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PubMed1993

Characterization of CA 125 synthesized by the human epithelial amnion WISH cell line.

CA 125 has been established as an important tumor marker for monitoring patients diagnosed with nonmucinous ovarian cystadenocarcinoma although it has also been shown to be expressed by other carcinomas, normal epithelial tissues, and fetal tissues. Current evidence implicates a role for CA 125 during early fetal development. The human epithelial amnion WISH cell line is a known secretor of CA 125. WISH cells have been investigated as a model system to characterize the structure of cell-associated and secreted CA 125 of fetal origin. CA 125 secretion was maximal in confluent monolayers of WISH cells where it averaged 2,081 units/ml/24 h. Secretion ranges from 600 to 900 units/10(6) cells/24 h. [35S]-Methionine-labelled CA 125 can be detected by 4 h and reached maximal levels of radioactive incorporation in tissue culture medium by 12 h when analyzed by immunoprecipitation with the M11 anti-CA 125 monoclonal antibody and SDS-PAGE, followed by autoradiography. Both cycloheximide and actinomycin D inhibited CA 125 synthesis. CA 125 was demonstrated to incorporate [3H]-galactose but the level of radioactive incorporation was greatly reduced when WISH cells were incubated in the presence of phenyl N-acetyl-alpha-D-galactosaminide (an inhibitor of O-linked glycosylation) or monensin (an inhibitor of intracellular protein transport within the Golgi complex). Treatment of WISH cells with tunicamycin (an inhibitor of N-linked glycosylation) only slightly decreased label incorporation.

Amnion↗
PubMed1997

CA125 phosphorylation is associated with its secretion from the WISH human amnion cell line.

Evidence is presented suggesting that CA125 is a serine and/or threonine phosphoprotein and that its secretion from the human amnion WISH cell line is closely linked to its phosphorylation. It is also indicated that regulation of CA125 secretion requires protein(s) tyrosine phosphorylation. WISH cells treated with a tyrosine phosphatase inhibitor, vanadate/ H2O2, resulted in increased levels of CA125 secretion. Exposure of vanadate-treated cells to epidermal growth factor further enhanced this secretory activity. Immunohistochemistry of vanadate-treated cells resulted in a substantial increase in not only cytoplasmic tyrosine phosphoproteins but also in membrane-associated CA125 when stained with the PY20 anti-phosphotyrosine and M11 anti-CA125 monoclonal antibodies, respectively. M11 immunoprecipitation of CA125 from cells labelled with [32P]-orthophosphate was analyzed by SDS-PAGE and autoradiography. Immunoprecipitates from cell lysates demonstrated that a phosphoprotein of > 200 kD was isolated and immunoreacted with both the OC125 and M11 anti-CA125 monoclonal antibodies by Western blotting. CA125 immunoprecipitated from vanadate-treated cells showed a marked increase in cell-associated CA125 phosphorylation. Although CA125 could be immunoprecipitated from WISH cell media incubated with [32P]-orthophosphate in the presence or absence of vanadate as detected by Western blotting, autoradiographic analysis of the Western blots revealed no [32P]-labelled CA125 co-migrating with the 200-kD plus molecule detected by M11. When the PY20 anti-phosphotyrosine monoclonal antibody was used as the probe, no tyrosine-phosphorylated CA125 was detected in cell lysates.

Amnion↗
PubMed1986

Expression of the antigens recognized by monoclonal antibodies to human amnion (GB4, GB9, GB11) on the rabbit eye during embryonic development.

Three monoclonal antibodies to human amnion (GB4, GB9 and GB11) which recognized well-defined structures of the adult rabbit eye were used to study the embryonic eye development. The reactivity of GB4 could be identified on the migrating neural crest cells under the corneal epithelium at 2 weeks of gestation. At 3 weeks of gestation, the conjunctival epithelium, corneal epithelium and corneal endothelium reacted with GB4. In the newborn rabbit, the corneal epithelium was reactive with GB9 instead of GB4; corneal endothelium was positive with GB4 at this stage, but gradually became negative in the adult rabbit; the subcapsular epithelium of the lens was recognized by GB4 when it changed from pseudostratified to simple cuboidal epithelium. During early embryonic life, GB11 was positive on the external retinal layer of the optic cup and lens vesicle. In the adult, GB11 reacted only with the pigmented epithelium of the ciliary processes. The results of this study demonstrated that the expression of antigens recognized by GB4, GB9 and GB11 could be mapped according to the epithelial differentiation of the rabbit eye during embryogenesis.

Amnion↗
PubMed1995

Prostaglandin G/H synthase-1 messenger RNA relative abundance in human amnion, choriodecidua and placenta before, during and after spontaneous-onset labour at term.

