奥利司他逆转肺腺癌A549/DDP细胞株顺铂耐药及机制研究

本研究的目的是观察奥利司他(Orlistat)逆转肺腺癌耐顺铂细胞株A549/DDP的耐药作用并考察其可能的分子机制。应用CCK-8法检测并计算肺腺癌耐药细胞株A549/DDP对顺铂(cisplatin, DDP)的耐药指数(resistance index, RI),筛选奥利司他的最佳实验浓度并观察其对A549/DDP细胞的耐药逆转效果;倒置荧光显微镜下观察各实验组细胞的形态学变化及Hoechst 33258荧光染色后细胞凋亡形态学改变;采用流式细胞术检测不同药物处理对细胞凋亡率的影响;Western blotting检测各实验组细胞P-gp、FASN、PI3K、Akt、p-Akt、NF-κB、Bcl-2和cleved-caspase-3的表达情况。结果显示,奥利司他抑制了A549/DDP耐药细胞株的增殖,且具有浓度依赖性。奥利司他与DDP联用增加了耐药株A549/DDP细胞对DDP的敏感性,耐药逆转倍数为5.02。倒置荧光显微镜观察到联合用药组细胞出现了明显的凋亡形态学改变。流式细胞术结果表明,与对照组和单药组比较,两种药物联用后细胞的凋亡率显著提高(p0.05)。Western blotting检测结果显示,相较于其它3组,联合用药组中耐药相关蛋白P-gp表达下调(p0.01),PI3K、p-Akt、NF-κB表达水平均显著降低(p0.01),抗凋亡蛋白Bcl-2表达下降(p0.01),凋亡相关蛋白cleved-caspase-3表达上调(p0.01);虽然FASN的表达在单用奥利司他组及联合用药组中均降低,但这两组间的FASN表达水平并无统计学差异。以上结果表明,奥利司他能够提高A549/DDP耐药细胞株对化疗药物DDP的敏感性,具有逆转肺腺癌细胞耐药性的作用,其机制与降低耐药蛋白P-gp的表达、抑制PI3K/Akt/NF-κB信号通路以及其下游凋亡相关蛋白有关。     

Wnt5a通过PI3K/Akt/GSK3β信号通路调控人卵巢癌SKOV3/VCR细胞对长春新碱的敏感性

本研究旨在探究Wnt5a对人卵巢癌SKOV3细胞长春新碱(vincristine, VCR)耐药性的影响,并探讨其分子机制。采用浓度梯度递增法构建耐药株SKOV3/VCR细胞,将人WNT5A基因的干扰质粒转染SKOV3/VCR细胞并筛选出稳定干扰Wnt5a的细胞株,用RT-PCR检测Wnt5a的mRNA表达水平,用CCK-8法检测细胞活力,用流式细胞术检测细胞凋亡,用Western blot法检测Wnt5a、MDR1、Survivin、β-catenin、Akt、p-Akt(S473)、GSK3β和p-GSK3β(Ser9)的蛋白表达水平。结果显示,SKOV3/VCR细胞中Wnt5a、MDR1、β-catenin和Survivin蛋白的表达水平、Akt和GSK3β蛋白的磷酸化水平以及Wnt5a mRNA表达水平均显著高于其亲代SKOV3细胞;WNT5A基因沉默使VCR抑制SKOV3/VCR细胞活力的IC50从38.412降至9.283 mg/L,提高SKOV3/VCR细胞凋亡率并协同增强VCR诱导的细胞凋亡(P 0.05),下调MDR1、β-catenin和Survivin蛋白的表达水平(P 0.05),抑制Akt和GSK3β蛋白磷酸化(P 0.05);此外,PI3K抑制剂LY294002也可下调SKOV3/VCR细胞中MDR1、β-catenin和Survivin蛋白表达水平,并降低Akt和GSK3β蛋白磷酸化水平(P 0.05)。以上结果提示,沉默WNT5A基因可在体外逆转人卵巢癌耐药株SKOV3/VCR细胞耐药性,作用机制可能与其抑制PI3K/Akt/GSK3β/β-catenin通路,继而下调MDR1和Survivin蛋白表达有关。     

