Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP

Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1^KD cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1^KD cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1^KD cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1^KD cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell–cell fusion. Toca-1^KD cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASP^KO MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1^KD cells restored myotube formation of Toca-1^KD cells. Thus, our results suggest that Toca-1^KD cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.     

肺癌组织中岩藻糖化糖链结构免疫组化研究

Lewis X(Le~x)、唾液酸化的Lewis X(Sialyl Lewis X,SLe~x)和唾液酸化的双岩藻糖Lewis X(Sialyl Dimeric Lewis X,SDLe~x)是细胞表面外侧带α1,3岩藻糖的糖链结构。本文用免疫组化ABC法研究了肺癌原发灶、转移灶和癌旁组织中这三种抗原结构的表达。结果发现这三种抗原在肺癌细胞表面及胞浆中均有不同程度的表达,而在肺癌癌旁组织及正常肺组织中未见表达。有转移的肺癌和(或)低分化肺癌中这三种抗原结构的表达要明显高于未发生转移和高、中分化肺癌中相同抗原结构的表达。其中以SLe~x的表达与肺癌细胞的转移能力和分化程度关系最为密切。另外,肺癌浸润转移的淋巴结中也有Le~x、SLe~x的明显表达和SDLe~x的少量表达,而未被肺癌浸润转移的淋巴结中就没有它们的表达。     

刚毛柽柳S-腺苷甲硫氨酸合成酶(ThSAMS)基因的克隆及表达分析

通过对刚毛柽柳转录组分析,克隆获得了一条与S-腺苷甲硫氨酸合成酶(SAMS)基因同源性高的基因,命名为ThSAMS。序列分析结果表明:ThSASM基因全长cDNA为1185bp,编码394个氨基酸,编码蛋白相对分子质量为97.85kDa,理论等电点为5.02。通过生物信息学分析表明,ThSASM基因编码的氨基酸与其他物种SAMS基因编码的氨基酸具有很高的同源性,其中与枣的同源性最高,达95%。实时荧光定量PCR(quantitativereal-timePCR,qRT-PCR)分析表明,ThSASM表达受NaCl、聚乙二醇(PEG)和ABA处理做出应答,暗示ThSASM可能参与了刚毛柽柳对盐和干旱的胁迫应答,为进一步研究SAMS基因在植物胁迫应答中的功能及作用机制提供了参考依据。     

Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.     

2011年、2013年和2016年医院内获得性血流感染常见病原菌分布及其耐药性分析

动态监测2011年、2013年和2016年我国不同地区医院内获得性血流感染病原菌分布及耐药进展趋势。从全国10个城市回顾性收集血流感染病原菌非重复性株,采用琼脂稀释法或微量肉汤稀释法进行药物敏感性试验,采用Whonet 5.6软件对药敏试验结果进行分析。收集的2 248株血流感染病原菌中革兰阴性杆菌为1 657株 (占73.7%),革兰阳性球菌为591株 (占26.3%)。分离率排名前五的病原菌依次为大肠埃希菌 (32.6%,733株/2 248株)、肺炎克雷伯菌 (14.5%,327株/2 248株)、金黄色葡萄球菌 (10.0%,225株/2 248株)、鲍曼不动杆菌 (8.7%,196株/ 2 248株) 和铜绿假单胞菌 (6.2%,140株/2 248株)。血流感染分离的革兰阴性杆菌对抗菌药物体外敏感率较高的抗菌药物依次为粘菌素 (96.5%,1 525株/1 581株,不包括天然耐药菌株)、替加环素 (95.6%,1 375株/1 438株,不包括天然耐药菌株)、头孢他啶/克拉维酸 (89.2%,1 112株/1 246株)、阿米卡星 (86.4%,1 382株/1 599株) 和美罗培南 (85.7%,1 376株/1 605株);革兰阳性球菌对抗菌药物体外敏感率较高的抗菌药物依次为替加环素、替考拉宁和达托霉素 (敏感率均为100.0%)、万古霉素和利奈唑胺 (敏感率均为99.7%)。2011年、2013年和2016年产超广谱β-内酰胺酶肠杆菌科细菌分离率分别为50.6% (206株/407株)、49.8% (136株/273株) 和38.9% (167株/429株);碳青霉烯不敏感肠杆菌科细菌分离率分别为2.2% (9株/408株)、4.0% (16株/402株) 和3.9% (17株/ 439株);多重耐药鲍曼不动杆菌分离率分别为76.4% (55株/72株)、82.7% (43株/52株) 和87.5% (63株/72株),多重耐药铜绿假单胞菌分离率分别为9.8% (5株/51株)、20.0% (7株/35株) 和13.0% (7株/54株);甲氧西林耐药金黄色葡萄球菌的分离率分别为51.9% (41株/79株)、29.7% (19株/64株) 和31.7% (26株/82株)。屎肠球菌和粪肠球菌中高水平庆大霉素耐药株分离率分别为43.2% (48株/111株) 和40.9% (27株/66株)。碳青霉烯不敏感肠杆菌科细菌中肺炎克雷伯菌居首位,占57.1% (24株/42株) 。肠杆菌科细菌中分离出30株替加环素不敏感株,其中肺炎克雷伯菌占76.7% (23株/30株);分离出粘菌素耐药肠杆菌科细菌39株,其中大肠埃希菌、阴沟肠杆菌和肺炎克雷伯菌分别占43.6% (17株/39株)、35.9% (14株/39株) 和15.4% (6株/39株)。医院获得性血流感染病原菌主要为革兰阴性杆菌 (以大肠埃希菌和肺炎克雷伯菌为主),其对替加环素、粘菌素和碳青霉烯类药物的敏感率较高;革兰阳性球菌中分离率最高的为金黄色葡萄球菌,其次为屎肠球菌,这两种细菌对替加环素、达托霉素、利奈唑胺、万古霉素和替考拉宁的敏感率较高。粘菌素耐药肠杆菌科细菌、替加环素不敏感肠杆菌科细菌、利奈唑胺或万古霉素不敏感革兰阳性球菌的分离,警示临床高度关注,仍需动态监测耐药进展趋势。     

