Identification of a new mutation in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

A mutation involving an A-to-G nucleotide replacement at position 985 of the medium-chain acyl-CoA dehydrogenase (MCAD) cDNA was found in homozygous form in 18 unrelated MCAD-deficient families and in heterozygous form in 4 families. By PCR amplification and sequencing of cDNA from a compound heterozygote, we have detected a new mutation in an MCAD-deficient patient in whom one MCAD allele produces mRNA that is missing 4 bp in the MCAD cDNA, while the other allele carries the A-to-G-985 mutation. The presence of this 4-bp deletion was confirmed in the patient's genomic DNA by dot-blot hybridization with allele-specific oligonucleotide probes and by restriction analysis of PCR products. A rapid screening test for this 4-bp deletion was developed, based on mismatched primer PCR amplification. The deletion created a new restrictive-enzyme site which yielded two DNA fragments. The 4-bp deletion was not found in the three remaining MCAD chromosomes not harboring the A-to-G-985 mutation, nor it was present in 20 chromosomes from 10 unrelated normal Caucasians. The PCR-based method for screening these two mutations can detect over 93% of all MCAD mutations.     

不同科属来源的六株Frankia代表菌株碳源利用谱的研究

对来自赤杨属、木麻黄属、异木麻黄属、沙棘属和杨梅属的六株Frankia代表菌株进行了二十四种碳源利用谱的比较研究(包括简单有机酸、单糖、双糖、三糖和糖醇在内)。结果表明,各菌株在碳源利用种类和程度上有明显差异;简单有机酸盐特别是丙酸钠是所有菌株的良好碳源;菌株Cc01、A11I1和Hr16还能很好地利用丙酮酸钠;除了菌株Hr16能很好地利用纤维二糖,菌株A11I1利用葡萄糖外;糖醇类很少被利用。如果以丙酮酸钠、丙酸钠和乙酸为“诊断性”碳源,则可以将供试菌株分为三个类群,即赤杨——杨梅类群、沙棘类群和木麻黄类群;这与交叉接种和血清学方法得出的结论相吻合。     

Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

一种宫内节育器的染色体畸变试验研究

目的检测一种宫内节育器的体外细胞的染色体畸变作为遗传毒性评价的一部分。方法在加和不加S9活化系统条件下,试验组用三种不同浓度的节育器浸提液处理CHL细胞20h,对照组分别加入阴性、阳性进行交换,各组置37℃培养箱中培养。24h后采集细胞并分析中期细胞染色体畸变情况,计算染色体畸变率。结果在4g/20mL的浓度下受试物对细胞有明显的细胞毒性,其稀释浸提液的畸变率与阴性对照相比,在统计学上无显著性差异(P>0.05)。结论在该试验条件下,受试物稀释浸提液未诱发CHL细胞染色体畸变。     

Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system

BACKGROUND: Polyethylenimines (PEIs) with high molecular weights are effective nonviral gene delivery vectors. However, the in vivo use of these PEIs can be hampered by their cellular toxicity. In the present study we developed and tested a new PEI polymer synthesized by linking less toxic, low molecular weight (MW) PEIs with a commonly used, biocompatible drug carrier, beta-cyclodextrin (CyD). METHODS AND RESULTS: The terminal CyD hydroxyl groups were activated by 1,1'-carbonyldiimidazole. Each activated CyD then linked two branched PEI molecules with MW of 600 Da to form a CyD-containing polymer with MW of 61 kDa, in which CyD served as a part of the backbone. The PEI-CyD polymer developed was soluble in water and biodegradable. In cell viability assays with sensitive neurons, the polymer performed similarly to low-MW PEIs and displayed much lower cellular cytotoxicity compared to PEI 25 kDa. The gene delivery efficiency of the polymer was comparable to, and at higher polymer/DNA ratios even higher than, that offered by PEI 25 kDa in neural cells. Attractively, intrathecal injection of plasmid DNA complexed by the polymer into the rat spinal cord provided levels of gene expression close to that offered by PEI 25 kDa. CONCLUSIONS: The polymer reported in the current study displayed improved biocompatibility over non-degradable PEI 25 kDa and mediated gene transfection in cultured neurons and in the central nervous system effectively. The new polymer would be worth exploring further as an in vivo delivery system of therapeutic genetic materials for gene therapy of neurological disorders.     

Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.     

Analysis of bacterial communities in rhizosphere soil of continuously cropped healthy and diseased konjac

The bacterial community and diversity in healthy and diseased konjac rhizosphere soils with different ages of continuous cropping were investigated using next-generation sequencing. The results demonstrated that the number of years of continuous cropping significantly altered soil bacterial community and diversity. Soil bacterial Shannon diversity index and Chao 1 index decreased with the increasing cropping years of konjac. After 1 year of cropping, the soil exhibited the highest bacterial relative abundance and diversity. Of the 44 bacterial genera (relative abundance ratio of genera greater than 0.3%), 14 were significantly affected by the duration of continuous cropping and plant status. With increasing continuous cropping, Alicyclobacillus decreased, while Achromobacter, Lactobacillus, Kaistobacter, Rhodoplanes increased after 3 years continuous cropping. Continuous cropping altered the structure and composition of the soil bacterial community, which led to the reduction in the beneficial bacteria and multiplication of harmful bacteria. These results will improve our understanding of soil microbial community regulation and soil health maintenance in konjac farm systems.     

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1

Aims: To characterize a robust NAD⁺‐dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD⁺‐dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL‐PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half‐life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n‐hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi‐aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.     

Polymorphisms of 5,10-methylenetetralydrofolate reductase (MTHFR), fruit and vegetable intake, and the risk of stomach cancer

Stomach cancer is a serious public health problem in China. 5,10-Methylenetetralydrofolate reductase (MTHFR) may be involved in both DNA methylation and DNA synthesis. Folate deficiency is associated with cancer risk that may be modulated by a genetic variation in the MTHFR gene in folate metabolism. The main goal of this study was to evaluate the association between polymorphisms of the MTHFR gene and the risk of stomach cancer. This study also explored the modification effects of fruit and vegetable intake (one of the main constituents is folate) on the risk of this disease. A population-based case-control study was conducted in Taixing, China, consisting of 206 newly diagnosed cases with primary stomach cancer and 415 healthy population controls. Polymorphisms of MTHFR C677T and A1298C were assayed by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) techniques. The data were analysed using the logistic regression model. No obvious association between the MTHFR A1298C polymorphism and the risk of stomach cancer was observed in this study. The frequencies of 677 C/C, C/T, and T/T were 34.5, 50.9, and 14.6%, respectively, in controls. The frequency of the MTHFR 677 wild homozygotic genotype was 25.8% in cases, which was lower than that in controls (34.5%). The adjusted odds ratio (OR) for the MTHFR 677 any T genotype was 2.05 (95% confidence interval (CI), 1.26-3.34) when compared with the C/C genotype. In the low fruit and vegetable intake group an increasing trend was observed with the T allele exposure, p=0.0056. The adjusted ORs were 1.68 (95% CI = 0.86-3.29) for the C/T genotype and 3.58 (95% CI = 1.46-8.75) for the T/T genotype, respectively. The MTHFR 677 any T genotype was associated with an increased risk of primary stomach cancer among the Chinese population. Folate deficiency might modify the MTHFR gene polymorphism and influence the risk of stomach cancer.     

木质纤维素预处理技术研究进展

详细评述了木质纤维素的预处理工艺研究进展,特别是浓酸低温水解-酸回收工艺、稀酸二阶段水解工艺、金属离子在稀酸水解过程中的助催化作用以及水蒸汽爆裂、氨纤维爆裂、CO2爆裂、酶催化水解等方法的研究进展情况。木质纤维素原料预处理技术发展为发酵生产乙醇技术的研究开发奠定了坚实基础。