异氟烷对小鼠星型胶质细胞Sirt1和MAO-A基因表达的影响

目的:探讨异氟烷对小鼠星形胶质细胞Sirt1和MAO-A基因表达的影响。方法:给予体外培养的新生小鼠原代星形胶质细胞不同浓度异氟烷处理,实验分为对照组和异氟烷处理组(0.5ISO、1.0ISO、1.5ISO),其中异氟烷组细胞分别给予0.5 MAC、1.0MAC和1.5 MAC三个浓度的异氟烷处理2小时,对照组给予O_2处理2小时,然后提取细胞RNA和蛋白检测Sirt1和MAO-A的mRNA和蛋白表达变化。结果:与对照组相比,异氟烷下调小鼠星形胶质细胞的Sirt1和MAO-A mRNA和蛋白水平,异氟烷浓度越大下调越明显。结论:异氟烷可抑制小鼠星形胶质细胞的Sirt1和MAO-A基因表达。     

异氟醚对小鼠神经干细胞活力及内大麻素系统相关基因表达的影响

目的:探讨异氟醚对小鼠神经干细胞活力及内大麻素系统相关基因表达的影响。方法:给予体外培养的小鼠海马神经干细胞不同浓度异氟醚处理,实验分为对照组和异氟醚处理组(0.5ISO,1.0ISO),其中异氟醚组细胞分别给予0.5 MAC和1.0 MAC两个浓度的异氟醚处理2小时,对照组给予O_2处理2小时,随后检测细胞活力并提取细胞RNA检测其内源性大麻素受体1(Cannabinoid receptor1,CB1)、脂肪酰胺水解酶(Fatty acid amide hydrolase,FAAH)、单酰甘油脂肪酶(Monoacylglycerol lipase,MAGL)及二酰基甘油脂肪酶(Diacylglycerol lipase alpha,DAGLα)的mRNA表达的变化。结果:与对照组相比,(1)异氟醚处理明显抑制了体外培养神经干细胞的活力,且高浓度异氟醚抑制作用强于低浓度异氟醚;(2)异氟醚上调神经干细胞的内大麻素受体CB1 mRNA水平,且高浓度异氟醚作用强于低浓度异氟醚;(3)异氟醚促进神经干细胞FAAH和MAGL的mRNA表达而抑制DAGLα的mRNA表达,且高浓度异氟醚作用强于低浓度异氟醚。结论:异氟醚可能通过影响小鼠神经干细胞的内大麻素系统影响神经干细胞的活力。     

不同剂量异氟醚对中年小鼠认知功能及海马MBP和pNF-H表达的影响

目的:探讨异氟醚对中年小鼠认知功能及海马髓磷脂碱性蛋白(MBP, myelin basic protein)和磷酸化神经丝重亚单位(pNF-H,phosphorylated neurofilament heavy chain)表达的影响。方法:给予中年小鼠不同浓度的异氟醚处理,实验分为对照组和异氟醚处理组(0.5ISO,1.0ISO,1.5ISO),其中异氟醚组小鼠细胞分别给予0.5 MAC,1.0 MAC和1.5 MAC三个浓度的异氟醚处理4小时,对照组给予O_2处理4小时,随后通过水迷宫测试检测其学习记忆能力变化,通过免疫荧光检测海马形态结构及髓鞘相关蛋白MBP和pNF-H表达变化。结果:与对照组相比,(1)类临床浓度的异氟醚处理不影响中年小鼠的自发运动能力(总运动距离:sham:7275.17±1732.58; 0.5ISO:8057.58±1732.58; 1.0ISO:7540.98±1401.61; 1.5ISO:8243.79±1257.65;运动速度:sham:116.75±22.35; 0.5ISO:135.45±32.84; 1.0ISO:130.16±21.38; 1.5ISO:142.31±20.58),但1.0 MAC和1.5 MAC的异氟醚处理明显降低了中年小鼠在水迷宫目标象限的活动时间百分比(sham:58.62±13.70; 0.5ISO:48.92±7.22; 1.0ISO:31.23±13.16; 1.5ISO:30.29±15.76)(P 0.05),且高浓度异氟醚作用强于低浓度异氟醚;(2) 1.0 MAC和1.5 MAC的异氟醚处理明显下调了海马的MBP和p NF-H表达(MBP:sham:60.48±8.20; 0.5ISO:56.69±7.86; 1.0ISO:40.15±4.50; 1.5ISO:31.66±5.46; pNF-H:sham:62.23±9.45; 0.5ISO:55.47±6.98; 1.0ISO:40.16±6.97; 1.5ISO:30.94±5.89)(P 0.05),造成了小鼠海马髓鞘结构损伤,且高浓度损伤强于低浓度。结论:异氟醚可能通过下调中年小鼠海马MBP和pNF-H表达,破坏海马髓鞘完整性而损伤小鼠的学习认知能力。     

