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.

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.

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.

[Variability of biological glucose transport activity in human erythrocytes]. / Variabilité de l'activité biologique du transporteur du glucose dans les érythrocytes humains.

Glucose transport activity was determined on erythrocytes from healthy adult and children. A large variability of values was found between each donor, but transport rate was significantly higher in erythrocytes from adults than those from children. In opposite, the same number of glucose transport sites was found on erythrocytes from adult and children, suggesting that transporters from adult erythrocytes exhibited an higher own activity than transporters from child erythrocytes. Involvement of various factors in glucose transport activity was discussed.

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.

Glycosylation of human leukocyte locus A molecules is dependent on the cell type.

Peripheral blood monocytes and B cells were isolated from a normal donor, and a portion of the B cells was transformed by the Epstein-Barr virus (EBV). Human leukocyte locus A (HLA) class-I and class-II molecules were immunoprecipitated by specific monoclonal antibodies after cell labeling with [3H]mannose. Glycopeptides of HLA molecules were obtained by pronase digestion and were analysed by lectin-affinity chromatography. Complex structures were hydrazinolysed, and their sialic acid content was analysed by ion-exchange chromatography, whereas the high-mannose structures were separated by HPLC. In normal cells, class-I antigens bear principally fucosylated biantennary structures while HLA-DR class-II antigens bear bi-, tri- and tetra-antennary structures and high-mannose structures. HLA antigens are more sialylated on normal B cells than on normal monocytes. An EBV cell line had a very different pattern of HLA-antigen glycosylation when compared with the original B cells. In the transformed cells, the fractions containing biantennary structures are largely decreased. In contrast, an increase of the tri- and tetra-antennary structure fractions is noticed, particularly in class-II molecules, while both triantennary and high-mannose structures are increased in class-I molecules. Moreover, when compared to normal B cells, the complex structures of class-I antigens in the EBV-transformed B-cell line are undersialylated while they are oversialylated in the case of the class-II molecules.

Differential entry of ricin into malignant and normal rat hepatocytes.

We have compared the mechanisms of ricin binding to and entry into Zajdela hepatoma cells (ZHC) and normal rat hepatocytes (HyC). Lactose but not mannan was found to inhibit ricin binding to and toxicity on ZHC and HyC. This finding suggests that ricin binding, entry, and toxicity are expressed only through the galactose binding sites on ZHC and HyC. Nevertheless, the characteristics of ricin binding and its entry pathway appeared to be different in several respects in ZHC and HyC. Scatchard analysis of equilibrium data determined over a wide range of 125I-labeled ricin concentrations yielded a curvilinear plot for ZHC, while a straight line was obtained for HyC. These results indicate that only ZHC possess high-affinity receptors for ricin. Analysis of ricin toxicity on ZHC and HyC, in the presence of ammonium chloride or after K+-depletion in both cell types, suggests that the ricin bound to galactose receptors entered through neutral vesicles in ZHC, and through both neutral and acidic vesicles in HyC. The qualitative and quantitative differences found between the process of receptor-mediated endocytosis of ricin in ZHC and HyC might explain the differential sensitivity of the two cell types toward the toxin.

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.

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.

[Interaction between ricin and Zajdela ascitic hepatoma cells]. / Interactions entre la ricine et les cellules de l'hépatome ascitique de Zajdela.

The binding to and toxicity of ricin on Zajdela hepatoma ascites cells were studied. The kinetic analysis of [125I]-ricin binding to hepatoma cells indicated that maximal specific binding was reached within 30 min. at 4 degrees C and 60 min. at 25 degrees C and that toxin binding to hepatoma cells was saturable. When the binding data were plotted according to the method of Scatchard, curvilinear graphs were obtained suggesting that hepatoma cells have both high and low affinity receptors for ricin. The number of high and low affinity receptors was identical at 4 and 25 degrees C, i.e., 8 x 10(5) and 1.2 x 10(7) sites per cell respectively. However, the capacity of hepatoma cells to bind ricin is stronger at 4 degrees C than at 25 degrees C. The toxic activity of ricin was totally abolished in the presence of lactose suggesting that ricin binding to cells occurs through binding sites containing galactosyl residues.

Plasma lipids in juvenile chronic granulocytic leukaemia.

Plasma lipid parameters (triglycerides, total cholesterol and high density lipoprotein cholesterol) were measured in 7 children with juvenile chronic granulocytic leukaemia, of whom 3 were with and 4 without xanthomas. In all cases, whatever the stage of the disease, these parameters were extremely altered. Plasma triglycerides were generally increased, total and HDL cholesterols were very low. No relationship seems to exist between these values and the occurrence of xanthomas.

