of mtDNA in the particulate fraction. Thus, TFAM and mtDNA are tightly associated with each other, and it is likely that few TFAM or mtDNA molecules exist in an unbound form in mitochondria. Based on the fact that TFAM is abundant enough to wrap mtDNA entirely, these results suggest that human mtDNA is packaged with TFAM.