MicroRNA-221/222对乳腺癌MDA-MB-231/DOX细胞阿霉素耐药性的影响

目的:探讨微小RNA-221/222(miR-221/222)对乳腺癌MDA-MB-231/阿霉素(DOX)细胞DOX耐药性的影响。方法:采用脂质体法转染miR-221/222抑制物(miR-221/222 inhibitor)至MDA-MB-231/DOX细胞内(Inhibitor组),同时设立空白对照组和转染无关序列的阴性对照组,采用实时荧光定量PCR (qRT-PCR)检测MDA-MB-231细胞株及MDA-MB-231/DOX细胞株的miR-221/222表达水平及转染效率;CCK-8法检测转染48 h后MDA-MB-231/DOX细胞对DOX药物敏感性的变化;流式细胞术(FCM)检测转染MDA-MB-231/DOX细胞的细胞凋亡率;蛋白免疫印迹实验(WB)检测转染后MDA-MB-231/DOX细胞内促凋亡蛋白p53上调凋亡调控因子(PUMA),Bcl2蛋白修饰因子(BMF)以及细胞周期蛋白激酶抑制因子p27(p27Kip1)的表达情况。结果:MDA-MB-231/DOX细胞中的miR-221/222表达水平高于亲本MDA-MB-231细胞(P0.05);MDA-MB-231/DOX细胞转染miR-221/222 inhibitor 96 h后,miR-221/222的表达水平低于空白对照组和阴性对照组(P0.05);与空白对照组相比,MDA-MB-231/DOX细胞转染miR-221/222 inhibitor 48h后,DOX继续处理48 h后,细胞的凋亡率明显升高,且细胞内的促凋亡蛋白PUMA,BMF以及p27Kip1的表达均增加(P0.05);DOX对inhibitor组耐药细胞的半数抑制浓度(IC50)显著低于空白对照组细胞及阴性对照组(P0.05)。结论:miR-221/222能够增加MDA-MB-231/DOX细胞对DOX的耐药性,这可能与下调促凋亡蛋白的表达有关;降低miR-221/222水平可诱导MDA-MB-231/DOX凋亡,并且上调促凋亡蛋白的表达,从而部分逆转MDA-MB-231/DOX对DOX的耐药性。     

miR-21与DNA甲基化在不同乳腺癌细胞中的相互作用

目的:探究不同乳腺癌细胞(MCF-7、MDA-MB-231)中miR-21与DNA甲基化相互调节作用。方法:将荧光标记的miR-21抑制剂及阴性对照瞬时转入MCF-7、MDA-MB-231细胞中,用荧光显微镜观察其转染效率,以Real-time PCR检测miR-21的敲低水平,并以bisulfite-q MSP法检测基因组DNA甲基化水平。同时,以2.5μmol/L DNA甲基化酶抑制剂5-AZA处理细胞72 h,以单纯二甲基亚枫(DMSO)处理做为阴性对照,观察DNA甲基化改变对miR-21表达水平的影响,接着以Western blot检测miR-21下游基因人第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)、蛋白激酶B(AKT)蛋白的表达水平。结果:miR-21抑制剂可敲低MCF-7细胞中miR-21的表达水平(P0.01),并引起基因组DNA甲基化水平的显著升高以及DNA甲基化转移酶Dnmt1、Dnmt3a以及Dnmt3b的普遍升高(P0.05,P0.01)。而在MDA-MB-231细胞中瞬转miR-21抑制剂则引起miR-21表达水平的小幅度升高(P0.01)以及整体DNA甲基化水平的降低(P0.05),并伴随有Dnmt3a的升高及Dnmt3b的降低。使用5-AZA处理后发现,其可显著上调MCF-7以及MDA-MB-231细胞中miR-21的表达(P0.01),并引起其下游基因PTEN在MCF-7细胞内的表达升高,进而下调AKT的蛋白水平。结论:瞬转miR-21抑制剂对MCF-7与MDA-MB-231细胞DNA甲基化水平的调节截然相反,而DNA甲基化的降低则可使miR-21的表达一致上调。本研究可为以后不同类型乳腺癌的临床治疗提供一定的实验依据。     

