苍耳子对慢性鼻-鼻窦炎大鼠炎症反应、NLRP3炎症小体及JAK2STAT3信号通路的影响

摘要 目的：探讨苍耳子对慢性鼻-鼻窦炎大鼠炎症反应、NLRP3炎症小体及JAK2STAT3信号通路的影响。方法：选择清洁级SD大鼠40只作为研究对象。将40只大鼠随机分为5组。采用鼻腔内注射金黄色葡萄球菌悬浊液的方法构建慢性鼻-鼻窦炎大鼠模型。空白对照组（8只）、模型组（8只）、低剂量组（7.5 mg/kg,8只）、中剂量组（15 mg/kg,8只）和高剂量组（30 mg/kg,8只）。采用埋藏食物小球实验进行嗅觉功能检查,采用qRC-PCR检测JAK2/STAT3信号通路和NLRP3、ACS、caspase-1的mRNA表达水平并采用ELISA法检查大鼠血液样本中肿瘤坏死因子-α（TNF-α）和IL-1β、IL-18的表达水平。结果：建模前,各组大鼠找到食物小球的时间比较（P＞0.05）;治疗后7 d和治疗完成时,与对照组相比,模型组、低剂量组、中剂量组和高剂量组找到食物小球的时间明显更长（P＜0.05）,高剂量组找到食物小球的时间明显短于模型组、低剂量组、中剂量组（P＜0.05）。与对照组相比,模型组、低剂量组、中剂量组和高剂量组JAK2 mRNA、STAT3 mRNA相对表达水平明显更高（P＜0.05）,高剂量组JAK2 mRNA、STAT3 mRNA相对表达水平明显低于模型组、低剂量组、中剂量组（P＜0.05）。与对照组相比,模型组、低剂量组、中剂量组和高剂量组TNF-α、IL-1β、IL-18表达水平明显更高（P＜0.05）,高剂量组TNF-α、IL-1β、IL-18表达水平明显低于模型组、低剂量组、中剂量组（P＜0.05）。与对照组相比,模型组、低剂量组、中剂量组和高剂量组NLRP3 mRNA、ACS mRNA、caspase-1 mRNA相对表达水平明显更高（P＜0.05）,高剂量组NLRP3 mRNA、ACS mRNA、caspase-1 mRNA相对表达水平明显低于模型组、低剂量组、中剂量组（P＜0.05）。结论：CRS大鼠采用苍耳子挥发油进行治疗可通过调控JAK2/STAT3信号通路的表达和NLRP3炎症小体的表达,从而抑制下游炎症因子的过度分泌,最终为缓解CRS病情和改善嗅觉功能做出贡献。     

外泌体miR-338对骨质疏松大鼠骨代谢水平、骨小梁微结构和骨生物力学的影响

摘要 目的：探讨外泌体miR-338对骨质疏松大鼠骨代谢水平、骨小梁微结构和骨生物力的影响。方法：采用健康成年SPF级SD雄性大鼠进行骨髓间充质干细胞（BMSCs）分离。采用双侧卵巢摘除手术方法构建了骨质疏松大鼠模型。采用qRT-PCR法检测miR-338的表达水平;检测大鼠的骨密度,骨小梁微结构和骨生物力学指标。结果：与空白对照组相比,OP模型组、OP+ ExoBMSCs、抑制组和过表达组miR-338的表达水平明显更高（P＜0.05）;抑制组的miR-338的表达水平低于OP模型组、OP+ExoBMSCs和过表达组（P＜0.05）;与空白对照组相比,OP模型组、OP+ ExoBMSCs、抑制组和过表达组OC、PINP、BALP的表达水平明显更低（P＜0.05）;抑制组的OC、PINP、BALP的表达水平明显高于OP模型组、OP+ ExoBMSCs和过表达组（P＜0.05）;与空白对照组相比,OP模型组、OP+ ExoBMSCs、抑制组和过表达组BV/TV、Th.N、Tb.Th、Conn.D水平更低,而Tb.Sp、SMI明显更高（P＜0.05）;抑制组组的BV/TV、Th.N、Tb.Th、Conn.D水平明显高于OP模型组、OP+ ExoBMSCs和过表达组,而Tb.Sp、SMI更低（P＜0.05）;与空白对照组相比,OP模型组、OP+ ExoBMSCs、抑制组和过表达组BMD、最大荷载、最大应力、最大位移、刚度水平更低（P＜0.05）;抑制组的BMD、最大荷载、最大应力、最大位移、刚度水平高于OP模型组、OP+ExoBMSCs和过表达组（P＜0.05）。结论：BMSCs源性的miR-338可影响骨质疏松大鼠骨代谢、骨小梁微结构和骨生物力学状态。     

