Raymond Julien

10-N Nonyl-Acridine Orange: A fluorescent probe which stains mitochondria independently of their energetic state

The specificity of binding of 10-N Nonyl Acridine Orange to mitochondria, and more precisely to inner membranes, is demonstrated by subcellular fractionation of hepatocytes. Unlike Rhodamine 123, which is a preferential marker of the transmembrane potential, Nonyl Acridine Orange binding is essentially independent of the mitochondria energization state although a low uptake of this dye, in response to the potential, may be measured. So 10-N Nonyl acridine orange is an appropriate marker of the mitochondial membrane surface per unit of cell mass.

Molecular Cloning and Expression of a Bovine α(1,3)-Fucosyltransferase Gene Homologous to a Putative Ancestor Gene of the Human FUT3-FUT5-FUT6 Cluster

Only one bovine gene, corresponding to the human cluster of genes FUT3-FUT5-FUT6, was found by Southern blot analysis. The cognate bovine α(1,3)-fucosyltransferase shares 67.3, 69.0, and 69.3% amino acid sequence identities with human FUC-T3, FUC-T5, and FUC-T6 enzymes, respectively. As revealed by protein sequence alignment, potential sites for asparagine-linked glycosylation and conserved cysteines, the bovine enzyme is an intermediate between FUC-T3, FUC-T5, and FUC-T6 human enzymes. Transfected into COS-7 cells, the bovine gene induced the synthesis of an α(1,3)-fucosyltransferase enzyme with type 2 substrate acceptor pattern specificity and induced expression of fucosylated type 2 epitopes (Lex and sialyl-Lex), but not of type 1 structures (Lea or sialyl-Lea), suggesting that it has an acceptor specificity similar to the human plasma FUC-T6. However, no enzyme activity was detected in bovine plasma. Gene transcripts are detected on tissues such as bovine liver, kidney, lung, and brain. The type 2 sialyl-Lex epitope was found in renal macula densa and biliary ducts, and Lex and Ley epitopes were detected on the brush border of epithelial cells of small and large intestine, suggesting a tissue distribution closer to human FUC-T3, but fucosylated type 1 structures (Lea, Leb, or sialyl-Lea) were not detected at all in any bovine tissue. Analysis of genetic distances on a combined phylogenetic tree of fucosyltransferase genes suggests that the bovine gene is the orthologous homologue of the ancestor of human genes constituting the present FUT3-FUT5-FUT6 cluster.

A Single Amino Acid in the Hypervariable Stem Domain of Vertebrate α1,3/1,4-Fucosyltransferases Determines the Type 1/Type 2 Transfer CHARACTERIZATION OF ACCEPTOR SUBSTRATE SPECIFICITY OF THE LEWIS ENZYME BY SITE-DIRECTED MUTAGENESIS

Alignment of 15 vertebrate α1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis α1,3/1,4-fucosyltransferase (Fuc-TIII) andFUT5-encoded α1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in α1,4-linkage. The single amino acid substitution Trp111 → Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 acceptors. The additional mutation of Asp112 → Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue Asp112 in Fuc-TIII by Glu decreased the activity of the enzyme and did not interfere with H-type 1/H-type 2 specificity. In contrast, substitution of Arg115 in bovinefutb-encoded α1,3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type 2 acceptors. However, the double mutation Arg115 → Trp/Glu116 → Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid Trp/Arg seems to modulate the relative type 1/type 2 acceptor specificity, and its presence is necessary for enzyme activity since its substitution by the corresponding amide inactivated both Fuc-TIII and Fuc-Tb enzymes.

