一氧化氮通过环磷酸鸟苷激活细胞外信号调节激酶／P^90RSK信号通路在大鼠脑缺血再灌注损伤中的作用

目的：观察大鼠脑缺血再灌注损伤时诱导型一氧化氮合酶源性一氧化氮、细胞外信号调节激酶激酶／细胞外信号调节激酶的信号转导通路，分析神经细胞损伤之间的联系。 方法：实验于2005-03／2006-03在中国医科大学第一临床学院麻醉实验室完成。32只Wistar大鼠随机摸球法分为4组，即对照组、模型组、诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组，每组各8只。采用4血管阻断法制作大鼠全脑缺血再灌注模型，对照组行假手术。诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组夹闭两侧颈总动脉前30min分别腹腔注射N-3-苯甲基-乙脒（诱导型一氧化氮合酶特异性抑制剂）45μmol／kg或SL327（细胞外信号调节激酶激酶特异性抑制剂）100mg／kg，对照组和模型组腹腔注射等量生理盐水。缺血20min再灌注24h后处死大鼠取海马，检测大鼠海马组织中一氧化氮、环磷酸鸟苷量的变化及诱导型一氧化氮合酶mRNA和细胞外信号调节激酶1／2、p^90RSK蛋白表达水平；电镜观察海马线粒体的变化。 结果：Wistar大鼠32只全部纳入结果分析。①模型组大鼠海马中一氧化氮、环磷酸鸟苷的量、诱导型一氧化氮合酶mRNA水平比对照组增高[（0．42&;#177;0．03），（0．21&;#177;0．02）μmol／g；（44．7&;#177;4．1），（19．6&;#177;1．8）nmol／L：1．07&;#177;0．04，0．25&;#177;0．02；P〈0．01]；诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组大鼠海马中一氧化氮、环磷酸鸟苷、诱导型一氧化氮合酶mRNA水平比模型组降低[（0．31&;#177;0．02），（0．44&;#177;0．03）μmol／g；（29．5&;#177;2．4），（46．3&;#177;4．2）nmol／L；0．70&;#177;0．03，1．10&;#177;0．04；P〈0．011。②模型组大鼠海马中细胞外信号调节激酶1／2，p^90RSK蛋白表达水平较对照组升高；诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组较模型组降低（P〈0．01）。③电镜观察模型组大鼠海马线粒体变性率比对照组升高（P〈0．01），诱导型一氧化氮合酶抑制剂组和细胞外信号调节激酶激酶抑制剂组大鼠海马线粒体变性率比模型组低（P〈0．01）。 结论：脑缺血再灌注损伤时，诱导型一氧化氮合酶源性的一氧化氮可能通过环磷酸鸟苷激活了细胞外信号调节激酶激酶／细胞外信号调节激酶/p^90RSK信号转导通路诱导了神经细胞的损伤。     

脑损伤大鼠皮质神经细胞凋亡过程中细胞外调节激酶通路的作用

目的:细胞外调节激酶通路在凋亡信号转导中具有重要作用,观察细胞外调节激酶信号转导通路在颅脑损伤中的作用。方法:实验于2004-09/2006-01在华北煤炭医学院中心实验室完成。选用SD大鼠70只,按随机数字表法分为2组,即对照组(n=28)和模型组(n=42),每组又分为伤后10min,30min,3h,6h,24h,48h和72h7个时相点,对照组每个时相点4只大鼠,模型组每个时相点6只大鼠。按照Mamarou方法建立大鼠重型弥漫性颅脑损伤模型,在不同时相点采用免疫组化、Western-blot法分别检测两组大鼠颅脑损伤后皮质细胞外调节激酶的表达情况,另外分别应用原位杂交方法、原位末端标记法检测半胱氨酸天冬氨酸酶3及凋亡细胞,并进行比较。结果:70只大鼠全部进入结果分析,无脱失。①模型组大鼠皮质p-细胞外调节激酶1/2蛋白在伤后10min时已较对照组明显升高(P<0.05),逐渐增强,伤后6h达高峰,24h仍有大量表达,明显高于对照组(P<0.01),至伤后48h降至对照组水平(P>0.05)。②模型组大鼠皮质半胱氨酸天冬氨酸酶3mRNA杂交阳性信号表达在伤后3h开始升高,48h达高峰,72h仍明显高于对照组(P<0.01)。③模型组大鼠皮质伤后3h偶见原位末端标记阳性细胞,6h开始增加,明显高于对照组(P<0.01),48h达高峰。④p-细胞外调节激酶1/2蛋白与半胱氨酸天冬氨酸酶3mRNA及原位末端标记阳性细胞均主要分布于损伤中心区及其周围,在分布区域上呈现很大的相似性。结论:脑损伤时p-细胞外调节激酶1/2蛋白表达呈一种过度激活状态,而且与半胱氨酸天冬氨酸酶3mRNA及原位末端标记阳性细胞在分布区域上呈现很大的相似性,推测大鼠脑损伤后细胞外调节激酶通路的过度激活是导致神经细胞凋亡的作用途径之一。     