PRMT5调控三阴性乳腺癌对多西他赛敏感性的研究

目的:探讨蛋白质精氨酸甲基转移酶5(protein arginine methyltransferase 5,PRMT5)对三阴性乳腺癌对多西他赛敏感性的影响,为三阴性乳腺癌的治疗提供新的思路。方法:通过包装慢病毒构建PRMT5过表达稳转细胞系和PRMT5敲除细胞系。采用平板克隆测定不同浓度下多西他赛对于MDA-MB-231细胞和PRMT5过表达细胞增殖的影响。采用流式周期和凋亡检测不同浓度多西他赛对PRMT5过表达和PRMT5敲除以及MDA-MB-231亲本细胞的周期以及凋亡的影响。采用Western Blot方法检测PARP以及p21、p27及LC3等表达。结果:成功构建PRMT5过表达及敲除稳转系。与对应对照组(MDA-MB-231细胞)比较,4nM和8nM的多西他赛处理的PRMT5过表达细胞系(MDA-MB-231-PRMT5细胞)形成克隆率明显降低(P0.05),细胞凋亡率明显增加(P0.05),细胞周期p21分子表达增多(P0.05),但未随浓度增加增高(P0.05),cycling D1、PARP剪切体、LC3-Ⅱ的表达均明显增加(P0.05)。4nM和8nM的多西他赛处理的PRMT5敲除稳转系(MDA-MB-231-sh PRMT5)细胞凋亡率、p21、p27、cyclin D1、LC3-Ⅱ的表达均较对应对照组(MDA-MB-231细胞)明显降低(P0.05),LC3-Ⅱ表达水平较亲本低(P0.05)。结论:PRMT5高表达可增加MDA-MB-231细胞对多西他赛的敏感性,PRMT5有望成为多西他赛临床治疗敏感性预测和方案选择的关键分子,并有望成为调控多西他赛等化疗药物耐药的新靶点。     

miR-429对乳腺癌干细胞干性维持和体内成瘤能力的影响

目的:探究miR-429在乳腺癌干性维持中所发挥的作用,并探索miR-429对乳腺癌干细胞体内成瘤能力的影响。方法:无血清悬浮培养法用于培养经流式细胞仪分选得到的CD44~+CD24~-表型乳腺癌细胞系干细胞MCF-7-S、SKBR3-S、MDA-MB-231-S及乳腺正常上皮干细胞MCF-10A-S,实时荧光定量聚合酶链式反应(qRT-PCR)用于检测miR-429在上述4株干细胞中的表达。将包含miR-429的重组慢病毒质粒及其阴性对照空载体质粒vector分别以病毒:细胞数量为15:1的比例感染MDA-MB-231细胞,经2.0μg/m L嘌呤霉素筛选,成功构建稳定表达miR-429或vector的MDA-MB-231细胞,经流式分选出上述两株稳转细胞株的CD44~+CD24~-表型干细胞MDA-MB-231-Svector和MDA-MB-231-SmiR-429。无血清悬浮培养后,镜下观察过表达miR-429对肿瘤球形成能力的影响,流式细胞术检测过表达miR-429对CD44~+CD24~-表型细胞亚群比例的影响,Western Blot检测过表达miR-429对乳腺癌干细胞干性相关因子ALDH1、SOX2和Bmi1蛋白表达的影响,将MDA-MB-231-Svector和MDA-MB-231-SmiR-429干细胞分别注射到BALB/c裸鼠右侧胸壁第二对乳腺脂肪垫中,构建乳腺癌干细胞裸鼠移植瘤模型,观察过表达miR-429对裸鼠体内成瘤能力的影响。结果:与MCF-10A-S相比,miR-429在MCF-7-S、SKBR3-S和MDA-MB-231-S细胞系中的表达水平均异常降低,其中,miR-429在MDA-MB-231-S细胞中表达最低(P0.05)。与MDA-MB-231-Svector细胞相比,经流式分选后的CD44~+CD24~-表型MDA-MB-231-SmiR-429干细胞形成的肿瘤球的大小和数量、分选时CD44~+CD24~-表型细胞亚群的比例、ALDH1、SOX2和Bmi1的蛋白表达水平以及裸鼠体内成瘤的体积和重量均显著降低(P0.05)。结论:miR-429可降低乳腺癌干细胞的干性和体内成瘤能力,其可能是抑制乳腺癌转移和耐药的关键分子。     

