Biomedical subjects

R Julien

Publications and source records attributed to R Julien.

At least 19 recordsLinked to original sources

PubMed1995

Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis.

Flow cytometry can measure variations in DNA content and chromatin structure as well as dramatic changes in the mitochondria of germ cells during maturation from spermatogonia to elongated spermatids. Using 10-N nonyl acridine orange (NAO), an inner mitochondrial membrane dye, it is easy to follow mitochondria rearrangements. Mouse testis cells stained with the DNA fluorescent probe propidium iodide (PI) and analyzed by flow cytometry can be discriminated on the basis of their ploidy levels into five main regions corresponding to elongated spermatids, round spermatids, diploid, S-phase, and tetraploid cells. The simultaneous use of PI and NAO demonstrated the presence of cells having low and high mitochondrial content in the haploid, diploid, and tetraploid compartments. Eleven sorting windows were selected from the bivariate analysis (PI/NAO) and the corresponding cells were identified by microscopic observation. Cells were also discriminated by two parameter analysis of DNA content vs. cell diameter. The definition of seven different regions allowed us to determine NAO or rhodamine 123 (Rh 123) uptakes in each compartment. We observed that the ratio (Rh 123/NAO) dramatically changed according to the progression of cell differentiation which occurs during spermatogenesis.

Acridine Orange↗

PubMed1995

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).

Acridine Orange↗

PubMed1995

Endo-N-acetyl-beta-D-glucosaminidase and peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase activities during germination of Raphanus sativus.

Endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) activities were monitored during germination and postgerminative development in Raphanus sativus. The PNGase activity was found in dry seeds and its level was constant during germination and postgermination. The ENGase activity was first detected about 18 hr after the start of imbibition (HAI) and displayed a maximum level at 36 HAI. After 36 HAI the production of both enzymes was constant until days 4-5. Both enzymes displayed substrate specificities corresponding to the potential glycoprotein substrates found in plants. They are in agreement (i) with the hypothesis that ENGase and PNGase are at the origin of the production of 'unconjugated N-glycans' and (ii) with the possibility that protein activity could be regulated by the removal of N-glycans.

Amidohydrolases↗

PubMed1995

Use of porcine fibrinogen as a model glycoprotein to study the binding specificity of the three variants of K88 lectin.

Known glycoproteins were used to determine the differences occurring in the binding specificities of the three variants of the K88 lectin in an approach essentially based on lectin blotting. During the screening, it was demonstrated that each variant of the K88 lectin biotinylated via its amino groups (NbioK88) exhibited a characteristic binding to the three chains of porcine fibrinogen. NbioK88ab weakly bound to A alpha chains, NbioK88ac bound to B beta and gamma chains, and NbioK88ad bound only to the gamma chain. To validate this model, the oligosaccharide moieties of porcine fibrinogen were analyzed with glycosidases and by lectin blotting and sugar composition. Both the B beta chain and gamma chain carry biantennary N-glycans of the N-acetyllactosamine type that are not recognized by K88 lectins. A alpha chains are substituted by sialylated T antigen. O-glycans were also detected on B beta and gamma chains of porcine fibrinogen and contribute to the recognition of these chains by K88ac and K88ad fimbriae.

Animals↗

PubMed1995

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.

Disaccharides↗

PubMed1995

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.

Acridine Orange↗

PubMed1994

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.

Adolescent↗

PubMed1994

Characterization and use of biotinylated Escherichia coli K99 lectin.

K99 lectin from Escherichia coli was purified and biotinylated via the amino groups of lysine residues using N-biotinyl-6-amino-caproic acid N-hydroxysuccinimide ester (BcapNHS). Biotin was detected on Lys-47 and Lys-87. It was previously demonstrated (Jacobs, A.A.C., Van den Berg, P.A., Bak, H.J. and De Graaf, F.K. (1986) Biochim. Biophys. Acta 872, 92-97) that modification of lysine residues 132 and 133 with 4-chloro-3,5-dinitrobenzoate (CDNB) resulted in the loss of the binding capacity of K99 fimbriae. Due to the higher size of the biotin derivative compared to CDNB, Lys-132 or Lys-133, essential for the biological activity, were not modified. The biotinylation did not cause the loss of the haemagglutinating activity but was sufficient to permit detection of the lectin by streptavidin. A flow cytometric analysis was used for the detection of the receptors on the surface of erythrocytes.

Adhesins, Escherichia coli↗

PubMed1994

Purification and characterization of an endo-N-acetyl-beta-D-glucosaminidase from the culture medium of Stigmatella aurantiaca DW4.