BNP/NPR-A/BKCa信号通路在大鼠神经痛形成中的作用及机制研究

摘要 目的：探讨BNP/NPR-A/BKCa信号通路在大鼠神经痛形成中的作用及机制研究。方法：选取SPF级大鼠作为研究对象,并构建神经痛病理性疼痛大鼠模型。并分为空白对照组、假手术组、A组（20 ng/mL BNP梢内注射）、B组（50 ng/mL BNP梢内注射）和C组（100 ng/mL BNP梢内注射）。采用qRT-PCR和Western blot检测NPR-A和BKCa的mRNA和蛋白表达水平。采用ELISA法检查炎症因子。全细胞膜片钳技术检测痛觉神经元BKCa通道电流;对大鼠进行机械性痛觉过敏测试和温度性痛觉敏感测试。结果：与空白组相比,模型组、A、B和C组PWT和PWL明显更低（P＜0.05）;与模型组相比,A、B和C组PWT和PWL明显更高,且C组大于B和A组,B组大于A组（P＜0.05）。与空白组相比,模型组NPR-A的蛋白和mRAN水平明显更高,而BKCa-α明显更低（P＜0.05）;与模型组相比,A、B和C组NPR-A和BKCa-α的蛋白和mRNA明显更高,且C组大于B和A组,B组大于A组（P＜0.05）。各电压水平,与空白组相比,模型组、A、B和C组BKCa-α电流水平明显更低（P＜0.05）;与模型组相比,A、B和C组BKCa-α电流水平明显更高,且C组大于B和A组,B组大于A组（P＜0.05）。与空白组相比,模型组、A、B和C组TNF-α、IL-6和IL-18水平明显更高（P＜0.05）;与模型组相比,A、B和C组TNF-α、IL-6和IL-18水平明显更低,且C组小于B和A组,B组小于A组（P＜0.05）。结论：靶向上调BNP的表达水平可增加BKCa的表达及BKCa电流,同时BNP的表达上调还有助于抑制炎症因子水平,从而达到多途径缓解疼痛的目的。     

Snail1基因表达在妊娠期糖尿病大鼠氧化应激及肝脏损伤的作用机制

摘要 目的：探讨Snail1基因表达在妊娠期糖尿病大鼠氧化应激及肝脏损伤的作用机制。方法：健康成年C57BL/6雌性大鼠32只作为研究对象。采用高脂喂养联合小剂量链脲佐菌素注射的方式构建妊娠期糖尿病大鼠模型。将所有大鼠分为正常妊娠组,正常妊娠+Snail1过表达组,妊娠期糖尿病组,妊娠期糖尿病+Snail1过表达组。采用qRT-PCR检测大鼠Snail1的mRNA表达水平,并检测大鼠妊娠期体重、氧化应激指标、炎症指标和肝功能指标。结果：与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的孕鼠体重、胎鼠体重、胎盘重量、Snail1 mRNA,明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组孕鼠体重、胎鼠体重、胎盘重量、Snail1 mRNA均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的FBG、FINS、HOMA-IR明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组FBG、FINS、HOMA-IR均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的ROS、GSH-Px、MDA明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组ROS、GSH-Px、MDA均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的TNF-α、IL-1β、IL-18明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组TNF-α、IL-1β、IL-18均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的GPT、GOT、ALP明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组GPT、GOT、ALP均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）。结论：妊娠期糖尿病大鼠Snail1基因的表达上调可加重糖脂代谢紊乱,并激活下游氧化应激和炎症反应,进而加重大鼠肝脏损伤。     

