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Autoradiographic distribution of 14C-labeled 3H-imidazo[4,5-f]quinoline-2-amines in mice.

The highly mutagenic heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), are formed during heating of protein-rich foods. In order to gain information about the distribution and fate of IQ and MeIQ in vivo, a whole-body autoradiographic study of i.v.-injected 14C-labeled IQ and MeIQ has been performed in male NMRI, pregnant NMRI, and female C3H mice. IQ and MeIQ showed similar distribution patterns. At short survival times, the autoradiograms were characterized by an accumulation of radioactivity in metabolic and excretory organs (liver, kidney, bile, urine, gastric and intestinal contents, salivary glands, nasal mucosa, and Harder's gland), as well as in lymphomyeloid tissues (bone marrow, thymus, spleen and lymph nodes) and in endocrine and reproductive tissues (adrenal medulla, pancreatic islets, thyroid, hypophysis, testis, epididymis, seminal vesicles, ampulla, and prostate). The liver and kidney cortex were identified as sites of retention of nonextractable radioactivity. IQ and MeIQ showed a strong affinity for melanin. IQ and MeIQ passed the placenta, but no radioactivity was retained in fetal tissues. The results pinpoint the liver as a site of IQ- and MeIQ-mediated toxicity. Future studies of IQ and MeIQ may be guided by and clarify the role of other tissue localizations in the toxicity of IQ and MeIQ.

Animals↗

Soluble HLA-G in human placentas: synthesis in trophoblasts and interferon-gamma-activated macrophages but not placental fibroblasts.

The HLA class Ib antigen, HLA-G, is highly expressed in early gestation placentas where it is believed to modulate maternal-fetal immunological interactions. In this study, soluble isoforms (sHLA-G) encoded by intron 4-retaining transcripts were identified in first trimester placentas by immunohistochemistry using a mAb specific for the C-terminus of sHLA-G. Immunoreactive sHLA-G protein was localized to trophoblast cells and to villous mesenchymal cells with the morphological features of macrophages. Reverse transcriptase polymerase chain reaction analysis which used primers specific for intron 4 and the 3' untranslated region of the HLA-G gene showed that transcripts encoding sHLA-G were present in the trophoblast-derived Jeg-3 cells as well as interferon-gamma-activated myelomonocytic U937 cells but were absent and uninducible in placental fibroblasts. These results indicate that placental sHLA-G is synthesized in trophoblast cells and activated placental macrophages and support the postulate that placenta-derived sHLA-G modulates maternal and fetal immune cell functions during pregnancy.

Amino Acid Sequence↗

Pregnancy in a patient with essential thrombocytosis.

An insurgent case of pregnancy in a patient in whom essential thrombocytosis was diagnosed five years earlier is described. Pregnancy was confirmed and therapy with platelet aggregation inhibitor was introduced. The pregnancy reached full term notwithstanding a positive result of the "Triple Test" during the 15th week of gestation. A histology exam of the placenta revealed an ischemic lesion. We retain that platelet aggregation inhibitor therapy remains an important aid in eliminating the risk of thrombosis determined by the presence of two conditions that are predisposed to these risks, such as pregnancy and essential thrombocytosis.

Aspirin↗

Isolation and characterization of the human gonadotropin-releasing hormone gene in the hypothalamus and placenta.

The GnRH gene has been cloned in several species, but the location of the promoter and the exact start of transcription have not previously been determined. To characterize the low abundance human GnRH mRNA in the hypothalamus and placenta, we have employed the polymerase chain reaction. The hypothalamus was found to have a 61-base pair first exon, and its transcriptional start site was determined. The human hypothalamic GnRH cDNAs isolated thus far have all contained a short 5' untranslated region which would correspond to this start site. However, all human placental GnRH cDNAs reported to date have a long 5' untranslated region, which extends more than 140-base pairs 5' to this start site in the hypothalamus, suggesting the utilization of an alternative promoter in the placenta. In addition, the human GnRH gene undergoes differential splicing in these tissues. The first intron is removed from the hypothalamic, but retained in the placental, GnRH mRNA. Thus, the placenta has a very long first exon, while the hypothalamus has a comparatively short first exon, followed by a long first intron. This characterization of the human GnRH gene will now allow hormonal regulatory studies to be performed using gene transfer techniques.

