CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

重组人组蛋白H3和H4的表达纯化及其细胞毒性分析

细胞外组蛋白在脓毒症、类风湿性关节炎、急性肺损伤等多种疾病的发生发展中起关键作用,但由于缺乏合适的标准品,至今无法对患者体内的胞外组蛋白进行精确定量,导致在多种感染性疾病中无法根据血清组蛋白含量对疾病进行精确分级,也无法据此合理用药。同时,对患者体内胞外组蛋白精确定量也有助于确定细胞毒性机制研究的使用剂量。本研究用大肠杆菌表达单体变性组蛋白H3和H4,亲和纯化后用梯度稀释和透析方法,可以得到复性的组蛋白单体H3、H4以及H3/H4复合物。通过对蛋白质在纯化过程中稳定性的比较,发现H3/H4复合物较单体更为稳定。 以该复合物（50 μg/mL）处理HUVEC细胞,细胞存活率约为20%,与小牛胸腺组蛋白（200 μg/mL）的毒性类似。 该复合物引起的细胞毒性可被人血清白蛋白以浓度依赖的形式（0.625~10 mg/mL）缓解,提示其构象基本正确。 因此,重组组蛋白H3/H4复合物可以作为精确定量组蛋白的标准品,对基于组蛋白含量的疾病分级和组蛋白毒性机制的研究均有应用价值。     

氦氖激光对绵羊精清中酶活性效应的研究

本文研究结果表明低剂量的氦氖激光可以提高绵羊精清中GOT和LDH酶的活性,并对其机制作了初步的探讨。     

鲈鱼IgM重链基因cDNA的克隆及其原核表达

采用同源克隆和RACE技术扩增到鲈鱼 (Lateolabrax japonicus) 免疫球蛋白M (Immunoglobulin M,IgM) 重链 (Heavy chain,H) 基因全长cDNA序列。鲈鱼IgM cDNA全长为1 901 bp,开放阅读框包含1 749 bp,编码582个氨基酸。根据鲈鱼IgM和其他硬骨鱼免疫球蛋白重链恒定区的氨基酸序列构建的系统发育树表明IgM、IgZ和IgD分别聚为一枝,其中IgM与IgZ分支的进化关系较近,而与IgD分支的进化关系较远。RT-PCR检测IgM在鲈鱼各组织器官的表达情况,其中在头肾及脾脏中表达量最高,心脏、肌肉及脑中几乎不表达。利用已获得的鲈鱼IgM cDNA序列,构建原核表达载体pQE30-IgM,并在M15大肠杆菌中成功诱导表达了分子量为63kD的重组蛋白His-IgM,Western-blotting显示鲈鱼IgM重组蛋白能与鼠源抗6×His的单克隆抗体特异性结合,说明已经获得了基因工程表达IgM重链蛋白。     

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

Circ_0075829 facilitates the progression of pancreatic carcinoma by sponging miR‐1287‐5p and activating LAMTOR3 signalling

Pancreatic cancer (PC) is a leading cause of cancer‐related mortality globally. Though increasing evidence has demonstrated that circular RNAs (circRNAs) are linked to the development and progression of cancers, the biological functions of circRNAs in PC remain largely unexplored so far. Based on previous studies, Hsc_circ_0075829 (circ_0075829) was screened out and then further identified in PC clinical specimens and cell lines by real‐time PCR. After the stability tests, a series of in vitro and in vivo functional experiments were performed to investigate the role of circ_0075829 in PC development. Furthermore, fluorescent in situ hybridization (FISH), bioinformatics tools, dual‐luciferase assays and rescue experiments were conducted to clarify the regulatory mechanisms of circ_0075829 in SW1990 and BxPC‐3 cells. Compared with paracancerous tissues, the expression of circ_0075829 was increased in PC tissues, which was positively correlated with the clinical features of PC. Knockdown of circ_0075829 significantly suppressed the proliferative, migratory and invasive rates of SW1990 and BxPC‐3 cells both in vitro and in vivo. Bioinformatics analysis and dual‐luciferase reporter gene assay indicated that circ_0075829 could bind to miR‐1287‐5p. Mechanism research and rescue experiments demonstrated that circ_0075829 could regulate the LAMTOR3/p‐ERK signalling pathway via sponging miR‐1287‐5p in PC cell lines. Our data reveal that the circ_0075829 could facilitate the proliferation and metastasis of PC through circ_0075829/miR‐1287‐5p/LAMTOR3 axis.     