基于JAK2/STAT3信号通路探讨头针结合艾灸对缺血缺氧脑瘫幼鼠模型的影响及作用机制

摘要 目的：基于JAK2/STAT3信号通路探讨头针结合艾灸对缺血缺氧脑瘫幼鼠模型的影响及作用机制。方法：选取40只7 d龄的清洁级SD幼鼠,按随机数字表法分为A组、B组、C组、D组、E组,每组8只。E组不予以任何处理,其余各组幼鼠均以改良的缺氧缺血性脑病造模方法构建缺血缺氧脑瘫模型。造模成功后A组、B组、C组分别给予头针结合艾灸治疗、头针治疗、艾灸治疗,D组、E组均给予50 ?滋L生理盐水。对比各组幼鼠脑组织Janus蛋白酪氨酸激酶2（JAK2）及转录活化因子3（STAT3）蛋白表达量、血清炎症因子[白介素-6（IL-6）、肿瘤坏死因子--α（TNF-α）、白介素-1（IL-1）]水平、神经递质[去甲肾上腺素（NE）、5-羟色胺（5-HT）]水平、记忆功能。结果：A组脑组织JAK2、STAT3 蛋白表达量低于B、C、D组,但高于E组（P<0.05）;A组血清IL-6、IL-1和TNF-α水平低于B、C、D组,但高于E组（P<0.05）;A组血清NE、5-HT水平低于B、C、D组,但高于E组（P<0.05）;A组Y迷宫实验正确次数多于B、C、D组（P<0.05）,而少于E组（P<0.05）。结论：头针结合艾灸可通过靶向抑制JAK2/STAT3信号通路相关蛋白的表达下调机体炎症反应水平,从而降低缺血缺氧脑瘫幼鼠脑组织损伤,促进其记忆功能恢复。     

儿茶素经PI3K-Akt-eNOS信号通路对冠心病大鼠心肌损伤及抗炎作用分析

摘要 目的：探讨儿茶素经PI3K-Akt-eNOS信号通路对冠心病大鼠心肌损伤及抗炎作用分析。方法：健康成年SPF级SD雄性大鼠,经腹腔注射垂体后叶素构建冠心病模型。采用Western blot法检测大鼠PI3K-Akt-eNOS信号通路相关蛋白的表达情况。采用ELISA法检测大鼠炎症因子和氧化应激指标的表达。观察并记录大鼠心肌损伤情况。结果：与空白组相比,模型组、儿茶素小剂量组、儿茶素中剂量组、儿茶素大剂量组的心肌缺血和心肌梗死面积均明显更高（P＜0.05）;儿茶素大剂量组心肌缺血和心肌梗死面积明显小于模型组、儿茶素小剂量组、儿茶素中剂量组（P＜0.05）;与空白组相比,模型组、儿茶素小剂量组、儿茶素中剂量组、儿茶素大剂量组的PI3K、p-Akt /Akt、p-eNOS /eNOS均明显更高（P＜0.05）;儿茶素大剂量组PI3K、p-Akt /Akt、p-eNOS /eNOS明显高于模型组、儿茶素小剂量组、儿茶素中剂量组（P＜0.05）;与空白组相比,模型组、儿茶素小剂量组、儿茶素中剂量组、儿茶素大剂量组的TNF-α、IL-1β、IL-18均明显更高（P＜0.05）;儿茶素大剂量组TNF-α、IL-1β、IL-18明显低于模型组、儿茶素小剂量组、儿茶素中剂量组（P＜0.05）;与空白组相比,模型组、儿茶素小剂量组、儿茶素中剂量组、儿茶素大剂量组的ROS、GSH-Px、MDA均明显更高（P＜0.05）;儿茶素大剂量组TNF-α、IL-1β、IL-18明显低于模型组、儿茶素小剂量组、儿茶素中剂量组（P＜0.05）。结论：儿茶素可通过PI3K-Akt-eNOS信号通路靶向改善冠心病大鼠心肌炎症和氧化应激状况,进而发挥心肌保护作用。     

罗氟司特对脓毒症炎症大鼠炎症因子及淋巴细胞的调控机制分析

摘要 目的：探究罗氟司特通过JAK/STAT途径对脓毒症炎症大鼠炎症因子及淋巴细胞的调控机制分析。方法：将54只SD大鼠随机分为正常组（n=18）,模型组（n=18）和罗氟司特组（n=18）,正常组正常喂养,模型组和罗氟司特组建立脓毒症大鼠模型。模型组和罗氟司特组腹膜内注射0.9 %的氯化钠和罗氟司特。干预7 d后,对大鼠取样。测定灌洗液中炎症细胞的数量、JAK和STAT-3的蛋白、AST和ALT、IL-6和TNF-α的mRNA表达。结果：在观察的7 d中,正常组大鼠均存活,生存率高于模型组和罗氟司特组（P<0.05）,罗氟司特组生存率高于模型组（P<0.05）。与正常组相比,模型组和罗氟司特组术后症状评分均增加（P<0.05）,罗氟司特组术后症状评分低于模型组（P<0.05）。模型组和罗氟司特组的miRNA-218表达低于正常组（P<0.05）,而罗氟司特组高于模型组（P<0.05）;模型组和罗氟司特组淋巴细胞,中性粒细胞和单核细胞总数,JAK和STAT-3的蛋白质表达,AST、ALT、IL-6和TNF-α水平高于正常组（P<0.05）,罗氟司特组上述指标低于模型组（P<0.05）。结论：罗氟司特通过JAK / STAT途径阻断miRNA-218表达,抑制促炎性细胞因子IL-6和TNF-α的表达,减轻脓毒症炎症的机制和肝脏损害并改善多发性脓毒症的生存率。     

