FoxO1在胰岛β细胞代谢灵活性受损及失代偿进程中的作用

葡萄糖及脂肪酸是胰岛β细胞的关键代谢底物,葡萄糖刺激胰岛β细胞分泌胰岛素是维持机体血糖稳态平衡的关键。胰岛素抵抗发生时,β细胞对能量代谢底物的选择失调,加速胰岛β细胞由代偿到胰岛β细胞失代偿的进程,是肥胖胰岛素抵抗最终发展为2型糖尿病的始动因素。核转录因子FoxO1属于Fox家族成员,在胰腺内广泛表达,在β细胞的代谢,发育,增殖过程中发挥着重要的调节作用。鉴于FoxO1在维持胰岛β细胞功能中的关键作用,现着重对FoxO1在胰岛β细胞代谢灵活性受损及失代偿过程发生中的作用调节进行阐述。为其作为调控胰岛β细胞功能的关键靶点提供参考。     

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B₂) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-l-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wild-type strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

促性腺激素释放激素Buserelin 稳定性的研究

目的:研究Buserelin原料药的性质在温度、湿度、光线等条件的影响下随时间变化的规律,为该原料药的生产、包装、储存、运输及有效期的制定提供依据。方法:根据中国药典2005版二部附录XIX C药物稳定性试验指导原则及化学药物稳定性研究技术指导原则进行强光照射、高温(60℃、40℃)、高湿(RH92.5%±5%、RH75%±5%)影响因素试验,加速试验(40℃±2℃、RH75%±5%;25℃±2℃、RH60%±10%);按Buserelin原料药标准规定的质量指标及相关的检验方法对产品在试验条件下的主要质量指标进行检测。结果:强光照射、高温、高湿等影响因素对Buserelin的稳定性有明显影响,故应密封、于干燥、阴凉处保存。在加速试验中,Buserelin原料药的各项质量指标发生了小的变化,但均在质量标准规定的范围内。结论:强光照射、高温、高湿等影响因素对Buserelin的稳定性有明显影响,应在阴凉干燥处避光密封保存和运输。加速试验结果证明:在此条件下,它的各项质量指标变化均在质量标准范围内,符合Buserelin原料药质量标准规定的要求;故将其保质期暂定为两年。     

Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii

Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene.     

Improved method for expression and isolation of the Mycoplasma hominis arginine deiminase from the recombinant strain of Escherichia coli

Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl β-d-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30–34 U/mg for 12 months when stored at 4 °C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.     

Treatment outcomes in locally advanced colorectal carcinoma

BACKGROUND: Locally advanced colorectal cancers form a distinct subgroup where contiguous organs could be involved without distant metastases and so may be amenable to curative surgical resection. It was our objective to report our experience in treating six such patients with operable locally advanced colorectal carcinomas. METHODS: We retrospectively reviewed the case notes of 47 patients who were diagnosed with colorectal cancers at M S Ramaiah Medical Teaching Hospital between the years 1996 - 2001. Six patients were identified with T4 lesions, adjacent organ involvement and with no nodal involvement. The treatments and outcomes for these patients were then reviewed. RESULTS: Two of three patients with rectal malignancies who underwent pelvic exenteration succumbed to disease recurrence within the first 18 months. One of the three patients with colonic cancers died of non malignant causes. The other two are disease free till date. CONCLUSIONS: Aggressive multivisceral resections for locally advanced colonic cancers might be appropriate. Rectal cancers when locally advanced may be considered for pelvic exenteration, but a more guarded prognosis may apply.     

从茶尺蠖中分离到一株微小RNA病毒

从核型多角体病毒(PV)感染致死的茶尺蠖幼虫尸体中分离到一株微小RNA病毒(oPV)透射电镜观察纯化的病毒粒子为无囊膜、无表面特征、直径约26nm的球状颗粒.16%的SDS-PAGE显示它有两个分子量为31.5 kDa和28.8 kDa的衣壳蛋白,后者的含量是前者的2.5倍.3′端克隆序列分析表明EoPV基因组3′有poly(A)尾,编码RNA聚合酶(dRp)含有微小RNA病毒RNA聚合酶的八个保守基序,同源性分析表明它与榕透翅毒蛾微小RNA病毒(nPV)亲缘关系最近.这些特点表明该病毒为一株新的微小RNA病毒.     

Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

The ability to propagate under anaerobic conditions is an essential and unique trait of brewers or bakers yeast (Saccharomyces cervisiae). To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species. While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen. From the phylogenetic point of view, this enzyme is closely related to a bacterial DHODase from Lactococcus lactis. Here we show that S. kluyveri, which separated from the S. cerevisiae lineage more than 100 million years ago, represents an evolutionary intermediate, having both cytoplasmic and mitochondrial DHODases. We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired a bacterial gene for DHODase, which subsequently allowed cell growth gradually to become independent of oxygen.Communicated by C. P. Hollenberg