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PubMed1996

Increased placental antifibrinolytic potential and fibrin deposits in pregnancy-induced hypertension and preeclampsia.

Preeclampsia is characterized by maternal hypercoagulable state and intravascular coagulation, microthromboses in several organs, and impairment of uteroplacental circulation. Excessive fibrin deposition occurs in the placenta, suggesting that disorders of placental coagulation and fibrinolysis physiologic systems may have a role in hemostasis activation. Term placentas were collected from 17 hypertensive/preeclamptic women and from 17 healthy pregnant women, and processed for both histologic and hemostasis studies. Placental fibrinoid deposition was visualized by cresyl-violet staining and quantified by histomorphometric analysis. The content in hemostasis factors was measured on extracts from homogenized placentas treated by a nonionic detergent. The percentage of villi with fibrinoid deposits was higher in the diseased placentas than in controls: 13.2 +/- 11.2 versus 6.75 +/- 2.7% (p < 0.001) for the total amount of deposits; 4.8 +/- 6.7 versus 1.5 +/- 1.0% (p = 0.04) for perivillous fibrinoid deposits, which are considered as histologic markers of intraplacental fibrin. The content in type 2 plasminogen activator inhibitor (PAI-2) antigen was higher in the diseased placentas than in controls: 124 +/- 8 versus 104 +/- 6 ng/mg placental protein (p = 0.046); there was a negative correlation between PAI-2 antigen and thrombomodulin activity (r = -0.57, p = 0.02) in the diseased placentas. No significant differences were found between the two groups for placental procoagulant tissue factor and anticoagulant thrombomodulin activities, and for the content in plasminogen activators and PAI-1 antigens. Placental antifibrinolytic potential is increased in pregnancy-induced hypertension and preeclampsia. This change, and the association of the highest PAI-2 placental concentrations with the lowest concentrations of thrombomodulin, may contribute to the prethrombotic state and to the excessive placental perivillous fibrin deposition observed in these situations.

Adult↗
PubMed1983

In vitro translation of human placental pregnancy-specific beta 1-glycoprotein (SP1).

Synthesis of SP1-glycoprotein by the human placenta was directly demonstrated, by in vitro translation of RNA extracted from full term and from early placentas in a cell-free wheat germ system followed by specific immunoprecipitation of the radioactively labeled nascent peptides. De novo synthesized SP1-glycoprotein in both RNA preparations accounted for 1-1.3% of total protein synthesis.

Cell-Free System↗
PubMed1983

Radioimmunoassay of 5 alpha-pregnane-3,20-dione. A metabolite of placental progesterone.

A radioimmunoassay (RIA) procedure has been developed for the measurement of 5 alpha-pregnane-3,20-dione (DHP) in human plasma after ether extraction of the plasma-samples, followed by column chromatography. The antiserum was generated in rabbits with 6 alpha-carboxyethylmer-captoprogesterone-BSA conjugate. The high affinity (Ka = 3.72 X 10(9) l/mol) antiserum binds 40% of 40 picograms of tritiated 5 alpha-pregnane-3,20-dione at working dilutions of 1 : 2800. Negligible cross-reactivity of the antiserum was detected with 5 beta-pregnane-3,20-dione (1.8%). Other hydroxylated pregnanes showed minor cross-reactivity (5-40%). The cross-reacting steroids were all separated from pregnanedione by one chromatographic step. The plasma levels of 5 alpha-pregnane-3,20-dione were measured from 22nd to 42nd week of normal pregnancy and compared to published data.

Female↗
PubMed1978

Physical evidence for the assembly of A and B chains of human placental collagen in a single triple helix.

Native collagen molecules containing A and B chains were isolated from pepsin-solubilised human chorionic and amniotic membrane extracts by fractional salt precipitation and DEAE-cellulose chromatography. They exhibited a circular dichroism spectrum, and a melting curve, characteristic for a triple-helical structure. Electron microscopical investigations of their segment-long-spacing crystallites revealed a molecule similar to those of the interstitial types I, II and III collagens. After denaturation, the A and B chains were separated by DEAE-cellulose chromatography and were consistently recovered in a ratio of 1:2. Renaturation experiments indicated that only the B chains are able to reform triple-helical molecules which are stable under conditions in vivo. The data support a molecular formula A(B)2 for the native collagen molecule.

