阿尔茨海默病血清miR-137、miR-138表达与认知功能损害和外周血淋巴细胞PI3K/Akt信号通路的关系研究

摘要 目的：探讨阿尔茨海默病（AD）患者血清微小核糖核酸（miR）-137、miR-138表达与认知功能损害和外周血淋巴细胞磷脂酰肌醇-3激酶/蛋白激酶（PI3K/Akt）信号通路的关系。方法：选取2020年1月至2022年5月青岛大学附属医院神经内科收治的95例AD患者（AD组）,根据临床痴呆评定量表（CDR）评分将患者分为轻度组（1分,35例）、中度组（2分,42例）、重度组（3分,18例）,另选取63例体检健康志愿者为对照组。检测AD组、对照组血清miR-137、miR-138表达水平以及外周血淋巴细胞PI3K/Akt信号通路相关蛋白表达,采用简易智能精神状态检查量表（MMSE）、蒙特利尔认知评估量表（MoCA）评估认知功能。分析血清miR-137、miR-138与MMSE、MoCA评分以及外周血淋巴细胞PI3K、Akt、B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白基因（Bax）表达的相关性。结果：AD组血清miR-137水平、外周血淋巴细胞PI3K、Akt、Bcl-2蛋白表达水平及MMSE、MoCA评分低于对照组（P＜0.05）,血清miR-138、外周血淋巴细胞Bax蛋白表达水平高于对照组（P＜0.05）。重度组血清miR-137水平、外周血淋巴细胞PI3K、Akt、Bcl-2蛋白表达水平及MMSE、MoCA评分低于中度组和轻度组（P＜0.05）,且中度组低于轻度组（P＜0.05）;重度组血清miR-138、外周血淋巴细胞Bax蛋白表达水平高于中度组和轻度组（P＜0.05）,且中度组高于轻度组（P＜0.05）。AD患者血清miR-137水平与MMSE、MoCA评分、外周血淋巴细胞PI3K、Akt、Bcl-2蛋白表达呈正相关（P＜0.05）,与外周血淋巴细胞Bax蛋白表达呈负相关（P＜0.05）;AD患者血清miR-138水平与MMSE、MoCA评分、外周血淋巴细胞PI3K、Akt、Bcl-2蛋白表达呈负相关（P＜0.05）,与外周血淋巴细胞Bax蛋白表达呈正相关（P＜0.05）。结论：AD患者的血清miR-137表达水平降低、miR-138表达水平增高,与认知功能障碍有关,且miR-137、miR-138可能通过调控PI3K/Akt信号通路参与AD发病过程。     

miR-145、miR-186表达与非小细胞肺癌组织临床病理特征和PI3K/Akt/ mTOR信号通路的关系

摘要 目的：探讨微小核糖核酸（miR）-145、miR-186表达与非小细胞肺癌（NSCLC）临床病理特征和磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路的关系。方法：选择2020年3月至2023年3月我院收治的128例行肺癌根治手术治疗的NSCLC患者,取其术中癌组织和癌旁组织（距离癌组织5 cm以上）,采用实时荧光定量聚合酶链式反应（qRT-PCR）检测miR-145、miR-186以及PI3K mRNA、Akt mRNA、mTOR mRNA表达。分析miR-145、miR-186表达与NSCLC患者临床病理特征的关系;Pearson检验分析NSCLC患者癌组织miR-145、miR-186表达与PI3K mRNA、Akt mRNA、mTOR mRNA表达的相关性。结果：癌组织miR-145、miR-186表达低于癌旁组织（P＜0.05）,低分化、TNM ⅢA期、淋巴结转移的NSCLC组织miR-145、miR-186表达低于中高分化、TNM Ⅰ～Ⅱ期、未发生淋巴结转移的NSCLC组织（P＜0.05）。癌组织PI3K mRNA、Akt mRNA 、mTOR mRNA表达均高于癌旁组织（P＜0.05）,癌组织miR-145、miR-186表达与PI3K mRNA、Akt mRNA、mTOR mRNA表达均呈负相关（P＜0.05）。结论：NSCLC组织中miR-145、miR-186表达下调,miR-145、miR-186表达下调可能激活PI3K/ Akt/mTOR信号通路促使NSCLC进展,且与NSCLC患者癌组织低分化、TNM ⅢA期、淋巴结转移有关。     

