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See detailOccurrence and subcellular distribution of the NAD(P)HX repair system in mammals.
Marbaix, Alexandre Y.; Tyteca, Donatienne; Niehaus, Tom D. et al

in The Biochemical journal (2014), 460(1), 49-58

Hydration of NAD(P)H to NAD(P)HX, which inhibits several dehydrogenases, is corrected by an ATP-dependent dehydratase and an epimerase recently identified as the products of the vertebrate Carkd ... [more ▼]

Hydration of NAD(P)H to NAD(P)HX, which inhibits several dehydrogenases, is corrected by an ATP-dependent dehydratase and an epimerase recently identified as the products of the vertebrate Carkd (carbohydrate kinase domain) and Aibp (apolipoprotein AI-binding protein) genes respectively. The purpose of the present study was to assess the presence of these enzymes in mammalian tissues and determine their subcellular localization. The Carkd gene encodes proteins with a predicted mitochondrial propeptide (mCARKD), a signal peptide (spCARKD) or neither of them (cCARKD). Confocal microscopy analysis of transfected CHO (Chinese-hamster ovary) cells indicated that cCARKD remains in the cytosol, whereas mCARKD and spCARKD are targeted to the mitochondria and the endoplasmic reticulum respectively. Unlike the other two forms, spCARKD is N-glycosylated, supporting its targeting to the endoplasmic reticulum. The Aibp gene encodes two different proteins, which we show to be targeted to the mitochondria (mAIBP) and the cytosol (cAIBP). Quantification of the NAD(P)HX dehydratase and epimerase activities in rat tissues, performed after partial purification, indicated that both enzymes are widely distributed, with total activities of approximately 3-10 nmol/min per g of tissue. Liver fractionation by differential centrifugation confirmed the presence of the dehydratase and the epimerase in the cytosol and in mitochondria. These data support the notion that NAD(P)HX repair is extremely widespread. [less ▲]

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See detailGreen fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb or beta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect of beta3GFP on alphaIIb(beta3) ligand binding.
Plançon, Sébastien UL; Morel-Kopp, M. C.; Schaffner-Reckinger, Elisabeth UL et al

in The Biochemical journal (2001), 357(Pt 2), 529-36

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either ... [more ▼]

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions. [less ▲]

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See detailStereospecific modulation of the calcium channel in human erythrocytes by cholesterol and its oxidized derivatives.
Neyses, Ludwig UL; Locher, R.; Stimpel, M. et al

in The Biochemical journal (1985), 227(1), 105-12

To study the effect of cholesterol and its pathophysiologically important oxidized derivatives (OSC) on the calcium entry channel, the human red blood cell was used as a model system. The calcium ejecting ... [more ▼]

To study the effect of cholesterol and its pathophysiologically important oxidized derivatives (OSC) on the calcium entry channel, the human red blood cell was used as a model system. The calcium ejecting adenosinetriphosphatase (ATPase) was inhibited by vanadate. The cells were loaded with OSC at concentrations between 1.25 X 10(-5) and 25 X 10(-5) mol/l. 22-Hydroxycholesterol, cholestan-3 beta,5 alpha,6 beta-triol, 5 alpha-cholestan-3 beta-ol,3 beta,5 alpha-dihydroxycholestan-6-one and 3 beta-hydroxy-5 alpha-cholestan-7-one stimulated 45Ca2+ influx by up to almost 90%, whereas 25-hydroxycholesterol, 7 beta-hydroxycholesterol, 20 alpha-hydroxycholesterol and 7-oxocholesterol inhibited influx by up to 75%. Both stimulation and inhibition were dependent on the amount of OSC incorporated into the membrane. More than 90% of the total modification of calcium influx by OSC was accounted for by an influence on the nitrendipine-inhibitable part of influx. Enrichment of cholesterol in the membrane greatly stimulated, and cholesterol depletion inhibited, Ca2+ influx. These results demonstrate that cholesterol and its oxidized derivatives are able to modulate the calcium channel in human red blood cells in a highly stereospecific manner. [less ▲]

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