Amino Acids↗
PubMed1989

Placental protein 14 (PP14) inhibits the synthesis of interleukin-2 and the release of soluble interleukin-2 receptors from phytohaemagglutinin-stimulated lymphocytes.

Crude human decidual extracts containing up to 15.0 mg/l PP14 were investigated for their effects on the release of interleukin-2 (IL-2) and of IL-2 receptor (IL-2R) from phytohaemagglutinin (PHA) stimulated lymphocytes. The crude decidual extract suppressed IL-2 production over the culture period investigated (0-90 h) with maximal suppression observed at around 66 h of culture. The suppression was dose dependent over the range of PP14 concentrations used (0-7.0 mg/l). Decidual extracts also inhibited the release of soluble IL-2R into the culture supernatants over the same period. In both cases, the specific reduction of the PP14 content of the extracts by a monoclonal anti-PP14 immunoadsorbent reduced the observed suppression. These results suggest that PP14 inhibits the production of IL-2 from mitogenically stimulated lymphocytes, and leads to a reduced IL-2R release. The mechanism for such an effect is as yet unknown; however, it may explain the previously reported immunosuppressive activity of PP14.

Cells, Cultured↗
PubMed1976

[Further purification and immunohistological localization of human placental lactogen in syncytiotrophoblasts (author's transl)].

In 1936, Ehrhadt reported a prolactin-like substance which has been identified as a second protein hormone exists in extracted substance from the placenta. To date various reports have been published, but ther are still unsolved problems concerning the specifity of the substance, and its clinical significance is unclear. The purpose of this project was to purify and investigate the biochemical, immunological properties of HPL, and try to localize HPL secretion immunohistologically. The biological properties examined by intramuscular and intradermal pigeon crop sac reactions, both showed positive results. Biochemical properties were tested by 7.5% polyacrylamide disc electrophoresis and Free Fatty Acid mobilization. By these methods, the former showed a single band by electrophoresis with purified HPL 200 mug, and the latter showed a strong positive reaction both in vivo and in vitro. Immunological propert iese were examined by immunoelectrophoresis and the Ouchterlony method. In immunoelectrophoresis, the reaction of purified HPL 80 mug and anti-HPL serum resulted in a single strong zone. In constrast, anti-serum and HGH resulted in a weak zone of reaction. We confirmed that HGH cross reacts with our anti-HGH serum to HPL. The cross reaction of HGH with anti-HPL serum was reported by Josimovich et al, and other workers. In the Ouchterlony method, no precipitation line was detected between anti-HPL serum and 50-300 iu of HCG (PREGNYL). The results of investigating the localization of secritions of HPL immunohistologically, show that the specific fluorescence was limited to the strong reacting syncytial layer in the both the early chorionic villi of normal pregnancy and in the late stages of pregnancy. And the results of examining the localization with enzyme reactions with peroxdase from horseradish Type II, the reaction of benzidine brown was limited to the syncytial trophoblastic layer as in the case of FITC. These results suggests that the localization of the secretion of HPL exists in the syncytial layer of the chorionic villi.

Animals↗
PubMed1991

[Study of trans-placental transport of methylamphotericin B in albino rats after intravenous administration].

Transplacental penetration of amphotericin B, an methyl derivative, was studied on rats after its intravenous administration. Microbiological and radioisotopic methods were used. When the microbiological method was applied the drug was administered on days 16 to 20 or on day 20 of pregnancy in a dose of 4 mg/kg. For extraction of the antibiotic dimethylformamide was added to the substrates. The labeled antibiotic was administered in a dose of 3.3 mg/kg on days 6 to 16 and on day 20 of pregnancy. It was noted that the antibiotic accumulated in the placenta. The accumulation was more pronounced after antibiotic use in the course doses. A significant part of the antibiotic was in the placenta in the bound state. The methyl derivative amphotericin B was not detected microbiologically in the umbilical cord serum, fetal organs and amniotic fluid. Neither was it detected by extraction with ++dimethylformamide. The labeled antibiotic was neither detected in the amniotic fluid and fetal organs during the whole observation period. Therefore, the methyl derivative amphotericin B did not penetrate through the placental barrier either in the free or bound state. The direct teratogenic action of amphotericin B, a methyl derivative, after its intravenous administration to female rats is likely possible.

