HEF1 promotes epithelial mesenchymal transition and bone invasion in prostate cancer under the regulation of microRNA‐145

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bones, and it's crucial to understand the mechanism of tumor progression to metastasis in order to develop therapies that may reduce the morbidity and mortality of PCa patients. Although we had identified that microRNA(miR)‐145 could repress bone metastasis of PCa via regulating epithelial–mesenchymal transition (EMT) in previous study, it is still unknown how miR‐145 regulated EMT. In the present study, we constructed a luciferase reporter system and identified HEF1 as a direct target of miR‐145. More importantly, HEF1 was shown to promote migration, invasion and EMT of PC‐3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. And HEF1 was also shown to partially mediate miR‐145 suppression of EMT and invasion. Furthermore, inhibition of HEF1 repressed bone invasion of PC‐3 cells in vivo. Expression of HEF1 was negatively correlated with miR‐145 in primary PCa and bone metastatic specimens, but HEF1 was higher in samples which were more likely to commit to bone metastasis or those with higher free prostate‐specific antigen (fPSA) levels and Gleason scores. Taken together, these findings indicate that HEF1 promotes EMT and bone invasion in prostate cancer by directly targeted by miR‐145, and miR‐145 suppresses EMT and invasion, at least in part, through repressing HEF1. J. Cell. Biochem. 114: 1606–1615, 2013. © 2013 Wiley Periodicals, Inc.     

Neotyphodium endophyte strain and superoxide dismutase activity in perennial ryegrass plants under water deficit

The presence of Neotyphodium endophyte in forage grass tillers has been associated with increased tolerance of abiotic stresses. The effect of four endophyte treatments (plants with three different strains of Neotyphodium lolii compared with plants without endophyte) on superoxide dismutase (SOD) (EC1.15.1.1) activity in Lolium perenne cv ‘Grasslands Samson’ was measured under high and low dehydration regimes in a glasshouse experiment. SOD activity was assayed by a microplate method utilising the inhibition of reduction of a tetrazolium dye by superoxide radicals. A progressive increase in dehydration over 2 weeks reduced shoot fresh weight, dry weight and shoot water content for high, compared with low, moisture-stressed plants. Mean shoot fresh weight was significantly lower for plants with strain AR37 endophyte than for plants with strain AR1 endophyte, wild-type or endophyte-free plants, but there was no interaction between endophyte treatment and dehydration treatment. There were no differences in mean SOD activity between the dehydration treatments, and the four endophyte treatments at any of the harvests. All harvest mean SOD levels for plants in both stress groups, however, were significantly different from the preceding harvest value. Between the first and second week of moisture stress there was a significant endophyte by harvest interaction for mean percentage change in SOD activity when activity in plants with wild-type strain endophyte increased more rapidly than in AR1, AR37 or endophyte-free plants. The results are in agreement with earlier reports suggesting that Neotyphodium endophytes do not have major effects on the water stress physiology of perennial ryegrass, although water deficits applied were not extreme.     

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

The NEDD8 protein and neddylation levels in cells are modulated by NUB1L or NUB1 through proteasomal degradation, but the underlying molecular mechanism is not well understood. Here, we report that NUB1L down-regulated the protein levels of NEDD8 and neddylation through specifically recognizing NEDD8 and P97/VCP. NUB1L directly interacted with NEDD8, but not with ubiquitin, on the key residue Asn-51 of NEDD8 and with P97/VCP on its positively charged VCP binding motif. In coordination with the P97-UFD1-NPL4 complex (P97^UFD1/NPL4), NUB1L promotes transfer of NEDD8 to proteasome for degradation. This mechanism is also exemplified by the canonical neddylation of cullin 1 for SCF (SKP1-cullin1-F-box) ubiquitin E3 ligases that is exquisitely regulated by the turnover of NEDD8.     

