转化生长因子β1对乳鼠心肌细胞转化生长因子结合蛋白2表达的影响

 目的：探讨转化生长因子β1（TGF-β1）在诱导心肌细胞表达转化生长因子结合蛋白2（LTBP2）中的作用及信号传导通路。 方法：培养乳鼠心肌细胞;实时定量聚合酶链式反应（Real-time PCR）、蛋白质印迹和免疫细胞化学方法检测不同时间和不同浓度的TGF-β1对大鼠乳鼠心肌细胞LTBP2基因及蛋白表达的影响;用TGF-β1相关信号通路阻断剂探讨TGF-β1调节LTBP2表达改变的信号传导机制。 结果：LTBP2基因表达随着TGF-β1浓度增加（0、2、5、10 ng/mL）而明显升高,在5 ng/mL时刺激最强（P < 0.05）;5 ng/mL的TGF-β1刺激下心肌细胞内LTBP2基因和蛋白表达的升高呈时间依赖性,均在12 h最高,24 h开始呈下降趋势（P < 0.05或P<0.01）;免疫细胞化学结果显示TGF-β1明显升高LTBP2的表达。信号传导通路研究显示TGF-β1在心肌细胞内主要通过ERK信号通路和PI3K信号通路诱导LTBP2的表达。 结论：TGF-β1在乳鼠心肌细胞内通过ERK信号通路和PI3K信号通路上调LTBP2的表达。     

大肠癌中胰岛素样生长因子2基因的印迹状态和表达

目的：研究胰岛素样生长因子2（insulin-like growth factor 2,IGF2)基因的印迹状态和表达与大肠癌的关系，为研究大肠癌的发生机理提供线索。方法：用逆转录－聚合酶链反应半定量检测IGF2的表达量，比较其在大肠癌及癌旁组织中有无差异，用限制性片段长度多态检测IGF2的印迹状态，分析印迹状态、表达量与大肠癌的关系。结果：82．4％（28／34）大肠癌有IGF2的表达增加，IGF2的表达量在肿瘤组织与癌旁组织中差异有显著性（P＜0．01，t=3.01)。IGF2在87．5％（14／16）大肠癌组织中发生了印迹丢失，但其相对应的癌旁组织也有71．4％（10／14）存在IGF2的印迹丢失。结论：IGF2的表达增加是大肠癌发生的相关因素，IGF2的印迹丢失可能是大肠癌发生的前期表现。     

Characterization of the nuclear localization signal of high risk HPV16 E2 protein

The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an alpha helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.     

Phenotypic effects of HPV-16 E2 protein expression in human keratinocytes

Expression of the HPV E2 open reading frame in cervical cancer cells has been shown to affect the expression of both viral and cellular genes. We have examined the phenotypic effects of the expression of human papillomavirus 16 E2 open reading frame in the human keratinocyte cell line HaCaT. Increased levels of apoptotic cell death were seen within 24 h of the transfection of HPV-16 E2 expression constructs. However, in those cells which survived selection and retained the intact E2 ORF, long-term stable expression of E2, as detected by RT-PCR, produced cells which developed phenotypes typical of terminally differentiated cells. These included characteristic morphological changes and expression of involucrin, filaggrin and senescence markers. This provides the first evidence of a role for E2 in stimulation of the normal epithelial differentiation programme, which would promote the progression of the HPV life cycle.     

猪瘟病毒E2蛋白重复多表位基因的融合表达及其兔体免疫保护研究

目的：研究猪瘟病毒(CSFV)E2蛋白重复多抗原表位基因的融合表达及其免疫攻毒保护作用。方法：应用PCR方法扩增猪瘟病毒E2蛋白重复多抗原表位基因，构建重复多表位基因的原核重组表达质粒PGEX-3E，进行融合蛋白的表达和纯化。ELISA和Western blot方法测定单表位融合蛋白GST-E和多表位融合蛋白GST-3E与猪抗CSFV的阳性血清和兔抗E2阳性血清的反应性，并进行融合蛋白的兔体免疫及免疫攻毒保护的比较研究。结果：分子克隆和构建了原核重组表达质粒pGEX-3E，表达和纯化了融合蛋白GST-3E；单表位融合蛋白与重复多表位融合蛋白均能够与猪抗CSFV的阳性血清反应和兔抗E2的阳性血清产生免疫反应。在刺激兔体产生抗体方面，单表位融合蛋白刺激兔体产生抗体的能力较弱，而多表位融合蛋白则能够使兔体产生高效价的抗体。接种100MID50剂量猪瘟兔化弱毒(HCLV)进行的兔体免疫攻毒保护试验表明，空白对照组和载体蛋白GST免疫组无保护作用，单表位融合蛋白免疫组具有一定的免疫保护作用，而重复多表位融合蛋白免疫组则完全能够抵抗猪瘟病毒的攻击。结论：猪瘟病毒E2蛋白重复多表位的融合表达具有免疫保护作用，本实验的成功完成为猪瘟病毒多表位抗原的串联及多表位疫苗的研究奠定了基础。     