Amphotericin B↗
PubMed1995

Peroxidase activity and glutathione content in the human first-trimester placenta and decidua.

OBJECTIVE: Recently, a novel pathway of xenobiotic oxidation by peroxidase in the placenta at term was described. Herein, we aim to determine the potential of the first-trimester placenta and decidua to activate carcinogens and mutagens by peroxidase and to scavenge free radicals by glutathione. METHODS: Placental and decidual peroxidase activity was measured using a sensitive, quantitative colorimetric kinetic assay, with O-phenylenediamine dihydrochloride (OPD) as substrate and H2O2 as co-substrate. Glutathione levels were measured using a colorimetric assay. RESULTS: Peroxidase activity in cytosolic and CaCl2-extracted (membrane-bound) fractions was inhibited by a specific inhibitor, NaN3. The membrane-bound peroxidase activity was maximal at 12 weeks of gestation while cytosolic peroxidase activity did not change. Placental glutathione content remained unchanged during the first trimester. Decidual and placental peroxidase activities were similar; however decidual glutathione content was 15-fold lower, resulting in a higher decidual peroxidase activity/glutathione ratio (p < 0.03). CONCLUSIONS: We report for the first time that peroxidase may be an important pathway for xenobiotic activation at the maternal-embryonal interface. It remains to be established whether the low glutathione content limits the ability of the decidua but not placenta to protect against genomic damage induced through xenobiotic oxidation.

Calcium Chloride↗
PubMed2004

Human placenta secretes apolipoprotein B-100-containing lipoproteins.

Supply of lipids from the mother is essential for fetal growth and development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-containing lipoproteins results in embryonic lethality. In humans, the yolk sac is vestigial. Nutritional functions are instead established very early during pregnancy in the placenta. To examine whether the human placenta produces lipoproteins, we examined apoB and microsomal triglyceride transfer protein (MTP) mRNA expression in placental biopsies. ApoB and MTP are mandatory for assembly and secretion of apoB-containing lipoproteins. Both genes were expressed in placenta and microsomal extracts from human placenta contained triglyceride transfer activity, indicating expression of bioactive MTP. To detect lipoprotein secretion, biopsies from term placentas were placed in medium with [(35)S]methionine and [(35)S]cysteine for 3-24 h. Upon sucrose gradient ultracentrifugation of the labeled medium, fractions were analyzed by apoB-immunoprecipitation. (35)S-labeled apoB-100 was recovered in d approximately 1.02-1.04 g/ml particles (i.e. similar to the density of plasma low density lipoproteins). Electron microscopy of negatively stained lipoproteins secreted from placental tissue showed spherical particles with a diameter of 47 +/- 10 nm. These results demonstrate that human placenta expresses both apoB and MTP and consequently synthesize and secrete apoB-100-containing lipoproteins. Placental lipoprotein formation constitutes a novel pathway of lipid transfer from the mother to the developing fetus.

Animals↗
PubMed2005

Global gene expression analysis of the living human fetus using cell-free messenger RNA in amniotic fluid.

