Different pathways of uptake and degradation of sphingomyelin by lymphoblastoid cells and the potential participation of the neutral sphingomyelinase.

The metabolism of sphingomyelin (SPM) was investigated in Epstein-Barr virus-transformed lymphoid cell lines from normal individuals and from patients with Niemann-Pick disease Type A (deficient in the acid, lysosomal sphingomyelinase) and familial hypercholesterolemia (lacking the low density lipoprotein receptor). Cells were incubated with the following radioactive or fluorescent SPMs: [choline-methyl-14C] SPM, [oleoyl-3H]SPM, pyrene-propenoyl-SPM (P3:1-SPM), pyrene-butanoyl-SPM (P4-SPM), pyrene-dodecanoyl-SPM (P12-SPM), and pyrene-sulfonylamino-undecanoyl-SPM (PSA11-SPM). Several pathways of uptake and subsequent metabolism of SPM in the lymphoblastoid cells were identified. [choline-methyl-14C]SPM and the P12-analog, administered to the cells in the presence of lipoproteins, were taken up through the apoB/E receptor-dependent pathway of endocytosis and degraded solely by the lysosomal sphingomyelinase. Under similar conditions, the other sphingomyelins, i.e. [oleoyl-3H]SPM, P3:1-SPM, P4-SPM, and PSA11-SPM, were taken up by a low density lipoprotein receptor-independent pathway and degraded mostly by a nonlysosomal sphingomyelinase which also catalyzed their hydrolysis in Niemann-Pick cells. In the absence of serum, all sphingomyelins were taken up by an apoB/E receptor-independent pathway and hydrolyzed by a nonlysosomal sphingomyelinase. Indeed, in vitro assays demonstrated the presence, in lymphoblastoid cells, of the neutral magnesium-activated sphingomyelinase, which was also fully active in the Niemann-Pick cells. In conclusion, our observations are consistent with: (i) the existence in lymphoblastoid cells of several pathways for the uptake and subsequent utilization of SPM; (ii) a major role of lipoproteins for the metabolic routing of the SPM; and (iii) the effect of the structure of the fatty acyl residue of SPM on its possible association with lipoproteins and/or cell membranes.     

Biochemical and ultrastructural studies on an Epstein-Barr virus-transformed lymphoid cell line from a Niemann-Pick disease type C patient

Human lymphoid cell lines established from normal subjects and from a Niemann-Pick disease type C patient were investigated from a triple point of view of enzymology, metabolism and ultrastructure: Sphingomyelinase activities, isoenzyme electrofocusing profiles and properties of the major enzyme were quite similar in type C and normal lymphoid cell lines. Similarly, no significant difference was observed in non-specific phosphodiesterases hydrolysing bis(methylumbelliferyl)phosphate and bis(methylumbelliferyl)pyrophosphate. The study of the lipid composition of type C cells showed no obvious accumulation of sphingomyelin or other phospholipid, but only a higher amount of glycolipids (mainly GlcCer and GbOse3Cer), as visualized by bidimensional thin-layer chromatography. Ultrastructural studies demonstrated, in type C cells, the presence of an obvious lysosomal storage of amphiphilic lipids quite similar to that observed in tissues of type C patients. These studies, which demonstrate the validity of lymphoid cell lines as an experimental model system for type C disease, agree with the current opinion that an impairment of sphingomyelin catabolism is not the primary defect in type C disease.     

Separation by electrofocusing of the molecular forms of splenic beta-glucosidase in normal subjects and Gaucher's disease

Electrofocusing allows to separate the beta-glucosidases from normal spleen in several molecular forms: a non specific beta-glucosidase form (pl 4.6) and two forms of beta-glucocerebrosidase (pl 5.0 and pl 6.5). beta-glucosidase and beta-glucocerbrosidase differ with regard to their thermal stability. In a spleen from a patient with non neuronopathis Gaucher's disease (type I or adult form), molecular whereas the non specific beta-glucosidase is not significantly decreased. Thus, the beta-glucocerebrosidase forms and the non specific beta-glucosidase are not coded by the same gene. The separation of molecular forms of beta-glucosidase allows to measure the part of specific beta-glucosidase activity in the assay of beta-glucocerebrosidase by artificial substrate, 4-methylumbeliferyl-beta-D-glucopyranoside.     

Modifications of the molecular weight of membrane-bound nonspecific β-glucosidase in type 1 gaucher disease determined in situ by the radiation inactivation method

The radiation inactivation method has been used to compare the molecular weight of the nonspecific membrane-bound β-glucosidase in situ in normal human spleen and in that of two patients with Gaucher diease type 1. We report, in type 1 Gaucher spleen, the presence of a high molecular weight component (557 000) in addition to the normal low molecular weight component (97 800). The various possible hypotheses explaining this high molecular weight component are discussed.     

Picomolar concentrations of free zinc(II) ions regulate receptor protein-tyrosine phosphatase β activity

As key enzymes in the regulation of biological phosphorylations, protein-tyrosine phosphatases are central to the control of cellular signaling and metabolism. Zinc(II) ions are known to inhibit these enzymes, but the physiological significance of this inhibition has remained elusive. Employing metal buffering for strict metal control and performing a kinetic analysis, we now demonstrate that zinc(II) ions are reversible inhibitors of the cytoplasmic catalytic domain of the receptor protein-tyrosine phosphatase β (also known as vascular endothelial protein-tyrosine phosphatase). The K(i)((Zn)) value is 21 ± 7 pm, 6 orders of magnitude lower than zinc inhibition reported previously for this enzyme. It exceeds the affinity of the most potent synthetic small molecule inhibitors targeting these enzymes. Inhibition is in the range of cellular zinc(II) ion concentrations, suggesting that zinc regulates this enzyme, which is involved in vascular physiology and angiogenesis. Thus, for some enzymes that are not recognized as zinc metalloenzymes, zinc binding inhibits rather than activates as in classical zinc enzymes. Activation then requires removal of the inhibitory zinc.     

