香草醛对新生大鼠缺氧缺血性脑损伤的神经保护作用研究

摘要 目的：探讨香草醛对新生大鼠缺氧缺血性脑损伤(HIBI)的神经保护作用及机制。方法：参考Rice-Vannucci方法建立HIBI大鼠模型。HIBI大鼠建模后立即腹腔注射20 mg/kg（HIBI+20Van组）或40 mg/kg（HIBI+40Van组）的香草醛,每隔12 h给药,连续7 d。然后评估大鼠的神经行为及脑组织中IL-1β、IL-6和TNF-α的水平。对BV2小胶质细胞进行氧糖剥夺/复氧(OGD/R)处理,并用20 μM香草醛培养。通过Western blot及免疫荧光检测HMGB1、NF-κB p65、SIRT1、MyD88和TLR4的表达水平。通过乳酸脱氢酶（LDH）释放测定试剂盒测定用不同BV2细胞培养基处理的原代神经元的LDH释放。结果：与HIBI组比较,HIBI+20Van组和HIBI+50Van组新生大鼠的前肢悬吊时间和旷场得分均升高,脑组织中的IL-1β、IL-6和TNF-α的水平均降低。香草醛均升高了HIBI大鼠和OGD/R处理的BV2细胞质中的SIRT1的表达水平,降低了TLR4、MyD88和HMGB1的表达水平及细胞核中NF-κB p65的表达水平（P<0.05）。香草醛降低了原代神经元的LDH释放量（P<0.05）。结论：香草醛通过调节SIRT1/HMGB1/TLR4/MyD88/NF-κB信号通路抑制HIBI引起的神经炎症,从而提高HIBI大鼠的神经功能。     

基于HMGB1/TLR4/NF-κB信号通路探讨忍冬苷对脓毒症肝损伤大鼠的影响

摘要 目的：基于高迁移率族蛋白B1（HMGB1）/Toll样受体4（TLR4）/核因子κB（NF-κB）信号通路探讨忍冬苷对脓毒症肝损伤大鼠的影响。方法：60只雄性SD大鼠随机分为模型组、对照组、阳性对照组（地塞米松10 mg/kg）、忍冬苷低剂量（7.5 mg/kg）、忍冬苷中剂量（15 mg/kg）、忍冬苷高剂量（30 mg/kg）组,每组10只。采用盲肠结扎穿刺法建立大鼠脓毒症模型。实验结束后麻醉大鼠取血制备血清,检测血清中超氧化物歧化酶（SOD）活性和丙二醛（MDA）、白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、白介素-10（IL-10）含量;分离肝组织称重,计算肝脏指数,一部分用于HE染色观察肝组织病理变化,一部分用于制备组织匀浆检测组织中肝功能指标谷丙转氨酶（ALT）、天冬氨酸转氨酶（AST）活性及HMGB1、TLR4、NF-κB蛋白表达。结果：与对照组比较,模型组大鼠血清中MDA、TNF-α、IL-6含量、肝脏指数以及肝组织中AST、ALT活性、HMGB1、TLR4、NF-κB蛋白表达显著增加（P＜0.05）,SOD活性与IL-10含量显著下降（P＜0.05）;且肝组织出现明显病灶,肝细胞水肿变性,大量炎性细胞浸润。与模型组比较,阳性对照组与忍冬苷各剂量组大鼠血清中MDA、TNF-α、IL-6含量、肝脏指数以及肝组织中AST、ALT活性、HMGB1、TLR4、NF-κB蛋白表达显著降低（P＜0.05）,SOD活性与IL-10含量显著升高（P＜0.05）;忍冬苷低、中剂量组仍可见病灶和水肿,但病变减轻;地塞米松组与忍冬苷高剂量组肝细胞结构趋于正常,未发现病灶。与阳性对照组比较,忍冬苷低、中剂量组大鼠血清中MDA、TNF-α、IL-6含量、肝脏指数以及肝组织中AST、ALT活性、HMGB1、TLR4、NF-κB蛋白表达显著升高（P＜0.05）,SOD活性与IL-10含量显著降低（P＜0.05）;忍冬苷高剂量组上述指标无显著差异（P＞0.05）。结论：忍冬苷通过下调HMGB1/TLR4/NF-κB信号通路的表达,抑制氧化应激,减轻炎症反应,改善肝功能,发挥对脓毒症肝损伤的保护作用。     

