Biomedical subjects

M Reynier

Publications and source records attributed to M Reynier.

At least 19 recordsLinked to original sources

PubMed1997

The hog intestinal mucosa acylase I: subcellular localization, isolation, kinetic studies and biological function.

The soluble acylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) from hog intestinal mucosa was 11,000-fold purified for the first time using a new four-step procedure involving an immunoaffinity chromatography. The resulting protein, which had an isoelectric point of 5.2 and a M(r) of 90,000 was composed of two apparently identical N-acylated polypeptide chains. Its amino acid composition was comparable to that of hog kidney acylase I. The enzyme had a pH optimum at 8.0 and required Zn2+ or Co2+. The optimal temperature for the acylase reaction was 40 degrees C and the activation energy of thermodenaturation was estimated at 260 kJ mol-1. The enzyme was strongly inhibited when preincubated with chelating agents, by diethyl pyrocarbonate under histidine-modifying conditions as well as by sulfhydryl compounds. The reaction of the purified enzyme with the synthetic substrate furylacryloyl-L-methionine was partly characterized as follows: Km = 0.22 +/- 0.03 mM, kcat = 128.0 +/- 17.8 s-1 and kcat/Km = 5.8 +/- 1.6 x 10(5) M-1 s-1. The L-stereoisomer of methionine competitively inhibited the enzyme reaction with a Ki of 3.4 +/- 0.2 mM. It is suggested that acylase I might not only be involved in the catabolism of intracellular N-acylated protein but also be responsible for the biological utilization of N-acylated food proteins.

Amidohydrolases↗

PubMed1995

Protective effect of gamma-hydroxybutyrate and nicotinamide on low-dose streptozotocin-induced diabetes in mice.

The purpose of this study was to investigate the effect of a treatment by gamma-hydroxybutyrate (GHB) and nicotinamide (NA) on low-dose streptozotocin (STZ)-induced diabetes in mice. Mean plasma glucose level was significantly elevated in mice given STZ by day 12 after the first STZ injection compared to controls (15.0 +/- 4.7 vs 8.0 +/- 1.6 mmol/l, p < 0.001) and 100% of the animals were severely diabetic by day 18. Plasma glucose levels remained in the normal range and no diabetic values were found in mice treated with combined treatment by GHB and NA for 25 days. However, hyperglycemia and glycosuria appeared within one week after discontinuation of the treatment. Treatment by either GHB or NA alone had only a slight and transient effect in preventing hyperglycemia. In vitro experiment on isolated pancreatic islets demonstrated that STZ-induced loss of insulin response to glucose was also counteracted by incubation with GHB and NA (Peak insulin response to 16.4 mM glucose: 0.69 +/- 0.31 vs 3.03 +/- 0.67 microU/islet/min), but not by GHB or NA alone. These results indicate that GHB and NA have complementary effects in preventing STZ-induced beta cell damage both in vivo and in vitro. This should be taken into account for future preventive strategies in human insulin-dependent diabetes mellitus.

Animals↗

PubMed1993

Carnitine action on neuromuscular disturbances in the fasting rat: potentiation by L-lysine.

L-Carnitine (oral route) significantly corrects the muscle hypocontractility and hypoexcitability induced in the rat after 5 consecutive days of fasting. This effect is interpreted on the basis of the dual role of L-carnitine as cofactor in the transport of long-chain fatty acids into the mitochondria and as a detoxifying agent of intracellular acyl-CoA. The activity of L-carnitine is increased with the concomitant administration (oral route) of an equimolar dose of L-lysine. In the present experimental conditions, there is a threefold potentiation by comparison with L-carnitine alone. This result is discussed on the basis of the combined effects of an exogenous supply of L-carnitine and endogenous synthesis of L-carnitine from the L-lysine administered at the same time.

Administration, Oral↗

PubMed1991

Differences in lipid characteristics of undifferentiated and enterocytic-differentiated HT29 human colonic cells.