CONTEXT: No molecular biological tests are available to monitor the ongoing development of human fetuses in vivo. OBJECTIVE: To determine whether cell-free fetal messenger RNA (mRNA) in amniotic fluid can be detected using oligonucleotide microarrays to study large-scale gene expression in living human fetuses, with analysis of sex, gestational age, and fetal pathology as variables. DESIGN, SETTING, AND PATIENTS: Four samples of cell-free amniotic fluid were analyzed from pregnant women between 20 and 32 weeks' gestation and undergoing amnioreduction for polyhydramnios associated with twin-twin transfusion syndrome or hydrops fetalis (cases). The control consisted of 6 pooled amniotic fluid samples from women at 17 weeks' gestation and undergoing genetic amniocentesis. After extraction from the normally discarded fraction of amniotic fluid, RNA was amplified twice, labeled, and analyzed using gene expression microarrays. MAIN OUTCOME MEASURE: Relative mRNA expression in cell-free samples of amniotic fluid from fetuses with polyhydramnios at different gestational ages vs cell-free amniotic fluid from a pooled control. RESULTS: Thirty-six percent of 22,283 probe sets represented on the arrays were present in the cell-free amniotic fluid, and a median of 20% of all probe sets differed between cases and the pooled control. Only male samples expressed 1 Y chromosome transcript. The expression of some developmental transcripts, such as surfactant proteins, mucins, and keratins, changed with gestational age by up to 64-fold. A water transporter gene transcript was increased up to 18-fold in both twin-twin transfusion samples. Placental gene transcripts were not present in any samples. CONCLUSIONS: This pilot study demonstrates that cell-free fetal mRNA can be extracted from amniotic fluid and successfully hybridized to gene expression microarrays. Preliminary analysis suggests that gene expression changes can be detected in fetuses of different sexes, gestational age, and disease status. Cell-free mRNA in amniotic fluid appears to originate from the fetus and not the placenta.

Amniocentesis↗
PubMed1997

Ontogenesis and cytomorphology of the nasal olfactory organs in the Oman shark, Iago omanensis (Triakidae), in the Gulf of Aqaba, Red Sea.

BACKGROUND: Sharks (Selachi) are among the largest predators in deep and shallow seas, feeding on live and dead prey. Olfaction is one of the central senses by which they forage, especially at night and in deep water. The organs responsible for this function are the olfactory rosettes, which are situated in their nares. This study follows the ontogenesis and cytological development of the olfactory rosettes of the Oman shark, Iago omanensis, found in the Gulf of Aqaba, Red Sea, at depths of 150-1500 m. METHODS: The sharks were collected bimonthly by means of a specially designed vertical standing net and sacrificed by an overdose of MS222. The olfactory rosettes were extracted from the adults and embryos, then fixed and prepared for EM and LM studies. RESULTS: Iago is a placental, matrotrophic species with a maximal dimension of 800 mm TL (total length). It reproduces all year round, giving birth to a maximum of four (occasionally five) young of 170-180 mm TL. In newborn and adult fish the nasal olfactory organs are as described for other sharks, composed of olfactory lamellae with secondary folds. The number of lamellae increases during embryogenesis up to a maximum of 28-32 in adults. The primary nasal placodes first appear in larvae of 10-14 mm on the dorso-lateral part of the head and then become gradually displaced to the ventral position, typical for adults. Ontogenesis of the nasal rosettes is characterized by a gradual development of the lamellae and their secondary folds, with a concomitant ripening of the sensory elements (ciliated, microvillar, and rod-like bearing cells), as well as glandular and supporting cells and cells containing kinocilia that agitate the nasal water flow. CONCLUSIONS: The released young possess functional olfactory organs and developed neural transmission across the olfactory bulb and tract, to the olfactory lobes in the brain, enabling them to forage from birth. Presented data show the occurrence in I. omanensis of two types of ciliated and microvillar cells. Ciliated and rod-bearing sensory neurons are described for the first time in sharks.

Animals↗
PubMed1997

A high-performance liquid chromatography-electrospray-tandem mass spectrometry analysis of cortisol and metabolites in placental perfusate.

A reversed-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry assay (HPLC-ESI-MS/MS) was developed to quantitate cortisol, cortisone, 20 alpha- and beta-dihydrocortisol, 20 alpha- and beta-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortisone. The technique was used to analyze perfusate from the isolated human placental lobule for cortisol and its metabolites. Analytes were prepared from the perfusion medium using C18 solid-phase extraction cartridges. The internal standard for the analyses was 6 alpha-methylprednisolone. Chromatography was performed on a Novapak C18 column at ambient temperature using 53% methanol and 47% 10 mM ammonium formate buffer (pH 4.0) as mobile phase, at a flow rate of 80 microL/min. A PE-SCIEX API III triple quadrupole instrument was used for mass spectrometric detection. An ionspray (pneumatically assisted electrospray) interface was used in negative and positive ionization mode. The assay was linear over the range 100-2000 micrograms/L for each analyte. The instrumental limit of detection was 50 pg. Assay imprecision at 400 and 800 micrograms/L was < or = 10% (total coefficient of variation). Accuracy ranged between 83.2% for 20 beta-dihydrocortisone to 102.6% for cortisone. Recovery of 1000 micrograms/L analyte ranged from 91.3% for cortisone to 109.7% for tetrahydrocortisol.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

Three-dimensional structure of membrane-bound annexin V. A correlative electron microscopy-X-ray crystallography study.

