Evaluation of a fetus at risk for dihydropteridine reductase deficiency by direct mutation analysis using denaturing gradient gel electrophoresis

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of tetrahydrobiopterin (BH₄), which is an obligate co-factor of the aromatic amino acid hydroxylases. DHPR deficiency is a rare, autosomal recessive disorder caused by mutations in the QDPR gene. DHPR-deficient patients are diagnosed by a lack of response to a low phenylalanine diet and by severe neurological symptoms. Final diagnosis is made by measurements of neurotransmitters and pterin metabolites in cerebrospinal fluid (CSF) and urine, in addition to DHPR enzyme activity, which can be assessed in whole red blood cells. Treatment of DHPR deficiency can be difficult and the outcome is not always satisfying, even if all treatment strategies are followed. Therefore prenatal diagnosis is of great importance in affected families. Prenatal diagnosis is possible by measuring DHPR activity in different cell types but this is time consuming. More than 25 different mutations have to date been identified in the QDPR gene and direct identification of a mutation in a fetus would be easy and rapid. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) for the analysis of the QDPR gene. The method is useful for rapid and simultaneous scanning of all exons and flanking intronic sequences of the QDPR gene. We describe the first prenatal diagnosis conducted using this method. Copyright © 2001 John Wiley & Sons, Ltd.     

Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.     

脱氮副球菌W12降解吡啶的影响因素及其赋存质粒特性

从实验室的废水生物处理反应器内的活性污泥样本,筛选出来一株以吡啶为唯一碳、氮源的脱氮副球菌(Paracoccus denitrificans),命名为W12;研究了温度、pH值、吡啶初始浓度和投菌量对W12降解吡啶的影响.结果显示,在试验温度范围内高温有利于W12降解吡啶;同时W12降解吡啶的最适pH值范围在7-9;吡啶初始浓度越大降解时间越长,且投菌量越大吡啶降解越快.此外还研究了W12菌上赋存的质粒特性.脉冲场试验表明,W12菌上2个质粒的大小分别为169 kb和182 kb,并通过质粒消除试验证实了质粒参与了编码吡啶降解基因.     

Diversity of methanotrophs in Zoige wetland soils under both anaerobic and aerobic conditions

Zoige wetland is one of the most important methane emission centers in China. The oxidation of methane in the wetland a ects global warming, soil ecology and atmospheric chemistry. Despite their global significance, microorganisms that consume methane in Zoige wetland remain poorly characterized. In this study, we investigated methanotrophs diversity in soil samples from both anaerobic site and aerobic site in Zoige wetland using pmoA gene as a molecular marker. The cloning library was constructed according to the pmoA sequences detected. Four clusters of methanotrophs were detected. The phylogenetic tree showed that all four clusters detected were a liated to type I methanotrophs. Two novel clusters (cluster 1, cluster 2) were found to relate to none of the recognized genera of methanotrophs. These clusters have no cultured representatives and reveal an ecological adaptation of particular uncultured methanotrophs in Zoige wetland. Two clusters were belonging to Methylobacter and Methylococcus separately. Denaturing gradient gel electrophoresis gel bands pattern retrieved from these two samples revealed that the community compositions of anaerobic soil and aerobic soil were di erent from each other while anaerobic soil showed a higher metanotrophs diversity. Real-time PCR assays of the two samples demonstrated that aerobic soil sample in Zoige wetland was 1.5 times as much copy numbers as anaerobic soil. These data illustrated that methanotrophs are a group of microorganisms influence the methane consumption in Zoige wetland.     

MBR和CAS工艺污泥在贫营养培养条件下的微生物群落结构研究

采用聚合酶链式-变性梯度凝胶电泳（PCR-DGGE）技术,研究了膜生物反应器（MBR）和传统活性污泥工艺（CAS）反应器中微生物在贫营养条件下的总细菌群落结构.结果表明,在培养过程中,污泥的微生物种群经历了一个比较明显的变化过程,且以CAS污泥微生物种群的变化更为明显,演替过程中既有原始优势种群的消亡,又有新的优势种群...     

Glutathione S-transferase gene polymorphism,cigarette smoking and DNA damage

Several factors such as smoking habits, diet, occupational, and environmental exposure to chemical carcinogens influence the overall level of DNA damage. In 69 healthy adult volunteers’ polymorphisms of glutathione S-transferase (GST), an enzyme which participates in the metabolism of a broad range of carcinogens and endogenous compounds were determined. The level of DNA damage was assessed by comet assay and classified according to GSTT1/M1 genotype and smoking habits. GSTM1 null genotype was recognized in 48% of studied subjects and GSTT1 null genotype in only two cases (3%). In subjects carrying GSTT1/M1 alleles a significantly lower degree of DNA damage, determined as % DNA in the comet's tail, than in null individuals was noted. However, the results obtained did not indicate that in studied subjects an elevated endogenous level of DNA damage may be significantly related to smoking habits.     

