Genome-wide analysis highlights genetic dilution in Algerian sheep.

Algeria represents a reservoir of genetic diversity with local sheep breeds adapted to a large range of environments and showing specific features necessary to deal with harsh conditions. This remarkable diversity results from the traditional management of dryland by pastoralists over centuries. Most of these breeds are poorly productive, and the economic pressure leads farmers to realize anarchic cross-breeding (that is, not carried out in the framework of selection plans) with the hope to increase animal's conformation. In this study, eight of the nine local Algerian sheep breeds (D'men, Hamra, Ouled-Djellal, Rembi, Sidaoun, Tazegzawt, Berber and Barbarine) were investigated for the first time by genome-wide single-nucleotide polymorphism genotyping. At an international scale, Algerian sheep occupied an original position shaped by relations with African and European (particularly Italian) breeds. The strong genetic proximity with Caribbean and Brazilian breeds confirmed that the genetic make-up of these American breeds was largely influenced by the Atlantic slave trade. At a national scale, an alarming genetic dilution of the Berber (a primitive breed) and the Rembi was observed, as a consequence of uncontrolled mating practices with Ouled-Djellal. A similar, though less pronounced, phenomenon was also detected for the Barbarine, another ancestral breed. Genetic originality appeared to be better preserved in Tazegzawt, Hamra, D'men and Sidaoun. These breeds should be given high priority in the establishment of conservation plans to halt their progressive loss. For Berber and Barbarine that also occur in the bordering neighbor countries, urgent concerted transnational actions are needed.

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.

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.

Human epidermal cells progressively lose their cardiolipins during ageing without change in mitochondrial transmembrane potential.

Mitochondria dysfunction is considered to be a major cause of the modifications that occur during cell ageing. For this reason, cardiolipin, a suitable marker of the chondriome, as well as the mitochondrial transmembrane potential were examined in keratinocytes obtained from 9- to 75-year-old women. The study was carried out by flow cytometry using two fluorescent mitochondria probes: nonyl acridine orange, which binds specifically to cardiolipin, and rhodamine 123, which is incorporated mainly in response to transmembrane potential. Cardiolipin levels in cells from elderly donors (75 years old) would be 57% lower (r = 0.540; P = 0.0002) than those in children (9 years old), while the inner transmembrane potential remained unchanged (r = 0.0394; P = 0.8017). The stability of the membrane potential may be explained by either or both of the following hypotheses: (i) the same pool of organelles able to maintain membrane potential is conserved even when cardiolipin levels decrease (ii) mitochondria membrane potential does indeed decrease with age but is compensated by glycolysis energy production. Finally, it may be stated that the fluorescent probes nonyl acridine orange and rhodamine 123 might be of interest in testing the phenotype of senescent cells and would be useful in screening the role of certain specific genes in cell ageing.

An improved method to obtain a single recombinant vasoactive intestinal peptide (VIP) analog.

The vasoactive intestinal peptide (VIP) is an ubiquitous peptide of great potential for applications. Development of new bioactive VIP analogs using production in recombinant E coli has been carried out in our laboratory. This work presents a new multimeric fusion protein expressing several VIP units separated by factor Xa cleavage site linkers. The steps leading from the affinity purification of the fusion protein and its processing by the factor Xa to the full characterization of the new bioactive improved VIP analog are also described.

Involvement of reactive oxygen species in the bactericidal activity of activated carbon fibre supporting silver; Bactericidal activity of ACF(Ag) mediated by ROS.

An activated carbon fibre supporting silver (ACF(Ag)) was tested for its antibacterial capacity against Escherichia coli (E. coli). Water that has passed through ACF(Ag) demonstrated strong bactericidal ability. This activity decreased over the time suggesting that generated bactericidal species were short lifespan. Since formation of reactive oxygen species (ROS) might be catalysed by silver impregnated and/or ACF itself, implication of ROS and silver was evaluated by the use of ROS scavengers and a silver ions neutralizing agent. The role of ROS in the E. coli mortality was confirmed by the use of a molecular approach which revealed a strong expression of oxidative stress genes.

Mutagenesis of the bovSERPINA3-3 demonstrates the requirement of aspartate-371 for intermolecular interaction and formation of dimers.

