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Effect of restriction of placental growth on fetal and utero-placental metabolism.

The effect of restriction of placental growth on the supply of glucose to the gravid uterus and fetus and on fetal and utero-placental metabolism of glucose and lactate was examined in this study. Endometrial caruncles were removed from 13 sheep (caruncle sheep) prior to mating, which restricted placental growth in the subsequent pregnancy. Half the fetuses of caruncle sheep were small or growth retarded, with the remainder normal in size. After insertion of vascular catheters at 110 days gestation, the caruncle sheep, together with 16 control sheep, were studied between 121 and 130 days of gestation. Glucose delivery to and consumption by the gravid uterus and its contents, both as a total and per kg of tissue mass, was significantly lower in caruncle ewes with small fetuses, although glucose extraction was similar to that in controls. Utero-placental glucose consumption was significantly lower in caruncle ewes carrying small fetuses compared to that in control ewes, both as a total and per kg of placenta. Small caruncle fetuses were hypoxaemic and hypoglycaemic and the lactate concentration in the common umbilical vein was significantly higher than in control sheep. Glucose delivery to and consumption by the fetus was significantly lower in normal-sized and in small caruncle fetuses compared to controls. Fetal glucose consumption per kg of fetus was similar in control and caruncle sheep. Fetal glucose extraction increased as fetal weight decreased. Utero-placental production of lactate was similar in control and caruncle ewes. However, uterine output of lactate decreased as placental weight fell. Utero-placental production of lactate per kg of placenta was significantly higher in caruncle ewes compared to controls and increased as oxygen content in blood from the fetal femoral artery decreased. Fetal lactate consumption per kg of fetus increased as the concentration of lactate in blood from the common umbilical vein increased. It is concluded that intrauterine growth retardation due to restriction of placental growth is associated with a reduced supply of glucose to both the pregnant uterus and fetus and a redistribution of glucose therein to the fetus, both directly as glucose and indirectly as lactate. This reflects the disproportionate maintenance of fetal weight relative to that of the placenta, reduced utero-placental consumption of glucose per kg of placenta, conversion of a greater proportion of that glucose or other substrate(s) to lactate by the placenta and an increase in the fraction of the lactate produced by utero-placental tissues that is secreted into the fetal circulation.

Animals↗

The transfer, of cocaethylene across the human term placental cotyledon perfused in vitro.

Cocaethylene is produced by transesterification of cocaine in the presence of ethanol, and there is evidence that it is more neurotoxic than cocaine. Because many women of reproductive age use cocaine and because many cocaine users also consume alcohol, the fetal toxicology of cocaethylene is of great concern. At the present time the placental transfer of cocaethylene has not been fully characterized. The objective of this article was to measure the transfer of cocaethylene across the human term placenta. The transfer of cocaethylene was measured using the in vitro dual perfusion of the human term placental cotyledon. Using a "closed-circuit" design, the extraction fraction of cocaethylene was measured to be 0.009 microgram/mL.min-1 and the transfer fraction was measured to be 0.013 microgram/mL.min-1, suggesting that the placental tissue retained some of the administered dose. Using an "open circuit" design, the clearance of the compound by the placenta was found to be 78 +/- 14% that of antipyrine clearance. The metabolism of cocaethylene by the perfused placental cotyledon was also measured using an "open circuit" design and was found to be negligible. These results indicate that the placenta does not serve as a significant physical or metabolic barrier to cocaethylene transfer from mother to child. As compared to previously reported results, the transfer of this compound across the human placenta is similar to that of cocaine. Variability in placental handling of cocaethylene may therefore determine fetal exposure to this compound.

Cocaine↗

RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris.

Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.

Adenosine Triphosphate↗

The measurement of propofol in human blood samples by liquid chromatography.

