Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity.

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the Vmax. In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity.

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.

Analysis of HLA-DR antigen glycosylation by serial lectin affinity chromatography.

The structure of the N-linked oligosaccharides of HLA-DR molecules from an HLA-DR homozygous B-lymphoblastoid cell line (CA) was characterized by serial lectin affinity analysis. Glycans lectin affinity profile were obtained for the HLA-DR complex and separated alpha- and beta-chains. Two structurally distinct glycosylation patterns were detected for the alpha-chain species, the alpha 1 with high-mannose- and complex-type oligosaccharides and alpha 2 with a complex-type one. In contrast, the beta-chain species contains only complex-type oligosaccharides. Further oligosaccharide heterogeneity is found for each alpha 1-, alpha 2- and beta-chain described. In contrast to murine and guinea-pig Ia antigens, no significant amount of glycopeptides with biantennary structure was found in HLA-DR.

Nature of the interaction between Ricinus communis agglutinin and blood cells.

Binding of Ricinus communis agglutinin (RCA 120) to carbohydrate receptors of human lymphocytes and erythrocytes is enthalpically driven. As in the case of simple saccharides, the delta S contribution is always unfavorable to the interaction. This result is different from that observed for other lectins and might indicate that hydrophobic interactions do not play a dominant role in binding of RCA 120 to cell surfaces.

Interaction between the Ricinus sanguineus agglutinin and receptor sites isolated from normal human lymphocytes.

Solubilized surface proteins from normal human lymphocytes were obtained by mild trypsin digestion. The binding of membrane components to labelled Ricinus sanguineus agglutinin (molecular weight = 120,000 daltons) was studied by a gel filtration method. The bound and unbound lectin amounts were determined from the gel filtration patterns. The binding parameters were calculated from Scatchard plots. They were compared to the parameters obtained at the same temperature for the lectin-intact lymphocyte system. The respective values for the affinity constant, were 3.5 x 10(6) M-1 and 6 x 10(6) M-1. The calculation of the number of sites per cell in each system specifies the yield of the trypsin digestion.

The impact of clinical biochemistry on university education in France.

In France, clinical biochemistry, similar to other disciplines of laboratory medicine, is taught in both the regular medical and pharmacy curricula, but medical teaching is oriented more towards the interpretation of laboratory findings than test performance. At present, there is no compulsory program of lifelong continuing education, but it is planned to introduce such an obligation in the near future. The practice of laboratory medicine is regulated strictly by the national Health Administration. Clinical laboratories are multidisciplinary, covering simultaneously clinical biochemistry, microbiology, parasitology, hematology and immunology. The only officially recognized laboratory profession is that of 'Director of a Laboratory for Medical Analysis'. The practice of this profession is only open to physicians and pharmacists, provided they graduated in 'Medical Biology' after 4 years of specialized training through a particular type of residency called the 'internat'. The 'interns' are selected by competitive examination. After completing their curriculum, specialized physicians or pharmacists can without further examination or certification either enter a career in a hospital, a university, or both, or direct or co-direct a private laboratory. In this scheme, clinical biochemistry exists as a separate academic discipline, but barely as a distinct profession.

The cholesterol content of HDL2 and HDL3 subfractions of high density lipoproteins in different normocholesterolemic populations.

Two normocholesterolemic populations, selected for either high triglyceridemia or low HDL cholesterol content, both known to have increased artery disease risks, were studied for their cholesterol content in HDL2 and HDL3 subfractions. These subfractions were isolated by a precipitation method. The results showed that, in both populations, total HDL cholesterol values were similar and HDL2 and HDL3 cholesterol content were decreased when compared with a control population. In percentage of total HDL cholesterol, the HDL2 cholesterol subfraction appeared significantly diminished only in female subjects for both populations studied.

'Pseudohypertriglyceridemia' caused by hyperglycerolemia due to congenital enzyme deficiency.