调节TREK1对小鼠海马神经干细胞增殖和BDNF表达的影响

目的:观察调节TWIK相关的K+通道1(TREK1)对小鼠海马神经干细胞增殖和脑源性神经营养因子(brain derived neurotrophic factor,BDNF)表达的影响。方法:从孕10.5 d C57bl/6J小鼠胚胎海马中分离出神经干细胞并培养,待细胞达到70%～80%融合后,对细胞进行慢病毒干预,细胞分为sham组,Ctrl组(转染对照病毒),Plenti-TREK-1组(转染携带TREK1高表达载体病毒)和sh-TREK-1组(转染携带TREK1-shRNA病毒)。采用CCK-8法检测病毒干预后3天和7天各组神经干细胞的细胞活力,采用q RT-PCR检测病毒干预后7天各组神经干细胞TREK-1基因表达情况,并通过Elisa检测各组神经干细胞上清液BDNF表达水平。结果:与sham组相比较,(1)Plenti-TREK-1组TREK1基因表达上调,神经干细胞的神经球体积减小,细胞活力降低,细胞增殖减少,细胞上清BDNF水平下降;(2)sh-TREK-1组TREK1基因表达下调,神经干细胞的神经球体积增加,细胞活力增高,细胞增殖增加,细胞上清BDNF水平上调;(3)上述各项指标Ctrl组与sham组之间无统计学差异。结论:神经干细胞的增殖与TREK1通道密切相关,上调TREK-1可以抑制神经干细胞增殖及其BDNF的表达水平;下调TREK-1则可以促进神经干细胞增殖并上调其BDNF的表达水平。     

Notch信号通路参与卵巢生殖干细胞的调控及卵巢的衰老进程

卵巢生殖干细胞(ovarian germline stem cells, OGSCs)的发现,打破了生殖医学领域传统的"固定卵泡池"理论。近年来,OGSCs新的研究成果不断涌现,但关于OGSCs体内调控机制的研究仍然较少。Notch通路广泛参与多种成体干细胞不对称分裂的过程,并与细胞衰老密切相关,但其是否参与OGSCs的体内调控机制及卵巢的衰老进程尚不清楚。本研究以原代培养技术提取OGSCs,通过荧光双标染色发现,OGSCs标志基因MVH、Oct4与Notch信号通路相关分子Notch1、Hes1在OGSCs中存在共表达;抑制Notch信号通路活性后,cck-8检测发现,OGSCs的增殖活性呈下降趋势;而以免疫组化、荧光双标、Western印迹法检测性成熟期(2月龄)、不孕和衰老(20月龄)小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达变化,发现2月龄小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达量较高(P<0.05),而不孕和衰老小鼠卵巢皮层中,MVH、Oct4、Notch1和Hes1的表达量均明显下降。上述结果表明,Notch信号通路在小鼠OGSCs中高表达,并可能参与调控OGSCs的增殖机制及卵巢的衰老进程。     