Base Sequence↗

Experimental transplantation study for possible transformation of bone marrow cells in the mouse placenta.

The aim of the present study is to establish a mouse model of the transplantation of bone marrow cells into the placenta in mid-gestation. The mononuclear fraction of bone marrow cells was isolated by Ficoll gradient centrifugation from the femur bones of C57BL/6 green fluorescent protein (GFP) gene transgenic (Tg) mice. After intraperitoneal injection of pentobarbital sodium, the abdominal cavities of pregnant non-Tg (C57BL/6 or ICR) mice were opened at 9.5 days postcoitum (dpc). The mononuclear fraction of bone marrow cells from Tg mice (3-5 x 10(5)cells/3 microl) was directly injected into the placental portion of the pregnant uterus, at a depth of approximately 3 mm, using a 31-gauge injector. The placenta was sampled at 14.5 dpc. Confocal laser scanning microscopic analysis of the serial sections of the sampled placenta (150-250 sections/placenta) was carried out to detect GFP-positive cells and to assess immunostaining for cytokeratin, CD34, p57(Kip2) and prolactin. Most pregnant mice survived until sampling of the placenta at 14.5-18.5 dpc (88.9% for C57BL6 and 100% for ICR). The survival rate of fetuses from mice in which the placenta was transplanted with GFP-positive bone marrow cells was approximately 50%. A small population (0.154%) of injected bone marrow cells was retained in the placental tissue. Immunohistochemically, cytokeratin, CD34 and p57(Kip2) were positively stained in 0.062%, 4.5% and 2.1% of GFP-positive cells, respectively, while prolactin was not positive in any of the cells examined. GFP-positive bone marrow cells were successfully transplanted to the murine placenta. Future investigations of the specific antigens in bone marrow cells retained in the placenta may enable a better understanding of the local regulation of placental development.

Animals↗

Proliferation and apoptosis in bovine placentomes during pregnancy and around induced and spontaneous parturition as well as in cows retaining the fetal membranes.

Placental growth can be achieved by either cellular proliferation or hypertrophy. Tissue regeneration and the nutrition of the fetus via embryotrophe require high rates of cellular turnover and the so-called pre-term 'maturation' of the placenta is correlated with a reduction of maternal crypt epithelial cells. Placentomes of 45 pregnant cows were collected from an abattoir to assess the role of proliferation and apoptosis in placental physiology and pathology. Placentomes were also taken from five cows undergoing premature Caesarean section and from ten naturally calving cows immediately after the expulsion of the fetus. Five of these animals had not released the fetal membranes after 12 h. Tissue sections of placentome were assessed for the Ki-67 protein; the TUNEL procedure was performed and verified by transmission electron microscopy. The maternal crypt epithelium and the fetal chorionic epithelium had a higher percentage of Ki-67-positive cells than the stroma. The percentage of Ki-67-positive cells increased significantly during pregnancy in fetal chorionic epithelium and was significantly decreased in fetal chorionic epithelium and maternal crypt epithelium after the expulsion of the fetus in comparison with tissue from month 9 of pregnancy. The number of apoptotic cells increased significantly during pregnancy in maternal crypt epithelium, maternal stroma and fetal chorionic epithelium as detected in slaughtered animals. Significantly more apoptotic fetal chorionic epithelial cells were found in animals retaining their fetal membranes in comparison with prepartum cattle during month 9 of pregnancy, at premature section and in animals releasing the fetal membranes completely. The results strongly indicate that bovine placentomes have cell type-specific rates of cellular turnover reflecting tissue growth, embryotrophe and placental maturation. Retention of fetal membranes is characterized by a large number of fetal chorionic epithelial cells undergoing apoptosis immediately after the expulsion of the fetus. This finding indicates that incomplete maturation of placentomes plays an important role in fetal membrane retention and that massive apoptosis after the expulsion of the fetus should be the consequence of diminished blood supply to the uterus, as verified in a recent study.

Animals↗

Appearance of the uterus by ultrasound immediately after placental delivery with pathologic correlation.

