Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy

The centromere and its binding proteins constitute the kinetochore structure of metaphase chromosomes, which is crucial for the high accuracy of the chromosome segregation process. Isolation and analysis of the gene encoding a centromere binding protein from the yeast S. cerevisiae, CBF1, are described in this paper. DNA sequence analysis of the CBF1 gene reveals homology with the transforming protein myc and a family of regulatory proteins known as the helix-loop-helix (HLH) proteins. Disruption of the CBF1 gene caused a decrease in the growth rate, an increase in the rate of chromosome loss/nondisjunction, and hypersensitivity to the antimitotic drug thiabendazole. Unexpectedly, the cbf1 null mutation concomitantly resulted in a methionine auxotrophic phenotype, which suggests that CBF1, like other HLH proteins in higher eukaryotic cells, participates in the regulation of gene expression.     

不同细胞周期大鼠肝实质细胞癌细胞粘弹特性研究

以胸腺嘧啶核苷和秋水仙碱顺序阻断法及胸腺嘧啶核苷双阻断法分别获得同步化Ｇ１期和Ｓ期细胞,从细胞周期角度出发,采用微管吸吮技术对大鼠肝实质细胞癌细胞的粘弹特性进行了测定并以标准线性固体模型对实验数据进行了拟合,结果表明：该细胞具有高弹性和低粘性的总体特征;Ｇ１期细胞与Ｓ期细胞相比具有高Ｋ１值和低μ值的特点,从而显示Ｇ１期细胞比Ｓ期细胞具有更大的强度和更快的被动变形能力。这些结果不仅反映了同步化细胞存在的细胞骨架状态的周期性差异,也提示Ｇ１期细胞可能比Ｓ期细胞更适于在血流中存活和转移。     

Density functional calculations of 15N chemical shifts in solvated dipeptides

We performed density functional calculations to examine the effects of solvation, hydrogen bonding, backbone conformation, and the side chain on 15N chemical shielding in proteins. We used N-methylacetamide (NMA) and N-formyl-alanyl-X (with X being one of the 19 naturally occurring amino acids excluding proline) as model systems. In addition, calculations were performed for selected fragments from protein GB3. The conducting polarizable continuum model was employed to include the effect of solvent in the density functional calculations. Our calculations for NMA show that the augmentation of the polarizable continuum model with the explicit water molecules in the first solvation shell has a significant influence on isotropic 15N chemical shift but not as much on the chemical shift anisotropy. The difference in the isotropic chemical shift between the standard beta-sheet and alpha-helical conformations ranges from 0.8 to 6.2 ppm depending on the residue type, with the mean of 2.7 ppm. This is in good agreement with the experimental chemical shifts averaged over a database of 36 proteins containing >6100 amino acid residues. The orientation of the 15N chemical shielding tensor as well as its anisotropy and asymmetry are also in the range of values experimentally observed for peptides and proteins.     

Copper-1,10-Phenanthroline-Induced Apoptosis in Liver Carcinoma Bel-7402 Cells Associates with Copper Overload,Reactive Oxygen Species Production,Glutathione Depletion and Oxidative DNA Damage

The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)₂, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)₂ or Cu²⁺ alone. It was found that the apoptosis induced by Cu(OP)₂ could not be induced by Cu²⁺ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)₂ in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)₂.     

Regulation of arginase I activity and expression by both PD-1 and CTLA-4 on the myeloid-derived suppressor cells

An elevated number of Gr-1⁺CD11b⁺ myeloid-derived suppression cells (MDSCs) has been described in mice and human bearing tumor and associated with immune suppression. Arginase I production by MDSCs in the tumor environment may be a central mechanism for immunosuppression and tumor evasion. In this study and before, we found that Gr-1⁺CD11b⁺ MDSCs from ascites and spleen of mice bearing ovarian 18D carcinoma express a high level of PD-1, CTLA-4, B7-H1 and CD80 while other co-stimulatory molecules, namely CD40, B7-DC and CD86 are not detected. Further studies showed that PD-1 and CTLA-4 on the Gr-1⁺CD11b⁺ MDSCs regulated the activity and expression of arginase I. The blockage and silencing of PD-1, CTLA-4 or both PD-1 and CTLA4 molecules could significantly reduce arginase I activity and expression induced with tumor-associated factor. Similar results were also observed while their ligands B7-H1 and/or CD80 were blocked or silenced. Furthermore, CD80 deficiency also decreased the arginase I expression and activity. Antibody blockade or silencing of PD-1, CTLA-4 or both reduced the suppressive potential of PD-1+CTLA-4+MDSCs. Blockade of PD-1, CTLA-4 or both also slowed tumor growth and improved the survival rate of tumor-bearing mice. Thus, there may exist a coinhibitory and costimulatory molecules-based immuno-regulating wet among MDSCs. This research was supported by Nankai University grant, NSFC grant “30771967”, “985” grant,The Ministry of Science and Technology grant “2006AA020502”“06C26211200695”, Tianjin Grant “07JCZDJC03300” and “06ZHCXSH04800”.     

New stemona alkaloids from the roots of Stemona sessilifolia

From the roots of Stemona sessilifolia, three new stemona-type alkaloids, namely stemosessifoine (1), isooxymaistemonine (2), and isomaistemonine (3), along with eight known alkaloids (bisdehydrostemoninine, isobisdehydrostemoninine, tuberostemonine, bisdehydrotuberostemonine, bisdehydrostemoninine, isobisdehydrostemoninine, stemoninine, and protostemonine), were isolated. Their structures were determined on the basis of extensive 2D-NMR spectroscopic-data analysis and by comparison with reported values in the literature. Compound 1 is a structurally unprecedented alkaloid, and it is depicted to be bioconverted from tuberostemonine as the precursor. Isooxymaistemonine (2) showed a positive effect on the human high-density lipoprotein (HDL) receptor gene CD36 and LIMP II analogous-1 (CLA-1) at the dosage of 10 microg/ml.