濒危物种金斑喙凤蝶的行为特征及其对生境的适应性

金斑喙凤蝶(Teinopalpus aureus)1985年被IUCN列为红色名录种,1989年被我国列为一级保护种,此后便日益受到人们关注,然而,至今为止对其野外生存、行为及生境适应等认识仍很模糊。在广西金秀县大瑶山设定4个研究区域,以线路调查法计数成虫与幼期个体数,定点跟踪观察法记录成虫交配行为、产卵行为、幼虫取食行为等。结果显示:金斑喙凤蝶主要在湿季(4—10月底,月降水量>50 mm)生长、发育与繁殖后代。金斑喙凤蝶在行为上表现出对温度的主动选择性,幼虫在17—24℃时取食行为活跃,雄蝶在19—26℃时山顶行为活跃,均表现出中温选择性;然而,雌蝶多选择在正午时刻产卵,期间温度为27—30℃,表现出高温选择性。雄蝶活动对生境地形表现出主动选择性,(87.34±7.58)%(n=339)的雄蝶选择飞向山顶,他们每日上午6:00至11:00在山顶聚集,绕圈飞行或停息,而以山顶停息为主,占山顶活动时间的(77.87±19.32)%。雄蝶通常停息在山顶的高枝位叶片上或山顶周缘的叶片上,以便迅速发现并拦截飞经的雌蝶,获得交配机会。因而,金斑喙凤蝶在交配策略上主要采取雄蝶等候的方式。停息期间,雄蝶表现出明显的占区行为,首先停息在某一区域的雄蝶在领域权竞争中通常都是最后的胜利者,赢得领域,获得更多交配机会。野外观察发现,金斑喙凤蝶的天敌种类较多,野外存活率偏低,最后羽化率仅为38.9%(n=20)。对于存活个体而言,他们已明显进化形成一套复杂的防御体系,主要包括由保护色、颜色拟态、形状拟态等组建的初级防御体系和由眼斑展示、身体晃动、Y-腺伸出等组建的次级防御体系。另外,老熟幼虫多选择在林下层的灌木丛或竹丛的隐蔽枝条上化蛹,化蛹高度为(1.82±1.58)m(n=20),这种对化蛹场所的主动选择行为可提高其蛹期的防御能力。研究结果说明,在自然选择作用下,金斑喙凤蝶对其阔叶林生境的适应性行为特征非常明显,然而,生境破坏(砍伐等)或人类强度干扰(林下层垦殖等)将使这些适应性行为失效,并威胁到该珍稀蝴蝶种群的繁衍生息,甚至导致局部灭绝的发生。     

太平洋中西部海域浮游植物营养盐的潜在限制

2009年8月至9月期间在太平洋西部N1站和中部N2站进行现场营养盐加富培养实验。结果显示:N1站,浮游植物生物量对N或者P添加都有较强的响应,其中N+P+Si组和N+P组浮游植物长势迅速,叶绿素a从初始的0.03μg/L分别达到2.12μg/L和1.83μg/L,同时P先于N和Si之前被耗尽;说明N1站为N、P共同限制,P是首要限制因子。而N2站,浮游植物生物量仅对N、P共同添加有明显响应,N先于P和Si被浮游植物消耗殆尽。利用培养过程中营养盐比值变化推断,N1站浮游植物以低于Redfield ratio(16N∶1P)吸收N和P;而N2站浮游植物以高于Redfield ratio(16N∶1P)吸收N和P。这可能解释了太平洋西部的寡营养盐海域为潜在P限制,而在太平洋中部海域则为潜在N限制。     

High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor

Since it was first introduced in late 1990s Wave bioreactor has been used for protein production by mammalian and insect cell lines. However, using Wave bioreactor to produce human monoclonal antibody by stable Drosophila Schneider 2 (S2) cell transfectants has not been reported before. In this study, S2 cells were co-transfected with an inducible vector expressing human monoclonal antibody heavy and light chains, respectively, specific for hemagglutinin (HA) of H5N1 influenza virus. Stable S2 transfectant clone was selected by limiting dilution assay. Stable S2 transfectant clone that produce the highest amount of human monoclonal antibody was inoculated into two 2-l disposable cellbags, where cell growth and antibody production were compared between batch and perfusion cultures using Wave bioreactor. Here, we report that maximum viable cell density reached 1.06?×?10(7) cells/ml in batch culture; whereas 1.04?×?10(8)?cells/ml was achieved in perfusion culture. The maximum volumetric antibody productivity in batch culture was 52?mg/l/day; while perfusion culture yielded 1,437?mg/l/day. As a result, the total antibody production was 201?mg in batch culture and 8,212?mg in perfusion culture. The antibody produced by both cultures displays full neutralizing activity. Thus, our results provide strong support for using Wave bioreactor in perfusion culture for a large-scale production of human monoclonal antibody by stable S2 cell transfectants.     