骨骼肌缺血再灌注损伤中血管功能障碍与丹参的干预效应

目的:观察骨骼肌缺血再灌注损伤血清一氧化氮、诱导型一氧化氮合酶、内皮素1含量的变化及中药丹参的干预作用。方法:实验于2005-01/12在福建中医学院骨伤系实验室完成。实验分组:选用雄性SD大鼠84只,按随机数字表法分为丹参组、生理盐水组,每组42只,每组又分缺血再灌注10,20,30,40,50,60,90min7个时间点,每个时间点6只。实验方法:①制备大鼠左侧提睾肌缺血再灌注损伤模型。②丹参组于缺血2h时腹腔注射丹参注射液,生理盐水组给予相应剂量生理盐水;分别于缺血再灌注10,20,30,40,50,60,90min抽取腹主动脉血。实验评估:①采用硝酸还原酶法测定血清一氧化氮浓度。②采用比色法测定诱导型一氧化氮合酶活性。③采用放射免疫法测定内皮素1含量。结果:纳入大鼠84只,均进入结果分析。①一氧化氮、诱导型一氧化氮合酶的表达量:缺血再灌注10,20min两组一氧化氮、诱导型一氧化氮合酶的表达量无明显差异(P>0.05);缺血再灌注30,40,50,60,90min丹参组一氧化氮和诱导型一氧化氮合酶的表达量均高于生理盐水组[一氧化氮分别为(70.95±2.10),(68.21±2.23)μmol/L;(77.05±2.28),(72.20±1.56)μmol/L;(81.12±2.74),(74.60±1.90)μmol/L;(68.81±2.32),(62.03±2.80)μmol/L;(57.08±3.02),(46.77±3.01)μmol/L;诱导型一氧化氮合酶分别为(515.17±47.54),(459.78±37.27)μkat/L;(629.46±44.19),(499.37±29.46)μkat/L;(673.73±29.96),(584.77±58.48)μkat/L;(590.62±31.96),(507.78±31.82)μkat/L;(485.33±38.27),(378.64±38.04)μkat/L],差异有非常显著性意义(t=2.238,4.332,4.783,4.569,5.922;2.246,6.000,3.317,4.499,4.843,P<0.05,0.01)。缺血再灌注50min两组一氧化氮、诱导型一氧化氮合酶的表达量均达到最高峰,此后表达量开始下降。②内皮素1的表达量:缺血再灌注40min两组内皮素1的表达量均达到最高峰,此后表达量开始下降;缺血再灌注10min两组间内皮素1的表达量无明显差异(P>0.05),缺血再灌注20,30,40,50,60,90min丹参组内皮素1的表达量低于生理盐水组[分别为(145.77±26.54),(237.76±14.41)ng/L;(197.32±21.80),(258.50±40.20)ng/L;(124.44±6.00),(189.58±7.24)ng/L;(115.88±10.84),(165.93±10.43)ng/L;(103.96±3.84),(158.05±10.62)ng/L;(84.42±6.16),(113.69±10.41)ng/L],差异有非常显著性意义(t=7.462,3.278,16.959,8.149,11.731,5.926,P<0.01)。结论:骨骼肌缺血再灌注损伤导致血清中一氧化氮、诱导型一氧化氮合酶、内皮素1的表达量发生改变,丹参可能通过调整一氧化氮和内皮素1的平衡,改善缺血再灌注损伤造成的血管内皮功能障碍。     

