Short-term variations of serum glycated apolipoprotein B.

Serum glycated apolipoprotein B (apo B-G) levels were determined in 31 non-insulin-dependent diabetic patients and 13 control subjects by an enzyme-linked immunosorbent assay. Apo B-G was increased in diabetic patients compared to non-diabetic subjects, whether expressed as a serum concentration (57.7 vs 36.1 mg/l) or a percentage of total apolipoprotein B (4.42 vs 3.14%). Apo B-G, together with other markers (mean daily plasma glucose, serum fructosamines, triglycerides, total cholesterol, glycated haemoglobin), was measured before and after 5 days of therapeutic adjustment in diabetic patients. In 20 patients with a favourable course of glycaemic control, the mean decrease of apo B-G concentration reached nearly 16%. In 11 patients with an unfavourable course, the increase of apo B-G concentration was about 14%. Therefore, variation of serum apo B-G concentration could serve as an additional short-term marker for glycaemic control, although possible concomitant variations of serum triglycerides or total apolipoprotein B concentrations should also be considered.

Low density lipoprotein oxidation is inhibited by extracts of Ilex paraguariensis.

Some dietary polyphenolic substances have been shown to inhibit oxidation of LDL. "Mate" is a polyphenol-containing beverage, brewed from the dried and minced leaves of Ilex paraguariensis. In the present work we studied the effect of water and alcohol extracts of Ilex paraguariensis on the initiation and propagation of LDL copper or H2O2-induced autoxidation. Our data show that substances in water extracts of Ilex paraguariensis are capable of inhibiting the initiation and the propagation of LDL oxidation. They inhibit lipid peroxidation, monitored by diene conjugates and thiobarbituric acid-reactive substances, as well as protein modification as shown through direct measurement of free amino groups, electrophoretic mobility, and fluorescence. This inhibition is a concentration dependent effect that becomes already apparent at concentrations of extracts as low as 7.5 micrograms/ml. Inhibition is almost complete at 37.5 micrograms/ml. Alcohol extracts show similar effects though with less potency. The substances implicated in this antioxidant activity are largely nondializable. In terms of mass, water extracts of Ilex paraguariensis were more potent antioxidants than either ascorbic acid, or butylated hydroxytoluene.

Susceptibility to copper-enhanced autoxidation of VLDL+LDL fractions from diabetic patients.

We describe a simplified method for studying the susceptibility to in vitro autoxidation of lipoproteins containing apolipoprotein B. It comprises copper induced autoxidation of VLDL+LDL plasma fractions and an assay for thiobarbituric acid reactive soluble substances. We studied autoxidation of VLDL+LDL in a population of 30 healthy subjects and a population of 30 diabetic patients. No significant difference in susceptibility to autoxidation could be detected. Protection afforded by ascorbic acid amounted to more than 95% at concentrations easily attainable in vivo by oral supplementation. Thiobarbituric acid reactive substances were higher in plasma from diabetic patients: 0.51 +/- 0.25 mumol/l vs 0.35 +/- 0.07 mumol/l for control subjects (p < 0.001). Our data confirm the presence of higher levels of lipid peroxides in diabetic patients, but fail to demonstrate any difference in susceptibility to oxidation of apolipoprotein B containing particles between control and diabetic subjects.

Glycation and oxidation of human low density lipoproteins reduces heparin binding and modifies charge.

The effects of glycation and oxidation of human low density lipoproteins (LDL) on heparin binding were studied and compared with modifications in the charge of the particles. Glycation of LDL at a molar ratio of 4 mol glucose mol-1 apoB, decreases affinity for heparin, as shown by heparin-agarose affinity chromatography since salt molarity needed for elution decreases from 550 mmol l-1 for control LDL (c-LDL) to 350 mmol l-1 for glycated LDL (glc-LDL). Oxidized LDL (oxi-LDL) shows marked heterogeneity, most of the fractions having decreased affinity. Heparin-agarose affinity chromatography of LDL preparations shows the presence of a small (5-7%), low-affinity fraction in euglycaemic human plasma LDL (c-LDL). Its elution volume coincides with both glc-LDL and a fraction of oxi-LDL, suggesting it may contain glycated and oxidized molecules present in plasma. DEAE-Trisacryl anion exchange chromatography elution profiles of c-LDL preparations shows the presence of a more electronegative fraction accounting for about 10% of total protein. This fraction elutes with 260 mmol l-1 NaCl instead of 130 mmol l-1 for the main fraction, it roughly coincides with elution volumes of main peaks of glc-LDL and oxi-LDL. Results indicate that glycated particles may be present in this fraction. Our data demonstrate then that glycation, and to a lesser degree, oxidation of LDL reduce affinity to heparin. From an analytical approach, modified LDL can be separated from the bulk of native LDL both by DEAE and heparin-agarose chromatographies.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycated immunoglobulin M in diabetic patients.