PCR detection of enzyme-encoding genes in <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strains of wine origin

Fifteen isolates of lactic acid bacteria originating from South African grape and wine samples were identified as Leuconostoc mesenteroides subsp. mesenteroides through the taxonomic analysis of their 16S rDNA gene sequences. These isolates were further tested for the presence of genes coding for enzymes of oenological relevance using PCR detection technique. A type strain of Leuc. mesenteroides (NCDO 529^T) was also incorporated for comparative analysis. From the PCR detection results, the estA, prtP, alsD, alsS, metK, metC and metB genes were present in all the strains tested. The bgl and gshR genes encoding β-glucosidase and glutathione reductase, respectively, were not detected in some strains. On the other hand, none of the tested strains possessed the genes encoding phenolic acid decarboxylase (padA), citrate permease (citP), citrate lyase (citD, citE and citF) and arginine deiminase pathway enzymes (arcA, arcB and arcC). The verification of PCR-generated fragments was performed by sequencing. GenBank database was used to search for homologous DNA sequences. Neighbour-joining trees based on nucleotide sequences of alsS, estA, metK and mleA genes were also constructed in order to study the phylogenetic relationship between Leuc. mesenteroides strains and closely related species. The phylogenetic analyses revealed that there are genetic heterogeneities between strains of Leuc. mesenteroides species. In conclusion, this study has improved our knowledge on the genetics of oenological strains of Leuc. mesenteroides and their genetic potential to contribute to certain wine aroma compounds.     

Surviving in the presence of sulphur dioxide: strategies developed by wine yeasts

Sulphur dioxide has been used as a common preservative in wine since at least the nineteenth century. Its use has even become essential to the making of quality wines because of its antioxidant, antioxidasic and antiseptic properties. The chemistry of SO₂ in wine is fairly complex due to its dissociation into different species and its binding to other compounds produced by yeasts and bacteria during fermentation. The only antiseptic species is the minute part remaining as molecular SO₂. The latter concentration is both dependent on pH and concentration of free bisulphite. However, certain yeast species have developed cellular and molecular mechanisms as a response to SO₂ exposure. Some of these mechanisms are fairly complex and have only been investigated recently, at least for the molecular mechanisms. They include sulphite reduction, sulphite oxidation, acetaldehyde production, sulphite efflux and the entry into viable but not culturable state, as the ultimate response. In this review, the chemistry of SO₂ in wine is explained together with the impact of SO₂ on yeast cells. The different defence mechanisms are described and discussed, mostly based on current knowledge available for Saccharomyces cerevisiae.     

Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation

Vitamin E disappearance is accelerated in cigarette smokers due to their increased oxidative stress and is inversely correlated with plasma vitamin C concentrations. Therefore, we hypothesized that ascorbic acid supplementation (500 mg, twice daily; 2 weeks) would normalize smokers' plasma alpha- and gamma-tocopherol disappearance rates and conducted a double-blind, placebo-controlled, randomized crossover investigation in smokers (n=11) and nonsmokers (n=13) given a single dose of deuterium-labeled alpha- and gamma-tocopherols (50 mg each d6-RRR-alpha and d2-RRR-gamma-tocopheryl acetate). During the placebo trial, smokers, compared with nonsmokers, had significantly (P<0.05) greater alpha- and gamma-tocopherol fractional disappearance rates and shorter half-lives. Ascorbic acid supplementation doubled (P<0.0001) plasma ascorbic acid concentrations in both groups and attenuated smokers', but not nonsmokers', plasma alpha- and gamma-tocopherol (P<0.05) fractional disappearance rates by 25% and 45%, respectively. Likewise, smokers' plasma deuterium-labeled alpha- and gamma-tocopherol concentrations were significantly higher (P<0.05) at 72 h during ascorbic acid supplementation compared with placebo. Ascorbic acid supplementation did not significantly change (P>0.05) time of maximal or maximal-labeled alpha- and gamma-tocopherol concentrations. Smokers' plasma F2alpha-isoprostanes were approximately 26% higher than nonsmokers (P>0.05) and were not affected by ascorbic acid supplementation in either group (P>0.05). In summary, cigarette smoking increased plasma alpha- and gamma-tocopherol fractional disappearance rates, suggesting that the oxidative stress from smoking oxidizes tocopherols and that plasma ascorbic acid reduces alpha- and gamma-tocopheroxyl radicals to nonoxidized forms, thereby decreasing vitamin E disappearance in humans.     

Dynamics of crowing development in the domestic Japanese quail (Coturnix coturnix japonica)

Species-specific behaviours gradually emerge, via incomplete patterns, to the final complete adult form. A classical example is birdsong, a learned behaviour ideally suited for studying the neural and molecular substrates of vocal learning. Young songbirds gradually transform primitive unstructured vocalizations (subsong, akin to human babbling) into complex, stereotyped sequences of syllables that constitute adult song. In comparison with birdsong, territorial and mating calls of vocal non-learner species are thought to exhibit little change during development. We revisited this issue using the crowing behaviour of domestic Japanese quail (Coturnix coturnix japonica). Crowing activity was continuously recorded in young males maintained in social isolation from the age of three weeks to four months. We observed developmental changes in crow structure, both the temporal and the spectral levels. Speed and trajectories of these developmental changes exhibited an unexpected high inter-individual variability. Mechanisms used by quails to transform sounds during ontogeny resemble those described in oscines during the sensorimotor phase of song learning. Studies on vocal non-learners could shed light on the specificity and evolution of vocal learning.