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。     

反复呼吸道感染儿童血清维生素A、维生素E水平与免疫球蛋白、T淋巴细胞亚群、NK细胞及骨密度的关系分析

摘要 目的：探讨反复呼吸道感染（RRTI）儿童血清维生素A、维生素E水平与免疫球蛋白（Ig）、T淋巴细胞亚群、NK细胞及骨密度的关系。方法：选择2018年2月至2020年12月我院儿科收治的107例RRTI患儿（感染组）和83例同期于我院体检的健康儿童（对照组）为研究对象,检测两组血清维生素A、维生素E水平、Ig水平,外周血T淋巴细胞亚群、NK细胞占比以及骨密度。分析维生素A、维生素E与Ig、T淋巴细胞亚群、NK细胞及骨密度的相关性。结果：感染组血清维生素A、维生素E、IgG、IgA、IgM及外周血CD3⁺T细胞百分比、CD4⁺T细胞百分比、CD3^-CD56⁺ NK细胞百分比、CD56brightNK细胞百分比、CD56dimNK细胞百分比、桡骨和胫骨骨密度均低于对照组（P＜0.05）,外周血CD8⁺T细胞百分比高于对照组（P＜0.05）。血清维生素A及维生素E水平与外周血CD8⁺T细胞百分比呈负相关（P＜0.05）,与IgG、IgA、IgM水平,外周血CD3⁺ T细胞百分比、CD4⁺T细胞百分比、CD3^-CD56⁺ NK细胞百分比、CD56brightNK细胞百分比、CD56dimNK细胞百分比、桡骨和胫骨骨密度呈正相关（P＜0.05）。结论：RRTI患儿血清维生素A、维生素E水平明显降低,且与免疫功能障碍和骨密度降低有关。     

顺铂联合VEGF抗体对食管癌移植瘤小鼠免疫调节以及癌细胞增殖和肺转移的影响

摘要 目的：研究顺铂联合血管内皮生长因子（vascular endothelial growth factor,VEGF）治疗对食管癌移植瘤小鼠免疫功能、癌细胞增殖以及肺转移的影响。方法：30只BALB/c小鼠通过皮下注射食管癌移植瘤模型。一周后,30只食管癌移植瘤模型小鼠被随机均分为3组,即模型组、顺铂组和联合组。模型组不进行治疗,顺铂组腹腔注顺铂治疗,联合组腹腔注射顺铂联合尾静脉注射VEGF抗体进行治疗,共治疗7周。比较各组小鼠体重,食管癌移植瘤体积和重量,卵巢癌细胞肺组织转移结节数、癌细胞转移面积和转移病灶总数,以及食管癌移植瘤外周血CD4⁺、CD8⁺以及CD4⁺/CD8⁺ T淋巴细胞比例。结果：（1）顺博组和联合组小鼠体重均显著高于对照组,而联合组小鼠体重显著高于顺铂组（P<0.05）;（2）顺铂组和联合组小鼠CD4+和CD8+细胞比例均显著低于对照组（P<0.05）,而CD4⁺/CD8⁺却显著高于对照组（P<0.05）;（3）联合组小鼠CD4⁺和CD8⁺细胞比例均显著高于对照组（P<0.05）,而CD4⁺/CD8⁺却显著低于顺铂组（P<0.05）;（4）顺铂组和联合组小鼠食管癌肿瘤组织体积和重量,肺转移结节数、转移面积和转移病灶数均显著低于对照组（P<0.05）,而联合组小鼠显著低于顺铂组（P<0.05）。结论：VEGF抗体可以显著增强顺铂在体内对食管癌的抗癌特性,并有助于增强食管癌移植瘤小鼠免疫功能、抑制癌细胞体内增殖和肺部转移。     

