MAT Loci Play Crucial Roles in Sexual Development but Are Dispensable for Asexual Reproduction and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae, a fungal pathogen that causes rice blast, which is the most destructive disease of rice worldwide, has the potential to perform both asexual and sexual reproduction. MAT loci, consisting of MAT genes, were deemed to determine the mating types of M. oryzae strains. However, investigation was rarely performed on the development and molecular mechanisms of the sexual reproduction of the fungus. In the present work, we analyzed the roles of two MAT loci and five individual MAT genes in the sex determination, sexual development and pathogenicity of M. oryzae. Both of the MAT1-1 and MAT1-2 loci are required for sex determination and the development of sexual structures. MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes are crucial for the formation of perithecium. MAT1-1-2 impacts the generation of asci and ascospores, while MAT1-2-2 is dispensable for sexual development. A GFP fusion experiment indicated that the protein of MAT1-1-3 is distributed in the nucleus. However, all of the MAT loci or MAT genes are dispensable for vegetative growth, asexual reproduction, pathogenicity and pathogenicity-related developments of the fungus, suggesting that sexual reproduction is regulated relatively independently in the development of the fungus. The data and methods of this work may be helpful to further understand the life cycle and the variation of the fungus.

Complete Genome Sequences of Two Pseudomonas Species Isolated from Marine Environments of the Pacific Ocean.

Pseudomonas marincola YsY11 and Pseudomonas oleovorans T9AD were both isolated from marine environments of the Pacific Ocean. Here, we report the whole-genome sequences of these two organisms. Pseudomonas marincola YsY11 consists of a single 4.77-Mb chromosome, and Pseudomonas oleovorans T9AD consists of a 5.57-Mb chromosome and a 2.8-kb plasmid.

Strategies and perspectives of assembling multi-enzyme systems.

Multi-enzyme complexes have the potential to achieve high catalytic efficiency for sequence reactions due to their advantages in eliminating product inhibition, facilitating intermediate transfer and in situ regenerating cofactors. Constructing functional multi-enzyme systems to mimic natural multi-enzyme complexes is of great interest for multi-enzymatic biosynthesis and cell-free synthetic biotransformation, but with many challenges. Currently, various assembly strategies have been developed based on the interaction of biomacromolecules such as DNA, peptide and scaffolding protein. On the other hand, chemical-induced assembly is based on the affinity of enzymes with small molecules including inhibitors, cofactors and metal ions has the advantage of simplicity, site-to-site oriented structure control and economy. This review summarizes advances and progresses employing these strategies. Furthermore, challenges and perspectives in designing multi-enzyme systems are highlighted.

Establishing a Mathematical Equations and Improving the Production of L-tert-Leucine by Uniform Design and Regression Analysis.

L-tert-Leucine (L-Tle) and its derivatives are extensively used as crucial building blocks for chiral auxiliaries, pharmaceutically active ingredients, and ligands. Combining with formate dehydrogenase (FDH) for regenerating the expensive coenzyme NADH, leucine dehydrogenase (LeuDH) is continually used for synthesizing L-Tle from α-keto acid. A multilevel factorial experimental design was executed for research of this system. In this work, an efficient optimization method for improving the productivity of L-Tle was developed. And the mathematical model between different fermentation conditions and L-Tle yield was also determined in the form of the equation by using uniform design and regression analysis. The multivariate regression equation was conveniently implemented in water, with a space time yield of 505.9 g L-1 day-1 and an enantiomeric excess value of >99 %. These results demonstrated that this method might become an ideal protocol for industrial production of chiral compounds and unnatural amino acids such as chiral drug intermediates.

Aspirin inhibits the proliferation and migration of gastric cancer cells in p53-knockout mice.

The aim of the present study was to investigate the effect of aspirin on the cell proliferation and migration of gastric cancer cells in p53-knockout mice. Twenty p53-/- male mice aged 6 to 7 weeks, with an average weight of 20±3 g were used. The model of gastric cancer was established by the implantation of a mouse forestomach carcinoma cell line, subcutaneously, at the back of the neck, and then the mice were randomly divided into two groups after establishment of the model (control group, n=10; experimental group, n=10). Aspirin (250 mg/kg) was added to the food in the experimental group one day before model establishment, until the end of the experiment. Mice in the control group were given regular food without aspirin. All mice were sacrificed 3 months afterwards, and gastric cancer tissues were harvested. MTT assay was used to detect the proliferation of the tumor cells. Tumor cell number was also observed. Migration ability was detected by scratch assay, and E-cadherin protein expression was evaluated by immunofluorescence. The results revealed that the proliferation ability of tumor cells in the experimental group was lower than that in the control group. In addition, cell numbers were significantly decreased and the migration ability was diminished. The expression of E-cadherin was also increased in the experimental group, and the differences were statistically significant (P<0.05). In conclusion, aspirin inhibited the cell proliferation and migration of gastric cancer cells in mice.

