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PubMed1994

Expression of retinoic acid receptor subtypes and cellular retinoic acid binding protein-II mRNAs during differentiation of human trophoblast cells.

An in vitro model of trophoblast cell differentiation has been used to examine the developmental expression of retinoid receptor mRNAs during the differentiation of human placental cytotrophoblast cells to syncytiotrophoblast cells. The levels of RAR alpha, RAR beta, RXR alpha mRNA increased by 12, 7, and 3 fold, respectively, during trophoblast differentiation, while the levels of RAR gamma mRNA remained relatively constant; and the increase in retinoid receptor mRNA expression preceded the increase in the expression of placental lactogen (hPL), a specific marker of syncytiotrophoblast cells. CRABP-II mRNA levels were lower in cytotrophoblast cells than in syncytiotrophoblast cells. Mobility shift assays demonstrated the appearance of a specific band that bound to a labeled DNA probe containing a retinoic acid responsive element (RARE) that was most intense from nuclear extracts from syncytiotrophoblast cells. These results suggest possible roles for these receptors in the regulation of trophoblast differentiation and hPL gene expression.

Base Sequence↗
PubMedDate not supplied

Epidermal growth factor binding and receptor distribution in term human placenta.

Human placenta has a large number of epidermal growth factor (EGF) receptors when measured either by [125I]iodoEGF binding or by protein yield after purification. To localize EGF receptors in situ in normal human term placenta, two different light microscopic methods were used. To detect unoccupied, accessible EGF binding sites on the extracellular surface of placental cells in intact blocks of tissue, samples were incubated with [125I]iodoEGF, sectioned and autoradiography performed. To detect the total pool of intracellular and extracellular EGF receptors, placental tissue was sectioned, treated with detergent, and then anti-EGF receptor antibody was localized by immunohistoperoxidase techniques. Both [125I]iodoEGF and anti-EGF receptor antibody methods showed that EGF receptors were primarily present on syncytiotrophoblast cells of placental villi. Smooth muscle cells of placental blood vessels also contained EGF receptors. Neither connective tissue cells within the core of terminal chorionic villi nor endothelium of fetal blood vessels had detectable [125I]iodoEGF binding or immunoreactive EGF receptors. Since the quantity of placental smooth muscle cells is only a small fraction compared to trophoblast cells, we conclude that syncytiotrophoblast cells are primarily responsible for the high levels of EGF receptors found in extracts prepared from human term placenta.

Autoradiography↗
PubMed2000

Modifying effects of propolis on MeIQx promotion of rat hepatocarcinogenesis and in a female rat two-stage carcinogenesis model after multiple carcinogen initiation.

The modifying effects of the dietary administration of water- and ethanol-extracted propolis produced in Brazil (WB and EB, respectively) on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) promotion of rat hepatocarcinogenesis were investigated in a medium-term liver bioassay system with use of male Fischer 344 rats. The number and area of glutathione S-transferase placental form (GST-P)-positive foci in rats given 0.5% WB were significantly increased compared with the group given MeIQx alone. Furthermore, the numbers of GST-P-positive foci were higher in rats given 0.1% WB or EB than in those given the basal diet alone. The modifying effects of propolis on other organs were also examined in female Fischer 344 rats given multiple carcinogens for initiation. Rats received water- and ethanol-extracted propolis produced in Brazil and Uruguay (WB, EB, WU, and EU, respectively) in the diet after exposure to three different carcinogens. The incidence of total mammary tumors was significantly lower in rats given EU than in the control group. These results indicate that a water extract of propolis exerts a cocarcinogenic effect on MeIQx hepatocarcinogenesis while promoting the effect at low dose in a two-stage hepatocarcinogenesis model. Moreover, they suggest that ethanol-extracted propolis may be an inhibitor of mammary gland carcinogenesis.

Alkylating Agents↗
PubMed1997

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

Amino Acid Sequence↗
PubMed2003

Rapid polymerase chain reaction-based confirmation of cat scratch disease and Bartonella henselae infection.