miR-21在大鼠心肌细胞缺血再灌注损伤中的作用机制研究

摘要 目的：探讨微小RNA-21（miR-21）在大鼠心肌细胞缺血再灌注损伤中的作用机制。方法：选取50只SPF级Wistar大鼠并随机分为5组（n=10）,分别为对照组、模型组、模型+阴性对照组、模型+ miR-21组和模型+ miR-21抑制物组。通过结扎大鼠左冠前降支进行建模。建模成功后采用高频彩色超声诊断仪检查各组大鼠的心脏功能指标：心脏射血分数（EF）、左心室收缩期峰值压力（LVSP）、左室舒张末压（LVEDP）和缩短分数（FS）。检测各组大鼠心肌梗死面积和心肌细胞凋亡率。采用酶联免疫吸附试验（ELISA）测定各组心肌组织中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-10（IL-10）的含量。采用反转录聚合酶链式反应（RT-PCR）检测心肌组织中miR-21的表达水平。蛋白免疫印迹试验（Western Blot）检测各组大鼠心肌凋亡蛋白和TLR4/NF-κB表达水平。结果：模型组大鼠出现心肌梗死,提示建模成功,建模后大鼠心肌组织中miR-21的表达水平显著下降,提示miR-21可能具有保护心肌细胞的作用。建模成功后,EF、LVSP和FS下降,LVEDP升高,心肌细胞凋亡率显著升高,TNF-α和IL-6表达水平显著升高,IL-10显著下降,Bcl-2/Bax表达下降,Caspase-3表达升高,大鼠心肌细胞TLR4和NF-κB蛋白磷酸化表达水平升高,而模型+ miR-21组上述指标均得到改善。结论：大鼠心肌细胞缺血再灌注损伤导致miR-21表达降低,而过表达miR-21能有效抑制TLR4/NF-κB信号通路,降低大鼠心肌凋亡水平和炎症因子的释放,从而发挥保护心肌细胞的作用。     

基于NF-κB通路探讨IRAK3基因表达对脂多糖诱导的心肌细胞损伤的作用及机制

摘要 目的：探讨白介素1受体相关激酶3（IRAK3）基因表达对脂多糖（LPS）诱导的心肌细胞损伤的作用及机制。方法：分离培养SD乳鼠原代心肌细胞,随机分为对照组、LPS组、siIRAK3组和siIRAK3+LPS组。siIRAK3组和siIRAK3+LPS组心肌细胞转染IRAK3沉默核糖核酸（siRNA）,对照组和LPS组转染阴性对照siRNA。转染48 h后LPS组和siIRAK3+LPS组分别用LPS（10 μg/mL）处理心肌细胞6 h,对照组和siIRAK3组加入等量的PBS溶液。采用免疫蛋白印迹法（Western blot）检测LPS对心肌细胞IRAK3表达的影响,采用细胞计数试剂盒-8（CCK-8）法检测四组心肌细胞增殖,采用原位末端转移酶标记技术（TUNEL）检测四组心肌细胞凋亡。采用酶联免疫吸附测定（ELISA）检测四组心肌细胞上清液中炎症因子白介素（IL）-6、肿瘤坏因子（TNF）-α的水平。Western blot检测核转录因子-κB（NF-κB）蛋白和NF-κB抑制蛋白α（IκB-α）的表达。结果：Western bolt结果显示,LPS使原代心肌细胞IRAK3的蛋白表达升高（P<0.05）,CCK-8和TUNEL结果显示,与对照组相比,LPS组心肌细胞活力降低,心肌细胞凋亡比例升高（P<0.05）;siIRAK3组细胞活力和细胞凋亡比例与对照组相比均无明显差异（P>0.05）。与LPS组相比,siIRAK3+LPS组心肌细胞活力升高,心肌细胞凋亡比例降低（P<0.05）。与对照组相比,LPS组心肌细胞分泌的IL-6、TNF-α升高,NF-κB蛋白表达升高而IκB-α蛋白表达降低（P<0.05）。与LPS组相比,siIRAK3+LPS组心肌细胞炎症因子分泌减少,NF-κB蛋白表达降低,IκB-α蛋白表达升高（P<0.05）。结论：干扰IRAK3基因表达通过负向调控NF-κB通路减轻LPS诱导的大鼠原代心肌细胞损伤。     