基于PI3K/AKT/mTOR信号通路探讨二甲双胍对结肠癌HCT116细胞的作用

摘要 目的：研究二甲双胍通过磷脂酰肌醇3激酶（PI3K）/蛋白激酶 B(Akt)/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路对结肠癌HCT116 细胞的作用。方法：体外培养结肠癌HCT116细胞,分别加入二甲双胍(20,40,80 μmol/L)处理HCT116细胞48 h,另设对照组。MTT法检测各组细胞增殖能力。Transwell实验检测各组细胞侵袭能力的变化。Annexin-FITC/PI 双染法分别检测各组处理48 h后细胞凋亡情况。免疫印迹法检测48 h后PI3K/Akt/mTOR通路蛋白表达水平。结果：相比于对照组,二甲双胍20,40,80 μmol/L各处理组对HCT116细胞的增殖具有明显的抑制作用,且呈浓度依赖效应,差异具有统计学意义(P＜0.05)。与对照组比较,二甲双胍20,40,80 μmol/L各处理组细胞凋亡率明显较高,且呈浓度依赖效应,差异具有统计学意义(P＜0.05)。相比于对照组,二甲双胍20,40,80 μmol/L各处理组HCT116细胞侵袭能力明显减弱,且呈浓度依赖效应,差异具有统计学意义(P <0.05)。与对照组比较,二甲双胍20,40,80 μmol/L各处理组Bax蛋白表达水平明显升高,而Bcl-2、p-Akt及p-mTOR蛋白表达水平明显降低,且呈浓度依赖效应,差异具有统计学意义(P＜0.05)。结论：二甲双胍在体外可抑制人结肠癌HCT-116细胞的增殖,促进其凋亡,抑制其侵袭能力,其抗肿瘤机制可能与抑制PI3K/Akt/mTOR 信号通路激活相关。     

血清miR-26a、miR-130a与老年急性脑梗死患者PI3K/Akt信号通路和预后的关系研究

摘要 目的：研究血清miR-26a、miR-130a与老年急性脑梗死（ACI）患者磷酸酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路和预后的关系。方法：选取2020年3月-2022年10月我院收治的98例老年ACI患者作为研究组,同期选取60例于本院体检健康的老年人作为对照组。采用实时荧光定量聚合酶链式反应（qRT-PCR）检测血清miR-26a、miR-130a表达水平,蛋白免疫印迹法检测外周血单个核细胞中PI3K、Akt蛋白表达;采用Pearson相关性分析miR-26a、miR-130a表达水平与PI3K、Akt蛋白表达的相关性。患者治疗后行1个月随访,根据改良Rankin量表（mRS）评分分为预后良好组（n=55例,mRS评分≤2分）和预后不良组（n=43例,评分＞2分）。收集患者的一般资料,采用Logistic回归模型分析老年ACI患者预后的影响因素。结果：研究组血清miR-26a表达水平高于对照组,miR-130a表达水平低于对照组（P＜0.05）。研究组PI3K蛋白表达量低于对照组,Akt蛋白表达量高于对照组（P＜0.05）。Pearson相关性分析显示,miR-26a表达水平与PI3K蛋白表达量呈负相关、与Akt蛋白表达量呈正相关,miR-130a表达水平与PI3K蛋白表达量呈正相关、与Akt蛋白表达量呈负相关（P＜0.05）。预后良好组与预后不良组在年龄、房颤占比、入院时美国国立卫生研究院卒中量表（NIHSS）评分、血糖及血清miR-26a、miR-130a表达水平方面比较具有统计学意义（P＜0.05）。Logistic回归分析显示,年龄偏大、入院时NIHSS评分偏高、血糖偏高及血清miR-26a表达水平升高为老年ACI患者预后不良的危险因素,miR-130a表达水平升高为其保护因素（P＜0.05）。结论：老年ACI患者血清miR-26a表达水平升高、miR-130a表达水平下降,与PI3K/Akt信号通路密切相关,是老年ACI患者预后的影响因素。     

姜黄素衍生物减缓大鼠糖尿病周围神经病变进展的作用及其机制研究

摘要 目的：探讨姜黄素衍生物减缓大鼠糖尿病周围神经病变（DPN）进展的作用及其机制。方法：30只健康成年雄性SD大鼠随机分为3组，每组10只；构建链脲佐菌素（STZ）诱导的DPN大鼠模型；研究组1按10 mg/kg体重喂养10 μM的姜黄素衍生物；研究组2按10 mg/kg体重喂养100 μM的姜黄素衍生物；对照组喂养同等量的生理盐水。利用Von Frey电子测痛仪检测各组大鼠痛觉机械戒断阈值（MWT）。采用实时定量聚合酶链反应（qRT-PCR）和蛋白免疫印迹试验检测AMPK、mTOR mRNA和蛋白表达水平。最后测定活化剂AICAR处理的RSC96细胞中mTOR的表达水平。结果：喂养姜黄素衍生物大鼠的痛觉机械戒断阈值（MWT）随时间推移逐渐降低，与对照组相比，研究组1大鼠的MWT显著降低，而研究组2大鼠的MWT也显著低于研究组1，差异均有统计学意义（P＜0.05）。与对照组相比，研究组1的AMPK mRNA和蛋白表达水平显著升高，mTOR mRNA和蛋白表达水平表达显著降低（P＜0.05）；而研究组2的AMPK mRNA和蛋白表达水平显著高于研究组1，mTOR mRNA和蛋白表达水平显著低于研究组1，差异均有统计学意义（P＜0.05）。使用活化剂AICAR处理RSC96细胞后，活化剂AICAR组的mTOR mRNA和mTOR蛋白表达水平明显低于非活化剂组，差异有统计学意义（P＜0.05）。结论：姜黄素衍生物可能通过AMPK调节mTOR信号通路改善DPN，其可能具有潜在的治疗DPN的能力。     

达格列净对高血压小鼠小动脉重构的影响及机制

摘要 目的：探究钠葡萄糖共转运蛋白2抑制剂（达格列净）对高血压小鼠小动脉重构的影响及机制。方法：30只12周龄的C57BL / 6雄性小鼠纳入本研究,根据实验目的将实验小鼠分为对照组、模型组和达格列净组。检测并比较各组小鼠心脏肥大、心脏纤维化、动脉重塑、炎症因子mRNA表达、PI3K和Akt蛋白质表达以及细胞活力和细胞迁移。结果：与对照组相比,模型组收缩压和心脏/体重增加（P<0.05）。与模型组相比,达格列净组收缩压和心脏/体重降低（P<0.05）。与对照组相比,模型组RV/（LV+S）和得分增加（P<0.05）。与模型组相比,达格列净组RV/（LV+S）和得分降低（P<0.05）。与对照组相比,模型组显示血管壁增厚,透明质改变,脑小动脉管腔狭窄或闭塞（P<0.05）。与模型组相比,达格列净组降低高血压引起的小动脉重塑（P<0.05）。与对照组相比,模型组IL-1β,IL-6和TNF-α的mRNA表达水平增加（P<0.05）。与模型组相比,达格列净组IL-1β,IL-6和TNF-α的mRNA表达水平降低（P<0.05）。与对照组相比,模型组PI3K和Akt蛋白质表达水平增加（P<0.05）。与模型组相比,达格列净组PI3K和Akt蛋白质表达水平降低（P<0.05）。与对照组相比,模型组VEZF1,Angpt-1和IGF1表达水平降低（P<0.05）。与模型组相比,达格列净组VEZF1,Angpt-1和IGF1表达水平增加（P<0.05）。与对照组相比,模型组细胞活力和细胞迁移降低（P<0.05）。与模型组相比,达格列净组细胞活力和细胞迁移增加（P<0.05）。结论：达格列净通过抑制PI3K / Akt信号通路,降低炎症反应,增加血管生成能力,降低高血压小鼠小动脉重构。     