The family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A-sheet of another. This "A-sheet polymerization" has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3-3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site-directed mutagenesis of recombinant bovSERPINA3-3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3-3. By structural modeling of mature bovSERPINA3-3, we propose a new "non-A-sheet swap" model of serpin homodimer in which D371 is involved at the molecular interface.

Molecular evolution of protein O-fucosyltransferase genes and splice variants.

O-Fucose has been described on both epidermal growth factor-like (EGF-like) repeats and Thrombospondin type 1 repeats (TSRs). The enzyme adding fucose to EGF-like repeats, protein O-fucosyltransferase 1 (Pofut1), is a soluble protein located in the lumen of endoplasmic reticulum (ER). A second protein O-fucosyltransferase, Pofut2, quite divergent from its homolog Pofut1, has recently been shown to O-fucosylate TSRs but not EGF-like repeats. To date, Pofut1 genes have only been characterized in human, mouse, and fly, and Pofut2 in mouse, fly, and partially in the nematode Caenorhabditis elegans. Here, we report cDNA sequences and genomic structures of bovine Pofut1 and Pofut2 genes and describe for the first time five alternative spliced transcripts for each gene. Only one transcript for both Pofut1 and Pofut2 encodes an active bovine O-fucosyltransferase. Variant transcript distribution was examined in 13 bovine tissues. Transcripts encoding active forms are ubiquitous, whereas other forms possess a more restricted tissue-expression profile. Sequence comparison and phylogenetic analyses revealed that both Pofut genes are present as a single copy in animal genomes, and their exon-intron organizations are conserved among vertebrates. The last common ancestor of all analyzed bilaterian species would be predicted to possess polyexonic Pofut genes in their genome.

Biochemical characterization of Silene alba alpha4-fucosyltransferase and Lewis a products.

alpha1,4-Fucosylation has been recently detected in Arabidopsis thaliana [Leonard et al. (2002), Glycobiology 12: 299-306], and corresponding enzymes have also been characterized in Beta vulgaris [Bakker et al. (2001), FEBS Lett, 507: 307-312], and Lycopersicum aesculentum [Wilson (2001), Glycoconjugate J., 18: 439-447]. Here we demonstrated fucosyltransferase activity (FucT) in Silene alba cells and tissues. The Fuc linkage to GlcNAc residues of the lactosamine moiety of the Type I acceptor was confirmed by mass spectrometry experiments. Le(a)-glycoconjugates are found in the Golgi apparatus and plasma membrane of plant cells. In planta, the highest levels of activity were detected in seedlings, young roots and male flowers. The enzyme was stable up to 45( composite function)C and the optimum pH of reaction was 8.0. The enzyme required Mg(2+) or Mn(2+) for activity and was inhibited by Zn(2+) and ethylenediaminetetraacetic acid. Chemical modification of the enzyme with group-selective reagents revealed that selective modifications of arginine and lysine residues had no effect on enzyme activity. However the enzyme contains histidine and tryptophan residues that are essential for its activity. In contrast to human FUT3, the S. alba alpha4-FucT was insensitive to N-ethylmaleimide (NEM) treatment. Measurement of enzyme activity in S. alba cell fractions indicated that the enzyme is bound to microsomal membranes, furthermore a soluble isoform of the protein may be present.

Linear correlation between bacterial overexpression of recombinant peptides and cell light scatter.

Fusion of multiple copies of a test peptide leads to insoluble inclusion bodies. Their presence within bacteria increases either forward-angle light scattering or, to a lesser extent, right-angle light scattering. A linear correlation has been established between cell forward-angle scattering and the level of overexpression of atrial natriuretic peptide. The correlation is valid only for unlysed cells and is protein product specific.

Flow cytometry's contribution to the measurement of cell functions.

Flow cytometry has important advantages over conventional techniques. It is rapid, highly sensitive and allows multi-parametric analysis and cell sorting. Potential exists for the measurement of many cell functions by flow cytometry. The technique can be used to determine cell viability, intracellular calcium and pH, membrane potential, enzyme activities, membrane fluidity and endocytosis. Numerous examples are given on the applications of flow cytometry for cell functions measurements in the fundamental and biomedical fields.