常压低氧小鼠氧耗量测定新装置的研究及应用

目的：建立一种实时记录常压低氧环境中动物氧耗量的方法。方法：本实验装置由动物舱、补水控制系统、天平、软管、装有体重记录软件的电脑等组成。为了实现常压低氧,用水补充动物消耗的氧气以保持动物舱内压力恒定,这个过程由气液联动装置控制;补充的水量由天平测量并同步输出信号至excel文档中。用注射器抽气校准方法检测了装置的准确性和精度。利用该装置观察了6只急性重复低氧小鼠（处理组）和6只未经低氧处理的小鼠（对照组）的常压低氧过程的氧耗量特征。结果：不同体积抽气量与相应补水量两组数据配对t检验P=1;重复抽1 ml氧气6次的补水量变异系数为4%。处理组小鼠的存活时间为（58.8±6.8）min,显著高于对照组（46.0±8.7）min（P〈0.05）。处理组小鼠的总氧耗量为（85.1±8.5）ml,显著高于对照组（73.6±5.4）ml（P〈0.05）。结论：处理组小鼠摄取氧总量增多从而显著延长其存活时间。氧耗量测定装置准确度和精密度较高,可用于低氧研究中氧耗量的测定。     

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.     

干预措施对噪声污染大鼠脑组织基因表达及去甲肾上腺素水平的影响

为了探索干预措施对噪声污染大鼠脑组织基因表达水平的影响, 将50只SPF级Wistar大鼠随机分为空白对照组、噪声污染组(分为30、60、80 dB三个组)、干预组(利血平+80 dB), 每组10只动物。每天刺激1次, 每次刺激30 min, 连续刺激15 d。第16天解剖出脑组织用酶联免疫吸附法(ELISA)检测基因表达水平。结果发现, 噪声污染组大脑前额叶皮质(PFC)和海马(Hipp)组织中去甲肾上腺素(noradrenaline,NA)水平比对照组分别升高了22.87%、50.35%、94.65%和 12.00%、31.76%、61.83%; 干预组NA水平比对照组分别降低了33.66%和52.06%; 去甲肾上腺素转运蛋白(noradrenaline transporter, NAt)水平比对照组分别升高了22.87%、50.35%、94.65%和12.00%、31.76%、61.83%, 干预组NAt水平比对照组分别降低了33.66%和52.06%; 脑源性神经营养因子(brain derived neurotrophic factor, BDNF)水平比对照组分别升高了24.87%、39.27%、67.41%和44.97%、80.81%、95.84%, 干预组BDNF水平比对照组分别升高了16.36%和14.34%, 升高程度明显低于噪声污染组; 酪氨酸激酶受体B(Tyrosine kinase B, TrkB)水平比对照组分别升高了32.64%、59.95%、82.64%和31.02%、57.31%、80.23%, 干预组TrkB水平比对照组分别升高了4.75%和10.52%, 升高程度明显低于噪声污染组。结果显示, 噪声污染使动物体内去甲肾上腺素等水平升高, 去甲肾上腺素是噪声污染引起组织器官损伤的主要因素, 脑源性神经营养因子和酪氨酸激酶受体B防止神经元受损死亡, 改善神经元的病理状态, 利血平使去甲肾上腺素耗竭, 保护组织器官免受噪声污染的损伤。     

