Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic beta cells

Several nuclear and cytoplasmic proteins in metazoans are modified by O-linked N-acetylglucosamine (O-GlcNAc). This modification is dynamic and reversible similar to phosphorylation and is catalyzed by the O-linked GlcNAc transferase (OGT). Hyperglycemia has been shown to increase O-GlcNAc levels in pancreatic beta cells, which appears to interfere with beta-cell function. To obtain a better understanding of the role of O-linked GlcNAc modification in beta cells, we have isolated OGT interacting proteins from a cDNA library made from the mouse insulinoma MIN6 cell line. We describe here the identification of Ataxin-10, encoded by the SCA10 (spinocerebellar ataxia type 10) gene as an OGT interacting protein. Mutations in the SCA10 gene cause progressive cerebellar ataxias and seizures. We demonstrate that SCA10 interacts with OGT in vivo and is modified by O-linked glycosylation in MIN6 cells, suggesting a novel role for the Ataxin-10 protein in pancreatic beta cells.     

Metagenomic gene discovery: past, present and future

It is now widely accepted that the application of standard microbiological methods for the recovery of microorganisms from the environment has had limited success in providing access to the true extent of microbial biodiversity. It follows that much of the extant microbial genetic diversity (collectively termed the metagenome) remains unexploited, an issue of considerable relevance to a wider understanding of microbial communities and of considerable importance to the biotechnology industry. The recent development of technologies designed to access this wealth of genetic information through environmental nucleic acid extraction has provided a means of avoiding the limitations of culture-dependent genetic exploitation.     

Tesaglitazar, a dual PPAR{alpha}/{gamma} agonist, ameliorates glucose and lipid intolerance in obese Zucker rats

Insulin resistance, impaired glucose tolerance, high circulating levels of free fatty acids (FFA), and postprandial hyperlipidemia are associated with the metabolic syndrome, which has been linked to increased risk of cardiovascular disease. We studied the metabolic responses to an oral glucose/triglyceride (TG) (1.7/2.0 g/kg lean body mass) load in three groups of conscious 7-h fasted Zucker rats: lean healthy controls, obese insulin-resistant/dyslipidemic controls, and obese rats treated with the dual peroxisome proliferator-activated receptor alpha/gamma agonist, tesaglitazar, 3 mumol.kg(-1).day(-1) for 4 wk. Untreated obese Zucker rats displayed marked insulin resistance, as well as glucose and lipid intolerance in response to the glucose/TG load. The 2-h postload area under the curve values were greater for glucose (+19%), insulin (+849%), FFA (+53%), and TG (+413%) compared with untreated lean controls. Treatment with tesaglitazar lowered fasting plasma glucose, improved glucose tolerance, substantially reduced fasting and postload insulin levels, and markedly lowered fasting TG and improved lipid tolerance. Fasting FFA were not affected, but postprandial FFA suppression was restored to levels seen in lean controls. Mechanisms of tesaglitazar-induced lowering of plasma TG were studied separately using the Triton WR1339 method. In anesthetized, 5-h fasted, obese Zucker rats, tesaglitazar reduced hepatic TG secretion by 47%, increased plasma TG clearance by 490%, and reduced very low-density lipoprotein (VLDL) apolipoprotein CIII content by 86%, compared with obese controls. In conclusion, the glucose/lipid tolerance test in obese Zucker rats appears to be a useful model of the metabolic syndrome that can be used to evaluate therapeutic effects on impaired postprandial glucose and lipid metabolism. The present work demonstrates that tesaglitazar ameliorates these abnormalities and enhances insulin sensitivity in this animal model.     

Food and feeding habits of pearly razorfish, <Emphasis Type="Italic">Xyrichtys novacula </Emphasis>(Linnaeus, 1758), in the southern Tyrrhenian Sea: variation by sex and size

We collected pearly razorfish, Xyrichtys novacula (Labridae), from a sandy bottom of Capo dOrlando (Tyrrhenian Sea) to study their feeding habits. We caught fish by hand-lines and seine nets and identified, counted and weighed food items in their stomachs. We evaluated the importance of the different prey types by calculating the frequency of occurrence, abundance and mass. We used these values to calculate the index of relative importance (IRI) for each taxonomic category and a modified index (MI) which did not incorporate %N into the formula. Gammarideans were dominant food items in terms of %F but showed a very low MI value; the bivalve Acanthocardia tubercolata was the dominant species in terms of %W and MI value. The prey items were mostly benthic organisms belonging to the assemblage of fine, well-sorted sands showing that X. novacula is a benthic feeder. Despite the large number of prey taxa found, few species accounted for most of the prey consumed, indicating specialist feeding, as confirmed by the low value of the Levins standardised index. We found significant differences in prey distribution among size classes, with the highest prey diversity recorded in 120–140 mm TL size classes. Correspondence analysis showed a trend of increasing predator size classes among prey items, with small prey, such as copepods and amphipods, in the smallest predator size classes and bigger prey in the larger ones. Prey caught by larger predators differed significantly in weight from those taken by smaller specimens. There was a significant positive correlation of fish length with bivalves, decapods and echinoids, and a negative one with copepods, ostracods, caprellids, gammarideans. There were no significant differences between males and females of overlapping sizes. Juveniles are able to exploit only small, vulnerable prey, while adults take few, large prey, thereby avoiding competition with juveniles.     

