Use of galactosyltransferase to assess the biological function of O-linked N-acetyl-d-glucosamine: a potential role for O-GlcNAc during cell division

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl-d-glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes beta1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.     

巴氏杆菌糖酵解酶的黏附作用研究

巴氏杆菌（主要是多杀性巴氏杆菌）可以引起多种动物疫病（巴氏杆菌病）,同时也引起人类感染发病。[目的] 研究巴氏杆菌糖酵解酶对宿主细胞（兔肾细胞）和两种常见分子[纤连蛋白（fibronectin,Fn）和血浆纤维蛋白溶解酶原（plasminogen,Plg）]的黏附作用。[方法] 采用原核表达系统对多杀性巴氏杆菌的糖酵解酶进行表达并纯化及制备多克隆抗体,通过菌体表面蛋白定位检测、黏附与黏附抑制等实验探究巴氏杆菌糖酵解酶的黏附作用。[结果] 菌体表面蛋白检测结果显示除烯醇化酶和丙酮酸激酶外的7个糖酵解酶在多杀性巴氏杆菌表面存在。这7个糖酵解酶均能黏附兔肾细胞,但仅有磷酸葡萄糖异构酶的多克隆抗体能对多杀性巴氏杆菌黏附宿主细胞产生抑制作用。Far Western blotting结果显示9个糖酵解酶均能结合宿主Fn和Plg。招募抑制实验结果显示磷酸葡萄糖异构酶、醛缩酶、磷酸甘油酸变位酶的抗体对多杀性巴氏杆菌结合Fn和Plg都有抑制作用,磷酸果糖激酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶抗体仅对多杀性巴氏杆菌结合Fn或Plg有抑制作用。[结论] 多杀性巴氏杆菌糖酵解酶成员葡萄糖异构酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶、磷酸甘油酸变位酶在多杀性巴氏杆菌黏附宿主细胞或分子过程中发挥作用。该研究的完成将加深巴氏杆菌病分子发病机制的认识,并为巴氏杆菌病的诊断标识筛选、新型疫苗创制和药物靶标筛选等提供基础数据。     

Identification of tumor metastasisrelated gene TMSG-1 by mRNA differential display

To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression of two cancer sublines derived from prostate carcinoma cell PC-3M that had different metastatic potentials. The differentially expressed genes were confirmed by Northern blot, and sequenced. The full-length cDNA of a tumor metastasis suppressor gene (TMSG-1) was obtained by using EST assembling and verified by RT-PCR and sequencing. The results showed that expression levels of TMSG-1 were lower in the highly metastatic cell line 1E8, compared with the non-metastatic cell line 2B4. The difference was significant. Full-length cDNA of TMSG-1 was about 2 kb, containing an open reading frame that encoded a protein of 230 amino acids. GenBank Blastn showed no marked homology with known genes. The functional prediction of amino acids sequence encoded by TMSG-1 gene indicated TMSG-1 protein was transmembrane protein, with 3 transmembrane domains, 3 putative protein kinase phosphorylatio     

Aromatic‐interaction‐mediated inhibition of β‐amyloid assembly structures and cytotoxicity

Abnormal aggregation of β‐amyloid (Aβ) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aβ aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic‐interaction‐mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aβ42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the β‐sheet formation of Aβ42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aβ42 through π–π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aβ42 to SH‐SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide‐based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

转基因高蛋氨酸大豆对根际土壤主要有机元素和酶活性的影响

【目的】为了评估转基因高蛋氨酸大豆ZD91对土壤生态系统的安全性,开展了其对土壤主要有机元素和酶活性影响的实验。【方法】连续2年,在大豆苗期、花期、鼓粒期和成熟期,采用抖落法采集根际土壤样品,通过室内测定,分析了转基因大豆ZD91对根际土壤含水量、pH、主要有机元素和酶活性的影响。【结果】转基因大豆ZD91较之对应的非转基因大豆对根际土壤含水量、pH、主要有机元素和酶活性无显著影响,但同一种酶活在不同年份和不同生育期存在显著差异。【结论】转基因大豆ZD91对土壤生态系统具有安全性。     

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Expression,purification and characterization of anti-BAFF antibody secreted from the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

B-Cell activating factor (BAFF) is critical for B cell survival and maturation; excessive expression of it corrupts B-cell tolerance and may lead to autoimmunity. The gene, scFv-Fc, coding for the antibody of BAFF was inserted into the eukaryotic expression vector, pPICZαA, and transformed into Pichia pastoris. A high-level expression strain was obtained using a ‘yeastern blotting’ method. The scFv-Fc antibody was purified and 56 mg was obtained from 1 l of culture supernatant. It retained high binding activity to both soluble BAFF and membrane-bound BAFF.     

VEGF siRNA降低人肝癌细胞株SMMC7721的致瘤性

本研究应用RNA干扰（RNAi）技术高效抑制VEGF的表达,明显降低了人肝癌细胞株SMMC7721的致瘤性。化学合成针对人VEGF基因的siRNA,体外瞬时转染人肝癌细胞株SMMC7721,RT-PCR和Elisa法检测表明VEGFsiRNA实验组与对照组相比,细胞内VEGFmRNA表达下降了76．16％,VEGF分泌蛋白表达则下降了96．28％,而MTT法结果显示VEGFsiRNA对SMMC7721细胞增殖没有明显作用。体内实验中,不同时间对荷SMMC7721细胞瘤裸小鼠进行siRNA直接瘤内注射,同时测量瘤体积,最后取瘤块进行组织切片观察并进行分子生物学分析,结果显示,与对照组相比,VEGFsiRNA实验组肿瘤体积明显缩小,瘤内出现细胞坏死,同时肿瘤组织中VEGFmRNA和蛋白表达水平均明显降低。     

PERMANENT GENETIC RESOURCES: A set of primer pairs for amplifying the complete mitochondrial DNA of endangered Chinese egret (Aves, Ardeidae, Egretta eulophotes)

The Chinese egret is a globally endangered species. Here we describe a set of primer pairs to amplify its entire mtDNA. The polymerase chain reaction products (1000–2000 bp) were successfully amplified by using this primer set and were then sequenced and aligned. The contiguous mtDNA sequences of the Chinese egret were assembled to be a circular molecule (17 579 bp). This primer set was also confirmed to be useful for six other species of ardeid birds. The versatility of this primer set will provide a groundwork for further studies on the genetic structure and molecular evolution of the ardeid species.     

Treatment of coal gasification wastewater by a two-continuous UASB system with step-feed for COD and phenols removal

A two-continuous mesophilic (37 ± 2 °C) UASB system with step-feed was investigated as an attractive optimization strategy for enhancing COD and total phenols removal of the system and improving aerobic biodegradability of real coal gasification wastewater. Through the step-feed period, the maximum removal efficiencies of COD and total phenols reached 55-60% and 58-63% respectively in the system, at an influent flow distribution ratio of 0.2 and influent COD concentration of 2500 mg/L; the corresponding efficiencies were at low levels of 45-50% and 43-50% respectively at total HRT of 48 h during the single-feed period. The maximum specific methanogenic activity and substrate utilization rate were 592 ± 16 mg COD-CH₄/(gVSS d) and 89 ± 12 mg phenol/(gVSS d) during the step-feed operation. After the anaerobic digestion with step-feed, the aerobic effluent COD concentration decreased from 270 ± 9 to 215 ± 10 mg/L. The results suggested that step-feed enhanced the degradation of refractory organics in the second reactor.