Purification and Comparison of Peroxisomal and Cytosolic Catalases from a Methanol-Grown Yeast, Kloeckera sp. 2201

Catalases were purified from a peroxisome-containing particulate fraction and a cytosolic fraction of methanol-grown Kloeckera sp. 2201 cells after subcellular fractionation. No difference was observed between the enzymes in the behaviours on column chromatographies, molecular mass of the subunits (M, 62, 000 daltons), and terminal amino acids, alanine. In addition, similar patterns were obtained with the peroxisomal and cytosolic enzymes on sodium dodecylsulfate/polyacrylamide slab-gel electrophoresis of the peptide fragments prepared by partial digestion with Staphylococcus aureus V 8 protease and papain. These results indicate that cytosolic catalase, even if functional, essentially has identical properties with the peroxisomal one in spite of the different subcellular distribution

Isolation of the Gene Encoding Yeast Peroxisomal Isocitrate Lyase by a Combination of the Plaque Hybridization with Non-Radioactive Probes and the Amplification of Phages in a Small Scale

A genomic DNA encoding isocitrate lyase, one of peroxisomal enzymes, was successfully isolated from an n-alkane-utilizable yeast genomic library prepared in a λEMBL phage by a combination of the plaque hybridization with a non-radioactive, biotin labeled, cDNA and the amplification of the phages in a small scale. Three clones, partially overlapping, with sizes of about 11, 13 and 16 kbp respectively were finally obtained. The genomic Southern blot analysis using the biotin-labeled probe suggested the presence of one isocitrate lyase gene in the genomic DNA

Expression Cloning of Catalase Genomic Gene : Genomic DNA Expression Library of Candida boidinii in Saccharomyces cerevisiae

The genomic DNA expression library of a methylotrophic yeast, Candida boidinii (Kloeckera sp.) 2201, was prepared in Saccharomyces cerevisiae by the electroporation method. Five transformants harbouring a catalase gene were independently isolated with an anti-C. boidinii catalase antibody from this library. Furthermore, exhibition of catalase activity in these transformants demonstrated that C. boidinii genes could sufficiently function even in S. cerevisiae

IL-18 ; a cytokine translates a stress into medical science

Psychological/physical stresses have been reported to exacerbate auto-immune and inflammatory diseases. To clarify a mechanism by which non-inflammatory stresses disrupt host defenses, responses to immobilization stress in mice were investigated, focusing on the role of a multifunctional cytokine, interleukin-18 (IL-18). In the adrenal cortex, the stress induced IL-18 precursor proteins (pro-IL-18) via ACTH and a superoxide-mediated caspase-1 activation pathway, resulting in conversion of pro-IL-18 to the mature form which was released into plasma. Inhibitors of caspase-1, reactive oxygen species and P38 MAPK prevented stress-induced accumulation of plasma IL-18. These inhibitors also blocked stress-induced IL-6 expression. This, together with the observation that IL-6was not induced in stressed-IL-18 deficient mice, showed that IL-6 induction by stress is dependent on IL-18. In stressed organisms, IL-18 may influence pathological and physiological processes. Controlling the caspase-1 activating pathway to suppress IL-18 levels may provide preventative means against stress-related disruption of host defenses

Multicenter evaluation of Verigene Enteric Pathogens Nucleic Acid Test for detection of gastrointestinal pathogens

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter　Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes.Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice

Prospective intervention study with a microarray-based, multiplexed, automated molecular diagnosis instrument (Verigene system) for the rapid diagnosis of bloodstream infections, and its impact on the clinical outcomes

The Verigene Gram-positive blood culture test (BC-GP) and the Verigene Gram-negative blood culture test (BC-GN) identify representative Gram-positive bacteria, Gram-negative bacteria and their antimicrobial resistance by detecting resistance genes within 3 h. Significant benefits are anticipated due to their rapidity and accuracy, however, their clinical utility is unproven in clinical studies. We performed a clinical trial between July 2014 and December 2014 for hospitalized bacteremia patients. During the intervention period (N = 88), Verigene BC-GP and BC-GN was used along with conventional microbiological diagnostic methods, while comparing the clinical data and outcomes with those during the control period (N = 147) (UMIN registration ID: UMIN000014399). The median duration between the initiation of blood culture incubation and the reporting time of the Verigene system results was 21.7 h (IQR 18.2-26.8) and the results were found in 88% of the cases by the next day after blood cultures were obtained without discordance. The hospital-onset infection rate was higher in the control period (24% vs. 44%, p = 0.002), however, no differences were seen in co-morbidities and severity between the control and intervention periods. During the intervention period, the time of appropriate antimicrobial agents\u27 initiation was significantly earlier than that in the control period (p = 0.001) and most cases (90%; 79/88) were treated with antimicrobial agents with in-vitro susceptibility for causative bacteria the day after the blood culture was obtained. The costs for antimicrobial agents were lower in the intervention period (3618 yen vs. 8505 yen, p = 0.001). The 30-day mortality was lower in the intervention period (3% vs. 13%, p = 0.019)

Genetics of rheumatoid arthritis contributes to biology and drug discovery

A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological datasets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA)1. Here, we performed a genome-wide association study (GWAS) meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ~10 million single nucleotide polymorphisms (SNPs). We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 1012–4. We devised an in-silico pipeline using established bioinformatics methods based on functional annotation5, cis-acting expression quantitative trait loci (cis-eQTL)6, and pathway analyses7–9 – as well as novel methods based on genetic overlap with human primary immunodeficiency (PID), hematological cancer somatic mutations and knock-out mouse phenotypes – to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery