BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.     

The distribution of Athetis lepigone and prediction of its potential distribution based on GARP and MaxEnt

Athetis lepigone (Möschler) is a new agronomic pest which has caused serious damages to summer maize in China. In order to effectively monitor it, it is necessary to carry‐out a worldwide investigation on its potential geographical distribution. In this study, we give two ecological niche models, Genetic Algorithm for Rule‐set Production and Maximum Entropy (MaxEnt), to predict the potential geographical distribution of A. lepigone. The results indicate that the suitable areas for A. lepigone are mainly in China (Beijing, Tianjin, central and southern Hebei, northern Jiangsu, Shandong, most of Henan, northern Anhui, central and southern Shanxi, central and southern Shaanxi, small parts of north‐west in Hubei, eastern Gansu, parts of Ningxia, western of Xinjiang and Dandong, and Liaoning) and other Asian countries (South Korea, North Korea and Japan). Parts of Europe (south‐western Russia, Ukraine, Moldova, Romania, Hungary, Bulgaria, Slovenia, and Croatia, Bosnia and Herzegovina, Serbia, parts of Austria, western Poland, the Czech Republic, Germany, northern Italy, Denmark, western Lithuania, Latvia, Estonia, southern Finland and Sweden) are also highly suitable for A. lepigone. In addition, in the states of the USA including Michigan, New York, Chicago, Missouri, Ohio, Illinois and Indiana are also favourable for its occurrence although till now no reports about of this species has been recorded. A jackknife test in MaxEnt showed that the mean temperature of the driest quarter was the most important environmental variable affecting the distribution of this pest.     

免疫磁珠分选人角朊干细胞的实验研究

目的:探索人角朊干细胞(keratinocyte stem cells,KSCs)的免疫磁珠分选(Magnetic Activated Cell Sorting,MACS)的方法。方法:从人包皮组织获得角朊细胞悬液,然后利用人角朊干细胞表面CD49f(整合素α6)和CD71的表达情况(CD49fbriCD71dim)通过MACS法将KSCs分选出来,在荧光显微镜下观察其荧光标记情况,同时将分选所得的CD49fbriCD71dim细胞进行体外无血清培养,观察其形态及克隆形成。结果:人角朊细胞经MACS法分选后所得的CD49fbriCD71dim细胞比率可达12．02(±6．92)%。CD49fbriCD71dim细胞经培养可见细胞为典型的上皮样特征,呈铺路石样形态,高核浆比例,细胞紧密排列,轮廓清楚,折光性好,并可在5-7天左右形成全克隆,符合KSCs的特点。结论:研究表明MACS分选法可以得到较高比例的人角朊干细胞,并能进行后续培养研究,其不失为一种较理想的人角朊干细胞的分选方法。     

遗传算法在转录因子结合位点识别中的应用

遗传算法是模拟生物进化过程的计算模型,是一种全局优化搜索算法。将遗传算法与转录因子结合位点识别问题相结合的新方法,以一致性序列模型作为保守motif的描述模型,通过对motif序列与待测序列的比对问题进行编码,将其转化成搜索空间中的优化问题,利用遗传算法来搜索最优解,预测转录因子的结合位点。实验结果表明,这种新的方法是有效的,它在占用少量内存的情况下能够准确地识别出待测转录因子结合位点。     

蛋白质的二级结构预测研究进展

认识蛋白质的二级结构是了解蛋白质的折叠模式和三级结构的基础,并为研究蛋白质的功能以及它们之间的相互作用模式提供结构基础,同时还可以为新药研发提供帮助。故研究蛋白质的二级结构具有重要的意义。随着后基因组时代的到来,越来越多的蛋白质序列不断被发现,给蛋白质的二级结构研究带来巨大的挑战和研究空间。而依靠传统的实验方法很难获取大规模蛋白质的二级结构信息。目前,采用生物信息学手段仍然是获得大部分蛋白质二级结构的途径。近年来,许多研究者通过构建用于二级结构预测的蛋白质数据集,计算、提取蛋白质的各种特征信息,并采用不同的预测算法预测蛋白质的二级结构得到了快速的发展。本文拟从蛋白质的特征信息的提取与筛选、预测算法以及预测效果的检验方法等方面进行综述,介绍蛋白质二级结构预测领域的研究进展。相信随着基因组学、蛋白质组学和生物信息学的不断发展,蛋白质二级结构预测会不断取得新突破。     