miR-22-3p通过激活PTEN/PI3K/AKT/NF-κB信号通路加重慢性阻塞性肺疾病

摘要 目的：探索miR-22-3p对慢性阻塞性肺疾病（COPD）的影响及作用机制。方法：将7周龄（体重250~280 g）雄性SD大鼠随机分为5组（n=12）：对照组（Con组）、COPD组、COPD+NC-agomir组、COPD+miR-22-3p-agomir组、COPD+NC-antagomir组和COPD+miR-22-3p-antagomir组。Con组大鼠为正常饲养的大鼠,其他组大鼠均通过香烟烟雾（CS）和脂多糖（LPS）诱导COPD动物模型。在CS暴露当天,Con组和COPD组大鼠鼻腔内注射50 μL生理盐水,COPD+NC-agomir组、COPD+miR-22-3p-agomir组、COPD+NC-antagomir组和COPD+miR-22-3p-antagomir组大鼠分别鼻腔内注射50 μL 10 nmoL的NC-agomir、miR-22-3p-agomir、NC-antagomir和miR-22-3p-antagomir,每2周注射1次,共注射6次。CS暴露90 d后,检测各组大鼠的最大自主分钟通气量（MVV）、0.3 s用力呼气量（FEV0.3）、用力肺活量（FVC）和最大呼气峰流速（PEF）。通过ELISA法测定支气管肺泡灌洗液（BALF）中白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和巨噬细胞炎性蛋白-2（MIP-2）水平。通过苏木精-伊红（HE）染色评价肺组织损伤。根据试剂盒说明检测肺组织丙二醛（MDA）和超氧化物歧化酶（SOD）水平。通过RT-PCR检测肺组织miR-22-3p、磷酸酶与张力蛋白同源物（PTEN）、诱导型一氧化氮合酶（iNOS）、CD86、CD206和精氨酸酶1（ARG1）mRNA水平。通过Western blot检测肺组织PTEN、磷脂酰肌醇3-激酶（PI3K）、p-PI3K、蛋白激酶B（AKT）、p-AKT、核因子-κB（NF-κB） p65和p-NF-κB p65的蛋白表达水平。结果：与COPD组和COPD+NC-antagomir组相比,COPD+miR-22-3p-antagomir组大鼠的MVV、FEV0.3/FVC和PEF均升高（P<0.05）;肺泡出血、肺泡壁断裂、肺泡间隔水肿和炎性细胞浸润等病变减轻;BALF中的IL-1β、TNF-α和MIP-2水平均降低（P<0.05）;肺组织SOD水平升高,MDA水平降低（P<0.05）;肺组织iNOS和CD86 mRNA相对表达量降低,CD206和ARG1 mRNA相对表达量升高（P<0.05）;肺组织miR-22-3p相对表达量降低,PTEN mRNA和蛋白相对表达量升高（P<0.05）;肺组织p-PI3K/PI3K、p-AKT/AKT和p-NF-κB p65/NF-κB p65的蛋白相对表达量降低（P<0.05）。结论：miR-22-3p在COPD大鼠肺组织中上调,可能通过激活PTEN/PI3K/AKT/NF-κB信号通路,促进肺组织炎症反应和氧化应激,加重大鼠肺功能障碍和肺组织结构损伤。     

