Pruning and loss of excitatory synapses by the parkin ubiquitin ligase

Mutations in the PARK2 gene cause hereditary Parkinson disease (PD). The PARK2 gene product, termed parkin, is an E3 ubiquitin ligase that mediates the transfer of ubiquitin onto diverse substrate proteins. Despite progress in defining the molecular properties and substrates of parkin, little is known about its physiological function. Here, we show that parkin regulates the function and stability of excitatory glutamatergic synapses. Postsynaptic expression of parkin dampens excitatory synaptic transmission and causes a marked loss of excitatory synapses onto hippocampal neurons. Conversely, knockdown of endogenous parkin or expression of PD-linked parkin mutants profoundly enhances synaptic efficacy and triggers a proliferation of glutamatergic synapses. This proliferation is associated with increased vulnerability to synaptic excitotoxicity. Thus, parkin negatively regulates the number and strength of excitatory synapses. Increased excitatory drive produced by disruption of parkin may contribute to the pathophysiology of PD.     

过氧化氢对人视网膜色素上皮细胞ARPE-19中20S蛋白酶体表达的上调作用

目的:探讨过氧化氢诱导的氧化应激对人视网膜色素上皮细胞ARPE-19中20S蛋白酶体表达的影响。方法:分别选择0,100,300μmol/L过氧化氢作用ARPE-19细胞4h后收集样本,Western blot方法检测细胞中20S蛋白酶体表达水平,细胞免疫荧光方法检测20S蛋白酶体在细胞中的分布。结果:100和300μmol/L过氧化氢溶液处理的细胞中20S蛋白酶体的表达和对照组相比明显增加(100μmol/L过氧化氢处理组vs0和300μmol/L过氧化氢处理组,P<0.05),并呈现剂量依赖性。正常培养情况下,细胞内20S蛋白酶体在核周微量表达,100和300μmol/L过氧化氢溶液处理的细胞内20S蛋白酶体和对照组相比明显增加,其分布集中在核内及核膜周围。结论:过氧化氢对ARPE-19的20S蛋白酶体表达有上调作用。     

Therapeutic Targets for Hypoxia-Elicited Pathways

Diminished oxygen supply to tissues (hypoxia) can stem from many sources, and is a contributing factor to diverse disease processes. Cell and tissue responses to hypoxia are diverse and include dramatic changes in metabolic demand, regulation of cellular gene products, and release of lipid and protein mediators. Surprisingly little attention has been paid to targeted development of therapeutics for hypoxia-related disease processes. This review will focus on recent advances in cellular and molecular biology pertaining to the hypoxia response, and will discuss paradigms used to study hypoxia and the potential targets for therapeutic intervention.     

Characterization of the proteasomeβ2 subunit gene and its mutant allele in the tephritid fruit fly pest, Anastrepha suspensa

In Drosophila melanogaster the β2 proteasome subunit gene, Prosβ2 , was first identified as a dominant temperature sensitive mutant, DTS-7, that causes pupal lethality at 29 °C but allows survival to adulthood at 25 °C. To explore the use of proteasome mutations for a conditional lethal system in insect pests, we identified and isolated the β2 subunit gene of the 20S proteasome from the Caribbean fruit fly, Anastrepha suspensa. The caribfly ortholog AsProsβ2 was isolated from pupal cDNA by 5' and 3' RACE. The AsProsβ2 protein has high amino acid sequence similarity to predicted insect Prosβ2 subunits and homologs from yeast and mammals, and it contains the well conserved amino acids that confer catalytic activity and substrate specificity. AsProsβ2 is a single copy gene and its RNA accumulates throughout all developmental stages of the caribfly. For functional studies a point mutation, analogous to the Prosβ2¹ mutation in D. melanogaster , was introduced into AsProsβ2 to create an aberrant protein with a Gly170Arg substitution. Consistent with the DTS-7 mutation, transgenic insects carrying the mutant allele undergo normal metamorphosis at the permissive temperature (25 °C) but at the non-permissive temperature (29 °C) they exhibit effective pupal lethality. This is the first report of a functional characterization of a Prosβ2 cognate based on the creation of a dominant temperature-sensitive mutation. This type of temperature-dependent lethality could be used for biological control, where transgenic insects are reared to adulthood at 25 °C or lower and then released into the field where ambient temperatures averaging 29 °C or greater cause lethality in their progeny.     