人巨细胞病毒感染对体外培养成纤维细胞E2F1表达的时序性影响

目的 研究人巨细胞病毒（human cytomegalovirus,HCMV）感染对体外培养成纤维细胞E2F1表达的时序性影响,进一步揭示HCMV的致病机制.方法 用高和低感染复数（multiplicity of infection,MOI）HCMV病毒感染体外培养的成纤维细胞,在不同时间点用Western blot法检测细胞E2F1蛋白表达水平.结果 低MOI和高MOI感染都可时序性上调宿主细胞E2F1蛋白表达,均以感染后2h上调幅度最高（2～3倍）.高MOI感染的上调作用强于低MOI感染.结论 HCMV在感染初期即可激发宿主细胞E2F1表达明显增加,并在24h内呈时序性上调,提示E2F1在病毒感染早期诱导宿主细胞向S期和G2/M期偏移,进而在营造有利于病毒复制微环境的机制中起关键作用.     

Bone morphogenetic protein is required for fibroblast growth factor 2-dependent later-stage osteoblastic differentiation in cranial suture cells

Background: Understanding the pathophysiological process of calvarial bones development is important for the treatments on relative diseases such as craniosynostosis. While, the role of fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) and how they interacted in osteoblast differentiation remain unclear. Methods: we digested bone fragments around the coronal and sagittal sutures from newborn rats to harvest suture cells. Markers expression at different osteoblast differentiation stage was analyzed by increasing FGF2 concentration and BMP2 blocking in these cells. Results: BMP2 expression could be stimulated by FGF2 in a dose and time dependent manner. FGF2 stimulation may decrease early marker of osteoblast differentiation (collagen type-1, COL-1) and increase the expression of continuously-expressed or late markers (alkaline phosphatase, ALP; osteocalcin, OC and bone sialoprotein, BSP) to accelerate mineralization. Inhibition of BMP2 signaling by Noggin weakens the effect of FGF2 on induction of later-stage osteoblastic differentiation of cranial suture cells. Conclusion: Our data suggest that BMP2 signaling is required for FGF2-dependent induction of later-stage of cranial suture cell osteoblastic differentiation.     

The Drosophila CG1674 gene encodes a synaptopodin 2-like related protein that localizes to the Z-disc and is required for normal flight muscle development and function

Background

To identify novel myofibrillar components of the Drosophila flight muscles, we carried out a proteomic analysis of chemically demembranated flight muscle myofibrils, and characterized the knockdown phenotype of a novel gene identified in the screen, CG1674.

Results

The CG1674 protein has some similarity to vertebrate synaptopodin 2-like, and when expressed as a FLAG-tagged fusion protein, it was localized during development to the Z-disc and cytoplasm. Knockdown of CG1674 expression affected the function of multiple muscle types, and defective flight in adults was accompanied by large actin-rich structures in the flight muscles that resembled overgrown Z-discs. Localization of CG1674 to the Z-disc depended predominantly upon presence of the Z-disc component alpha-actinin, but also depended upon other Z-disc components, including Mask, Zasp52, and Sals. We also observed re-localization of FLAG-CG1674 to the nucleus in Alpha-actinin and sals knockdown animals.

Conclusions

These studies identify and characterize a previously unreported myofibrillar component of Drosophila muscle that is necessary for proper myofibril assembly during development.

     

Hepatocyte growth factor suppresses tumor cell apoptosis in nasopharyngeal carcinoma by upregulating Bcl-2 protein expression

Hepatocyte growth factor (HGF) is a multifunctional cytokine, but cell apoptosis related to HGF in nasopharyngeal carcinoma (NPC) and the potential mechanisms involved have not yet been identified. In this study, we aimed at determining whether HGF is a potent inhibitor of cell apoptosis in NPC, and tried to find out which antiapoptotic or proapoptotic protein is involved in this process.     

Phylogenetic diversity of GB virus C at the antigenic site of E2 protein

Amino acids at position 267–298 in E2 protein of GB virus C (GBV-C) were recognized as the antigenic site, and peptides within the region were previously reported to have inhibitory effect on HIV entry. The effect of sequence variability between different types of GBV-C on the antigenic region of the E2 protein was studied by using phylogenetic analysis. Eighty-one unique sequences encompassing this region derived from all seven GBV-C genotypes were compared to each other in this study. The results showed that GBV-C E2 antigenic nucleotide sites belonging to genotype 3 clustered together regardless of synonymous or nonsynonmous sites in the region, whereas, GBV-C E2 antigenic nucleotide sites belonging to the other 6 genotypes clustered together regardless of genotypes. Despite the fact that GBV-C genotypes might confer different degree of ‘protection’ against HIV, the lack of clustering as a unique group based on the amino acid differences in the antigenic site among the six genotypes suggested some other genomic regions or secondary structure of E2 protein might have played a crucial role in determining the variable protection effect of GBV-C on HIV infection.     