Notch3对促进胰腺星形细胞活化的基因表达及信号通路的影响

目的: 分离培养小鼠胰腺星形细胞(PSCs),检测Notch3 对促进PSCs活化的基因表达及信号通路的影响。方法: 对小鼠PSCs进行分离培养及传代。采用免疫荧光染色检测活化的小鼠PSCs中α-SMA, fibronectin及collagen I的表达;细胞分组为空白对照组(MOCK组),阴性对照组(转染Notch3 siRNA negative control,NC组),Notch3 siRNA组(转染Notch3 siRNA,N3 siRNA组)及Notch3 siRNA-1组(转染Notch3 siRNA-1,N3 siRNA-1组),提取各组总RNA,测定RNA浓度及纯度后,送至安诺优达基因科技(北京)有限公司进行转录组测序。结果: 免疫荧光结果显示,在活化的PSCs中α-SMA,fibronectin及collagen I都有明显的表达。测序结果分析表明,与NC组相比较,在N3 siRNA组与N3 siRNA-1组,α-SMA基因,collagen I基因,fibronectin基因及CTGF基因均表达下调,与胶原蛋白代谢过程相关的基因表达上调,正向调节胶原生物合成的基因表达下调,而负向调节胶原生物合成的基因表达上调,PCNA基因表达下调;在N3siRNA组与N3siRNA-1组,调节细胞聚集的基因表达下调;在细胞组分部分,细胞外基质的基因表达下调;抑制PSCs中Notch3的表达可对细胞粘附分子信号通路,MAPK信号通路及TGF-β信号通路的组成成员的基因表达产生影响。结论: 抑制Notch3的表达可抑制PSCs的活化,降低细胞增殖能力,降低迁移聚集能力及ECM合成的能力;抑制Notch3的表达可对其他的信号如细胞粘附分子信号通路,MAPK信号通路及TGF-β信号通路产生影响。     

天麻素对镉诱导的星形胶质细胞损伤的保护效应

目的:探讨氯化镉(CdCl_2)和天麻素(GAS)对小鼠星形胶质细胞活力及神经营养因子GDNF和抗氧化基因Nrf2,HO-1,SOD-1表达的影响。方法:首先,给予体外培养的小鼠星形胶质细胞不同浓度的CdCl_2(Con,2.5μM,5μM,10μM,20μM)处理24 h或48 h,随后检测细胞活力筛选出造成星形胶质细胞损伤的CdCl_2浓度和时间。然后使用上述筛选的CdCl_2作用浓度(5μM)构建星形胶质细胞损伤的同时再给予不同浓度的天麻素(0,20μg/m L,30μg/m L,40μg/m L, 50μg/m L)处理24 h或48 h,随后检测细胞活力并提取细胞RNA检测其caspase3,GDNF(胶质源性神经营养因子Glial cell-derived neurotrophic factor,GDNF)和Nrf2(Nuclear factor erythroid2-related factor2),HO-1(Heme oxygenase 1),SOD-1(superoxide dismutase 1)等抗氧化基因的m RNA表达的变化。结果:(1) 2.5μM CdCl_2处理24 h后星形胶质细胞活力已经有明显下降(P0.05),5μM CdCl_2处理24 h后,星形胶质细胞活力显著下降(P0.01);(2) CdCl_2浓度越大,细胞损伤严重;(3)一定浓度的天麻素处理可以缓解CdCl_2造成的星形胶质损伤,恢复其细胞活力,下调caspase3 m RNA水平;(4) CdCl_2下调了星形胶质细胞的GDNF, Nrf2, HO-1和SOD-1的m RNA水平,天麻素可以抑制Cd Cl_2对上述基因的m RNA水平的调节作用,且浓度越高调节作用越强。结论:天麻素可能通过调节小鼠星形胶质细胞的GDNF, Nrf2, HO-1和SOD-1基因表达缓解CdCl_2导致的细胞损伤。     

RNA干扰Mcl-1基因表达对淋巴瘤Raji细胞增殖和凋亡的机制研究

目的:探讨抑制Mcl-1基因表达对淋巴瘤Raji细胞增殖和凋亡的影响及机制。方法:NC-siRNA、Mcl-1-siRNA转染Raji细胞,以不作任何处理的细胞作为空白对照组,48h后Western blot检测各组细胞中Mcl-1的蛋白表达;CCK8实验和流式细胞仪分别检测细胞的增殖和凋亡情况;Western blot检测Cleaved caspase3、Notch1、Hes1蛋白表达。结果:转染Mcl-1-siRNA后Mcl-1的蛋白表达显著降低;与对照组及NC-siRNA组比较,Mcl-1-siRNA组细胞存活率显著降低,细胞凋亡率显著升高,Cleaved caspase3蛋白显著上调表达,Notch1和Hes1蛋白显著下调表达。结论:RNA干扰抑制Mcl-1基因表达可显著降低Raji细胞增殖及诱导细胞凋亡,其机制与抑制Notch1信号通路有关。     