生物样品体外致突变高通量检测方法研究进展

评述了几种常用的体外致突变检测方法及其用于生物样品检测的可行性,有些方法经改进可用于高通量检测生物样品体外致突变性.经典Ames实验受生物样品中组氨酸的影响,易产生假阳性结果,尽管经过修正可以排除组氨酸的干扰,但操作繁琐,不适合高通量检测.基于SOS反应的检测体系避开了组氨酸的影响,且简单易行,适合高通量检测:以β-半乳糖苷酶基因(lacZ)作为报告基因的检测体系灵敏度高,且经过离心洗涤或后培养的方式可降低样品颜色的影响;以绿色荧光蛋白(GFP)基因作为报告基因的检测体系避开了颜色的干扰,但这类方法灵敏度普遍不高,可以寻找信号更强的荧光蛋白以替代GFP;荧光素酶(lux)基因集lacZ和GFP的优点于一身,但检测时需要额外添加辅助因子,限制了其应用.也对单细胞凝胶电泳、tk基因突变实验、染色体损伤检测等方法进行了分析,有些适合生物样品高通量检测,但由于缺少国际通用的标准,很难推广使用.     

石油烃对栉孔扇贝血淋巴细胞DNA损伤的初步研究

为研究石油烃对海洋生物的毒性效应,将栉孔扇贝(Chlamys farreri)暴露于0.08、0.21和0.88mg·L-1石油烃中,采用单细胞凝胶电泳实验(彗星实验)技术检测不同暴露时间扇贝血淋巴细胞的DNA损伤程度,对照组中石油烃背景浓度为0.04mg·L-1。结果显示,低浓度(0.08mg·L-1)的石油烃短期(<7d)内即可导致栉孔扇贝血淋巴细胞的DNA损伤,并且随石油烃浓度的增大和暴露时间的延长,DNA损伤程度增加,石油烃浓度达0.88mg·L-1时,DNA损伤程度已非常严重。3d恢复实验后,各浓度组DNA损伤又均有不同程度的恢复。研究表明,彗星实验是检测石油烃对海洋贝类DNA损伤的一种有效手段,贝类血淋巴细胞DNA损伤有望成为石油烃污染的一种生物标志物,用于海洋污染的早期预警监测。     

亚麻温水脱胶液中细菌菌群结构分析

为了探讨实验室条件下模拟的工厂亚麻温水脱胶液中细菌菌群结构,采用纯培养技术和PCR-DGGE技术(Denaturing gradient gel electrophoresis)对细菌菌群结构进行了研究.用纯培养方法分离获得9类菌落,其中假单胞菌属(Pseudomonas)在有氧培养条件下总是处于优势.梭菌属(Clostridium)在厌氧培养过程中总是处于优势.微球菌属(Micrococcus)和葡萄球菌属(Staphylococcus)只有亚麻脱胶初期才有发现.PCR-DGGE指纹图谱显示,亚麻温水脱胶过程中条带数量较少且没有明显种群群落结构演替过程.通过对不同时期沤麻液中16S rDNAV3片段PCR产物d、e两个DGGE条带进行分子克隆、序列测定和Blas份析,发现e条带包含的16S rDNAV3片段除e 35外均属于假单胞菌属.d条带包含着较多不同的16S rDNAV3片段,其中有传统方法没有分离到的泛菌属(Pantoea)细菌、一些NCBI未收录的序列及一些非可培养微生物序列.纯培养技术和PCR-DGGE技术的共同使用,可以更全面准确地提供细菌多样性方面的信息.图4参15     

Application of proteomics in environmental science

Proteomics involves the separation of proteins, identification of the amino acid sequence of the interested or target proteins, study of the function of the proteins, modification, structure and ultimate assignments to functional pathways in the cell. The proteomic investigations have contributed greatly to human diseases studies, new drugs discovery researches, and environmental science in recent years. This article provides a review on the development of the main proteomic technologies, including both the gel based and non-gel based technologies, and their applications in environmental science. Proteomic technologies have been utilized in the environmental stresses studies to analyze the induction or reduction of proteins at expression level and identify the target proteins to investigate their function in response to environmental stresses, such as high or low pH, oxidation stress, and toxic chemicals. Such protein responses are also helpful to understand the mechanisms of some cellular activities and the functions of some proteins.