Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.[32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)-domain-containing PtdIns(3)P binding protein early endosomal antigen 1 as well as of a gluathione-S-transferase-FYVE probe directed to PtdIns(3)P. Expression of wt myotubularin, although not affecting activation of proximal insulin signal transduction targets such as protein kinase B and MAPK, induced a decrease in insulin-induced glucose uptake, whereas basal glucose uptake was augmented by expression of D278A (DA) and C375S (CS) mutants. Moreover, overexpression of myotubularin in 3T3-L1 adipocytes impaired insulin-induced translocation at the plasma membrane of green fluorescent protein-tagged glucose transporter 4. These data indicate that PtdIns(3)P is required to direct glucose transporter 4 to insulin-responsive compartments and/or to allow the translocation of the latter at the plasma membrane.We conclude that myotubularin, by modulating the intracellular levels of PtdIns(3)P, plays a role in the control of vesicular traffic related to glucose transport, by counteracting the activities of the PtdIns(3)P-producing phosphatidylinositol 3-kinases.     

Nitric oxide affects plant mitochondrial functionality in vivo

In this report, we show that nitric oxide affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway. The residual respiration depends on the alternative pathway and novel synthesis of alternative oxidase occurs. These modifications are associated with depolarisation of the mitochondrial membrane potential and release of cytochrome c from mitochondria, suggesting a conserved signalling pathway in plants and animals. This signal cascade is triggered at the mitochondrial level and induces about 20% of cell death. In order to achieve a higher level of cell death, the addition of H(2)O(2) is necessary.     

Hypobetalipoproteinemia with an apparently recessive inheritance due to a "de novo" mutation of apolipoprotein B

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder either linked or not linked to apolipoprotein (apo) B gene. Abetalipoproteinemia (ABL) is a recessive disorder due to mutations of microsomal triglyceride transfer protein (MTP) gene. We investigated a patient with apparently recessive hypobetalipoproteinemia consistent with symptomatic heterozygous FHBL or a "mild" form of ABL. The proband had fatty liver associated with LDL-cholesterol (LDL-C) and apo B levels <5th percentile but no truncated apo B forms detectable in plasma. MTP gene sequence revealed that he was a carrier of the I128T polymorphism and an unreported amino acid substitution (V168I) unlikely to be the cause of hypobetalipoproteinemia. Apo B gene sequence showed that he was heterozygous for two single base substitutions in exon 9 and 22 resulting in a nonsense (Q294X) and a missense (R1101H) mutation, respectively. Neither of his parents carried the Q294X; his father and paternal grandmother carried the R1101H mutation. Analysis of polymorphic genetic markers excluded non-paternity. In conclusion, the proband has a "de novo" mutation of apo B gene resulting in a short truncated apo B form (apo B-6.46). Sporadic cases of FHBL with an apparently recessive transmission may be caused by "de novo" mutations of apo B gene.     

DKT1, a novel K+ channel from carrot, forms functional heteromeric channels with KDC1

We report the isolation and characterisation of DKT1, a new carrot K+ channel alpha-subunit belonging to the Shaker-like family. DKT1 is expressed in many tissues of the adult plant, suggesting that it may play important roles in both nutrition and other important physiological processes. During embryo development, DKT1 is expressed at later phases implying the involvement of K+ in embryo maturation. When co-expressed with KDC1 in Xenopus oocytes, DKT1 is able to form functional, heteromeric channels, suggesting that possible interactions between these two ion channels in plant tissues may modulate K+ uptake.     

Trojan horse-like behavior of a biologically representative mixture of oxysterols

Oxysterols, 27-carbon atoms cholesterol oxidation products, are consistently detectable in minimally oxidized low density lipoproteins (oxLDLs) and accumulate in the core of fibrotic plaques. Several oxysterols of pathophysiological interest have been shown to possess many and diverse biochemical activities. In particular, 7-ketocholesterol (7K), a major cholesterol oxide both in oxLDLs and in atherosclerotic lesions, is able to lead vascular cells to apoptosis. Indeed, when 7K is added to cells of the macrophage lineage, in a concentration range actually detectable in hypercholesterolemic patients, a marked apoptotic effect was observed. However, when identical concentrations of 7K are given to the same cells in a mixture with other oxysterols, also detectable in human low density lipoprotein (LDL), cell apoptosis was dramatically reduced. Of note, identical amounts of unoxidized cholesterol did not show any significant pro-apoptotic effect. With the aim to investigate the mechanisms underlying the quenching of 7K-dependent apoptosis by the oxysterol mixture, we found that the combined oxysterol mixture counteracted the ability of 7K given alone to strongly increase the steady-state level of reactive oxygen species (ROS) in macrophages as well as the up-regulation of the pro-apoptotic factor p21 and the triggering of the mitochondria-dependent pathway of apoptosis. Competition among oxysterols, apparently at NADPH oxidase level, diminishes the macrophage ROS production and direct toxicity that is evoked by defined oxysterols, as for instance, 7-ketocholesterol.     

4-Hydroxynonenal is markedly higher in patients on a standard long-term home parenteral nutrition

Parenteral nutrition, a commonly used procedure in patients with gastrointestinal disorders, may lead with time to liver steatosis and fibrosis, whose pathogenesis has yet to be elucidated. Oxidative stress and particularly lipid peroxidation likely contribute to the expression of such hepatobiliary complications, by means of their recognized proinflammatory and profibrogenic effects. To evaluate the adequacy against oxidative insult of a standard micronutrient supplementation in patients under long term parenteral nutrition, a comprehensive patterns of redox indices has been determined on peripheral blood samples from forty one adults in comparison to fifty eight blood donors taken as controls. A sustained oxidative stress in peripheral blood of home parenteral patients was observed. Of the two lipid peroxidation markers found to be markedly increased, namely fluorescent plasma protein adducts with malondialdehyde and 4-hydroxynonenal, respectively, only the second was statistically correlated with all the antioxidant-related changes consistently detected in the patients, namely decreased plasma alpha-tocopherol and selenium intake and higher erythrocyte oxidized glutathione. Plasma level of 4-hydroxynonenal-protein adducts appears to be a reliable and easily measurable marker of oxidative status, particularly indicated to monitor the adequacy of dietary regimen during parenteral nutrition.     

Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²⁺ handling relationship in plant cells

The existence of a transport protein that imports cytosolic NAD(+) into peroxisomes has been controversially discussed for decades. Nevertheless, the biosynthesis of NAD(+) in the cytosol necessitates the import of NAD(+) into peroxisomes for numerous reduction/oxidation (redox) reactions. However, a gene encoding such a transport system has not yet been identified in any eukaryotic organism. Here, we describe the peroxisomal NAD(+) carrier in Arabidopsis. Our candidate gene At2g39970 encodes for a member of the mitochondrial carrier family. We confirmed its peroxisomal localization using fluorescence microscopy. For a long time At2g39970 was assumed to represent the peroxisomal ATP transporter. In this study, we could show that the recombinant protein mediated the transport of NAD(+) . Hence, At2g39970 was named PXN for peroxisomal NAD(+) carrier. The loss of PXN in Arabidopsis causes defects in NAD(+) -dependent β-oxidation during seedling establishment. The breakdown of fatty acid released from storage oil was delayed, which led to the retention of oil bodies in pxn mutant seedlings. Based on our results, we propose that PXN delivers NAD(+) for optimal fatty acid degradation during storage oil mobilization.     

Molecular Identification of Collagen 17a1 as a Major Genetic Modifier of Laminin Gamma 2 Mutation-Induced Junctional Epidermolysis Bullosa in Mice

Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2^jeb mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2^jeb mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2^jeb mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.