建立稳定表达人RANTES基因的夏枯草细胞克隆

为了在中药细胞中表达人源有用基因 ,以增强其特异药理活性 ,将克隆自人外周血淋巴细胞 (PBL)mRNA的RANTES基因经Ti质粒衍生的中间表达载体pROKII导入携带pAL44 0 4质粒的根癌农杆菌LBA44 0 4菌株中 ,并采用叶盘共培养法转化离体培养夏枯草细胞 ,经Southern杂交确认RANTES基因在转化细胞基因组中的整合 ,用RT -PCR扩增、Western印迹杂交和酶联免疫吸附测定 (ELISA)分析转化细胞中RANTES基因的表达 ,以PBL的过氧化物酶活性作为重组RANTES对细胞趋化性诱导的检测指标。结果表明 ,RANTES基因已在转基因夏枯草细胞中整合 ,并已形成稳定表达RANTES基因的细胞克隆 ,为进一步培育具有特异药理活性的转基因夏枯草植株打下了基础。     

建立稳定表达人RANTES基因的夏枯草细胞克隆

为了在中药细胞中表达人源有用基因,以增强其特异药理活性,将克隆自人外周血淋巴细胞（PBL）mRNA的RANTES基因经Ti质粒衍生的中间表达载体pROKII导入携带pAL4404质粒的根癌农杆菌LBA4404菌株中,并采用叶盘共培养法转化离体培养夏枯草细胞,经Southern杂交确认RANTES基因在转化细胞基因组中的整合,用RT-PCR扩增、Western印迹杂交和酶联免疫吸附测定（ELISA）分析转化细胞中RANTES基因的表达,以PBL的过氧化物酶活性作为重组RANTES对细胞趋化性诱导的检测指标。结果表明,RANTES基因已在转基因夏枯草细胞中整合,并已形成稳定表达RANTES基因的细胞克隆,为进一步培育具有特异药理活性的转基因夏枯草植株打下了基础。     

NF-κB的生物学功能

NF—κB因其广泛参与机体的免疫及其它应激反应而受到人们的关注,其常见的形式是由p50和p65组成的异源二聚体。细胞受到外部因素刺激后,NF-κB由细胞质转移到细胞核中,并发生磷酸化和乙酰化,启动相关基因的表达。目前研究表明受NF-κB调节的基因有200多种,其激活因子不少于150种,所以对NF-κB在各种条件下的激活过程及信号传导网络的研究具有重要的意义。本文综合了当前关于NF—κB研究的最新进展,着重阐述了由TNF-α、IL-Ⅰ及LPS刺激而引起NF—κB激活的信号传导通路,并进一步阐述了其在人类某些疾病当中的生物学功能。     

药用植物内生真菌的多样性及生物功能研究进展

药用植物内生真菌资源丰富,其代谢产物常具有抗肿瘤、抗氧化、抑菌等作用,能产生药用植物生长调节物质及与宿主相同或类似的次生代谢产物,从而成为近年来的研究热点。本文对药用植物内生真菌的分离鉴定、多样性、生物活性及生物学功能等方面进行综述,以期为今后筛选及利用有效的药用植物内生真菌奠定基础。     

生物多样性的生态系统功能

本文从以下几个方面综述了生物多样性对生态系统功能和作用的影响：第一,几个关于物种在生态系统中的不同地位和生物多样性如何影响生态系统功能的假说;第二,生物多样性与生态系统的稳定性;第三,生物多样性如何影响生态系统的生产力;第四,生物多样性对生态系统可持续性的影响。此外还提出了几个需要继续探讨和关注的问题。     

生物多样性的生态系统功能

本文从以下几个方面综述了生物多样性对生态系统功能和作用的影响 :第一 ,几个关于物种在生态系统中的不同地位和生物多样性如何影响生态系统功能的假说 ;第二 ,生物多样性与生态系统的稳定性 ;第三 ,生物多样性如何影响生态系统的生产力 ;第四 ,生物多样性对生态系统可持续性的影响。此外还提出了几个需要继续探讨和关注的问题     

