Tumor necrosis factor-α accelerates apoptosis of steatotic hepatocytes from a murine model of non-alcoholic fatty liver disease

Non-alcoholic steatohepatitis (NASH) develops in a subset of patients with non-alcoholic fatty liver disease (NAFLD), but the exact mechanisms involved in the progression of NAFLD to NASH remain poorly understood. We investigated the role of tumor necrosis factor-α (TNF-α) in the apoptosis of hepatocytes that is related to the severity of NASH. We separated primary hepatocytes from the NAFLD liver caused by a high-fat diet. The production of intracellular reactive oxygen species was increased in steatotic hepatocytes, which were also sensitive to TNF-α. This factor induced significant apoptosis through the signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) pathway. We describe here a novel culture model of steatotic hepatocytes separated from the NAFLD liver, and demonstrate that TNF-α induces their apoptosis in vitro.     

CD11b表达与人工髋关节置换术后无菌性松动的相关性分析

目的:分析髋关节周围滑膜组织CD11b表达水平与人工髋关节置换术后无菌性松动的相关性。方法:以2006年5月至2016年5月于西京医院接受人工髋关节置换的患者为研究对象,对其髋关节周围滑膜组织进行CD11b免疫组化染色,并随访术后5年和10年无菌性松动的发生情况,通过单因素分析及logistic回归分析讨论CD11b表达与无菌性松动之间的相关性。结果:共300例患者纳入研究,全部获得随访,CD11b表达阳性患者163例,阳性率为54.33%;术后5年松动患者29例,术后5年无菌性松动发生率为9.67%;术后10年无菌性松动患者49例,术后10年无菌性松动发生率为16.33%;单因素分析结果表明CD11b表达阳性患者5年及10年松动率均高于CD11b表达阴性患者(P0.05);logistic回归分析结果表明CD11b过表达是髋关节置换术后无菌性松动发生的危险因素。结论:髋关节周围滑膜组织CD11b过表达是人工髋关节置换术后无菌性松动发生的危险因素,该分子或可作为无菌性松动的辅助诊断指标及潜在治疗靶点。     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37 ℃下在pH8～10的缓冲液中保温1 h酶活几乎不改变。重组酶反应的最适温度为75 ℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的K_m^KG为7.559mmol/L,V_max^KG为0.086mmol/(L·min),K_m^Asp为2.031mmol/L,V_max^Asp为0.024mmol/(L·min)。Ca²⁺、Fe³⁺、Mn²⁺等金属离子对酶活性有微弱抑制作用。     

A NPxY-independent beta5 integrin activation signal regulates phagocytosis of apoptotic cells

Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the β chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 (β5) integrin cDNA was expressed in αv positive, β5 and β3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, β5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing β5 mutant (Y750A) abrogated adhesion and β5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of β5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a β5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 μm diameter microspheres developed as apoptotic cell mimetics. The critical sequences in β5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of β5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the β5 cytoplasmic tail for adhesion and phagocytosis.     

新城疫病毒F蛋白在昆虫细胞中的表达及其融细胞作用

用RT-PCR方法扩增出新城疫病毒标准强毒株F48E8的F基因,并将其克隆到pGEM-T载体,命名为pGEM-NDF.鉴定正确后,以BamHI和XbaI双酶切将F基因从pGEM-NDF中释放出来,并插入到pFast Bac I载体中,得到重组转移载体pFast-NDF.然后将该重组质粒转入含有穿梭质粒的感受态DH10Bac中,通过转座作用获得重组穿梭质粒reBacmid-NDF.再用reBacmid-NDF转染Sf9昆虫细胞,获得含有新城疫病毒F48E8株F基因的重组杆状病毒.间接免疫荧光和Western-blot分析结果表明F蛋白在昆虫细胞中获得表达,而且主要表达于细胞膜上,并使感染重组杆状病毒的昆虫细胞在96h发生融合作用.动物试验表明,表达的F蛋白能够产生中和抗体.本文的研究结果为F蛋白的进一步开发奠定了基础.     

Reciprocal influence of connexins and apical junction proteins on their expressions and functions

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another.     

慢性胃炎肠化生CD_(44)、CD_(44V6)及Cyclin D_1、Cyclin E的表达和意义

目的探讨慢性胃炎胃粘膜肠化生CD44、CD44V6及cyclin D1、Cyclin E表达的意义。方法利用免疫细胞化学技术对39例伴有肠化生的慢性胃炎和5例正常人胃窦粘膜的活检组织进行检查。结果正常人胃窦粘膜上皮,腺上皮对CD44、CD44V6、Cyclin D1和Cyclin E均为阴性,但在有神经内分泌样细胞的粘膜,CD44V6和cyclin D1为阳性。慢性胃炎肠化生区和不典型增生区除CD44为阴性外,CD44V6、cyclin D1和cyclin E均呈现不同的阳性反应,但未见有阳性的神经内分泌样细胞。间质细胞大都呈阳性反应。结论CD44V6、cyclin D1和cyclin E可能是胃癌前状态的早期事件,而CD44可能为胃癌晚期的标志物。     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.