扶正方对Lewis肺癌小鼠免疫功能、PI3K/AKT信号通路和外周血IL-2、IL-6、INF-γ的影响

摘要 目的：观察扶正方对Lewis肺癌小鼠免疫功能、磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）信号通路和外周血白细胞介素（IL）-2、IL-6、γ干扰素（INF-γ）的影响。方法：将40只Lewis肺癌小鼠随机分为模型组（M组）、扶正方低剂量组（A组）、扶正方高剂量组（B组）、顺铂组（S组）,每组10只,A组、B组分别给予扶正方0.4 mL/20 g、0.8 mL/20 g灌胃,M组给予生理盐水0.4 mL/20 g灌胃,S组给予顺铂1 mg/mL,0.4 mL灌胃,连续14d,比较各组小鼠一般情况、肿瘤重量,胸腺指数、脾脏指数、脾脏CD3⁺细胞、CD4⁺细胞、CD8⁺细胞比例细胞百分比,鼠肿瘤组织PI3K/AKT信号通路蛋白表达水平及外周血IL-2、IL-6、INF-γ水平。结果：A组、B组、S组小鼠肿瘤重量低于M组,S组小鼠肿瘤重量低于A组、B组（P<0.05）,治疗前各组小鼠体重比较无统计学差异（P>0.05）,治疗后A组、B组小鼠体重高于S组、M组（P<0.05）。A组、B组小鼠胸腺指数显著高于M组、S组（P<0.05）。A组、B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于M组、S组,CD8⁺显著低于M组、S组（P<0.05）,B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于A组,CD8⁺低于A组（P<0.05）。A组、B组、S组小鼠肿瘤组织PI3K蛋白、AKT蛋白表达水平显著低于M组（P<0.05）。A组、B组、S组小鼠外周血IL-2、INF-γ水平显著高于M组,IL-6水平显著低于M组（P<0.05）。结论：扶正方可以提升Lewis肺癌小鼠免疫功能,调节IL-2、IL-6、INF-γ细胞因子水平,抑制PI3K/AKT信号通路起到抗肺癌的作用。     

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。     

老年支气管哮喘患者血清 MC-CP、S1P水平与 T细胞免疫分子及气道基底膜厚度的关系

摘要 目的：探讨老年支气管哮喘患者血清肥大细胞羧肽酶（MC-CP）、1-磷酸鞘氨醇（S1P）水平,并分析与 T细胞免疫分子及气道基底膜厚度的关系。方法：选择 2016年 8月至 2018年 12月我院收治的老年支气管哮喘患者 86例,根据哮喘控制情况分为急性发作期组 48例和缓解期组 38例,选择同期医院体检的健康老年人 40例作为对照组,检测并比较各组血清 MC-CP、S1P水平、肺功能、T细胞免疫分子、支气管黏膜网状基底膜厚度（BMT）,并分析血清 MC-CP、S1P水平与肺功能指标、T细胞免疫分子及BMT的相关性。结果：急性发作组、缓解组患者血清 MC-CP、S1P水平及 BMT、CD8⁺均高于对照组,且急性发作组患者血清MC-CP、S1P、CD8+水平高于缓解期组（P＜0.05）。急性发作期组和缓解期组 FEV1%pred、CD4⁺、CD4⁺/ CD8⁺,血清免疫球蛋白 A（IgA）、免疫球蛋白 M（IgM）、免疫球蛋白 G（IgG）水平低于对照组,且急性发作期组上述指标低于缓解期组（P＜0.05）。老年支气管哮喘患者血清 MC-CP、S1P与 CD8⁺、BMT呈正相关（P＜0.05）,与 FEV1%pred、CD4⁺、CD4⁺/CD8⁺、IgA、IgM、IgG呈负相关（P＜0.05）。结论：老年支气管哮喘患者的血清 MC-CP、S1P水平显著升高,其水平与与肺功能、免疫功能和气道重塑相关,且血清MC-CP与 S1P水平密切相关。     

