Schizosaccharomyces pombe: A novel transport vehicle of functional DNA and mRNA into mammalian antigen-presenting cells

Vaccine vehicles based on recombinant yeasts have become promising candidates for the induction of cellular immune responses. In this study, we investigated the capacity of the fission yeast Sz. pombe for the delivery of functional nucleic acids into murine and human antigen-presenting cells. We demonstrate that Sz. pombe cells effectively induce maturation of human dendritic cells (DC), an important prerequisite for T-cell activation. Further, recombinant fission yeast efficiently delivers functional DNA and mRNA into murine macrophages and human DC resulting in the expression of the model antigen eGFP in these cells. Thus, Sz. pombe suggests itself as a promising candidate for a novel live vaccine.     

人CDK2-AP1(DOC-1)酵母双杂交诱饵质粒自激活作用鉴定

目的验证诱饵质粒pBD—DOC-1在酵母双杂交系统的自激活性及毒性作用，为应用酵母双杂交系统（yeasttwo—hybrid system）筛选与p12DOC-1／CDK2-AP1相互作用的蛋白建立实验基础。方法将诱饵质粒pBD—DOC-1转化到酵母细胞MAV203中，检测诱饵蛋白有无毒性和自激活作用。同时利用对照质粒组筛选组氨酸（His）本底表达抑制剂3AT（3-氨基．1，2，4-三唑）的合适工作浓度。结果诱饵质粒pBD．DOC—1成功转化到酵母细胞MAV203中，对宿主酵母细胞无毒性，对报告基因无自激活作用。确定了组氨酸（His）本底表达抑制剂3AT（3-氨基-1，2，4-三唑）的合适工作浓度。结论诱饵质粒pBD—DOC-1可以用于酵母双杂交实验，为进-步运用酵母双杂交技术在人类组织cDNA文库中筛选与之相互作用的蛋白奠定了基础。     

Prediction of protein functions based on function-function correlation relations

A protein function pair approach, based on protein-protein interaction (PPI) data, is proposed to predict protein functions. Randomization tests are performed on the PPI dataset, which resulted in a protein function correlation scoring value which is used to rank the relative importance of a function pair. It has been found that certain classes of protein functions tend to be correlated together. Scoring values of these correlation pairs allow us to predict the functionality of a protein given that it interacts with proteins having well-defined function annotations.The jackknife test is used to validate the function pair method. The protein function pair approach achieves a prediction sensitivity comparable to an approach using more sophisticated method. The main advantages of this approach are as follows: (i) a set of function-function correlation relations are derived and intuitive biological interpretation can be achieved, and (ii) its simplicity, only two parameters are needed.     

Identification of a novel protein binding to hepatitis C virus core protein

Background:  Hepatitis C virus (HCV) core protein is a multi-functional viral protein that interacts with several target proteins of both viral and cellular origin.
Aim and Methods:  To gain insight into the mechanism of action of HCV core protein, we used a yeast two-hybrid system to identify the core protein-interacting cellular targets.
Results:  A cDNA clone encoding an aspartoacylase was obtained, termed aspartoacylase 3 (ACY3). Interaction between ACY3 and HCV core protein was verified using a co-immunoprecipitation assay in vitro , and a mammalian two-hybrid system in vivo . Fluorescence microscopy showed green fluorescence protein-fused ACY3 localized in the cytoplasm.
Conclusion:  Our data suggest that ACY3 is an HCV core binding protein, which may play a role in the development of HCV-associated diseases.     

Protective effect of basil (Ocimum basilicum L.) against oxidative DNA damage and mutagenesis.

Mutagenic and antimutagenic properties of essential oil (EO) of basil and its major constituent Linalool, reported to possess antioxidative properties, were examined in microbial tests. In Salmonella/microsome and Escherichia. coli WP2 reversion assays both derivatives (0.25–2.0 μl/plate) showed no mutagenic effect. Salmonella. typhimurium TA98, TA100 and TA102 strains displayed similar sensitivity to both basil derivatives as non-permeable E. coli WP2 strains IC185 and IC202 oxyR. Moreover, the toxicity of basil derivatives to WP2 strains did not depend on OxyR function. The reduction of t-BOOH-induced mutagenesis by EO and Linalool (30–60%) was obtained in repair proficient strains of the E. coli K12 assay (Nikoli?, B., Stanojevi?, J., Miti?, D., Vukovi?-Ga?i?, B., Kne?evi?-Vuk?evi?, J., Simi?, D., 2004. Comparative study of the antimutagenic potential of vitamin E in different E. coli strains. Mutat. Res. 564, 31–38), as well as in E. coli WP2 IC202 strain. EO and Linalool reduced spontaneous mutagenesis in mismatch repair deficient E. coli K12 strains (27–44%). In all tests, antimutagenic effect of basil derivatives was comparable with that obtained with model antioxidant vitamin E. Linalool and vitamin E induced DNA strand breaks in Comet assay on S. cerevisiae 3A cells, but at non-genotoxic concentrations (0.075 and 0.025 μg/ml, respectively) they reduced the number of H₂O₂-induced comets (45–70% Linalool and 80–93% vitamin E). Obtained results indicate that antigenotoxic potential of basil derivatives could be attributed to their antioxidative properties.     

