Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate‐induced colitis in mice due to increased intestinal permeability

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long‐chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very‐long acyl chain ceramides with concomitant increase of long chain bases and C16‐ceramides, were more susceptible to dextran sodium sulphate‐induced colitis, and their survival rate was markedly decreased compared with that of wild‐type littermates. Using mixed bone‐marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule‐A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco‐2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2‐knockdown via CRISPR‐Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long‐chain bases and C16‐ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC‐dextran levels, indicating that altered SLs including deficiency of very‐long‐chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.     

溃疡性结肠炎小鼠肠道通透性改变与TNF-α及NF-κB P65的关系

目的：采用2.5%葡聚糖硫酸钠（DSS）定量灌胃诱导小鼠溃疡性结肠炎（UC）,观察小鼠结肠通透性改变与肿瘤坏死因子α（TNF-α）及NF-κB p65的关系。方法：48只ICR小鼠随机分为2组（n=24）：对照组和模型组。模型组小鼠给予2.5% DSS定量灌胃诱发小鼠急性UC,对照组小鼠予同体积的蒸馏水灌胃代替。记录两组小鼠疾病活动指数（DAI）,9 d后测定两组小鼠结肠组织病理学评分、结肠通透性、TNF-α及NF-κB p65。统计分析DAI、结肠通透性、TNF-α与NF-κB p65之间的相关性。结果：与对照组比较,模型组小鼠DAI、结肠病理学评分、结肠通透性、TNF-α、NF-κB p65均显著增高（P均<0.01）。小鼠DAI增高与结肠通透性密切相关（P均<0.01）,结肠通透性增高与TNF-α、NF-κB p65密切相关（P均<0.01）。结论：与对照组小鼠相比,DSS造模小鼠的结肠通透性显著增高,并与TNF-α、NF-κB p65增高呈正相关。TNF-α、NF-κB p65增高导致结肠通透性增高,进而导致炎症免疫反应过度增强,可能是UC发病的重要环节。     

Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase

Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28°–30°C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4°C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100°C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.     

右旋糖酐-α-1,6键水解酶的家族、结构及其应用

右旋糖酐（dextran）水解酶种类繁多,其中右旋糖酐-α-1,6键水解酶（D-α-1,6 H）是主要的水解酶类.该类酶包括右旋糖酐酶（EC 3.2.1.11）、葡萄糖右旋糖酐酶（EC 3.2.1.70）、异麦芽糖右旋糖酐酶（EC 3.2.1.94）等,分属不同糖苷水解酶家族.D-α-1,6 H的结构和催化方式多样,分类和进化关系复杂,是糖苷水解酶催化机制研究和酶蛋白分子进化研究的好材料.D-α-1,6H及该类酶的催化产物在工业和医学中均有重要而广泛的应用.近年来对D-α-1,6H的理论和应用研究逐渐增加,但仍缺乏深入的系统性研究.本文对D-α-1,6H的家族、结构和功能进行分析,并对其在工业和医学中的最新应用研究作以总结.     

一种葡聚糖芯片的再生方法、表征及其应用*

表面等离子体共振（surface plasmon resonance, SPR）生物传感器,作为一种适时快捷,无需标记的生物分子相互作用研究工具,已广泛应用于生物化学分析与研究。羧甲基化葡聚糖修饰的CM5传感芯片是Biacore 系列仪器应用最为普遍的核心部件,目前CM5芯片主要从法玛西亚公司购买,价格昂贵,且一旦共价交联的受体分子失活,就不能重复利用。阐述了一种简便、低成本、用于SPR生物传感器的葡聚糖修饰金膜芯片的再生方法及其表征和应用。用此方法再生的芯片能被循环伏安法和原子力显微镜很好地表征,并成功地用于抗前列腺特异性抗原(prostate-specific antigen,PSA)固定和PSA检测, 同时测定了PSA与其抗体之间的动力学和亲和常数。     

Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography

Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl₂. These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6–1.0 M arginine at pH 3.0–3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1536–1541, 2015     

荧光素标记的庚型肝炎病毒基因探针的制备及应用

参考国内外已完成测序的庚型肝炎病毒（ＧＢＶＣ／ＨＧＶ）基因序列,选取毒株间系列较保守的基因片段,并合成与之互补的核苷酸序列（Ａｎｔｉｓｅｎｓｅ）,用末端转移酶将荧光素Ｎ６ｄｄＡＴＰ标记该片段,制成庚肝病毒基因探针。在严格控制温度的条件下,与固定于硝酸纤维膜（ＮＣ膜）上血清斑点杂交,洗膜后与抗荧光素碱性磷酸酶（ＡＰ）结合,加底物后化学发光自显影判断结果。该方法检测与套式逆转录聚合酶链反应（ＮｅｓｔｅｄＲＴＰＣＲ）检测结果的阳性符合率为８８．２％,阴性符合率为１００％;并且与其他相关病毒基因无交叉反应,具有较好的特异性与灵敏性。其检测结果比ＥＩＡ法检测庚肝病毒抗体更具临床意义。     

Fluorescent-fipronil: Design and synthesis of a stable conjugate

Fipronil is a phenyl pyrazole molecule widely used across the world as both insecticide and veterinary drug. The main goal of this work was to synthesize a fluorescently labeled fipronil derivative for cellular imaging without affecting its intrinsic properties. We selected fluorescein as fluorescent probe and we investigated different strategies for stable chemical ligation between both entities, such as thiourea and direct peptide bond. While thiourea bond displayed low stability, direct peptide bond was difficult to achieve due to problems of steric hindrance. The best result was obtained by conjugation using click chemistry, which allowed to obtain fipronil stably labeled with the fluorescent probe.