In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (＞1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.     

黄色黄素抑制人白血病HL-60细胞内蛋白激酶CK2的实验研究

蛋白激酶CK2在寻找抗肿瘤及抗病毒药物方面是一个具有吸引力的分子靶点,其特异性抑制剂具有潜在的临床应用价值.通过测定药物作用后转移到CK2底物上[γ-32P]ATP的放射性活度,探讨黄色黄素对重组人CK2全酶以及细胞内CK2活性的影响;采用多重RT-PCR检测CK2α、α'和β亚基的mRNA表达水平;Lineweaver-Burk作图法分析CK2的酶动力学机制.发现黄色黄素能显著抑制重组人CK2全酶(IC50=0.86μmol/L)以及HL-60细胞内的CK2活性,其作用效果均强于阳性对照TBB.另外,黄色黄素作用2h可使CK2α和α'亚基的mRNA表达下降,对β亚基则无明显的影响.酶动力学分析表明,黄色黄素与ATP呈竞争性抑制CK2的活性,与酪蛋白则呈混合性抑制CK2的活性.研究说明,黄色黄素是一种有效的细胞内蛋白激酶CK2抑制剂.     

香菇基因组中EST-SSR的构成和分布

从真菌基因组计划网站(FGP)和NCBI网站数据库下载了符合条件的总长度为8.1×106bp的11150条香菇的EST(包括10条cDNA)序列,通过SSRhunter1.3软件结合手工查找,从中发现2.83%即316条EST含有一共469个SSR,平均每17.3kb出现一个EST-SSR。在所有EST-SSR中,三碱基和六碱基SSR出现最多,分别占EST-SSR总数的38.00%和20.00%。出现较多的基序为(A)n、(T)n、(GA)n、(AG)n、(TGA)n、(GAT)n和(TCTTT)n,占所有EST-SSR的35.39%。     

TTC-CaCO3复合平板法快速筛选丙酮酸高产菌株

设计出一种用于快速筛选丙酮酸高产菌株的方法。这是一种理性化筛选方法。其原理是菌落产生的丙酮酸与碳酸钙反应形成可溶性的丙酮酸钙而出现透明圈,比较透明圈直径可以直观的选择出产酸较多的菌株;TTC与ADH作用而显色,颜色的深浅与ADH活性呈正比,这样可监测丙酮酸向乙醇的转化,筛选出低转化率的菌株。通过此方法,筛选出产酸多、ADH酶活力弱、发酵副产物少的目的菌株。     

龙眼果实采后果肉自溶过程中细胞壁组分及其降解酶活性的变化

在(10±1)℃下贮藏的‘福眼’龙眼果实果肉自溶指数和自溶程度随着贮藏时间的延长而增加。果肉细胞壁干重、原果胶、纤维素、半纤维素和细胞壁蛋白含量不断减少。果肉果胶酯酶(PE)活性下降;多聚半乳糖醛酸酶(PG)活性在贮藏6～12d以及纤维素酶活性在贮藏0～12d期间均明显增强,到第12天达到活性高峰,之后下降。但在贮藏0～24d期间,PE、PG和纤维素酶仍然保持较高活性,贮藏24d之后快速下降。β-半乳糖苷酶活性在贮藏0～24d期间略有下降,而在贮藏24d后,活性增强,尤其是贮藏30d后,活性急剧升高。     

中国遗传性胰腺炎患者胰蛋白酶原基因多位点杂合突变及其临床特征

用基因产物直接测序法对2个遗传性胰腺炎家系中胰腺炎患者(共有4例成员)的胰蛋白酶原基因(cationic trypsinogen,PRSS1)5个外显子进行测序,并分析其各自的临床特征.在4例胰腺炎患者中均出现了PRSS1基因杂合突变,但两家系PRSS1基因突变的位点不同,且临床表现差异较大,其中家系1出现6例糖尿病患者且发病年龄较家系2明显延迟,平均发病年龄为29岁,分析其PRSS1基因发现3号外显子336位碱基存在G→A杂合性突变,为中性突变,表达的氨基酸从赖氨酸(Lys)→赖氨酸(Lys),同时在同一外显子的361位碱基还存在另一个G→A杂合性突变,造成121位的丙氨酸(Ala)被苏氨酸(Thr)所取代,胰蛋白酶原的空间结构发生改变,其与抑制因子的结合位点消失,"保护失败"而产生有活性的胰蛋白酶,造成胰腺自身的消化.而家系2未发现糖尿病患者,其胰腺炎患者的血清肿瘤标志物不增高,先证者(Ⅲ8)在胰腺炎发病过程中表现为CD4 T/CD8 Tcell和乙肝表面抗体(anti-HBs)随病程进展逐渐降低,而Ⅲ7不表现出此现象,分析其PRSS1基因发现3号外显子361位碱基同样存在G→A(c.361G→A)突变,而且在415位还存在一个杂合性突变点T→A(c.415T→A),其中c.415T→A不存在于Ⅲ7.胰蛋白酶原基因存在多种形式的突变,而且与临床表型相关.     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electrophoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd 2 h at 25℃ and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips of Vicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd to plants, and that the combined pollution with imidacloprid can enhance the genotoxicity of Cd.     

Survival and DNA Repair of Somatic Cell Hybrids after Ultraviolet Irradiation

THE technique of somatic cell hybridization has opened up studies on genetic regulation¹ and human genetic analysis^2–5. Hybrid cells are isolated in conditions that select against parental cells while allowing hybrids to survive by genomic complementation. In xeroderma pigmentosum (XP), a human disease with an autosomal recessive defect in an early stage of DNA repair⁶, the skin is extremely sensitive to sunlight in vivo⁷ and skin fibroblasts show sharply reduced survival following ultraviolet irradiation in vitro^8,9. This communication concerns the use of ultraviolet irradiation in combination with a chemical method to produce hybrids between fibroblasts from XP and a hamster line, followed by analysis of these cells for their capacity to survive and repair DNA after exposure to ultraviolet. Methods for initiation and propagation of skin fibroblasts from two subjects, male and female siblings with XP, have been described⁸. Details on the origin of the TG2 line of golden hamster fibroblasts, which has a non-reverting mutation in the gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the general hybridization procedure¹⁰ and methods for cell survival and DNA repair by unscheduled synthesis⁸ were also described previously. Hybrids were produced by fusion with Sendai virus and selected by ultraviolet irradiation followed by culture on HAT medium (Fig. 1).     

应用白僵菌防治玉米螟的田间试验

玉米螟是我省粮食作物上的重要害虫,利用化学农药(滴滴涕、六六六)治螟,已在生产上推广使用(邱式邦等,1963;农安县柴岗公社农业站等,1966)。随着近年来防治面积的扩大,使用农药数量日益增多。同时,从国内、外发展趋势表明,由于大量生产和使用有机氯等化学农药防治害虫带来了一系列不良后果(如残毒,抗药性及环境污染等问题),因此,迫切要求研究治螟的新方