细粒棘球绦虫EgAgB8/1重组抗原和EgAgB8/1-EgAgB8/2重组嵌合抗原的表达及血清学诊断价值的比较

目的诱导表达并纯化细粒棘球绦虫EgAgB8/1重组抗原和EgAgB8/1-EgAgB8/2重组嵌合抗原,比较两个重组蛋白对囊型包虫病(CE)的血清学诊断价值。方法 IPTG诱导转染至E.coliBL21(DE3)LysS的pET32a-EgAgB8/1和pET32a-EgAgB8/1-EgAgB8/2重组原核表达质粒,表达和纯化EgAgB8/1重组蛋白和EgAgB8/1-EgAgB8/2重组嵌合蛋白,用SDS-PAGE电泳分析鉴定重组蛋白,并通过超声裂解法进行纯化,以CE病人及囊虫病人血清为一抗,Western blot法检测两个重组蛋白免疫反应性。结果细粒棘球绦虫重组抗原pET32a-EgAgB8/1和pET32a-EgAgB8/1-EgAgB8/2重组原核表达质粒得到成功诱导表达,经SDS-PAGE电泳分析,重组蛋白分子质量单位为28 ku和38 ku;Western blot结果表明,EgAgB8/1重组蛋白和EgAgB8/1-EgAgB8/2重组嵌合蛋白均能被CE病人血清特异性识别,16份CE病人血清中,以EgAgB8/1重组蛋白为抗原时11份阳性,以EgAgB8/1-EgAgB8/2重组蛋白为抗原时...     

布鲁氏菌TIR结构域蛋白TcpB的研究进展

布鲁氏菌病是由布鲁氏杆菌感染引起的一种人兽共患病,其感染的重要特征是机体的免疫功能受到抑制。布鲁氏菌TIR结构域蛋白TcpB具有多种免疫抑制功能,在逃避宿主免疫应答中具有重要作用。谨就布鲁氏菌TcpB的基本结构、免疫调节功能和分子机制的研究进展综述,以便理解TIR结构域蛋白TcpB在布鲁氏菌致病中介导的免疫抑制机制,有助于更好理解布鲁氏菌的致病机理,为诊治布鲁氏菌病提供新思路。     

恶性疟原虫FCC-1/HN株环子孢子蛋白基因片段的亚克隆及表达

目的 构建恶性疟原虫FCC－1／HN株CSP基因的重组真核表达质粒pBK－CSP,在大肠杆菌中进行表达,并进行鉴定。方法 采用限制性内切酶法从重组的大肠杆菌－分枝杆菌穿梭质粒pBCG5.6/CSP中分离出经过测序鉴定的CSP基因片段,将其亚克隆于pBK－CMV真核表达载体,构建重组真核表达质粒pBK-CSP.经IPTG诱导,重组质粒在大肠杆菌DH5α中进行表达,并进行SDS－PAGE及免疫印迹分析。结果 从pBCG5.6/CSP中分离出SP基因片段,成功构建pBK-CSP重组质粒;SDS－PAGE及免疫印迹分析结果显示特异性蛋白条带的相对分子质量约为42000。结论 从pBCG5．6／CSP中成功分离出CSP基因片段,并成功构建pBK-CSP重组质粒,诱导表达CSP非融合蛋白,为恶性疟原虫DNA疫苗的研制奠定了基础。     

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.     

Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1 and p53 expression in gastric cancer. METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed. RESULTS: TP53INP1 was expressed in 98% (139/142 cases) of non-cancerous gastric tissues and was down-expressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%). Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3% vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients). P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues. A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed, loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P=0.0006). CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1 is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may reflect the malignant grade of gastric cancer and is regarded as an adverse prognostic factor.     

The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.     