Destabilization of Rb by human papillomavirus E7 is cell cycle dependent: E2-25K is involved in the proteolysis

The HPV oncoprotein E7 promotes proteasomal degradation of the tumor suppressor protein Rb. In this study, we analyzed the regulation of E7-induced Rb proteolysis in HPV-containing Caski cervical cancer cells. We show that the Rb proteolysis is cell cycle dependent; in S phase Rb is stable while in post-mitotic early G1 phase cells and in differentiated cells, Rb is unstable. Similarly, the in vivo Rb/E7 interaction is not detected in S-phase cells, but is readily detected in differentiating Caski cells. The ubiquitinating enzymes involved in Rb proteolysis have not been identified. We find that the E3 ligase MDM2 is not involved in the Rb proteolysis in Caski cells. An in vivo analysis using multiple catalytic site mutant dominant negative E2 enzymes show that the C92A E2-25K most effectively blocks E7-induced Rb proteolysis. Taken together, these results show that E7 induces Rb proteolysis in growth-arrested cells and E2-25K is involved in the proteolysis.     

Impaired proteolysis by SPPL2a causes CD74 fragment accumulation that can be recognized by anti-CD74 autoantibodies in human ankylosing spondylitis

Ankylosing spondylitis (AS) is associated with autoantibody production to class II MHC-associated invariant chain peptide, CD74/CLIP. In this study, we considered that anti-CD74/CLIP autoantibodies present in sera from AS might recognize CD74 degradation products that accumulate upon deficiency of the enzyme signal peptide peptidase-like 2A (SPPL2a). We analyzed monocytes from healthy controls (n = 42), psoriatic arthritis (n = 25), rheumatoid arthritis (n = 16), and AS patients (n = 15) for SPPL2a enzyme activity and complemented the experiments using SPPL2a-sufficient and -deficient THP-1 cells. We found defects in SPPL2a function and CD74 processing in a subset of AS patients, which culminated in CD74 and HLA class II display at the cell surface. These findings were verified in SPPL2a-deficient THP-1 cells, which showed expedited expression of MHC class II, total CD74 and CD74 N-terminal degradation products at the plasma membrane upon receipt of an inflammatory trigger. Furthermore, we observed that IgG anti-CD74/CLIP autoantibodies recognize CD74 N-terminal degradation products that accumulate upon SPPL2a defect. In conclusion, reduced activity of SPPL2a protease in monocytes from AS predisposes to endosomal accumulation of CD74 and CD74 N-terminal fragments, which, upon IFN-γ-exposure, is deposited at the plasma membrane and can be recognized by anti-CD74/CLIP autoantibodies.     

Graphene nanoribbons elicit cell specific uptake and delivery via activation of epidermal growth factor receptor enhanced by human papillomavirus E5 protein

Ligands such as peptides, antibodies or other epitopes bind and activate specific cell receptors, and are employed for targeted cellular delivery of pharmaceuticals such as drugs, genes and imaging agents. Herein, we show that oxidized graphene nanoribbons, non-covalently functionalized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethyleneglycol)]) (O-GNR-PEG-DSPE) activate epidermal growth factor receptors (EGFRs). This activation generates a predominantly dynamin-dependent macropinocytosis-like response, and results in significant O-GNR-PEG-DSPE uptake into cells with high EGFR expression. Cells with an integrated human papillomavirus (HPV) genome also show increased uptake due to the modulation of the activated EGFR by the viral protein E5. We demonstrate that this cell specific uptake of O-GNR-PEG-DSPE can be exploited to achieve significantly enhanced drug efficacies even in drug resistant cells. These results have implications for the development of active targeting and delivery agents without ligand functionalization for use in the diagnosis and treatment of pathologies that overexpress EGFR or mediated by HPV.     

猪链球菌2型四川人源分离株ZYH24细胞外蛋白因子的原核表达及活性分析

目的：克隆、表达猪链球菌2型四川人源分离株ZYH24细胞外蛋白因子基因片段，分析蛋白活性。方法：根据GenBank S.suis 2 epf基因序列设计引物，克隆ZYH24株epf基因片段并进行序列分析；构建原核表达质粒pGEX4T-2-epf，在大肠杆菌中诱导带有谷胱苷肽转移酶（GST）标签的融合蛋白EF-GST的表达；亲和层析法纯化融合蛋白EF-GST，用凝血酶切除重组蛋白中的GST，获得纯化的EF抗原；SDS-PAGE和Western blot分析诱导表达及纯化的重组蛋白。结果：序列分析表明，获得的epf基因片段长895bp；原核表达的融合蛋白EF-GST分子量约62000，凝血酶处理后的EF抗原分子量约35000，两者均可与制备的EF多克隆抗血清发生特异性反应。结论：成功克隆了人源分离株ZYH24 epf基因片段，在原核系统实现高效的功能性表达，为开展EF蛋白的相关研究奠定了基础。