The purpose of this study was to compare the ultrasound appearance of the uterus immediately after the placenta was delivered with the gross and histologic findings obtained by manual exploration and sponge curettage. One hundred thirty-one patients underwent a sonographic assessment of the uterus within 5 minutes of placental delivery. A manual exploration and sponge curettage were performed with 2 minutes of the ultrasound examination, and the specimens were assessed for gross and histologic evidence of retained products of conception. Twenty-four (18.8%) of the patients had documented evidence of retained products of conception either by gross inspection or histologic assessment. Nineteen (15%) of the patients had what appeared to be retained products on ultrasound examination. Using our sonographic description of retained products of conception, the sensitivity, specificity, positive predictive value, and negative predictive value of ultrasound in detecting retained products was 44%, 92%, 58%, and 87%, respectively. The ultrasound findings in patients with retained products of conception were: a normal endometrial cavity in 9 (37.5%), echogenic mass in 6 (25%), heterogenous mixed density mass in 5 (21%), and fluid only in 4 (16.6%). Of the 6 cases with an echogenic mass, all were associated with retained products of conception. The ultrasound appearance of retained products of conception in the immediate time period after placental delivery is highly variable.

Adult↗

Examination of the placenta.

A one-minute examination of the placenta performed in the delivery room provides information that may be important to the care of both mother and infant. The findings of this assessment should be documented in the delivery records. During the examination, the size, shape, consistency and completeness of the placenta should be determined, and the presence of accessory lobes, placental infarcts, hemorrhage, tumors and nodules should be noted. The umbilical cord should be assessed for length, insertion, number of vessels, thromboses, knots and the presence of Wharton's jelly. The color, luster and odor of the fetal membranes should be evaluated, and the membranes should be examined for the presence of large (velamentous) vessels. Tissue may be retained because of abnormal lobation of the placenta or because of placenta accreta, placenta increta or placenta percreta. Numerous common and uncommon findings of the placenta, umbilical cord and membranes are associated with abnormal fetal development and perinatal morbidity. The placenta should be submitted for pathologic evaluation if an abnormality is detected or certain indications are present.

Female↗

Matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor-2 of matrix metalloproteinases (TIMP-2) in the placenta and interplacental uterine wall in normal cows and in cattle with retention of fetal membranes.

Matrixmetalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a key role in tissue re-modelling in the placenta. In the present study, distribution of MMP-2, MMP-9 and TIMP-2 was demonstrated immunohistochemically in the bovine placenta and interplacentomal tissue. Specimens representing the whole gestation until parturition were processed. Additionally, materials from cows with and without retention of fetal membranes were compared. MMP-2 expression was abundant in the maternal septae of the placentome in early gestation, with ongoing pregnancy immunoreactivity was restricted to the stromal tissue at the openings of maternal crypts. The chorionic epithelium opposite to these regions was also positive for MMP-2. MMP-9 expression was observed in the chorionic epithelium, except in the giant binucleate cells. In addition, the maternal epithelium and stroma showed immunoreactivity for MMP-9. No differences in MMP-2 and MMP-9 distribution could be observed between cows with proper release of fetal membranes and cows with retained fetal membranes. Giant binucleate cells expressed TIMP-2 during the whole gestation. Immunostaining for alpha-smooth muscle actin revealed contractile elements in the bovine placentome. Balance between proteolytic enzymes and their activators and inhibitors is essential for regular development of the placenta. The expression of TIMP-2 in the giant binucleate cells indicates an essential role of inhibitory factors during gestation. It is likely that less TIMP-2 is produced at the end of pregnancy as the number of binucleate cells is diminished.

Actins↗

Permeability and metabolic properties of a trophoblast cell line (HRP-1) derived from normal rat placenta.