CONTEXT: Cat scratch disease (CSD) commonly occurs secondary to Bartonella henselae infection, and the diagnosis has traditionally been made by microscopic findings, the identification of organisms by cytochemistry, and clinical history. However, cytochemical analysis tends to be very difficult to interpret, and histology alone may be insufficient to establish a definitive diagnosis of CSD. OBJECTIVE: To demonstrate the presence of B henselae in tissue suspected of involvement by CSD, using a novel polymerase chain reaction (PCR) assay. DESIGN: Isolates of B henselae (American Tissue Culture Collection 49793) and Afipia felis (American Tissue Culture Collection 49714) were cultured on blood agar and buffered charcoal yeast extract agar, respectively. DNA was isolated from these organisms and from formalin-fixed, paraffin-embedded tissue sections with involvement by CSD (8 patients). Negative controls included water, human placental tissue, and lymph node specimens from 6 patients with reactive lymphoid hyperplasia and from 2 patients with granulomatous lymphadenitis. A primer complementary to B henselae citrate synthase gltA gene sequence was designed to perform a seminested PCR amplification. For restriction fragment length polymorphism analysis, PCR products were digested by TaqI restriction enzyme and analyzed by gel electrophoresis. RESULTS: Seminested PCR analysis of the cultured isolates of B henselae, but not of A felis, showed specific amplification. However, nonnested PCR did not provide consistently positive results in tissue sections with CSD. Therefore, we used a seminested PCR, which revealed positivity in all of the cases with clinicopathologic diagnoses of CSD. None of the negative controls showed positivity. Restriction enzyme provided confirmation of the specific PCR amplification of the B henselae sequence. CONCLUSIONS: Since the amplification product has a low molecular size (<200 base pairs), this assay is useful for detection of B henselae in formalin-fixed, paraffin-embedded tissues. The seminested PCR protocol described here can be used for rapid and reliable confirmation of B henselae in samples that are histologically suggestive of CSD.

Adolescent↗
PubMed1990

Distribution of free amino acids in streptozotocin-induced diabetic pregnant rats, their placentae and fetuses.

Amino acid levels in the non-pregnant streptozotocin (STZ)-induced diabetic rat have been shown to be abnormal. Our preliminary studies showed that placental transport, fetal serum levels and tissue uptake of the non-metabolizable amino acid alpha-amino isobutyric acid (AIB) were decreased in STZ-diabetic pregnant rats. In the present experiments, amino acid concentrations were measured in maternal (MS) and fetal (FS) sera and placentae (PL) by high performance liquid chromatography (HPLC) after triple extraction in 80% ethanol. Control (C), STZ-diabetic (D) and insulin-treated diabetic (DI) animals were studied at 22 days gestation. Pregnant diabetic rats had low serum levels of Gln, Lys, and Ser and insulin treatment corrected Gln and Ser but not Lys levels. Branched-chain amino acids did not show the large elevation characteristic of the non-pregnant diabetic rat. Placental levels of Tau, Gln, HPr, Thr and Lys were depressed in the diabetic animals and insulin treatment only partially improved these amino acid profiles. Placental amino acid levels did not always reflect maternal serum levels. Serum levels of most amino acids were lower in the fetus of the diabetic rat than in the fetus of the control rat. The notable exception was Ala which was higher in the fetuses of the diabetic animals. Insulin treatment of the mother did not correct many of the fetal amino acid levels even though maternal and fetal serum glucose levels at the time of autopsy were normal. The ability to maintain normal serum levels of many amino acids is impaired in the fetus of the diabetic rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Specific binding of cardiac glycoside drugs and endogenous digitalis-like substances to particulate membrane fractions from human placenta.

We studied the characteristics of binding of cardiac glycosides to particulate membrane fractions from human placenta, to demonstrate that placental tissue is a suitable source of receptors for digitalis drugs. Moreover, we performed preliminary experiments with 125I-labeled digoxin and placental particulates to develop a radioreceptor assay for measurement of endogenous substances with activity similar to cardiac glycoside drugs (EDLS). Placental membrane fractions were incubated with [3H]ouabain (10 nmol/L) or 125I-labeled digoxin (50 pmol/L). With both ligands, binding followed a pseudo-first-order reaction kinetics and was saturable. Scatchard analysis revealed a single class of sites [for ouabain, KD = 20.2 +/- 5.8 nmol/L (mean +/- SEM), Bmax = 3.1 +/- 0.9 nmol per gram of protein; for digoxin, KD = 29.7 +/- 1.9 nmol/L, Bmax = 24.3 +/- 1.1 nmol per gram of protein]. As expected, digoxin was less potent than ouabain in displacing both tracers from digitalis drugs receptors; progesterone, cortisone, digitoxose, furosemide, bumetanide, and propranolol had no or little effect. Specific 125I-labeled digoxin binding was competitively inhibited by plasma and (or) urine extracts from newborns, adults, pregnant women, and patients with renal insufficiency. Inhibition of binding and volume of plasma and urine assayed were linearly related. These findings support the hypothesis that cardiac glycosides and EDLS can interact with the human placenta and suggest placental tissue to be a suitable source of receptors for cardiac glycosides.

Binding Sites↗
PubMed1984

Multiple origins of transcription for the human placental lactogen genes.

