Biomedical subjects

G C Douglas

Publications and source records attributed to G C Douglas.

42 records · Page 3Linked to original sources

PubMedDate not supplied

Expression of transferrin receptors during differentiation of human placental trophoblast cells in vitro.

In most cell types, transferrin receptor expression is correlated with the proliferation rate, being increased by growth stimulation, or decreased by induction of terminal differentiation. In the human placenta the multinucleated syncytiotrophoblast, in direct contact with maternal blood, is derived by differentiation from mononucleated cytotrophoblast. In this study we examined changes in transferrin receptor expression during in vitro differentiation of trophoblast. Cells cultured in Ham's/Waymouth's medium (HWM) remained primarily mononuclear throughout the study, whereas incubation in keratinocyte growth medium (KGM) led to formation of multinucleate masses within 2-3 days of culture. Cell surface binding of 125I-labelled transferrin increased fivefold between days 1-5 of culture in both media and surface receptors were saturated at 7-14 micrograms/ml (90-200 fM). At saturation, the amount of transferrin bound to syncytiotrophoblast was 37 per cent lower than in cytotrophoblast. Scatchard analysis revealed a reduction in the number of surface transferrin receptors in syncytiotrophoblast compared to cytotrophoblast. A corresponding 29 per cent reduction in the binding of transferrin to intracellular sites was observed in syncytiotrophoblast. Distribution of receptors between surface and intracellular sites was therefore similar in both cytotrophoblast and syncytiotrophoblast. The affinity of transferrin for transferrin receptors was 3.7-fold higher in syncytiotrophoblast when compared to cytotrophoblast. Observed differences between the two cell types were not due to the presence of growth factors or higher iron levels in KGM. Expression of a high number of surface transferrin receptors in syncytiotrophoblast (1.5 x 10(12)/mg protein), along with a high affinity of these receptors for iron-saturated transferrin, could help explain the efficient transport of large amounts of iron from mother to fetus.

Binding Sites↗

PubMedDate not supplied

Interaction of 125I-labelled ceruloplasmin with human trophoblast cells in vitro.

In vitro binding, internalization and release of the plasma protein ceruloplasmin was investigated using primary cultures of human placental trophoblast cells. Binding of 125I-labelled ceruloplasmin at 4 degrees C reached equilibrium by 5-6 h; binding was linear throughout all concentrations tested (1 nM-3.3 microM). Addition of greater than 5-10 microM unlabelled ceruloplasmin or a variety of other proteins (albumin, transferrin, IgG) were equally effective in displacing bound ceruloplasmin in a concentration-dependent manner. When cells were incubated at 37 degrees C, the majority of surface-bound 125I-labelled ceruloplasmin was released directly to the extracellular medium. Trypsin-resistant radioactivity increased to 18 per cent of initially bound ceruloplasmin within 1 min, declining to 5 per cent by 2 h. The acquisition of trypsin-resistant radioactivity was unaffected by the addition of a variety of metabolic inhibitors and no evidence of intracellular degradation of ceruloplasmin was found. In summary, our results suggest that the majority of ceruloplasmin binding to trophoblast cells is nonspecific, of low affinity, and easily dissociable at 4 degrees C. Only a small amount of ceruloplasmin appeared to be internalized, by an as yet unknown mechanism.

Ammonium Chloride↗

PubMedDate not supplied

Colchicine inhibits human trophoblast differentiation in vitro.

When cultured in keratinocyte growth medium, mononuclear cytotrophoblast cells aggregate into multicellular colonies which then fuse to form multinucleated syncytiotrophoblast. In an attempt to characterize better the mechanism of human trophoblast differentiation and to obtain information about the role of the cytoskeleton, experiments were performed using cytoskeletal-disrupting drugs and primary cultures of cytotrophoblast cells from term placentae. Addition of colchicine 6 h after plating permitted aggregation but the cells did not form syncytiotrophoblast, as revealed by staining for desmosomes and nuclei. Staining with an anti-tubulin antibody showed that microtubules were present in untreated control cells but absent in colchicine-treated cultures. If colchicine was added 24 h after plating, the cells also failed to differentiate. When cells were exposed to colchicine for the first 24 h after plating and then cultured in the absence of the drug, differentiation proceeded normally. Cells exposed to colchicine for 48 h and then incubated in the absence of the drug failed to form syncytiotrophoblast. The results suggest that a decision to differentiate is made between 24 and 48 h after plating. The effects of colchicine were observed between 2.5 and 250 microM. Beta-lumicolchicine blocked differentiation at 250 microM but was ineffective at lower concentrations. Colchicine also inhibited HCG secretion in a dose-dependent manner; beta-lumicolchicine only caused inhibition at 250 microM. Staining with antitubulin antibody revealed that lumicolchicine-treated cells had intact microtubules. These results suggest a role for microtubules in trophoblast differentiation.

