Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling

Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.     

The effects of beta acids from hops (Humulus lupulus) on mortality of Varroa destructor (Acari: Varroidae)

Hop (Humulus lupulus L.) beta acids (HBA) were tested for miticidal effects on varroa destructor Anderson and Trueman, a parasitic mite of the honey bee (Apis mellifera L.). When varroa were placed on bees that had topical applications of 1?% HBA, there was 100?% mite mortality. Bee mortality was unaffected. Cardboard strips saturated with HBA and placed in colonies resulted in mite drop that was significantly greater than in untreated hives. HBA was detected on about 60?% of the bees in colonies during the first 48?h after application. Mite drop in colonies lasted for about 7?days with the highest drop occurring in the first 2–3?days after treatment. There was a reduction in the percentages of bees with HBA and in the amounts on their bodies after 7?days. Bee and queen mortality in the colonies were not affected by HBA treatments. When cardboard strips saturated with HBA were put in packages of bees, more than 90?% of the mites were killed without an increase in bee mortality. HBA might have potential to control varroa when establishing colonies from packages or during broodless periods.     

Detection of quantitative trait loci for resistance to gastrointestinal nematode infections in Creole goats

This study aimed to identify regions of the genome affecting resistance to gastrointestinal nematodes in a Creole goat population naturally exposed to a mixed nematode infection (Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum) by grazing on irrigated pasture. A genome‐wide quantitative trait loci (QTL) scan was performed on 383 offspring from 12 half‐sib families. A total of 101 microsatellite markers were genotyped. Traits analysed were faecal egg count (FEC), packed cell volume (PCV), eosinophil count and bodyweight (BW) at 7 and 11 months of age. Levels of activity of immunoglobulin A (IgA) and activity of immunoglobulin E (IgE) anti‐Haemonchus contortus L3 crude extracts and adult excretion/secretion products (ESPs) were also analysed. Using interval mapping, this study identified 13 QTL for parasite resistance. Two QTL linked with FEC were found on chromosomes 22 and 26. Three QTL were detected on chromosomes 7, 8 and 14 for eosinophil counts. Three QTL linked with PCV were identified on chromosomes 5, 9 and 21. A QTL for BW at 7 months of age was found on chromosome 6. Lastly, two QTL detected on chromosomes 3 and 10 were associated with IgE anti‐L3, and IgE anti‐ESP was linked with two QTL on chromosomes 1 and 26. This study is the first to have identified regions of the genome linked with nematode resistance in a goat population using a genome scan. These results provide useful tools for the understanding of parasite resistance in small ruminants.     

Genetic mapping at 3-kilobase resolution reveals inositol 1,4,5-triphosphate receptor 3 as a risk factor for type 1 diabetes in Sweden

We mapped the genetic influences for type 1 diabetes (T1D), using 2,360 single-nucleotide polymorphism (SNP) markers in the 4.4-Mb human major histocompatibility complex (MHC) locus and the adjacent 493 kb centromeric to the MHC, initially in a survey of 363 Swedish T1D cases and controls. We confirmed prior studies showing association with T1D in the MHC, most significantly near HLA-DR/DQ. In the region centromeric to the MHC, we identified a peak of association within the inositol 1,4,5-triphosphate receptor 3 gene (ITPR3; formerly IP3R3). The most significant single SNP in this region was at the center of the ITPR3 peak of association (P=1.7 x 10(-4) for the survey study). For validation, we typed an additional 761 Swedish individuals. The P value for association computed from all 1,124 individuals was 1.30 x 10(-6) (recessive odds ratio 2.5; 95% confidence interval [CI] 1.7-3.9). The estimated population-attributable risk of 21.6% (95% CI 10.0%-31.0%) suggests that variation within ITPR3 reflects an important contribution to T1D in Sweden. Two-locus regression analysis supports an influence of ITPR3 variation on T1D that is distinct from that of any MHC class II gene.     

Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.     

Oxidative/nitrative stress in the pathogenesis of systemic sclerosis: are antioxidants beneficial?

Abstract

Systemic sclerosis (SSc) is a multisystem autoimmune disease: characterised from the clinical side by progressive vasculopathy and fibrosis of the skin and different organs and from the biochemical side by fibroblast deregulation with excessive production of collagen and increased expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). The latter contributes to an overproduction of reactive oxygen species that through an autocrine loop maintains NOX4 in a state of activation. Reactive oxygen and nitrogen species are implicated in the origin and perpetuation of several clinical manifestations of SSc having vascular damage in common; attempts to dampen oxidative and nitrative stress through different agents with antioxidant properties have not translated into a sustained clinical benefit. Objective of this narrative review is to describe the origin and clinical implications of oxidative and nitrative stress in SSc, with particular focus on the central role of NOX4 and its interactions, to re-evaluate the antioxidant approaches so far used to limit disease progression, to appraise the complexity of antioxidant treatment and to touch on novel pathways elements of which may represent specific treatment targets in the not so distant future.