Quaternary structure and apical membrane sorting of the mammalian NaSi-1 sulfate transporter in renal cell lines

NaSi-1 encodes a Na⁺-sulfate cotransporter expressed on the apical membrane of renal proximal tubular cells, which is responsible for body sulfate homeostasis. Limited information is available on NaSi-1 protein structure and the mechanisms controlling its apical membrane sorting. The aims of this study were to biochemically determine the quaternary structure of the rat NaSi-1 protein and to characterize its expression in renal epithelial cell lines. Hexahistidyl-tagged NaSi-1 (NaSi-1-His) proteins expressed in Xenopus oocytes, appeared as two bands of about 60 and 75 kDa. PNGase F treatment shifted both bands to 57 kDa while endoglycosidase H treatment led to a downward shift of the lower molecular mass band only. Mutagenesis of a putative N-glycosylation site (N591S) produced a single band that was not shifted by endoglycosidase H or PNGase F, confirming a single glycosylation site at residue 591. Blue native-PAGE and cross-linking experiments revealed dimeric complexes, suggesting the native form of NaSi-1 to be a dimer. Transient transfection of EGFP/NaSi-1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane sorting, which was insensitive to tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant also showed apical expression, suggesting N591 is not essential for apical sorting. Treatment with cholesterol depleting compounds did not disrupt apical sorting, but brefeldin A led to misrouting to the basolateral membrane, suggesting that NaSi-1 sorting is through the ER to Golgi pathway. Our data demonstrates that NaSi-1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is brefeldin A-sensitive and independent of lipid rafts or glycosylation.     

蓝藻水华预报模型及基于遗传算法的参数优化

蓝藻水华预报是应对水危机,保障水资源供给的一项重要工作。以太湖北部三湾(竺山湖、梅梁湾、贡湖)为研究对象,采用动态空间环境建模技术,构建了蓝藻水华预报模型,并通过实地观测建立了模拟的初始参数集。利用2008年04-09月太湖水环境、气象等实测数据,采用遗传算法优化叶绿素a浓度预报模型中敏感度较高的4个参数。研究结果表明,该模型在蓝藻水华空间分布的预报上达到了一定的精度;采用遗传算法能全面、高效地进行参数优化,降低了模拟结果的相对残差,提高了模型预报精度。     

Current and future applications of flow cytometry in aquatic microbiology

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.     

N-聚糖和O-聚糖在极性化细胞蛋白质分拣中的作用

极性化上皮细胞的质膜因其所含蛋白质、脂质等组分不同,可以分为细胞膜顶端和细胞膜基底侧端两个区域,而新合成的蛋白质向这两个区域的有效分拣是上皮细胞维持其自身极性及正常功能所必需的。细胞膜基底侧端蛋白质的分拣主要由位于该蛋白质胞质区的信号肽所介导,关于这方面的研究是比较深入的;而细胞膜顶端蛋白质的分拣机制目前尚未阐明,因而显得比较复杂。近年来,糖类分子作为生物体内细胞识别和调控过程的信息分子日益受到关注,人们通过干扰聚糖合成、基因突变以及构建糖基化缺陷细胞株等实验方法,逐渐地认识到糖类分子在极性化上皮细胞的蛋白质分拣调节中起重要作用。由于糖分子本身结构非常复杂,而且目前缺乏研究糖类分子的有效手段,使得糖生物学的研究远远落后于蛋白质和核酸的研究。从而导致探讨糖类分子在蛋白质分拣过程的具体机制相对来说比较困难。本综述拟简要概括糖类分子中N-聚糖和O-聚糖在极性化上皮细胞的蛋白质分拣过程中的作用,以及两种聚糖在此过程中行使分拣信号功能的可能机制。