Transcriptional activity of the three human placental lactogen (hPL) genes was compared in vitro and their relation to the hPL RNA species obtained from term placenta was analyzed. We used in vitro transcription system containing the HeLa cell crude extract as the source for RNA polymerase II and initiation factors. Gene fragments of identical length of 0.96 kilobase pair made from hPL1, hPL3, and hPL4 each contained the 5' flanking sequence of 497 base pairs, the first exon, the first intron, and a portion of the second exon. More than 90% transcripts of the hPL1 template were 470 nucleotides long, indicating that transcription was initiated at the proposed cap site. hPL3 and hPL4 genes generated heterogeneous RNA products of about 430, 470, 520, and 680 nucleotides suggesting that multiple start points were recognized for RNA synthesis in vitro. alpha-Amanitin sensitivity of transcription indicated that the DNA-dependent RNA synthesis was carried out by RNA polymerase II. These results show that hPL1, hPL3, and hPL4 genes have functional promoters and multiple initiation sites for transcription. Primer extension analysis of the 5' termini of hPL RNA isolated from term placenta shows that 82-83% of the transcripts are initiated at a region 29 base pairs downstream from a "TATA" sequence. This origin is observed in vitro for the transcript 470 nucleotides long. An additional upstream initiation region (-53) accounts for 8% of transcripts in term placenta and corresponds to the origin for the in vitro transcript of 520 nucleotides. At least three other sites 15, 23, and 39 base pairs downstream from the major cap site are functional in vivo. The initiation site at +40 is utilized preferentially for transcription from hPL3 and hPL4 genes in vitro. We have mapped the different transcription origins on hPL genes.

Base Sequence↗
PubMed1996

Protein kinase C in cultured human placental trophoblasts: identification of isoforms and role in cAMP signalling.

The protein kinase C (PKC) superfamily is a family of serine/threonine kinases involved in the regulation of many cell functions. The objective of the present work was to identify the PKC isoenzymes present in human placental trophoblasts and to compare their relative responses to acute and chronic phorbol 12-myristate 13-acetate (PMA) treatment. In addition, the effect of PMA treatment on ligand-stimulated cAMP production was determined. In total extracts prepared from cultured or freshly purified trophoblasts, PKC isoforms alpha, epsilon and zeta were detected. Following acute treatment with PMA, PKC alpha and PKC epsilon were translocated from the cytosol to the particulate fraction. Prolonged treatment (up to 36 h) with PMA resulted in the temporal down-regulation of PKC alpha and PKC epsilon. PKC zeta did not respond to either acute or chronic treatment with PMA. An acute 10 min treatment of the cells with PMA (10(-10)-10(-6) M) enhanced isoprenaline- and adrenaline-stimulated cAMP production. An enhanced response was observed at all concentrations of isoprenaline tested (10(-9)-10(-4) M), suggesting an increased capacity to respond. The acute PMA effect was evident within 2 min and near maximal by 5 min. The acute response to PMA was lost in cells where PKC was down-regulated by prior PMA treatment. Epidermal growth factor, a potential ligand for the activation of PKC in trophoblasts, enhanced isoprenaline-stimulated cAMP production. In summary, activation of PMA-responsive PKCs (PKC alpha or PKC epsilon) appears to enhance ligand-stimulated cAMP production in trophoblasts. This may be a physiologically important example of 'cross-talk' between various signalling pathways in human placental trophoblasts.

Animals↗
PubMed1991

[The effect of dietary Omega 6 polyunsaturated fatty acids on the activity of enzymes associated with liver mitochondrial and placental function in rats].

Female virgin rats, Wistar strain, divided into three groups of 18 each, were fed either a diet containing 45% of calories as fat (45 g%), the second received low-fat diet (15 g%), before and throughout pregnancy, and the third served as control. For both dietary fat levels, the polyunsaturated to saturated fatty acid (P/S) ratio was adjusted by substitution of saturated fatty acids for corn oil, to provide P/S of 2.0. The control group was fed a diet containing 30% of calories, as fat, with a P/S ratio of 1.0. Rats were sacrificed by decapitation 20 days after mating: fetuses, placenta and liver were then removed and weighed. Liver and placenta mitochondria were isolated. Phospholipids were extracted from mitochondrial membranes, and fatty acid tail composition was determined. Cytochrome c oxidase (a3) and rotenone-insensitive-NADH cytochrome c reductase (NADH cyt c red) in mitochondria subfractions were also assayed. The high-fat diet (45 g%) resulted in an increase in both liver and placental a3, but NADHcyt-c-red, activity did not change. The low fat diet (15 g%) reduced the activity of insensitive rotenone-NADH cytochrome c reductase. The fetal weight of the mothers fed the high-fat diet was higher (p less than 0.001) than in the other groups. No difference in fetal weight was observed between the pregnant groups fed 30% or 15% of calories (fat diets). These results suggest that changes in the fatty acid mitochondrial phospholipids which reflects the composition of dietary fat can result in changes in lipid-dependent function of integral membrane-bound enzymes. Therefore, it can be postulated that the increase of membrane fatty acid Omega 6 enhanced the a3 enzyme activity, which correlated with an increase in fetal growth.