中国河南两株庚型肝炎病毒（HGV）NS5区部分cDNA的克隆与序列分析

通过逆转录－聚合酶链反应从我国河南省２例重叠感染ＨＣＶ或ＨＢＶ／ＨＤＶ的献血啼中,分离到ＨＢＶＮＳ５区的部分ｃＤＮＡ,对其进行序列分析比较,结果表明,河南株ＨＧＶＮＳ５工核苷酸与两中国ＨＧＶ主同源性高于国外代表株（８８．５－９０．６％）,但由核苷酸推导的氨基酸的同源性都无明显的地区性区别。ＨＧＶＮＳ５区氨基酸序列较保守,缺乏明显高变区,中国４株ＨＧＶ在７３８４位发生了由Ｃ→Ｔ的变异,从而导致一个人     

CDK5-Mediated Phosphorylation-Dependent Ubiquitination and Degradation of E3 Ubiquitin Ligases GP78 Accelerates Neuronal Death in Parkinson’s Disease

The molecular mechanisms responsible for the loss of dopaminergic neurons in Parkinson’s disease (PD) remain obscure. Loss of function of E3 ubiquitin ligases is associated with mitochondria dysfunction, dysfunction of protein degradation, and α-synuclein aggregation, which are major contributors to neurodegeneration in PD. Recent research has thus focused on E3 ubiquitin ligase glycoprotein 78 (GP78); however, the role of GP78 in PD pathogenesis remains unclear. Notably, cyclin-dependent kinase 5 (CDK5) controls multiple cellular events in postmitotic neurons, and CDK5 activity has been implicated in the pathogenesis of PD. Thus, we addressed the relationship between CDK5 and GP78 in MPTP-based PD models. We found that GP78 expression is decreased in MPTP-based cellular and animal PD models, and CDK5 directly phosphorylated GP78 at Ser516, which promoted the ubiquitination and degradation of GP78. Importantly, overexpression of GP78 or interference of GP78 Ser516 phosphorylation protected neurons against MPP⁺-induced cell death. Thus, our research reveals that the CDK5-GP78 pathway is involved in the pathogenesis of PD and could be a novel candidate drug target for the treatment of PD.     

禽致病性大肠杆菌毒力基因多重PCR方法的建立和应用

【目的】建立禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)黏附相关基因、侵袭及毒素相关基因、抗血清存活相关基因及铁转运相关基因的多重PCR方法,实现禽致病性大肠杆菌毒力基因的简便、快速检测。【方法】根据GenBank公布的基因序列,设计合成18对特异性引物,通过条件优化,建立四组多重PCR体系,并通过模板倍比稀释检测各组多重PCR的灵敏性。利用多重PCR检测100株APEC毒力基因的分布,验证多重PCR方法的可行性。【结果】根据PCR扩增片段大小判定,上述四组多重PCR体系均能同时扩增出该组中的各个毒力基因,且灵敏度分别为:103CFU、103CFU、105CFU、105CFU细菌和1ng、1ng、10ng、10ng DNA。100株APEC的毒力因子检测结果显示,多重PCR和单基因PCR结果一致。【结论】建立的四组多重PCR方法能够简便、快速地检测禽致病性大肠杆菌的毒力基因,可用于毒力基因的鉴定以及流行病学调查。     

艾滋病病毒蛋白酶高效表达载体及体外活性检测系统的构建

艾滋病是本世纪80年代初发现的一种烈性传染病,5年病死率为100％,致病因子为人免疫缺陷病毒,该病毒的蛋白酶在病毒复制和成熟中具有决定性的意义。由于目前国内外尚未获得艾滋病病毒蛋白酶的高效表达的重组子及活性检测系统,限制了它的研究与应用。本文利用PCR技术修饰了艾滋病病毒蛋白酶的基因,使其具有便于克隆及表达用的限制酶切位点及转录终止码,井在其C末端设置了一个可用于检验该酶活性的特殊序列。DNA序列分析揭示上述突变策略成功,将修饰后的艾滋病病毒蛋白酶基因克隆入大肠杆菌表达系统,并获得高效表达(>30％),Western-Bolt鉴定结果表明所表达的蛋白为艾滋病病毒所特有,并具有较好的生物活性。