MiR-205 in cancer: an angel or a devil?

MicroRNAs (MiRNAs) are small non-coding RNAs that regulate their target genes expression at the post-transcriptional level. As accumulating properties of miR-205 have been identified, complex roles of miR-205 in tumor initiation and progression are emerging. MiR-205 acts either as a tumor suppressor through inhibiting proliferation and invasion, or as an oncogene through facilitating tumor initiation and proliferation, depending on the specific tumor context and target genes. In this review, we focus on the properties of miR-205 in cancers to shed light on better management of various fatal malignancies. Moreover, we discuss epigenetics that may account for the fluctuation of miR-205 expression. In addition, we sketch a network of miR-205 and its targets to further elucidate the mechanisms through which miR-205 exerts its multiple functions.

Expressions of nuclear factor-kappa B p50 and p65 and their significance in the up-regulation of intercellular cell adhesion molecule-1 mRNA in the nasal mucosa of allergic rhinitis patients.

The purpose of the study was to investigate expressions of nuclear factor-kappa B (NF-κB) and intercellular cell adhesion molecule-1 mRNA (ICAM-1 mRNA) in the nasal mucosa of allergic rhinitis (AR) patients. Expressions of NF-κB and ICAM-1 mRNA were studied using immunohistochemistry and reverse transcription-PCR (RT-PCR) in AR tissues and corresponding normal nasal mucosa. The correlation between NF-κB and ICAM-1 mRNA was studied using linear correlation analysis. The results of immunohistochemistry showed that expression of NF-κB was significantly up-regulated in the nasal mucosa of AR compared with that in normal tissue (P < 0.01), over-expression of NF-κB p50 was found in the cytoplasm and nucleus (P < 0.01), and NF-κB p65 was mainly expressed in the cytoplasm (P < 0.01). ICAM-1 mRNA was strongly expressed in the nasal mucosa of AR compared with that in normal tissue as shown by RT-PCR (P < 0.01). Up-regulation of ICAM-1 mRNA was significantly correlated with over-expressions of NF-κB p50 and NF-κB p65 (r = 0.8995, P < 0.01; r = 0.7601, P < 0.01). In conclusion, NF-κB plays a key role in AR. Excessively activated NF-κB promotes the transcription of ICAM-1 mRNA. ICAM-1 is related to the pathogenesis and development of AR.

Chemo-enzymatic asymmetric synthesis of S-citalopram by lipase-catalyzed cyclic resolution and stereoinversion of quaternary stereogenic center.

A chemo-enzymatic synthesis method of S-citalopram was developed to overcome the disadvantage of relatively low selectivity of enzyme towards tertiary alcohols. The combination of kinetic resolution, cyclic resolution and stereoinversion synthesis was successfully applied in the asymmetric synthesis of the S-citalopram. Using the kinetic model to predict the cyclic resolution, R-diol with high ee value was obtained by controlling the conversion rate. Subsequently, the unwanted R-diol was inverted to S-citalopram by stereoinversion of chiral quaternary center with 98.0% yield and ee value of 91.0%. Based on dynamic simulation and experiments, the kinetic resolution was scaled up from 10 mL to 1 L and 14 L, gradually. There was no significant scale-up effect and the dynamic simulation result fitted the experimental data well, with an error of 12.5 and 14.0%, respectively. This chemo-enzymatic synthesis route is a promising model system for the production of pharmaceuticals with the chiral tertiary alcohols intermediate.

Lipase-catalyzed remote kinetic resolution of citalopram intermediate by asymmetric alcoholysis and thermodynamic analysis.

Lipase-catalyzed remote resolution of the tertiary alcohol, citalopram intermediate (diol acetate), has been achieved. The chiral discrimination was obtained by the Novozym435-catalyzed alcoholysis of the primary hydroxyl ester which was four bonds away from the center. The influence of acyl acceptor structure and the organic solvents on the reaction rate and enantioselectivity were investigated. Based on the thermodynamic analysis, the difference of activation free energy between the two enantiomers which dominated the enantioselectivity was significantly affected by the organic solvents, while the acyl acceptor showed less effect. In addition, the enantiomer discrimination was driven by both the difference of activation enthalpy and activation entropy. The thermodynamic analysis provides further insights into the prediction and optimization of enantioselectivity and reaction rate in remote resolution.

