cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Cloning and evolutionary analysis of tobacco MAPK gene family

The mitogen-activated protein (MAP) kinase cascade is an important signaling module which is involved in biotic and abiotic stress responses as well as plant growth and development. In this study, we identified 17 tobacco MAPKs including 11 novel tobacco MAPK genes that have not been identified before. Comparative analysis with MAPK gene families from other plants, such as Athaliana thaliana, rice and poplar, suggested that tobacco MAPKs (such as NtMPK1, NtMPK3 and NtMPK8) might play similar functions in response to abiotic and biotic stresses. QRT-PCR analysis revealed that a total of 14 NtMPKs were regulated by SA and/or MeJA, suggesting their potential roles involved in plant defense response. In addition, 6 NtMPKs were induced by drought treatment, implying their roles in response to drought stress. Our results indicated that most of tobacco MAPK might be involved in plant defense response, which provides the basis for further analysis on physiological functions of tobacco MAPKs.     

Neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside against sodium nitroprusside-induced neurotoxicity in HT22 cells

In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 °C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 °C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.     

Overexpression of Smad7 suppressed ROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1

We previously reported that AngiotensinII receptor blocker effectively inhibited TGF-β1-mediated epithelial-to-mesenchymal transition progress through regulating Smad7. However, the underlying mechanism by which Smad7 exerted in regulating MMP9 and fibrogenic response has not been fully elucidated. In the current study, we proved that NADPH p47^phox-dependent reactive oxygen species (ROS) production contributed to MMP9 activation and collagen expression, which was suppressed by transfecting pcDNA3–Smad7 in cardiac fibroblasts. The effect of Smad7 overexpression on MMP9 activity and collagen expression was further reversed by adding H₂O₂ (10 μmol/L). In contrast, knockdown of Smad7 caused the enhanced collagen synthesis in cardiac fibroblasts, which was also reversed by treating cells with a ROS inhibitor, YCG063 (2 μmol/L). Further investigation showed that Smad7 regulated NADPH-mediated ROS production through activating Heme oxygenase-1 (HO-1). Meanwhile, the intercellular level of bilirubin (product of hemin) and nitric oxide (NO) in cell supernatant were not significantly increased in cells treated with AngII or transfected with Smad7. Knockdown of HO-1 in Smad7-overexpressed cardiac fibroblasts or cells pretreated with SnPP IX, a competitive inhibitor of HO-1 activity, resulted in increased productions of ROS and NADPH p47^phox, and abolished the inhibitory effects of Smad7 on MMP9 activity and collagen expression. Our results indicated that HO-1 might be critically involved in Smad7-mediated regulation of MMP9 activity and fibrogenic genes expression via antagonizing the enhanced myocardial oxidative stress.     

Association between rs10118757(A/G) in methylthioadenosine phosphorylase gene and coronary artery disease in Chinese Hans

Studies focusing on the association of gene methylthioadenosine phosphorylase (MTAP) with the risk of coronary artery disease (CAD) and myocardial infarction (MI) are limited.     

Microarray analysis of gene expression profile in resistant and susceptible Bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus

To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we constructed a near-isogenic line (BC8) with BmNPV resistance using highly resistant (NB) and highly susceptible parental strains (306). We investigated variations in the gene expression in the midguts of BmNPV-infected BC8 and 306 at 12 h pi using the microarray. 92 differentially expressed genes were identified. Real-time qPCR analysis confirmed that 10 genes were significantly up-regulated or down-regulated in the midguts of BC8 and NB compared to 306. To our knowledge, we first defined the role of the amino acid transporter and 26S proteasome in insect antiviral. However, serine protease was not completely consistent with data of reported previously in insect antiviral. The role of the 5 genes (Bm123, Bm122, COP β′, aquaporin, glycoside hydrolases) was also demonstrated in insect antiviral. Our results provided new insights into the molecular mechanism of the Bombyx mori immune response against BmNPV infection.