Cloning, expression, purification, and activity assay of proteins related to D-lactic acid formation in Lactobacillus rhamnosus

Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet™-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.     

Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress

Diets containing 8% salt or 4% fructose (FR) cause insulin resistance and increase tissue methylglyoxal and advanced glycation end products (AGEs), platelet cytosolic-free calcium, and systolic blood pressure (SBP) in rats. In WKY rats, we have shown that moderately high salt, 4% NaCl (MHS) alone in diet does not cause hypertension, and when given along with 4% FR it does not have an additive effect. N-acetylcysteine (NAC) or l-arginine (ARG), treatment alone does not prevent hypertension in this model. The objectives of this study were to investigate the effect of NAC plus ARG in diet on SBP, platelet cytosolic-free calcium in a MHS + FR model, and to measure the plasma levels of methylglyoxal and the AGE, methylglyoxal-derived hydroimidazolone (MGH). At 7 weeks of age, WKY rats were divided into three groups: control group was given regular rat chow (0.7% NaCl) and water; MHS + FR group, diet containing 4% NaCl and 4% FR in drinking water; and MHS + FR + NAC + ARG group, MHS diet supplemented with 1.5% N-acetylcysteine (NAC) and 1.5% l-arginine (ARG), and 4% FR in drinking water, and followed for 6 weeks. NAC + ARG prevented the increase in platelet cytosolic-free calcium and SBP in MHS + FR treated rats. There was no difference in mean values of plasma methylglyoxal and MGH among the groups. In conclusion, NAC + ARG treatment is effective in preventing hypertension in a moderately high salt + FR-induced animal model. Plasma methylglyoxal and MGH may not represent tissue modification or, alternatively, other tissue AGEs, derived from methylglyoxal or other aldehydes, may be involved in hypertension in this model.     

谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析

以一株由自然界筛选获得的能够利用糖质原料直接产L-丝氨酸的谷氨酸棒杆菌Corynebacterium glutamicum SYPS-062为研究对象,考察了一碳单元循环中的辅因子—叶酸和维生素B12对菌株生长、蔗糖消耗及L-丝氨酸生成的影响,同时对处于对数生长期的菌株进行了代谢流量分析。结果发现,添加扰动因子叶酸和维生素B12对磷酸戊糖途径(HMP)碳流影响较大,碳源主要用于细胞生长及合成能量,而流向目的产物L-丝氨酸的碳流减少。同时在添加维生素B12时,增大了G3P节点的L-丝氨酸合成途径的分流比,但造成三羧酸循环(TCA)的流量不足,需要大量回补,从而限制了产物合成速率的进一步提高。     

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island “shores” and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations—the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.     

“分离以尿素为氮源的微生物”的实验改进

实验过程中,对尿素培养基的配方进行了改进,并思考如何更好地体现实验的重复性原则,如何更好地观察实验结果。改进后实验严谨合理,结果明显。     

Mimotopes selected with a neutralizing antibody against urease B from <Emphasis Type="Italic">Helicobacter pylori</Emphasis>induce enzyme inhibitory antibodies in mice upon vaccination

Background  

Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the immunogenicity of the selected mimotopes in vivo.     

Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure

This study investigated how Cd exposure affected oxidative biomarkers in Japanese flounder, Paralichthys olivaceus, at early life stages (ELS). Fish were exposed to waterborne Cd (0–48 µg L^− 1) from embryonic to juvenile stages for 80 days. Growth, Cd accumulation, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18), and levels of glutathione (GSH) and lipid peroxidation (LPO) were investigated at three developmental stages. Flounder growth decreased and Cd accumulation increased with increasing Cd concentration. In metamorphosing larvae, CAT and SOD activities were inhibited and GSH level was elevated, while LPO was enhanced by increasing Cd concentrations. CAT and GST activities of settling larvae were inhibited but GSH level was elevated at high Cd concentrations. In juveniles, SOD activity and LPO level were increased but GST activity was inhibited as Cd concentration increased. Antioxidants in flounder at ELS were able to develop ductile responses to defend against oxidative stress, but LPO fatally occurred due to Cd exposure. These biochemical parameters could be used as effective oxidative biomarkers for evaluating Cd contamination and toxicity in marine environments: CAT, SOD, GSH, and LPO for metamorphosing stage; CAT, GSH, and GST for settling stage; and SOD, GST, and LPO for juvenile stage.     

AMPA-mediated excitotoxicity in oligodendrocytes: role for Na(+)-K(+)-Cl(-) co-transport and reversal of Na(+)/Ca(2+) exchanger

We investigated the role of Na(+)-K(+)-Cl(-) co-transporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchanger (NCX(rev)) in glutamate-mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6-8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na(+)/Ca(2+) exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1-mediated Na(+) influx. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ-induced sustained rise led to mitochondrial Ca(2+) accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ-elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX(rev). These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Na(+) influx. The latter triggers NCX(rev) and NCX(rev)-mediated overload and compromises mitochondrial function and cellular viability.     

Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis

Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.