A simple, sensitive, reliable and rapid column liquid chromatographic method was developed for the measurement of propofol, a new anesthetic agent, in human maternal and placental blood. The assay involved a single extraction of the drug and internal standard, thymol, from blood buffered with 0.1 M sodium dihydrogen phosphate buffer into cyclohexane. The organic extract, basified with tetramethylammonium hydroxide, in evaporation tube was evaporated to dryness at 30 degrees C under nitrogen. The residue was redissolved in 80 microliters acetonitrile and an aliquot (25-50 microliters) of the concentrate was injected into a C18 reversed-phase column (4 microns, Nova-Pak) linked to a C18 pre-column. The mobile phase consisted of 60% (v/v) acetonitrile in distilled water containing 1% v/v glacial acetic acid and was eluted at 2.5 ml min-1. The components of the column effluent was monitored by a fluorometric detector with excitation and emission wavelengths set at 276 nm and 310 nm, respectively. The method has been used to measure propofol concentrations in blood from maternal veins and placental arteries and veins during cesarean section.

Adult↗

Increase in the relative level of type V collagen during development and ageing of the placenta.

AIM: To obtain some insight into the extracellular matrix in the placenta, changes in the composition of collagens during placental development were investigated. METHODS: Collagen was extracted from placentas (group 1, 25-30 weeks, n = 21; group 2, 31-36 weeks, n = 32; and group 3, 37-41 weeks of gestation, n = 40) and the relative concentrations of various collagens were evaluated by SDS-PAGE. RESULTS: The ratio of the intensity of the alpha 1 (III) band to that of alpha 1 (I) chain collagen in group 3 placentas were lower than those in group 1 placentas. In contrast, the ratio of the intensity of the alpha 1 (V) band to that of alpha 1 (I) chain collagen in group 3 placentas were higher than those in group 1 and group 2 placentas. CONCLUSIONS: These results suggest that type V collagen might play an important role in the function of the placenta and that an increased relative concentration of type V collagen might be closely associated with the development and ageing of the placenta.

Adult↗

Determination of Gaucher's disease phenotypes with monoclonal antibody.

Discrimination between the three clinical subtypes of Gaucher's disease based on the molecular forms of beta-glucocerebrosidase detected by monoclonal antibody is described. In normal fibroblast extracts, cross-reacting material (CRM) to human placental glucocerebrosidase is detected at Mr approximately equal to 63 000, 61 000 and 56 000. In Type 1 Gaucher's disease, the major fibroblast CRM has a Mr approximately equal to 56 000,, with less CRM seen at 61 000 and 56 000. Type 3 fibroblast extracts have a single CRM form at Mr approximately equal to 63 000. No CRM is found in Type 2 Gaucher's disease fibroblasts with monoclonal antiglucocerebrosidase antibody 8E4.

Antibodies, Monoclonal↗

Serum somatomedin levels in adults with chronic renal failure: the importance of measuring insulin-like growth factor I (IGF-I) and IGF-II in acid-chromatographed uremic serum.

Somatomedin levels measured by radioreceptor assay (RRA) or RIA on acid-ethanol-extracted or unextracted serum from patients with uremia are quite variable relative to normal values. We have investigated whether compounds accumulating in uremic serum were interfering with these RRA and RIA measurements. Insulin-like growth factor I (IGF-I) and IGF-II were separated from carrier proteins by either Sephadex G-50 acid chromatography or acid-ethanol extraction. IGF-I was measured by RIA, and IGF-II was determined by rat placental membrane RRA. IGF-I levels in the acid-ethanol extracts of serum from eight uremic adults were only 50% of the levels found in seven normal subjects, and IGF-II levels in these uremic patients were 350% of normal values. However, these significant differences were not found when comparable serum samples were acid chromatographed rather than acid-ethanol extracted; instead, IGF-I levels were 343 +/- 168 (mean +/- SD) ng/ml in uremic patients and 325 +/- 54 ng/ml in normal subjects, while IGF-II levels were 780 +/- 215 ng/ml in uremic patients and 588 +/- 46 ng/ml in normal subjects. To determine whether compounds that interfere with somatomedin assays were present in the acid-ethanol extracts of uremic serum, we acid chromatographed these extracts and found that a compound eluting in the carrier protein fraction of the uremic extract artifactually decreased IGF-I levels measured by RIA and increased IGF-II levels measured by RRA. We also found that unsaturated carrier protein binding of IGF-I and IGF-II was significantly increased in the carrier protein fraction of uremic acid-ethanol extracts at the dilutions used for somatomedin assays. We conclude that acid chromatography of unextracted or acid-ethanol-extracted uremic serum is necessary to remove a compound, probably unsaturated carrier protein, which interferes with RIA of IGF-I and RRA of IGF-II. Once this compound is removed, serum IGF-I levels (RIA) in uremic patients are not different from normal, and serum IGF-II levels (RRA) are not lower than normal.

