盟发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L-苹果酸、α-酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP/O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

布氏田鼠对低温的适应性产热

将布氏田鼠置１到３０天的低温（６±１℃）环境下暴露,其体重与体温没有明显变化;静止代谢率（ＲＭＲ）、非震颤性产热（ＮＳＴ）及血清中三碘甲腺原氨酸（Ｔ３）含量,随冷暴露时间的延长而增加;肝线粒体状态３呼吸及细胞色素Ｃ氧化酶活力亦随冷暴露时间的延长而升高。肝线粒体蛋白质含量经冷暴露３０天时有明显升高,表明动物在低温适应中肝线粒体蛋白合成增加与呼吸功能增强是个体ＲＭＲ提高的细胞学机制之一。布氏田鼠褐色脂     

绿豆幼苗线粒体氧化磷酸化偶联因子

分离和初步提纯了绿豆黄化幼苗线粒体氧化磷酸化偶联因子 F_1。结果表明,得到的偶联因子 F_1具有 ATP 酶活性。初步提纯的制剂活性比线粒体的活性提高约54倍,达到水解 ATP生成无机磷2.14微克分子/分钟/毫克蛋白,其最适 pH 为8.5,最适温度为45℃。绿豆线粒体的偶联因子是冷不稳定的;当它与膜分离处于溶解状态时,失去对二环己基碳二亚胺(DCCD)的敏感性;它水解 ATP 需要 Mg~(++),能为2,4-二硝基酚(DNP)激活,但 Ca~(++),NaCl和 KCl 的激活作用没有观察到。利用凝胶电泳证明,它的分子量约为380,000。     

萌发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L—苹果酸、a—酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP／O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

光合磷酸化偶联机制的研究寡霉素对PSP、ATP酶和ATP_b-Pi交换反应的作用

在过去工作基础上,我们进一步研究类囊体膜上牢固结合的ATP_b 与Pi 的交换反应和PSP 的关系。主要结果是:①在线粒体中对ATP 酶复合体的疏水蛋白专一敏感的寡霉素,对叶绿体中ATP 的光下形成和水解均表现为抑制,其中对ATP 酶活力的抑制要比对ATP 形成的抑制强烈得多,对核苷酸交换和光下H~ 吸收也有明显的抑制作用(表1)。②去除内源游离核苷酸的叶绿体悬浮液,与Pi(~(31)Pi ~(32)Pi)照光(不外加ADP),发现在适当的寡霉素浓度(20微克/毫克蛋白)下显著促进此系统ATP 中~(32)Pi 参入的数量;并且在所测温度下均促进,温度升高(30℃),促进作用更为明显(表2,3)。③用荧光素酶测定ATP 的方法对上述系统的反应产物进行鉴别,并与~(32)Pi酯化法相比较,证明寡霉素促进的是ATP_b-Pi 交换(图1,2;表4,6)。④ATP_b-Pi 交换反应与类囊体膜的能量转换有密切的关系。这交换反应需光、需辅助因子,也受解联剂的影响(表5),是需能反应。这ATP_b-Pi 交换,较之PSP 受解联剂的影响要小得多,可能它与膜上高能态有更为直接的联系。     

薄膜氧电极的制作与呼吸或光合控制的测定

线粒体的呼吸耗氧或叶绿体的光合放氧,都偶联着腺二磷(ADP)与无机磷(Pi)合成腺三磷(ATP)的磷酸化反应。ADP∶O值是指线粒体每吸收或叶绿体每释放一克原子氧的同时,酯化转变ADP成ATP的克分子数的比值,它反映这两种细胞器的能量转化效率。自从Chance根据线粒体系统的底物呼吸水平,氧吸收速率受到ADP和Pi促进而提出呼吸控制的观念以后,叶绿体的希尔放氧反应中也以同样的观念提出了光合控制。这些控制数值与ADP∶O比值一起,都成为衡量线粒体或叶绿体机构完善与否的重要生化指标。这些反应     