Therapeutic effect of anti-amyloid β peptide single-chain antibody gene mediated by adeno-associated virus on animal Alzheimer's disease / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the therapeutic effect of recombinant adeno-associated virus carrying anti-amyloid β peptide single-chain antibody gene on Alzheimer's disease (AD) in animal models.</p><p><b>METHODS</b>The recombinant adeno-associated viruses were injected to the leg muscle of mutant amyloid precursor protein transgenic AD mice. The latency of the mice in Morris water maze was tested before and 3, 7, 10 months after drug administration. The animal brains were harvested 10 months after drug administration and sectioned for amyloid plaques staining.</p><p><b>RESULTS</b>The learning and memory abilities of AD model mice were improved significantly 3 months after gene drug administration. Ten months after gene therapy, the numbers of amyloid plaque in hippocampus of model mice decreased.</p><p><b>CONCLUSION</b>The adeno-associated virus carrying anti-amyloid β peptide single-chain antibody gene has therapeutic effect on AD in model mice.</p>

[Application of 99Tc(m)-annexinV in predicting the therapeutic effect of chemical agent on lung cancer].

OBJECTIVE: To prepare 99Tcm-annexinV and evaluate its application in the early prediction of therapeutic effect of chemical agent on lung cancer. METHODS: Annexin V was obtained by recombinant pichia pastoris expression, ammonium sulfate precipitation, and size-exclusion chromatography. The purified protein was labeled with 99Tc(m) at the N-terminal site by stannous chloride reduction and purified by desalting. The labeling yield and radiochemical purity of 99Tc(m)-annexinV were determined by instant thin-layer chromatography. The biological activity of 99Tc(m)-annexinV was tested by phosphatidylserine-exposed erythrocytes bound radioactivity counting. The lung cancer mice models were established by inoculating LA795 cells to right flank of 615 mice subcutaneously and tumor tissue transplantation. The biodistribution of 99Tc(m)-annexinV in lung cancer mice models were investigated at 6, 12, 24, and 48 h after cyclophosphamide administration. RESULTS: The annexin V was secreted from pichia pastoris and purified by ammonium sulfate precipitation and size-exclusion chromatography with high yield. The annexin V could be labeled at room temperature with 50.2% radioactivity yield. The radiochemical purity of 99Tc(m)-annexinV reached up to 93.9% with intact biological activity. The biodistribution analysis demonstrated that 99Tc(m)-annexinV was excreted from kidney. The uptake of 99Tc(m)-annexinV at tumor reached maximum 48 h after cyclophosphamide administration while tumor to muscle ratio was 6.34 and tumor to blood ratio was 4.09. CONCLUSIONS: 99Tc(m)-annexinV derived from pichia pastoris was successfully prepared. It is useful in predicting the therapeutic effect of chemical agent on lung cancer.

Lack of association of functional variants in alpha-ENaC gene and essential hypertension in two ethnic groups in China.

BACKGROUND: It has been proposed that subtle genetic changes in epithelial sodium channel (ENaC) subunits might be at the origin of less rare forms of hypertension. In some populations, subtle functional genetic changes in ENaC genes associated with essential hypertension were indeed observed. To further test this hypothesis, we observed the role of three functional variants G2139A, A334T and A663T in the alpha-ENaC gene on essential hypertension in two Chinese minority groups, the Kazaks and Uyghurs. METHODS: A population-based case-control study was carried out in the two populations mentioned above. RESULTS: The distribution of genotype and allele frequencies of G2139A, A334T and A663T did not differ significantly between hypertensive subjects and control subjects in both Kazak and Uyghur populations. No significant associations of the three polymorphisms with hypertension were observed in both populations in univariate and multivariate logistic regression analysis by applying dominant, additive and recessive models. Haplotype-based association analysis based on G2139A, A334T and A663T did not show significant association between hypertensive subjects and control subjects in both populations. CONCLUSIONS: For the above variants, we did not confirm the hypothesis that subtle genetic changes in alpha-ENaC subunits might be at the origin of essential hypertension in our populations.

A common polymorphism is associated with body mass index in Uyghur population.

We aimed at observing the association of a common genetic variant near the INSIG2 gene (rs7655505) with body mass index (BMI) related obesity in a population-based association study in Uyghur population. We observed a significant association of rs7566605 polymorphism with BMI related obesity in Uyghurs with an odds ratio of 1.47 (95% CI 1.11-1.95, P=0.006) under a dominant model (CC+GC versus GG). The mean BMI of rs7566605 CC+GC was 0.59kg/m(2) higher than for GG genotype (P=0.024), regardless of sex and age.