诱导型一氧化氮合酶在关节软骨缺血再灌注损伤及软骨细胞凋亡中的作用

目的:观察兔膝关节软骨缺血再灌注损伤过程中诱导型一氧化氮合酶的表达规律及软骨细胞的凋亡情况,探讨诱导型一氧化氮合酶在关节软骨缺血再灌注损伤中的作用。方法:实验于2004-03/09在泰山医学院形态学研究室完成。①选择健康新西兰白兔45只,随机分为假手术组5只,暴露股动静脉但不阻断血运,术后4h取材;缺血组5只,缺血4h不恢复血运即取材;缺血再灌注组35只,阻断股动静脉4h后恢复血运,于缺血再灌注2,4,8,24h,3,7,14d取材,每时点各5只。②采用免疫荧光法测定软骨内诱导型一氧化氮合酶的表达阳性细胞,染色成功后用激光共聚焦显微镜观察并扫描记录。阳性细胞标记为红色荧光。③采用原位末端标记法测定凋亡软骨细胞数目,染色成功后在显微镜下观察并记数。细胞核中有棕黄色颗粒者为阳性细胞,即凋亡细胞。结果:纳入动物45只,均进入结果分析。①缺血再灌注组诱导型一氧化氮合酶阳性细胞数高于缺血组和假手术组[假手术组、缺血组、缺血再灌注2,4,8,24h,3,7,14d分别为0,(1.20±0.40),(22.20±1.30),(33.00±1.58),(40.00±1.58),(33.80±1.30),(18.20±1.48),(11.20±1.67),(3.00±1.00)个/mm2,P<0.01]。②缺血再灌注组软骨细胞凋亡数高于缺血组和假手术组[假手术组、缺血组、缺血再灌注2,4,8,24h,3,7,14d分别为[(0.50±0.40),(1.20±0.45),(7.80±1.30),(12.60±1.14),(16.60±1.12),(17.80±1.30),(15.20±0.87),(13.60±0.89),(4.40±1.67)个/mm2,P<0.01]。③诱导型一氧化氮合酶的表达与软骨细胞凋亡数高度相关(r=0.716,P=0.046)。结论:诱导型一氧化氮合酶参与了软骨缺血再灌注损伤,并可能是触发软骨细胞凋亡的重要因素。     

Aβ1～42致老年性痴呆大鼠海马神经元细胞外信号调节激酶1,2的表达

目的:建立老年性痴呆大鼠模型,观察老年性痴呆大鼠海马神经元细胞外信号调节激酶1,2表达的变化,探讨其在老年性痴呆发病机制中的作用。方法:采用立体定向下双侧海马注射Aβ1~42,建立老年性痴呆动物模型,经Y型电迷宫试验测试其行为学,采用免疫组织化学、蛋白印迹等方法观察细胞外信号调节激酶1,2表达变化。结果:模型组大鼠学习记忆能力较对照组显著下降,免疫组化显示海马CA1区细胞外信号调节激酶1,2的免疫反应阳性神经元数目及细胞平均光密度值均显著减少,蛋白印迹显示模型组较对照组条带变细,灰度值变小,差异均有统计学意义(P<0.05)。结论:海马神经元细胞外信号调节激酶1,2表达的减少可能参与了老年性痴呆的发病机制。     

睡眠剥夺引起大鼠海马神经元凋亡与丝裂素活化蛋白激酶表达的变化

背景:丝裂素活化蛋白激酶是一组与神经元的存活凋亡有关的蛋白激酶,睡眠剥夺可以引起神经元凋亡。目的:观察睡眠剥夺大鼠丝裂素活化蛋白激酶表达的变化并分析其可能的意义。设计:完全随机分组的前瞻性研究。单位:郑州大学生理学教研室神经研究室。材料:实验于2000-06/2002-10在郑州大学完成。选取成年健康SD大鼠24只。方法:24只大鼠随机分为快眼动睡眠剥夺组、快眼动睡眠剥夺对照组和正常对照组3组,每组8只。睡眠剥夺组从早晨8点始,连续剥夺睡眠72h。正常对照组则置饲养笼中饲养,维持正常的睡眠-觉醒周期。观察其形态学变化。另取24只大鼠分组同上用于丝裂素活化蛋白激酶的检测。采用TUNEL染色法观察睡眠剥夺大鼠的海马神经元形态学变化,观察细胞外信号调节激酶活性的变化和c-Jun氨基末端激酶蛋白表达量的变化。主要观察指标:①观察睡眠剥夺大鼠海马神经元的形态学变化。②观察海马神经元细胞外信号调节激酶和c-Jun氨基末端激酶表达的变化。结果:①海马神经元形态学变化:快眼动睡眠剥夺组大鼠CA1和CA3区可见较多的凋亡阳性细胞,主要分布在海马的锥体细胞层。CA2区仅见极少量凋亡细胞,CA4区偶见凋亡细胞。正常对照组和快眼动睡眠剥夺对照组海马组织切片中未见明显阳性细胞。②细胞外信号调节激酶活性的变化:快眼动睡眠剥夺组明显低于快眼动睡眠剥夺对照组和对照组(1764.00±941.56,6139.67±2863.62,566.700±2763.41,t=3.2111,0.9863,P<0.05)。③c-Jun氨基末端激酶的阳性表达:快眼动睡眠剥夺组明显高于快眼动睡眠剥夺对照组和对照组(87.5%,25%,75%,t=3.4121,P<0.05)。结论:睡眠剥夺可引起大鼠海马神经元丝裂素活化蛋白激酶活性的变化,可能与神经元的凋亡有关。     

