Store-dependent and -independent modes regulating Ca2+ release-activated Ca2+ channel activity of human Orai1 and Orai3

We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca(2+)-selective current with Ca(2+)-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca(2+) release-activated Ca(2+) (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca(2+) influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca(2+) entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.     

Combined effect of sodium dodecyl sulfate and enzyme on waste activated sludge hydrolysis and acidification

The combined effect of sodium dodecyl sulfate (SDS) and enzyme system on hydrolysis and acidification of waste activated sludge (WAS) was investigated. The results showed that the combined system was more effective in the promotion of sludge hydrolysis than sole SDS and sole enzyme, and the SDS + mixed-enzymes (ME) system had better hydrolysis performance than SDS + single enzyme system. Compared with SDS + protease and SDS + amylase systems, the soluble protein concentration in SDS + ME system increased respectively by 20.0% and 44.4%, and the soluble carbohydrate concentration increased by 78.3% and 37.0%, respectively. During the WAS acidification stage, the SDS, ME and SDS + ME system could make the maximum short-chain fatty acids (SCFAs) concentration increased by 1.82 (6th day), 2.04 (5th day), 2.32 (7th day) times, respectively. The composition analysis of SCFAs produced in SDS + ME system indicated that acetic acid was the most prevalent product and propionic acid was the second one.     

HIF-1α acts downstream of TNF-α to inhibit vasodilator-stimulated phosphoprotein expression and modulates the adhesion and proliferation of breast cancer cells

Metastasis is the leading cause of death in breast cancer patients. Recent evidence suggests that inflammation-related cytokine tumor necrosis factor-alpha (TNF-α) is implicated in tumor invasion and metastasis, but the mechanism of its involvement remains elusive. In this study, we employed MCF-7 breast cancer cells as an experimental model to demonstrate that TNF-α inhibits breast cancer cell adhesion and cell proliferation through hypoxia inducible factor-1alpha (HIF-1α) mediated suppression of vasodilator-stimulated phosphoprotein (VASP). We observed that TNF-α treatment attenuated the adhesion and proliferation of MCF-7 cells it also dramatically increased HIF-1α expression and decreased VASP expression. Through a variety of approaches, including promoter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified VASP as a direct target gene of HIF-1α. In addition, we confirmed that HIF-1α mediated the repression of VASP expression by TNF-α in MCF-7 cells. We also demonstrated that exogenous VASP expression or knockdown of HIF-1α relieved TNF-α induced inhibition of cell adhesion and proliferation. We identified a novel TNF-α/HIF-1α/VASP axis in which HIF-1α acts downstream of TNF-α to inhibit VASP expression and modulate the adhesion and proliferation of breast cancer cells. These data provide new insight into the potential anti-tumor effects of TNF-α.     

Mitochondrial tRNA mutations associated with deafness

Mitochondrial tRNA mutations are one of the important causes of both syndromic and non-syndromic deafness. Of those, syndromic deafness-associated tRNA mutations such as tRNA(Leu(UUR)) 3243A>G are often present in heteroplasmy, while non-syndromic deafness-associated tRNA mutations including tRNA(Ser(UCN)) 7445A>G often occur in homplasmy or in high levels of heteroplasmy. These tRNA mutations are the primary mutations leading to hearing loss. However, other tRNA mutations such as tRNA(Thr) 15927G>A and tRNA(Ser(UCN)) 7444G>A may act in synergy with the primary mitochondrial DNA mutations, modulating the phenotypic manifestation of the primary mitochondrial DNA mutations. Theses tRNA mutations cause structural and functional alteration. A failure in tRNA metabolism caused by these tRNA mutations impaired mitochondrial translation and respiration, thereby causing mitochondrial dysfunctions responsible for deafness. These data offer valuable information for the early diagnosis, management and treatment of maternally inherited deafness.     

听神经元的频率调谐

频率作为声音的一个重要参数,在听敏感神经元对声音进行分析和编码过程中扮演重要角色。一般用频率调谐曲线来表示听敏感神经元的频率调谐特性,并用Qn(10,30,50)值表达频率调谐曲线的尖锐程度,Qn值越大,频率调谐曲线也越尖锐,神经元的频率调谐能力越好,对频率的分辨能力越高。从听觉外周到中枢,听敏感神经元的频率调谐逐级锐化,而这种锐化主要是由听中枢的多种抑制性神经递质的作用而产生的,其中起主要作用的是GABA能和甘氨酸能神经递质。此外,离皮层调控,双侧下丘间的联合投射以及弱噪声前掩蔽等因素也会影响听敏感神经元的频率调谐特性。     