A new methodology for testing chemicals and drugs on cell activity.

Synopsis A new method for testing the effects of cosmetics and various chemicals on cell activity is described. It is based on the study of mitochondria compartments and combines the use of fluorescent probes which are accumulated in a potential-dependent (Rhodamine 123) or potential-independent (Nonyl Acridine Orange) manner with flow cytometric analysis. Measured fluorescences allow the determination of a new parameter indicating mitochondria efficiency. This new methodology for the evaluation of cell activity has been applied in three different situations: during cell ageing, during cell cycle and when cells are in the presence of drugs.

10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria.

The acridine orange derivative, 10N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/mol cardiolipin and 1 mol dye/mol phosphatidylserine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 10(6) M-1 for cardiolipin-10N-nonyl-acridine-orange association and only 7 x 10(4) M-1 for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction between the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.

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.

Use of nonyl acridine orange and rhodamine 123 to follow biosynthesis and functional assembly of mitochondrial membrane during L1210 cell cycle.

Specific mitochondrial incorporation of 10 N-nonyl acridine orange (NAO) is demonstrated by subcellular fractionation of rat hepatocytes. Moreover, comparative studies with NAO and rhodamine 123 (Rh 123) prove that acridine orange-derivative uptake is independent of transmembrane mitochondrial potential, a property allowing its utilization for the assessment of mitochondrial membrane mass modifications under various physiological states. Using NAO and Rh 123, we have respectively followed the biosynthesis of mitochondrial membrane and its assembly under a functional state during the L1210 cell cycle. Their evolution occurs in two stages according to a well-defined sequential order. Mitochondrial biogenesis, as revealed by NAO incorporation, occurs essentially in the G1 phase (probably mitochondrion enlargement) but also starts in late S phase (probably mitochondrion division). The increased amount of functional mitochondrial membrane, monitored by Rh 123 uptake, is emphasized in late G1 (prerequisite to DNA synthesis) and during G2M phases (prerequisite to mitosis). This alternative succession of phases displays the existence of a time-lag between the biosynthesis of mitochondrial membrane and its functional organization. Such an analysis confirms the potential of the NAO probe to evaluate mitochondrial membrane mass changes in various biological fields.

Asymmetrical distribution of cardiolipin in yeast inner mitochondrial membrane triggered by carbon catabolite repression.

Transmembrane asymmetry of cardiolipin in yeast was monitored during the switch from fermentative to gluconeogenic growth and the reverse. As soon as cells used ethanol as an electron donor to produce ATP by oxidative phosphorylation, rapid and abundant cardiolipin synthesis was observed on the matrix side of the inner mitochondrial membrane followed by a transverse rearrangement between the two leaflets. The cardiolipin distribution changed from about 20:80 (in/out) to 70:30 (in/out), and after translocation towards the outer leaflet it finally became 37:63 (in/out). At the same time, cytochrome c oxidase activity remained stable, then increased as a possible result of the topographical rearrangement. During the reverse process from gluconeogenic to fermentative growth, the amount of cardiolipin rapidly decreased by half, its bilayer distribution apparently changing to a monolayer organization before the 20:80 (in/out) asymmetry of repressed cells was re-established. Experimental impairment of cardiolipin topography by antibiotic inhibition of gene expression or in situ dissipation of mitochondrial membrane potential produced data that prove that the amount and transmembrane distribution of the phospholipid are two specific parameters of the mitochondrial inner membrane organization in both fermentative (2.2 fmol/cell and 20:80, in/out) and gluconeogenic (4.2 fmol/cell and 37:63, in/out) growing yeast cells. Finally, the inner mitochondrial membrane topography of cardiolipin appeared to be closely associated with the transmembrane redox potential.

Flow cytometric analysis of human epidermal cell ageing using two fluorescent mitochondrial probes.