内皮细胞的免疫学功能

血管内皮细胞（Endothelial cell,EC)通过表达多种免疫相关分子与或影响免疫过程,能以MHC－II类分子限制性方式提呈抗原,同时可通过B7／CD28,CD40／CD40L,CD58／CD2等途径向T细胞提供活化所必需的共刺激信号,EC表达的粘附分子介导EC与不同白细胞亚群间相互作用,对白细胞粘附穿过EC进入组织间隙参与炎症反应,淋巴细胞归巢或再循环等过程有重要意义,EC可表达补体调节因子调节补体系统活化,EC受多种因素激活后所表达的免疫相关分子表达上调,并产生多种细胞因子,参与机体的炎症反应及免疫应答,是重要的免疫调节细胞,本文将对EC免疫学方面的功能作简要综述。     

RANTES的多聚化及其意义

RANTES可以单体,双体和多聚体等形式存在,单体和双体形式的RANTES受体为CCR1,CCR3,CCR4和CCR5,而多聚体GAG。Glu26和Glu66在RANTES的多聚化过程中起着重要的作用。多聚体的RANTES与Jak2,Jak3和Src kinase p56(lck)等酪氨酸磷酸化有关,活化T细胞,单个核细胞及巨噬细胞,并可增强HIV的复制。     

人RANTES基因克隆及其体外表达

1995年,Cocchi等[1]发现RANTES、MIP-1α和MIP-1β等β-趋化因子具有抗HIV-1感染活性.1997年,Feng等[2]和Deng等[3]证实β-趋化因子受体CXCR4和CCR5分别是HIV-1侵染T淋巴细胞和巨噬细胞的辅助受体(co-receptor).T淋巴细胞嗜性(T-tropism)分离株被称为X4毒株,巨噬细胞嗜性(M-tropism)分离株则被称为R5毒株[4].RANTES与CCR5有着高度的亲和力,二者的结合可对HIV-1的细胞附着产生空间位阻效应,并下调CCR5在细胞表面的表达.这一结果使RANTES抗HIV-1感染机制在分子水平上得到合理的解释.最近,Garzino-Demo等[5]证明,β-趋化因子的诱导分泌与HIV-1感染后疾病进程的控制有着密切的关系,而且人群中β-趋化因子水平存在着显著的个体差异,表明β-趋化因子对艾滋病具有潜在的预防和治疗价值.为此,我们在克隆人RAN-TES基因的基础上,在体外转录与翻译系统中实现了该基因的表达,有利于今后进一步开展艾滋病的基因治疗.     

Chemokine expression in CF epithelia: implications for the role of CFTR in RANTES expression

To delineate themechanisms that facilitate leukocyte migration into the cystic fibrosis(CF) lung, expression of chemokines, including interleukin-8 (IL-8),monocyte chemoattractant protein-1 (MCP-1), andRANTES, was compared between CF and non-CF airway epithelia. Thefindings presented herein demonstrate that, under either basalconditions or tumor necrosis factor- (TNF-)- and/or interferon- (IFN-)-stimulated conditions, a consistent pattern ofdifferences in the secretion of IL-8 and MCP-1 between CF and non-CFepithelial cells was not observed. In contrast, CF epithelial cellsexpressed no detectable RANTES protein or mRNA under basal conditionsor when stimulated with TNF- and/or IFN-(P  0.05), unlike their non-CFcounterparts. Correction of the CF transmembrane conductance regulator(CFTR) defect in CF airway epithelial cells restored the induction ofRANTES protein and mRNA by TNF- in combination with IFN-(P  0.05) but had little effect onIL-8 or MCP-1 production compared with mock controls. Transfection studies utilizing RANTES promoter constructs suggested that CFTR activates the RANTES promoter via a nuclear factor-B-mediated pathway. Together, these results suggest that1) RANTES expression is altered inCF epithelia and 2) epithelialexpression of RANTES, but not IL-8 or MCP-1, is dependent on CFTR.     