Adult↗

Microsatellite analysis provides efficient confirmation of fetal trophoblast isolation from maternal circulation.

Fetal trophoblasts can be found in maternal circulation from an early stage of pregnancy and thus provide a potential source of DNA for non-invasive prenatal diagnosis. We have developed a two-step method for trophoblast isolation between the 8th and 12th week of pregnancy. Blood was sampled from 14 women undergoing termination of pregnancy or spontaneous abortion. Immunomagnetic beads precoated with HLA class I and II, and with anti-cytokeratin-18 monoclonal antibodies, were used to remove CD8+ and other maternal cells, and to select for fetal trophoblasts, respectively. Microsatellite analysis was performed on DNA extracted from the isolated, maternal, paternal and placental cells after PCR amplification. Recovery of the trophoblasts was confirmed in 13/14 cases (93%) by the identification of an identical microsatellite pattern for fetal and placental cells. Further evidence was the presence of heterozygous alleles of both maternal and paternal origin. The correct prediction of gender in all five male fetuses was an additional confirmation of trophoblast recovery. We conclude that trophoblasts can be effectively isolated from maternal blood in the first trimester, and by using polymorphic microsatellite markers to confirm sample purity, this method has potential future application in prenatal diagnosis.

Adolescent↗

Properties of hepatic binding sites for prolactin in ring doves (Streptopelia risoria).

Specific binding of 125I-labeled ovine prolactin (125I-oPRL) was detected in crude membrane fractions prepared from ring dove (Streptopelia risoria) liver homogenates. In characterization studies, specific binding was found to depend upon pH, incubation time, incubation temperature, and membrane protein concentration. Competitive inhibition of specifically bound 125I-oPRL was observed with human growth hormone, human and rat prolactin, and dove pituitary extract but not with turkey prolactin, human placental lactogen, and several nonlactogenic hormone preparations. Dove liver membranes showed high affinity (Kd = 3 X 10(-10) M) for binding to oPRL but had relatively low binding capacity (Bmax less than 20 fmol/mg protein). PRL binding activity in pooled liver fractions from breeding doves during early stages of incubation prior to crop sac growth did not differ markedly from that observed in doves sampled at the end of incubation when crop sac weight and serum PRL were elevated. However, binding activity was higher in pooled male liver fractions than in pooled female liver fractions at both reproductive stages. A two- to threefold increase in binding capacity was observed in pooled liver fractions from late-incubating doves following MgCl2-induced binding site desaturation. The MgCl2 treatment did not eliminate the differences in specific binding observed between male and female liver fraction pools, thus suggesting the possibility of sex-specific mechanisms of hepatic PRL binding site regulation in this species. It is concluded that the dove liver possesses specific binding sites for PRL with properties similar, but not identical, to those found in other vertebrate target tissues.

Animals↗

Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens.

We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.

Antigens, Viral↗

Identification of placental protein 14 as an immunosuppressive factor in human reproduction.

Extracts of human decidual tissue obtained in the first trimester of pregnancy showed potent suppressive activity in mixed lymphocyte cultures. These extracts contained substantial amounts of the decidual protein PP14. Purified PP14 also exhibited in vitro immunosuppressive activity, and such activity in decidual extract and purified PP14 preparations was removed by treatment with a monoclonal anti-PP14 antibody-based immunoadsorbent. PP14 was present in seminal plasma, which also exhibited immunosuppressive activity that could be reduced, but not removed, by the immunoadsorbent. PP14 may be an important immunomodulator in the human reproductive system.

Animals↗

Pro-inflammatory maternal cytokine profile in preterm delivery.