Lipoprotein receptor associated protein (LRPAP1) insertion/deletion polymorphism: association with gallbladder cancer susceptibility

Background Low-density lipoprotein receptor-related protein associated protein (LRPAP1) insertion/deletion polymorphism influences cholesterol homeostasis and may confer risk for gallstone disease and gallbladder carcinoma (GBC) incidence usually parallels with the prevalence of cholelithiosis. Aim We aimed to examine the role of LRPAP1 polymorphism in susceptibility to GBC. Methods Present case control study included 129 proven GBC patients, 183 gallstone patients, and 208 healthy controls. Genotyping was done by polymerase chain reaction–restriction fragment length polymorphism method. Results The D allele of LRPAP1 was significantly higher in GBC patients as compared to gallstone patients (p = 0.013; OR = 1.6, 95% CI = 1.1–2.4). However, II genotype and I allele was associated with reduced risk of GBC as compared to gallstone patients (p = 0.002; OR = 0.1, 95% CI = 0.1–0.6; p = 0.013; OR = 0.6, 95% CI = 0.4–0.8) The increased risk due to D allele was limited to female GBC patients (p = 0.021; OR = 1.8, 95% CI = 1.1–3.0). However, reduced risk due to II genotype and I allele was observed which was also confined to female GBC patients (p = 0.005; OR = 0.1, 95% CI = 0.1–0.6; p = 0.021; OR = 0.5, 95% CI = 0.3–0.8). On comparing GBC patients having gallstone with gallstone patients, high risk was observed in the GBC patients having gallstone due to the presence of D allele (p = 0.032; OR = 1.7, 95% CI = 1.0–2.8). However, low risk was observed because of I allele in GBC patients with gallstone in comparison to gallstone patients (p = 0.032, OR = 0.6, 95% CI = 0.4–0.9). Conclusion It appears that ‘D’ allele may modulate the susceptibility of GBC, and the risk is independent to genetic risk of gallstone.     

Mitochondrial phosphoglycerate mutase 5 uses alternate catalytic activity as a protein serine/threonine phosphatase to activate ASK1

Phosphoglycerate mutase (PGAM) is an enzyme of intermediary metabolism that converts 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. Here, we discovered PGAM5 that is anchored in the mitochondrial membrane lacks PGAM activity and instead associates with the MAP kinase kinase kinase ASK1 and acts as a specific protein Ser/Thr phosphatase that activates ASK1 by dephosphorylation of inhibitory sites. Mutation of an active site His-105 in PGAM5 abolished phosphatase activity with ASK1 and phospho-Thr peptides as substrates. The Drosophila and Caenorhabditis elegans orthologs of PGAM5 also exhibit specific Ser/Thr phosphatase activity and activate the corresponding Drosophila and C. elegans ASK1 kinases. PGAM5 is unrelated to the other known Ser/Thr phosphatases of the PPP, MPP, and FCP families, and our results suggest that this member of the PGAM family has crossed over from small molecules to protein substrates and been adapted to serve as a specialized activator of ASK1.     

Y-box binding protein 1 augments sorafenib resistance via the PI3K/Akt signaling pathway in hepatocellular carcinoma

BACKGROUNDSorafenib is the first-line treatment for patients with advanced hepatocellular carcinoma (HCC). Y-box binding protein 1 (YB-1) is closely correlated with tumors and drug resistance. However, the relationship between YB-1 and sorafenib resistance and the underlying mechanism in HCC remain unknown.AIMTo explore the role and related mechanisms of YB-1 in mediating sorafenib resistance in HCC.METHODSThe protein expression levels of YB-1 were assessed in human HCC tissues and adjacent nontumor tissues. Next, we constructed YB-1 overexpression and knockdown hepatocarcinoma cell lines with lentiviruses and stimulated these cell lines with different concentrations of sorafenib. Then, we detected the proliferation and apoptosis in these cells by terminal deoxynucleotidyl transferase dUTP nick end labeling, flow cytometry and Western blotting assays. We also constructed a xenograft tumor model to explore the effect of YB-1 on the efficacy of sorafenib in vivo. Moreover, we studied and verified the specific molecular mechanism of YB-1 mediating sorafenib resistance in hepatoma cells by digital gene expression sequencing (DGE-seq).RESULTSYB-1 protein levels were found to be higher in HCC tissues than in corresponding nontumor tissues. YB-1 suppressed the effect of sorafenib on cell proliferation and apoptosis. Consistently, the efficacy of sorafenib in vivo was enhanced after YB-1 was knocked down. Furthermore, KEGG pathway enrichment analysis of DGE-seq demonstrated that the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was essential for the sorafenib resistance induced by YB-1. Subsequently, YB-1 interacted with two key proteins of the PI3K/Akt signaling pathway (Akt1 and PIK3R1) as shown by searching the BioGRID and HitPredict websites. Finally, YB-1 suppressed the inactivation of the PI3K/Akt signaling pathway induced by sorafenib, and the blockade of the PI3K/Akt signaling pathway by LY294002 mitigated YB-1-induced sorafenib resistance.CONCLUSIONOverall, we concluded that YB-1 augments sorafenib resistance through the PI3K/Akt signaling pathway in HCC and suggest that YB-1 is a key drug resistance-related gene, which is of great significance for the application of sorafenib in advanced-stage HCC.     