KDM2A对人卵巢癌顺铂耐药细胞株A2780增殖和凋亡的影响及机制研究

目的:探讨赖氨酸脱甲基酶2A(Lysine-specific demethylase 2A,KDM2A)对顺铂(cisplatin,DDP)耐药的人卵巢癌细胞A2780细胞增殖和凋亡的影响及其可能作用机制。方法:通过构建慢病毒载体转染A2780/DDP,分为A2780、A2780/DDP、A2780/DDP/KDM2A(转染KDM2A)、A2780/DDP/Jagged1(转染Jagged1)以及A2780/DDP/NC(转染病毒载体)组。采用Western blot检测3组KDM2A、Jagged1、Bcl2和BAX蛋白表达,CCK8和平板克隆形成实验检测细胞对顺铂的敏感性,流式细胞术检测细胞凋亡情况。结果:A2780/DDP细胞KDM2A和Jagged1的蛋白表达水平均显著高于A2780细胞(P0.05),且A2780/DDP/KDM2A细胞中KDM2A和Jagged1的蛋白表达均低于A2780/DDP以及阴性对照组A2780/DDP/NC(P0.05);A2780/DDP/Jagged1细胞的Jagged1蛋白表达低于A2780/DDP以及A2780/DDP/NC(P0.05),而其KDM2A蛋白的表达比较差异无统计学意义(P0.05)。不同浓度DDP处理的A2780/DDP/KDM2A细胞的生长抑制率均显著高于A2780/DDP/NC和A2780/DDP细胞(P0.05),A2780/DDP/KDM2A细胞克隆形成数量亦明显高于A2780/DDP/NC和A2780/DDP细胞(P0.05)。A2780/DDP/KDM2A细胞凋亡率为(25.84±3.27)%,明显高于A2780/DDP细胞[(14.29±1.96)%](P0.05)和A2780/DDP/NC细胞[(12.46±2.15)%](P0.05)。A2780/DDP/KDM2A细胞中Bcl-2蛋白表达明显低于A2780/DDP细胞(P0.05),而A2780/DDP/KDM2A细胞Bax的表达水平却高于A2780/DDP细胞(P0.05)。结论:KDM2A可能通过上调Jagged1的表达,促进人卵巢癌细胞A2780的增殖并抑制其凋亡,进而降低人卵巢癌耐药细胞A2780的顺铂敏感性。     

miR-425-5p调节PTEN/PI3K/AKT轴对卵巢癌细胞顺铂敏感性的影响

该文探究微小RNA-425-5p(miR-425-5p)调节磷酸酶及张力蛋白同源基因(phosphatase and tensin homolog, PTEN)/磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase, PI3K)/蛋白激酶B(protein kinase B, AKT)轴对卵巢癌(ovarian cancer, OC)细胞顺铂(cisplatin, DDP)敏感性的影响。将人OC耐药细胞A2780/DDP随机分为DDP组、DDP+miR-425-5p抑制剂(inhibitor)组、DDP+阴性对照(NC) inhibitor组、DDP+miR-425-5p inhibitor+过氧钒(PIC, PTEN抑制剂)组,另设A2780细胞为对照组(Control组)。CCK-8法检测各组细胞的增殖能力。细胞划痕实验检测各组细胞的迁移能力。Transwell实验检测各组细胞的侵袭能力。流式细胞术测定各组细胞的凋亡率。荧光实时定量PCR法测定各组细胞中miR-425-5p以及PTEN、PI3K和AKT mRNA的表达情况。Western blot法测定...     