PROBLEM: The objective of this study was to determine the levels of cytokines produced by maternal peripheral blood mononuclear cells (PBMC) upon stimulation with a mitogen, with autologous placental cells and with a trophoblast antigen extract. METHOD OF STUDY: Peripheral blood mononuclear cells from 54 women with a history of successful pregnancy and 30 women undergoing preterm delivery (PTD) were stimulated with the mitogen and antigens, and the cytokine levels in mitogen-stimulated culture supernatants assessed. RESULTS: Significantly higher levels of the type 1 cytokines, interferon (IFN)-gamma and interleukin (IL)-2, were produced by the PTD group than by the normal pregnancy group, which on the contrary showed significantly greater production of the type 2 cytokines, IL-4, IL-5 and IL-10. A comparison of the ratios of type 2 to type 1 cytokines is indicative of a type 1 cytokine bias in PTD. CONCLUSIONS: These data are suggestive of a maternal type 1 cytokine bias in PTD.

Cytokines↗

The 15-deoxy-delta(12,14)-prostaglandin J(2)receptor, peroxisome proliferator activated receptor-gamma (PPARgamma) is expressed in human gestational tissues and is functionally active in JEG3 choriocarcinoma cells.

RNA was extracted from human gestational membranes and villous placental tissue following spontaneous delivery (n = 15) or elective caesarean section (n = 15) at term. The samples were subjected to Northern analysis, using a 2 kb cDNA probe for peroxisome proliferator activated receptor (PPAR)-gamma. The mRNA was detectable in all choriodecidual and villous placental samples, irrespective of mode of delivery, but was only rarely detectable in the amnion. The JEG3 choriocarcinoma cell line also expressed PPARgamma. In order to evaluate PPAR mediated transcriptional activation in JEG3 cells, the cells were transfected with pTK-PPREx3-luc, a PPAR response element (PPRE) containing luciferase reporter construct. Subsequent treatment with 10 microm 15-deoxy-delta(12,14)prostaglandin J(2)(15dPGJ(2)) resulted in an eight-fold stimulation of luciferase production relative to controls transfected with the same construct lacking the PPRE. This stimulation was concentration-dependent. These results suggest roles for PPARgamma and its ligand in lipid, steroid and inflammatory mediator homeostasis and in remodelling of gestational tissues.

Adult↗

Human placental alkaline phosphatase in liver and intestine.

Three distinct forms of human alkaline phosphatase, presumably isozymes, are known, each apparently associated with a specific tissue. These are placental, intestinal, and liver (kidney and bone). We have used a specific immunoassay and HPLC to show that placental alkaline phosphatase is also present in extracts of liver and intestine in appreciable amounts.

Alkaline Phosphatase↗

Purification and characterization of 1,25(OH)2D3 receptor from human placenta.

OBJECTIVE: To characterize the receptor for 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], we purified it from nuclear fractions of human placentae. METHODS: Human placental fractions were concentrated with ammonium sulfate, extracted from hydroxylapatite, and then chromatographed on Sepharcryl S-200 and DEAE-cellulose. RESULTS: The receptor for [1,25(OH)2D3] was purified approximately 1,500-fold. The molecular weight of the receptor was estimated to be 55 K dalton by gel filtration. The receptor fractions showed a dissociation constant (Kd) of 3.0 x 10(-10) mol/l, and adsorbed to the DNA cellulose column. D3 analogs, estradiol, and progesterone had almost no effect on 1,25(OH)2D3 binding. CONCLUSION: These properties of the 1,25(OH)2D3 receptor in human placenta are similar to those of the chicken intestinal 1,25(OH)2D3 receptor.

Female↗

Immunoregulatory activities of human trophoblasts mediated by polyamine complexes.

