L－缬氨酸菌种选育

以硫酸二乙酯（ＤＥＳ）诱变处理黄色短杆菌（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍｆｌａｖｕｍ）ＸＱ５１２２，得到突变株Ｖ３－３６＜Ｌｅｕ￣１、α－ＡＢ￣ｒ、ＡＨＶ￣ｒ），在１０％葡萄糖培养基中可积累２．３％Ｌ－缬氨酸。以亚硝基胍（ＮＴＧ）诱变Ｖ４－１５３，得到一株突变株（Ｌｅｕ￣１、α－ＡＢ￣ｒ、ＡＨＶ￣ｒ、２－ＴＡ￣ｒ），再进行单菌落分离，得到突变株ＺＱ－２，能在培养基中积累Ｌ－缬氨酸４．２％～４．５％，最高达５．５７％．     

Fifty years of research onN-acetyl-2-aminofluorene,one of the most versatile compounds in experimental cancer research

Summary It is just about 50 years since the publication of the report on the toxicity and carcinogenicity of the potent carcinogenN-acetyl-2-aminofluorene (AAF). In 1940 very few reports on the carcinogenic activity of chemical compounds in experimental animals were available. The discovery of pure chemicals as carcinogens, such as AAF, azo dyes and benzo[a]pyrene, provided cancer researchers with a number of tools whereby the progressive changes involved in the induction of cancer could be studied in experimental systems. Contrary to the results with other carcinogens then known, AAF induced numerous types of tumors, but not at the site of application. This finding stimulated a great deal of interest in its use as an experimental carcinogen to study its metabolic fate and mechanism of action. During the following years an ever increasing number of reports appeared on the carcinogenicity of AAF in various species, on its metabolic fate, on the interaction of reactive metabolites with nucleic acids and proteins, and on its mutagenic activity. Particularly studies on the metabolism of AAF and the interaction with nucleic acids have contributed appreciably to our understanding of the mechanism of action of aromatic amines and also of other chemical carcinogens. It can be expected that AAF and its derivatives will continue to be used for specific applications in experimental cancer research. One of the most recent achievements is the preparation of site-specific AAF- and aminofluorene-modified DNA sequences for mutagenesis studies.Abbreviation AAF N-acetyl-2-aminofluorenem The Journal of Cancer Research and Clinical Oncology publishes in loose succession Editorials and Guest editorials on current and/or controversial problems in experimental and clinical oncology. These contributions represent exclusively the personal opinion of the author The Editors     

用点突变的方法组建巨细胞病毒抗原决定簇基因的表达克隆

研究资料表明，人巨细胞病毒（ＨＣＭｖ）单一蛋白的单一抗原决定簇只能被部分患者阳性血清识别。组建在血清学诊断中能够替代全病毒抗原的基因工程抗原，需要含有病毒多种主要抗原蛋白的抗原决定簇。为搞清在表达载体中重复插入某一抗原决定簇基因是否能表达出更高抗原效价的融会蛋白，我们用点突变的方法，在表达载体中分别插入了人ＨＣＭｖ的ｐｐＵＬ３２蛋白羧基端一个抗原决定簇基因的１个、２个和３个拷贝。在免疫转印检测中，这些克隆表达的融合蛋白与特异性阳性血清的反应性差别不明显。这表明，插入表达载体中目的基因的多寡对表达蛋白的抗原效价没有显著影响。     

Site-specific homologous recombination mutagenesis in group B streptococci

We describe the use of suicide vectors to generate site-specific mutations in group B streptococcus. This is accomplished by cloning gene-specific sequences into a temperature sensitive plasmid and selecting for clones which have undergone homologous recombination. The recombinan clones can be easily isolated by selecting for clones which have retained the antibiotic resistance markers that are present on the vector or cloned into the gene-specific sequences. To confirm the fidelity of the recombination events, PCR analysis is performed on chromosomal DNA isolated from the recombinant clones. Using this strategy, we have generated site- specific insertions in several capsule genes and have found that these insertions occurred as expected and that the mutations result in loss of capsule expression. In this report, we specifically detail the construction of cpsB mutants by single and double cross-over recombination and demonstrate that the resulting strains are acapsular.     

Mutagenicity studies with praziquantel,a new anthelmintic drug: Tissue-, host-, and urine-mediated mutagenicity assays

Praziquantel, a new anthelmintic drug with activity against all species of schistosomes pathogenic to man, and against a wide range of Cestodes, was tested for mutagenic potential. For the detection of both base substitutions and frameshift mutations, Salmonella typhimurium TA 100 and TA 98 were used as tester strains. Using the plate assay with and without added S-9, host-mediated assay and urine-mediated assay without and after incubation with -glucuronidase/arylsulfatase, no mutagenic activity could be detected.     

Comparative proteomics reveals essential mechanisms for osmotolerance in Gluconacetobacter diazotrophicus

Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.     

Three C-terminal phosphorylation sites in the Abutilon mosaic virus movement protein affect symptom development and viral DNA accumulation

The Abutilon mosaic virus (AbMV, Geminiviridae) DNA B component encodes a movement protein (MP), which facilitates viral transport within plants and affects pathogenicity. The presence of phosphorylated serine and threonine residues was confirmed for MP expressed in yeast and Nicotiana benthamiana by comparative Western blot analysis using phospho-amino acid- and MP-specific immunodetection. Mass spectrometry of yeast-derived MP identified three phosphorylation sites located in the C-terminal domain (Thr-221, Ser-223 and Ser-250). To assess their functional relevance in plants, several point mutations were generated in the MP gene of DNA B, which replace Thr-221, Ser-223 and Ser-250, either singly or in combinations, with either an uncharged alanine or a phosphorylation-mimicking aspartate residue. When co-inoculated with DNA A, all mutants were infectious. In systemically infected plants the symptoms and/or viral DNA accumulation were significantly altered for several of the mutants.     

重组人源化ING4基因的构建及其对肺癌NCI-H460细胞生长抑制作用

目的:构建重组人源化hING4基因并探究其对肺癌细胞的生长抑制效应.方法:以pcDNA3.0-mING4重组质粒为模板.通过定点突变技术构建带有绿色荧光蛋白(GFP)的重组腺病毒Ad-hING4基因.用荧光显微镜和RT-PCR法检测hING4在肺癌细胞中的表达;Westem印迹法检测hING4对肺癌细胞中Bax、Bcl-2表达的影响;集落形成试验和FCM法检测hING4对肺癌细胞增殖和凋亡影响.结果:基因测序和PCR结果提示成功构建人源化Ad-hING4基因;并能在肺癌细胞中表达,对肺癌细胞有细胞毒作用;Westem印迹法检测结果提示基因在上调Bax表达同时下调Bel-2的表达;集落形成试验、FCM检测结果提示hING4基因可抑制肺癌细胞增殖并诱导凋亡.结论:成功构建了Ad-hING4基因,并能在体外抑制肺癌细胞生长.