传染性单核细胞增多症患儿NLR、CD4+/CD8+比值、ADA与EBV-DNA载量的相关性分析及对肝损害的影响

摘要 目的：探讨传染性单核细胞增多症（IM）患儿外周血中性粒细胞/淋巴细胞比值（NLR）、CD4⁺/CD8⁺比值、腺苷脱氨酶（ADA）与EB病毒（EBV）-脱氧核糖核酸（DNA）载量的相关性,分析其对IM患儿肝损害的影响。方法：选择2019年1月至2022年4月我院儿科收治的102例IM患儿（IM组）,另选择同期我科收治的95例EB病毒检测阴性的发热患儿（非IM组）和体检健康的73例健康儿童（对照组）。根据是否发生肝损害将IM患儿分为肝损害组（61例）和非肝损害组（41例）。比较外周血NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量,Pearson法分析NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量的相关性。多因素Logistic回归分析IM患儿发生肝损害的影响因素。结果：IM组ADA高于非IM组和对照组（P＜0.05）,且非IM组高于对照组（P＜0.05）,NLR、CD4⁺/CD8⁺比值低于非IM组和对照组（P＜0.05）,且非IM组低于对照组（P＜0.05）,IM组EBV-DNA载量高于非IM组（P＜0.05）。IM患儿ADA与EBV-DNA载量呈正相关（r=0.493,P＜0.05）,NLR、CD4⁺/CD8⁺比值与EBV-DNA载量呈负相关（r=-0.419、-472,P＜0.05）。肝损害组ADA、EBV-DNA载量高于非肝损害组（P＜0.05）,NLR、CD4⁺/CD8+⁺比值低于非肝损害组（P＜0.05）。肝脏肿大、高EBV-DNA载量、高ADA是IM患儿肝损害的危险因素（P＜0.05）,高NLR、高CD4⁺/ CD8⁺比值是保护因素（P＜0.05）。结论：IM患儿ADA增高,NLR、CD4⁺/CD8⁺比值降低,与EBV-DNA载量增加以及肝损害有关。     

贮存式自体成分输血对胃肠肿瘤根治术患者T淋巴细胞亚群、血液流变学和预后的影响

摘要 目的：研究贮存式自体成分输血对胃肠肿瘤根治术患者T淋巴细胞亚群、血液流变学以及预后的影响。方法：选取2016年12月~2017年12月我院收治的95例行胃肠肿瘤根治术的胃肠肿瘤早期患者作为研究对象。按随机数表法分为A组（n=47,贮存式自体成分输血）和B组(n=48,异体成分输血)。比较两组患者血常规指标[红细胞计数(RBC)、血红蛋白（Hb）、血细胞比容（Hct）]、免疫功能指标（CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺）、血液流变学指标[红细胞沉降率、平均血液黏度、红细胞刚性指数及红细胞变形指数]的变化。随访患者2年,采用Kaplan-Meier曲线分析两组预后情况。结果：术前至术后7 d 时间段A组的CD3⁺、CD4⁺水平及CD4⁺/CD8⁺呈先降低后升高趋势,且术后7 d已恢复至术前水平;B组术后1 d、3 d、7 d的CD3⁺、CD4⁺水平及CD4⁺/CD8⁺明显低于A组(P＜0.05),两组CD8⁺水平相比无差异(P＞0.05) 。两组术后7 d的RBC、Hct、Hb分别较输血前下降（P＜0.05）,但两组间比较差异无统计学意义(P＞0.05);两组患者术后7 d的红细胞沉降率均升高,但A组低于B组(P＜0.05) ,两组患者术后的红细胞变形指数、平均血液黏度和红细胞刚性指数比较无差异 (P＞0.05) 。Kaplan-Meier检验结果显示,A组较B组生存率明显升高（P＜0.05）。结论：贮存式自体成分输血对胃肠肿瘤根治术患者红细胞沉降率、细胞免疫功能的影响程度较异体成分输血减轻,且贮存式自体成分输血可以明显提高患者的术后生存率。     

