Cyanide detoxification in an insect herbivore: Molecular identification of β-cyanoalanine synthases from Pieris rapae

Cyanogenic compounds occur widely in the plant kingdom. Therefore, many herbivores are adapted to the presence of these compounds in their diet by either avoiding cyanide release or by efficient cyanide detoxification mechanisms. The mechanisms of adaptation are not fully understood. Larvae of Pieris rapae (Lepidoptera: Pieridae) are specialist herbivores on glucosinolate-containing plants. They are exposed to cyanide during metabolism of phenylacetonitrile, a product of benzylglucosinolate breakdown catalyzed by plant myrosinases and larval nitrile-specifier protein (NSP) in the gut. Cyanide is metabolized to β-cyanoalanine and thiocyanate in the larvae. Here, we demonstrate that larvae of P. rapae possess β-cyanoalanine activity in their gut. We have identified three gut-expressed cDNAs designated PrBSAS1-PrBSAS3 which encode proteins with similarity to β-substituted alanine synthases (BSAS). Characterization of recombinant PrBSAS1-PrBSAS3 shows that they possess β-cyanoalanine activity. In phylogenetic trees, PrBSAS1-PrBSAS3, the first characterized insect BSAS, group together with a characterized mite β-cyanoalanine synthase and bacterial enzymes indicating a similar evolutionary history.     

Association of the Breast Cancer Antiestrogen Resistance Protein 1 (BCAR1) and BCAR3 Scaffolding Proteins in Cell Signaling and Antiestrogen Resistance

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3. BCAR1 and BCAR3 bind tightly to each other through their C-terminal domains, thus potentially connecting their associated signaling networks. However, recent studies using BCAR1 and BCAR3 interaction mutants concluded that association between the two proteins is not critical for many of their interrelated activities regulating breast cancer malignancy. We report that these previously used BCAR mutations fail to cause adequate loss-of-function of the complex. By using structure-based BCAR1 and BCAR3 mutants that lack the ability to interact, we show that BCAR3-induced antiestrogen resistance in MCF7 breast cancer cells critically depends on its ability to bind BCAR1. Interaction with BCAR3 increases the levels of phosphorylated BCAR1, ultimately potentiating BCAR1-dependent antiestrogen resistance. Furthermore, antiestrogen resistance in cells overexpressing BCAR1/BCAR3 correlates with increased ERK1/2 activity. Inhibiting ERK1/2 through overexpression of the regulatory protein PEA15 negates the resistance, revealing a key role for ERK1/2 in BCAR1/BCAR3-induced antiestrogen resistance. Reverse-phase protein array data show that PEA15 levels in invasive breast cancers correlate with patient survival, suggesting that PEA15 can override ERK1/2 activation by BCAR1/BCAR3 and other upstream regulators. We further uncovered that the BCAR3-related NSP3 can also promote antiestrogen resistance. Thus, strategies to disrupt BCAR1-BCAR3/NSP3 complexes and associated signaling networks could ultimately lead to new breast cancer therapies.     

针对NSP4基因的shRNA抑制牛轮状病毒的体外增殖

本研究根据牛轮状病毒(Bovine rotavirus,BRV)的NSP4基因序列,设计并构建了shRNA重组慢病毒载体RNAi-H1-89,利用脂质体介导法,将RNAi-H1-89与辅助质粒共转染293细胞,包装shRNA重组慢病毒;重组慢病毒感染MA104细胞24h后继续接种BRV,以针对LacZ基因的shRNA重组慢病毒为对照,48h后与LacZshR-NA对照比较,RNAi-H1-89明显抑制MA104细胞病变;RT-PCR检测结果表明,针对NSP4基因的shRNA可特异性抑制牛轮状病毒NSP4基因表达;病毒滴度测定表明,在转染50%、75%、95%RNAi-H1-89质粒的细胞培养上清液中,病毒滴度分别是对照组的50%、20%和10%。上述实验结果表明,针对NSP4基因的shRNA能高效地沉默NSP4基因,并且能阻断牛轮状病毒增殖。     

轮状病毒不同基因型NSP4的免疫原性研究

NSP4作为轮状病毒的致泻相关蛋白,其在疫苗研究中的作用近年来深受瞩目。为比较不同基因型非结构蛋白NSP4的免疫原性,我们构建了四种基因型的重组表达质粒pCI-97B6、pCI-97S36、pCI-97S34和pCI-97SZ8,在细胞水平进行瞬时表达检测后,进一步免疫小鼠,检测血清IgG抗体滴度和亚型。结果表明利用真核表达质粒表达的NSP4蛋白既可以诱导体液免疫反应,又可以诱导细胞免疫反应,但以体液免疫为主,不同基因型NSP4可具有不同的免疫原性。     

Structural Insights into the Association between BCAR3 and Cas Family Members, an Atypical Complex Implicated in Anti-Oestrogen Resistance

The association between novel Src homology 2-containing protein (NSP) and Crk-associated substrate (Cas) family members contributes to integrin and receptor tyrosine kinase signalling and is involved in conferring anti-oestrogen resistance to human breast carcinomas. The precise role of this association in tumorigenesis remains controversial, and the molecular basis for the complex NSP and Cas protein form is unknown. Here we present a pluridisciplinary approach, including small-angle X-ray scattering, that provides first insights into the structure of the complex formed between breast cancer anti-oestrogen resistance 3 (BCAR3, an NSP family member) and human enhancer of filamentation 1 (HEF1, also named NEDD9 or Cas-L, a Cas family protein). Our analysis corroborates a four-helix bundle structure for the NSP-binding domain of HEF1 and a Cdc25-like guanine nucleotide exchange factor (GEF) fold for the Cas-binding domain of BCAR3. Using residues located on helix 2 of the four-helix bundle, HEF1 binds very tightly to a site on BCAR3 that is remote from the putative guanosine triphosphatase binding site of the GEF domain, but similar to a site implicated in allosteric regulation of the homologous SOS (Son of Sevenless) GEF domain. Thus, the association between NSP and Cas proteins might not only create a very stable link between these molecules, co-localising their cellular functions, but also modulate the function of the NSP GEF domains. Such modulation may explain, at least in part, the controversial results published for NSP GEF function.     