We have used electron microscopy to analyse the structure of wild-type human annexin V (recombinant and placental) and of several mutants (single and double point mutants) bound to monolayers composed of DOPS, DOPE, or brain extract (Folch fraction III). On these phospholipids and on DOPS/DOPC (3:1, w/w) protein trimers, as also found in 3-D crystals, assemble to form a hexagonal lattice with a unit vector length of about 18 nm. The resolution obtained in projection is 1.7 to 2.2 nm for wild-type and mutants. There are no significant differences between the annexin V mutants and the wild-type protein at this resolution. All proteins bind as trimers with their convex side harbouring the Ca(2+)-binding sites facing the membrane. A comparison of the 3-D reconstruction of annexin V wild-type with the high resolution crystal structure shows that the domain structure is preserved but the relative orientation of the modules (II/III) and (I/IV) is slightly changed so that the Ca(2+)-binding sites in all four domains (including the recently observed binding site in domain III) become coplanar to the membrane. The thickness of the molecule obtained in the 3-D reconstruction corresponds well with the thickness of the high resolution crystal structure indicative of peripheral binding of annexin V without substantial penetration of the membrane.

Annexin A5↗
PubMed1996

Molecular genetic etiology of twin reversed arterial perfusion sequence.

OBJECTIVE: Our purpose was to determine whether the twin reversed arterial perfusion sequence (acardiac anomaly) results from fertilization of the first or second polar body. STUDY DESIGN: Placental or fetal tissue was obtained from nine twin sets discordant for twin reversed arterial perfusion. After deoxyribonucleic acid extraction, the polymerase chain reaction was used to amplify five polymorphic microsatellite repeats. The products were differentiated by polyacrylamide gel electrophoresis, and patterns were compared within twin sets. RESULTS: Deoxyribonucleic acid fingerprinting patterns were identical in all twin sets for all primer pairs. It is calculated that the chance that any of the acardiac twins resulted from fertilization of either the first or second polar body is <4% and the chance that they all resulted from polar body fertilization is <0.001%. CONCLUSION: Twins discordant for the twin reversed arterial perfusion sequence anomaly are monozygous. Our results exclude polar body fertilization as a likely cause of this condition.

Autoradiography↗
PubMed1977

Human chorionic gonadotropin-like substance in nonendocrine tissues of normal subjects.

By means of two assay systems, a beta chain human chorionic gonadotropin radioimmunoassay and a radioreceptor gonadotropin assay, a chorionic gonadotropin-like substance was demonstrated in extracts of liver and colon obtained at autopsy from three patients who died of nonneoplastic disease. In contrast to placental chorionic gonadotropin, colon and liver chorionic gonadotropin was not bound to concanavalin A-Sepharose columns, indicating that this substance possessed little or no carbohydrate. Previous workers demonstrated that desialylated human chorionic gonadotropin possesses little or no bioactivity in vivo but retains full radioreceptor and radioimmunoassay activity in vitro. Our data suggest that the genome responsible for the human chorionic gonadotropin production is not completely suppressed in adult nonendocrine tissues, and that the chorionic gonadotropin produced by colon and liver has little or no bioactivity in vivo because of its low carbohydrate content. Since many normal tissues produce chorionic gonadotropin, bioactivity may be modulated by regulation of carbohydrate content.

Chorionic Gonadotropin↗
PubMed2002

Tumor-specific regulation of angiogenic growth factors and their receptors during recovery from cytotoxic therapy.