The aim of this study was to determine the relative abundance of prostaglandin G/H synthase-1 (PGHS-1) mRNA in human amnion, choriodecidua and placenta obtained before (n = 5), during (n = 5) and after spontaneous-onset labour and delivery at term (n = 5). PGHS-1 mRNA relative abundance was not affected by labour status (p > 0.1) nor differently expressed between gestational tissues (p > 0.05). These data are consistent with the hypothesis that PGHS-1 is a constitutively expressed isozyme and that an increase in the relative abundance of mRNA encoding this enzyme is not necessary for the labour-associated increase in prostaglandin formation.

Amnion↗
PubMed1994

Epidermal growth factor stimulation of lecithin release by human amnion.

Lecithin release from human amnion disks was assayed in basal conditions as well as under epidermal growth factor administration. The tissue responded to the peptide by increasing the phospholipid release. A significant effect was observed only after 30 min of treatment. A possible role of epidermal growth factor-induced amniotic lecithin release in fetal lung maturation as well as in the mechanism of delivery is discussed.

Amnion↗
PubMed1994

Modulation of amnionic adenylate cyclase and cAMP phosphodiesterase by prostaglandins E1 and F2 alpha.

The action of prostaglandins E1 and F2 alpha on adenylate cyclase and cAMP phosphodiesterase was assayed in cell membrane fractions of amnion obtained before and after labor from term physiological pregnancies. Both prostaglandins stimulate the activity of adenylate cyclase in a dose-dependent manner. After spontaneous delivery both prostaglandins stimulate the activity of phosphodiesterase at lower doses and inhibit it at higher ones while the stimulatory effect of low doses is missing before labor. Such a behavior could represent a mechanism for the preservation of the cAMP-mediated processes throughout the entire event of delivery.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Activity of lysosomal hydrolases in human placenta, amnion, decidua and myometrium in the 2nd and 3rd trimester.

Six lysosomal hydrolases were studied in tissues of human placenta, amnion, decidua, and myometrium obtained in the 2nd and 3rd trimester. No significant increase in the activity of any lysosomal hydrolase was found in the 3rd-trimester values compared with those obtained in the 2nd trimester. Thus, the increased activity of lysosomal hydrolases in maternal serum at term, previously described in several studies, seems not to be paralleled by a corresponding increase in the activity of these enzymes in the tissues of the pregnant uterus. We speculate that the increase in maternal serum towards term may reflect a macrophage activation initiated by elevated estrogen concentrations in the 3rd trimester.

Amnion↗
PubMed2003

17Beta-eEstradiol stimulates arachidonate release from human amnion-like WISH cells through a rapid mechanism involving a membrane receptor.

17beta-Estradiol (17beta-E(2)) greatly and dose-dependently stimulates [(3)H]arachidonic acid (AA) release from the human amnion-like Wistar Institute Susan Hayflick (WISH) cells. This action is abolished by the phospholipase A(2) inhibitor AACOCF(3), significantly reduced by the estrogen receptor (ER) antagonist ICI 182,780, and uninfluenced by cycloheximide. The estradiol-BSA conjugate E(2)coBSA, which binds putative membrane ERs and is unable to enter the cell, also highly stimulates [(3)H]AA release from WISH cells, although to a lesser extent compared with 17beta-E(2). The fluorescent conjugate E(2)coBSA-FITC specifically binds to the surface of a subset of intact WISH cells, and labeling intensity appears dose and time dependent. Cell permeabilization results in a dense intracellular staining, mainly in the peripheral cytoplasm. H-150, an antibody against the N terminus of human ERbeta, also labels the plasma membrane of intact WISH cells and the cytoplasm of permeabilized cells. Almost no labeling is observed using ER-21, an antibody against the N terminus of human ERalpha. RT-PCR evidences the presence of mRNA for ERbeta, not for ERalpha. Our data suggest that 17beta-E(2) stimulates [(3)H]AA release from WISH cells through an apparently nongenomic pathway and interaction with membrane binding sites. These last are, at least in part, similar if not identical to ERbeta.

Amnion↗
PubMed1983

Relaxin stimulates collagenase and plasminogen activator secretion by dispersed human amnion and chorion cells in vitro.

Dispersed cells from human amnion and chorion were cultured with and without relaxin. The addition of this hormone caused an increased secretion of both collagenase and plasminogen activator into the culture medium over a 32 h period, but had no effect on proteoglycanase or beta-glucuronidase secretion. The increase in plasminogen activator was dose-related to the amount of relaxin added in vitro. The results show that the fetal membranes are a novel target tissue for relaxin in the human, and suggest that relaxin in vivo may cause a similar release of collagenolytic enzymes, leading to the weakening and eventual rupture of the fetal membranes.