荧光偏振法测定肺细胞线粒体膜的流动性

本文在线粒体膜能量偶联ATP酶与矽肺发病关系研究的基础上进一步作矽尘对肺细胞线粒体膜流动性的影响。 材料和方法 1.动物 纯系Wistar雄性大白鼠体重250g左右,每个剂量组40只,按10 mg/ml、30 mg/ml和50mg/ml三种不同剂量的石英粉尘一次气管注入,染尘     

食物水限制对子午沙鼠肾和肝组织线粒体呼吸链复合物活性及相关自由基代谢的影响

目的观察食物水限制条件下对肾和肝组织线粒体呼吸链复合物活性的影响,研究组织线粒体能量代谢相关的响应特征。方法以健康成年子午沙鼠Meriones meridianus为材料,运用分光光度法测定了食物水限制3 d、6 d、9 d后子午沙鼠肾脏与肝脏组织线粒体呼吸链复合物Ⅰ~Ⅳ活性及超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量变化。结果水限制胁迫可引起肾和肝组织中线粒体呼吸链4种复合物的活性明显升高(P<0.05,P<0.01),体质量逐渐降低。其中水限制3 d是其适应性反应的重要阶段,3 d时呼吸链复合物活性升高幅度较大,9 d时活性均降低,但仍高于对照组。肾组织线粒体SOD活性呈不同程度升高,肾与肝组织线粒体MDA含量在水限制下显著升高。结论食物水限制引起肾和肝组织线粒体呼吸链复合物活性的升高与肾对水的重吸收和肝代谢增加有关,长时间水限制诱导自由基水平升高,对代谢酶活性的维持可能产生不利的影响。     

生物膜能量偶联ATP酶的比较研究——Ⅱ.鼠肝线粒体ATP酶(F_1)与人原发性肝癌线粒体内膜的杂交重组及其对肝癌发生的遗传控制的初步分析

1974到1975年,我们用人原发性肝癌细胞的线粒体内膜进行ATP酶活力测定,结果证明人肝癌线粒体ATP酶活力极低(0.04～0.1微克分子/分/毫克蛋白)只相当于正常大鼠线粒体的1/10～1/25(0.49～1.07微克分子/分/毫克蛋白)。Walker肉瘤和人肝硬变组织的线粒体与人肝癌的酶活力相近。电镜负染标本观察证明肝癌线粒体内膜大部分失去特征性的直径为90(?)的ATP酶颗粒,表现为光滑膜。ANS萤光探针的发射萤光光谱测定和2,4-二硝基酚的激活试验均证明人肝癌细胞线粒体内膜的ATP酶大量消失是肝癌细胞的特征之一。用提取的大鼠肝线粒体ATP酶(F_1)与人肝癌线粒体内膜进行人工杂交重组,结果证明,重组后的杂交膜的ATP酶活力比人肝癌线粒体内膜高6～11倍;寡霉素敏感性也显著提高。电镜负染标本观察表明杂交膜出现了典型的直径为90(?)的ATP酶的颗粒形态;ANS萤光增强效应测定证明杂交膜的萤光强度比肝癌膜高276%(相对单位);0℃低温处理2小时,ANS萤光强度不变;酶活力在0℃2小时后,仍相当于原来活力的90%。此项试验结果证明杂交重组获得成功。鼠肝线粒体ATP酶与人肝癌线粒体内膜杂交后的特性表现了与天然线粒体内膜的ATP酶的一系列相似的特性。讨论了ATP酶复合体杂交重组试验在探索肝癌发生与细胞中两个遗传系统控制的可能关系问题。     

基于H原子和O原子代谢的葡萄糖有氧氧化解析

通过解析葡萄糖有氧氧化偶联电子传递链合成ATP过程中的物质代谢与能量代谢,特别是基于H原子和O原子的来源与去路的全面追踪,明晰了葡萄糖有氧氧化途径不仅仅只是葡萄糖分子彻底分解代谢为CO2和H2O并合成ATP,还有葡萄糖和O2以外的物质(如Pi、Pi+GDP和H2O等)提供H原子和O原子参与合成ATP和脱羧生成CO2。     