柚皮素通过Notch1/Hes1通路抑制肺癌干细胞增殖、迁移和分化

为探讨柚皮素对肺癌干细胞增殖、迁移和分化的分子机制,本研究应用免疫磁珠法分选肺癌干细胞(A549-CSCs),并通过流式细胞术进行表面分子的鉴定;通过CCK8法检测不同浓度的柚皮素(25μg/m L,50μg/mL, 100μg/mL)对肺癌干细胞(A549-CSCs)活力的影响,Transwell检测柚皮素对A549-CSCs细胞迁移能力的影响,Q-PCR检测柚皮素对肺癌干细胞分化相关因子Sox2和Oct4 m RNA表达的影响,Western blotting法检测柚皮素对细胞内Notch1和Hes1蛋白表达的影响。流式细胞术检测结果显示,A549-CSCs细胞表面分子CD133呈阳性表达,符合肺癌干细胞特征。CCK8结果显示,与对照组(control)比较,25μg/m L、50μg/mL、100μg/mL柚皮素处理A549-CSCs 24 h,细胞活力显著降低(p<0.05);Transwell检测结果显示,与对照组比较,不同浓度柚皮素处理组A549-CSCs迁移能力显著降低(p<0.05);定量PCR (real-time polymerase chain reaction, Q-PCR)结果显示,与对照组比较,柚皮素处理组细胞Sox2和Oct4 m RNA表达水平显著降低(p<0.05);蛋白质印迹法(Western blotting)结果显示,与对照组相比柚皮素处理组细胞Notch1和Hes1蛋白表达水平均降低。本研究发现柚皮素可能通过抑制Notch1/Hes1通路抑制肺癌干细胞增殖、迁移和分化。这为柚皮素治疗肺癌提供临床依据。     

活化的肝星状细胞RBP-J可诱导性基因敲除小鼠的构建及应用

目的:利用Cre-loxp可诱导系统构建活化的肝星状细胞RBP-J可诱导性基因敲除小鼠模型,以实现肝纤维化过程中活化的肝星状细胞Notch信号通路的特异性阻断。方法:将Sm22αCre ERT2和RBP-Jflox/flox转基因小鼠杂交,得到F1小鼠,将繁殖的F1小鼠继续进行交配,得到F2小鼠,通过PCR鉴定出基因型为Sm22αCre ERT2,RBP-Jflox/flox的小鼠。通过腹腔注射四氯化碳建立肝纤维化模型,检测四氯化碳注射不同时间段后肝脏纤维化进展情况和肝星状细胞活化过程中Sm22α的表达情况。注射他莫昔芬诱导活化肝星状细胞中RBP-J基因的特异性敲除。分选肝星状细胞,q-PCR和Western Blot检测活化肝星状细胞中Notch下游靶基因Hes1、Hey1表达情况以验证敲除效果。结果:通过连续交配我们获得了Sm22αCre ERT2,RBP-Jflox/flox小鼠。四氯化碳腹腔注射4周即可诱导肝脏发生较为明显的纤维化,同时肝星状细胞活化并逐渐高表达Sm22α。给予他莫昔芬诱导Cre重组酶发挥作用后,敲除了活化的肝星状细胞中的RBP-J基因,其下游基因Hes1、Hey1表达降低70%以上,阻断了Notch信号通路。结论:我们成功构建了RBP-J可诱导性条件性基因敲除小鼠模型,实现了小鼠肝纤维化过程中活化肝星状细胞Notch信号通路的阻断,为深入研究Notch信号通路在肝纤维化中的作用和机制奠定了坚实的基础。     

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.     

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.