Cardiolipin, mitochondrial transmembrane potential, cell refringence and cell diameter were examined in epidermal cells obtained from 42 women between 9- to 75-year-old. The study was carried out in situ by flow cytometry on cells having incorporated either Nonyl Acridine Orange or Rhodamine 123, 2 mitochondria-specific dyes. Cardiolipin levels, determined by the binding of the cardiolipin-specific probe Nonyl Acridine Orange, decreased significantly with age, especially in young individuals. This suggests 2 stages in the age-dependent transformation of mitochondria (organelle number and/or size): one during childhood development and to adulthood (9 to 27 years) in which cardiolipin levels decrease dramatically (slope: -3.742; p = 0.0243) and the other corresponding to senescence (35 to 75 years) in which this decrease is less pronounced (slope: -0.618; p = 0.0467). These changes have no effect on mitochondrial potential, measured by Rhodamine 123 incorporation, which remained constant with age. This function, controlling calcium partitioning within the cell, might allow keratinocytes to differentiate and maintain the skin barrier function of the epidermis. Like cardiolipin, intrinsic parameters such as cell size and refringence also significantly decreased in epidermal cells from elderly subjects. The methodology can be used to determine physiological ageing in various cell types and to analyse human ageing and related parameters.

Direct cardiolipin assay in yeast using the red fluorescence emission of 10-N-nonyl acridine orange.

The dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) has been recently identified as a specific probe for cardiolipin (Ka = 2 x 10(6) M-1). It also interacts, at lower affinity (Ka = 7 x 10(4) M-1), with other acidic phospholipids [Petit, J. M., Maftah, A., Ratinaud, M. H. & Julien, R. (1992) Eur. J. Biochem. 209, 267-273]. In order to reduce the interference corresponding to monoacidic phospholipid binding, we have quantified cardiolipin by using a fluorimetric method based on the red fluorescence of the dye dimers formed at the diacidic phospholipid contact. Hence we have demonstrated that: (a) in yeast, the mitochondrion is the target of the dye whatever the cell metabolism; (b) membrane or protein organization and fatty acid unsaturation do not significantly modify the binding of 10-N-nonyl acridine orange. Using thin-walled vesicles, a linear relationship was established between the amount of cardiolipin and the red fluorescence emitted by the dye. Low red fluorescences were also observed with vesicles containing phosphatidylserine and phosphatidylinositol. However, at the same acidic phospholipid concentration, the fluorescence was much higher using cardiolipin-containing vesicles (fivefold that observed with phosphatidylserine-containing vesicles). Thus, 10-N-nonyl acridine orange was applied to cardiolipin quantification in yeast. This new method revealed that cells growing with a high glucose concentration contained 2.2 +/- 0.3 nmol cardiolipin/10(6) cells, whereas with lactate they contained about twice this amount (3.9 +/- 0.3 nmol cardiolipin).

A single amino acid in the hypervariable stem domain of vertebrate alpha1,3/1,4-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 alpha1,3-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 alpha1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha1, 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 bovine futb-encoded alpha1, 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.

Ancestral exonic organization of FUT8, the gene encoding the alpha6-fucosyltransferase, reveals successive peptide domains which suggest a particular three-dimensional core structure for the alpha6-fucosyltransferase family.

Based on PCR strategies and expression studies, we define the genomic organization of the FUT8b gene. This gene encodes the only known mammalian enzyme transferring fucose in an alpha1-->6 linkage on the asparagine-branched GlcNAc residue of the chitobiose unit of complex N:-glycans. The intron/exon organization of the bovine coding sequence determines five successive functional domains. The first exon encodes a domain homologous to cytoskeleton proteins, the second presents a proline-rich region including a motif XPXPPYXP similar to the peptide ligand of the SH3-domain proteins, the third encodes a gyrase-like domain (an enzyme which can bind nucleotides), and the fourth encodes a peptide sequence homologous to the catalytic domain of proteins transferring sugars. Finally, the last exon encodes a domain homologous to the SH3 conserved motif of the SH2-SH3 protein family. This organization suggests that intramolecular interactions might give a tulip-shaped scaffolding, including the catalytic pocket of the enzyme in the Golgi lumen. Deduced from the published sequence of chromosome 14 (AL109847), the human gene organization of FUT8 seems to be similar to that of bovine FUT8b, although the exon partition is more pronounced (bovine exons 1 and 2 correspond to human exons 1-6). The mosaicism and phylogenetic positions of the alpha6-fucosyltransferase genes are compared with those of other fucosyltransferase genes.