Autographa californica Nucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif

Our previous study showed that the Autographa californicaNucleopolyhedrovirus (AcMNPV) ac76 gene is essential for both budded virion (BV) and occlusion-derived virion (ODV) development. More importantly, deletion of ac76 affects intranuclear microvesicle formation. However, the exact role by which ac76 affects virion morphogenesis remains unknown. In this report, we characterized the expression, distribution, and topology of Ac76 to further understand the functional role of Ac76 in virion morphogenesis. Ac76 contains an α-helical transmembrane domain, and phase separation showed that it was an integral membrane protein. In AcMNPV-infected cells, Ac76 was detected as a stable dimer that was resistant to SDS and thermal denaturation, and only a trace amount of monomer was detected. A coimmunoprecipitation assay demonstrated the dimerization of Ac76 by high-affinity self-association. Western blot analyses of purified virions and their nucleocapsid and envelope fractions showed that Ac76 was associated with the envelope fractions of both BVs and ODVs. Immunoelectron microscopy revealed that Ac76 was localized to the plasma membrane, endoplasmic reticulum (ER), nuclear membrane, intranuclear microvesicles, and ODV envelope. Amino acids 15 to 48 of Ac76 were identified as an atypical inner nuclear membrane-sorting motif because it was sufficient to target fusion proteins to the ER and nuclear membrane in the absence of viral infection and to the intranuclear microvesicles and ODV envelope during infection. Topology analysis of Ac76 by selective permeabilization showed that Ac76 was a type II integral membrane protein with an N terminus exposed to the cytosol and a C terminus hidden in the ER lumen.     

Determination of zeranol and its metabolites in bovine muscle and liver by a chemiluminescence enzyme immunoassay: compared to an ultraperformance liquid chromatography tandem mass spectroscopy method

A chemiluminescent enzyme immunoassay (CLEIA) was compared to an ultraperformance liquid chromatography tandem mass spectroscopy (UPLC‐MS/MS) procedure for the analysis of zeranol and its metabolites in bovine tissue samples. Apparent recoveries from fortified samples by both methods were comparable at 0.5–4.0 µg/kg and a significant correlation was obtained. For CLEIA analysis, hapten mimicking the analyte was first synthesized and conjugated with the carrier protein bovine serum albumin as the immunogen to produce monoclonal antibody. The obtained antibody showed extensive cross‐reactivity toward zeranol metabolites (zearalanone). The limit of detection of CLEIA and UPLC‐MS/MS was 0.05 µg/kg and 0.5 µg/kg, respectively. Recoveries of both methods for fortified samples were higher than 75.0% with the coefficient of variation less than 15%. These results indicated that the combination of screening with CLEIA and confirmation with UPLC‐MS/MS for zeranol and its metabolites would be a reliable method for a large number of bovine samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Developmentally regulated sphingolipid synthesis in African trypanosomes

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.     

云南省爬行动物名录和地理区划更新

云南省作为中国生物多样性最高的省份, 其详实的物种本底资料对我国生物多样性研究和保护具有重要意义。本文在前期研究的基础上, 结合实体标本, 汇总编制了云南省现生、原生爬行动物更新名录。截至2021年12月31日, 云南省记录爬行动物25科82属235种, 其中龟鳖目4科12属16种, 有鳞目蜥蜴亚目6科20属72种, 蛇亚目15科50属147种。较《云南两栖爬行动物》确认新增82种, 存疑收录21种, 移除23种。基于先前云南省爬行动物区划和更新后的物种分布信息, 将云南省爬行动物地理分为6个动物地理区, 即滇西北横断山区、滇西山地区、滇南山地区、滇东南山地区、滇中高原区以及滇东北山地区; 其中滇西北横断山区、滇西山地区、滇中高原区和滇东南山地区的范围与先前研究相比有所调整。结合调整后的爬行动物地理区划, 对物种分布、物种特有性、受威胁状况等给出了统计结果。云南省爬行动物特有物种、国内仅见于云南的非特有物种数量较多, 受威胁等级高。建议今后继续加大分类学研究投入, 对滇西北、滇中特有爬行动物分布集中的区域积极开展栖息地保护工作, 同时在最新调整的《国家重点保护野生动物名录》基础上, 定期组织专家研讨, 对《云南省省级重点保护动物名录》提出更新建议。     

An apicoplast‐resident folate transporter is essential for sporogony of malaria parasites

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co‐factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2‐deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single‐membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2‐deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.     

珍稀药用真菌桑黄的国内外研究进展

桑黄是一种十分珍贵的药用真菌,对增强人体免疫功能及治疗疾病等方面都有明显作用。综述了近年来国内外学者对桑黄的名称、地区分布、化学成分、药理作用等方面的研究进展。     

胶孢炭疽菌生防放线菌SD-29的鉴定及抗菌活性评价

【背景】胶孢炭疽菌是引起橡胶炭疽病的一种重要病原菌,可导致橡胶树产胶量下降。【目的】从山东青岛一农田土壤中分离出一株胶孢炭疽菌生防放线菌SD-29,并对其进行鉴定及抗菌活性评价。【方法】采用对峙生长法及菌丝生长速率法对菌株SD-29的拮抗活性进行鉴定;利用乙酸乙酯萃取法提取菌株SD-29发酵液粗提物并进行活性评价;根据菌株SD-29的形态特征、生理生化及16S rRNA基因序列进行鉴定。【结果】菌株SD-29对胶孢炭疽菌具有较强的抑制活性,皿内抑制活性达到82.6%。发酵液粗提物对菌丝生长的EC50为13.6μg/mL,100μg/mL的粗提物对胶孢炭疽菌孢子萌发抑制率达到63.16%,其对感炭疽病橡胶叶片的防治效果达到48.96%。根据该菌的形态特征、生理生化特征及16S rRNA基因序列分析鉴定菌株SD-29为Streptomyces yatensis。【结论】菌株SD-29对胶孢炭疽菌有较强的防治效果,具有潜在的应用价值。     

Tetracycline inducible gene manipulation in serotonergic neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.