Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cmr1 protein preferentially binds to UV-damaged DNA <Emphasis Type="Italic">in vitro</Emphasis>

DNA metabolic processes such as DNA replication, recombination, and repair are fundamentally important for the maintenance of genome integrity and cell viability. Although a large number of proteins involved in these pathways have been extensively studied, many proteins still remain to be identified. In this study, we isolated DNA-binding proteins from Saccharomyces cerevisiae using DNA-cellulose columns. By analyzing the proteins using mass spectrometry, an uncharacterized protein, Cmr1/YDL156W, was identified. Cmr1 showed sequence homology to human Damaged-DNA binding protein 2 in its C-terminal WD40 repeats. Consistent with this finding, the purified recombinant Cmr1 protein was found to be intrinsically associated with DNA-binding activity and exhibited higher affinity to UV-damaged DNA substrates. Chromatin isolation experiments revealed that Cmr1 localized in both the chromatin and supernatant fractions, and the level of Cmr1 in the chromatin fraction increased when yeast cells were irradiated with UV. These results suggest that Cmr1 may be involved in DNA-damage responses in yeast.     

Association between the CTLA-4 +49 A/G polymorphism and susceptibility to rheumatoid arthritis: a meta-analysis

The aim of this study was to explore whether the cytotoxic T lymphocyte antigen-4 (CTLA-4) +49 A/G polymorphism confers susceptibility to rheumatoid arthritis (RA). A meta-analysis was conducted on the associations between CTLA-4 +49 A/G polymorphism and RA using; 1) allele contrast, 2) the recessive model, 3) the dominant model, and 4) an additive model. A total of 19 studies, 5,752 RA patients and 5,508 controls, encompassing 9 Caucasian, 8 Asian, 1 Mexican, and 1 Tunisian population were included in this meta-analysis. Ethnicity-specific meta-analysis was performed on Caucasian and Asian populations. Meta-analysis of the CTLA-4 +49 A/G polymorphism revealed an association between RA and the CTLA-4 +49 G allele in all 11,260 study subjects (odds ratio (OR) 1.118, 95% confidence interval (CI) 1.033–1.210, P = 0.005). Stratification by ethnicity showed an association between the CTLA-4 +49 G allele and RA in Asians (OR 1.164, 95% CI 1.056–1.283, P = 0.002), but no evidence of an association in Caucasians (OR 1.085, 95% CI 0.973–1.209, P = 0.431). Furthermore, associations were found between RA and the CTLA-4 +49 A/G polymorphism in Asians using the dominant and additive models, but not using the recessive model. On the other hand, no association was found between RA and the CTLA-4 +49 A/G polymorphism using the recessive, dominant, or additive models in Caucasians. This meta-analysis demonstrates that the CTLA-4 +49 A/G polymorphism confers susceptibility to RA in Asians, but not in Caucasians.     

Genetic variations in <Emphasis Type="Italic">KIFC1</Emphasis> and the risk of aspirin exacerbated respiratory disease in a Korean population: an association analysis

Modest effects of genes in various pathways are significant in the etiology of complex human diseases, including aspirin exacerbated respiratory disease (AERD). By functioning as a relevant component of respiratory processes, the human kinesin family member C1 (KIFC1) is hypothesized to play a role in AERD pathogenesis. A case–control analysis was carried out by comparing the genotype distribution of six KIFC1 single-nucleotide polymorphisms between 93 AERD cases and 96 aspirin-tolerant asthma controls in a Korean population. After controlling for confounds, logistic and regression models via various modes of genetic inheritance facilitated the association analysis. Initial results revealed significant association at 0.05 level of significance between several KIFC1 variations and AERD (P = 0.01–0.05, OR = 1.81–1.90) as well as fall rate of forced expiratory volume in the 1st second, an important diagnostic marker of airways constriction (P = 0.04–0.05). However, the signals were not deemed significant after multiple testing corrections (P ^corr > 0.05). Although the results do not support a major role of KIFC1 in AERD pathogenesis in a Korean asthma cohort, further replication and validation studies are required to clarify the current findings.     