Actin Cytoskeleton↗

PubMedDate not supplied

Uptake and processing of 125I-labelled transferrin and 59Fe-labelled transferrin by isolated human trophoblast cells.

Trophoblast cells isolated from term human placenta and maintained as an adherent culture express surface receptors for transferrin as indicated by quantitative binding studies using 125I-labelled transferrin. The Kd was 5.3 x 10(-9) M. About 36 per cent of the total cell receptor population was found at the cell surface, the remainder being intracellular. Both 125I-labelled and 59Fe-labelled transferrin were internalized by receptor-mediated endocytosis with similar rates. Pulse-chase experiments showed that 125I-labelled transferrin was recycled and released back to the medium, whereas 59Fe accumulated intracellularly and was released slowly. Polyacrylamide gel electrophoresis followed by autoradiography revealed that 59Fe was accumulated by cells largely in the form of ferritin. A small intracellular pool of low molecular weight 59Fe was also detected. In the presence of monensin, the transfer of 59Fe to ferritin was greatly reduced. The nature and amount of 59Fe released from cells could be modulated by the incubation conditions. In the absence of chelating agents and iron salts, released 59Fe was found to be associated with a low molecular weight fraction as well as with transferrin and ferritin. The low molecular weight 59Fe readily formed a complex with added chelators such as apotransferrin, DTPA or desferrioxamine. The release of 59Fe could be increased by repeatedly changing the medium during the course of the incubation. 59Fe release from trophoblast cells exceeded the release of lactate dehydrogenase and also exceeded the release of 59Fe from 3T3 fibroblasts, suggesting a cell-specific process.

Cell Survival↗

PubMedDate not supplied

Secretion and biosynthesis of N-acetyl-beta-D-hexosaminidases by human placental tissues in vitro.

Chorionic villi selectively secrete the B isoenzyme and, to a lesser extent, the A isoenzyme of lysosomal beta-hexosaminidase when incubated in vitro, suggesting that this tissue could contribute to maternal serum beta-hexosaminidase B and A activities in vivo. The results make it unlikely, however, that the placenta is the source of the I2 isoenzyme found in maternal serum. Samples of amnion and term chorion laeve secreted predominantly the B isoenzyme whereas first-trimester chorion laeve secreted the A, B and I2 isoenzymes. Amnion and chorion laeve may, therefore, be a source of the A, B and I2 activities found in amniotic fluid. Chorionic villi supported the cycloheximide-sensitive incorporation of [3H]leucine into immunoreactive beta-hexosaminidase thereby providing direct evidence for enzyme biosynthesis. Newly synthesized beta-hexosaminidase was detectable in the medium only after extended incubation and the secretion of beta-hexosaminidase activity continued at normal rates for several hours in the absence of protein synthesis. These results indicate that enzyme synthesis is not a primary factor in the regulation of enzyme secretion.

Chromatography, DEAE-Cellulose↗

PubMedDate not supplied

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on chorionic gonadotropin secretion by human trophoblasts.

This study tests the idea that the environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), affects human trophoblast differentiation and alters the secretion of chorionic gonadotropin (CG). Primary cultures of cytotrophoblast cells were incubated under differentiation-inducing and nondifferentiation-inducing conditions in the presence or absence of different concentrations of TCDD. Levels of immunoreactive CG as well as bioactive CG were measured in culture supernatants. TCDD caused a significant increase in the secretion of immunoreactive CG from differentiated trophoblast cultures but had no effect on the secretion of bioactive hormone. The net effect was a TCDD-dependent reduction in the CG bioactive/immunoreactive (B/I) ratio for differentiated trophoblast cultures. TCDD had no effect on immunoreactive or bioactive CG secretion by undifferentiated trophoblasts. Immunocytochemical studies showed that TCDD had no effect on the morphologic differentiation of trophoblast cells as determined by staining nuclei and desmosomal proteins. On the other hand, immunocytochemical staining for CG was increased in cells exposed to TCDD compared to control cells. These in vitro results support earlier in vivo studies in macaques suggesting that trophoblast is a target for TCCD and that TCDD-induced early pregnancy loss is accompanied by a decrease in the CG B/I ratio.

Cell Nucleus↗