转化生长因子β1影响三阴性乳腺癌顺铂耐药的机制研究

摘要 目的：建立三阴性乳腺癌MDA-MB-231/顺铂（DDP）耐药细胞株,探讨转化生长因子β1（TGF-β1）调控三阴性乳腺癌DDP耐药的机制。方法：采用小剂量间歇诱导法建立MDA-MB-231耐药细胞株（MDA-MB-231/DDP）,在MDA-MB-231/DDP中构建TGF-β1沉默细胞并分为TGF-β1沉默组（sh-TGF-β1）、阴性对照组以及对照组,实时定量聚合酶链反应（RT-qPCR）检测TGF-β1含量。另取MDA-MB-231细胞和MDA-MB-231/DDP细胞分为MDA-MB-231组（正常培养MDA-MB-231敏感细胞）、MDA-MB-231-DDP组（正常培养MDA-MB-231 DDP耐药细胞）、TGFβ1-shRNA组（MDA-MB-231 DDP细胞转染TGFβ1-shRNA慢病毒载体）和MDA-MB-231-DDP+3-MA组（MDA-MB-231 DDP细胞给予5mM 3-MA处理2 h）。细胞计数试剂盒（CCK-8）法检测耐药株的半数抑制浓度（IC₅₀）,并计算耐药指数及逆转耐药指数,RT-qPCR检测TGF-β1含量,蛋白印迹法（Western blot）检测TGF-β1、自噬相关蛋白LC3-I、LC3-II表达量,激光共聚焦显微镜观察自噬流的变化,应用SPSS 20.0软件进行统计分析。结果：成功建立DDP耐药细胞株MDA-MB-231/DDP,耐药指数为5.231;MDA-MB-231/DDP细胞的TGF-β1 mRNA表达和蛋白表达较MDA-MB-231细胞显著上调（P<0.05）。DDP耐药细胞MDA-MB-231/DDP中自噬相关蛋白LC3-II/LC3-I表达较MDA-MB-231细胞显著升高（P<0.05);应用自噬抑制剂3-甲基腺嘌呤（3-MA）后MDA-MB-231/DDP细胞自噬相关蛋白LC3-II/LC3-I表达显著下降（P<0.05);沉默MDA-MB-231/DDP细胞的TGF-β1基因后,DDP耐药细胞株的耐药指数从5.231下降到3.404,同时自噬相关蛋白LC3-II/LC3-I表达降低（P<0.05）,且激光共聚焦显微镜观察到黄色和红色斑点的显著减少,表明自噬受到抑制。结论：TGF-β1与三阴性乳腺癌DDP耐药有关,其机制可能是增加自噬引起MDA-MB-231细胞DDP耐药。通过沉默TGF-β1可降低自噬水平,恢复三阴性乳腺癌细胞对DDP的敏感性。     

大豆苷元增强多西紫杉醇体外杀伤三阴性乳腺癌作用研究

目的:研究大豆苷元通过调节BRCA1表达逆转多西紫杉醇耐药的具体机制。方法:应用三阴性人乳腺癌细胞系MDA-MB-231、SUM-1315作为研究对象,通过MTT法评估分析不同治疗组的抑瘤率,应用流式细胞仪检测各治疗组细胞的凋亡情况;应用western-blot分析各治疗组BRCA1及凋亡相关蛋白的表达情况。结果:研究发现大豆苷元、多西紫杉醇及联合组对乳腺癌MDA-MB-231、SUM1315细胞的抑制呈浓度依赖的原则。并且BRCA1表达的细胞系MDA-MB-231比BRCA1突变的细胞系SUM1315抑瘤率更高,两个细胞系不同治疗组分别与对照组相比较,凋亡率明显升高,均具有统计学意义;且随着大豆苷元的加入,可使多西紫杉醇的凋亡率显著增加,并且可减少多西紫杉醇的用量。在SUM-1315细胞系,大豆苷元逆转多西紫杉醇耐药,增加凋亡比率更为显著。大豆苷元可使BRCA1突变型的三阴性乳腺癌细胞系SUM-1315表达BRCA1,并具有浓度依赖性。对各组细胞的凋亡蛋白的变化进行免疫印迹分析表明大豆苷元联合多西紫杉醇可通过调节caspase依赖的凋亡通路活性来增强多西紫杉醇的凋亡作用,从而增强乳腺癌细胞杀伤。结论:大豆苷元可通过调节BRCA1表达量,协同增强多西紫杉类药物杀伤三阴性乳腺癌细胞,促进凋亡进而逆转化疗耐药。     

靶向下调FOXM1 基因表达对乳腺癌MDA-MB-231 细胞增殖的影响

目的：探讨靶向抑制FOXM1 对乳腺癌细胞增殖能力的影响,为乳腺癌的个性化靶向治疗提供理论依据。方法：利用重组真 核转录载体pSilencer1.0-U6-FOXM1-shRNA,脂质体法转染乳腺癌细胞株MDA-MB-231,下调其FOXM1基因表达。采用四甲基 偶氮唑盐(MTT) 比色法、平板克隆形成实验观察细胞增值曲线以及克隆形成能力;采用实时定量- 聚合酶链反应(Real-time qPCR)、蛋白免疫印迹法（Western blot）分别检测FOXM1 基因在mRNA、蛋白水平的表达变化。结果：重组载体pSilencer1. 0-U6-FOXM1-shRNA转染MDA-MB-231细胞后,与对照组相比,增殖速率明显下降（P<0.05）,平板克隆形成显著减少（P<0.05）, 重组载体转染后显著抑制MDA-MB-231 细胞中FOXM1 基因在mRNA、蛋白水平的表达。结论：沉默FOXM1 基因对乳腺癌细胞 株MDA-MB-231 生长具有抑制作用,为阐明乳腺癌发病机制提供了新的切入点,也为临床抑制肿瘤生长提供了新的作用靶点。     