The HRP-1 cell line is derived from normal rat placenta and appears morphologically similar to and retains characteristic expression of cellular markers of labyrinthine trophoblast cells. In this study, monolayers of HRP-1 cells grown on permeable supports were evaluated as a potential in vitro system to study trophoblast transport and metabolism. The cell line was shown to express and retain functional activity of the predominant placental cytochrome P450 isozyme, CYP1A1. Additionally, the HRP-1 cells retain functional activity of angiotensin I converting enzyme and carboxypeptidase N-like enzyme, peptidases characteristic of the trophoblast. The permeation of several hydrophilic, inert markers across the HRP-1 monolayers was observed to be dependent on effective molecular size and to be passive in nature. Functional asymmetry of the HRP-1 cells was illustrated by the predominant permeation of linoleic acid in the apical-to-basolateral direction across the monolayers. Transferrin passage across HRP-1 monolayers was concentration-dependent, was bidirectional, and could be inhibited by unlabeled transferrin, features typical of the trophoblast transport system for transferrin. Collectively, these properties suggest that the HRP-1 cell line may provide a useful tool for evaluating some of the permeability and metabolic properties of the trophoblast.

Animals↗

A soluble isoform of the rhesus monkey nonclassical MHC class I molecule Mamu-AG is expressed in the placenta and the testis.

The nonclassical MHC class I locus HLA-G is expressed primarily in the placenta, although other sites of expression have been noted in normal and pathological situations. In addition, soluble HLA-G isoforms have been detected in the serum of pregnant and nonpregnant women as well as men. The rhesus monkey placenta expresses a novel nonclassical MHC class I molecule Mamu-AG, which has features remarkably similar to those of HLA-G. We determined that the rhesus placenta expresses Mamu-AG mRNA (Mamu-AG5), retaining intron 4 as previously noted in HLA-G5. Immunostaining experiments with Ab 16G1 against the soluble HLA-G5 intron 4 peptide demonstrated that an immunoreactive protein(s) was present in the syncytiotrophoblasts of the chorionic villi of the rhesus placenta, within villous cytotrophoblasts, and occasionally within cells of the villous stroma. The Mamu-AG5 mRNA was readily detected in rhesus testis (although not in ejaculated sperm). Whereas an Ab against membrane-bound Mamu-AG stained few cells, primarily in the interstitium of the testis, there was consistent immunostaining for Mamu-AG5 in cells within the seminiferous tubules, which was corroborated by localization of Mamu-AG mRNA by in situ hybridization. While primary spermatocytes were negative, Sertoli cells, spermatocytes, and spermatids were consistently positive for 16G1 immunostaining. The specific recognition of the soluble Mamu-AG isoform was confirmed by Western blotting of Mamu-AG5 expressed in heterologous cells. The results demonstrate that a soluble nonclassical MHC class I molecule is expressed in the rhesus monkey placenta and testis, and confirm and extend the unique homology between HLA-G and the rhesus nonclassical molecule Mamu-AG.

Amino Acid Sequence↗

Free amino acid distribution inside the first trimester human gestational sac.

The trophoblast functions of nutrient transport and protein synthesis generate high concentrations of amino acids in the placenta and in fetal blood during the second half of pregnancy, but little is known about these metabolic processes in embryonic and early fetal periods. The aim of this study is to compare the distribution of amino acids inside the first trimester gestational sac. Free amino acid concentrations were measured in homogenates of placental villi, in samples of coelomic and amniotic fluid, and in the maternal serum from 17 normal pregnancies between 7 and 11 weeks of gestation. Significant positive relationships between maternal serum and placental tissue were found for 10 amino acids, indicating that active amino acid transport and accumulation by the human syncytiotrophoblast occurs as early as 7 weeks of gestation. The transplacental flux of most amino acid transport from maternal blood to the exocoelomic cavity was against a concentration gradient. The highest placental amino acid concentrations were found for taurine, glutamic acid, glycine and alanine. The amniotic fluid contained lower mean concentration of all amino acids than coelomic fluid and maternal serum. The concentration distribution of individual amino acids in coelomic and amniotic fluid were related indicating a passive transfer through the amniotic membrane. A coelomic-maternal gradient was observed in 19 out of 24 amino acids measured and positive correlations were found between maternal serum and coelomic fluid for concentrations of alpha-aminobutyric acid, tyrosine and histidine, suggesting that these amino acids are only partially retained and/or transferred more rapidly by the early placenta.