HIV-1 Tat蛋白对人类疱疹病毒8型复制的影响

用HindⅢ将HIV-1Tat101蛋白编码基因从pEV质粒中切出,BamHI、NotⅠ将绿色荧光蛋白(GFP)编码基因从表达质粒pcDNA3．1 ／GFP中切出,分别插入到质粒LZRSpBMN-Z中,构建成重组反转录病毒表达质粒LZRS—Tat101和LZRS—GFP。采用磷酸钙转染法将两重组质粒转染到含反转录病毒env,gal和pol编码基因的包装细胞Phoenix(φNX)中,嘌呤霉素筛选获得稳定细胞系。分别收集稳定细胞系分泌的病毒上清,并感染体外培养的原发性渗出性淋巴瘤(PEL)BC2BL-1细胞。收集LZRS—GFP重组病毒感染的BCBL-1细胞进行流式细胞计数,检测GFP表达水平。收集LZRS—Tat101重组病毒感染的BCBL-1细胞,提取蛋白作Western blot,检测Tat蛋白表达状况;取细胞总RNA作Northem blot和定量PCR,检查HHV-8次要衣壳蛋白ORF26 mRNA转录水平。重组LZRS—Tat101病毒进一步感染HL3T1细胞(HeLa细胞包含HIV-1-LTR／CAT报告基因),收集感染细胞提取蛋白,检测CAT活性,评价Tat生物学功能。PCR扩增HHV-8复制和转录激活蛋白Rta启动子区上游序列,并克隆至pGL-3载体中,构建Rta启动子 虫荧光素酶(Luciferase)报告基因重组质粒。此重组质粒进一步电转染预先感染了LZRS—Tat101病毒的BC-3细胞,TPA刺激后收集细胞,检测Luciferase活性。结果显示：①重组反转录病毒感染BCBL-1细胞,一次感染效率达56％;②重组LZRS—Tat101毒能够在其感染的BCBL-1细胞中表达Tat蛋白,且表达蛋白具有转录激活功能;③Tat蛋白不能有效上调HHV-8Rta启动子活性;④细胞内HIV-1Tat蛋白诱导HHV-8可溶性周期复制的能力较弱。提示,单纯HIV-1Tat蛋白并不能激活潜伏感染的HHV-8。     

Perceptions of Workplace Heat Exposure and Controls among Occupational Hygienists and Relevant Specialists in Australia

With warmer weather projections, workplace heat exposure is presenting a growing challenge to workers’ health and safety. Occupational hygienists are the specialist group conducting measurements and providing advice on heat stress management to industry. In order to provide insights into hygienists perceptions on workplace heat exposure, current and future preparedness for extreme heat, and barriers to possible heat adaptation strategies, a self-administered questionnaire survey was conducted during a national conference of the Australian Institute of Occupational Hygienists. Nearly 90% of the 180 respondents were at least moderately concerned about extreme heat and 19% were dissatisfied with current heat stress prevention measures. Barriers recognized by the participants were lack of awareness (68%), insufficient training (56%), unsatisfactory management commitment (52%), and low compliance with prevention policies (40%). The findings suggest a need to refine occupational heat management and prevention strategies.     

Identification of Pseudomonas bacteriophage PaP3 lysozyme

The complete genome of bacteriophage PaP3 was sequenced in a previous study by our laboratory; however, the PaP3 lysozyme gene could not be identified by homology search. In this study, based on bioinformatic analysis of its secondary structure, we have determined that the protein encoded by the p02 gene of PaP3 is likely to be a lysin. To confirm the function of the p02 gene, a recombinant expression plasmid was constructed by inserting the p02 gene into a pQE-31 plasmid; the recombinant construct was cloned and expressed in Escherichia coli JM109. The lytic activity of the expressed, purified product was observed by gel diffusion assay. The result showed that the recombinant plasmid successfully expressed 6 × his-tagged p02 protein. The expressed product had a growth inhibitory effect on Staphylococcus aureus but not on Pseudomonas aeruginosa or E. coli. However, it retained lytic activity against peptidoglycan from cell walls of P. aeruginosa and E. coli. Therefore, it is supposed that this lysozyme requires the help of holin or other punching proteins to exert lytic effects on live gram-negative bacteria. The results suggest that the p02 protein of PaP3 is a new member of the lysozyme family, which is not completely host-specific and might serve as an anti-staphylococcal agent.     

Suppression subtractive hybridization identifies an autotransporter adhesin gene of <Emphasis Type="Italic">E. coli</Emphasis> IMT5155 specifically associated with avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (APEC)

Background  

Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group.