急性热暴露大白鼠肝脏热休克蛋白形成的研究

本研究采用人工气候室模拟自然热环境对大白鼠进行急性热暴露实验。用苯酚法提取大白鼠肝脏总RNA;用 Oligo(dT)-纤维素亲和层析柱分离出Poly(A)⁺mRNA。将各条件下的大白鼠肝脏Poly(A)⁺mRNA在麦胚无细胞体外转译系统中表达。结果证明急性热暴露大白鼠肝脏同样能生成分子量分别为71kD、90kD、98kD和 110kD的一组热休克蛋白。     

CHROMOSOMAL ANALYSIS OF HEAT-SHOCK TOLERANCE IN DROSOPHILA MELANOGASTER EVOLVING AT DIFFERENT TEMPERATURES IN THE LABORATORY

We investigated the heat tolerance of adults of three replicated lines of Drosophila melanogaster that have been evolving independently by laboratory natural selection for 15 yr at “nonextreme” temperatures (18°C, 25°C, or 28°C). These lines are known to have diverged in body size and in the thermal dependence of several life-history traits. Here we show that they differ also in tolerance of extreme high temperature as well as in induced thermotolerance (“heat hardening”). For example, the 28°C flies had the highest probability of surviving a heat shock, whereas the 18°C flies generally had the lowest probability. A short heat pretreatment increased the heat tolerance of the 18°C and 25°C lines, and the threshold temperature necessary to induce thermotolerance was lower for the 18°C line than for the 25°C line. However, neither heat pretreatment nor acclimation to different temperatures influenced heat tolerance of the 28°C line, suggesting the loss of capacity for induced thermotolerance and for acclimation. Thus, patterns of tolerance of extreme heat, of acclimation, and of induced thermotolerance have evolved as correlated responses to natural selection at nonextreme temperatures. A genetic analysis of heat tolerance of a representative replicate population each from the 18°C and 28°C lines indicates that chromosomes 1, 2, and 3 have significant effects on heat tolerance. However, the cytoplasm has little influence, contrary to findings in an earlier study of other stocks that had been evolving for 7 yr at 14°C versus 25°C. Because genes for heat stress proteins (hsps) are concentrated on chromosome 3, the potential role of hsps in the heat tolerance and of induced thermotolerance in these naturally selected lines is currently unclear. In any case, species of Drosophila possess considerable genetic variation in thermal sensitivity and thus have the potential to evolve rapidly in response to climate change; but predicting that response may be difficult.     

热休克T细胞中mRNA的表达

 富集的人外周血T淋巴细胞经40—42℃保温(热休克)可诱导产生90kd和71kd的两种主要热休克蛋白(HSP),此外,62和34kd HSP也可在不同条件下诱导。从不同处理的T淋巴细胞中提取mRNA并在免网织细胞裂解液系统中进行体外转译,显示出相同的主要HSP。其中71和62kdHSP不仅在升温时还可在低温(4℃)下诱导,表明淋巴细胞中几种主要HSP的诱导机制不完全相同。比较L-~(35)S-Met参入实验和体外转译的结果提示淋巴细胞中HSP基因表达主要在转录水平调控。     

