Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.     

DNA vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicity

BACKGROUND: Foot-and-mouth disease virus (FMDV) affects susceptible livestock animals and causes disastrous economic impact. Immunization with plasmid expressing VP1 that contains the major antigenic epitope(s) of FMDV as cytoplasmic protein (cVP1) failed to elicit full protection against FMDV challenge. MATERIALS AND METHODS: In this study, mice were immunized via electroporation with four cDNA expression vectors that were constructed to express VP1 of FMDV, as cytoplasmic (cVP1), secreted (sVP1), membrane-anchored (mVP1) or capsid precursor protein (P1), respectively, to evaluate whether expression of VP1 in specific subcellular compartment(s) would result in better immune responses. RESULTS: Electroporation enhanced immune responses to vectors expressing cVP1 or P1 and expedited the immune responses to vectors expressing sVP1 or mVP1. Immunization of mice via electroporation with mVP1 cDNA was better than sVP1 or cVP1 cDNA in eliciting neutralizing antibodies and viral clearance protection. Vaccination with P1 cDNA, nonetheless, yielded the best immune responses and protection among all four cDNAs that we tested. CONCLUSIONS: These results suggest that the antigenicity of a VP1 DNA vaccine can be significantly enhanced by altering the cellular localization of the VP1 antigen. Electroporation is a useful tool for enhancing the immune responses of vectors expressing VP1 or P1. By mimicking FMDV more closely than that of transgenic VP1 and eliciting immune responses favorably toward Th2, transgenic P1 may induce more neutralizing antibodies and better protection against FMDV challenge.     

研究生病理学实验带教的探索与实践

医学研究生教育的目的是培养高素质创新型医学人才,创新离不开科研,病理学是多学科科研究工作的基础。建立科研平台提高科研能力是研究生教育十分关注的问题。如何培养研究生病理学实验能力,使学到知识技术在今后的科研活动中得以运用,本文就近几年我们在研究生病理学实验带教实践过程中的一些方法作一介绍。     

对TMV不同抗性番茄品种的叶绿体DNA限制性内切酶酶谱分析

选用对TMV有抗性和敏感的番茄品种、制备其ct-DNA, 用限制性内切酶BumHI、EcoRI和PstI完全酶解, 三种酶切图谱与前人报道一致, 由酶切片段计算番茄ct-DNA。分子量约为156.9kb。比较抗性和敏感品种的ct-DNA图谱, 发现三种酶切图谱均存在差异, 但由差异片段计算分子量之和又很除近。我们推测这是由于检基顺序变异或小段DNA顺序插入或缺失所造成, 由此证明, 叶绿体基因组与核中的TMV抗性基因, 共同决定着植物体对TMV的抗性。     

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.     

Effects of Revegetation on Soil Microbial Biomass,Enzyme Activities,and Nutrient Cycling on the Loess Plateau in China

Revegetation is a traditional practice widely used for soil and water conservation on the Loess Plateau in China. However, there has been a lack of reports on soil microbial–biochemical indices required for a comprehensive evaluation of the success of revegetation systems. In this study, we examined the effects of revegetation on major soil nutrients and microbial–biochemical properties in an artificial alfalfa grassland, an enclosed natural grassland, and an artificial shrubland (Caragana korshinskii), with an abandoned cropland as control. Results showed that at 0–5, 5–20, and 20–40 cm depths, soil organic carbon, alkaline extractable nitrogen and available potassium were higher in natural grassland and artificial shrubland compared with artificial grassland and abandoned cropland. Soil microbial biomass C (Cmic) and phosphorous (Pmic) substantially decreased with depth at all sites, and in abandoned cropland was significantly lower than those of natural grassland, artificial grassland, and artificial shrubland at the depth of 0–5 cm. Soil microbial biomass N (Nmic) was higher in artificial shrubland and abandoned cropland compared with that in natural and artificial grasslands. Both Cmic and Pmic were significantly different between the 23‐year‐old and the 13‐year‐old artificial shrublands at the 0–5 cm depth. The activities of soil invertase, urease, and alkaline phosphatase in natural grassland and artificial shrubland were higher than those in artificial grassland and abandoned cropland. This study demonstrated that the regeneration of both natural grassland and artificial shrubland effectively preserved and enhanced soil microbial biomass and major nutrient cycling, thus is an ecologically beneficial practice for recovery of degraded soils on the Loess Plateau.     