葛根素对正常产后小鼠泌乳作用的影响

摘要 目的：探究葛根素对产后正常小鼠泌乳作用的影响及其机制,并初步探究葛根素对产后正常小鼠的安全性。方法：将雌、雄KM小鼠以3:1比例合笼配种,得到孕鼠饲养至分娩。分娩后的小鼠随机分为正常对照组、葛根素低剂量（18 mg?kg^-1）、高剂量组（72 mg?kg^-1）,每组8只。从产后第3 d起,每天灌胃一次,共10 d。观察小鼠每日泌乳量变化,ELISA法检测血清中催乳素（PRL）、孕酮（P4）、雌二醇（E₂）含量,HE染色观察乳腺、肝、肾、子宫、卵巢组织病理学形态,Western Blot法检测乳腺组织中催乳素受体（PRLR）、酪氨酸激酶 2（JAK2）和信号传导与激活因子5a（STAT5a）的表达。结果：与正常对照组相比,从给药的第6天起,葛根素低剂量组的泌乳量显著升高（P＜0.05）;葛根素低、高剂量组均可见乳腺小叶内腺泡明显变大,分泌物明显增多,且低剂量组更为明显;葛根素低、高剂量组血清PRL水平明显升高（P＜0.01或P＜0.05）;葛根素低剂量组PRLR的蛋白表达明显增加（P＜0.01）,而葛根素高剂量组PRLR、JAK2的蛋白表达明显降低（P＜0.01）。葛根素低剂量组PRLR、JAK2、STAT5a的蛋白表达明显高于葛根素高剂量组（P＜0.01或P＜0.05）。结论：葛根素低剂量对产后正常小鼠有一定促进泌乳作用,高剂量时对泌乳作用不明显。葛根素低、高剂量均未对产后正常小鼠的肝、肾、卵巢和子宫产生明显的病理学改变。     

参苓白术散联合针刺对溃疡性结肠炎大鼠内质网应激、炎症反应的作用机制

摘要 目的：探讨参苓白术散联合针刺对溃疡性结肠炎大鼠内质网应激、炎症反应的作用机制。方法：采用三硝基苯磺酸的方法构建溃疡性结肠炎大鼠模型。给予大鼠参苓白术散联合针刺干预。采用ELISA法检测大鼠病变结肠的炎症反应和氧化应激水平;采用肉眼观察各组结肠组织形态学评分;采用Western blot 检测大鼠内质网应激相关蛋白的表达。结果：与空白组相比,模型组、针刺组、参苓白术散、参苓白术散组联合针刺组大鼠体质量更低（P＜0.05）;与模型组相比,针刺组、参苓白术散、参苓白术散组联合针刺组溃疡指数评分和大鼠体质量更低（P＜0.05）,且参苓白术散组联合针刺组明显低于针刺组和参苓白术散组;与空白组相比,模型组、针刺组、参苓白术散、参苓白术散组联合针刺组ROS、GSH-Px、MDA更高（P＜0.05）;与模型组相比,针刺组、参苓白术散、参苓白术散组联合针刺组ROS、GSH-Px、MDA更低（P＜0.05）,且参苓白术散组联合针刺组明显低于针刺组和参苓白术散组;与空白组相比,模型组、针刺组、参苓白术散、参苓白术散组联合针刺组TNF-α、IL-1β、IL-18更高（P＜0.05）;与模型组相比,针刺组、参苓白术散、参苓白术散组联合针刺组TNF-α、IL-1β、IL-18更低（P＜0.05）,且参苓白术散组联合针刺组低于针刺组和参苓白术散组;与空白组相比,模型组、针刺组、参苓白术散、参苓白术散组联合针刺组GRP78、p-PERK、p-eIF2α更高（P＜0.05）;与模型组相比,针刺组、参苓白术散、参苓白术散组联合针刺组GRP78、p-PERK、p-eIF2α更低（P＜0.05）,且参苓白术散组联合针刺组明显低于针刺组和参苓白术散组。结论：参苓白术散组联合针刺可有效抑制溃疡性结肠炎大鼠内质网应激,进而抑制炎症反应和氧化应激反应,并促进溃疡性结肠炎大鼠病变病情转归。     