Animals↗
PubMed1999

Ocular development in the oman shark, Iago omanensis (Triakidae), Gulf of Aqaba, Red Sea.

Ocular ontogenesis was studied in embryos of the placental viviparous shark, Iago omanensis, abundant in the Gulf of Aqaba, Red Sea, at depths of 150-1500 meters. Samples of gravid females were collected bi-monthly, and their embryos extracted. The eyes of 220 of those embryos of various dimensions were dissected and routinely prepared for histological and electron microscopic studies. The initial signs of eyes appear in embryos of 8 mm total length (TL). The primordial zone of germinal neural cells appears in 12 mmTL embryos and at 26 mm separation of the visual layer of the retina and the plexiform layers is initiated. From this stage on until 60 mmTL the nuclear and plexiform parts of the retina continue to develop and outer segments of the visual cells begin to form. Concomitant with ripening of the inner and outer plexiform layers, the tapetal layers of melanocytes and tapetal platelets of reflecting guanine also begin to ripen. The tapetum in Iago is of the cellular type. In embryos of 140-145 mmTL (6-7 months old), as they approach term, the visual cells, their synaptic connections and the intermediate cell types of the retina are all full developed. The melanocytes, rich in pigmentation, and sacs of tapetal platelets, penetrate deeply between the lamellated outer segments of the visual cells. Data are provided on growth parameters of the retinal cell layers and growth of the eyes during embryonic development. According to the position of the nuclei of the visual cells, the retina of Iago appears to be duplex, with rods and cones.

Animals↗
PubMedDate not supplied

Purification of DNA topoisomerase I from the spleen of a patient with non-Hodgkin's lymphoma.

DNA topoisomerase I (topo I) was purified 124-fold from the spleen of a patient which was diffusely infiltrated by malignant lymphoma. Extracts prepared from the lymphoma tissue demonstrated elevated topo I activity. The purification uses a new two step chromatographic method involving hydroxylapatite and Fast Protein Liquid Chromatography mono S. The purified enzyme has a molecular weight of 67 kilodaltons as determined by sodium dodecyl sulfate denaturing gel electrophoresis. Western blotting confirms the identity of the protein as topo I. The purified lymphoma topo I has a similar specific activity as topo I isolated by the same procedure from normal placenta. By using a new quantitative radiolabeled DNA relaxation assay, it was estimated that each molecule of topo I is able to relax 9.6 molecules of supercoiled DNA per min at 30C. In addition it was found that both the placental topo I and the lymphoma topo I were each 50% inhibited by 8 microns camptothecin and that the drug stimulates the lymphoma topo I to cleave supercoiled plasmid DNA. These findings indicate that the elevation of topo I measured in crude extracts of this human malignant lymphoma can be entirely accounted for by the increase in topo I protein in the tumor. Furthermore, the sensitivity of the lymphoma topo I towards camptothecin suggests that therapy with topo I directed agents might be beneficial in this tumor.

Aged↗
PubMed2005

Severe intrauterine growth restriction and trisomy 15 confined placental mosaicism: a case report and review of literature.

OBJECTIVES: To correlate confined placental mosaicism (CPM) for trisomy 15 with severe intrauterine growth restriction (IUGR) and early death after birth (age of 6 months). METHODS: Chromosome analysis was performed on amniotic fluid at 21st week of gestation, on peripheral blood at birth and on fibroblasts at death using conventional techniques. FISH was performed with Chromoprobe I-Multiprobe System kit and commercial probes according to manufacturer's protocol. DNA was extracted from parental and child peripheral blood, placenta tissue and skin fibroblasts, and UPD tests were done with microsatellites selected from Genome Data Base. RESULTS: The child presented severe hypospadias, micropenis, bilateral cryptorchidism and bifid scrotum, inguinal hernia, dolichomegacolon, severe thymic lymphatic depletion and heart hypertrophy, mainly involving right ventricle. Mosaic trisomy 15 (84%) was discovered by FISH on placental biopsy at term. Uniparental disomy (UPD) for chromosome 15 was excluded. Prenatal (amniotic fluid), postnatal (peripheral blood) karyotypes and analysis on skin fibroblast metaphases were normal. Since the autopsy showed some features suggesting genetic syndromes, such as CATCH22, Williams-Beuren syndrome, matUPD2, we excluded the presence of all these diseases. CONCLUSIONS: The only explanation for this clinical case seems to be the presence of tris15CPM that is reported as one of IUGR causes.

