(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.     

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.     

Translational accuracy and sexual differentiation in Chlamydomonas reinhardtii

Summary A renewal of ribosomes has been previously reported to occur during gametogenesis in C. reinhardtii. In order to further characterize these new ribosomes, we performed pulse-labelling experiments on whole cells of C. reinhardtii, during gametogenesis and in the presence of various aminoglycosides known to alter translational accuracy: Hygromycin and Paromomycin are assumed to increase the rate of translational errors at the level of 80S and 70S ribosomes whereas Kasugamycin is assumed to induce the opposite effect. Three lines of evidence support an increased inaccuracy in protein translation during gametogenesis: (1) gamete cells displayed a higher sensitivity than vegetative cells to Hygromycin and Paromomycin; 4 g/ml Hygromycin cancelled cytoplasmic protein synthesis in gametes but not in vegetative cells; Paromomycin induced the synthesis of new polypeptides of high molecular weight and of nuclear origin in gametes but not in vegetative cells. In addition, chloroplast protein synthesis was more sensitive to Hygromycin and Paromomycin in gametes than in vegetative cells. (2) Kasugamycin-sensitive alterations of thylakoid membranes were detected during gametogenesis. (3) ³⁵S-misincorporation in the OEE3 polypeptide, of nuclear origin and normally devoid of sulphur containing amino acids, was more than three times higher in gametes than in vegetative cells. This increase was prevented by Kasugamycin, suggesting that 80S translation in gametes was more inaccurate than in vegetative cells. The possible significance of these changes occurring during gametogenic differentiation is discussed in light of the importance of a modulation of translational accuracy at particular stages of the life cycle in other lower eukaryotes.Abbreviations Hm Hygromycin B - Pm Paromomycin - Ks Kasugamycin - CAP Chloramphenicol - OEE Oxygen evolving enhancer     

A Kiswahili version of the SF-36 Health Survey for use in Tanzania: translation and tests of scaling assumptions

The objective of the study was to translate and adapt the SF-36 Health Survey for use in Tanzania and to test the psychometric properties of the Kiswahili SF-36. A cross-sectional study was conducted as part of a household survey of a representative sample of the adult population of Dar es Salaam, Tanzania. The IQOLA method of forward and backward translation was used to translate the SF-36 into Kiswahili. The translated questionnaire was administered by trained interviewers to 3,802 adults (50% women, mean (SD) age 31 (13) years, 50% married and 60% with primary education). Data quality and psychometric assumptions underlying the scoring of the eight SF-36 scales were evaluated for the entire sample and separately for the least educated subgroup (n=402), using multitrait scaling analysis. Forward and backward translation procedures resulted in a Kiswahili SF-36 that was considered conceptually equivalent to the US English SF-36. Data quality was excellent: only 1.2% of respondents were excluded because they answered less than half of the items for one or more scales; ninety percent of respondents answered mutually exclusive items consistently. Median item–scale correlations across the eight scales ranged from 0.47 to 0.81 for the entire sample. Median scaling success rates were 100% (range 87.5–100.0). The median internal consistency reliability of the eight scales for the entire sample was 0.81 (range 0.70–0.92). Floor effects were low and ceiling effects were high on five of the eight scales. Results for n=402 people without formal education did not differ substantially from those of the entire sample. The results of data quality and psychometric tests support the scoring of the eight scales using standard scoring algorithms. The Kiswahili translation of the SF-36 may be useful in estimating the health of people in Dar es Salaam. Evidence for the validity of the SF-36 for use in Tanzania needs to be accumulated.     

A napin‐like polypeptide with translation‐inhibitory,trypsin‐inhibitory,antiproliferative and antibacterial activities from kale seeds

Abstract: A heterodimeric napin‐like polypeptide with translation‐inhibiting and antibacterial activities has been isolated from kale seeds. The purification procedure entailed ion‐exchange chromatography on dielthylaminoethyl (DEAE)‐cellulose, affinity chromatography on Affi‐gel blue gel, ion‐exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on Superdex 75. The napin‐like polypeptide was unadsorbed on DEAE‐cellulose but adsorbed on Affi‐gel blue gel and Mono S. Its 7‐kDa large subunit differs in N‐terminal amino acid sequence from the 4‐kDa small subunit. The polypeptide inhibited translation in the rabbit reticulocyte lysate system with an IC₅₀ of 37.5 nm . This activity was preserved between pH 5 and pH 11, and between 10 and 40 °C. It fell to a low level at pH 3 and pH 13 and at 70 °C. Antibacterial activity against Bacillus, Megabacterium, and Pseudomonas species and antiproliferative activity against leukemia L1210 cells were observed. However, the polypeptide did not exert antifungal, ribonuclease, or protease activity.