In a previous publication we described the presence in human placenta (HP) of immunosuppressive factors inhibiting the lymphoproliferative responses to mitogen. The results of further study reported herein indicate that the substance involved is of a syncytiotrophoblastic origin, that it is thermostable to 100 degrees C for 1 hr, and of low molecular weight, i.e. 3,500. It was defined as a polyamine conjugate with nucleic acids. Trophoblast cell extracts lost their immunosuppressive ability after heating in cultures of human lymphocytes supplemented with 5% autologous serum. These effects were, however, preserved both in cultures assayed in 5% fetal calf serum and in those to which purified polyamine oxidase (PAO) was added to autologous serum. Trophoblast cell extract was found to contain polyamine oxidases. Placental PAO can be inhibited by quinacrine a typical inhibitor of flavoprotein enzymes but not by isoniazid, an inhibitor of pyridoxal enzymes; this would suggest that the enzymes in human placenta are of a tissular rather than seric origin. The implication of these observations is that immunosuppression is mediated by oxidative products issued from an interaction between polyamine and polyamine oxidase in the syncytiotrophoblast cytosol. This interaction may constitute the basis for a local immunological barrier and may be involved in the protection of the fetus against maternal immune rejection.

Animals↗

Basic peptide with insulin-like activity in human serum.

A peptide with insulin-like activity was isolated from human plasma. In the purification, insulin-like activity (ILA) was monitored by radioreceptor assay for insulin, using human placental membrane and [125I]-insulin. ILA was extracted from Cohn fraction III with acid-ethanol and chromatographed on Sephadex G-75 in 1% formic acid. When the active fractions were subjected to ion-exchange chromatography with CM-cellulose, the ILA was adsorbed to the column at pH 5.0 and was eluted with a gradient of ammonium acetate. The chromatographic behavior of the ILA was not identical to that of somatomedin A as determined by radioreceptor assay (RRA) for the latter. On isoelectric focusing of the ILA from the CM-cellulose column, insulin-like activity was distributed over a wide pH range. The ILA that was focused at pH 7.5-9.0 was further purified by gel filtration on Sephadex G-50 in 1 M acetic acid. The specific activity of the basic ILA was approximately 200 mU insulin equivalent /mg protein. The apparent molecular weight of the material was estimated to be 7,000. It stimulated [14C]-glucose oxidation in rat epididymal fat cells and had sulfation activity in chick chondrocytes. Furthermore, the basic ILA had a potent mitogenic activity in Balb/c-3T3 cells. Thus, the basic ILA is qualified as an 'insulin-like growth factor' (IGF). It is obviously different from somatomedin A, and may be closely related to IGF-I or somatomedin C. Subsequently, RRA for the basic ILA was developed in serum concentration of the basic ILA were determined. The serum concentrations of the basic ILA were high in acromegalics and low in patients with hypopituitarism. Thus, the basic ILA is entitled to be one of the IGFs.

Chromatography, Gel↗

Calcium-dependant binding proteins associated with human placental syncytiotrophoblast microvillous cytoskeleton.

Isolated human placental syncytiotrophoblast microvillous plasma membrane vesicles were extracted with Triton X-100 to yield a detergent-insoluble residue. The residue contained approx. 50% of the total membrane protein and was qualitatively different from untreated trophoblast on SDS-polyacrylamide gel electrophoresis, Western blots and dot-immunobinding assay. Three major proteins, with molecular weights of 68, 36 and 34 kDa, dissociated from this non-ionic detergent-insoluble submembranous cytoskeletal fraction in the presence of calcium chelators. They were immunologically related to human lymphocyte cytoskeletal calcium-binding proteins, and the 36 kDa component reacted with antisera to the phospholipase A2 inhibitor, lipocortin II. Anti-lipocortin I sera did not recognise the 34 kDa protein, but did react with a series of trophoblast cytoskeletal proteins in the 34-37 kDa region. Incubation of epidermal growth factor with isolated trophoblast membrane vesicles stimulated the phosphorylation of a 36 kDa protein on tyrosine residues. Immunoprecipitation studies further showed there was no phosphorylation of the 34 kDa protein, but the 68 kDa protein was a major phosphorylated component of isolated syncytiotrophoblast membranes. p68 was principally phosphorylated on serine with slight tyrosine phosphorylation which showed an apparent increase after epidermal growth factor treatment. These results indicate a family of calcium-dependant binding proteins, some of which are phosphorylated, associated with the submembranous cytoskeleton of syncytiotrophoblast microvilli.

Calcium-Binding Proteins↗