A novel endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans, was characterized in the culture medium of Stigmatella aurantiaca DW4. Purified to homogeneity by ammonium sulfate precipitation, gel filtration, and chromatofocusing, this ENGase presents, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a molecular mass near 27 kDa. Optimal pH and pI were 4.0 and 6.8, respectively. The enzyme, named ENGase St, exhibits high activity on oligomannoside-type glycoasparagines and glycoproteins and could also hydrolyze hybrid- and complex-type glycoasparagines but does not acts as a murein hydrolase.

Asparagine↗

PubMed1992

Immunochemical studies of pancreatic colipase-lipase interaction employing immobilized synthetic peptides.

In view to study the possible participation of the sequence portions of colipase including or close to the free carboxyl groups at positions 15 and/or 72 to the binding with pancreatic lipase, we have used three synthetic peptides matching portions 8-16, 59-67 and 67-72 of the amino acid sequence. Polyclonal rabbit anticolipase immune serum, which cross-reacts with peptides in ELISA, was fractionated on columns of peptide coupled to Sepharose. Of the three fractions of antibodies, only that interacting with peptide 8-16 had the capacity to inhibit colipase-dependent lipase activity by specifically preventing the association of lipase with its protein cofactor previously bound to lipid. We conclude that the region spanning residues 8-16 of colipase is of importance for colipase-lipase interaction in the active complex formed at interface.

Amino Acid Sequence↗

PubMed1992

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.

Acridine Orange↗

PubMed1992

NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

MTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark-blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a non-fluorescent dye usable by this technique to study an intracellular NADH-dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature-dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 microM and 500 microM. The inhibition of mitochondrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.

Animals↗

PubMed1992

Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation of Flavobacterium meningosepticum.

Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F (PNGase F) and endo-beta-N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation of Flavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate. The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase. PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase.

Amidohydrolases↗

PubMed1992

Peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension.

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducing N-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man3(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and 1H- and 13C-NMR assignments for the oligosaccharide Man3(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).

Amidohydrolases↗

PubMed1992

Detection of the ganglioside N-glycolyl-neuraminyl-lactosyl-ceramide by biotinylated Escherichia coli K99 lectin.

K99 lectin from Escherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognize N-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay.

Antigens, Surface↗

PubMed1992

On-line visualization of the competitive behavior of antagonistic bacteria.

To study the interaction between cocultured Listeria monocytogenes and an antagonistic Leuconostoc strain producing an anti-Listeria bacteriocin, flow cytometry, a technique allowing on-line and real-time analysis, was used along with classical microbiological methods. Culture methods and flow cytometric measurements of the mixed culture over time point to a bactericidal action of the lactic acid-producing bacterial strain against L. monocytogenes cells.

Bacteriocins↗

PubMed1991

Evidence for a ribosome-associated thiol protease cleaving wheat germ methionyl-tRNA synthetase.

A wheat germ protease is responsible for Mr 105,000 methionyl-tRNA synthetase hydrolysis, generating two fragments of Mr 82,000 (harbouring the catalytic domain) and 20,000, respectively. Specificity of the protease was sought for using different kinds of protein substrates. It turned out that charged peptides were preferentially cleaved and that no proteolysis occurred when proteins were replaced by small synthetic substrates, harbouring target sites similar to those cleaved in proteins. The protease could be a ribosomal protein, since it remained associated to ribosomal structure, even after treatment by deoxycholate, Triton X-100, 800 mM KC1 and puromycin. Nevertheless, it was still active after ribonuclease treatment of the ribosomes. An identical protease activity was found in rat liver, but not in E. coli.

Chromatography, Gel↗

PubMed1991

Evaluation of 2-methoxyacetic acid toxicity on mouse germ cells by flow cytometry.

Flow cytometric (FCM) DNA content measurements were carried out on testicular monocellular suspensions obtained from mice exposed per os to a single dose of 50, 100, 300, 600, and 900 mg/kg body weight (b.w.) of 2-methoxyacetic acid (MAA) in order to investigate its cytotoxic action on germ cells. The effects of MAA were evaluated 2, 7, 14, 28, and 45 d after treatment in terms of altered cell type ratios in FCM fluorescence distribution histograms. Testis weight and histological tissue sections were also analyzed. MAA induced marked changes in the relative percentages of tetraploid and haploid cells, indicating the occurrence of cytotoxic damage on primary spermatocytes. Multiparameter FCM analysis showed that, besides its action on nucleic acid synthesis, MAA can also affect the cellular energy metabolism reflected in an altered mitochondrial mass distribution on round spermatids surviving the MAA treatment. This study demonstrates that rapid and unique FCM procedures can be usefully applied in reproductive toxicology.

Acetates↗