波摩那型钩端螺旋体外膜蛋白LipL32基因片段的克隆表达

目的　构建L3 2_pGEX_5x_2重组质粒 ,诱导表达重组钩端螺旋体外膜脂蛋白LipL3 2。方法　PCR获取编码LipL3 2的基因片段 ,构建重组克隆载体和表达载体 ,转化受体菌 ,诱导表达重组LipL3 2蛋白。将重组LipL3 2蛋白和钩体抗血清进行Western_blot。结果　扩增出约 750bp的LipL3 2成熟蛋白基因 ,LipL3 2基因插入pGEX_5x_2表达载体 ,表达产物谷胱甘肽S_转移酶 (GST ,2 6× 10 3)与LipL3 2蛋白的融合蛋白的相对分子质量约为 53× 10 3 ,与预期大小一致。Western_blot显示重组LipL3 2蛋白能与钩体抗血清特异结合。结论　LipL3 2蛋白能在大肠埃希菌中表达 ,重组LipL3 2蛋白具有免疫反应性     

PCR-DGGE技术解析东北稻田蓝藻群落结构

[目的]明确东北稻田生态系统蓝藻群落结构组成变化及主要驱动因子。[方法]采用蓝藻特异性引物GC-Cya-b-F371和Cya-R783对采自东北不同土壤类型稻田土壤及田面水提取的DNA进行PCR扩增,采用DGGE电泳和条带测序技术解析稻田蓝藻群落结构。[结果]多样性指数表明,土壤中蓝藻群落结构比田面水体丰富。图谱冗余分析(RDA)显示,稻田土壤中蓝藻群落结构具有明显的地域差异,土壤碳氮比值是驱动土壤蓝藻分布的主要环境因素;而田面水体蓝藻群落结构地域差异不明显,p H值是驱动水体蓝藻分布的主要环境因素。DGGE条带测序发现,稻田蓝藻群落结构组成以丝状颤藻亚纲蓝藻(Oscillatoriophycideae)为主,一些条带基因序列与已知蓝藻相似度低于97%。[结论]东北稻田生态系统蓝藻群落结构复杂多样,生存着一些未知的蓝藻种群。     

Oxidative stress and NF-κB signaling are involved in LPS induced pulmonary dysplasia in chick embryos

Inflammation or dysbacteriosis-derived lipopolysaccharides (LPS) adversely influence the embryonic development of respiratory system. However, the precise pathological mechanisms still remain to be elucidated. In this study, we demonstrated that LPS exposure caused lung maldevelopment in chick embryos, including higher embryo mortality, increased thickness of alveolar gas exchange zone, and accumulation of PAS⁺ immature pulmonary cells, accompanied with reduced expression of alveolar epithelial cell markers and lamellar body count. Upon LPS exposure, pulmonary cell proliferation was significantly altered and cell apoptosis was inhibited as well, indicating a delayed progress of pulmonary development. LPS treatment also resulted in reduced CAV-1 expression and up-regulation of Collagen I, suggesting increased lung fibrosis, which was verified by Masson staining. Moreover, LPS induced enhanced Nrf2 expression in E18 lungs, and the increased reactive oxygen species (ROS) production was confirmed in MLE-12 cells in vitro. Antioxidant vitamin C restored the LPS induced down-regulation of ABCA3, SP-C and GATA-6 in MLE-12 cells. Furthermore, LPS induced activation of NF-κB signaling in MLE-12 cells, and the LPS-induced decrease in SP-C expression was partially abrogated by blocking NF-κB signaling with Bay-11–7082. Bay-11–7082 also inhibited LPS-induced increases of ROS and Nrf2 expression. Taken together, we have demonstrated that oxidative stress and NF-κB signaling are involved in LPS induced disruption of pulmonary cell development in chick embryos.     

中药"神曲"对脾虚小鼠肠道菌群的调整及肠保护作用研究

目的研究中药"神曲"对脾虚小鼠肠道菌群的调整及肠保护作用.方法1.昆明小白鼠,分成4组:A组(正常对照组),B组(脾虚自然恢复组),C组(神曲治疗组),D组(丽珠肠乐治疗组).2.检测结肠内容物和粪便标本的肠杆菌、肠球菌、双歧杆菌、类杆菌和乳酸杆菌数量.3.结肠组织光镜和电镜观察.结果脾虚小鼠给予神曲水煎剂治疗后,肠杆菌、肠球菌、双歧杆菌、类杆菌和乳酸杆菌数量逐渐恢复正常,肠壁肌层厚度增加,杯状细胞数量增多,肠黏膜微绒毛排列紊乱、线粒体肿胀显著改善,明显好于自然恢复组,与丽珠肠乐治疗组相比差异无显著性.结论中药神曲对脾虚小鼠具有肠道菌群调整及促进损伤肠组织恢复的作用.