Specific interaction of the new fluorescent dye 10-N-nonyl acridine orange with inner mitochondrial membrane: A lipid-mediated inhibition of oxidative phosphorylation

The fluorescent dye 10‐N‐nonyl acridine orange (NAO), known as specifically associated with mitochondria, has been reported to have a cytotoxic effect when high doses were applied to cells. Presently, the biochemical basis of its toxicity was investigated on isolated rat liver mitochondria. At low concentrations, NAO strongly inhibited state 3 respiration and ATP synthesis. At high concentrations, electron transport, ATP hydrolysis, Pi‐transport and adenine nucleotide activities were also decreased. All these inhibitions can be explained by probe‐cardiolipin interactions which could induce the collapse of energy conversion and/or the modification of membrane fluidity.

Transbilayer movement and distribution of spin-labelled phospholipids in the inner mitochondrial membrane

The transmembrane diffusion and equilibrium distribution of spin-labelled phosphatidylethanolamine (PE*), phosphatidylcholine (PC*) and cardiolipin (CL*) were investigated in purified mitochondrial inner membranes using electron spin resonance spectroscopy. Using the back exchange technique, we found that the outside-inside movement of PE* and PC* in beef-heart inner mitochondrial membranes was rapid (t1/2 in the range 10-15 min at 30 degrees C). The steady-state distributions in non-energised mitoplasts were approximately 30% in the inner leaflet for PC* and 39% for PE*. Within the limits of probe concentration that can possibly be used in these experiments, the initial velocity of the inward movement was not saturable with respect to the amount of analogue added to the membranes, suggesting that the spin-labelled phospholipids diffused passively between the two leaflets of the inner mitochondrial membrane. In energised mitoplasts, PC* behaviour was not affected, PE* diffused approximately two times faster toward the inner monolayer but reached the same plateau. Treatment of energised mitochondria with N-ethylmaleimide did not affect PC* diffusion, while the kinetics of PE* internalisation became identical to that of PC*. Similar results were found when PC* and PE* movements were studied in mitoplasts from beef heart, rat liver or yeast. The spin-labelled cardiolipin, which possesses four long chains, had to be introduced in the mitoplast with some ethanol. After equilibration (t1/2 of the order of 13 min at 30 degrees C), the transmembrane distribution suggested that approximately half of the cardiolipin analogue remained in the outer leaflet. These results do not allow us to determine if a specific protein (or flippase) is involved in the phospholipid transmembrane traffic within inner mitochondrial membranes, but they show that lipids can rapidly flip through the mitochondrial membrane.

A new method for testing cell ageing using two mitochondria specific fluorescent probes

Cell culture techniques have considerably improved our understanding of the numerous changes related to aging. For instance, murine lymphocytes obtained from animals older than 6 months progressively lose their, in vitro, proliferative capacity. Numerous studies have shown that this loss is due to changes in the mitochondrial compartment such as reduction in the transmembrane potential and/or membrane mass. Using two mitochondria specific probes with a potential-dependent (Rhodamine 123) or independent (Nonyl Acridine Orange) uptake, we found that the decline in the respiratory activity in the mouse occurred approximately 6 months prior to the decrease in mitochondrial membrane mass. The analysis of the Rh 123/NAO fluorescence ratio measured in splenocytes obtained from mice aged more than 6 months, showed that there was a linear loss of respiratory efficiency per unit of mitochondrial membrane mass. Moreover, cells with a ratio of less than 0.85 were incapable of proliferating and remained quiescent. The time separating the infection points of the two dye uptake curves might provide informations about the regulation and coordination of nuclear and/or mitochondrial genomes. Moreover, the ratio between the two fluorescent probes, in particular during the linear phase, may also have a predictive value.

Adhesion of K99 fimbriated Escherichia coli to pig intestinal epithelium: correlation of adhesive and non-adhesive phenotypes with the sialoglycolipid content.