北沙参对肺癌大鼠PI3K/Akt信号通路及免疫炎症反应的影响

摘要 目的：探讨北沙参对肺癌大鼠PI3K/Akt信号通路及免疫炎症反应的影响。方法：从60只Wistar大鼠中随机选取15只作为对照组,其余45只大鼠采用气管内灌注致癌碘油液建立肺癌模型,按照随机数字表法将其分为模型组（n=15）、环磷酰胺组（n=15）、北沙参组（n=15）,开展前瞻性研究。对照组、模型组给予生理盐水灌胃,环磷酰胺组给予环磷酰胺干预,北沙参组给予北沙参干预。比较各组大鼠免疫功能、炎症因子水平及PI3K/Akt表达。结果：与对照组相比,环磷酰胺组、北沙参组、模型组脾脏指数依次降低,而肺脏指数依次升高（P<0.05）。与对照组相比,环磷酰胺组、北沙参组、模型组CD3⁺、CD4⁺百分比及CD4⁺/CD8⁺依次降低,而CD8⁺百分比依次升高（P<0.05）。与对照组相比,环磷酰胺组、北沙参组、模型组白介素6（IL-6）、白介素1β（IL-1β）、肿瘤坏死因子-α（TNF-α）水平依次升高（P<0.05）。与对照组相比,环磷酰胺组、北沙参组、模型组PI3K、Akt表达水平依次升高（P<0.05）。结论：北沙参能够有效调节肺癌大鼠的脏器指数,促进免疫功能改善,抑制炎症反应,调控PI3K/Akt信号通路可能是其发挥作用的重要机制。     

LncRNA-NEAT1对妊娠期高血压大鼠JAK2/STAT3信号通路、炎症反应和妊娠结局的影响

摘要 目的：探讨LncRNA-NEAT1对妊娠期高血压大鼠JAK2/STAT3信号通路、炎症反应和妊娠结局的影响。方法：采用注射L-精氨酸甲酯的方法构建妊娠期高血压大鼠模型。采用Western blot检测JAK2/STAT3信号通路蛋白表达;采用ELISA法检测炎症因子和血管内皮损伤因子。观察并记录大鼠24 h蛋白尿、尾静脉压和死胎率。结果：与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组JAK2、STAT3的蛋白表达水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组JAK2、STAT3的蛋白表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组JAK2、STAT3的蛋白表达水平明显更高（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组ET-1和sICAM-1水平明显更高,而NO水平明显更低（P＜0.05）;与模型组相比,LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组ET-1和sICAM-1水平明显更高（P＜0.05）,而NO水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组ET-1和sICAM-1表达水平明显更高,而NO明显更低（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组TNF-α、IL-1β、IL-18表达水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组TNF-α、IL-1β、IL-18表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组TNF-α、IL-1β、IL-18表达水平明显更高（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组尾静脉压和24h蛋白尿水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组尾静脉压和24 h蛋白尿表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组尾静脉压和24 h蛋白尿表达水平明显更高（P＜0.05）;LncRNA-NEAT1过表达组（21.56%）死胎率显著高于模型组（16.72%）和LncRNA-NEAT1抑制组（5.65%）。结论：妊娠期糖尿病大鼠LncRNA-NEAT1的表达下调可抑制JAK2/STAT3信号通路的表达并下调下游促炎因子的表达,进而缓解血管内皮损伤降低死胎率。     

Nrf2激动剂CDDO-Im对高脂饮食诱导的肥胖小鼠肝脏脂肪变性的影响

摘要 目的：探究Nrf2激动剂CDDO-Im对高脂饮食诱导的肥胖小鼠肝脏脂肪变性的作用。方法：33只雄性C57BL/6J小鼠随机分为两组：一组16只饲喂普通饲料,另一组17只饲喂高脂饲料建立肥胖模型。造模成功后将小鼠随机分成四组：普通饲料溶剂对照组（Control ND组）、普通饲料Nrf2激动剂组（Nrf2(+) ND组）、高脂饲料溶剂对照组（Control HFD组）和高脂饲料Nrf2激动剂组（Nrf2(+) HFD组）。分别给予Nrf2激动剂CDDO-Im和等体积溶剂灌胃干预6周后,检测各组小鼠血清甘油三酯（TG）、总胆固醇（T-CHO）和低密度脂蛋白-胆固醇（LDL-C）。苏木素-伊红（HE）染色观察肝脏组织形态学变化。RT-qPCR检测肝脏Nrf2下游抗氧化基因Nqo1、Ho1和Gclc的mRNA表达水平,Western Blot检测肝脏NQO1、HO-1和GCLC的蛋白表达水平。结果：与正常小鼠相比,肥胖小鼠的体重、TG和LDL-C升高（P＜0.05）,肝脏脂肪变性增加,GCLC的蛋白表达水平降低（P＜0.05）。在肥胖小鼠中,与溶剂对照组相比,Nrf2激动剂组小鼠的体重、血清TG降低（P＜0.05）,肝脏脂肪变性减轻,Nqo1和Gclc的mRNA表达水平升高（P＜0.05）,NQO1和GCLC的蛋白表达水平升高（P＜0.05）。结论：Nrf2激动剂CDDO-Im可改善高脂饮食诱导的肥胖小鼠肝脏脂肪变性,可能与Nrf2激动剂CDDO-Im激活抗氧化基因的表达来减轻肝细胞氧化应激有关。     