A 76-year-old man was found to have a false hypertriglyceridemia due to a 40-fold increased glycerolemia. This metabolic change was due to a deficiency in glycerol kinase (ATP:glycerol phosphotransferase, EC 2.7.1.30) activity in the cells of this patient as shown by incubation of his white blood cells with [14 C]glycerol. Several chromatographic analyses and quantitative assays were performed on plasma and urine of this patient and of his relatives. The small number of this family's members did not allow to specify the mode of transmission of this genetic trait.

Structural analysis of the carbohydrate chain of glycopeptides isolated from Robinia pseudoacacia seed lectins.

Robinia pseudoacacia seeds contain lectins which are closely related. Pronase digestion of the dimeric and tetrameric lectins, RPA1 and RPA3, gave glycopeptides. The structure of the oligosaccharide was determined by 1H NMR spectroscopy and carbohydrate determination as alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-[alpha-D-Manp+ ++-(1-->6)]-beta- D-Manp-(1-->4)-beta-D-GlcpNAc-(1-->4)-[alpha-L-Fucp-(1-->3)] -beta-D-GlcpNAc - (1-->4)-Asn. It appears that the 34-kDa constitutive polypeptide of RPA1 contains 4-5 carbohydrate chains whereas the 30.5-kDa and 29-kDa subunits of RPA3 contain two and one oligosaccharide chains, respectively.

Biosynthesis, in calf pancreas microsomes, of three lipid-linked oligosaccharide diphosphates from a synthetic dolichyl diphosphate tetrasaccharide.

Incubation of calf pancreas microsomes with synthetic alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1 ----4)-alpha-D- GlcpNAc-PP-Dol and GDP-D-[14C]-mannose gave three major lipid-linked oligosaccharide diphosphates. After release of the phospholipid residue by mild acid hydrolysis, the corresponding [14C]oligosaccharides were analyzed by gel-filtration, liquid chromatography, degradation by endo-N-acetyl-beta-D-glucosaminidases D and H, by jack bean alpha-D-mannosidase and Aspergillus oryzae (1----2)-alpha-D-mannosidase, acetolysis, and binding to concanavalin A-Sepharose. From the results it could be inferred that the following reaction took place in calf pancreas microsomes: alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1 ----4)-alpha-D- GlcpNAc-PP-Dol + GDP-D-Man gave GDP + alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)- beta-D-GlcpNAc-(1----4)-alpha-D-GlcpNAc-PP-Dol. The next products to be formed were alpha-D-Manp-(1----2)-alpha-D-Manp-(1----3)-[alpha-D-Manp -(1----6)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-alpha-D-GlcpNAc-PP-Dol, followed by alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)- [alpha-D-Manp-(1----6)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-alpha- D-GlcpNAc-PP-Dol. The mannose incorporation was enhanced by Triton X-100 and inhibited by Mn2+, and it occurred in the presence of either Mg2+ or EDTA. It is likely that the mannose donor was GDP-mannose since, under the conditions used, the formation of dolichyl mannosyl phosphate was negligible and the dolichyl heptasaccharide diphosphate accumulated.

[Modifications of peptide hydroxyprolinuria during Paget's disease and osteoma (author's transl)]. / Modifications de l'hydroxyprolinurie peptidique au cours de la maladie de Paget et des ostéomes.

The values of hydroxyprolinuria were determined in 314 cases of Paget's disease and 91 cases of osteoma. During Paget's disease, the values found were related to the degree of the bony disorder. A more detailed study of the hydroxyproline polypeptides was undertaken, comparatively in a few cases of these two diseases. In Paget's disease, there was shown a relative increase in the oligopeptide fraction, whereas the polypeptide fraction was increased in osteoma. By chromatographic and electrophoretic technics, it was shown that the acid oligopeptides (containing mainly the dipeptide (Glu-Hyp) are in percentage, reduced in Paget's disease and increased in osteoma, whereas the neutral oligopeptides (containing mainly Pro-Hyp) undergo the opposite variation in these two diseases.

