Biomedical subjects

E Gouin

Publications and source records attributed to E Gouin.

43 records · Page 3Linked to original sources

PubMed1993

Release of carcinoembryonic antigen from human tumor cells by phosphatidylinositol-specific phospholipase C: highly effective extraction and upregulation from LS-174T colonic adenocarcinoma cells.

Carcinoembryonic antigen (CEA), produced by gastrointestinal tumor cells, is anchored to cell membrane by a glycosyl-phosphatidylinositol moiety which can be cleaved with phosphatidylinositol-specific phospholipase C (PI-PLC). We studied the extraction of CEA from living human colon carcinoma (LS-174T, HT-29, COLO-205, and HRT-18) and pancreatic carcinoma (CAPAN) cells by PI-PLC from Bacillus cereus. The total CEA content of LS-174T cells, quantitated by Triton X-114 extraction followed by radioimmunoassay or by immunohistochemistry, was 3.5-fold higher than that of other cells (P < 0.001). The spontaneous release of CEA from LS-174T cells into culture medium was also higher than from other cells (P < 0.001), reaching 620 ng/10(7) cells (approximately 28% of cellular content) after 24 h. Overall, living LS-174T cells were highly susceptible to CEA extraction by PI-PLC, which was dependent on PI-PLC dose and on treatment time, leading in optimal conditions to the solubilization of 4100 ng/10(7) cells after 24 h (approximately 75% of total CEA). After 24 h treatment at the highest PI-PLC dose, cell lines remained viable and growing, and membrane CEA expression was not exhausted but only reduced as compared to untreated cells. At the same time, the amount of CEA solubilized by PI-PLC exceeded the CEA reduction in membranes, suggesting that enzyme treatment increased CEA turnover. This was particularly true for LS-174T cells which maintained 54% of the expression of untreated cells, whereas the amount of CEA extracted by PI-PLC reached 190% of this expression. Growing LS-174T cells thus constitute an effective material for producing high quantities of CEA by PI-PLC cleavage, especially since these cells probably "regenerate" because of enhanced turnover during PI-PLC action, thus allowing continuous CEA production. These experimental conditions also provide an interesting model for studying the modulation of CEA expression and release.

Adenocarcinoma↗

PubMed1992

L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein.

The intracellular pathogenic bacterium L. monocytogenes can spread directly from cell to cell without leaving the cytoplasm. The mechanism of this movement, generated through bacterially induced actin polymerization, is not understood. By analyzing an avirulent Tn917-lac mutant defective for actin polymerization, we have identified a bacterial component involved in this process. The transposon had inserted in actA, the second gene of an operon. Gene disruption of downstream genes and transformation of the mutant strain with actA showed that the actA gene encodes a surface protein necessary for bacterially induced actin assembly. Our results indicate that it is a 610 amino acid protein with an apparent molecular weight of 90 kd.

3T3 Cells↗

PubMed1991

Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci.

We report the identification of a previously unknown gene, inlA, which is necessary for the gram-positive intracellular pathogen Listeria monocytogenes to invade cultured epithelial cells. The inlA region was localized by transposon mutagenesis, cloned, and sequenced. inlA was introduced into Listeria innocua and shown to confer on this normally noninvasive species the ability to enter cells. Sequencing of inlA predicts an 80 kd protein, internalin. Two-thirds of internalin is made up of two regions of repeats, region A and region B, and the C-terminus of the molecule is similar to that of surface proteins from gram-positive cocci. Internalin has a high content of threonine and serine residues, and the repeat motif of region A has regularly spaced leucine residues. As evidenced by Southern blot analysis, inlA is part of a gene family. One of them is the gene situated directly downstream of inlA, called inlB, which also encodes a leucine-rich repeat protein.

Amino Acid Sequence↗

PubMed1991

Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated.

