Identification of a alpha-NeuAc-(2----3)-beta-D-galactopyranosyl N-acetyl-beta-D-galactosaminyltransferase in human kidney

Microsomal preparations from human kidney were found to contain enzymic activity capable to transfer N-acetylgalactosamine from UDP-N-acetylgalactosamine to native bovine fetuin. The acceptor structures on the fetuin molecules were identified as N- as well as O-linked glycans with a markedly higher incorporation into the N-linked carbohydrate chains. Analysis of the alkali-labile transferase products by thin-layer chromatography indicated that the enzyme is able to synthesize structures having mobilities identical with those found on glycophorin from Cad erythrocytes. Mild acid treatment and enzymic hydrolysis with N-acetylhexosaminidase from jack beans of the N-linked transferase products suggested that beta-D-GalpNAc-(1----4)-[alpha-NeuAc-(2----3)]-beta-D-Galp-(1----s tructures were formed by the enzymic reaction on both N- and O-linked acceptors. The enzyme might, therefore, be involved in the biosynthesis of Sda (and Cad) antigenic structures. By use of various oligosaccharides, glycopeptides, and glycolipids having well characterized carbohydrate sequences, the acceptor-substrate specificity of the N-acetylgalactosaminyltransferase was determined. The enzyme generally recognized alpha-NeuAc-(2----3)-beta-D-Gal groups as acceptors, but in a certain conformation. Thus, tri- and tetra-saccharide alditols, native human glycophorin A, and GM3 were not acceptor substrates although they carry the potential disaccharide acceptor unit. When these structures were presented as sialyl-(2----3)-lactose or as a tryptic peptide from glycophorin A, they were shown to be rather good acceptor substrates for the N-acetyl-beta-D-galactosaminyltransferase from human kidney.     

Bax activation and mitochondrial insertion during apoptosis

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells.The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization.Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.     

Establishment of a DNA bank of human origin

High molecular weight DNA extracted from 50 unrelated parisian individuals constitutes a "DNA-thèque" which can be used at the population level. This procedure allows preparation from a variety of tissues such as liver, placenta and leucocytes. The "quality" of the DNA obtained (about 50 Kb, RNA and protein free) and the efficiency of the method (in terms of the yield of DNA obtained) are considered. Blood is the easiest material to obtain and permits the obtaining of a reasonable amount of human and primate DNA, particularly when familial investigation is planned for polymorphism studies. One of the objects of this article is to encourage others to make use of our bank, for studies in population and evolutionary genetics.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.     

Structure of a ganglioside with Cad blood group antigen activity

The Cad antigen is a rare erythrocyte blood group antigen expressed on both sialoglycoprotein and ganglioside structures. It is related both serologically and biochemically to the Sda blood group antigen expressed on over 90% of Caucasian erythrocytes. We reported previously that Cad erythrocytes contain a novel ganglioside that binds Helix pomatia lectin and inhibits human anti-Sda antibody. We have now purified the Cad ganglioside and determined its structure. The ganglioside contained Glc-Gal-GlcNAc-GalNAc-NeuAc in a molar ratio of 1.00:1.94:0.95:0.93:1.05. Its chromatographic mobility was between that of GM1 and GD3. After treatment with beta-hexosaminidase (human placenta Hex A), the product migrated with 2-3-sialosylparagloboside (IV3NeuAcnLc4OseCer), it no longer bound H. pomatia lectin, and it acquired the ability to bind an antibody to sialosylparagloboside. Treatment of this material with neuraminidase (Vibrio cholerae) yielded a product with the mobility of paragloboside (nLc4OseCer) that bound monoclonal antibody 1B2, which is specific for terminal N-acetyllactosaminyl structures. Treatment of the Cad ganglioside with Arthrobacter ureafaciens neuraminidase yielded a product reactive with monoclonal antibody 2D4, which is specific for terminal GalNAc beta (1-4)Gal structures. These data provide strong evidence that the Cad ganglioside structure is GalNAc beta (1-4)[NeuAc alpha (2-3)]Gal beta (1-3)Gal beta (1-4)GlcCer. 1H NMR analysis also supports the conclusion that the terminal GalNAc is linked beta (1-4) to Gal. High-performance thin-layer chromatographic ganglioside patterns from three blood group Cad individuals showed a direct correlation between the quantity of Cad ganglioside and the strength of Cad antigen expression on the erythrocytes, as measured by hemagglutination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Miltenberger Class I and II erythrocytes carry a variant of glycophorin A.

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.