Characterization of N-linked oligosaccharides of an HLA-DR molecule expressed in different cell lines.

In order to determine the factors that influence the glycosylation of an integral membrane protein, we investigated the N-glycosylation of a molecule of the human major histocompatibility complex (MHC) class II, the HLA-DR antigen. This glycoprotein was studied in a human Epstein-Barr-virus-transformed B cell line and in a mouse fibroblastic cell line co-transfected with DR alpha and DR beta genes. We observed that the HLA-DR-antigen glycosylation pattern depends on the cell line in which processing takes place and is closely related to the glycosylation pattern of the overall cellular glycoproteins. Furthermore, when comparing the glycosylation of the separated alpha- and beta-chains, differences were noticed within the same molecule, showing the importance of the individual peptide backbone for the glycosylation process.

Purification and characterization of Robinia pseudoacacia seed lectins. A re-investigation.

Two lectins, RPA 1 and RPA 3, were purified from Robinia pseudoacacia seeds. These two lectins differ in their physicochemical and biological properties. By analytical ultracentrifugation the Mr values of RPA 1 and RPA 3 were estimated to be 59,000 and 105,000 respectively. From SDS/polyacrylamide-gel-electrophoresis data it was estimated that RPA 1 consisted of two subunits of Mr 34,000, and RPA 3 of two types of subunits (Mr 30,500 and 29,000). RPA 1 and RPA 3 were found to be glycoproteins of comparable amino acid composition. RPA 1 was the more highly glycosylated molecule (11.6% versus 4.3%). The carbohydrate-specificity of RPA 1 appears to be complex. RPA 3 was inhibited by N-acetyl-D-galactosamine and human alpha-glycoproteins. Both lectins exerted a mitogenic effect on human peripheral-blood lymphocytes. Concentrations between 0.5 and 1 microgram of RPA 3/ml gave optimal proliferative responses, whereas for RPA 1 concentrations higher than 10 micrograms/ml were needed for these responses.

In vitro study of the comparative behaviour of a human acute lymphoblastic leukemia cell line and a reference normal cell line towards the effects of a mitogenic lectin.

An in vitro study of the behaviour of a human acute lymphoblastoid leukemia cell line (REH) towards the action of a mitogenic lectin of Robinia pseudoacacia was carried out. The results were compared with those a reference cell line (LHN13) established from normal human lymphocytes. In both cell lines, the lectin induces agglutination (measured by counting the number of aggregates as well as the number of cells in each aggregate) and decrease of growth (measured by counting the number of cells and the incorporation of tritiated thymidine into TCA-precipitable material per 10(6) cells). The agglutination and the decrease of growth are produced at the doses of 0.5 and 1 microgram/ml of culture medium and after 4 h of exposure of cells to the lectin, respectively. These effects increase progressively with higher doses of lectin and continues throughout the culture. However, the REH line is less sensitive than the LHN13 line to the effects of lectin. Both agglutination and growth decrease of REH as well as LHN13 cell lines by the lectin are reversible; this is confirmed by the fact that the monospecific anti-Robinia lectin serum suppresses these effects.

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.

Effect of pH on oligomeric equilibrium and saccharide-binding properties of peanut agglutinin.

The conformation and saccharide-binding properties of peanut agglutinin (PNA) depend on pH as studied by analytical ultracentrifugation, fluorescence, circular dichroism, equilibrium dialysis, and absorption spectroscopy. PNA is tetrameric in neutral solution and dissociates reversibly into dimers below pH 5.1. Below pH 3.4, the lectin is totally dimeric. Lowering of the pH induces reversible changes in the tertiary and secondary structures of PNA. Binding of saturating amounts of lactose to tetrameric (pH 6.9) or dimeric (pH 3.2) PNA resulted in identical ultraviolet difference spectra. Fluorescence studies of PNA as a function of pH in the presence of lactose indicated that tryptophanyl residues, present at or near the saccharide binding site, are more accessible to the ligand in dimeric than in tetrameric PNA. For solutions of dimeric PNA, containing only minor amounts of tetramers (pH 3.6), equilibrium dialysis with MeUmb-beta Gal beta(1----3)GalNac showed that the binding capacity of PNA was the same as for tetrameric PNA (one binding site per protomer) but the apparent association constant was one order of magnitude lower than for tetrameric PNA. The enhancement of MeUmb-beta Gal beta(1----3)GalNac fluorescence upon binding to PNA was pH dependent: 50% at neutrality, 16% at pH 3.7, and unobservable at pH 3.0, suggesting that the microenvironment of this PNA-bound chromophore changed progressively with pH and was dependent on ionization of an acidic amino acid residue.

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.