双歧杆菌脂磷壁酸与5-氟尿嘧啶联用的抗肿瘤研究

目的探讨双歧杆菌脂磷壁酸与5-氟尿嘧啶（5-Fu）联用对H22荷瘤小鼠的抗肿瘤作用及免疫功能的影响。方法双歧杆菌脂磷壁酸单独或联合5-Fu处理H22荷瘤Balb／c小鼠,定期测量肿瘤大小,观察小鼠一般状况;计算抑瘤率、血红细胞数和白细胞数,取脾和胸腺计算脏器指数;HE染色分析肿瘤组织变化;MTT法检测小鼠脾T淋巴细胞增殖转化功能以及ELISA法检测小鼠脾淋巴细胞分泌IFN-γ含量。结果双歧杆菌脂磷壁酸及5-Fu单独应用均可抑制肿瘤生长,但单独5-Fu处理组小鼠一般状况差,毒性反应重;双歧杆菌脂磷壁酸与5-Fu联合应用,与单独5-Fu处理组比较,不仅抑瘤率明显提高（P〈0．01）,且荷瘤小鼠一般状况改善,白细胞数升高,脏器指数增加,小鼠脾T淋巴细胞增殖能力强,脾淋巴细胞分泌IFN-γ,水平提高;光镜观察HE染色瘤体组织,双歧杆菌脂磷壁酸处理组可见大量炎症细胞浸润。结论双歧杆菌脂磷壁酸联合5-FU能增强化疗的抑瘤作用,并能扭转化疗引起的免疫低下现象,起到增效减毒作用。     

自然保护地生物多样性保护研究进展

建立自然保护地是保护生物多样性最为重要的措施之一。总体来看, 自然保护地生物多样性保护研究主要围绕关键生态系统以及珍稀濒危物种等保护对象的状态以及变化两个层面进行, 并重点关注自然保护地数量与面积、保护了多少重要生态系统和物种、能否有效保护生物多样性等一系列科学问题。然而, 在自然保护地生物多样性保护研究方面, 还缺少针对上述研究领域的系统性综述。为此, 本文系统梳理了自然保护地空间布局及其与生物多样性分布的关系、自然保护地生物多样性变化及其保护成效等近20年来相关领域的研究进展。自然保护地的空间布局以及与生物多样性分布的关系主要围绕自然保护地与生物多样性在某一阶段的状态开展研究, 致力于探究自然保护地“保护多少” “代表性如何” “在哪儿保护”等一系列关键科学问题。同时, 自然保护地内的生物多样性会随着气候变化、人类活动以及自身演替等发生时空动态变化, 基于自然保护地生物多样性变化分析, 各国学者在全球尺度、国家尺度和单个自然保护地进行了大量的保护成效评估研究, 并逐渐发展出了自然保护地内外配对分析方法以提升保护成效评估的精度, 进而识别出不同自然保护地的主要影响因素。在此基础上, 本文进一步对自然保护地生物多样性保护研究提出了展望, 主要包括: (1)综合考虑自然保护地生物多样性状态和变化; (2)开展多目标协同的自然保护地空间优化布局; (3)强化自然保护地主要保护对象的识别、调查与监测; (4)提升自然保护地的质量和连通性; (5)探究自然保护地管理措施与保护成效的关联机制。本文可为“2020年后全球生物多样性框架”的制定与实施特别是在自然保护地体系建设与优化方面提供参考与借鉴。     

脑型一氧化氮合成酶的钙调蛋白结合区的表达及活性鉴定

用PCR法克隆出nNOS的CaM结合区基因(nNOS 2455～2988bp),并在大肠杆菌中进行了高效表达。经金属离子螯合亲和层析得到纯度为90％以上的重组蛋白．分子量为22kDa,CaM Oveday assay证实该蛋白具有CaM的结合活性。由于所表达的重组蛋白既具有序列特异性又具有CaM的结合活性．因此。可将它作为筛选nNOS特异性抑制肽的靶蛋白,亦可用于特异性抗体的制备。