Association study of variants in two ion-channel genes (TSC and CLCNKB) and hypertension in two ethnic groups in Northwest China.

BACKGROUND: Abnormalities in renal sodium and water retention and membrane ion transport play important roles in development of hypertension. Recently, Arg904Gln variant of thiazide-sensitive NaCl-cotransporter gene (TSC) and Thr418Ser variant of renal epithelial Cl-channel ClC-Kb gene (CLCNKB) were found implicated in the prevalence of essential hypertension (EH). The objective of this study was to examine the role of these two variants on EH in two Chinese minorities -- Kazaks and Uyghurs. METHODS: A case-control study was conducted in Kazak herdsmen and Uyghur farmers live in Xinjiang Uyghur Autonomous Region of Northwest China. RESULTS: For the Arg904Gln polymorphism in the TSC gene, we observed a stronger trend of 904Gln allele in controls than hypertensives in Uyghurs (p=0.058), but the greater prevalence of genotype of 904Gln carrier reached significance (p=0.015). No association or trend with hypertension was observed at Arg904Gln in Kazaks. For the Thr418Ser variant of the CLCNKB gene in Kazaks, we observed a significantly higher prevalence of 418Ser allele frequencies in hypertensives than controls (p=0.037), but the higher prevalence of genotype of 418Ser carrier did not reach significance (p=0.08). No association with hypertension was observed at Thr418Ser in Uyghurs. CONCLUSION: The risk reduction effect of 904Gln to hypertension in Uyghurs and the association of 418Ser to hypertension in Kazaks are weak. Given that we have explored two polymorphisms in each of two populations, for a total of four independent tests, a Bonferroni correction for multiple tests (0.05/4) renders non-significant of our results in our two Chinese populations (alpha=0.0125). The roles of Thr418Ser polymorphism of the CLCNKB gene and Arg904Gln polymorphism in the TSC gene on essential hypertension need to be explored in other ethnic groups.

[Biological activity of human single-chain antibody against amyloid beta peptide involved in Alzheimer's disease].

OBJECTIVE: To explore the biological activity of recombinant human single-chain antibody against amyloid beta peptide in vitro. METHODS: Human single-chain antibody against amyloid beta peptide was obtained from recombinant bacteria. The antigen-binding activity of this antibody was measured by enzyme-linked immunosorbent assay (ELISA) and competitive ELISA. Human neuroblastoma SH-SY5Y cells were used as cell models to test the protective role of human single-chain antibody against amyloid beta peptide. RESULTS: Recombinant human single-chain antibody was mainly located in the insoluble inclusion bodies of bacteria. The antibody was dissolved by urea and purified by metal affinity chromatography as active form to bind synthetic amyloid beta peptide 40 or amyloid beta peptide 42. The improvement of the survival rates of human neuroblastoma cells was significantly superior in amyloid peptide 42 plus equimolar antibody group than in amyloid peptide 42 group (P < 0.05), and was significantly superior in the amyloid peptide 40 plus equimolar antibody group than in amyloid peptide 40 group (P < 0.01). CONCLUSION: The recombinant human single-chain antibody against beta amyloid peptide 40 from E. coli can partially inhibit the neurotoxicity effect of amyloid beta peptide in vitro.

[Bioluminescent imaging monitoring of a anti-angiogenesis therapeutic gene vasostatin in tumor cell PC3].

OBJECTIVE: To generate a sensitive tool for noninvasive monitoring of a therapeutic gene vasostatin. METHODS: We fused the bioluminescent reporter gene firefly luciferase to the therapeutic transgene vasostatin and ensured that these two proteins would not interrupt each other and kept their own natural character. RESULTS: We therefore examined clones of PC3 cells stably expressing fusion gene and positive controlfluc with bioluminescence. In vivo imaging of PC3-Fluc subcutaneous tumors showed that the mean tumor bioluminescence increased in animals over several weeks. CONCLUSION: Noninvasive monitoring facilitates the detection of gene expression in vivo and in vitro.

Escherichia coli CorA periplasmic domain functions as a homotetramer to bind substrate.

CorA is a primary Mg2+ transporter in bacteria, which also mediates influx of Ni2+ and Co2+. Topological studies suggested that it could be divided into a large soluble periplasmic domain (PPD) and three membrane-spanning alpha-helixes. In the present study, glutathione S-transferase (GST) fusion Escherichia coli CorA PPD was purified by GST affinity chromatography, and PPD was obtained by on-column thrombin digestion. Size-exclusion chromatography indicated that purified PPD exists as a homotetramer. Single particle electron microscopy analysis of PPD and two-dimensional crystals of GST-PPD indicated that E. coli CorA PPD is a pyramid-like homotetramer with a central cavity. Comparison of the CD spectra of full-length CorA and PPD also suggested that PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified PPD retains its substrate binding ability as native CorA. The CorA PPD structure described here may provide structural information for the E. coli CorA functional oligomeric state.

