The chaperonin-related protein Tcm62p ensures mitochondrial gene expresssion under heat stress

Tcm62p, distantly related to chaperonins, is required for the assembly of succinate dehydrogenase in mitochondria of Saccharomyces cerevisiae and was proposed to exert chaperone activity. We demonstrate here crucial functions of Tcm62p under heat stress. It ensures mitochondrial gene expression at elevated temperatures and prevents heat-aggregation of the ribosomal subunit Var1p. Similar to chaperonins, Tcm62p forms a high molecular mass protein complex of approximately 850 kDa in the mitochondrial matrix space. These results suggest a more general chaperone function of Tcm62p in mitochondria.     

Detection of Two Fungal Biocontrol Agents against Root-knot Nematodes by RAPD Markers

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 μg, respectively.     

巴西固氮螺菌Yu62在玉米根的定植

将GFPmut2质粒中的gfp基因(编码绿色荧光蛋白)克隆到载体pVK100中,构建成重组质粒pVK1001。将pVK1001通过电转化方法导入到联合固氮菌巴西固氮螺菌Yu62中,获得GFP)标记的巴西固氮螺菌Yu62菌株。用标记菌株接种限菌培养条件下生长的玉米(农大3318)幼苗,在接种后8d、12d,用激光共聚焦扫描显微镜进行观测,结果表明巴西固氮螺菌Yu62菌株能定植于玉米根部皮层的薄壁细胞间隙。用扫描电镜和超薄切片电镜观察表明,大多数细菌主要定植于根表,少数菌可进入玉米根组织内。     

人工注射Dnmt3 siRNA对意大利蜜蜂雌蜂发育的影响

为了研究人工注射DNA甲基化转移酶3 (DNA methyltransferase3, Dnmt3) siRNA对意大利蜜蜂Apis mellifera ligustica雌蜂的Dnmt3酶活性、 Dnmt3 mRNA表达量、 dynactin p62基因甲基化水平以及蜜蜂形态指标之间的影响和关系, 分别给3组3日龄的意大利蜜蜂幼虫人工注射50 ng Ringer, uth siRNA和Dnmt3 siRNA溶液。在幼虫的发育过程中, 测定每组幼虫头部的Dnmt3酶活性、 Dnmt3 mRNA表达量、 dynactin p62基因甲基化水平以及刚羽化雌性蜜蜂的初生重、 体长、 前翅长、 前翅宽、 吻长、 第3背板长等形态指标。结果表明: 人工注射Dnmt3 siRNA不仅可以显著降低意大利蜜蜂幼虫头部Dnmt3酶活性、 Dnmt3 mRNA表达量和dynactin p62基因整体甲基化水平, 同时显著提高了刚羽化雌性蜜蜂的初生重、 体长、 第3背板长。结果说明Dnmt3的改变可以影响雌性意蜂幼虫的发育。     

Physiological significance of selective degradation of p62 by autophagy

Autophagy is a highly conserved bulk protein degradation pathway responsible for the turnover of long-lived proteins, disposal of damaged organelles, and clearance of aggregate-prone proteins. Thus, inactivation of autophagy results in cytoplasmic protein inclusions, which are composed of misfolded proteins and excess accumulation of deformed organelles, leading to liver injury, diabetes, myopathy, and neurodegeneration. Although autophagy has been considered non-selective, growing lines of evidence indicate the selectivity of autophagy in sorting vacuolar enzymes and in the removal of aggregate-prone proteins, unwanted organelles and microbes. Such selectivity by autophagy enables diverse cellular regulations, similar to the ubiquitin-proteasome pathway. In this review, we introduce the selective turnover of the ubiquitin- and LC3-binding protein ‘p62’ through autophagy and discuss its physiological significance.     

Celastrol targets proteostasis and acts synergistically with a heat-shock protein 90 inhibitor to kill human glioblastoma cells

Glioblastoma multiforme is a devastating disease of the central nervous system and, at present, no effective therapeutic interventions have been identified. Celastrol, a natural occurring triterpene, exhibits potent anti-tumor activity against gliomas in xenograft mouse models. In this study, we describe the cell death mechanism employed by celastrol and identify secondary targets for effective combination therapy against glioblastoma cell survival. In contrast to the previously proposed reactive oxygen species (ROS)-dependent mechanism, cell death in human glioblastoma cells is shown here to be mediated by alternate signal transduction pathways involving, but not fully dependent on, poly(ADP-ribose) polymerase-1 and caspase-3. Our studies indicate that celastrol promotes proteotoxic stress, supported by two feedback mechanisms: (i) impairment of protein quality control as revealed by accumulation of polyubiquitinated aggregates and the canonical autophagy substrate, p62, and (ii) the induction of heat-shock proteins, HSP72 and HSP90. The Michael adduct of celastrol and N-acetylcysteine, 6-N-acetylcysteinyldihydrocelastrol, had no effect on p62, nor on HSP72 expression, confirming a thiol-dependent mechanism. Restriction of protein folding stress with cycloheximide was protective, while combination with autophagy inhibitors did not sensitize cells to celastrol-mediated cytotoxicity. Collectively, these findings imply that celastrol targets proteostasis by disrupting sulfyhydryl homeostasis, independently of ROS, in human glioblastoma cells. This study further emphasizes that targeting proteotoxic stress responses by inhibiting HSP90 with 17-N-Allylamino-17-demethoxygeldanamycin sensitizes human glioblastoma to celastrol treatment, thereby serving as a novel synergism to overcome drug resistance.