运动结合补充葛根素干预糖尿病大鼠一氧化氮及一氧化氮合酶的变化

目的:分析有氧运动结合补充葛根素对链脲佐菌素所致糖尿病大鼠血清及肝脏中的一氧化氮含量和一氧化氮合酶活性的影响。方法:实验于2005-08/2006-01在江西师范大学体育学院完成。①选取8周龄雄性Sprague-Dawley大鼠60只,随机数字表法分为5组:正常对照组、模型对照组、运动组、葛根素组、运动 葛根素组,12只/组。②除正常对照组外,其余各组均喂以高脂饲料,腹腔注射链脲佐菌素建立糖尿病模型。血糖值≥14.7mmol/L且尿糖为～者确定为造模成功。正常对照组仅以普通饲料喂养,腹腔注射等体积0.1mmol/L的枸椽酸盐缓冲液。③造模后,运动组进行跑台训练,跑台坡度为0°,每天以10～15m/s的速度运动30min,共训练8周。葛根素组每天按500mg/kg体质量给予葛根素灌胃,共8周。运动 葛根素组跑台训练的同时给予葛根素灌胃。正常对照组、模型对照组不进行任何干预。各组均以普通饲料喂养。④分别于造模后第3天、造模后第8周末对各组大鼠尾静脉取血测定空腹血糖。造模后第8周末,各组大鼠乙醚麻醉,心脏取血,离心取上层无溶血血清,硝酸还原酶法测定一氧化氮含量。取新鲜肝脏组织1g,用40mmol/L的磷酸钾缓冲液制成匀浆,离心取上清检测一氧化氮合酶活性。结果:共54只大鼠进入结果分析。①空腹血糖检测结果:造模后第3天～第8周末,各模型组空腹血糖值均明显高于正常对照组(P<0.01)。与模型对照组比较,造模后第3天葛根素组、运动组、运动 葛根素组空腹血糖值无明显变化[(18.3±4.7),(18.6±3.0),(18.9±4.6),(19.1±2.9)mmol/L,P>0.05],第8周末均不同程度下降,尤以运动 葛根素组明显[(22.3±4.2),(9.4±2.0)mmol/L,P<0.01]。②造模后各组大鼠血清中一氧化氮含量及一氧化氮合酶活性的比较:与模型对照组比较,运动组、葛根素组一氧化氮含量及一氧化氮合酶活性均明显降低(P<0.01或0.05),运动 葛根素组降低的幅度尤为显著[(58.58±8.97),(30.80±14.80)μmol/L;(2.0±0.23),(0.92±0.13)μkat/L;P均<0.01]。③造模后各组大鼠肝脏组织中一氧化氮含量及一氧化氮合酶活性的比较:与模型对照组比较,运动组、葛根素组、运动 葛根素组一氧化氮合酶活性略有下降,但差异无显著性意义[(0.26±0.02),(0.23±0.03),(0.21±0.01),(0.19±0.03)μkat/L,P>0.05];运动组、葛根素组一氧化氮含量均明显降低[(4.21±0.83),(2.86±0.64),(3.39±0.86)μmol/L,P<0.05],运动 葛根素组降低至(1.75±0.31)μmol/L,差异尤为显著(P<0.01)。结论:有氧运动结合补充葛根素,能够有效降低糖尿病大鼠血清及脏肝中一氧化氮含量及一氧化氮合酶的活性。     