Lithium Down-regulates Histone Deacetylase 1 (HDAC1) and Induces Degradation of Mutant Huntingtin

Lithium is an effective mood stabilizer that has been clinically used to treat bipolar disorder for several decades. Recent studies have suggested that lithium possesses robust neuroprotective and anti-tumor properties. Thus far, a large number of lithium targets have been discovered. Here, we report for the first time that HDAC1 is a target of lithium. Lithium significantly down-regulated HDAC1 at the translational level by targeting HDAC1 mRNA. We also showed that depletion of HDAC1 is essential for the neuroprotective effects of lithium and for the lithium-mediated degradation of mutant huntingtin through the autophagic pathway. Our studies explain the multiple functions of lithium and reveal a novel mechanism for the function of lithium in neurodegeneration.     

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.     

荒漠破碎化生境中长爪沙鼠集合种群野外验证研究

近年来,人类活动和自然干扰,导致内蒙古阿拉善荒漠区生境的破碎化,出现了长爪沙鼠在不同斑块间的不连续分布,每一斑块内可能存在一个局域种群,而集合种群建立的前提条件,是局域种群斑块状分布在离散的栖息地环境中。2002~2012年每年的4~10月,在阿拉善荒漠区禁牧、轮牧、过牧和开垦4种人为不同利用方式形成的生境斑块中,采用标志重捕法对长爪沙鼠（Meriones unguiculatus）种群进行定点监测。通过分析长爪沙鼠种群动态,计算各局域种群的灭绝风险,利用Spearman秩相关系数检验种群动态的空间同步性,同时以种群周转率对长爪沙鼠扩散能力进行评估,以检验阿拉善荒漠区长爪沙鼠种群空间结构是否具有经典集合种群的功能。结果表明：（1） 不同生境斑块可被长爪沙鼠局域种群占据,11年间捕获长爪沙鼠2~7次不等;（2） 长爪沙鼠所有局域种群均具有灭绝风险,在轮牧区和禁牧区灭绝率高达1.000 0,开垦区灭绝率最低,也达到0.333 4,而本研究期间最大局域种群（2008年过牧区,26只/hm²）,在2010年发生了局域灭绝;（3） 不同生境斑块间没有明显的空间隔离而阻碍局域种群的重新建立,长爪沙鼠扩散能力较强,绝大部分月份的种群周转率在50.0%以上,特别是周转率达到100.0%的月份较多;（4） 不同生境斑块间仅轮牧区和禁牧区中长爪沙鼠种群密度显著正相关（P<0.05）,而其他生境斑块间相关性均不显著（P >0.05）,长爪沙鼠局域种群整体显示出明显的非同步空间动态。阿拉善荒漠区长爪沙鼠种群满足作为经典集合种群物种区域续存的4个条件,具有作为研究小哺乳动物集合种群的潜在价值。     

里氏木霉细胞表面表达系统的构建

[目的]烟曲霉(Aspergillus fumigatus)的AfMp1p是一种通过糖基磷脂酰肌醇( glycosylphosphatidylinositol,GPI)修饰定位于细胞壁上的蛋白,其细胞壁定位信号位于蛋白质的C末端.里氏木霉(Trichoderma reesei)是一种重要的工业生产菌种.构建里氏木霉的细胞表面表达系统具有十分重要的意义.[方法]我们将AfMp1p的细胞壁定位GPI信号肽和烟曲霉几丁质酶AfChiB1的N端信号肽分别与绿色荧光蛋白(green fluorescent protein,GFP)的C末端和N末端融合并转化里氏木霉.本文首先对木霉遗传转化系统进行了优化;随后通过Real-time PCR和蛋白定量,对GFP融合蛋白在里氏木霉中不同时期的表达情况进行了研究;最后对里氏木霉表达的GFP融合蛋白进行细胞定位研究.[结果]荧光观察结合Western blot的结果表明,在平台期中期和后期,带有GPI信号的GFP融合蛋白定位于细胞壁.[结论]烟曲霉来源的GPI信号可被里氏木霉识别,本论文所构建的表达系统可用于外源蛋白在里氏木霉中的细胞壁定位表达.