miR-19通过Keap-Nrf2/HO-1信号通路对卵巢癌细胞增殖的影响

摘要 目的：研究卵巢癌组织和细胞中miR-19的表达,探讨其异常表达对卵巢癌细胞Kelch样环氧氯丙烷相关蛋白-1（Kelch-like epichlorohydrin-associated protein1,Keap1）--核因子E2相关因子2（nuclearfactor-E2-relatedfactor2,Nrf2） /血红素氧合酶-1（heme oxygenase1,HO-1）信号通路及卵巢癌细胞增殖的影响。方法：回顾性收集2019年1月至2020年12月于我院就诊的患者经病理切片诊断为卵巢癌上皮细胞的手术标本30例,卵巢良性肿瘤标本30例,正常卵巢组织标本30例。免疫组化检测不同标本中Keap1、Nrf2、HO-1的表达,检测卵巢组织及细胞中miR-19、Keap1、Nrf2、HO-1的mRNA表达水平,及卵巢癌细胞中Keap1、Nrf2、HO-1的蛋白表达水平。在OVCAR-3细胞中沉默miR-19后,Western Blot检测细胞内Keap1、Nrf2、HO-1蛋白表达水平,收集沉默miR-19,对照组,沉默Nrf2、对照组的OVCAR-3细胞,继续培养0 h、24 h、48 h后,检测细胞增殖和凋亡。结果：Keap1蛋白在卵巢癌组织中的阳性表达显著低于良性卵巢肿瘤组织及正常卵巢组织;Nrf2和HO-1蛋白在卵巢癌组织中的阳性表达显著低于良性卵巢肿瘤组织及正常卵巢组织（P＜0.05）;沉默miR-19抑制其表达后,细胞内Keap1 mRNA、蛋白表达水平明显升高,Nrf2、HO-1 mRNA表达水平无明显变化,蛋白表达水平明显降低（P＜0.05）;沉默miR-19 组、沉默Nrf2组与转染阴性对照组相比,增殖能力明显降低,凋亡能力明显升高（P＜0.05）。结论：卵巢癌细胞中,miR-19表达水平升高,可通过调控Keap1-Nrf2/HO-1信号通路影响卵巢癌细胞的增值、凋亡能力。     

甲型H1N1流感病毒在季流病毒、禽流感病毒共感染时变异可能性的验证

目的 甲型H1N1流感病毒A/California/7/2009分别与A/Brisbane/10/07和A/ShenZhen/406H/06共感染小型香猪,预测甲流病毒在与季流H3N2病毒/甲流病毒与禽流感病毒共感染时是否会发生变异.方法 分别将A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)对5～6月龄小型猪共感染,小型猪经复方氯胺酮0.1 mL/kg麻醉后进行滴鼻感染,感染后第5天安乐死动物,取动物肺组织作病毒测序分析.结果 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2)共感染后,A/California/7/2009病毒PB1基因993位G→A突变,PA基因1659位G→A突变,没有氨基酸的变异.A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后A/California/7/2009病毒PB2基因1711位T→C突变.碱基的突变未引起氨基酸的变异.结论 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后在猪的体内没有发生病毒重组、变异.     

