腾冲嗜热厌氧杆菌Cas2原核表达及生物信息学分析

为研究CRISPR/Cas系统及其相关蛋白Cas2(TTE2657)在腾冲嗜热厌氧杆菌热适应中的作用,应用PCR技术构建了原核重组质粒pET-28a::cas2,并在大肠埃希菌BL21表达Cas2蛋白;结合生物信息学软件对cas2编码蛋白的基本理化性质、氨基酸同源性、空间结构及蛋白质相互作用网络进行预测和分析。结果显示,成功构建了原核表达载体pET-28a::cas2并在大肠埃希菌BL21中得到表达,Cas2分子质量大小为9.9 ku,主要以可溶性形式存在;qRT-PCR显示cas2 mRNA在60℃和75℃高表达;生物信息学分析显示cas2基因其完整的ORF全长264 bp,编码88个氨基酸,其中Ile(14)、Ser(14)、Phe (12)含量较高,等电点为9.31,不存在跨膜结构。其蛋白质二级空间结构以α-螺旋、无规则卷曲、β-折叠为主,蛋白互作预测网络显示Cas2与Cas3、Cas5、Cas7等其家族大部分蛋白存在相互作用。进化树分析显示腾冲嗜热厌氧杆菌cas2基因与厌氧菌芽胞杆菌B7M1同源性最高(39.5%)。腾冲嗜热厌氧杆菌cas2编码蛋白是一种亲水性蛋白,在原核系统能高效表达。本研究为嗜热蛋白质的热稳定性机制的研究提供参考。     

转基因植物中外源基因的沉默及应对策略

随着转基因技术在作物育种领域的应用,转基因植物中外源基因表达量低的现象较为普遍。导致外源基因表达量低的主要原因是基因沉默。外源基因沉默可分为转录水平的基因沉默和转录后水平的基因沉默。如何应对基因沉默,提高外源基因的表达量,是转基因技术发展亟待解决的问题。     

MicroRNA-409-3p inhibits migration and invasion of bladder cancer cells via targeting c-Met

There is increasing evidence suggesting that dysregulation of certain microRNAs (miRNAs) may contribute to tumor progression and metastasis. Previous studies have shown that miR-409-3p is dysregulated in some malignancies, but its role in bladder cancer is still unknown. Here, we find that miR-409-3p is down-regulated in human bladder cancer tissues and cell lines. Enforced expression of miR-409-3p in bladder cancer cells significantly reduced their migration and invasion without affecting cell viability. Bioinformatics analysis identified the pro-metastatic gene c-Met as a potential miR-409-3p target. Further studies indicated that miR-409-3p suppressed the expression of c-Met by binding to its 3′-untranslated region. Silencing of c-Met by small interfering RNAs phenocopied the effects of miR-409-3p overexpression, whereas restoration of c-Met in bladder cancer cells bladder cancer cells overexpressing miR-409-3p, partially reversed the suppressive effects of miR-409-3p. We further showed that MMP2 and MMP9 may be downstream effector proteins of miR-409-3p. These findings indicate that miR-409-3p could be a potential tumor suppressor in bladder cancer.     

Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.     

Hydrogen-rich saline protects against intestinal ischemia/reperfusion injury in rats

Hydrogen gas was reported to reduce reactive oxygen species and alleviate cerebral, myocardial and hepatic ischemia/reperfusion (I/R) injuries. This paper studied the effect of hydrogen-rich saline, which was easier for clinical application, on the intestinal I/R injury. Model of intestinal I/R injury was induced in male Sprague-Dawley rats. Physiological saline, hydrogen-rich saline or nitrogen-rich saline (5 ml/kg) was administered via intravenous infusion at 10 min before reperfusion, respectively. The intestine damage was detected microscopically and was assessed by Chiu score system after I/R injury. In addition, serum DAO activity, TNF-α, IL-1β and IL-6 levels, tissue MDA, protein carbonyl and MPO activity were all increased significantly by I/R injury. Hydrogen-rich saline reduced these markers and relieved morphological intestinal injury, while no significant reduction was observed in the nitrogen-rich saline-treated animals. In conclusion, hydrogen-rich saline protected the small intestine against I/R injury, possibly by reduction of inflammation and oxidative stress.     

The MRN-CtIP Pathway Is Required for Metaphase Chromosome Alignment

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Highlights? The MRN-CtIP pathway is required for metaphase chromosome alignment in egg extracts ? MRN inhibition interferes with spindle assembly around DNA-coated beads ? MRN inhibition in cells causes a metaphase delay and disrupts the RanGTP gradient ? The MRN complex regulates the stable association of RCC1 to chromatin     

Inhibition by N′-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in α-ketoglutarate amination

The effect of N′-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the α-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K_i, estimated as 199?μmol?l^?1 at 25°C and pH 8.0.