TLR4/P38/JNK信号通路在海马神经元凋亡中的作用

目的：探讨Toll样受体4（TLR4）/P38/JNK信号通路在海马神经元凋亡中的作用及其机制,为神经退行性疾病（ND）的发病机制与防治研究提供新的实验依据。方法：采用体外培养7 d的新生大鼠海马神经元,免疫荧光双标法鉴定海马神经元纯度。用TLR4配体脂多糖（LPS）或TLR4抗体预处理海马神经元,以激活或阻断TLR4的作用。实验1设正常对照组、LPS组及TLR4抗体+ LPS组;免疫荧光法检测P-P38,P-JNK的表达。实验2分为6组：正常对照组,LPS组,TLR4抗体+ LPS组,SB202190（抑制P38） + LPS组,SP600125（抑制JNK） + LPS组,PD98059（抑制ERK） + LPS组;分别用TLR4抗体、P38、JNK及ERK的抑制剂预处理海马神经元后再给以LPS刺激24 h,Western blot法检测Bcl-2,Bax,Active-caspase-3的表达变化;流式细胞术检测海马神经元凋亡率。结果：LPS组海马神经元P-P38、P-JNK的表达明显高于正常对照组（P < 0. 01）,TLR4抗体+ LPS组P-P38,P-JNK表达显著低于LPS组（P <0.01）。与正常对照组相比,LPS组海马神经元Bcl-2/Bax表达减少、Active-caspase-3表达增加,海马神经元凋亡率增加（P < 0.01）。而TLR4抗体+ LPS组、SB202190 + LPS组、SP600125 + LPS组Bcl-2/Bax显著高于LPS组、Active cas-pase-3显著低于LPS组（P < 0.01）,海马神经元凋亡率显著低于LPS组（P < 0. 05,P < 0. 01）。PD98059 + LPS组与LPS组海马神经元凋亡率无明显差异。结论：①海马神经元中有TLR4介导的P38/JNK信号通路。②海马神经元TLR4激活后,P-P38、P-JNK表达增加,使Bcl-2/Bax的比例降低和Active-caspase-3表达增加,从而促进海马神经元的凋亡。海马神经元凋亡过程中有TLR4介导的P38/JNK信号通路的参与。     

丹参酮IIA缓解大鼠心肌梗死后左心室重构的作用及其机制研究

摘要 目的：探讨丹参酮IIA（T-IIA）对于缓解大鼠心肌梗死（MI）后左心室重构（LVR）的作用及其机制。方法：选取SD雄性大鼠80只,通过结扎左前降支（LAD）建立MI大鼠模型。将大鼠随机分为8组,假手术组未结扎LAD,其余各组均结扎LAD;除假手术组和MI组腹腔注射生理盐水外,其余各组分别给予T-IIA、脂多糖（LPS）和TAK-242治疗。HE和马松（Masson）三色染色评估MI大小、组织病理改变和纤维化程度。末端dUTP镍末端标记（TUNEL）染色观察心肌细胞凋亡情况。采用反转录定量聚合酶链反应(RT-qPCR)和蛋白免疫印迹试验检测Toll样受体4（TLR4）、髓样分化蛋白88（MyD88）和核因子κB（NF-κB）的表达水平。结果：T-IIA能改善MI大鼠心功能,可降低MI大鼠心脏体积,改善心脏形态,减轻MI大鼠的组织病理学改变,并有效减轻MI和心肌纤维化。T-IIA抑制MI大鼠的TLR4/MyD88/NF-κB信号通路,且能有效减少MI大鼠梗死边缘区心肌细胞凋亡。结论：T-IIA通过抑制TLR4/MyD88/NF-κB信号通路的激活,改善心脏形态、功能和病理组织学变化,有效减轻MI的严重程度,预防LVR。     