Pathogenesis of primary defects in mitochondrial ATP synthesis

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often-fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum. These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline. Based on the accumulating data on both the structure of ATP synthase and the mechanism by which rotary catalysis couples proton flow to ATP synthesis, we propose a model that may help explain why mutations at codons 156 and 217 are pathogenic.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

A new kind of carbohydrate array,its use for profiling antiglycan antibodies,and the discovery of a novel human cellulose-binding antibody

In this study, we use a novel glycan array to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. The glycan array used is based on mono- and oligosaccharides covalently linked to the surface via a long linker at their reducing ends. They are thus presented to the medium with a well-defined orientation and are accessible for specific binding by glycan-binding proteins, such as antibodies and lectins. A novel anticellulose antibody was detected that binds specifically to beta4-linked saccharides with a preference for glucopyranose over galactopyranose residues. We also found previously known antiglycan antibodies against mono- and oligosaccharides that are constituents of commonly occurring bacterial polysaccharides. We propose that this array can facilitate high-throughput screening of glycan-binding proteins and the search for biomarkers for personalized medicine.     

Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.     

Heparin sequencing

Heparin is a highly sulfated glycosaminoglycan widely used as an anticoagulant. Modifications in its relatively uniform structure appear to be key to its recognition and modulation of serine proteases, growth factors, chemokines, and extracellular proteins, as has been most clearly demonstrated in the antithrombin binding site. We sequenced the major oligosaccharides released from mastocytoma heparin by partial nitrous acid using a highly sensitive technique tailored for sequencing of metabolically radiolabeled heparin. It utilizes partial nitrous acid cleavage to allow simultaneous sequencing of the internal components of the oligosaccharide under investigation by specific lysosomal exoenzymes. Sequencing revealed that although the majority of the heparin disaccharides are N-, 2-O-, and 6-O-sulfated, the less sulfated disaccharides (lacking 2-O- or 6-O-sulfates) seem to be spaced out along the chain. The technique may be particularly useful for characterizing heparin from novel sources, such as the glial progenitor cells and Ascidia, as well as for sequencing protein binding sites.     

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.     

Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry

In an effort to understand the role of molecular glycosylationin cancer a murine model has been used to characterize and fingerprintmalignancies in established cell lines that manifest all thehallmarks of metastatic disease: spontaneous development, localinvasion, intravasation, immune system survival, extravasation,and secondary tumor formation involving liver, kidney, spleen,lung, and brain. Using astrocyte cell controls, we comparedN-linked glycosylation from a nonmetastatic brain tumor cellline and two different metastatic brain tumor cells. Selectedions in each profile were disassembled by ion trap mass spectrometry(MSⁿ) which exhibited multiple structural differences betweeneach tissue. These unique structures were identified withinisomeric compositions as pendant nonreducing termini of di-and trisaccharide fragments, probably transparent to a tandemMS approach but distinctively not to sequential ion trap MSⁿdetection.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Glucan-like synthetic oligosaccharides: iterative synthesis of linear oligo-beta-(1,3)-glucans and immunostimulatory effects

Small reducing and linear oligo-beta-(1,3)-glucans, which are able to act as phytoallexin elicitors or as immunostimulating agents in anticancer therapy, were synthesized according to an iterative strategy that involved a unique key monosaccharidic donor. To avoid anomeric mixtures, the reducing entity of the target oligomers was first locked with benzyl alcohol and further selective deprotection of the 3-OH with DDQ afforded the desired building block as an acceptor. The latter was then used in a second cycle of glycosylation/deprotection to afford the desired disaccharide, and successive reiterations of this process provided the desired oligomers. Unusual conformational behaviors were observed by standard NMR sequences and supported by NOESY studies. Finally, removal of protecting groups afforded free tri-, tetra-, and pentaglucosides in good overall yields. Two oligosaccharides representing linear laminaritetraose and laminaripentaose were compared to the recently described beta-(1,3)-glucan phycarine. Following an intraperitoneal injection, the influx of monocytes and granulocytes into the blood and macrophages into the peritoneal cavity was comparable to that caused by phycarine. Similarly, both oligosaccharides stimulated phagocytic activity of granulocytes and macrophages. Using ELISA, we also demonstrated a significant stimulation of secretion of IL-1beta. Together these results suggest that the synthetic oligosaccharides have similar stimulatory effects as natural beta-(1,3)-glucans.