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。     

外周血T细胞CD38和HLA-DR的表达水平在儿童传染性单核细胞增多症中的临床意义

摘要 目的：探讨传染性单核细胞增多症（IM）患儿外周血T细胞活化分子CD38和人类白细胞抗原DR（HLA-DR）表达水平的临床意义。方法：采用流式细胞术分别检测45例IM患儿急性期和恢复期的活化分子CD38和HLA-DR在T细胞的表达水平,并与30例健康体检儿童进行对比。分析IM患儿急性期CD38和HLA-DR在T细胞的表达水平与EB病毒载量、肝功能指标、外周血异型淋巴细胞比例、淋巴细胞计数的相关性,并采用ROC曲线分析CD8⁺CD38⁺T和CD8⁺HLA-DR+T细胞百分比的诊断效能。结果：与对照组比较,IM急性期患儿的CD38和HLA-DR在T细胞的表达水平显著升高（P＜0.05）。CD8⁺CD38⁺T、CD8⁺HLA-DR+T细胞百分比分别与EBV-DNA、ALT、AST、LDH、异型淋巴细胞百分比、淋巴细胞计数呈正相关（P＜0.05）,与白蛋白（ALB）呈负相关（P＜0.05）;CD4⁺CD38⁺T、CD4⁺HLA-DR+T细胞百分比与上述指标无显著相关性（P均＞0.05）。IM恢复期CD38和HLA-DR在T细胞的表达水平较急性期明显降低（P＜0.05）。ROC曲线分析CD8⁺CD38⁺T、CD8⁺HLA-DR+T细胞百分比显示诊断儿童IM的AUC值分别为0.931和0.993,特异度均为100%,灵敏度分别为88.89 %和93.33 %。结论：流式法检测CD38和HLA-DR在T细胞的变化有助于判断病情变化。外周血CD8⁺CD38⁺T、CD8⁺HLA-DR+T细胞百分比不仅能反映出IM急性期肝功能损伤严重程度,还可作为儿童IM的流式诊断指标。     

黄芪多糖基于PI3K/Akt通路对食管癌大鼠抑瘤作用及免疫功能的影响

摘要 目的：探究黄芪多糖（APS）对食管癌（EPC）模型大鼠的抑瘤作用、免疫功能及磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（Akt）信号通路的影响。方法：选取6周龄SPF级健康SD大鼠50只,随机分为模型组(M组)、顺铂组（S组）、黄芪多糖低剂量组（APSL）、黄芪多糖中剂量组（APSM）、黄芪多糖高剂量组（APSH）,每组10只。通过移植人食管癌Eca109细胞建立EPC模型,分别给予3 mg/kg顺铂和100、200、400 mg/kg的APS干预。测量各组大鼠肿瘤体积和肿瘤质量,计算肿瘤生长抑制率、胸腺指数和脾指数,HE染色观察肿瘤组织形态学,采用流式细胞仪检测外周血CD3⁺、CD4⁺、CD8⁺T淋巴细胞群,Western blot测定各组肿瘤组织中PI3K和Akt蛋白的相对表达。结果：经干预后,S组和各APS组大鼠肿瘤体积和肿瘤质量均显著小于M组（P＜0.05）;S组和APSH组肿瘤体积和肿瘤质量相显著小于APSM和APSL组（P＜0.05）;APSM组大鼠肿瘤体积和肿瘤质量显著小于APSL组（P＜0.05）;APSH、APSM和APSL组的抑瘤率分别为45.59%、32.35%和17.65%。S组大鼠的胸腺指数和脾指数均显著低于M组（P＜0.05）;各APS组大鼠的胸腺指数和脾指数均显著高于S组（P＜0.05）;APSH和APSM组大鼠胸腺指数和脾指数均显著高于M组（P＜0.05）。各APS组大鼠CD3⁺、CD4⁺和CD4⁺/CD8⁺均显著高于S组和M组,而CD8+均显著低于S组和M组（P＜0.05）;APSH、APSM和APSL组之间两两相比亦均有统计学差异（P＜0.05）。S组和各APS组PI3K/Akt信号通路蛋白的相对表达均显著低于M组（P＜0.05）;APSH、APSM和APSL组之间两两相比亦均有统计学差异（P＜0.05）。结论：APS可通过调控PI3K/Akt 信号通路和改善免疫功能,发挥对EPC的抑制作用,为临床治疗EPC提供了新的思路和理论依据。     