Measurement of glycation levels on isolated immunoglobulin M (IgM), a short half-life protein, could be an index of glycaemic control. We determined glycated IgM levels in a diabetic patients population as compared to control non-diabetic subjects in a cross-sectional and longitudinal study. For that purpose we developed a precipitation method for IgM purification, measured glycation on the purified protein by a nitroblue tetrazolium assay and correlated glycated IgM, glycated haemoglobin (HbA1c) and fructosamine rates. The purification method we developed comprises dextran sulfate-CaCl2, ammonium sulfate and polyethylene glycol precipitations and it allows extraction of IgM free of contaminants as shown by immunoelectrophoresis. Glycated IgM levels were 8.65 +/- 0.15 nmol deoxymorpholinofructose (DOMF) equivalents/mg protein for non-diabetic subjects (n = 30) and 12.08 +/- 0.60 nmol DOMF equivalents/mg protein (n = 67) for diabetic patients. Diabetic patients had then a 40% rise in glycated IgM (p < 0.0005). In a longitudinal study with patients undergoing treatment aimed at improving their metabolic control, glycated IgM levels decreased significantly (p < 0.001) between the day of admission and the fifth day. Average rate of fall was 13% with a range of 3.0 to 22.0% (n = 28). Glycated IgM clearly responds more rapidly than fructosamines which fell by 7.0% and than HbA1c, which showed a rate of fall of 3.9% in the same period. A significant positive correlation was found between these parameters, being stronger between glycated IgM and fructosamines. This method could provide an alternative approach as a short-term marker of glycaemic control for clinical trials.

Polyclonal immunoglobulin M: location of glycation sites.

A four step purification procedure for polyclonal human serum IgM was elaborated, including ultracentrifugation, ammonium sulfate and polyethyleneglycol precipitations and diffusion-exclusion gel chromatography. IgM was glycated in vitro both in the presence of [14C]glucose and with unlabeled glucose. Influence of incubation time up to 10 days and of glucose concentration between 10 and 60 mmol/l were studied. With 10 mmol/l glucose, a molar ratio glucose/IgM of 5.7 was attained in 10 days. Increase of glucose concentration up to 60 mmol/l led to a molar ratio of 16.0. Both basal and in vitro glycation were evaluated by 3H-labeling by gel filtration and Concanavalin A-Sepharose chromatographies. Glycation occurs mainly on the heavy chains (> 85%), particularly on the Fd region.

In vitro glycated low-density lipoprotein interaction with human monocyte-derived macrophages.

Human low-density lipoprotein (LDL) was glycated in vitro (5 days, glucose 50 mmol/l), labelled with 125I, and its binding and uptake by human monocyte-derived macrophages studied. Glycation produced lower binding and lower uptake. Competition experiments using unlabelled LDL (control, glycated, and acetyl-LDL) showed that most glycated LDL was taken up by the apolipoprotein-B100: E receptor pathway. Results suggest that less of the glycated LDL may enter the cells via scavenger receptors, and very minute amount via non-saturable receptor-independent pathways.

Purification of cytoplasmic precursors of yeast mitochondrial phenylalanyl-tRNA synthetase subunits.

Two polypeptidic precursors of yeast mitochondrial phenylalanyl-tRNA synthetase subunits were purified from the cytoplasm by immunoprecipitation with an insolubilized glutaraldehyde-treated IgG fraction, followed by two chromatographies on Sephadex G-200 and on DEAE-cellulose. Methionine was found as the N-terminal residue in both precursors, which exhibited N-terminal extensions.

[Microassay for blood adenosine deaminase: study of the role of the enzyme in the ammoniagenesis of the blood sample]. / Microdosage de l'adénosine désaminase sanguine: étude du rôle de l'enzyme dans l'ammoniogénèse du sang prélevé.

A microassay for adenosine deaminase is elaborated. It is based on the colorimetric measurement of small amounts of ammonia raised during adenosine deamination and separated by continuous flow dialysis. The analytical qualities of the microassay are reported. The enzymic activity concentration in whole arterial blood of 30 control subjects is 2.54 +/- 1,68 micro katal/l (mean +/- 2ET). It is slightly lower in venous blood. The values are higher in man than in women. 92 p. cent of the enzymic activity of whole blood are due to the erythrocytes. The distribution of the arterial enzymic activity concentrations is not significantly different in a group of 85 cirrhotic patients when compared to the control group. No tight correlation can be found between blood adenosine deaminase concentrations and either blood ammonia nor shed blood ammoniagenesis. Inhibitors of adenosine deaminase have little effects on the in vitro ammoniagenesis.