Evidence for the existence of two phenotypes of piglets born to experimental herds was obtained based on the susceptibility of intestinal brush borders to adhesion of K99-positive Escherichia coli. The enterocytes of the K99-receptive piglets displayed a characteristic sialoglycolipid pattern, with a higher content of the monosialoglycolipids II3NeuGc-LacCer (GM3Gc), IV3NeuGc-nLcOse4Cer (SPGGc) and IV3NeuAc-nLcOse4Cer (SPG) and the oligosialogangliosides IV3NeuAc,II3NeuAc-GgOse4Cer (GD1a), II3(NeuAc)2-GgOse3Cer (GD2), II3(NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc,II3(NeuAc)2-GgOse4Cer (GT1b) when compared to the gangliosides of non-receptive piglets. The gangliosides from enterocytes of the non-receptive piglets were mainly the monosialogangliosides II3NeuAc-GgOse3Cer (GM2) and II3NeuAc-LacCer (GM3), only traces of the other sialoglycolipids being detected. Adhesion of 14C-labelled K99-positive E. coli cells to the piglet small intestinal sialoglycolipids, as tested by the thin-layer chromatogram overlay assay, revealed that the receptive enterocyte membrane was richer in glycolipids containing K99 receptor structures than the non-receptive enterocyte. Adhesion of K99-positive E. coli correlated with the degree of sialylation of the brush border glycolipids.

Do de-N-glycosylation enzymes have an important role in plant cells?☆

In this review de-N-glycosylation was defined as the removal of the glycan(s) from a N-glycosylprotein, by means of enzymes acting on the di-N-acetylchitobiosyl part of the invariant pentasaccharide inner-core of N-glycosylproteins. Peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidases (PNGase) and endo-N-acetyl-beta-D-glucosaminidases (ENGase) were both considered as de-N-glycosylation enzymes. A detailed description of the characterization and the function of plant PNGases and ENGases is presented, together with a brief presentation on the occurrence and the current knowledge on the function of microbial and animal enzymes. De-N-glycosylation of plant glycoproteins was proposed as a possible mechanism for the release of oligosaccharides displaying biological activities and the removal of N-glycans could also explain the regulation of protein activity. Each enzyme seems to have a specific function during germination and post-germinative development. All the arguments concur that de-N-glycosylation enzymes have an important role in plant cells and confirm that the N-glycosylation/de-N-glycosylation system should occur more commonly than presently recognized in living organisms.

Chemo-enzymatic synthesis of a selectin ligand using recombinant yeast cells

Abstract Functional soluble human α(1-3/4) fucosyltransferase was successfully expressed in Pichia pastoris cells. The recombinant protein was located in the periplasmic space; thus, incubation of the whole yeast cells with disulfated tetrasaccharide1 (SO3−3)Galβ1-4(SO3−-6)GlcNAcβ1-3Galβ1-4GlcβOMBn and GDP-fucose directly provided pentasaccharide 2 (SO3−3)Galβ1-4(Fucα1-3)(SO3−-6)GlcNAcβ1-3Galβ1-4GlcβOMBn in 70% yield.

The Saccharomyces cerevisiae MFS superfamily SGE1 gene confers resistance to cationic dyes.

A gene from Saccharomyces cerevisiae whose overexpression confers resistance to 10‐N‐nonyl acridine orange (NAO) has been isolated. This cationic dye binds acidic phospholipids and more specifically cardiolipin (Petit, J. M., Maftah, A., Ratinaud, M. H. and Julien, R. Eur. J. Biochem. 209, 267–273, 1992). The isolated gene was found to be identical to SGE1, a partial multicopy suppressor of the gal11 mutation (Amakasu, H., Suzuki, Y., Nishizawa, M. and Fukasawa, T. Genetics 134, 675–683, 1993), that also confers crystal violet resistance to a supersensitive strain (Ehrenhofer‐Murray, A. E., Wurgler, F. E. and Sengstag, C. Mol. Gen. Genet. 244, 287–294, 1994). The data presented in this paper show that the SGE1 gene product, a member of the major facilitator superfamily, confers a pleiotropic drug‐resistance phenotype when present in high copy number. The results also demonstrate that Sge1p acts as an extrusion permease whose specificity seems restricted to dye molecules possessing a large unsaturated domain that stabilizes a permanent positive charge such as NAO, crystal violet, ethidium bromide or malachite green.