Melatonin inhibits human fibroblast-like synoviocyte proliferation via extracellular signal-regulated protein kinase/P21CIP1/P27KIP1 pathways

Abstract:  The excessive proliferation and migration of synoviocytes are well-characterized phenomena that play key roles in the pathophysiology of rheumatoid arthritis (RA). Melatonin has been shown to have potent anti-proliferative effect in various cancer cells such as breast and prostate cancer cells. In this study, we examined the role of melatonin on synoviocyte proliferation in primary cultured human fibroblast-like synoviocytes (FLSs) by analyzing protein expression of P21^CIP1 (P21) and P27^KIP1 (P27), the cyclin-dependent kinase inhibitors that are important in cell cycle control, and the phosphorylation of mitogen-activated protein kinases (MAPKs). RA-FLS proliferation was determined by a [³H]-thymidine incorporation assay. Western blot analysis was applied to examine the underlying mechanisms of melatonin's effect. Melatonin inhibited RA-FLS proliferation in a dose-dependent manner. It reduced proliferation of passage 2 FLSs by 25% at 10  μ m and by nearly 40% at 100  μ m concentrations. The inhibitory effect of melatonin on RA-FLS proliferation was also observed in passages 4 and 6. Melatonin upregulated the expression levels of P21 and P27 dose-dependently (24 hr), induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) time-dependently (10  μ m ), but did not affect phosphorylation of P38 in RA-FLSs. In addition, the expression of P21 and P27 triggered by melatonin was inhibited by the pretreatment of the ERK inhibitor, PD98059 (10  μ m ). The anti-proliferative action of melatonin in RA-FLSs was also blocked by PD98059. Taken together, these results suggest that melatonin exerts the inhibitory effect of the proliferation of RA-FLSs through the activation of P21 and P27 mediated by ERK. Hence we suggest that melatonin could be used as a therapeutic agent for the treatment of RA.     

食管癌组织中CtBP1、p16蛋白的表达变化

目的观察食管癌组织中C端结合蛋白（CtBP）1和p16蛋白的表达变化。方法采用免疫组化法检测57例食管癌组织（观察组）及30例癌旁组织（对照组）中的CtBP1和p16蛋白,并分析其与食管癌临床病理参数的关系。结果观察组51例、对照组29例CtBP1蛋白表达阳性,两组CtBP1蛋白阳性表达率相比P〉0.05。观察组30例、对照组24例p16蛋白表达阳性,两组p16蛋白阳性表达率相比P〈0.05。食管癌组织中p16表达与临床分期、淋巴结转移有关（P均〈0.05）,与病理分型无关（P〉0.05）;CtBP1表达与临床分期、淋巴结转移、病理分型均无关（P均〉0.05）。结论食管癌组织中p16蛋白表达下调,CtBP1蛋白表达无明显变化。     