CD4＋与T淋巴细胞干扰素Y／诱导型一氧化氮合酶／一氧化氮通路在脐带间充质干细胞移植治疗重症肌无力中的作用

背景：有研究表明,CD4＋、干扰素γ/诱导型一氧化氮合酶/一氧化氮通路与重症肌无力的发生密切相关。 目的：探讨CD4＋ T细胞与干扰素γ/诱导型一氧化氮合酶/一氧化氮通路在脐带间充质干细胞移植治疗重症肌无力中的作用机制。方法：建立重症肌无力大鼠模型,并进行脐带间充质干细胞经静脉移植治疗,同时设立对照组。流式细胞术检测移植后大鼠腋窝淋巴结细胞CD4＋的表达,ELISA法检测其干扰素γ的表达,Griess试剂和比色法检测一氧化氮和一氧化氮合酶水平。结果与结论：移植1周后,移植组大鼠腋窝淋巴结的淋巴细胞CD4＋的表达显著高于模型组（P 〈 0.01）,干扰素γ、一氧化氮及诱导型一氧化氮合酶水平显著低于模型组（P 〈 0.01）。证实,脐带间充质干细胞移植可上调重症肌无力模型大鼠淋巴细胞CD4＋的表达,并调节干扰素γ/诱导型一氧化氮合酶/一氧化氮通路,下调一氧化氮水平,以减轻机体的免疫损伤。     

CD4~+与T淋巴细胞干扰素γ/诱导型一氧化氮合酶/一氧化氮通路在脐带间充质干细胞移植治疗重症肌无力中的作用

背景:有研究表明,CD4+、干扰素γ/诱导型一氧化氮合酶/一氧化氮通路与重症肌无力的发生密切相关。目的:探讨CD4+T细胞与干扰素γ/诱导型一氧化氮合酶/一氧化氮通路在脐带间充质干细胞移植治疗重症肌无力中的作用机制。方法:建立重症肌无力大鼠模型,并进行脐带间充质干细胞经静脉移植治疗,同时设立对照组。流式细胞术检测移植后大鼠腋窝淋巴结细胞CD4+的表达,ELISA法检测其干扰素γ的表达,Griess试剂和比色法检测一氧化氮和一氧化氮合酶水平。结果与结论:移植1周后,移植组大鼠腋窝淋巴结的淋巴细胞CD4+的表达显著高于模型组(P<0.01),干扰素γ、一氧化氮及诱导型一氧化氮合酶水平显著低于模型组(P<0.01)。证实,脐带间充质干细胞移植可上调重症肌无力模型大鼠淋巴细胞CD4+的表达,并调节干扰素γ/诱导型一氧化氮合酶/一氧化氮通路,下调一氧化氮水平,以减轻机体的免疫损伤。     

精氨酸强化全胃肠外营养对肠屏障功能的保护

目的:采用幼兔全胃肠外营养动物模型,观察精氨酸强化全胃肠外营养对肠屏障功能的保护作用。方法:实验于2004-06/2005-05在上海交通大学医学院附属新华医院中心实验室完成。选择新西兰幼兔24只,随机数字表法分为3组。正常对照组右颈静脉结扎后自由饮食,其余两组经右颈静脉插入硅胶管;标准全胃肠外营养组每天输注标准全胃肠外营养液(735kJ/kg,200mL/kg),精氨酸强化全胃肠外营养组输注精氨酸占总热量2%的等氮等热量的全胃肠外营养液。7d后分别取血浆及回肠标本进行检测:①肠黏膜形态学改变。②肠菌移位率。③血浆D-乳酸含量。⑤血浆和回肠组织一氧化氮含量、回肠组织一氧化氮合酶活性和诱导型一氧化氮合酶mRNA表达。结果:纳入动物24只,均进入结果分析。①肠黏膜在标准全胃肠外营养组明显变薄、萎缩(P<0.01),而精氨酸强化全胃肠外营养组肠黏膜的萎缩较标准全胃肠外营养组明显减轻[黏膜厚度分别为(333.12±36.29),(279.13±49.01)μm,P<0.05],与正常对照组差异无显著性意义(P>0.05)。②标准全胃肠外营养组细菌移位率明显高于正常对照组(分别为62.5%,0,P<0.01),精氨酸强化全胃肠外营养组肠菌移位率较标准全胃肠外营养组显著降低(分别为12.5%,62.5%,P<0.05),与正常对照组差异无显著性意义(P>0.05)。③精氨酸强化全胃肠外营养组血浆D-乳酸含量较标准全胃肠外营养组明显降低[分别为(3.886±1.243),(7.218±1.470)mg/L,P<0.01],但较正常对照组仍偏高(P<0.05)。④精氨酸强化全胃肠外营养组回肠组织的一氧化氮含量、一氧化氮合酶活性和诱导型一氧化氮合酶mRNA表达强度较标准全胃肠外营养组有显著增加[一氧化氮含量分别为(1.163±0.123),(0.901±0.252)μmol/L;一氧化氮合酶活性分别为(85.92±16.92),(67.76±15.57)μkat/g;诱导型一氧化氮合酶mRNA表达分别为71.0±10.1,60.4±9.4,P<0.05],与正常对照组差异无显著性意义(P>0.05)。各组血浆中一氧化氮含量差异无显著性意义(P>0.05)。结论:精氨酸对维持肠黏膜形态和功能的完整性具有重要作用,其作用机制与肠道诱导型一氧化氮合酶产生的一氧化氮有关,添加适量外源性的精氨酸具有改善全胃肠外营养所致小肠黏膜损伤的作用。     