肠内营养联合益生菌对直肠恶性肿瘤术后大鼠肠粘膜屏障及血清炎症因子的影响

摘要 目的：研究肠内营养（enteral nutrition, EN）联合益生菌对直肠恶性肿瘤术后大鼠肠粘膜屏障及血清炎症因子的影响。方法：选取32只SPF级雄性SD大鼠,随机分对照组、模型组、EN组和EC组,每组8只。模型组、EN组和EC组建立直肠恶性肿瘤术后模型,EN组灌胃给予肠内营养混悬液,EC组灌胃肠内营养混悬液加益生菌颗粒,对照组和模型组灌胃同体积的生理盐水,四组大鼠均连续灌胃7 d。观察各组大鼠小肠超微结构;检测occludin蛋白、D-乳糖及炎症因子的表达水平。结果：模型组大鼠小肠上皮细胞紧密连接和绒毛结构遭破坏,EN组和EC组与模型组相比有明显改善。与对照组相比,模型组大鼠occludin蛋白和抑炎因子IL-10的表达水平均较低,D-乳糖、血清白介素1β（interleukin-1β, IL-1β）和肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）的表达水平较高（P＜0.05）;与模型组相比,EN组和EC组的occludin蛋白和IL-10的表达水平较高,D-乳糖、IL-1β和TNF-α的表达水平较低（P＜0.05）;与EN组相比,EC组occludin蛋白和IL-10的表达水平较高,D-乳糖、IL-1β和TNF-α的表达水平较低（P＜0.05）。结论：肠内营养联合益生菌可有效改善直肠恶性肿瘤术后大鼠的肠道屏障功能,提高occludin蛋白的表达,降低D-乳糖的表达,降低促炎因子IL-1β和TNF-α的表达同时升高抑炎因子IL-10的表达。     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

Characterization of cytochrome P-450SCC-containing liposomes

Purified cytochrome $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}\text{-}\text{containing liposomes}$ showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was less stable than $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. Liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was subjected to $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ treatment. This reagent destroyed the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. These results suggest that the heme is located in the proximity of the $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.     

AutoFACT: AnAutomaticFunctionalAnnotation andClassificationTool

Background  

Assignment of function to new molecular sequence data is an essential step in genomics projects. The usual process involves similarity searches of a given sequence against one or more databases, an arduous process for large datasets.     

A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens

The partial purification of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in $\text{p-}\text{nitrophenylphosphatase}$ activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ which can be phosphorylated by reaction with [γ-³²P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the $\text{I}{}_{50}$ for $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and $\text{p-}\text{nitrophenylphosphatase}$ inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from other tissues such as oligomycin, Ca²⁺, vanadate, $\text{N-}\text{ethylmaleimide}$, $\text{p-}\text{chloromercuribenzenesulfonic acid}$ (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K⁺ are partially effective in activating the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-ATPase and $\text{p-}\text{nitrophenylphosphatase}$ activities. The K⁺ congeners were relatively more effective in supporting $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ compared to $\text{p-}\text{nitrophenylphosphatase}$ activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, $\text{p-}\text{nitrophenylphosphatase}$ activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Quenching of the fluorescence of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Comparison of parameters estimated from A/Ci and A/Cc curve analysis

The parameters estimated from traditional A/C _i curve analysis are dependent upon some underlying assumptions that substomatal CO₂ concentration (C _i) equals the chloroplast CO₂ concentration (C _c) and the C _i value at which the A/C _i curve switches between Rubisco- and electron transport-limited portions of the curve (C _i-t) is set to a constant. However, the assumptions reduced the accuracy of parameter estimation significantly without taking the influence of C _i-t value and mesophyll conductance (g _m) on parameters into account. Based on the analysis of Larix gmelinii’s A/C _i curves, it showed the C _i-t value varied significantly, ranging from 24 Pa to 72 Pa and averaging 38 Pa. t-test demonstrated there were significant differences in parameters respectively estimated from A/C _i and A/C _c curve analysis (p<0.01). Compared with the maximum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (V_cmax), the maximum electron transport rate (J_max) and J_max/V_cmax estimated from A/C _c curve analysis which considers the effects of g _m limit and simultaneously fits parameters with the whole A/C _c curve, mean V_cmax estimated from A/C _i curve analysis (V_cmax-C _i) was underestimated by 37.49%; mean Jmax estimated from A/C _i curve analysis (J_max-C _i) was overestimated by 17.8% and (J_max-C _i)/(V_cmax-C _i) was overestimated by 24.2%. However, there was a significant linear relationship between V_cmax estimated from A/C _i curve analysis and V_cmax estimated from A/C _c curve analysis, so was it J_max (p<0.05).     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.