pTWIN1-EgAgB8/3自剪切融合蛋白原核表达载体的构建及表达

目的 表达获得较纯的细粒棘球绦虫AgB8/3重组抗原(rEgAgB8/3)。方法 根据GeneBank登陆号(AF362442)下载目的基因核酸序列,利用DNAman软件设计引物,对EgAgB8/3编码分泌型多肽片段的核酸序列进行PCR扩增,测序鉴定其正确性后定向连入原核表达质粒pTWIN1上,并转化至大肠杆菌ER2566,IPTG诱导表达CBD-intein1-EgAgB8/3融合蛋白后进行纯化,用SDS-PAGE电泳和western blot分析鉴定融合蛋白与目的蛋白rEgAgB8/3的表达量和纯度。结果 成功克隆获得EgAgB8/3基因目的片段和具有蛋白自剪切功能的重组表达载体pTWIN1-EgAgB8/3,并鉴定其表达的融合蛋白主要以可溶形式存在;构建的重组载体中融合标签的几丁质结合域(CBD)和内含肽(intein1)分别使亲和层析纯化和融合标签自剪切一步完成。结论 成功的构建重组表达载体pTWIN1-EgAgB8/3,获得高表达融合蛋白CBD-intein1-EgAgB8/3,并经简单后续处理即可获得含极少任何额外氨基酸的可溶性目的蛋白rEgAgB8/3,尽可能保证了其原有的结构和活性,为抗rEgAgB8/3特异性单克隆抗体的快速制备奠定了基础。     

中国人甘露糖结合蛋白基因第1外显子核苷酸序列的测定

目的：为了阐明中国人甘露糖结合蛋白（MBP）的基因突变对健康的影响和疾病的关系,须测定中国人MBP基因的第1外显子基因序列。方法：应用PCR产物直接经310型全自动遗传分析仪进行DNA序列分析。结果：测定了中国人甘露糖结合蛋白基因第1外显子核苷酸序列,检测到密码子54例的纯合和杂合的点突变,未检测到密码子52例和57例的突变。结论我们检测出中国人MBP基因第1外显子的核苷酸序列,密码子54基因的突变。     

脂联素通过LKB1途径激活腺苷酸活化蛋白激酶

目的 探讨脂联素是否通过LKB1途径激活骨骼肌及肝脏中腺苷酸活化蛋白激酶(AMPK).方法 将28只6周龄雄性Sprague-Dawley大鼠分为普通饮食组(NC组,n=15)和高脂饮食组(HF组,n=13).喂养16 周后,取空腹静脉血测定血清游离脂肪酸(FFA)、甘油三酯(TG)、总胆固醇(TC)、空腹血糖(FPG)、空腹胰岛素(FINS)及脂联素.采用Western印迹法测定各组大鼠骨骼肌及肝脏组织中AMPKα、磷酸化的AMPKcα和LKB1蛋白的表达.将原代培养的骨骼肌细胞及肝细胞分别予以脂联素和根赤壳菌素干预,免疫荧光技术测定各组细胞中AMPKα、磷酸化AMPKα和LKB1蛋白的表达.结果 与NC组比较,HF组大鼠体重、FFA、TG、FPG、FINS均升高(均P＜0.05),脂联素水平降低(P＜0.05).骨骼肌及肝组织巾AMPKα磷酸化和LKB1蛋白表达水平降低(均P＜0.05).原代培养大鼠骨骼肌细胞及肝细胞中脂联素显著增加AMPKα磷酸化及LKB1表达水平(均P＜0.05).加入根赤壳菌素表达明显降低(均P＜0.05).结论 脂联素在大鼠骨骼肌和肝脏组织可能通过LKB1途径激活AMPK.     

弓形虫棒状体蛋白2和膜表面蛋白1重组复性后体外免疫反应性研究

目的分析基因工程生产的弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)的体外免疫反应性,并对表达产物进行复性处理以获得表达产物的天然构象,为体内免疫学活性的研究作准备。方法重组质粒pET28b/ROP2-P30转化大肠杆菌BL21-Codon Plus(DE3)-RIL菌株,经IPTG诱导的表达产物超声破壁后,SDS-PAGE分析表达产物的表达形式,对产生的重组蛋白ROP2-P30过Sephadex G-25交联葡聚糖柱,经尿素梯度洗脱进行目的蛋白的复性,通过免疫共沉淀反应及免疫印迹实验检测其复性后的重组蛋白的免疫反应性。结果重组质粒pET28b/ROP2-P30在大肠杆菌中以融合形式表达,融合表达的产物主要以包涵体形式存在,该重组蛋白复性后具有明显的免疫反应性。结论利用SephadexG-25交联葡聚糖柱尿素梯度可以复性重组的弓形虫复合蛋白ROP2-P30,该蛋白复性后体外具有免疫反应性,可用于进一步开展弓形虫病复合型疫苗的研制工作。