Distribution of ochratoxin A in plasma and tissues of rats fed a naturally contaminated diet amended with micronized wheat fibres: effectiveness of mycotoxin sequestering activity.

The effectiveness of micronized wheat fibres (MWF) alone or in association with yeast cell walls (YCW) as active adsorbents to decrease, in vivo, the levels of ochratoxin A (OTA) was checked in a total of 48 rats, equitably distributed into four groups: (1) control; (2) OTA naturally contaminated diet (2.2 microg/g); (3) OTA naturally contaminated diet (2.2 microg/g) amended with MWF (2%); (4) OTA naturally contaminated diet (2 microg/g) amended with MWF (1.8%) in association with YCW (0.2%). A 4 week experimental period corresponding to a daily intake in the range of 132.2-146.1 microg OTA/kg bw decreased the rat body weight gains, as compared to the controls. The adsorbents did not significantly alleviate the growth depression caused by the contaminated diet. However, a significant protective effect of MWF was observed in terms of OTA concentration in plasma (40.5% decrease), kidney (28.1% decrease) and liver (38.8% decrease). Mixing this sorbent with the YCW did not significantly improve its protective activity against OTA. The faecal OTA concentrations were higher for the MWF and MWF+YCW treated animals, as compared to the positive control (group II). Taken together, these results suggest that MWF are a promising tool to counteract the toxic effects of OTA naturally contaminated diets.     

FasL蛋白及其相互作用蛋白对直肠癌细胞耐药性影响的研究

目的:探讨FasL蛋白及其相互作用蛋白在直肠癌细胞耐药中的作用机制.方法:利用酵母双杂交系统在相互作用蛋白研究中的特性,筛选与FasL蛋白相互作用较强的蛋白质,通过细胞学实验(MTT法)研究FasL蛋白及其相互作用蛋白对直肠癌细胞耐药性的影响.结果:经过筛选发现,FasL蛋白与anchor attachment蛋白、金属硫蛋白1K具有较强的相互作用.经细胞耐药学实验证实.FasL蛋白、anchor attachment蛋白与金属硫蛋白1K组直肠癌HB-8348细胞的耐药性明显高于其他组细胞,差异具有非常显著性统计学意义(P<0.01),FasL蛋白组以及FasL蛋白和金属硫蛋白1K组直肠癌HP-8348细胞的耐药性均高于空白对照组,差异具有显著性统计学意义(P<0.05).结论:FasL蛋白与anchor attachment蛋白1K结合后,增强了金属硫蛋白1K在结直肠癌细胞中的耐药能力,可能促进了结直肠癌的复发与转移.     

Application of the yeast two-hybrid system in molecular gerontology

Most – if not all – proteins are bound to interact with other proteins to exert their function, and thus the identification of the interaction partners of a protein is vital in proteomics. The yeast two-hybrid system is a popular and effective tool for studyingprotein–protein interactions. Although the advantages of the system are manifold, it also has certain drawbacks and limitations. The two-hybrid system has been shown to be extremely useful for placing a protein of unknown function within a functional context, thereby providing information about a putative role of the uncharacterised protein. This concept has also been successfully applied in molecular gerontology. This revised version was published online in July 2006 with corrections to the Cover Date.     

心血管病医院临床检出酵母菌分布及耐药性分析

目的 了解心血管医院临床检出酵母菌的分布及其耐药性，供临床治疗参考。方法 对住院患者送检标本按常规作真菌分离培养，经Api20Aux真菌鉴定板条鉴定为酵母菌，经微量稀释法真菌药敏板测定药敏，再对其结果进行分析。结果共分离酵母菌196株，其中来自呼吸道标本152株占77．5％，检出的酵母菌中白假丝酵母菌112株占57、1％，其次为热带假丝酵母菌32株占16．3％，近平滑假丝酵母菌30株占15.3％；各种酵母菌对氟康唑总的敏感率为91．8％，对二性霉素B的敏感率为97.9％，而对特比奈芬的耐药率较高（64、3％）。结论 院内临床检出酵母菌以白假丝酵母菌为主，其次为热带假丝酵母菌和近平滑假丝酵母菌；对氟康唑敏感率高，氟康唑仍为酵母菌治疗的首选药物。     

CLN -encoded proteins do not interact with each other

The lysosomal storage of lipofuscins is the common pathological feature that characterizes the infantile, late-infantile, juvenile (Batten's disease), and Finnish-variant neuronal ceroid lipofuscinosis (INCL, LINCL, JNCL and FNCL), which are due to mutations in the genes CLN1 , CLN2 , CLN3 , and CLN5 , respectively. The CLN1 and CLN2 genes encode lysosomal enzymes, but the CLN3 and CLN5 genes encode membrane-spanning proteins. Why deficiencies of lysosomal enzymes and membrane-spanning proteins produce similar clinical phenotypes and pathological changes is still unanswered. We hypothesize that CLN -encoded proteins may comprise a functional pathogenic pathway, in which protein associations may play important roles. To test this hypothesis, we studied protein-protein interactions among the CLN1 -, CLN2 -, and CLN3 -encoded proteins using a yeast two-hybrid system. Our results provided no evidence that CLN -encoded proteins interact with each other. This suggests there may be unidentified components in NCL pathogenesis. Received: December 1, 1999 / Accepted: February 2, 2000 / Published online: May 9, 2000