Cas9蛋白对亲骨转移人乳腺癌细胞株生物学性质和超微结构的影响

目的：构建表达Cas9蛋白的亲骨转移人乳腺癌细胞株,并研究Cas9蛋白的表达对细胞生物学性质和超微结构的影响,为利用CRISPR/Cas9技术研究乳腺癌骨转移分子机制提供实验基础。方法：通过慢病毒载体介导Cas9蛋白基因转染亲骨转移人乳腺癌细胞株MDA-MB-231BO,通过G418筛选转染细胞,采用流式细胞术、Western blot、免疫细胞化学验证Cas9基因转染是否成功;运用实时无标记动态细胞分析技术和细胞划痕实验检测Cas9蛋白表达对细胞增殖及迁移的影响;透射电镜观察Cas9蛋白表达对细胞超微结构的影响。结果：成功构建稳定表达Cas9蛋白的亲骨转移人乳腺癌细胞株MDA-MB-231BO-Cas9,Cas9蛋白的表达对细胞的增殖、迁移和超微结构无明显影响。结论：MDA-MB-231BO-Cas9细胞可用于CRISPR/Cas9技术进一步研究。     

慢病毒介导的靶向c-Met可诱导shRNA稳定乳腺癌MDA-MB-231细胞系的构建及生物活性鉴定

目的:探讨适当敲除c-Met后对乳腺癌MDA-MB-231细胞增殖的影响。方法:设计3条针对c-Met基因不同位点的短发夹RNA(shRNA)片段,将其连接到TA载体上,瞬时转染MDA-MB-231细胞,采用RT-PCR、Western印迹检测c-Met mRNA及蛋白表达情况,挑选沉默效率最佳的shRNA。以scramble为阴性对照,MDA-MB-231细胞为空白对照,挑选沉默效率最佳的TA-shRNA,并将其与pSD400慢病毒载体连接,利用293T细胞进行慢病毒组装,收集病毒液并感染MDA-MB-231细胞,将嘌呤霉素与MDA-MB-231细胞共培养,筛选稳定表达shRNA的细胞株。利用多西环素对稳定细胞系进行诱导,Western印迹检测稳定细胞系c-Met蛋白的表达量,MTT法检测敲低c-Met对稳定细胞系增殖的影响。结果:测序结果表明模板序列与设计序列正确,并筛选出沉默效率最高的shRNA为shRNA1,慢病毒包装后筛选出稳定细胞株,适当敲除MDA-MB-231细胞c-Met基因后细胞的增殖明显低于对照组。结论:构建了针对c-Met基因可诱导shRNA的乳腺癌稳定细胞系,为探讨适当敲除c-Met基因后乳腺癌细胞对化疗、放疗敏感性检测奠定了基础。     

骨唾液酸蛋白通过整合素αvβ3调控ILK信号通路

旨在探究骨唾液酸蛋白(BSP)是否通过整合素αvβ3对整合素连接激酶(ILK)信号通路进行调控。BSP基因沉默乳腺癌MDA-MB-231细胞,流式细胞仪在细胞水平检测BSP不同水平的细胞株中整合素αvβ3的表达量。Western blotting检测磷酸化ILK水平的变化,MTT法检测细胞增殖能力。与对照组231BO-Scrambled细胞相比,BSP基因沉默组231BO-BSP27细胞中整合素αvβ3的表达水平明显下调(61.32±1.94)%(P<0.01)。整合素αvβ3鼠抗单克隆抗体(LM609)处理前的BSP基因沉默组231BO-BSP27细胞与21BO-Scrambled细胞相比,ILK磷酸化水平下调明显(39.38±1.38)%(P<0.01);LM609处理后的231BO-BSP27细胞与21BO-Scrambled细胞相比,ILK磷酸化水平下调明显(33.78±1.51)%(P<0.01)。向乳腺癌细胞231BO-scrambled和231BO-BSP27中添加LM609,MTT试验结果显示两株乳腺癌细胞的增殖能力均有降低(P<0.05)。BSP通过整合素αvβ3对乳腺癌MDA-MB-231细胞ILK信号通路进行调控,并影响细胞增殖。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.