Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.     

SAPl8和Sufu蛋白质复合物的表达纯化研究

通过分子克隆技术将Sufu和SAP18构建到原核载体,在原核表达系统进行外源表达,采用亲和层析和分子筛层析对Sufu和SAPl8及其复合物进行纯化。同时利用非变性胶的方法进一步确定该蛋白之间的相互作用及结合比例。结果表明,原核表达系统中Sufu和SAPl8蛋白表达量高,可以纯化到高纯度的蛋白质。su如和SAPl8结合的摩尔比为1：1,按摩尔比1：2混合、纯化可以得到高纯度、稳定的蛋白复合物,从而为进一步复合物的结构生物学研究奠定基础。     

Up-Regulation of MiR-300 Promotes Proliferation and Invasion of Osteosarcoma by Targeting BRD7

Increasing reports suggest that deregulated microRNAs (miRNAs) might provide novel therapeutic targets for cancers. However, the expression and function of miR-300 in osteosarcoma is still unknown. In our study, we found that the expression of miR-300 was up-regulated in osteosarcoma tissues and cells compared with paired adjacent non-tumor bone tissues and osteoblastic cells using RT-qPCR. The enforced expression of miR-300 could promote cell proliferation, invasion and epithelial-mesenchymal transition (EMT). Moreover, we identified that bromodomain-containing protein 7 (BRD7), a new tumor suppressor gene, was a direct target of miR-300. Ectopic expression of BRD7 could significantly inhibit miR-300-promoted proliferation, invasion and EMT. Therefore, our results identify an important role for miR-300 in osteosarcoma through regulating BRD7 expression.     

细叶黄芪叶肉原生质体发育早期细胞壁再生的研究

采用透射电镜术、电镜多糖细胞化学染色、细胞壁荧光染色以及香豆素抑制细胞壁再生等方法,对细叶黄芪（Ａｓｔｒａｇａｌｕｓｍ ｅｌｉｌｏｔｏｉｄｅｓ ｖａｒ．ｔｅｎｕｉｓ）叶肉原生质体细胞壁的再生及其化学特点进行了研究。结果表明,离体培养２４ 小时的原生质体表面产生一些突起小泡,有时可见少量纤维组分的形成。培养３ 天时这种纤维组分明显增多。至５ 天时可清楚看到再生壁是由纤维和颗粒构成。六亚甲四胺银染色证明它们都是由多糖组分组成的。另外,培养３６ 小时的原生质体有相互粘连的现象。电镜观察、荧光染色及香豆素处理的研究表明粘连与再生壁的形成有关。根据上述观察结果,对原生质体再生壁的结构及其化学性质等问题进行了讨论     

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.     

Role of exonic variation in chemokine receptor genes on AIDS: CCRL2 F167Y association with pneumocystis pneumonia

Chromosome 3p21–22 harbors two clusters of chemokine receptor genes, several of which serve as major or minor coreceptors of HIV-1. Although the genetic association of CCR5 and CCR2 variants with HIV-1 pathogenesis is well known, the role of variation in other nearby chemokine receptor genes remain unresolved. We genotyped exonic single nucleotide polymorphisms (SNPs) in chemokine receptor genes: CCR3, CCRL2, and CXCR6 (at 3p21) and CCR8 and CX3CR1 (at 3p22), the majority of which were non-synonymous. The individual SNPs were tested for their effects on disease progression and outcomes in five treatment-naïve HIV-1/AIDS natural history cohorts. In addition to the known CCR5 and CCR2 associations, significant associations were identified for CCR3, CCR8, and CCRL2 on progression to AIDS. A multivariate survival analysis pointed to a previously undetected association of a non-conservative amino acid change F167Y in CCRL2 with AIDS progression: 167F is associated with accelerated progression to AIDS (RH = 1.90, P = 0.002, corrected). Further analysis indicated that CCRL2-167F was specifically associated with more rapid development of pneumocystis pneumonia (PCP) (RH = 2.84, 95% CI 1.28–6.31) among four major AIDS–defining conditions. Considering the newly defined role of CCRL2 in lung dendritic cell trafficking, this atypical chemokine receptor may affect PCP through immune regulation and inducing inflammation.     

Structure-based discovery of a novel inhibitor targeting the β-catenin/Tcf4 interaction

Overactivation or overexpression of β-catenin in the Wnt (wingless) signaling pathway plays an important role in tumorigenesis. Interaction of β-catenin with T-cell factor (Tcf) DNA binding proteins is a key step in the activation of the proliferative genes in response to upstream signals of this Wnt/β-catenin pathway. Recently, we identified a new small molecule inhibitor, named BC21 (C(32)H(36)Cl(2)Cu(2)N(2)O(2)), which effectively inhibits the binding of β-catenin with Tcf4-derived peptide and suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor decreases the viability of β-catenin overexpressing HCT116 colon cancer cells that harbor the β-catenin mutation, and more significantly, it inhibits the clonogenic activity of these cells. Down-regulation of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin signaling, is confirmed by treating HCT116 cells with BC21. This compound represents a new and modifiable potential anticancer candidate that targets β-catenin/Tcf-4 interaction.     

高滴度单克隆抗体杂交瘤细胞系获得途径的探讨

为探索获得能分泌较高价单克隆抗体杂交瘤细胞系的途径,比较了用不同免疫方法得到的小鼠脾细胞制备杂交瘤,对其产生有效瘤和高价抗体分泌瘤的影响。用不使小鼠致病的病毒(如NDV)或巳灭活的病毒作为抗原时,以较高浓度加佐剂、长间隔(30天)、多次免疫效果较好。用能使小鼠感染的病毒作为免疫原时,以活病毒的亚致死剂量感染小鼠,取其脾细胞制备杂交瘤,效果最理想。有效杂交瘤产率高,分泌高效价抗体的杂交瘤细胞系也多。探讨了从免疫方法着手,定向研制高价单克隆抗体杂交瘤细胞系的可行性。     

抗菌肽的研究进展

抗菌肽又称抗微生物肽(antimicrobial peptide)或肽抗生素(peptide antibiotics),在动植物体内分布广泛,是天然免疫防御系统的一部分。抗菌肽不仅有广谱抗细菌能力,而且对真菌、病毒及癌细胞也有作用。对抗菌肽作用机理的研究是近来的热点之一,本文综述了此方面近来的进展,并对微生物针对抗菌肽的耐药性进行了讨论。