Evidence for pleiotropic activation of virulence genes in Listeria monocytogenes is presented. A complementation study of a spontaneous prfA-deletion mutant and analysis of cassette and transposon insertion mutants showed that the gene prfA activates the transcription of four independent genes which code for a phosphatidyl-inositol-specific phospholipase C (gene plcA), listeriolysin O (gene hlyA), a metallo-protease (gene prtA) and a lecithinase (gene prtC). Transcription of prfA is not constitutive. During the growth phase, two peaks of prfA transcript accumulation were observed: the first was during exponential growth, and the second was at the beginning of the stationary phase. In addition, two prfA-specific transcripts of 2.2 kb and 1 kb are detected. Early in exponential growth, prfA is co-transcribed with plcA which lies upstream prfA, giving rise to the 2.2 kb plcA-prfA transcript. In late-exponential growth and at the beginning of the stationary phase, prfA transcripts of 1 kb are predominantly detected. Our results demonstrate that since prfA controls plcA transcription, it also regulates its own synthesis.

Amino Acid Sequence↗

PubMed1986

Tumor cell biotransformation products of prostaglandin A1 with growth inhibitory activity.

The growth inhibitory effect and the fate of prostaglandin A1 (10(-6) M) were followed in cultures of rat B104 neuroblastoma and C6 glioma cells. More than 40% and 85% of the drug were neither recognized by a prostaglandin A1 antiserum nor extracted from the acidified medium with ethyl acetate, after 6 h and 24 h-incubation, respectively. When the supernatant of cells cultured in the presence of prostaglandin A1 during 24 hours was transferred to other cells and used as culture medium, the same growth inhibitory effect as with prostaglandin A1 was observed even when no prostaglandin A1 was added. After extensive purification and reverse phase HPLC of supernatant, four peaks more polar than prostaglandin A1 were shown; two of them were still active as growth inhibitors. This biotransformation was not observed with normal cells like L 929 or chick embryo fibroblasts, for which prostaglandin A1 had no inhibitory effect. The identification of these metabolites will allow the study of the structure-activity relationship.

Animals↗

PubMed1985

Growth hormone-releasing factor (GRF) stimulates PGE2 production in rat anterior pituitary. Evidence for a PGE2 involvement in GRF-induced GH release.

Rat anterior pituitaries were incubated over a 3-h period. Both PGE2 and GH were increased by GRF in a concentration-related manner (ED50: 3.5 nM and 6.5 nM, respectively). A significant correlation (r = 0.88, n = 127) was observed between GH and PGE2 release over the range of GRF concentrations tested. Among the five prostanoids analyzed, only PGE2 was selectively increased. Somatostatin lowered GH release, without any effect on PGE2 production. Indomethacin (Id) and Aspirin reduced significantly PGE2 synthesis and GRF-induced GH release. The inhibitory effect of Id was counteracted by addition of PGE2 to the medium. GRF and PGE2, at maximal concentrations, had a partial additive effect on GH release. The increase in PGE2 production and the reduced GH release in the presence of cyclooxygenase inhibitors suggest that PGE2 is involved in GRF-induced GH release.

Animals↗

PubMedDate not supplied

[Spontaneous animal models for insulin-dependent diabetes (type 1 diabetes)].

Insulin dependent (type 1) diabetes in humans is a polygenic, auto-immune disease that is characterized, among other things, by the infiltration of the islets of Langerhans by immune cells (insulite) as well as many serum auto-antibodies (including islet cell antibodies: ICA). The medical goal is to diagnose the condition at a sub-clinical stage and then to prevent the disease from developing. Spontaneous diabetic rodent models, in particular the NOD mouse and BB rat are invaluable to the continuing progress of the work aimed at better understanding the human disease. In addition to these models, the study of type 1 diabetes in larger animals, having a longer life-span would also be helpful. In dogs and cats, certain minor kinds of diabetes appear to be of type 1. The classification of diabetes types in carnivores remains poorly defined, however, epidemiological, genetic and metabolic studies are required before these diabetes can be used as operational models for the human pathology. Even if the classification of these diseases is clarified, the ethical and social considerations involved with the use of companion animals, will limit the use of these animals as models for spontaneous diabetes. The selection of a specific line of diabetic dogs should perhaps be considered.

Animal Welfare↗