热性发热兔血浆脂多糖（LPS）浓度与生理反应的变化

在干球温度４２℃、湿球温度３５℃、相对湿度６０％条件下,测定了２４只热暴露兔［分为肛温维持４３℃（Ⅰ）组和肛温持续上升（Ⅱ）组］的心率、平均动脉压、呼吸频率、肛温及血浆ＬＰＳ浓度等指标。结果显示：（１）Ⅰ、Ⅱ组当Ｔｒ升达４３℃,热暴露时间分别为１００ｍｉｎ、６０ｍｉｎ时,血浆ＬＰＳ浓度分别为０．１９５ｎｇ／ｍｌ、０．１８０ｎｇ／ｍｌ,与实验前比,Ｐ＜０．０５。两组临死前,热暴露时间分别为２２０ｍｉｎ、１２０ｍｉｎ（Ⅱ组Ｔｒ４４．１５℃）时,其血浆ＬＰＳ浓度分别为０．２８５ｎｇ／ｍｌ、０．２４９ｎｇ／ｍｌ,Ｐ＜０．０１;（２）Ｔｒ４３℃和临死前两阶段ＬＰＳ的上升速率,Ⅰ组分别为０．０００６６ｎｇ／ｍｉｎ和０．０００６７ｎｇ／ｍｉｎ,Ⅱ组分别为０．０００８３ｎｇ／ｍｉｎ和０．００１１３ｎｇ／ｍｉｎ;（３）动物受热过程,Ｔｒ升至４３℃时,ＨＲ和ＭＡＰ达峰值水平,而呼吸频率则开始下降。本文结果提示,ＬＰＳ在中暑的病理生理学过程中可能是一个值得重视的因素     

热应激心肌细胞损伤的线粒体机制探讨

目的：观察热应激对大鼠凡肌细胞线粒体氧化磷酸化和钙代谢功能的影响、研究线粒体膜渗透性转移（ＰＴ）的变化及其病理学意义、探索热应激心肌细胞损伤发生机制。方法：用Ｋｌａｒｋ氧电极极谱法测定线粒体呼吸功能,用生物发光法主肌ＡＴＰ含量及线粒体Ｃａ＾２＋。ＡＴＰ酶活性;用电感耦合等离子－原子发射光谱仪测定线粒体内Ｃａ＾２＋含量,用分光光度法测定线粒体膜ＰＴ。结果：热应激大鼠心肌细胞线粒体的呼吸控制率（ＲＣＰ     

COMPARISON OF PHYSIOLOGICAL REACTIONS AND PHYSIOLOGICAL STRAIN IN HEALTHY MEN UNDER HEAT STRESS IN DRY AND STEAM HEAT SAUNAS

The aim of the paper was to follow up major physiological reactions, provoked by heat stress during dry and wet sauna baths. A physical strain index and subjective estimation of heat comfort of subjects who had not taken sauna baths before was also evaluated. Ten healthy males aged 25-28 underwent a dry sauna bath and then after a one-month break they underwent a steam sauna bath. Each time, they entered the sauna chamber 3 times for 15 minutes with five-minute breaks. During breaks they cooled their bodies with a cold shower and then rested in a sitting position. Before and after the baths, body mass and blood pressure were measured. Rectal temperature and heart rate were monitored during the baths. The physiological strain index (PSI) and cumulative heat strain index (CHSI) were calculated. Subjects assessed heat comfort by Bedford''s scale. Greater body mass losses were observed after the dry sauna bath compared to the wet sauna (-0.72 vs. -0.36 kg respectively). However, larger increases in rectal temperature and heart rate were observed during the wet sauna bath (38.8% and 21.2% respectively). Both types of sauna baths caused elevation of systolic blood pressure, but changes were greater after the dry one. Diastolic pressure was reduced similarly. Subjective feelings of heat comfort as well as PSI (4.83 ± 0.29 vs. 5.7 ± 0.28) and CHSI (76.3 ± 18.4 vs. 144.6 ± 21.7) were greater during the wet sauna bath. It can be concluded that due to high humidity and reduction of thermoregulation mechanisms, the wet sauna is more stressful for the organism than the dry sauna, where the temperature is higher with low humidity. Both observed indexes (PSI and CHSI) could be appropriate for objective assessment of heat strain during passive heating of the organism.     

SELECTION FOR HEAT-SHOCK RESISTANCE IN LARVAL AND IN ADULT DROSOPHILA BUZZATII: COMPARING DIRECT AND INDIRECT RESPONSES

Direct and correlated responses in selection for heat-shock resistance in adult and in larval Drosophila buzzatii were studied. Two lines were artificially selected for higher survival to heat stress as adults, and two other lines were reared under a fluctuating thermal environment as larvae, 35°C for 6 h and 25°C for 18 h, to “naturally” select for higher resistance as larvae. The latter two lines were duplicated after nine generations to yield additional lines to be “naturally” selected as larvae at a higher temperature, 38.2°C for 6 h. Control lines were maintained separately for the adult and larval selection lines. A significant direct response to selection was found for the adult selection lines. However, larvae of these adult selection lines were no more heat resistant than were larvae of the control lines. One of the two larval selection lines increased significantly in heat resistance as larvae. However, adult heat resistance was similar for lines selected as larvae and the corresponding control lines maintained at 25°C. Changes in developmental time accompanied changes in survival after stress in both sets of lines selected for increased heat resistance.     