千金藤素腹腔注射对卵巢癌模型大鼠的作用及其机制研究

摘要 目的：研究千金藤素腹腔注射对卵巢癌模型大鼠的作用及其机制。方法：选取30只SD级清洁级健康雌性大鼠,按照随机数字表法分成模型组、实验组与空白对照组,每组各10只。空白对照组不予以处理,模型组与实验组建立卵巢癌模型,且实验组在造模后予以千金藤素腹腔注射干预,使用剂量为20 mg/kg每次,1次/d,给药30d,模型组与空白对照组予以等量生理盐水干预。比较各组大鼠荷瘤重量、腹水量以及肿瘤体积,癌胚抗原（CEA）、糖类抗原153（CA153）及人附睾蛋白（HE4）表达水平,磷脂酰肌醇3-激酶（PI3K）、雷帕霉素靶蛋白（p-mTOR）、张力蛋白同源缺失的染色体（PTEN）表达,免疫指标水平。结果：模型组与实验组大鼠荷瘤重量、腹水量、肿瘤体积及CEA、CA153、HE4表达水平均高于空白对照组,且实验组大鼠上述各项指标水平均低于模型组（均P＜0.05）。模型组及实验组大鼠的PI3K、p-mTOR、PTEN表达水平均低于空白对照组,且实验组大鼠的PI3K、p-mTOR、PTEN表达水平均高于模型组（均P＜0.05）。模型组及实验组大鼠的CD4⁺、CD4⁺/CD8⁺水平均低于空白对照组,且实验组大鼠CD4⁺、CD4⁺/CD8⁺水平均高于模型组（均P＜0.05）;模型组及实验组大鼠的CD8⁺水平均高于空白对照组,且实验组大鼠CD8⁺水平低于模型组（均P＜0.05）。结论：千金藤素腹腔注射对卵巢癌模型大鼠具有明显的抑癌作用,有利于下调肿瘤标志物水平,其主要作用机制可能与调控PI3K/mTOR信号通路、PTEN表达以及免疫功能有关。     

狼疮性肾炎患者血清NETs、TWEAK、外周血CD4+T/CD8+T比例与疾病活动度及肾脏预后的关系

摘要 目的：探讨狼疮性肾炎（LN）患者血清中性粒细胞胞外诱捕网（NETs）、肿瘤坏死因子样凋亡微弱诱导剂（TWEAK）、外周血分化簇（CD）4⁺T/CD8⁺T比例与疾病活动度及肾脏预后的关系。方法：选取2021年8月～2022年8月川北医学院附属医院肾内科收治的LN患者137例（LN组）,根据系统性红斑狼疮疾病活动指数（SLEDAI）-2000评分分为轻度活动组（52例）、中度活动组（45例）、重度活动组（40例）。随访1年,根据肾脏相关终点事件发生情况分为预后不良组（43例）和预后良好组（94例）,另选取同期76名体检健康志愿者（对照组）。采用酶联免疫吸附法检测血清NETs、TWEAK水平,流式细胞术检测外周血CD4⁺T/CD8⁺T比例。Spearman相关性分析LN患者血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T与SLEDAI-2000评分的相关性,多因素Logistic回归分析LN患者预后不良的因素,受试者工作特征曲线分析血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T对LN患者预后不良的预测价值。结果：与对照组比较,LN组血清NETs、TWEAK水平升高,外周血CD4⁺T/CD8⁺T降低（P＜0.05）。轻度活动组、中度活动组、重度活动组血清NETs、TWEAK依次升高,外周血CD4⁺T/CD8⁺T依次降低（P＜0.05）。LN患者SLEDAI-2000评分与血清NETs、TWEAK呈正相关,与外周血CD4⁺T/CD8⁺T呈负相关（P＜0.05）。慢性肾脏病分期4期、SLEDAI-2000评分升高、NETs升高、TWEAK升高为LN患者预后不良的独立危险因素,估算肾小球滤过率升高、CD4⁺T/CD8⁺T升高为独立保护因素（P＜0.05）。血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T联合预测LN患者预后不良的曲线下面积为0.943,大于血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T单独预测的0.790、0.788、0.799（P＜0.05）。结论：LN患者血清NETs、TWEAK水平升高,外周血CD4⁺T/CD8⁺T降低,与疾病活动度及肾脏预后不良密切相关,血清NETs、TWEAK联合外周血CD4⁺T/CD8⁺T预测LN患者肾脏预后的价值较高。     