血清与腹腔液中趋化因子RANTES水平在子宫内膜异位症患者中的临床应用

目的:探讨血清与腹腔液中趋化因子RANTES水平在子宫内膜异位症(EM)患者中的临床意义。方法:选取2012年5月-2013年5月本院收治的33例EM患者(观察组)、33例良性卵巢肿瘤患者(对照组)和33例健康体检者(健康对照组),应用ELISA法对血清与腹腔液中趋化因子RANTES水平进行检测,分析RANTES水平与患者r-AFS分期及痛经程度的相关性。结果:观察组血清RANTES水平明显高于对照组和健康对照组,差异均有统计学意义(t=7.163,6.743,均P0.05);观察组腹腔液RANTES水平亦高于对照组,两组比较差异有统计学意义(t=5.927,P0.05);观察组血清及腹腔液中RANTES水平与r-AFS分期呈正相关(r=0.975,0.893,均P0.05),且随分期增高而呈递增趋势;观察组血清RANTES水平与患者痛经评分无明显的相关性(r=-0.312,P0.05);而腹腔液中RANTES水平与患者痛经评分呈正关(r=0.517,P0.05)。结论:EM患者血清与腹腔液中趋化因子RANTES水平明显上升,应用ELISA法检测RANTES水平可辅助EM诊断,有利于提高诊断准确率。     

Human Cytomegalovirus Clinical Strain-Specific microRNA miR-UL148D Targets the Human Chemokine RANTES during Infection

The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b'' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.     

Increased Levels of C-C Chemokine RANTES in Asbestos Exposed Workers and in Malignant Mesothelioma Patients from an Hyperendemic Area

Background

Asbestos-induced mesothelial inflammatory processes are thought to be the basic mechanisms underlying Malignant Mesothelioma (MM) development. Detection of MM often occurs at late stage due to the long and unpredictable latent period and the low incidence in asbestos exposed individuals. The aim of this study was to investigate early immunological biomarkers to characterize the prognostic profile of a possible asbestos-induced disease, in subjects from a MM hyperendemic area.

Methods

The Luminex Multiplex Panel Technology was used for the simultaneous measurement of serum levels of a large panel of 47 analytes, including cytokines and growth factors, from workers previously exposed to asbestos (Asb-workers), asbestos-induced MM patients and healthy subjects. In addition, to explore the influence on serum cytokines profile exerted by SV40 infection, a cofactor in MM development, a quantitative real time PCR was performed for sequences detection in the N-terminal and intronic regions of the SV40 Tag gene. Statistical analysis was done by means of the Mann-Whitney test and the Kruskall-Wallis test for variance analysis.

Results

A variety of 25 cytokines linked to pulmonary inflammation and tumor development were found significantly associated with Asb-workers and MM patients compared with healthy controls. A specific pattern of cytokines were found highly expressed in Asb-workers: IFN-alpha (p<0.05), EOTAXIN (p<0.01), RANTES (p<0.001), and in MM patients: IL-12(p40), IL-3, IL-1 alpha, MCP-3, beta-NGF, TNF-beta, RANTES (p<0.001). Notably, the chemokine RANTES measured the highest serum level showing an increased gradient of concentration from healthy subjects to Asb-workers and MM patients (p<0.001), independently of SV40 infection.

Conclusion

This study shows that, in subjects from an hyperendemic area for MM, the C-C chemokine RANTES is associated with the exposure to asbestos fibres. If validated in larger samples, this factor could have the potential to be a critical biomarker for MM prognosis as recently reported for breast tumor.     

LucY: A Versatile New Fluorescent Reporter Protein

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.     

A Simple and Versatile Nebulizer

The nebulizer presented in this paper is of simple and rugged construction, permits easy control of cell concentrations, prevents sedimentation of the microorganisms, and permits the cleaning of the suspension needles even during the runs and under aseptic conditions. Fluid consumptions from 10 to 25 ml per hr were obtained with rates of primary air varying from 10 to 25 liters per min. The average diameter of the droplets varied from 1.5 to 2.7 μ.     

Salicylic Acid: A Versatile Signaling Molecule in Plants

Journal of Plant Growth Regulation -     

趋化因子及其受体基因家族的系统进化分析

通过分析现有的趋化因子和趋化因子受体的氨基酸序列,用距离法和最简约法构建了聚类图,探讨了趋化因子和趋化因子受体基因家族的系统演化特征。可见基因家族成员的分化早于脊椎动物的分化。不同物种的同一种基因的聚类关系能较好地反映物种经因子受体的进化速度不同,其中ＣＸＣＲ４的进化速率最低。趋化因子和趋化因子受体可能都起源于少数几个原始的基因,病毒编码与寄主相似的趋化因子或受体是进化过程中分子模拟的结果。