In this paper we compared several lipid characteristics of the homogenate and the corresponding plasma membrane in undifferentiated and differentiated HT29 human colon cancer cells, using normal human colonic cells as a reference. Electron microscopy showed that HT29 cells were morphologically undifferentiated when cultured in the presence of either glucose or inosine without glucose at early confluency. On the contrary, HT29 cells cultured at late confluency in a glucose-free medium containing inosine or grown in nude mice exhibited an enterocytic differentiation with the presence of tight junctions and an apical brush border. The cell homogenate and the plasma membrane were prepared from each cell type. The study of specific marker enzymes showed the same degree of purity in all plasma membranes, with a highly marked increase of brush border-associated hydrolases (N-aminopeptidase and alkaline phosphatase) only in the organelles isolated from differentiated HT29 and colonic cells. Respective similar increases in the amount of free cholesterol and phospholipid and in the free cholesterol:phospholipid molar ratio were found in the plasma membrane as compared with the homogenate in all HT29 cell types. This ratio, due to an increased phospholipid content in both homogenate and plasma membrane, was lowered in colonic cells. No differences in the phospholipid profile were found between the homogenates of all cell types and the plasma membrane of undifferentiated HT29 cells, with the exception of a decrease of cardiolipin in this organelle. On the contrary, the plasma membrane phospholipid composition was different from that of the corresponding homogenate in differentiated HT29 and colonic cells. The most striking changes were a highly increased sphingomyelin amount and concomitant decreases in phosphatidylethanolamine, phosphatidylserine, and cardiolipin. Moreover, differences in the percentage of phosphatidylcholine plus sphingomyelin as well as in phosphatidylcholine:sphingomyelin, phosphatidylethanolamine, and/or phosphatidylcholine molar ratios were also found. The monounsaturated:polyunsaturated fatty acid ratio in phosphatidylethanolamine was similar in differentiated HT29 and colonic cells and lower than in undifferentiated HT29 cells. A decrease in this latter ratio in phosphatidylcholine was also observed in colonic cells and HT29 cells grown in nude mice. These changes were essentially due to opposite variations in the percentage of palmitoleic acid and those of linoleic and/or arachidonic acids in both phospholipids. Thus, these data indicate that undifferentiated HT29 cells were characterized by the absence of a specific phospholipid composition in their plasma membrane, which is suggested to be related to altered phospholipid sorting. The plasma membrane phospholipid profile reversed essentially to the normal pattern when HT29 cells recovered the ability to differentiate.

5'-Nucleotidase↗

PubMed1990

WGA binding to the surface of two autologous human melanoma cell lines: different expression of sialyl and N-acetylglucosaminyl residues.

Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetyl-glucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity.

Acetylglucosamine↗

PubMed1990

Calmodulin content and distribution in six human melanoma cell lines.

Calmodulin content and distribution between soluble and particulate fractions were determined by radioimmunoassay in six human melanoma cell lines exhibiting differences in tumor origin (primary or metastatic), degree of tumorigenicity and of pigmentation (amelanotic or melanotic). The results indicate that a) total, soluble and particulate calmodulin levels expressed as ng/10(6) cells or ng/micrograms of proteins remained constant for five out of six cell lines when cells grew from subconfluency to confluency. For IGR 37 line, derived from metastatic melanoma, the calmodulin content decreases from 2.39 to 1.27 ng/micrograms protein for total calmodulin, from 2.17 to 1.52 ng/micrograms protein for soluble calmodulin and from 2.61 to 1.02 ng/micrograms protein for particulate calmodulin, b) total, soluble and particulate calmodulin levels expressed as ng/microgram proteins were twofold (at confluency) to fourfold (at subconfluency) higher in the two cell lines from metastatic origin, IGR 37 and IPC 167. As for example, for total calmodulin, values in IGR 37 and IPC 167 cell lines, were, respectively at subconfluency, 2.39 and 2.31 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.96 ng/micrograms protein and at confluency: 1.27 and 1.98 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.90 ng/micrograms protein, c) ratio of calmodulin between soluble and particulate fractions was about 1 for the two autologous cell lines IGR 37 and IGR 39 and varies from 2 to 3 for the four other cell lines.

Calmodulin↗

PubMed1989

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 1. Molecular characterization of the binding site.

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.

Binding, Competitive↗

PubMed1987

Differences in lipid characteristics of autologous human melanoma cell lines with distinct biological properties.