外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值研究

摘要 目的：分析外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值。方法：选择我院自2017年1月至2020年12月接诊的60例接受子宫颈癌根治术及盆腔淋巴清扫术的早期宫颈癌患者作为观察组,另选同期的60例健康体检者作为对照组。比较两组外周血Treg细胞、T淋巴细胞及其亚群水平,使用受试者工作特征曲线（ROC）下面积（AUC）评价外周血Treg细胞、T淋巴细胞及其亚群对淋巴结转移的预测效能。结果：观察组外周血Treg细胞、CD8⁺T细胞水平高于对照组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于对照组（P＜0.05）;观察组术后外周血Treg细胞、CD8⁺T细胞水平较术前降低,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均较术前升高（P＜0.05）;在60例早期宫颈癌患者中,发生淋巴结转移12例;淋巴结转移组术前外周血Treg细胞水平、CD8⁺T细胞高于非淋巴结转移组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于非淋巴结转移组（P＜0.05）;经多因素Logistic回归分析,外周血Treg细胞、CD3⁺T细胞、CD4⁺/CD8⁺比值均是早期宫颈癌患者发生淋巴结转移的独立预测因素（P＜0.05）;经ROC曲线分析,外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测早期宫颈癌患者发生淋巴结转移的AUC为0.910。结论：外周血Treg细胞、T淋巴细胞及其亚群水平与早期宫颈癌的病情演变有关,其中外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测淋巴结转移的效能较好,值得进一步研究应用。     

卡瑞利珠单抗联合卡培他滨用于一线治疗复发转移鼻咽癌患者维持治疗疗效及对患者免疫功能的影响

摘要 目的：探讨卡瑞利珠单抗联合卡培他滨用于一线治疗复发转移鼻咽癌（NPC）患者维持治疗的疗效及对患者免疫功能的影响。方法：选取2020年1月至2022年1月钦州市第一人民医院收治的80例复发转移NPC患者,按照随机数字表法分为对照组和观察组,每组各40例。两组均接受吉西他滨联合顺铂化疗,对照组化疗后接受卡瑞利珠单抗单药维持治疗至1年,观察组化疗后接受卡瑞利珠单抗联合卡培他滨维持治疗至1年。比较两组疗效,不良反应,治疗前后B淋巴细胞亚群和自然杀伤（NK）细胞所占百分比的差异。结果：观察组客观缓解率（ORR）、疾病控制率（DCR）高于对照组（P＜0.05）。两组治疗后外周血CD5⁺B细胞、CD5⁺CD19⁺ B细胞、CD3^-CD56⁺NK细胞占比降低（P＜0.05）,但观察组治疗后外周血CD5⁺B细胞、CD5⁺CD19⁺ B细胞、CD3^-CD56⁺NK细胞占比高于对照组（P＜0.05）。两组Ⅲ度以上不良反应发生率比较差异无统计学意义（P＞0.05）。结论：卡瑞利珠单抗联合卡培他滨一线维持治疗复发转移NPC可提高临床疗效,减轻对细胞免疫功能的影响,且安全可靠。     