Amnion↗
PubMed1984

Growth and function of human placenta and amnion grafted to nude mice. A preliminary communication.

Normal human placenta and amnion from the 20th week of pregnancy were transplanted into nude mice. The transplants retained the histological and immunohistochemical picture of the original tissue and maintained synthesis of human chorionic gonadotropin (hCG), pregnancy-specific beta 1-glycoprotein (SP1), GnRH and placental proteins PP5, PP11 and PP12 for 5 weeks. This experimental model has a wide potential application for studies on placental protein synthesis and the hormonal influence of human placenta on other simultaneously transplanted human tissues.

Amnion↗
PubMed1992

The mechanism of the action of IFN-gamma and TPA on the modulation of epidermal growth factor receptors of human amnion cells.

We have examined the mechanism of synergistic action occurring between interferon (IFN)-gamma and 12-0-tetradecanoylphorbol-13-acetate (TPA) with respect to the reduction of 125I-epidermal growth factor (125I-EGF) binding to human amnion (WISH) cells [Karasaki Y et al (1989) J Biol Chem 264: 6158-6163]. The cells were treated with protein kinase C (PKC) inhibitors (H7, staurosporine) to investigate the role of PKC in the synergism between IFN-gamma and TPA, since TPA is a strong activator of PKC. The combined effect of IFN-gamma and TPA was blocked by the PKC inhibitor, suggesting that PKC plays an important role in the synergistic action of TPA and IFN-gamma on the inhibition of EGF binding to the cells. The prolonged incubation (24 h) of the cells with TPA resulted in the restoration of EGF binding to the cells. A 24 h treatment of WISH cells with both IFN-gamma and TPA, however, still exhibited greater than 50% inhibition of EGF binding. No PKC activity, however, was observed in the WISH cells treated with both IFN-gamma and TPA for 24 h as well as with TPA alone for 24 h, indicating that IFN-gamma may synergize with the second mediator induced by PKC rather than PKC itself in the reduction of EGF binding to WISH cells. In addition, IFN-gamma showed the synergistic action with calcium ionophores on the reduction of EGF binding to the cells, suggesting that Ca2+ may be one of the second mediators which was induced by TPA and which cooperated with IFN-gamma.

Amnion↗
PubMed1976

[Ultrastructural studies on the permeability of the amnion-epithelium for peroxidase and ferritin. Contribution on paraplacental metabolism].

By means tracer substances (horseradish peroxidase, ferritin) the permeability of human amnion and umbilical cord (2. to 6. lunar month) has been investigated ultrastructurally. The structure of intercellular cleft permits a passiv transfer of the water and electrolytes in both ways. The findings indicated, that the passage of proteins with a molecular weight of peroxidase predominantly takes place through the intercellular clefts less by means of pinocytosis. Ferritin particles can not penetrate. The epithelium of umbilical cord seems to play no special role in protein and water transport. The results were discussed.

Amnion↗
PubMed2000

Influence of oxytocin on prostaglandin E2, intracellular calcium, and cyclic adenosine monophosphate in human amnion-derived (WISH) cells.

OBJECTIVE: This study was undertaken to investigate the regulation of prostaglandin release by oxytocin and the influence of oxytocin on intracellular calcium and on the cyclic adenosine monophosphate system in human amnion-derived WISH cells. STUDY DESIGN: We determined prostaglandin E(2) release from WISH cells treated with oxytocin, evaluated the cytosolic calcium concentration in single WISH cells by confocal microscopy, and measured both intracellular cyclic adenosine monophosphate levels and adenylyl cyclase activity after oxytocin treatment. RESULTS: Treatment of WISH cells with oxytocin resulted in a concentration-dependent release of prostaglandin E(2), which was increased by lithium chloride and inhibited by indomethacin and U-73122. In single WISH cells, oxytocin increased cytosolic calcium. Moreover, the hormone lowered levels of intracellular cyclic adenosine monophosphate but did not alter adenylyl cyclase activity. CONCLUSIONS: Our data demonstrate, for the first time, that WISH cells respond to oxytocin by increasing prostaglandin E(2) release. In addition to phospholipase C activation and cytosolic calcium increase, the hormone effect involves also a reduction of the cyclic adenosine monophosphate level.

Adenylyl Cyclases↗
PubMedDate not supplied

[Kinetic of beta1-adrenergic reaction of chicken embryo amnion].