槲皮素调节AMPK/SIRT1/NF-κB通路对乙型肝炎大鼠肝组织损伤的影响

摘要 目的：探究槲皮素调节AMPK/SIRT1/NF-κB通路对乙型肝炎（HB）大鼠肝组织损伤的影响。方法：采用随机数字表法将65只Wistar大鼠分为Ctrl组、HB组、槲皮素低剂量组（槲皮素L组,50 mg/kg）、槲皮素高剂量组（槲皮素H组,200 mg/kg）及槲皮素H+AMPK抑制剂组（200 mg/kg槲皮素+10 mg/kg Compound C）,每组各13只,采用尾静脉注射携带1.3拷贝HBV基因组的重组8型腺相关病毒（rAAV8-1.3HBV）法建立HB大鼠模型（Ctrl组除外）;酶联免疫吸附实验（Elisa）检测血清中HB表面抗原（HBsAg）、HB e抗原（HBeAg）、肝功能指标[谷丙转氨酶（ALT）、谷草转氨酶（AST）、总胆红素（TBIL）]水平及肝组织中炎性因子[白介素-1β（IL-1β）、肿瘤坏死因子α（TNF-α）]水平;实时荧光定量PCR（qRT-PCR）检测血清中HBV-DNA水平;苏木素-伊红（HE）染色、曼森氏（Masson）染色观察肝组织病理改变;免疫印迹法（WB）检测肝组织中AMPK/SIRT1/NF-κB通路蛋白表达水平。结果：与Ctrl组比较,HB组血清中HBsAg、HBeAg、HBV-DNA、ALT、AST、TBIL水平均升高（P<0.05）;肝组织可见肝静脉扩张、充血,肝细胞排列紊乱、水肿、坏死,同时发生明显纤维化;肝组织中AMPK磷酸化、SIRT1蛋白水平均降低,IL-1β、TNF-α水平及核NF-κB蛋白水平均升高;经槲皮素L、槲皮素H干预后上述情况均得到改善,且槲皮素H干预改善更明显（P<0.05）;而增加AMPK抑制剂干预后,槲皮素H干预的改善作用被削弱（P<0.05）。结论：槲皮素能够减轻HB大鼠肝组织损伤,保护其肝功能,其机制可能与调节AMPK/SIRT1/NF-κB通路有关。     

基于体外实验对牡荆素的结肠炎调控机制的研究

牡荆素是从牡荆叶子中提取出的一种天然化合物，具有多种生物活性。经研究发现牡荆素对结肠炎具有良好的调控作用，但其具体机制还有待深入探讨。文章通过建立LPS诱导的Caco-2细胞体外结肠炎模型研究牡荆素对肠上皮细胞炎症的直接调控作用机制；通过研究结肠炎患者粪便的体外发酵探讨牡荆素对肠道菌群的调控作用。细胞实验结果表明牡荆素下调了LPS导致炎症因子TNF-α和IL-1β的表达升高，提高了紧密连接蛋白Occludin的表达水平，并通过抑制TLR4/MYD88/NF-κB炎症信号通路的激活发挥抑制炎症作用；体外发酵实验结果表明，牡荆素处理改善了结肠炎患者肠道菌群结构，降低了有害菌Escherichia-Shigella、Enterococcus和Clostridioides的丰度。综上所述，牡荆素可以通过抑制肠上皮细胞TLR4/MYD88/NF-κB炎症信号通路的激活和调节肠道菌群发挥结肠炎调控作用。     

