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Yi-pin Lv Qian Wang# Chun-chen Wu Rong-juan Pei Yuan Zhou Yun Wang Xin-wen Chen 《Virologica Sinica》2011,(4)
Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 2... 相似文献
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对棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV)基因组中EcoR I-N片段进行序列分析,获得了完整的解螺旋酶基因(hel),其开放阅读框大小为3762bp,编码一个分子量为146kD的蛋白质。在hel起始密码子ATG上游50位有强晚期启动子转录起始信号ATAAG,在-112位和-189位存在两个TATA box,但未发现早期转录信号CAGT。其在终止密码子下游第12位有一PolyA终止信号AATAAA。在其它真核或原解螺旋酶中存在的7个保守基元(Ⅰ、Ⅰa、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ),只有5个(Ⅰ、Ⅰa、Ⅱ、Ⅲ、Ⅳ)在杆状病毒中保守。同源性比较发现,HaSNPV解螺旋酶的氨基酸序列与甜菜夜蛾核多角体病毒(Spodoptera exigus MNPV,SeMNPV)的解螺旋酶具有最高的同源性(66%),与Xestia c-nigrum颗粒体病毒(XcGV)解螺旋酶的同源性最低(43%)。HaSNPV解螺旋酶基因是第一个报道的单粒包埋核多角体病毒的解螺旋酶基因。 相似文献
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棉铃虫核型多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HearNPV)orf86是一个功能未知的基因。从HearNPV基因组中通过PCR得到orf86基因编码序列,将其构建于原核表达载体pGEX-4T-2,转化大肠杆菌BL21获得融合表达产物。融合表达蛋白经分离纯化后免疫新西兰大白兔,制备其多克隆抗体。用ELISA测定结果表明,ORF86多克隆抗体效价为1:5.12×105。利用该抗体Western印迹检测HearNPV感染的HZAM1细胞,检测到一个36kD的目的蛋白条带,与ORF86预期大小一致。结果表明该多抗可用于对orf86编码蛋白的检测和相关功能研究。 相似文献
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成纤维细胞生长因子8(fibroblast growth factor 8,FGF8)是成纤维细胞生长因子(FGFs)家族的成员之一。其在人胚胎时期多种组织内进行表达,对各种器官的形成中起着重要的作用。在正常成人体内,FGF8的表达水平受到严格的限制,然而在某些癌细胞或炎症部位中大量表达,特别是在激素类癌症的发生和发展中起着重要的作用。因此应用FGF8抗体治疗激素类癌症,为临床提供了新的治疗途径。 相似文献
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Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry. 相似文献
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Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry. 相似文献
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中国棉铃虫核型多角体病毒几丁质酶基因的定位与克隆 总被引:10,自引:2,他引:8
以α32PdATP标记含CfMNPV几丁质酶基因的重组质粒为探针,在68℃条件下对棉铃虫单粒包埋核型多角体病毒(HaSNPV)进行Southern杂交,将HaSNPV的几丁质酶基因分别定位在BamHIE、BglⅡE、EcoRIG、HindⅢF、XbaIH、BamHI+HindIIM和BamHI+XbaIH,并以pTZ19R为载体获得了XbaIH片段克隆。 相似文献
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成纤维细胞生长因子6(fibroblast growth factor 6,FGF6)是成纤维细胞生长因子家族(FGFs)的成员之一,主要通过与酪氨酸激酶受体(fibroblast growth factor receptor,FGFR)1和4结合发挥其生物学活性。研究发现,人FGF6几乎都积聚在肌源性细胞系中,参与肌源性细胞系的增殖及分化,在肌肉修复和再生过程中起重要作用,同时它还是一个重要的调节骨生成和骨重建的因子;FGF6在心脏中也有表达,进一步试验结果表明其具有促进心肌细胞增殖及保护心肌细胞凋亡的作用;在成体睾丸和乳腺癌中也检测到有FGF6的转录物,表明其在肿瘤发生发展中的作用。目前,FGF6在多种疾病中的功能和相关机制仍有待进一步研究和确认,但其所具备的生物学活性尤其是在肌肉再生方面具有重要的意义和巨大的应用潜力。 相似文献
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Cristina Ferreras Graham Rushton Claire L. Cole Muhammad Babur Brian A. Telfer Toin H. van Kuppevelt John M. Gardiner Kaye J. Williams Gordon C. Jayson Egle Avizienyte 《The Journal of biological chemistry》2012,287(43):36132-36146
Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor 165 (VEGF165) are potent pro-angiogenic growth factors that play a pivotal role in tumor angiogenesis. The activity of these growth factors is regulated by heparan sulfate (HS), which is essential for the formation of FGF2/FGF receptor (FGFR) and VEGF165/VEGF receptor signaling complexes. However, the structural characteristics of HS that determine activation or inhibition of such complexes are only partially defined. Here we show that ovarian tumor endothelium displays high levels of HS sequences that harbor glucosamine 6-O-sulfates when compared with normal ovarian vasculature where these sequences are also detected in perivascular area. Reduced HS 6-O-sulfotransferase 1 (HS6ST-1) or 6-O-sulfotransferase 2 (HS6ST-2) expression in endothelial cells impacts upon the prevalence of HS 6-O-sulfate moieties in HS sequences, which consist of repeating short, highly sulfated S domains interspersed by transitional N-acetylated/N-sulfated domains. 1–40% reduction in 6-O-sulfates significantly compromises FGF2- and VEGF165-induced endothelial cell sprouting and tube formation in vitro and FGF2-dependent angiogenesis in vivo. Moreover, HS on wild-type neighboring endothelial or smooth muscle cells fails to restore endothelial cell sprouting and tube formation. The affinity of FGF2 for HS with reduced 6-O-sulfation is preserved, although FGFR1 activation is inhibited correlating with reduced receptor internalization. These data show that 6-O-sulfate moieties in endothelial HS are of major importance in regulating FGF2- and VEGF165-dependent endothelial cell functions in vitro and in vivo and highlight HS6ST-1 and HS6ST-2 as potential targets of novel antiangiogenic agents. 相似文献