[Comparative study of total cholesterol, HDL cholesterol, LDL + VLDL cholesterol. Two methods: precipitation by concanavalin A and ultra centrifugation (author's transl)]. / Etude comparative des taux de cholestérol HDL, cholestérol LDL + VLDL et du rapport cholestérol HDL/cholestérol LDL + VLDL dans trois populations par deux méthodes: précipitation par la concanavaline A et ultracentrifugation.

The authors determined in 3 populations (37 controls, 59 asymptomatic hyperlipemias, and 20 cases of arterial hyperlipemia) the total cholesterol, the HDL cholesterol and the LDL + VLDL cholesterol and their ratio by two methods : precipitation by concanavalin A and ultracentrifugation (AirFuge). These various lipid parameters were compared between 3 populations in order to determine their more or less discriminant character; furthermore, was established the degree of correlation between the results obtained by precipitation with concanavalin A and by ultracentrifugation. The results show : firstly, that the LDL + VLDL was significantly higher and the ratio HDL/LDL + VLDL significantly lower in hyperlipemia and in arterial disease compared with controls ; the HDL was significantly lower in arterial disease than in controls. No lipid parameter permitted one to differentiate between asymptomatic hyperlipemia and arterial disease. Furthermore, the correlation determined by the chi square method was very strong between the results obtained by precipitation with concanavalin A and ultracentrifugation as the coefficient of simple linear regression is very little different from the value + 1. They emphasize the interest of the method of precipitation by concanavalin A which is simple, rapid and very reliable.

[Xanthelasma palpebrarum and dyslipoproteinaemia: two retrospective studies (author's transl)]. / Xanthelasma et dyslipoprotéinémies: présentation de deux études rétrospectives.

In a retrospective study about 600 hyperlipoproteinaemic patients (390 men and 210 women, mean age 47), xanthelasma palpebrarum was infrequent (25 cases or 4.16 p. 100), since it was only at the third rank of chronic extra-vascular lipid deposits after arcus corneus and tendineous xanthomas. It was twice as frequent in men (5.12 p. 100) as in women (2.38 p. 100) and of regularly increasing occurrence until the sixth decade. Among the atherosclerotic risk factors, only obesity was significantly associated. The existence of atherosclerotic diseases was observed more frequently in patients with xanthelasma palpebrarum (40 p. 100) than in total hyperlipoproteinaemic population (28 p. 100). In a second retrospective study concerning 29 patients (15 men and 14 women, mean age 50), selected from the only presence of one or several xanthelasma palpebrarum, a lipoproteinaemic abnormality was observed in 27 cases (93 p. 100): 23 patients (71 p. 100) were hyperlipoproteinaemic (type IIa, IIb or IV) and in 4 out of 6 normolipidemic patients we observed an abnormal lipoprotein cholesterol distribution (LDL + VLDL cholesterol increase and HDL cholesterol decrease). In this group, we observed in nine patients (31 p. 100) unknown atherosclerotic diseases and tendineous xanthomas in other nine patients (31 p. 100).

In vitro study of the modification by bleomycin of thymidine phosphorylation activity in lectin-stimulated normal human lymphocytes.

We have investigated the effect of bleomycin (BLM) on thymidine phosphorylation in lectin-stimulated normal human lymphocytes. BLM reduces thymidine phosphorylation by decreasing the activity of thymidine kinase (TK). Accordingly, polyacrylamide gel electrophoresis (PAGE) of extracts of cells incubated for 48, 72 and 96 h showed here that this activity dropped 48, 65 and 67% respectively. The electrophoretic profiles of TK activity were similar but different in amplitude. These effects of the BLM were confirmed firstly by direct measurement of TK activity, secondly by amount of 3H-thymidine incorporation in the cultures before cell lysis. Both the measurement of TK activity and 3H-thymidine incorporation were correlated.