热暴露大鼠心肌细胞钙稳态及其调节机制的研究

本文观察了离体成年大鼠心室肌肌质网、线粒体Ｃａ＾２＋－ＡＴＰ酶活性和总钙含是到及原代培养乳腺心肌细胞＾４６Ｃａ摄取、活性钙调素相对含量、胞浆内游离钙浓度在不同温度热暴露４０ｍｉｎ后的变化。结果表明：热暴露后,心肌奖浆、肌质网及线粒体中的Ｃａ＾２＋－ＡＴＰ酶活发表 所下降,心肌肌质网及线粒体中的总钙含量亦有降低趋势,心肌细胞活性钙调素相对含量显著下降,＾４６Ｃａ摄取量及胞浆内游离浓度显著升高。提示,     

KARYOGAMY BLOCK BY HEAT STRESS IN THE FERTILIZATION OF BROWN ALGAE

Karyogamy was inhibited by heat stress in zygotes of Scytosiphon lomentaria (Lyngbye) Link (isogamy), Cutleria cylindrica Okamura (anisogamy), and Fucus distichus subsp. evanescens (C. Agardh) Powell (oogamy). Although high temperatures did not inhibit migration of the male and female nuclei, nuclear envelope fusion was blocked. The ultrastructural stage at which karyogamy was inhibited varied among these species. In S. lomentaria, the outer membranes fused with each other, but the inner membranes did not fuse. Partial fusion of the nuclear envelope occurred in C. cylindrica. In F. distichus, the block of karyogamy at high temperature was incomplete, and nuclear fusion proceeded gradually. The block to karyogamy in S. lomentaria zygotes was reversible, and karyogamy proceeded when zygotes were transferred from 22° to 14° C. Experiments using inhibitors suggested that proteins that might be formed de novo after fertilization do not participate in karyogamy or its inhibition at either 14° or 22° C.     

热应激大鼠心肌钙代谢的变化及其机理探索

心肌细胞内钙离子对心功能的调节有着极其重要的作用。本研究观察了不同热应激强度下大鼠心肌细胞内质网、线粒体中钙含量,Ｃａ２＋－ＡＴＰ酶活力,内质网Ｃａ２＋主动转运速率及心肌ＡＴＰ含量的变化。研究结果表明,热应激大鼠心肌细胞内质网、线粒体钙含量随肛温升高显著下降;大鼠肛温达４２℃时,其钙含量分别较对照下降３２．２％和４６．５％;心肌细胞内质网和线粒体Ｃａ２＋－ＡＴＰ酶活力亦明显降低,线粒体Ｃａ２＋－ＡＴＰ酶活力下降幅度更为激烈。热应激大鼠心肌内质网Ｃａ２＋主动转运速率和Ｃａ２＋－ＡＴＰ酶活力变化趋势相同,且两者呈密切相关关系。热应激大鼠心肌ＡＴＰ含量亦随肛温升高大幅度降低,当动物肛温超过４２℃时,其可降至对照动物的３７．５％;热应激大鼠心肌ＡＴＰ含量的变化与内质网Ｃａ２＋－ＡＴＰ酶活力关系密切。实验结果提示：热应激心肌细胞质膜受损所致细胞Ｃａ２＋主动转运功能紊乱及心肌能量代谢障碍是心肌钙稳态失调的主要原因;心肌细胞钙代谢紊乱是诱导心肌细胞严重受损,导致热应激机体心血管功能失调,甚至心功能衰竭的重要机制