Yeast mitochondrial inner membrane 30 kd hydrophobic protein: properties and interaction with phospholipids.

A 30 kd hydrophobic protein is extracted from yeast mitochondrial inner membrane. It is present in wild yeast strains but absent in mitochondrial DNA lacking mutants. The isoelectric point of the protein and its solubility in various organic solvents are determined. The fluorescence of a tryptophan residue near the surface of the 30 kd protein dissolved in butanol-1, can be quenched by phospholipids containing unsaturated fatty acids. Results are in accordance with the 30 kd protein being an integral protein of the yeast mitochondrial inner membrane.

Effects of econazole nitrate on yeast cells and mitochondria.

The inhibitory effect of econazole nitrate on the growth of yeast Saccharomyces cerevisiae is proportional to the concentration of the product. It depends on the phase of culture and on the number of cells present at the moment of econazole addition into the medium. The most important inhibition is obtained in the exponential phase of growth with a low concentration of cells. It is enhanced with cells which were previously in contact with the product. There is no adaptation of the yeast toward increased concentrations of econazole. The product penetrates the cells and attaches first to particular fractions, later to soluble fractions. The highest concentration of econazole nitrate in cells lies in the mitochondria. No product of econazole metabolism by S. cerevisiae was uncovered. Econazole nitrate does not slow down the in vivo activities of mitochondrial enzymes (cytochrome c oxidase, succinate dehydrogenase and phenylalanyl-tRNA synthetase), but inhibits the biosynthesis of mitochondrial membrane enzymes without affecting that of the synthetase, a matrix enzyme.

Mitochondrial phenylalanyl t-RNA synthetase from yeast: formation of enzyme-substrate complexes shown by heat or SH reagent inactivation.

The binding of substrates to purified mitochondrial phenylalanyl-tRNA synthetase from yeast was examined using the kinetics of heat or p-hydroxymercurybenzoate inactivation. Individually magnesium chloride and each of the substrates protect the enzyme against thermal denaturation and p-hydroxymercurybenzoate inhibition. No enzyme protection is observed with ATP alone against p-hydroxymercurybenzoate inhibition. The combinations of the various substrates induce a synergistic protection effect. Protection constants of 31 microM and 0.3 microM were found for L-Phe and mt tRNAPhe respectively, from heat inactivation studies. The inhibition of the enzyme activity by p-hydroxymercurybenzoate can be reverted by 2-mercaptoethanol or dithiothreitol.

A yeast mitochondrial inner membrane 30K hydrophobic protein: comparison with subunit 32K of the cytochrome BC 1 complex.

A yeast mitochondrial inner membrane hydrophobic protein 30K has been isolated and compared to subunit 32K of the yeast cytochrome bc 1 complex. Both proteins are translated on mitochondrial ribosomes, have nearly the same molecular weight and similar aminoacid compositions. Comparison was carried out by immunological techniques with specific antibodies, and by studying 3 yeast strains having mutations in the COB region of the mitochondrial DNA. Results show that the two proteins are not identical.

Biosynthesis and transport of yeast mitochondrial phenylalanyl-tRNA synthetase.

The biosynthesis of yeast mitochondrial Phe-tRNA synthetase is studied in vivo. Antibodies against the enzyme are raised in rabbits. They precipitate two proteins in the post-ribosomal supernatant of the yeast cell homogenate. Immunoprecipitate analysis on SDS - gel electrophoresis shows that the two types of mitochondrial enzyme subunits with molecular weights of 57,000 and 72,000, respectively, are cytoplasmically synthesized as larger, individual precursors. Terminal extensions of the precursors prevent enzyme activity. Mitochondrial membranes linked protease(s) play(s) an active role in maturation.

[Isolation of yeast protoplasts using various preparations of the hepato-pancreatic juice of Helix pomatia]. / Obtention de protoplastes de levure à l'aide de différences préparations de suc hépato-pancréatique d'Helix pomatia.

Conversion of large amounts of Saccharomyces cerevisiae cells to protoplasts is studied using various preparations extracted from Helix pomatia hepato-pancreatic juice. The most favourable yield in two hours incubations (88 per cent) is obtained with 20 ml cytohelicase, a chitinase and glucanase enriched extract, per 400 g of yeast cells, harvested at the end of the logarithmic growth phase and preincubated in presence of 2-mercaptoethanol.