Biomedical subjects
Publications and source records attributed to R Bourrillon.
Normal human peripheral lymphocytes, cultured in serum-deprived medium, synthetized and released serum albumin and some glycoproteins into the culture supernatant. With the use of [3H]leucine, it was shown that this biosynthetic activity was increased about 2-3 times when the mitogenic lectin from Robinia pseudo acacia was added to the lymphocyte culture medium.
Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.
The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Concanavalin A decrease cell number and proportion of cells incorporating [3H] thymidine in case of 8- and 10-day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-day-old embryo cells. No effect was noticed in 12-day cells. These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8th to the 12th day of development, and slowly increases on the 16-day cells, the affinity constant remaining rather constant. The results indicate that the age-dependent effect of lectin on embryo cells could not be directly related to the number of lectin-binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of concanavalin A. Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.
A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.
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We report here the effect of Robinia lectin and Concanavalin A on fibroblasts and liver cells from chick embryos between the 8th and the 20th day of development. This was observed in vitro after different times of cultivation. There is evidence that these lectins decrease cell number in cultures from young embryo cells but that they stimulate the proliferation of older embryo cells. The optimum concentration of either lectin was 3 mug/ml. No effect was observed on 12- and 14-day cells at the different concentrations of lectin used. The agglutination of fibroblasts by these lectins regularly decreased from the 8th to the 16th day of development. Liver cells however were agglutinated at no stage. These results could perhaps be explained in terms of cell surface changes either during the course of ontogeny or as a result of lectin treatment.
A hexasaccharide containing sialic acid has been isolated from the urine of pregnant women. It contains D-glucose, N-acetyl-D-glucosamine, and sialic acid in the proportions of 2:1:1:2. Its structure, established by methylation analysis, enzymic digestion, and potassium borohydride reduction, is that of lacto-N-tetraose with one of the residues of sialic acid linked alpha-(2 leads to 3) to the terminal nonreducing residue of D-galactose, and the other sialic acid residue linked alpha-(2 leads to 6) to the N-acetyl-D-glucosamine residue. This hexasaccharide is related to human pregnancy, as it has not been found in nonpregnant, normal-woman urine and is present in human milk.
The isolation and the chemical composition of two low-molecular-weight carbohydrate-rich fractions individualized in non-pregnant and pregnant women urine is reported. The first one is almost composed of glycopeptides whereas the second one gathers the major part of oligosaccharides. It is shown that at the 8th month of pregnancy, both fractions contain a larger amount of neutral hexoses, N-acetylneuraminic acid, N-acetylhexosamines 6-deoxyhexoses and aminoacids than in normal urine. Further, a discrimination is observed in the enhancement of each fraction. The increased amount of fraction 2 is observed as soon as the 17th week of pregnancy, whilst the amount of fraction 1 increases slowly from the 17th week until the end of pregnancy. Both fractions exhibit a tremendous increase of all carbohydrates, one week after delivery. The increased amount of glycopeptide material could be related to glycoproteins catabolism, and the increased amount of oligosaccharide material could be related to the mammary tissue metabolism.
A lectin is isolated from Vicia graminea seeds; it is purified from a crude extract after a precipitation with ammonium sulfate "DEAE"-cellulose chromatography and "sephadex G 150" gel filtration. Its homogeneity is demonstrated by different methods. It is a glycoprotein with 7.3% of carbohydrates. The native lectin is found to have a molecular weight about 100 000 and the subunit molecular weight is estimated to be 25 000 daltons. The lectin agglutinates only N erythrocytes and it is not mitogenic. The Vicia graminea lectin is the first anti N lectin which has been purified and characterized.
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A new sialyl-hexassaccharide was isolated from human pregnancy urine. It has a number of structural similarities with a sialyl-hexasaccharide of human milk so that its presence in urine is likely related to the mammary gland emtabolism during the course of pregnancy.
The conformation of the lectins from Dolichos biflorus and Robinia pseudoacacia was studied by means of circular dichroism (CD). It was found that N-acetyl-D-galactosamine induced significant changes in the near-ultraviolet CD spectrum of Dolichos lectin but was ineffective with the lectin from Robinia. Tyrosine and tryptophan chromophores were chiefly involved in this saccharide-lectin interaction. The far-ultraviolet CD spectra indicated that both lectins have a significant content of the pleated sheet conformation, but not much, if any, alpha-helix. The predominant conformation in these lectins is the aperiodic bend structure which is stabilized chiefly by hydrophobic interactions. This was ascertained by the effect of sodium dodecylsulfate on these proteins.
The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number ((0.26 plus or minus 0.03)-10-6 sites/cell) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites fo Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N-acetylgalactosamine, the determinant of Dolichos lectin, as well as by Dolichos antiserum.
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We report here the effect of Dolichos lectin on chick embryo fibroblasts from embryos between 6th and 16th day of development. There is evidence that Dolichos lectin decreases cell number and proportion of cells incorporating tritium labelled thymidine in case of chick embryo fibroblasts of 6th, 8th and 10th day of development. Dolichos lectin stimulated the proliferation of 16-day old embryo cells. No effect was noticed on 12-day embryo cells at different concentrations of Dolichos lectin used. This lectin is specifically inhibited by N-acetyl-D-galactosamine and anti-Dolichos lectin serum. The difference in response by cells during different stages of embryonic development could perhaps be explained as some regulatory changes occurring on the cell surface.
Concanavalin A and Robinia pseudoacacia lectin decreased [(3)H]thymidine incorporation into acid-insoluble material of fibroblasts cultured from 6-10-day chick embryos. In contrast, these lectins stimulated [(3)H]thymidine incorporation in cells from 16-day embryos. These effects are due to neither [(3)H]thymidine permeability modification nor toxicity of the lectins. The specificity of lectin action was proved by blocking experiments with alpha-methyl mannopyranoside and with anti-(Robinia lectin) serum.