Pyruvate kinase electrophoresis in normal and pyruvate kinase deficient hemolysates

Two bands of pyruvate kinase activity were demonstrated in normal hemolysates with high voltage electrophoresis at pH 5.3 using cellulose acetate strips.Heterozygotes with red cell pyruvate kinase deficiency also exhibited two bands. Six of ten homozygous patients showed two bands, the remaining four only one band. No mutant enzyme with abnormal electrophoretic mobility could be demonstrated among the ten homozygous individuals with pyruvate kinase deficiency. High voltage electrophoresis may be an additional procedure for the characterization of PK variants in patients with congenital hemolytic anemia associated with PK deficiency.     

Sulfur specifically inhibits adenylate kinase in assays for creatine kinase

Elemental sulfur is a specific and potent inhibitor of the muscle-type isoenzyme of adenylate kinase (EC 2.7.4.3). We find inhibition by sulfur and by diadenosine pentaphosphate to be similarly potent and specific. Some properties of inhibition of adenylate kinase isoenzymes by sulfur are given. The adenylate kinase isoenzymes from skeletal muscle, brain, and heart muscle are inhibited by sulfur; those from liver and kidney are not. Other enzymes not inhibited by sulfur include the isoenzymes of creatine kinase (EC 2.7.3.2). We show that creatine kinase can be measured in serum when adenylate kinase is inhibited by sulfur, and that the sensitivity and specificity of this inhibition are of the same order as the inhibition of serum adenylate kinase activity by AMP plus diadenosine pentaphosphate.     

Rho-Rho kinase pathway

The serine/threonine protein kinase Rho kinase, a downstream effector of the small GTPase Rho, regulates actin cytoskeletal organization, cell adhesion, cell motility, smooth muscle contraction and gene expression. Rho kinase constitutes the essential regulatory system for myosin phosphatase, controlling myosin light chain phosphorylation and thereby vascular tone. Inhibitors of Rho kinase were shown to be effective in ameliorating hypertension, vasospasm, vascular injury and associated organ damages in various animal disease models. These recent observations imply potential usefulness of the Rho kinase inhibitors as novel therapeutic and preventive agents for vascular disorders and accompanying organ injury.     

Cytoplasmic kinase inhibitors

Protein kinases play essential roles in the regulation of cell proliferation. Point mutations or/and fusions of protein kinases are frequently identified in human cancers, and targeting such activated kinases provides us with a chance to eradicate tumor cells. This was first proved by imatinib mesylate that inhibits ABL tyrosine kinase and, thereby, efficiently kills malignant cells in chronic myeloid leukemia. In addition, other clinical trials are ongoing for kinase inhibitors against EML4--ALK in lung cancer, JAK2 in myeloproliferative disorders and BRAF in malignant melanoma. Early reports indeed reveal that such targeting compounds are promising drugs for human cancers with activated kinases.     