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG(beta)-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG(beta)-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG(beta)-specific and anti-hCG(beta)/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG(beta)-R68E was eliminated, mAbs reacting with hCG(beta)-specific epitopes bound to hCG(beta) and hCG(beta)-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG(beta)-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG(beta)-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.     

The minor receptor group of human rhinovirus (HRV) includes HRV23 and HRV25, but the presence of a lysine in the VP1 HI loop is not sufficient for receptor binding

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore "true" major group viruses.     

Gene expression profiling of serum- and interleukin-1 beta-stimulated primary human adult articular chondrocytes--a molecular analysis based on chondrocytes isolated from one donor

In order to understand the cellular disease mechanisms of osteoarthritic cartilage degeneration it is of primary importance to understand both the anabolic and the catabolic processes going on in parallel in the diseased tissue. In this study, we have applied cDNA-array technology (Clontech) to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one donor cultured under anabolic (serum) and catabolic (IL-1beta) conditions. Significant differences between the different in vitro cultures tested were detected. Overall, serum and IL-1beta significantly altered gene expression levels of 102 and 79 genes, respectively. IL-1beta stimulated the matrix metalloproteinases-1, -3, and -13 as well as members of its intracellular signaling cascade, whereas serum increased the expression of many cartilage matrix genes. Comparative gene expression analysis with previously published in vivo data (normal and osteoarthritic cartilage) showed significant differences of all in vitro stimulations compared to the changes detected in osteoarthritic cartilage in vivo. This investigation allowed us to characterize gene expression profiles of two classical anabolic and catabolic stimuli of human adult articular chondrocytes in vitro. No in vitro model appeared to be adequate to study overall gene expression alterations in osteoarthritic cartilage. Serum stimulated in vitro cultures largely reflected the results that were only consistent with the anabolic activation seen in osteoarthritic chondrocytes. In contrast, IL-1beta did not appear to be a good model for mimicking catabolic gene alterations in degenerating chondrocytes.     

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

Oxygen therapy at low flow causes oxidative stress in chronic obstructive pulmonary disease: Prevention by N-acetyl cysteine

Exposure to high oxygen concentration produces toxicity by free radical release. We aimed to study: whether stable chronic obstructive pulmonary disease (COPD) patients present an unbalance in the blood redox status; the effect of oxygen administration on blood redox balance; the efficacy of N-acetyl-cysteine (NAC) treatment against the oxidative stress-induced by oxygen administration and whether it is dose-related. To this, 45 stable state III COPD patients were recruited and reduced glutathione (GSH) and oxidised glutathione (GSSG) in erythrocytes and thiol proteins (P-SH) and carbonyl proteins (PC) in both erythrocytes and plasma were evaluated. All COPD patients underwent 2 l/m oxygen for 18 h and NAC at 1200 or 1800 mg/day or placebo for 48 h starting with oxygen administration. Blood samples were collected at basal conditions, after 8 and 18 h of oxygen administration and 24 h after oxygen withdrawal. Results: COPD patients present an unstable redox equilibrium mainly due to plasma sulphydryl protein depletion. Oxygen administration oxidize erythrocyte GSH, decrease P-SH and increase PC levels in both plasma and erythrocytes. NAC administration counteract the oxidative stress and at the highest dose completely prevent protein oxidation. In conclusion, stable state III COPD patients present an unstable redox balance; long term low flow oxygen administration induces systemic oxidative stress, which is prevented by NAC treatment.     

Root-derived cytokinins as long-distance signals for NO3--induced stimulation of leaf growth

Leaf growth of many plant species shows rapid changes in response to alterations of the form and the level of N supply. In hydroponically-grown tomato (Lycopersicon esculentum L.), leaf growth was rapidly stimulated by NO(3)(-) application to NH(4)(+) precultured plants, while NH(4)(+) supply or complete N deprivation to NO(3)(-) precultured plants resulted in a rapid inhibition of leaf growth. Just 10 microM NO(3)(-) supply was sufficient to stimulate leaf growth to the same extent as 2 mM. Furthermore, continuous NO(3)(-) supply induced an oscillation of leaf growth rate with a 48 h interval. Since changes in NO(3)(-) levels in the xylem exudate and leaves did not correlate with NO(3)(-)-induced alterations of leaf growth rate, additional signals such as phytohormones may be involved. Levels of a known inhibitor of leaf growth, abscisic acid (ABA), did not consistently correspond to leaf growth rates in wild-type plants. Moreover, leaf growth of the ABA-deficient tomato mutant flacca was inhibited by NH(4)(+) without an increase in ABA concentration and was stimulated by NO(3)(-) despite its excessive ethylene production. These findings suggest that neither ABA nor ethylene are directly involved in the effects of N form on leaf growth. However, under all experimental conditions, stimulation of leaf growth by NO(3)(-) was consistently associated with increased concentration of the physiologically active forms of cytokinins, zeatin and zeatin riboside, in the xylem exudate. This indicates a major role for cytokinins as long-distance signals mediating the shoot response to NO(3)(-) perception in roots.