脓毒症患者外周血T淋巴细胞PD-1表达变化及胸腺肽α-1的免疫调理作用研究

摘要 目的：探讨脓毒症患者外周血T淋巴细胞程序性细胞死亡受体1（PD-1）表达特点,分析胸腺肽?琢-1治疗对患者免疫功能的影响。方法：选择2018年3月至2020年6月我院重症医学科收治的140例脓毒症患者（脓毒症组）和同期于我院进行体检的95例健康志愿者（对照组）,根据急性生理与慢性健康评估Ⅱ（APACHE Ⅱ）、序贯器官衰竭评估（SOFA）评分结果将脓毒症患者分为APACHE Ⅱ 0~10分组（51例）、11~20分组（62例）和＞20分组（27例）;SOFA评分0~5分组（48例）、6~10分组（60例）和＞10分组（32例）。检测外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达,比较组间差异性。Pearson秩相关性分析外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达与APACHE Ⅱ、SOFA评分相关性。根据治疗方法将脓毒症患者分为A组（60例）和B组（80例）,A组给予常规综合治疗和乌司他丁治疗,B组在A组的基础上联合胸腺肽α-1治疗,比较两组治疗前后外周血T淋巴细胞（CD3⁺、CD4⁺、CD8⁺）、NK细胞（CD3-CD16⁺CD56⁺）差异。结果：脓毒症组外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达高于对照组（P＜0.001）,外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达随APACHE Ⅱ、SOFA评分的增加而增高,各组间差异显著（P＜0.05）。Pearson秩相关分析结果显示外周血CD4⁺T细胞上PD-1表达、CD8+T细胞上PD-1表达与APACHE Ⅱ评分、SOFA评分呈正相关（r=0.569、0.475;0.653、0.509,P均＜0.05）。B组治疗后CD3⁺、CD4⁺、CD3-CD16⁺CD56⁺高于A组（P＜0.05）,CD8⁺低于A组（P＜0.05）。结论：脓毒症患者外周血CD4⁺、CD8⁺T细胞上PD-1表达均增高,其表达与病情严重程度密切相关。给予胸腺肽α-1治疗可改善患者免疫功能。     

YKL-40、CD4+/CD8+比值与腺病毒肺炎患儿炎性因子和并发喘息的关系研究

摘要 目的：探讨血清壳多糖酶3样蛋白1（YKL-40）、外周血CD4⁺/CD8⁺比值与腺病毒肺炎（AP）患儿炎性因子和并发喘息的关系。方法：选取2019年10月～2022年10月湖北省妇幼保健院收治的97例AP患儿为AP组,根据是否并发喘息分别为喘息组和无喘息组,另选取同期50例体检健康儿童为对照组。收集AP患儿的临床资料,采用酶联免疫吸附法检测血清YKL-40和炎性因子[白细胞介素（IL）-6、IL-8、肿瘤坏死因子-α（TNFα）]水平,流式细胞术检测外周血CD4⁺、CD8⁺比例并计算CD4⁺/CD8⁺比值。采用Spearman相关性分析AP患儿血清YKL-40、CD4⁺/CD8⁺比值与炎性因子水平的相关性,多因素Logistic回归分析AP患儿并发喘息的影响因素。结果：与对照组比较,AP组血清YKL-40、外周血CD8⁺比例升高,CD4⁺比例、CD4⁺/CD8⁺比值降低（P＜0.05）。AP组血清IL-6、IL-8、TNF-α水平高于对照组（P＜0.05）。Spearman相关性分析显示,AP患儿血清YKL-40与IL-6、IL-8、TNF-α水平呈正相关,外周血CD4⁺/CD8⁺比值与IL-6、IL-8、TNF-α水平呈负相关（P＜0.05）。97例AP患儿住院期间喘息发生率为50.52%（49/97）。多因素Logistic回归分析显示,呼吸衰竭、小气道病变、特应性体质和血清IL-6、IL-8、TNF-α、YKL-40升高为AP患儿并发喘息的独立危险因素,外周血CD4⁺/CD8⁺比值升高为独立保护因素（P＜0.05）。结论：AP患儿血清YKL-40水平升高和外周血CD4⁺/CD8⁺比值降低,与炎性因子水平升高和并发喘息密切相关。     

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⁺.     

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.     

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.     

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.     

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.     

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.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca²⁺ and decreased approx. 1000-times when the Ca²⁺ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca_iZ) decreases in the order of $\text{Ca}{}_3\text{Z}\text{>}\text{Ca}{}_4\text{Z}\text{>}\text{Ca}{}_2\text{Z}\text{?}\text{CaZ}$. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca²⁺-ATPase in the range of 10^?7–10^?6 M Ca²⁺, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca²⁺-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca²⁺-transport ATPase in erythrocytes at the prevailing Ca²⁺ concentration (probably 10^?7 – 10^?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca²⁺-ATPase requires that the Ca²⁺ concentration rises to 10^?6 – 10^?5 M.