The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

MALE PEACH APHID ATTRACTION IN THE FIELD BY SEX PHEROMONES1)

Abstract Field trials by sex pheromone of aphid to trap peach aphids Myzus persicae have been carried out in 1995 and in 1996. Suitable time and the effect of ratio of two components nepetalactone and nepetalactol to apply the lure have been observed.     

四川自贡大山铺蜀龙动物群——简报Ⅴ 两栖类

本文对扁头中国短头鲵(新属新种) (Sinobrachyops placenticephalus gen. et sp. nov.) 的形态特征和分类位置进行简述.标本得自著名的恐龙化石产地——自贡大山铺,产出时代为中侏罗世.中国短头鲵是目前迷齿类中在地史上最年轻的一个属.它的发现使迷齿类在地球上生存时代的上限推移到中侏罗世.     

轮生钩藻的形态特征及其系统位置

十年前在云南省早泥盆世的地层中发现了轮生钩藻(Uncataella verticillata),限于当时的材料,其分类位置未定。本研究所用材料采自它的模式标本产地,化石植物保存良好,首次发现了着生在植物体上的雌性生殖器官——藏卵器。根据藏卵器和植物体其它部分显示的形态特征,本文修订了它的原始描述并对其系统位置进行了探讨,认为轮生钩藻是一种原始的轮藻植物,很可能属于直立轮藻目直立轮藻科中的一个成员。     

氚水在模拟水生－陆生生态系中的迁移与分布

利用同位素示踪技术研究了进入水体的ＨＴＯ向陆地的迁移。结果表明：１．进入水体的ＨＴＯ将向系统各组分转移,池水中的ＨＴＯ单调地减少,底泥及水生生物中的ＨＴＯ浓度皆在经历某一最大值后平缓地下降;２．ＨＴＯ中的而以自由水沉和结合态氚存在于水生动植物和底泥中;３．ＨＴＯ还明显地向毗邻的陆地和作物迁移,在一个半月期间,陆地系统中的总而约占水体的２４％。     

Modulation of SPARC Expression during Butyrate-Induced Terminal Differentiation of Cultured Human Keratinocytes: Regulation via a TGF-β-Dependent Pathway

Expression of SPARC (secreted protein acidic and rich in cysteine), a 43-kDa extracellular matrix-associated glycoprotein involved in tissue remodeling, was quantitated during normal human keratinocyte (NHK) growth in culture and as a function of sodium n-butyrate (NaB)-induced differentiation to mature enucleate cornified envelopes (CEs). Low levels of SPARC expression were observed in the basal-like cells of control NHKs, with isolated cells showing intense SPARC expression on the ventral surface. After addition of NaB, SPARC expression increased and the pattern of expression shifted to one involving predominantly suprabasal cells (i.e., spinous cells, pre-CEs, and mature CEs). Dense deposits of SPARC often surrounded the mature CEs. Flow cytometric analysis indicated that approximately 13% of NHKs expressed SPARC within 24 h of seeding into culture. This fraction of SPARC⁺ cells increased with time and peaked immediately postconfluence (31.3 ± 6.3% SPARC⁺). Cellular SPARC expression then decreased to baseline levels during entrance into plateau phase growth. SPARC was detectable in all phases of the cell cycle. SPARC levels were more intense and heterogeneous within the G₂/M and G₁ phases while S phase cells exhibited relatively homogeneous, low intensity, SPARC expression. During NaB-induced NHK differentiation, SPARC intracellular content increased prior to the onset of CE formation (i.e., 2 days after its addition) followed by a period of extracellular accumulation which coincided with the time of maximal CE generation (i.e., Days 4 and 5 after NaB addition). Correlation of cell size with anti-SPARC immunoreactivity revealed a predominance of SPARC expression in cells with a suprabasal phenotype. NHKs cultured on fibronectin (FN), an established modulator of epidermal cell maturation in vitro, showed a similar response to NaB. In general, however, the level of NaB-induced SPARC expression was considerably reduced in FN cultures correlating with a lower efficiency of CE formation. Induced SPARC expression was, in large part, dependent on autocrine transforming growth factor-β (TGF-β) production since incubation in the presence of NaB + neutralizing antibodies to TGF-β inhibited both the expression of SPARC by 72% and development of mature CEs.     

植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产g-氨基丁酸

γ-氨基丁酸可由谷氨酸脱羧酶(glutamate decarboxylase, GAD)催化谷氨酸一步合成,反应体系成分简单、环境友好。然而,绝大多数GAD酶催化pH偏酸性且反应范围狭小,需要加入无机盐维持最适催化环境,增加了生产附加成分。此外,随着产物γ-氨基丁酸的生成,溶液pH会逐渐上升,不利于GAD酶的持续转化。本研究首先从实验室保藏的一株高产γ-氨基丁酸的植物乳杆菌(Lactobacillus plantarum)中克隆得到谷氨酸脱羧酶LpGAD,基于酶蛋白表面电荷修饰,选择9个位点进行定点突变及组合突变,酶学性质表征结果显示三突变体LpGAD^{S24R/D88R/Y309K}在催化pH区间内酶活力整体提高,尤其拓宽了在偏中性pH 6.0下的酶活,为野生酶的1.68倍。接下来,通过分子动力学模拟解析了酶活提高的机理。此外,将Lpgad与Lpgad^{S24R/D88R/Y309K}突变基因分别在谷氨酸棒杆菌(Corynebacterium glutamicum) E01中过表达,通过优化确定了摇瓶最适转化条件为反应温度40 ℃,菌体量OD₆₀₀=20,底物L-谷氨酸100.0 g/L,5-磷酸吡哆醛添加量为100 μmol/L。5 L发酵罐中,不调节pH,通过分批投料底物L-谷氨酸,γ-氨基丁酸产量高达402.8 g/L,较对照菌株提高了1.63倍。本研究成功拓宽了LpGAD的pH催化范围及酶活,提高了γ氨基丁酸的转化效率,为实现其规模化工业生产奠定了基础。     

DNA末端硫修饰依赖的体外大片段DNA连接法

【背景】DNA组装技术是基因组合成中的一个关键技术。探索低成本、高效率的基因组合成技术一直是合成生物学的重要研究领域。在某些细菌如变铅青链霉菌中,DNA上有磷硫酰化修饰(简称硫修饰),而在另一些细菌如天蓝色链霉菌中存在一种含有硫修饰识别结构域(sulfur-binding domain, SBD)的识别蛋白,可以特异性识别DNA上的硫修饰,这启发了我们发展出一种新的DNA组装技术。【目的】探究在DNA末端硫修饰的连接中,T4 DNA连接酶与SBD相融合蛋白和单独的T4 DNA连接酶相比,是否有更高的连接效率。【方法】根据同源重组原理,设计硫修饰引物,扩增硫修饰的DNA片段。构建T4 DNA连接酶与SBD融合蛋白的3种表达载体T4-linker-SBD(Hga)、T4-linker-SBD(Spr)和T4-linker-SBD(Mmo),表达纯化以上3种融合蛋白。比较3组浓度梯度(2.4、0.24、0.024 mg/mL) T4 DNA连接酶与融合蛋白在2.5 kb和8.0 kb DNA片段连接上的差异。【结果】DNA末端硫修饰的2.5kb和8.0kb的两端片段均能扩增,而且3种融合蛋白...