Alanine↗

Different combinations of regulatory elements may account for expression of the glycoprotein hormone alpha-subunit gene in primate and horse placenta.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitaries of all mammals and in the placentas of primates and horses. In humans, tandem cAMP response elements (CREs), located in the proximal promoter-regulatory region of the alpha-subunit gene, act together with an adjacent upstream regulatory element to confer placenta-specific expression. Here, we report that the alpha-subunit genes of Old World Monkeys contain a single functional CRE. This suggests that tandem CREs are unique to higher primates and humans and are not absolutely required for placenta-specific expression. In contrast, the comparable promoter-regulatory region of the horse alpha-subunit gene lacks a functional CRE but appears to retain a functional upstream regulatory element. This suggests that acquisition of placenta-specific expression of the alpha-subunit gene occurred independently in these distantly related mammals. As a result, different combinations of cis-acting elements may explain why expression of the alpha-subunit gene only occurs in placenta of primates and horses.

Animals↗

Secretion of pro-apoptotic intron 4-retaining soluble HLA-G1 by human villous trophoblast.

One major materno-fetal interface in the human placenta is constituted by the syncytiotrophoblast, in contact with maternal blood of the intervillous space, which derives from differentiation and fusion of the villous cytotrophoblast (vct). In the present work, we purified vct from term placenta by depleting HLA class I- and class II-positive cells. We found by RT-PCR that both soluble intron 4-retaining HLA-G1 (sHLA-G1) and HLA-G2 isoforms were transcribed in purified vct. Using different HLA-G-specific mAb, we demonstrated by intracellular flow cytometry, Western blotting and ELISA, that sHLA-G1 but no soluble HLA class Ia molecule was secreted by vct. We then purified sHLA-G1 from vct culture supernatant and found that it exhibited an unusual glycosylation pattern. Finally, we showed that such trophoblast-derived sHLA-G1 triggered specific apoptosis of activated CD8+ T cells. Taken together, these results demonstrated that vct did secrete functional sHLA-G1 in primary culture and suggested that, in vivo, sHLA-G1 might be an important immunomodulatory molecule controlling the activity of maternal immune effector CD8+ cells circulating in the blood that immerses chorionic villi.

Apoptosis↗

Interaction with lectin of kappa opioid binding sites solubilized from human placenta.

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.

Acetylglucosamine↗

Amino acid pools in the feto-maternal system.

The determinations of the amino acid pools, including tryptophan (by fluorescence spectrometry), in the arterial and venous blood of the mother, the arterial and venous blood of the fetus (cord blood), the amniotic fluid and the placenta was done in eight women at the moment of delivery. Only nine amino acids (asp, try, met, phe, ser, cys, lys, gly, thr) were significantly retained and four (arg, glu, pro and glu-NH2) were significantly released by the fetal tissues. In contrast with this behavior most amino acids were retained by the maternal tissue, try, phe and hist showing the highest retention. When the amino acids are grouped as essential and nonessentials, the maternal tissues retained both categories without apparent discrimination, while the fetal tissues retained essential amino acids preferentially. Our results emphasize the importance of the placenta as the regulating system of the fetal milieu under normal conditions. Thus human fetal blood levels of amino acids are patterned after the placental ones and not after the maternal values obtained at the same time. It is apparent that the placenta seems to function as a nonspecific retention filter for outgoing amino acids, but that its function is selective in respect to the release of amino acids into the fetal circulation.

Amino Acids↗

The human placenta--a rich source of beta-adrenergic receptors: characterization of the receptors in particulate and solubilized preparations.

A crude particulate fraction of human placenta possesses a high concentration of beta-adrenergic receptors, as determined by (-)-[3H]dihydroalprenolol binding (approximately 240 fmol/mg protein; Kd approximately 2 nM). The sites display all the typical characteristics of beta-adrenergic receptors, including rapid and reversible kinetics, saturability, and appropriate specificity and stereospecificity. Computer modelling of ligand binding data indicate that the binding of (-)-[3H]dihydroalprenolol to these sites conforms closely to the pattern anticipated for interactions of the ligand with a homogeneous class of receptors according to the law of mass action. The rejeptors are readily solubilized with digitonin, retaining their typical beta-adrenergic characteristics. The human placenta is likely to be a particularly useful source of beta-adrenergic receptors for purification because of its high receptor content and its ready availability in substantial quantities.

Adrenergic beta-Antagonists↗