Dose-dependent curves of the inhibitory beta 1-adrenergic reaction of the chick embryo amnion were analyzed on the basis of different mathematical models. Selection of the optimal model, closest to the experimental data, allows us to obtain the reaction parameters with the smallest statistical error. Two main characteristics of the physiological reaction, EC50 and Pm, sufficed for the description of the action of agonists nonselective for beta 1-adrenoreceptors: adrenaline (EC50 0.19 +/- 0.02 microM, Pm 101.3 +/- 1.9%) and salbutamol (EC50 0.58 +/- 0.01 microM, Pm 42.7 +/- 0.3%). The Hill coefficient n in these experiments was close to 1 (1.06 +/- 0.14; 0.96 +/- 0.02). It was necessary to introduce fractional values 1 < n < 2 to describe the effects of noradrenaline and isopropyl noradrenaline.

Adrenergic beta-Agonists↗
PubMed1992

Oxytocin stimulates translocation of protein kinase C and induces antiviral state in human amnion cells.

Association of protein kinase C (PKC) activity to the membrane fraction was observed in oxytocin treated human amnion cells (UAC). In addition, oxytocin was shown to induce an antiviral state and to inhibit multiplication of vesicular stomatitis virus (VSV) in UAC. These observations together with earlier findings indicate that activation of inositol phospholipid breakdown with a consecutive activation of PKC is a common signal transduction pathway in interferon action and hormonal stimulation.

Amnion↗
PubMed1992

Migration of polymorphonuclear leukocytes through human amnion membrane--a scanning electron microscopic study.

Scanning electron microscopy has been used to examine the changes in the basement membrane structure induced by polymorphonuclear leukocytes during leukodiapedesis. A diapedesis model based on a Boyden chamber fitted with a human amnion membrane simulated inflammatory processes during which leukocytes are stimulated to leave blood vessels and to penetrate basement membranes. In the Boyden chamber, a chemotactic gradient was developed from a solution of 10(-7) M formyl-methionyl-leucyl-phenylalanine, which induced the cells to migrate through the membrane. Our observations suggest that leukocytes, like tumor cells, emigrate in a three-step process. In the first instance, they adhere to the basement membrane. A local partial proteolysis follows, which is caused by secreted metalloproteinases, especially by gelatinase. The partial dissolution of the matrix facilitates cell penetration. Active locomotion and squeezing through the residual membrane matrix allows the cells to penetrate without complete local membrane destruction. The last step involves migration of the cells through the connective tissue barrier to the site of infection. The type I collagen fibres which form this loose stroma tissue are no obstacle and can be pushed aside without proteolytic degradation.

Amnion↗
PubMed1992

Metabolic conversion of platelet-activating factor into ethanolamine plasmalogen in an amnion-derived cell line.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) labeled with 3H in the alkyl side chain was taken up rapidly by amnion-derived WISH cells in culture. The radioactivity was found in a number of cellular metabolites, principally 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) which was labeled at a rapid rate. No intracellular accumulation of lyso-PAF was detected. At longer time periods, a substantial proportion of the radioactivity was found in association with the phosphatidylethanolamine fraction extracted from the cells. This fraction contained a high proportion of the corresponding 1',2'-alkenyl derivative (plasmalogen), as judged by the formation of long-chain fatty aldehyde after exposure to acid. The magnitude of the conversion of PAF into ethanolamine plasmalogen is suggestive of a correlation between plasmalogen content and exposure to PAF in some tissues. The exact sequence of reactions leading from alkyl-acyl-GPC to the ethanolamine derivatives is yet to be established.

Amnion↗
PubMed2004

[Bridging rat sciatic nerve defects with the composite nerve-muscle autografts wrapped with human amnion matrix membrane].

OBJECTIVE: To explore the possibility of bridging the sciatic nerve defects with the composite nerve-muscle (NM) autografts wrapped with human amnion matrix membrane (HAMM) in rats. METHODS: Fifty-four Wistar rats were divided randomly into 3 groups (n = 18), and about 10 mm of right sciatic nerve defects were bridged with the composite NM autografts, the nerve autografts and the denatured skeletal muscle autografts respectively (Group A, B, and C), and all grafts were wrapped with HAMM. After the operation, the regenerated nerves were assessed by Fast blue retrograde tracing, neurofilament (NF) immnohistochemical staining, regenerated axons counting, measuring the diameter and myelin thickness of the regenerated axons, and quantifying the wet weight of tibialis anterior muscle. RESULTS: In Group A and B, there were more fluorescent-labeled cells in dorsal root ganglion and spinal cord than in Group C. The regenerated NFs were sparse and disordered in Group C, but dense and regular in Group A and B. Group A and Group B showed results superior to Group C as to other histologic and morphologic characters (P < 0.05), whereas there was no significant difference between Group A and B (P > 0.05). CONCLUSION: The composite NM autografts wrapped with HAMM in this study can well bridge and repair sciatic nerve defects in rats.

Amnion↗