Identification of Chromosomes from Multiple Rice Genomes Using a Universal Molecular Cytogenetic Marker System

To develop reliable techniques for chromosome identification is critical for cytogenetic research, especially for genomes with a large number and smaller-sized chromosomes. An efficient approach using bacterial artificial chromosome （BAC） clones as molecular cytological markers has been developed for many organisms. Herein, we present a set of chromosomal arm-specific molecular cytological markers derived from the gene-enriched regions of the sequenced rice genome. All these markers are able to generate very strong signals on the pachytene chromosomes of Oryza sativa L. （AA genome） when used as fluorescence in situ hybridization （FISH） probes. We further probed those markers to the pachytene chromosomes of O. punctata （BB genome） and O. officinalis （CC genome） and also got very strong signals on the relevant pachytene chromosomes. The signal position of each marker on the related chromosomes from the three different rice genomes was pretty much stable, which enabled us to identify different chromosomes among various rice genomes. We also constructed the karyotype for both O. punctata and O. officinalis with the BB and CC genomes, respectively, by analysis of 10 pachytene cells anchored by these chromosomal arm-specific markers.     

家蚕色氨酸羟化酶(TRH)基因的克隆及表达特性分析（英文）

5-羟色胺(5-hydroxytryptamine, 5-HT)是生物界广泛分布的信号分子,涉及动物的重要行为。5-HT是色氨酸羟化酶(Tryptophan hydroxylase, TRH)将L-色氨酸羟化为5-羟-L-色氨酸,5-羟-L-色氨酸随即被多巴脱羧酶(Aromatic L-amino acid decarboxylase, DDC)脱羧而成。TRH作为5-HT合成的限速酶,在无脊椎动物神经调控中具有重要地位。鳞翅目昆虫中TRH的功能研究并不多。在家蚕中克隆了家蚕TRH (Bombyx mori TRH, BmTRH)的cDNA序列1667bp,其中包含1632bp的开放读码框(Openreadingframe,ORF)。人类TPH或者果蝇TRH(Drosophila TRH, DmTRH)与BmTRH有高度相似性,尤其BmTRH和DmTRH之间大多数氨基酸保守说明它们在系统发育上的密切关系并可能有相似功能。基因表达分析显示BmTRH主要表达于头部和中枢神经组织,免疫组织化学和Western blotting结果显示BmTRH只存在于神经组织中,即BmTRH可能仅参与家蚕的神经活动。此外,家蚕DDC(B.moridecarboxylase,BmDDC)和蛋白具有TRH活性的苯丙氨酸羟化酶基因(Phenylalanine hydroxylase, BmPAH)也在中枢神经系统中有表达,暗示家蚕神经系统5-HT的合成与果蝇中不同,可能有两种不同的调控机制。     

线虫区系分析指示土壤食物网结构和功能研究进展

土壤食物网结构复杂,功能众多,直接测定土壤食物网各功能群生物量并结合数学模型来推断土壤食物网结构和功能,工作量大且分析过程繁琐。线虫生态学的发展为土壤食物网的研究开辟了一条新的思路,即利用线虫区系分析来定性推断食物网的结构和功能。线虫作为土壤中数量最丰富的后生动物,占据着土壤食物网的中心位置,其物种多样性、食性多样性、生活史策略多样性、功能团多样性奠定了其作为土壤食物网结构和功能指示生物的生态学基础。线虫区系分析根据发展历史可以分为个体分类、生活史策略分类、功能团分类和代谢足迹分类四个时期,其中后两个时期主要用于推断土壤食物网结构和功能。基于功能团的线虫区系分析将线虫的食性和生活史策略结合起来,发展出一系列指数来判断土壤食物网的连通性、食物网链长度、外界养分投入情况、分解途径及对外界干扰的响应等。基于代谢足迹的线虫区系分析在功能团分析基础上,加入线虫能流分析,从而定性反映了土壤食物网功能的大小。两者在指示土壤食物网自下而上调节及对植物线虫控制等方面起着重要的作用。     

Bio-Plex悬浮芯片系统检测两种实验兔白介素基因的差异表达

目的采用液相悬浮芯片系统同时测定实验兔圆小囊中IL-1β、IL-1R1、IL=8、IL-8RA和IL=15各基因的表达情况,并对该方法进行评价。方法利用Affymetrix的Panomics QuantiGene Plex2．0Assay中bDNA信号放大和多磁珠分析技术,来同时检测两种实验兔圆小囊中多重mRNA并定量。建立实验兔免疫相关白介素基因的液相悬浮芯片检测方法。结果可同时检测IL-1β、IL=1R1、IL-8、IL=8RA和IL-15各基因的含量,并发现WHBE兔IL-15基因的相对表达量显著高于JW兔（P〈0．05）,IL-1R1基因的相对表达量显著高于JW兔（P〈0．01）,IL-8RA基因在WHBE兔中的相对表达量也高于JW兔（P〈0．05）。结论建立了实验兔白介素基因的液相悬浮芯片检测方法,WHBE兔的IL-15、IL-1R1和IL-8RA基因表达量较高,可能与WHBE兔独特的免疫学特性有关。