大黄素对顺铂耐药卵巢癌细胞的逆转作用及其相关基因表达研究

为了研究大黄素对人卵巢癌耐药细胞株SKOV3/DDP细胞耐药逆转作用及其机制,本研究以卵巢癌SK-OV3和多药耐药细胞株SKOV3/DDP为研究对象,通过噻唑蓝(MTT)法测定SKOV3/DDP细胞的耐药指数和大黄素在无细胞毒浓度下对卵巢癌细胞耐受顺铂(DDP)的逆转作用;采用Real-time PCR技术检测耐药相关基因HIF-1α、STAT1、CK2α、GSTP1 mRNA表达情况。结果发现无细胞毒作用浓度的7.8 mg/L和3.9 mg/L大黄素能逆转SKOV3/DDP细胞对DDP的耐药性,对DDP的逆转倍数分别为1.91倍和1.30倍,与SKOV3比较,SKOV3/DDP细胞的HIF-1α、STAT1、CK2α、GSTP1 mRNA表达明显升高(P<0.01)。3.9 mg/L和7.8 mg/L大黄素作用均可下调HIF-1α、CK2α、STAT1 mRNA表达,存在剂量-效应依赖关系。7.8 mg/L大黄素作用可下调GSTP1 mR-NA表达,但3.9 mg/L大黄素作用不明显。7.8 mg/L和3.9 mg/L大黄素联合IC50浓度的DDP时,四个耐药相关基因的表达与单独化疗药组作用相比明显下调(P<0.01)。提示无细胞毒浓度的大黄素对卵巢癌细胞耐药逆转的作用可能是通过下调HIF-1α、STAT1、GSTP1、CK2α的表达起作用。     

顺铂短期诱导人腺样囊性癌细胞NACC 产生耐药性的研究

目的:探讨人腭部涎腺腺样囊性癌细胞(NACC)经顺铂(DDP)短期诱导后耐药性产生情况及耐药机制。方法:采用恒定浓度DDP反复间歇诱导法诱导NACC细胞,获得耐药细胞NACC/DDP3,MTT法检测细胞耐药指数;实时荧光定量PCR检测存活蛋白(Survivin)及核苷酸切除修复交叉互补基因1(ERCC1)的mRNA表达;裸鼠右侧腋部皮下接种NACC及NACC/DDP3细胞,成瘤后将荷瘤裸鼠随机分为生理盐水对照组和DDP 2 mg·kg~(-1)组,比较2 mg·kg~(-1) DDP对两组荷瘤裸鼠的抑瘤率,HE染色观察肿瘤组织形态。结果:诱导后的NACC/DDP3对DDP耐药性增强,耐药指数为1.44;在NACC/DDP3细胞中,Survivin及ERCC1的mRNA表达明显上调,分别为亲本细胞的2.02(P0.01)和1.59(P0.05)倍;2 mg·kg~(-1) DDP对NACC组抑瘤率为(31.64±1.15)%,对NACC/DDP3组抑瘤率为(16.66±0.76)%,两组间差异具有统计学意义(P0.01),HE染色观察两组瘤体标本均符合腺样囊性癌实体型组织学表现。结论:NACC细胞经DDP短期诱导即产生一定耐药性,NACC/DDP3细胞中Survivin及ERCC1的mRNA表达上调,提示Survivin及ERCC1可能参与了腺样囊性癌细胞对DDP的耐药。相同剂量DDP对NACC/DDP3所建立的皮下移植瘤模型抑瘤率显著低于NACC组,提示NACC/DDP3接种到裸鼠体内仍具有耐药性,这将为体内研究耐药腺样囊性癌的治疗提供良好的平台。     

白血病耐药细胞系U937/ADR的建立及其生物学性状

目的：建立白血病耐药细胞系U937/ADR模型,并检测其多药耐药相关基因及其生物学性状的改变。方法：以大剂量阿霉素(IC50浓度),短时间(2h)暴露法诱导人白血病细胞系U937细胞的阿霉素耐药性。检测细胞的生长曲线,计算阿霉素耐药倍数,流式细胞术分析细胞周期分布;罗丹明123检测药物外排功能;荧光定量PCR（FQ-PCR）检测MDR1、MRP1、NF-Κb、Bcl-2、Bax mRNA水平变化;Western blot 检测Akt、p-Akt、P65、P-gp、MRP1和Bcl-2蛋白水平变化。结果：成功构建耐阿霉素U937/ADR细胞系,对阿霉素耐药指数为亲代U937细胞的11倍,U937/ADR群体倍增时间为43.6h,高于亲代细胞8.9h;流式细胞分析显示与U937细胞相比,U937/ADR的G0/G1期细胞增多,而G2/M期细胞减少。并对多种化疗药物产生交叉耐药性。罗丹明123外排试验显示,U937/ADR细胞外排明显增加。U937/ADR细胞MDR1、NF-Κb、Bcl-2 mRNA表达水平明显增加,P-gp及p-Akt、P65表达水平增加。结论：成功构建的U937/ADR细胞系其生物学特性明显不同与亲代U937细胞,对多种化疗药物产生多药耐药,高表达多药耐药蛋白P-gp,同时激活p-Akt及NF-Kb。     

