扇贝大防御素和G型溶菌酶的基因克隆与重组表达

扇贝养殖是我国传统的海水养殖产业，但自1997 年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且严重影响了该产业的健康发展。扇贝病害的不断爆发以及病因的多样性迫切要求制定新的疾病防治措施和开发新型的抗菌物质。 从扇贝自身的免疫防御因子入手，筛选和克隆参与免疫防御的功能基因，一方面可以研究抗病功能基因在病原感染或环境胁迫条件下的表达规律，深入探讨扇贝的免疫防御机制，并可作为抗病良种选育的分子标记，指导扇贝的遗传改良和抗病品系的培育；另一方面，可对抗菌效应物实现重组表达，开发新型的病害预防治疗制剂，取代目前普遍使用的抗生素和化学药物。抗菌效应物是机体在免疫应答过程中产生的多肽类物质，对侵入生物体内的细菌、病毒具有很强的免疫杀灭作用，对抗菌效应物的研究有助于深入了解机体先天性免疫防御的机制。 本研究采用大规模EST测序方法，结合cDNA末端快速扩增（RACE）技术，从海湾扇贝血淋巴中克隆到了大防御素基因（big defensin, AiBD）的全长cDNA序列，该cDNA全长为531 bp，其中5' 非编码区（UTR）为24 bp，开放阅读框（Open Reading Frame, ORF）含有369 bp，编码122 个氨基酸残基；随后为138-bp 的3' UTR，包括一个多聚腺苷酸信号序列（AATAAA）和ploy A尾巴。分析表明，海湾扇贝大防御素是以前体的形式合成，前体分子包括信号肽、前域和成熟肽三部分。采用Northern blot方法，以DIG标记的DNA探针检测了 AiBD mRNA在不同组织中的表达。结果发现，AiBD 基因的转录本主要在血淋巴中表达，在鳃中也有微量的表达，而在外套膜、闭壳肌、性腺及肝胰腺中检测不到杂交信号。采用QRT-PCR（quantitative real time PCR）对鳗弧菌感染后海湾扇贝血淋巴中AiBD mRNA 的表达量进行了检测，结果发现在感染后8 h 内， AiBD mRNA 的相对表达量平缓升高；随着刺激时间的增长，AiBD基因的mRNA表达量急剧增加，在刺激后16 h 和32 h 分别达到了空白组的72.3 倍和131.1 倍。为了研究海湾扇贝大防御素的抗菌活性，将其成熟肽编码区克隆到毕赤酵母表达载体pPIC9K并实现了重组表达。抑菌实验表明，重组AiBD具有广谱的抗菌活性，其对供试的三株革兰氏阳性菌（藤黄微球菌、溶壁微球菌、金黄色葡萄球菌）都表现出显著的抗菌活性，而对革兰氏阴性菌（鳗弧菌、亮弧菌）的抑菌活性则相对较弱；此外，重组AiBD对表达宿主也表现出杀菌活性，证明其具有抗真菌活性。 根据栉孔扇贝G 型溶菌酶基因的cDNA序列，利用构建的Genome Walking 文库获得了栉孔扇贝G 型溶菌酶基因的全长序列，该基因序列全长为8131 bp，由六个外显子和五个内含子组成。六个外显子长度分别为55 bp，60 bp，90 bp，113 bp，145 bp 和140 bp；五个内含子的长度分别为1126 bp，2161 bp，2744 bp，750 bp和592 bp；内含子的两侧都具有RNA正确剪接所必需的识别位点（GT/AG）。利用TRANSFAC 软件对栉孔扇贝G 型溶菌酶基因的5' 侧翼序列分析发现，该基因的5' 侧翼具有 TATA box 和 CAAT box 的共有序列；此外，在该基因的5' 侧翼发现了C/EBP、NF-κB、OCT-1 和 NF-IL6 等参与免疫基因激活的转录因子潜在结合位点。采用Northern blot方法，以生物素标记的RNA 探针检测了栉孔扇贝G 型溶菌酶基因在不同组织中的表达。结果发现，该基因的转录本主要在鳃、性腺及肝胰腺中表达，在血细胞和外套膜中也有微量的的表达，而在闭壳肌中检测不到杂交信号，这表明栉孔扇贝G 型溶菌酶可能兼备参与机体免疫防御和消化的功能。为了研究栉孔扇贝G 型溶菌酶的抗菌活性，将其成熟肽编码区克隆到毕赤酵母表达载体pPIC9K并实现了重组表达。抑菌实验表明，重组产物具有显著的抗阳性菌活性，其对供试的藤黄微球菌、溶壁微球菌表现出明显的抑制作用，对金黄色葡萄球菌未检测到抑制活性；而对革兰氏阴性菌仅表现出微弱的抑菌活性（亮弧菌和鳗弧菌），对大肠杆菌则基本无抑制活性。

PRINCIPAIS INDICADORES: AGRÍCOLAS, v. 3, n. 20, 2010.

2010

PRINCIPAIS INDICADORES: LEITE E DERIVADOS, v. 3, n. 20, 2010.

2010

PRINCIPAIS INDICADORES: MACROECONÔMICOS, v. 3, n. 20, 2010.

2010

Controlling alloy formation and optical properties by galvanic replacement of sub-20 nm silver nanoparticles in organic media

Galvanic replacement is a versatile synthetic strategy for the synthesis of alloy and hollow nanostructures. The structural evolution of single crystalline and multiply twinned nanoparticles <20 nm in diameter and capped with oleylamine is systematically studied. Changes in chemical composition are dependent on the size and crystallinity of the parent nanoparticle. The effects of reaction temperature and rate of precursor addition are also investigated. Galvanic replacement of single crystal spherical and truncated cubic nanoparticles follows the same mechanism to form hollow octahedral nanoparticles, a mechanism which is not observed for galvanic replacement of Ag templates in aqueous systems. Multiply twinned nanoparticles can form nanorings or solid alloys by manipulating the reaction conditions. Oleylamine-capped Ag nanoparticles are highly adaptable templates to synthesize a range of hollow and alloy nanostructures with tuneable localised surface plasmon resonance.

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.

HBHA and ESAT-6 induced IFN-g responses in M. tuberculosis infected subjects from a low incidence country

info:eu-repo/semantics/nonPublished

WEBCOM-G: A candidate middleware for Grid-Ireland

WebCom-G is a fledgling Grid Operating System, designed to provide independent service access through interoperability with existing middlewares. It offers an expressive programming model that automatically handles task synchronisation – load balancing, fault tolerance, and task allocation are handled at the WebCom-G system level – without burdening the application writer. These characteristics, together with the ability of its computing model to mix evaluation strategies to match the characteristics of the geographically dispersed facilities and the overall problem- solving environment, make WebCom-G a promising grid middleware candidate.