After cytotoxic treatment, up-regulation of vascular growth factors and their receptors may be crucial for tumor relapse or progression. To determine how cytotoxic therapy (radioimmunotherapy) alters expression of angiogenic growth factors and receptors, athymic mice bearing LoVo, GW-39, HT-29, or Calu3 human tumor xenografts were treated with one dose (240 microCi) of (131)I-MN-14 anti-CEA IgG or (295 microCi) (131)I-RS-7-3G11 anti-EGP-1 IgG. Tumors removed at 1-week intervals up to week 6 were probed by immunohistochemistry (n = 3-11 samples) for vascular endothelial growth factor (VEGF), placental growth factor (PlGF), flk-1 and flt-1, angiopoietin-1 and -2, and Tie-1 and -2. Tumor extracts were also assayed for VEGF by immunoblot and for PlGF by comparative reverse transcription-PCR. During weeks 2-5 after radioimmunotherapy, significantly up-regulated tumor cell VEGF was only detected in HT-29 (immunohistochemistry; 2-fold; week 4; P < 0.05). The increased VEGF of HT-29 was paralleled by a 2-fold (week 4) rise in VEGF receptor flk-1 on vessels as well as increased ang-2/Tie-2. HT-29, GW-39, and Calu-3 increased tumor cell expression of PlGF by week 2 (HT-29 and Calu-3, P < 0.05). In Calu-3, PlGF mRNA increased 8-fold at week 5. PlGF was detected in hypoxic areas at week 2. Vascular expression of orphan receptor Tie-1 increased in all four of the tumors by week 2 (LoVo, GW-39, and Calu-3, P < 0.05). Regulation of angiogenic factors and angiogenesis after tumoricidal therapy may be tumor-specific. Antiangiogenic therapy may require a tumor-specific combination of inhibitors for PlGF, the angiopoietins, or their receptors, in addition to VEGF/flk-1.

Angiopoietin-1↗
PubMed1988

Production and characterization of human basic fibroblast growth factor from Escherichia coli.

The cDNA sequence coding for human basic fibroblast growth factor (bFGF) has been cloned downstream of a transcription promoter recognized by the bacteriophage T7 RNA polymerase. Initiation of translation at the fgf gene has been coupled to upstream translation of a fragment of the T7 phi 10 gene. Expression of the fgf gene in this system can lead to an accumulation of approximately 40 mg/liter/A600 unit of bFGF. This material can be purified close to homogeneity from a soluble protein extract on a heparin-Sepharose column. bFGF so obtained has been shown to have bioactivity indistinguishable from human placental fibroblast growth factor in mitogenicity, synthesis of plasminogen activator, and angiogenesis assays.

Amino Acid Sequence↗
PubMed1985

Purification and properties of mitochondrial monoamine oxidase type A from human placenta.

A high yield purification scheme for monoamine oxidase A from human placental mitochondria is described. The enzyme is solubilized by a combination of treatment with phospholipase A and C and extraction with Triton X-100 and further purified by partitioning between dextran and polyethylene glycol polymers. The enzyme was obtained in 35% yield and high purity on DEAE-Sepharose CL-6B chromatography. This product, 90% catalytically active, showed a single major and several minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further purification could be achieved by additional chromatography using Bio-Gel HTP, but concomitant loss of catalytic activity occurred (enzyme remained about 60% active). The difference extinction coefficient for flavinox--flavinred at 456 nm was 10,800 +/- 350 m-1 cm-1. A sulfhydryl to flavin ratio of 7.5 was obtained when enzyme was denatured with sodium dodecyl sulfate, reduced with 2-mercaptoethanol, and titrated with 2,2'-dipyridyl disulfide. Anaerobic titration with 0.5 eq of sodium dithionite gave rise to the red anionic flavin radical, and full reduction was observed on further addition of reagent. The Km value for kynuramine was essentially the same for mitochondria (0.12 mM) and enzyme after DEAE-Sepharose CL-6B chromatography (0.17 mM). The concentration of clorgyline and deprenyl required for 50% inactivation also remained essentially unchanged. Incubation of the enzyme with 2,2'-dipyridyl disulfide caused inactivation in a biphasic manner with apparent second-order rate constants of 1230 M-1 min-1 and 235 M-1 min-1 for the rapid and slow phase, respectively. This inactivation was largely abolished by the inclusion of the competitive inhibitor amphetamine (Ki = 20 microM) in the incubation mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit molecular mass of 60-64 kDa, about 1.5-2.5 kDa higher than human liver monoamine oxidase B.

2,2'-Dipyridyl↗