A组轮状病毒NSP1基因克隆表达及免疫学性质研究

轮状病毒（rotavirus, RV）非结构蛋白1（non structural protein 1, NSP1）在病毒与宿主的相互作用中发挥着重要的功能。运用基因克隆和表达技术在大肠杆菌中表达了TB-Chen株RV NSP1蛋白,进行了NSP1的免疫学性质和RV感染细胞中NSP1蛋白的合成与分布以及NSP1的系统进化和基因分型研究。结果表明,大肠杆菌BL21(DE3)能高效表达重组NSP1蛋白（rNSP1）,rNSP1表达量约占菌体总蛋白的34.4%。rNSP1能诱导免疫豚鼠产生特异性血清抗体。Western blot及免疫荧光检测结果表明,抗rNSP1血清抗体能特异性识别自身蛋白,对SA11、Wa株的NSP1蛋白有交叉反应性;免疫荧光结果还表明,SA11感染的MA104细胞中合成的NSP1蛋白在细胞质中区域化聚集形成辐射状排列的颗粒状结构,而Wa株的NSP1不能形成此样结构。至今发现的A组RV至少可以分为16个不同的NSP1基因型,TB-Chen株NSP1为A2型。不同基因型有独特的敏感宿主范围,同一基因型可能感染不同种动物,同一种动物也可能感染不同基因型。基因型A4型和A16型仅在鸟类病毒株中出现;而且鸟类中只有A4型和A16型。研究结果为进一步研究NSP1蛋白质的结构功能及其应用开发奠定了很好的基础。     

COMPARATIVE MORPHOLOGY AND MOLECULAR PHYLOGENETIC ANALYSIS OF THREE NEW SPECIES OF THE GENUS KARENIA (DINOPHYCEAE) FROM NEW ZEALAND1

Three new dinoflagellate species, Karenia papilionacea sp. nov., Karenia selliformis sp. nov., and Karenia bidigitata sp. nov., were compared with the toxic species Karenia mikimotoi (Miyake & Kominami ex Oda) G. Hansen & Moestrup, Karenia brevis (Davis) G. Hansen & Moestrup, and Karenia brevisulcata (Chang) G. Hansen & Moestrup using the same fixative. Distinguishing morphological characters for the genus Karenia included a smooth theca and a linear apical groove. The new species can be distinguished on the basis of morphological characters of vegetative cells that include the location and shape of the nucleus; the relative excavation of the hypotheca; the characteristics of apical and sulcal groove extensions on the epitheca; the cellular shape, size, and symmetry; the degree of dorsoventral compression; and the presence of an apical protrusion or carina. Species with pronounced dorsoventral compression swim in a distinctive fluttering motion. An intercingular tubular structure traversing the proximal and distal ends of the cingulum is common to the species of Karenia, Karlodinium micrum (Leadbeater & Dodge) J. Larsen, Gymnodinium pulchellum J. Larsen, and Gyrodinium corsicum Paulmier. Molecular phylogenetic analyses of rDNA sequence alignments show that the new species are phylogenetically distinct but closely related to K. mikimotoi and K. brevis.     

冠状病毒非结构蛋白13的研究进展

冠状病毒能够引发多种传染性疾病,给动物和人类的健康带来严重危害。研发有效的疫苗和抗病毒药物成为防治疾病的重要手段。冠状病毒基因组能够编码多种蛋白质,包括结构蛋白、非结构蛋白和辅助蛋白。解旋酶非结构蛋白13 (nonstructural protein 13, NSP13)是冠状病毒编码的一种关键非结构蛋白,能够调控病毒复制和宿主先天免疫反应。因此,NSP13被认为是研发抗冠状病毒药物的重要靶点。本文结合国内外现有NSP13相关研究成果,对冠状病毒解旋酶NSP13的来源与结构、序列保守性、解旋机制、酶抑制剂、蛋白互作以及免疫调控等方面进行综述,并且分析了NSP13研究目前面临的问题,为研发靶向NSP13的广谱抗冠状病毒药物提供了理论依据。     

口蹄疫病毒非结构蛋白3A、3B和2C基因的表达及产物纯化与活性检测

[目的]口蹄疫病毒(FMDV)非结构蛋白(NSP)3A、3B和2C基因的表达及产物纯化与活性检测.[方法]利用原核表达系统表达了FMDV NSP 3A、3B和富含B细胞抗原位点序列的2C蛋白.利用高浓度尿素裂解包涵体,采用稀释法和氧化型、还原型谷胱甘肽系统相结合方法对2C蛋白进行复性.用金属鳌合亲合层析的方法对表达的FMDV NSP 3A、3B和2C进行纯化.采用ELISA方法对比检测了3种纯化蛋白在检测羊血清NSP抗体的效果.[结果]检测得知3A和3B为可溶性表达蛋白,2C以包涵体形式表达.通过Western-blot分析,表明纯化后蛋白能与FMDV感染动物血清发生特异性反应.纯化的3A、3B和复性后的2C融合蛋白与3ABC抗原的检测结果具有很高的符合性.[结论]该研究为建立鉴别FMDV自然感染动物和灭活疫苗免疫动物的酶联免疫电转移印迹技术(EITB)提供了所需的材料.