急性高容量血液稀释联合控制性低中心静脉压对脊柱后路手术患者炎症免疫调节及血液保护作用研究

摘要 目的：探讨急性高容量血液稀释（AHH）联合控制性低中心静脉压（CLCVP）对脊柱后路手术患者炎症、免疫调节、血液保护的作用。方法：选择2020年1月至2022年12月于南京鼓楼医院行脊柱后路手术的120例患者作为研究对象,根据随机数字表法分为对照组、AHH组、AHH+CLCVP组,每组各40例。对照组给予乳酸钠林格氏液血液稀释,AHH组给予AHH血液管理,AHH+ CLCVP组给予AHH联合CLCVP血液管理。比较三组围术期指标、血流动力学指标、炎症因子指标、免疫功能指标的变化情况。结果： AHH+CLCVP组术中出血量、悬浮红细胞输注量低于AHH组和对照组（P＜0.05）,AHH组术中出血量、悬浮红细胞输注量低于对照组（P＜0.05）。与T1时间点相比,三组心率（HR）于手术开始后60 min（T2）~术毕（T3）先升高后降低（P＜0.05）,平均动脉压（MAP）先降低后升高（P＜0.05）,对照组和AHH组中心静脉压（CVP）于T2~T3先升高后降低（P＜0.05）,AHH+CLCVP组T0~T3 CVP保持稳定水平（P＞0.05）。AHH+CLCVP组T2~T3 HR低于AHH组和对照组（P＜0.05）,MAP高于AHH组和对照组（P＜0.05）,CVP低于AHH组和对照组（P＜0.05）,AHH组T2~T3 HR低于对照组（P＜0.05）,MAP高于对照组（P＜0.05）。与T1时间相比,三组血清降钙素原（PCT）、C反应蛋白（CRP）、白细胞介素-6（IL-6）水平、外周血CD8⁺于T2~术后24 h（T4）先升高后降低（P＜0.05）,外周血CD3⁺、CD4⁺、CD4⁺/CD8⁺于T2~T4先降低后升高（P＜0.05）。AHH+CLCVP组T2~T4血清PCT、CRP、IL-6水平、外周血CD8⁺低于AHH组和对照组（P＜0.05）,外周血CD3⁺、CD4⁺、CD4⁺/CD8⁺高于AHH组和对照组（P＜0.05）,AHH组T2~T4血清PCT、CRP、IL-6水平、外周血CD8⁺低于对照组（P＜0.05）,外周血CD3⁺、CD4⁺、CD4⁺/CD8⁺高于对照组（P＜0.05）。结论：AHH联合CLCVP可稳定脊柱后路手术患者血流动力学,减少术中出血量和输血量,降低炎症反应,提高免疫功能。     