[Association between endothelial nitric oxide synthase gene (G894T) polymorphism and essential hypertension in Uygur populations].

OBJECTIVE: To investigate the association between G894T polymorphism and essential hypertension (EH) in Uygur population in Xinjiang province. METHODS: In this case-control study, G894T genotypes in 375 hypertension patients (EH group) and 414 normotensive control subjects (NT group) from the rural area of Tuluafan of Xinjiang was investigated by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: (1) Significant differences were found in GG, GT and TT frequencies of G894T genotypes between the EH and NT groups (56.5%, 28.3%, 15.2% in EH group and 65.9%, 22.5%, 11.6% in NT group, OR = 2.97, 95% CI 1.393 - 6.358). T allele frequencies were significantly higher in EH group (29.33%) than that in NT group (22.83%, P < 0.05). (2) SBP, DBP in patients with T allele of eNOS gene [(171.36 +/- 22.30) mm Hg and (103.63 +/- 13.22) mm Hg] were significantly higher than that of GG genotype [(158.07 +/- 20.850) mm Hg and (89.90 +/- 10.39) mm Hg] (P < 0.01). CONCLUSIONS: eNOS gene exon7 G894T polymorphism might relate to essential hypertension in Uygur population in Xinjiang province.

[Synthesis and animal imaging of 99mTc-hydrazinonicotinamide-folate as a new folate receptor-targeted tumor imaging agent].

OBJECTIVE: The synthesis, biodistribution, and animal imaging of 99mTc- hydrazinonicotinamide-folate (99mTc-HYNIC-Folate) were studied as a folate receptor-targeted tumor imaging agent. METHODS: HYNIC-Folate was synthesized by a muti-step reaction and radiolabeled with 99mTc using tricine and trisodium phenylphosphine-3, 3', 3"-trisulfonate (TPPTS) as coligands. The radiochemical purity and stability of 99mTc HYNIC-Folate was measured. The biodistributions of 99mTc-HYNIC-Folate in normal mice and tumor-bearing mice were detected. Whole-body gamma imaging was performed using an athymic mouse tumor xenograft model. RESULTS: The ligand HYNIC-Folate was successfully synthesized and characterized by hydrogen nuclear magnetic resonance (1HNMR) and mass spectrometry (MS). The radiochemical purity of 99mTc-HYNIC-Folate was 96% under optimal conditions. Data from gamma scintigraphy and the biodistribution in tumor-bearing mice showed that 99mTc-HYNIC-Folate predominantly accumulated in tumor, its uptake rate per gram tissue alpham was 5. 620+/- 0. 753. The uptakes of 99mTc-HYNIC-Folate in the other non-target tissues were very low, except it was high in the kidneys ( am was 41. 959 +/-6. 759) . CONCLUSION: 99mTc-HYNIC-Folate has the potential to be used as a noninvasive radiodiagnostic imaging agent for the detection of folate receptor-positive human cancers.

[Association of the T(-344)C polymorphism of aldosterone synthase gene CYP11B2 with essential hypertension in Xinjiang Kazakh isolated group].

OBJECTIVE: To investigate the association of the T(-344)C polymorphism of aldosterone synthase gene CYP11B2 with essential hypertension in Xinjiang Kazakh isolated population. METHODS: The study covered 186 hypertensives and 168 normotensive controls in Xinjiang Kazakh population. The segment of CYP11B2 was amplified from DNA by polymerase chain reaction(PCR). The PCR products were digested by restriction endonuclease. RESULTS: The frequencies of C and T in hypertensive group (0.45 and 0.55) were not significantly different from those in the control group (0.43 and 0.57; chi-square test=0.380, P=0.537). The frequencies of CYP11B2 genotypes of CC, CT and TT were 0.20, 0.50 and 0.30 in hypertensives respectively, and 0.12, 0.61 and 0.27 in controls respectively. There was no significant difference in genotypes between hypertensive group and normotensive group (chi-square test=4.838, P=0.089). But the frequencies of CC genotype were higher in the female hypertensives than in the normotensive controls (chi-square test=6.104, P<0.05). CONCLUSION: The results suggested that the T(-344)C polymorphism of CYP11B2 gene may be associated with hypertension in female Kazakh population of Xinjiang Barlikun area.