Significant differences in lipid composition were found when six established human melanoma cell lines were compared. A pair of cell lines was initiated from a superficial spreading melanoma and the lymph node of the same patient; four others were also autologous, three of which originated from the same nodular melanoma and the other from its metastasis. Cell lines varied in pigmentation level and ability to grow in nude mice. Cell lines contained similar amounts of total cholesterol, glycerides, and phospholipids but different amounts of free cholesterol and cholesterol esters. In particular, the molar ratio of free cholesterol to phospholipid was increased in highly tumorigenic cell lines. No changes in phospholipid profiles were noted among cell lines, except an increase in sphingomyelin with a concomitant decrease in phosphatidylcholine in one cell line compared to the profiles of its counterpart cell line. The saturated-to-unsaturated fatty acid ratios in phosphatidylcholine and phosphatidylethanolamine were similar in all cell lines, but the monounsaturated-to-polyunsaturated fatty acid ratio in phosphatidylcholine was increased in highly tumorigenic cell lines. A significant variation in the latter ratio in phosphatidylethanolamine was also observed in the pair of autologous cell lines. These changes were unrelated to a depletion in linoleic acid in culture medium. Results obtained by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene were consistent with the differences in lipid composition between two autologous cell lines. The present results indicate that two lipid characteristics were significantly changed in highly tumorigenic cell lines as compared to cell lines with low tumorigenicity, but no correlation was found between either pigmentation level or origin (primary or metastatic) and lipid composition.

Cell Line↗

PubMed1985

[Demonstration of calmodulin in cultured human malignant melanocytes by indirect immunofluorescence].

Using calmodulin antibody, evidence for the presence of calmodulin, a calcium-binding protein modulator of numerous enzymes was shown by indirect immunofluorescence in 9 cell lines of cultured human malignant melanocytes. Calmodulin was localized in cytoplasm and in higher concentration in the perinuclear region. These observations agree with the distribution of calmodulin-binding proteins in cytoplasm and membranes.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1984

Differences in surface sialic acid and galactosyl residues of two autologous human melanoma cell lines.

Cell surface carbohydrate differences were observed between two human cell lines initiated from primary melanoma and metastasis of the same patient. Although total sialic acid content was similar in both cell lines, neuraminidase-released sialic acid was twice as high in metastatic cells than that of primary cells. One class of Concanavalin A binding sites with similar affinity constant was found in untreated and neuraminidase-treated cells in both cell lines. Before surface sialic acid release, the primary cell line expressed two classes of Ricinus lectin binding sites with high and low affinity; the cell line of metastatic origin had only one class of Ricinus lectin binding sites with low affinity. After neuraminidase treatment, the number of Ricinus lectin binding sites with low affinity increased two- or three-fold in both cell lines, whereas the high-affinity binding sites were not observed in primary cells. The present data indicated that differences in surface sialic acid level modified the Ricinus lectin binding in two human melanoma cell lines. However, the ability of the cells to bind Concanavalin A was not changed.

Adult↗

PubMed1984

Ia-like antigen expression on biologically different human melanoma cell lines.

Ia-like antigen binding of a large panel of monoclonal antibodies (six anti-human Ia-like monoclonal antibodies and ten murine anti-Ia monoclonal antibodies cross-reactive with human Ia-like antigens) were compared on seven permanent human melanoma cell lines by radioimmunoassay. Cell lines were initiated from primary or metastatic tumors and presented various levels of tumorigenicity (assessed by heterotransplantation in nude mice) and pigmentation (shown by 5-S-cysteinyldopa determination and cytological data). Two cell lines originated from the same primary melanoma, while two other pairs of cell lines originated from superficial spreading melanoma or metastatic lymph node of the same patients. Identical Ia-like allodeterminants were found in cell lines of the same individual origin. Quantitative expression of beta 2-microglobulin and Ia-like antigens was similar in all cell lines except for one, in which these molecules were expressed in lower amounts. These results indicate that Ia-like antigen expression of the cell lines is unrelated to primary or metastatic origin, degree of pigmentation and ability to grow in nude mice.

Adult↗

PubMed1977