Adult↗
PubMed2000

Differences in umbilical venous and arterial leptin levels by mode of delivery.

OBJECTIVE: To investigate the differences in umbilical venous and arterial leptin levels by mode of delivery. METHODS: Subjects were 30 mothers who had elective cesarean deliveries and 34 mothers who had vaginal deliveries. Umbilical venous and arterial leptin levels were measured immediately after delivery. Maternal age, neonatal gender, neonatal birth weight, placental weight, and gestational duration were recorded. Inter- and intragroup comparisons were made in umbilical venous and arterial leptin levels and obstetric variables. Significant determinants of differences in umbilical venous and arterial leptin levels were investigated. RESULTS: Umbilical venous and arterial leptin levels were higher in the vaginal delivery group (n = 34) than in the cesarean group (n = 30) (P <.01). In the vaginal delivery group, umbilical venous leptin levels were significantly higher than arterial leptin levels (P <.001). These differences were still significant after adjustment for neonatal gender, neonatal birth weight, and placental weight. However, in the cesarean group, leptin levels did not differ between umbilical vein and artery. CONCLUSION: Placental leptin release is augumented during advanced labor.

Adult↗
PubMedDate not supplied

The influence of protein hormones and conceptus extracts on sheep lymphocyte transformation induced in vitro.

The study describes the effects of crude extracts of the fetal membranes of day 25 sheep conceptuses and of certain purified protein hormones and pregnancy-associated antigen on the mitogen-induced response of sheep peripheral blood lymphocytes (PBL) in vitro. A dose-dependent suppression of the proliferative response of PBL to phytohaemagglutinin, pokeweed mitogen and concanavalin-A was seen when cultures were supplemented with a soluble extract of fetal membranes. Preliminary characterization suggested that the suppressive agent was heat stable and dialysable. These properties distinguished the immunosuppressive component of membrane extracts from ovine pregnancy-associated antigen, which is heat labile and non-dialysable. However, both high and low molecular weight forms of ovine pregnancy associated antigen were also shown to inhibit lymphocyte transformation in vitro. In contrast, ovine luteinizing hormone, prolactin, growth hormone and human placental lactogen were inactive. The findings are consistent with the hypothesis that immunosuppressive substances are present in the gravid uterus during early pregnancy in the sheep.

Animals↗
PubMed1986

Microheterogeneity of Kasahara isozyme.

The microheterogeneity of Kasahara isozyme was investigated by affinity electrophoresis with Con A as the affinity ligand in combination with polyacrylamide gradient gel electrophoresis. On two-dimensional Con A-containing agarose gel electrophoresis, the Kasahara isozyme was separated into three molecular species. Kasahara isozyme electrophoresed as two distinct bands with enzyme activity on polyacrylamide gradient gel, but liver, intestinal or placental alkaline phosphatase showed only one distinct spot or band on both electrophoreses. One of the three molecular species of Kasahara isozyme separated by Con A-containing agarose gel electrophoresis was extracted from the gel and applied to the polyacrylamide gradient gel electrophoresis again, resulting in the same electrophoretic pattern as that of the original Kasahara isozyme. These findings indicated that the Kasahara isozyme consists of at least four molecular species. The same analysis was conducted with alkaline phosphatase of the HuH-6 cl-5 cell line, which has been reported to release an alkaline phosphatase closely resembling the Kasahara isozyme, and the results were compared with those obtained with the Kasahara isozyme.

Alkaline Phosphatase↗
PubMed1985

Nucleotide sequences in human chromosomal DNA from nonhepatic tissues homologous to the hepatitis B virus genome.

DNA extracted from human nonhepatic tissues (placenta and kidney) have been digested with restriction endonucleases and examined by the Southern procedure with cloned 32P-labelled DNA of hepatitis B virus (HBV). In placental DNAs of women with the history of a hepatitis B infection and in one out of four cases of patients with no known HBV exposure or manifestation, HBV-related chromosomal nucleotide sequences were detected. The integration of HBV-related sequences was observed also in human kidney DNA. Moreover, in the placenta of women who had hepatitis B infection prior to delivery, unusual unintegrated forms of HBV have been found. We conclude that HBV sequences can be found not only in hepatic tissue but also in placental and kidney DNA, both of HBV-exposed and in one case even of a nonexposed patient.

Base Sequence↗