Isolation and RNA-binding analysis of NAD+-isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe

Krebs cycle NAD⁺-isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiaeCOX2 leaders. In contrast, Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA. Received: 19 January 2000 / 24 March 2000     

In vitro biosynthesis and core glycosylation of the histidine-rich protein of Plasmodium lophurae

We have characterized the early biosynthetic forms of the histidine-rich protein (HisRP), a major, granule-bound protein (Mr 58 000) of the avian malarial parasite Plasmodium lophurae. We have translated poly(A)-containing, size-selected parasite mRNA in the wheat germ cell-free system in the presence of [3H]histidine. HisRP was synthesized as a larger precursor (Mr 63 000). When dog pancreas microsomal membranes were present in the cell-free system during translation, a still larger form of HisRP (Mr 66 000) was detected. This larger form was segregated into the dog pancreas microsomal vesicles and was core glycosylated. Presumably, it corresponds to an intermediate form located in the parasite rough endoplasmic reticulum (RER). The difference in the Mr of approx. 8 000 between this RER associated 'pro' form and the granule-bound, mature form of HisRP suggests that proteolytic processing occurs upon transport from the RER to the granule. Segregation and core glycosylation were strictly coupled to translation and were not observed upon posttranslational addition of microsomal membranes. Thus, the early events in the biosynthesis of HisRP are similar to those established for secretory and lysosomal proteins.     

(His)6 -eIF3s4融合蛋白在人乳腺癌细胞Bcap37中的表达

目的：构建真核细胞翻译起始因子3第4亚单位（eIF3s4）真核表达载体用于进一步功能研究。方法：设计引物,以K562细胞的总RNA为模板,采用RT-PCR方法扩增eIF3s4的全长编码区cDNA,克隆于pGEM-TEasy载体,经DNA测序并与GenBank数据库序列进行比对后,将该cDNA序列亚克隆于真核表达载体pcDNA4/HisMaxB载体,构建成（His）6-eIF3s4融合蛋白的表达载体,用LipofectamineTM2000瞬时转染人乳腺癌细胞株Bcap37,利用Westernblot方法检测（His）6-eIF3s4融合蛋白的表达。结果：DNA序列测定表明,利用RT-PCR方法克隆的eIF3s4全长编码区cDNA序列与GenBank数据库序列一致,Westernblot结果证实（His）6-eIF3s4融合蛋白在Bcap37细胞中获得预期表达。结论：成功地构建了eIF3s4的真核表达载体并在人乳腺癌细胞Bcap37中表达,为建立稳定的eIF3s4高表达细胞系,进而研究eIF3s4与肿瘤细胞多药耐药相关性打下了基础。     

Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication

To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3' noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3'NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.     

In-vitro translation of mitochondrial mRNAs by yeast mitochondrial ribosomes is hampered by the lack of start-codon recognition

Summary In an attempt to reconstitute an homologous in-vitro translation system for yeast mitochondrial mRNAs, we have isolated ribosomes, supernatant factors, and tRNAs from mitochondria of Saccharomyces carlsbergensis. While poly(U) is translated faithfully in this system, no translation of in-vitro synthesised cytochrome c oxidase subunit II (COX2) mRNA could be detected. Formation of formylmethionyl-puromycin on mitochondrial ribosomes is stimulated by ApUpG, but not by COX2 mRNA, although mitochondrial small ribosomal subunits bind to this mRNA in vitro, even without added tRNA and initiation factors. We conclude, therefore, that the inability to faithfully translate mitochondrial mRNAs in vitro may be the result of an inability of mitochondrial ribosomes to recognize the initiation codon.     

Adriamycin-induced DNA strand breaks in HeLa and in P388 leukaemia cells detected using in situ nick translation

DNA strand breaks produced by adriamycin (ADR) were measured in HeLa cells and ADR-sensitive and -resistant P388 leukaemia cells, using the in situ nick translation method. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labelled dTTP, and were visualized by autoradiographic observation of the grains. The DNA strand breaks in the HeLa cells increased in a dose-dependent manner, compared with findings in the untreated control cells, i.e., 15.2 fold at 20 micrograms/ml of ADR for 1 h. This level correlated with DNA single-strand breaks detected by the alkaline elution method. DNA breaks were also noted in the ADR-sensitive P388 cells, but in the ADR-resistant cells the level of DNA strand breaks was low. The enhanced cytotoxicity is apparently the consequence of the enhanced potential of ADR to cause breaks in the DNA strands. Our findings show that the survival response of the cells decreases and the level of DNA strand breaks increases following exposure to ADR. ADR resistance may be mediated by a reduction in the level of DNA strand breaks.     

英文科技期刊促进公共卫生学术交流与传播的策略——以《生物安全与健康(英文)》的办刊实践为例

【目的】 探讨建设促进公共卫生学术交流与传播的国际化英文科技期刊的有效途径,为我国科技期刊,特别是新创办的英文科技期刊提供借鉴和参考。【方法】 结合《生物安全与健康(英文)》(Biosafety and Health,B&H)的办刊实践,归纳新创办的英文科技期刊的办刊背景、提升国际影响力的举措及获得的成效。【结果】 B&H创刊3 年以来,先后被国内外重要数据库,如DOAJ、RCCSE、CSCD、Scopus、CSTPCD等收录,期刊的国际影响力正在快速提升,期刊的开放获取政策、出版质量和规范性达到国际评估机构的认可标准。【结论】 B&H可填补我国生物安全学科领域期刊的空白,通过坚持国际化办刊之路、吸引优质稿源、积极策划专刊出版、多渠道宣传推广等特色举措,使期刊国际影响力得到快速提升,从而助力公共卫生领域的学术交流与传播。     

Ten years of Pan-InfORM: modelling research for public health in Canada

Modelling and simulation methods can play an important role in guiding public health responses to infectious diseases and emerging health threats by projecting the plausible outcomes of decisions and interventions. The 2003 SARS epidemic marked a new chapter in disease modelling in Canada as it triggered a national discussion on the utility and uptake of modelling research in local and pandemic outbreaks. However, integration and application of model-based outcomes in public health requires knowledge translation and contextualization. We reviewed the history and performance of Pan-InfORM (Pandemic Influenza Outbreak Research Modelling), which created a national infrastructure in Canada with a mandate to develop innovative knowledge translation methodologies to inform policy makers through modelling frameworks that bridge the gaps between theory, policy, and practice. This review demonstrates the importance of a collaborative infrastructure as a “Community of Practice” to guide public health responses, especially in the context of emerging diseases with substantial uncertainty, such as the COVID-19 pandemic. Dedicated resources to modelling and knowledge translation activities can help create synergistic strategies at the global scale and optimize public health responses to protect at-risk populations and quell socioeconomic and health burden.     

国际同行评议中审稿意见造假现象及相关的学术不端防范

【目的】 提升英文科技期刊在国际同行评议中对于审稿专家审稿意见造假这一学术不端行为的防范意识和能力,进一步完善其对学术不端的防范举措。【方法】 针对英文科技期刊办刊实践中所发现的审稿专家批量捏造或制造审稿意见的现象,探析其外在存续条件与内在动机,阐述这类造假意见的主要表现形式,进而总结出发现和防范此类学术不端审稿专家的有效方式。【结果】 在国际同行评议中,审稿专家审稿意见造假的学术不端情况并非个例,所提出的方式可有效发现和防范此类审稿专家。【结论】 在国际同行评议中,英文科技期刊需要针对审稿专家审稿意见造假的情况,进一步完善学术不端防范措施。