红景天苷对脂多糖诱导小鼠巨噬细胞炎性介质分泌的影响

本文旨在探讨红景天苷(salidroside,Sal)对脂多糖(lipopolysaccharide,LPS)诱导小鼠巨噬细胞系J774.1炎性活化的影响及其可能机制。J774.1细胞分为PBS对照组、LPS(0.5μg/m L)刺激组和不同剂量(5、25、125μg/m L)Sal预处理+LPS组。CCK-8比色法检测细胞活性,ELISA测定培养上清中TNF-α、MCP-1和MIP-2含量,硝酸还原酶法测定上清中NO含量,RT-PCR检测细胞i NOS m RNA表达,Western blot检测胞浆i NOS蛋白和胞浆与胞核NF-κB/p65蛋白表达,Trans AMTM NF-κB/p65活性检测试剂盒测定NF-κB/p65 DNA结合活性。结果显示,0.5μg/m L LPS以及不同剂量(5、25、125μg/m L)Sal处理细胞12 h对J774.1细胞活力无影响;与LPS刺激组比较,LPS刺激前Sal预处理J774.1细胞,培养上清中TNF-α、MCP-1、MIP-2和NO含量呈剂量依赖性降低(P0.05),细胞i NOS m RNA和蛋白表达水平下调(P0.05),胞核NF-κB/p65蛋白表达降低(P0.05)而胞浆NF-κB/p65蛋白相应增加(P0.05),且NF-κB/p65 DNA结合活性呈剂量依赖性降低(P0.05)。以上结果提示,Sal预处理能够降低LPS诱导的巨噬细胞炎性活化,其机制可能通过干扰LPS/TLR4/NF-κB信号通路,从而降低炎性介质及细胞因子的过度表达和分泌。     

过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。     

IGFBP3基因启动子高甲基化所致表达下降促进胃癌化疗耐药

目的:研究胰岛素样生长因子结合蛋白3(IGFBP3)在胃癌化疗敏感及化疗耐药细胞和组织中的表达情况,初步探讨其在胃癌化疗耐药中的作用及表达异常机制。方法:采用荧光实时定量PCR(qRT-PCR)及蛋白质免疫印迹实验(WB)的方法检测IGFBP3在化疗药物敏感的胃癌细胞AZ521、SC-M1,对应顺铂耐药细胞AZ521/cisplatin、SC-M1/cisplatin和胃癌组织中的表达水平;在降低和增加IGFBP3表达量后,采用细胞计数试剂盒-8(CCK-8)试剂及流式细胞术(FCM)检测胃癌细胞对化疗药物的敏感性的变化;甲基化特异性PCR(MSP)检测IGFBP3基因启动子区甲基化水平。结果:IGFBP3在化疗耐受的胃癌细胞及组织中表达低于化疗敏感的胃癌细胞及组织(P0.05);在敏感细胞中干扰IGFBP3表达可促进胃癌细胞的耐药性,而在耐药细胞中恢复IGFBP3表达可显著逆转耐药性;MSP结果显示,IGFBP3的表达受DNA甲基化调控,耐药细胞中IGFBP3启动子高甲基化导致其表达下降(P0.05);甲基转移酶抑制剂地西他滨(DAC)处理耐药的胃癌细胞,可恢复IGFBP3表达,并提高其对化疗药物的敏感性(P0.05)。结论:IGFBP3启动子区发生DNA高甲基化导致其表达下降,使得化疗药物诱导胃癌细胞发生凋亡的能力下降,最终导致胃癌细胞的化疗耐药。     

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.     

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).     

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⁺.     

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.     

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.     

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.     

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.     

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.