Pyruvate kinase deficiency

Pyruvate kinase (PK) deficiency was initially described by Valentine et al. in 1961. Since then, more than 300 cases have been described, including 65 in Japan. PK deficiency is the most common hereditary nonspherocytic hemolytic anemia among several red cell enzyme defects of the Embden-Meyerhof glycolytic pathway. The clinical manifestations are highly variable. Splenectomy usually increases the hemoglobin level by about 2 g/100 mL. Standardization of methods for characterization of PK variants was achieved in 1979. There are four PK isozymes, M1, M2, L and R, in mammalian tissues. We have clarified the switch from M2-type to L-type PK during maturation of erythroid precursor cells. Recently we cloned and sequenced a full length human L-type PK cDNA. It will be useful to clarify the molecular basis of PK deficiency.     

Creatine kinase isoenzymes

Creatine kinase (CK), a widely distributed enzyme in the body, has its highest activities in skeletal muscle and myocardium; when serum CK activities are abnormally increased, injury to these organs must be part of the differential diagnosis. The isoenzyme CK-MB is the most important biochemical test in the diagnosis of acute myocardial infarction.     

Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions.     

Effect of exercise on plasma pyruvate kinase and creatine kinase activity

Plasma pyruvate kinase (PK) and creatine kinase (CK) were measured in healthy subjects engaging in (a) mild exercise, 30 min on an exercise cycle maintaining a pulse rate of 150/min, (b) moderate exercise, squeezing a ball until exhaustion with a sphygmomanometer cuff inflated above systolic pressure around the arm (max. 2 min) and (c) severe exercise, completing a marathon race. Mild exercise resulted in no change in enzyme levels over 24 h. Moderate exercise produced a small increase in PK but no change in CK. PK activity rose from 35.3 +/- 10 U/l pre-exercise to 41.3 +/- 13 U/l 15 min post-exercise (n = 8, p less than 0.025). Severe exercise (completing a marathon race) resulted in a 3-fold increase in PK from 26 (4-87) U/l pre-race to 69 (21-156) U/l immediately post-race, and also, as expected, an increase in CK from 60 (15-164) U/l to 257 (72-1535) U/l (results are means and ranges, n = 69, p less than 0.001 for both enzymes). Runners showed parallel increases in PK and CK (p less than 0.05 by Spearman rank correlation). The mean post-race activity of CK-MB was less than 5% of total CK but 18 runners had values greater than 6% (mean 4.8, range 1-18). We conclude that PK, like CK, is increased following exercise due to liberation of muscle enzyme. However, only severe exercise is likely to lead to a substantial increase in plasma PK activity and therefore prejudice its clinical usefulness as a diagnostic test.     

Lithium protection of phencyclidine-induced neurotoxicity in developing brain: the role of phosphatidylinositol-3 kinase/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathways

Phencyclidine (PCP) and other N-methyl-D-aspartate (NMDA) receptor antagonists have been shown to be neurotoxic to developing brains and to result in schizophrenia-like behaviors later in development. Prevention of both effects by antischizophrenic drugs suggests the validity of PCP neurodevelopmental toxicity as a heuristic model of schizophrenia. Lithium is used for the treatment of bipolar and schizoaffective disorders and has recently been shown to have neuroprotective properties. The present study used organotypic corticostriatal slices taken from postnatal day 2 rat pups to investigate the protective effect of lithium and the role of the phosphatidylinositol-3 kinase (PI-3K)/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways in PCP-induced cell death. Lithium pretreatment dose-dependently reduced PCP-induced caspase-3 activation and DNA fragmentation in layers II to IV of the cortex. PCP elicited time-dependent inhibition of the MEK/ERK and PI-3K/Akt pathways, as indicated by dephosphorylation of ERK1/2 and Akt. The proapoptotic factor glycogen synthase kinase (GSK)-3beta was also dephosphorylated at serine 9 and thus activated. Lithium prevented PCP-induced inhibition of the two pathways and activation of GSK-3beta. Furthermore, blocking either PI-3K/Akt or MEK/ERK pathway abolished the protective effect of lithium, whereas inhibiting GSK-3beta activity mimicked the protective effect of lithium. However, no cross-talk between the two pathways was found. Finally, specific GSK-3beta inhibition did not prevent PCP-induced dephosphorylation of Akt and ERK. These data strongly suggest that the protective effect of lithium against PCP-induced neuroapoptosis is mediated through independent stimulation of the PI-3K/Akt and ERK pathways and suppression of GSK-3beta activity.