Associations between interleukin-10 polymorphisms and susceptibility to rheumatoid arthritis: a meta-analysis

The aim of this study was to determine whether the interleukin-10 (IL-10) polymorphisms confer susceptibility to rheumatoid arthritis (RA). A meta-analysis was conducted on the associations between the IL-10 −1082 G/A, −592 C/A, −892 C/T and IL-10.R polymorphisms and RA using; (1) allele contrast, (2) the recessive model, (3) the dominant model, and (4) the additive model. A total of 16 studies (19 comparisons) involving 2647 RA patients and 3383 controls were considered in the meta-analysis. Meta-analysis of the IL-10 −1082 G/A polymorphism showed no association with RA in the study subjects, or in European or Asian subjects. However, meta-analysis of the −1082 G allele in 4 studies in Hardy–Weinberg equilibrium showed a significant association with RA (OR = 1.217, 95% CI = 1.027–1.442, P = 0.0236). In contrast, meta-analysis of the C allele, the CC genotype, and of the CC versus the AA genotype of the IL-10 −592 C/A polymorphism showed significant associations with RA. The overall ORs of the associations between the C allele and RA were 0.684 and 0.758 (95% CI = 0.494–0.946, P = 0.022; 95% CI = 0.475–1.210, P = 0.045) in all study subjects and Asians. Meta-analysis of the CC + CT versus TT genotype and of the CC versus TT genotype of the IL-10 −892 C/T polymorphism revealed significant associations with RA. The overall OR of the association between the C allele carrier and RA was 0.552 (95% CI = 0.375–0.812, P = 0.003). No association was found between the IL10.R2 alleles and RA. This meta-analysis suggests that the IL-10 −592 C/A polymorphism confers susceptibility to RA in Asians and that the IL-10 −1082 G/A and −892 C/T polymorphisms are associated with RA susceptibility. These findings suggest the IL-10 genes confer susceptibility to RA.     

Phenelzine (Monoamine Oxidase Inhibitor) Increases Production of Nitric Oxide and Proinflammatory Cytokines via the NF-κB Pathway in Lipopolysaccharide-Activated Microglia Cells

Phenelzine is a potent monoamine oxidase inhibitor that is used in patients with depression. It is also well known that nitric oxide (NO) synthase inhibitors show preclinical antidepressant-like properties, which suggests that NO is involved in the pathogenesis of depression. The purpose of this study was to determine if phenelzine affects the production of NO and tumor necrosis factor-alpha (TNF-α) in activated microglia cells. BV-2 microglia cells and primary microglia cells were cultured in DMEM and DMEM/F12 and then cells were treated with LPS or LPS plus phenelzine for 24?h. The culture medium was collected for determination of NO, TNF-α, and IL-6 and cells were harvested by lysis buffer for Western blot analysis. Phenelzine increased the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS), as well as the release of TNF-α and IL-6 in BV-2 microglia cells. It is also confirmed that phenelzine increased the levels of NO, TNF-α and IL-6 in LPS-activated primary microglia cells. Phenelzine increased nuclear translocation of NF-κB by phosphorylation of IκB-α in LPS-activated microglia cells. These findings suggest that high doses of phenelzine could aggravate inflammatory responses in microglia cells that are mediated by NO and TNF-α.     

Confirmation of Frm2 as a novel nitroreductase in Saccharomyces cerevisiae

Nitroreductases comprise a group of FMN- or FAD-dependent enzymes that reduce nitrosubstituted compounds by using NAD(P)H, and are found in bacterial species and yeast. Although there is little information on the biological functions of nitroreductases, some studies suggest their possible involvement in oxidative stress responses. In the yeast Saccharomyces cerevisiae, a putative nitroreductase protein, Frm2, has been identified based on its sequence similarity with known bacterial nitroreductases. Frm2 has been reported to function in the lipid signaling pathway. To study the functions of Frm2, we measured the nitroreductase activity of purified Frm2 on 4-nitroquinoline-N-oxide (4-NQO) using NADH. LC-MS analysis of the reaction products revealed that Frm2 reduced NQO into 4-aminoquinoline-N-oxide (4-AQO) via 4-hydroxyaminoquinoline (4-HAQO). An Frm2 deletion mutant exhibited growth inhibition in the presence of 4-NQO. Thus, in this study, we demonstrate a novel nitroreductase activity of Frm2 and its involvement in the oxidative stress defense system.