欧前胡素调节Gas6/Axl轴对膀胱癌荷瘤小鼠免疫功能的影响

摘要 目的：探讨欧前胡素（IMP）调节生长停滞特异蛋白6（Gas6）/Axl轴对膀胱癌荷瘤小鼠免疫功能的影响。方法：从60只SPF级雄性C57BL/6小鼠中随机选取12只小鼠作为对照组（CON组）,其余小鼠平均分为Model组、IMP组、Gas6组、IMP+Gas6组,每组12只,往其左侧前肢皮下注射0.2 mL浓度为1×10⁷个/mL的鼠源性膀胱癌肿瘤细胞系T739细胞溶液（Model组和IMP组注射对照载体的T739稳转株细胞,Gas6组和IMP+Gas6组注射过表达Gas6的T739稳转细胞株）造模。造模成功后,IMP组及IMP+Gas6组通过腹腔注射5 mg/kg的IMP;CON组、Model组及Gas6组通过腹腔注射等量的生理盐水,2天1次,持续5次。测量小鼠肿瘤体积和重量;测定胸腺指数和脾脏指数;酶联免疫吸附测定（ELISA）法检测血清白介素2（IL-2）、γ干扰素（IFN-γ）、血管内皮生长因子（VEGF）水平;苏木精-伊红（HE）染色检查肿瘤组织病理变化;流式细胞术检测T细胞亚群;TUNEL染色检测细胞凋亡;蛋白质印迹法（Western blot）检测Gas6/Axl通路相关蛋白表达。结果：Model组肿瘤细胞整体上排列松散,出现不同程度的核固缩、核碎裂现象;IMP则可以逆转该现象,使肿瘤细胞排列重新变得紧密,细胞核固缩程度提升,核碎裂现象降低;Model组较CON组肿瘤体积、肿瘤质量、CD8⁺细胞百分比、Gas6、Axl蛋白水平、VEGF水平显著增加（P<0.05）,胸腺指数和脾脏指数、IL-2、IFN-γ、CD4⁺细胞百分比、CD4⁺/CD8⁺、肿瘤细胞凋亡率显著下降（P<0.05）;与Model组相比,IMP组胸腺指数和脾脏指数、IL-2、IFN-γ、CD4⁺细胞百分比、CD4⁺/CD8⁺、肿瘤细胞凋亡率显著升高（P<0.05）,肿瘤体积、肿瘤质量、CD8⁺细胞百分比、Gas6、Axl蛋白水平、VEGF水平显著下降（P<0.05）;而Gas6组结果与IMP组趋势相反;而IMP+Gas6组的结果显示：IMP能逆转Gas6过表达后对膀胱癌荷瘤小鼠的促瘤作用。结论：IMP可能通过下调Gas6/Axl信号通路改善膀胱癌荷瘤小鼠免疫功能,从而产生抑瘤效果。     

NLR、25-（OH）D3、IL-6、PCT与重症肺炎支原体肺炎患儿免疫功能和预后不良的关系研究

摘要 目的：探讨中性粒细胞与淋巴细胞比值（NLR）、25-羟维生素D3 [25-（OH）D3]、白细胞介素-6（IL-6）、降钙素原（PCT）与重症肺炎支原体肺炎（MMP）患儿免疫功能和预后不良的关系。方法：选取2019年2月至2021年12月我院收治的106例重症MMP患儿作为重症组,同期收治的101例轻症MMP患儿（轻症组）作为对照。检测外周血中性粒细胞计数、淋巴细胞计数、T淋巴细胞亚群以及血清25-（OH）D3、IL-6、PCT水平,计算NLR。分析NLR、25-（OH）D3、IL-6、PCT与T淋巴细胞亚群的相关性。重症MMP患儿治疗后随访半年,根据重症MMP患儿的预后情况分为预后良好组（75例）和预后不良组（31例）,多因素Logistic回归分析影响重症MMP患儿预后的因素。结果：重症组NLR、IL-6、PCT水平,CD8⁺高于轻症组（P＜0.05）,25-（OH）D3水平、CD3⁺、CD4⁺、CD4⁺/CD8⁺低于轻症组（P＜0.05）。NLR、IL-6、PCT水平与CD3⁺、CD4⁺、CD4⁺/CD8⁺呈负相关（P＜0.05）,与CD8⁺呈正相关（P＜0.05）;25-（OH）D3与CD3⁺、CD4⁺、CD4⁺/CD8⁺呈正相关（P＜0.05）,与CD8⁺呈负相关（P＜0.05）。多因素Logistic回归分析显示：肺大片实变影、NLR（较高）、IL-6（较高）、PCT（较高）是重症MPP患儿预后不良的危险因素（P＜0.05）,25-（OH）D3（较高）是保护因素（P＜0.05）。结论：重症MMP患儿NLR、IL-6、PCT水平升高,25-（OH）D3水平降低,且与细胞免疫功能低下以及预后不良有关,检测NLR、IL-6、PCT、25-（OH）D3有助于评估重症MMP患儿的预后。     

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⁺.     

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.     

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.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(1) ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C₁₂E₈) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K⁺-stimulated Na⁺-influx is found to be about 35 μmol Na⁺ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na⁺ transported per ATP molecule hydrolyzed.     

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.     

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.     

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.     

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.