血必净注射液对重症急性胰腺炎患者肾功能、炎性因子和外周血单核细胞TLR4/NF-κB信号通路的影响

摘要 目的：探讨血必净注射液对重症急性胰腺炎（SAP）患者肾功能、炎性因子和外周血单核细胞Toll 样受体4（TLR4）/NF-κB信号通路的影响。方法：采用随机数字表法,将南京医科大学附属宿迁第一人民医院于2019年4月~2021年12月期间收治的重症SAP患者108例分为对照组（常规治疗,n=54）和观察组（n=54,对照组基础上结合血必净注射液）。对比两组肾功能、炎性因子和外周血单核细胞TLR4/NF-κB信号通路相关指标的变化情况。同时观察两组疗效、临床症状改善情况和不良反应发生情况。结果：观察组临床总有效率较对照组高（P<0.05）。两组不良反应率比较无差异（P>0.05）。与对照组比较,观察组腹胀腹痛缓解时间、体温恢复正常时间、恶心呕吐消失时间、住院时间更短（P<0.05）。两组治疗7 d后尿素氮（BUN）、血肌酐（Scr）下降（P<0.05）,观察组治疗7 d后BUN、Scr低于对照组（P<0.05）。两组治疗7 d后白介素-6（IL-6）、白介素-1（IL-1）、肿瘤坏死因子-α（TNF-α）下降（P<0.05）,治疗7 d后观察组IL-6、TNF-α、IL-1较对照组低（P<0.05）。两组治疗7 d后TLR4、NF-κB下降（P<0.05）,观察组治疗7 d后TLR4、NF-κB低于对照组（P<0.05）。结论：血必净注射液可有效控制重症SAP患者炎性因子水平,减轻肾功能损伤,可能与调节外周血单核细胞TLR4/NF-κB信号通路有关。     

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).     

Occurrence of N-malonyl-D-alanine in pea seedlings

One of the ninhydrin-negative alanine conjugates isolated from pea seedlings was identified as $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$.The identification of this conjugate was carried out by a comparison of its gas-liquid chromatographic and mass spectrometric properties, and its nuclear magnetic resonance and infrared spectra with those of synthetic $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$. The alanine in the conjugate was shown to be present as the D-isomer by enzymatic and chromatographic analyses.     

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.     

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⁺.     

The effects of multiple features of alternatively spliced exons on the KA/KSratio test

Background  

The evolution of alternatively spliced exons (ASEs) is of primary interest because these exons are suggested to be a major source of functional diversity of proteins. Many exon features have been suggested to affect the evolution of ASEs. However, previous studies have relied on the K _A /K _S ratio test without taking into consideration information sufficiency (i.e., exon length > 75 bp, cross-species divergence > 5%) of the studied exons, leading to potentially biased interpretations. Furthermore, which exon feature dominates the results of the K _A /K _S ratio test and whether multiple exon features have additive effects have remained unexplored.     

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.     

Two-Electron Reactions S2QB → S0QB and S3QB → S1QB are Involved in Deactivation of Higher S States of the Oxygen-Evolving Complex of Photosystem II

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S₀-S₄), and dark incubation leads to 25% S₀ and 75% S₁. This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q_B⁻. We measured flash-induced oxygen evolution to understand how S₃ and S₂ are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q_B⁻. Thylakoid samples preconditioned to make the concentration of the S₁ state 100% and to oxidize tyrosine Y_D were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S₂ and S₃ are converted to lower S states via recombination from S₂Q_B⁻ and S₃Q_B⁻ and by a slow change of the state of oxygen-evolving complex from S₃ and S₂ to S₁ and S₀ in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S₂Q_B → S₀Q_B, and S₃Q_B → S₁Q_B. The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S₃Q_B → S₂Q_B → S₁Q_B in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H₂O₂ acts as an electron donor for the reaction S₂Q_B → S₀Q_B but added H₂O₂ did not enhance this reaction.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.     

Dermal cysts participate in reparative regeneration of epidermis in Hrhr/Hrhr mice

One of the phenotypic effects of mutation in the Hr gene in mice is disintegration of hair follicles and their degeneration into open funnel-shaped structures (utricles) opened on skin surface and cysts located in the depth of the dermis. The aim of the current study consists in analysis of the process of reparative regeneration of skin in homozygotous mice with one of the mutant alleles of the Hr gene—Hr ^hr . It is shown that epithelial cells that constitute the inner pavement of cysts take part in the process of epithelization of deep skin wounds. This indicates that the competence of ectodermal cells in relation to inductive signals from injured skin remains in Hr ^hr homozygote mice, in spite of the significant anatomic abnormalities of the hair follicles.     

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.