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Julia Yu Fong Chang Cong Wang Junchen Liu Yanqing Huang Chengliu Jin Chaofeng Yang Bo Hai Fei Liu Rena N. D'Souza Wallace L. McKeehan Fen Wang 《The Journal of biological chemistry》2013,288(40):28952-28961
A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated. 相似文献
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戊型肝炎病毒基因 ORF3编码蛋白在大肠杆菌中的表达及鉴定 总被引:3,自引:0,他引:3
实验以两种不同的表达策略构建了两个以大肠杆菌DE3为宿主的原核表达载体,由T7启动子启动外源基因的转录,在诱导剂IPTG诱导下成功地进行了戊肝病毒ORF3蛋白的原核表达。并通过SDS-聚丙烯酰胺凝胶电泳、免疫印迹、竞争抑制法酶联免疫等一系列实验对两种表达产物进行了鉴定和分析。综合分析两种表达结果发现,在融合型表达中ORF3蛋白与其融合标签蛋白(谷胱甘肽S一转移酶)之间存在免疫交叉反应,而且这种融合标签蛋白在空间结构上可能对ORF3蛋白中的抗体结合位点有掩盖作用。 相似文献
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成纤维细胞生长因子8b(fibroblast growth factor, FGF8b)在生长因子中与内分泌癌的发生和发展相关联,是一个有潜在临床应用价值的候选分子,为了满足重组人FGF8b的研发需要,论文采用分子克隆的方法构建了以包涵体形式表达FGF8b的重组大肠杆菌,并初步摸索出包涵体蛋白的纯化和复性工艺条件,通过Western blot和MTT法鉴定了FGF8b的生物化学特征和促增殖活性,表明利用包涵体复性技术初步得到了具有活性的蛋白,为后续研发进程奠定了基础。 相似文献
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Yanqing Huang Tomoaki Hamana Junchen Liu Cong Wang Lei An Pan You Julia Y. F. Chang Jianming Xu Chengliu Jin Zhongying Zhang Wallace L. McKeehan Fen Wang 《The Journal of biological chemistry》2015,290(29):17753-17761
Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, which include basal, luminal, and neuroendocrine cells. Two types of P-SCs have been identified in both human and mouse adult prostates based on prostasphere or organoid cultures, cell lineage tracing, renal capsule implantation, and expression of luminal- and basal-specific proteins. The sphere-forming P-SCs are from the basal cell compartment that express P63, and are therefore designated as basal P-SCs (P-bSCs). Luminal P-SCs (P-lSCs) express luminal cytokeratins and Nkx3.1. Herein, we report that the type 2 FGF receptor (FGFR2) signaling axis is crucial for preserving stemness and preventing differentiation of P-bSCs. FGFR2 signaling mediated by FGFR substrate 2α (FRS2α) is indispensable for formation and maintenance of prostaspheres derived from P63+ P-bSCs. Ablation of Fgfr2 in P63+ cells in vitro causes the disintegration of prostaspheres. Ablation of Fgfr2 in vivo reduces the number of P63-expressing basal cells and enriches luminal cells. This suggests a basal stem cell-to-luminal cell differentiation. In addition, ablation of Fgfr2 in P63+ cells causes defective postnatal development of the prostate. Therefore, the data indicate that FGFR2 signaling is critical for preserving stemness and preventing differentiation of P-bSCs. 相似文献
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Elizabeth W. Bradley Lomeli R. Carpio Alexandra C. Newton Jennifer J. Westendorf 《The Journal of biological chemistry》2015,290(26):16272-16280
Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1−/− chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair. 相似文献
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Jihun Lee 《Journal of molecular biology》2009,393(1):128-763
The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in ∼ 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine. 相似文献
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The molecular mechanism underlying unconventional secretion of Fibroblast Growth Factor 2 from tumour cells
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Julia P. Steringer Walter Nickel 《Biology of the cell / under the auspices of the European Cell Biology Organization》2017,109(11):375-380
Fibroblast Growth Factor 2 (FGF2) is a potent cell survival factor involved in tumour‐induced angiogenesis. FGF2 is secreted from cells through an unconventional secretory mechanism based upon direct translocation across the plasma membrane. The molecular mechanism underlying this process depends on a surprisingly small set of trans‐acting factors that are physically associated with the plasma membrane. FGF2 membrane translocation is mediated by the ability of FGF2 to oligomerise and to insert into the plasma membrane in a PI(4,5)P2‐dependent manner. Membrane‐inserted FGF2 oligomers are dynamic translocation intermediates that are disassembled at the extracellular leaflet mediated by membrane proximal heparan sulphate proteoglycans. This process results in the exposure of FGF2 on cell surfaces as part of its unconventional mechanism of secretion. Although the trans‐acting factors and cis‐elements in FGF2 required for unconventional secretion have been known for a while, the core mechanism of this mysterious process has now been reconstituted with purified components establishing the molecular basis of FGF2 secretion from tumour cells. 相似文献