泛素羧基末端水解酶-1抑制剂对多巴胺能神经元的神经毒性作用

目的 利用人神经母细胞瘤SK-N-SH细胞观察泛素羧基末端水解酶-1(OCH-L1)抑制剂对多巴胺能神经元的毒性作用并探讨其可能的毒性机制. 方法 用不同浓度(5、10、25、50、75、100 μmol/L)的UCH-L1抑制剂作用SK-N-SH细胞24h,MTT法检测细胞活力、Hoechst染色检测凋亡的细胞核及Western blot检测UCH-L1蛋白、单个泛素分子及多聚化泛素蛋白的表达、荧光检测泛素蛋白酶体系统(UPS)的功能. 结果经UCH-L1抑制剂处理24 h后SK-N-SH细胞突起样结构消失,细胞体积变小、形态变圆;随着UCH-L1抑制剂浓度的增加,细胞活性进一步下降;与对照组比较,细胞活力在抑制剂浓度为50μmol/L时.作用24h后即出现明显下降,差异有统计学意义(P<0.05);Hoechst染色可见凋亡细胞碎裂的细胞核;Western blot检测到细胞内UCH-L1蛋白表达没有变化、单个泛素分子水平下降、多聚泛素化蛋白增加;荧光检测显示UPS功能下降.结论 UCH-L1抑制剂在体外对多巴胺能神经元有毒性作用,可诱导细胞凋亡.在凋亡过程中,UPS功能下降、细胞内多聚泛素化蛋白堆积可能发挥了作用.     

蛋白酶体抑制剂硼替佐米联合柔红霉素对HL-60细胞的凋亡作用和Survivin mRNA表达的影响

目的：研究蛋白酶体抑制剂硼替佐米（Bor）单用或联合柔红霉素（DNR）对HL-60细胞凋亡和Survivin mRNA表达的影响。方法：将不同浓度Bor,DNR及两药联合组和对照组作用于HL-60细胞,采用MTT法测定细胞增殖活性,RT—PCR检测Survivin mRNA表达。结果：5nmol／L Bor作用24h对HL-60细胞有增殖抑制作用,并诱导其凋亡。随着浓度增加及其作用时间的延长可使细胞凋亡率明显增加,10nmol／LBor与1．0μmol／L DNR联合作用72h细胞凋亡率最高,与同剂量两药单独作用相比差异有显著性（P〈0．01）。RT—PCR检测结果表明：随着药物浓度的增加,Survivin mRNA的表达逐渐下降,10mmol／L Bor与1．0μmol／L DNR联合作用72h Survivin mRNA表达最低。结论：Bor能显著抑制HL-60细胞的增殖并诱导其凋亡,Bor联合DNR对HL-60细胞有协同作用,并能显著下调Survivin基因的表达,这可能是促使HL-60细胞凋亡的机制之一。     

Bortezomib/docetaxel combination therapy in patients with anthracycline-pretreated advanced/metastatic breast cancer: a phase I/II dose-escalation study

The aim of this study was to determine the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of bortezomib plus docetaxel in patients with anthracycline-pretreated advanced/metastatic breast cancer. Forty-eight patients received up to eight 21-day cycles of docetaxel (60-100 mg m(-2) on day 1) plus bortezomib (1.0-1.5 mg m(-2) on days 1, 4, 8, and 11). Pharmacodynamic and pharmacokinetic analyses were performed in a subset of patients. Five patients experienced DLTs: grade 3 bone pain (n=1) and febrile neutropenia (n=4). The MTD was bortezomib 1.5 mg m(-2) plus docetaxel 75 mg m(-2). All 48 patients were assessable for safety and efficacy. The most common adverse events were diarrhoea, nausea, alopecia, asthenia, and vomiting. The most common grade 3/4 toxicities were neutropenia (44%), and febrile neutropenia and diarrhoea (each 19%). Overall patient response rate was 29%. Median time to progression was 5.4 months. In patients with confirmed response, median time to response was 1.3 months and median duration of response was 3.2 months. At the MTD, response rate was 38%. Pharmacokinetic characteristics of bortezomib/docetaxel were comparable with single-agent data. Addition of docetaxel appeared not to affect bortezomib inhibition of 20S proteasome activity. Mean alpha-1 acid glycoprotein concentrations increased from baseline at nearly all time points across different bortezomib dose levels. Bortezomib plus docetaxel is an active combination for anthracycline-pretreated advanced/metastatic breast cancer. The safety profile is manageable and consistent with the side effects of the individual agents.