镰刀菌T-2毒素对培养软骨细胞的影响

在含有不同浓度 T-2 毒素培养液中离体培养鸡胚软骨细胞,当 T-2 浓度达到0.01ppm 时,可引起软骨细胞胞外基质胶原微原纤维减少,线粒体细胞色素 c 氧化酶和 H⁺-ATP 酶的比活力下降.表明此浓度 T-2 毒素引起了软骨细胞结构与功能的明显改变.文章报道的方法可为检验大骨节病致病因子是否直接作用于大骨节病易侵蚀的软骨细胞提供了一种有效的实验手段.     

菠菜能量转化复合体的人工组装

从菠菜叶绿体中分离了H~+-ATP酶复合体,其SDS-聚丙烯酰胺凝胶电泳呈九条亚单位带,为纯度较高的酶复合体。将H~+-ATP酶复合体重组于人工膜(脂质体)上,表现出PiATP交换活力。Mg~(++)-ATP酶活力也明显提高。并表现对DCCD,寡霉素的敏感性。 重组H~+-ATP酶复合体表现有ATP诱导的H~+转移。但脂质体也出现类似现象,为此对以pH改变为标准的检测方法提出商榷。     

氧化磷酸化中质子的转运机制介绍和讨论

氧化磷酸化过程中电子传递和磷酸化所伴随的质子（H＋）跨线粒体内膜转运,是生物化学教学中的一个重点和难点。该文介绍参与H＋跨膜（线粒体内膜或细菌质膜）转运的复合体Ⅰ（又称为NADH-Q还原酶或NADH脱氢酶）、复合体Ⅲ（又称为细胞色素还原酶或细胞色素bc1复合体）、复合体Ⅳ（又称为细胞色素氧化酶或细胞色素c氧化酶）和复合体Ⅴ（又称为F1F0-ATP合酶）跨膜转运H＋的机制。     

抗寒剂CR-4 对冬小麦幼苗质膜钙泵（Ca2 +-ATPase） 的稳定作用

通过磷酸铈沉淀的细胞化学观察揭示,常温下生长的冬小麦幼苗的Ca2+ -ATP酶活性主要定位在质膜上,同时,水浸种和抗寒剂浸种的小麦质膜Ca2+ -ATP酶活性没有差异。然而,小麦幼苗经-7℃冰冻处理12小时和24小时后,则表现明显的区别：水浸种的小麦幼苗质膜Ca2+ -ATP酶活性明显下降,直至完全失活,细胞的精细结构也同时被破坏;而经抗寒剂浸种的小麦幼苗质膜Ca2+ -ATP酶仍维持较高的活性,细胞结构也保持完整,显示抗寒剂对质膜Ca2+ -ATPase酶起着明显的稳定作用。     

“非双层脂结构形成的倾向性”对猪心线粒体H~+-ATP酶活性的影响

1、PC+PE重组的线粒体H~+-ATP酶,ATP水解活力及其对寡酶素的敏感性,ATP诱导电位随非双层脂PE含量增加显著增大,当PE含量为60%-80%时ATP诱导电位达最高值.2、用PC+DOPE和PC+DEPE重组的脂酶体,前者的ATP诱导电位显著高于后者,但是两种脂酶体的ATP水解活性和寡霉素敏感性没有明显差别.3、降低PH可以促进PA形式非双层结构,PC+20%PA或大豆磷脂重组的脂酶体H~+-ATP酶活性随pH降低显著增高.4、PC+20%PA和大豆磷脂重组的脂酶体膜表层流动性随透析液PH降低而降低,5、综合上述结果.我们认为“非双层脂结构形成的倾向性”的加强,可能导致一种不稳定的或柔性的膜结构的出现,使得H~+-ATP酶呈现发挥其活性的最适构象.     

外源绿原酸缓解黑大豆SB铝毒害的机理研究

该文研究了外源绿原酸(CGA)对Al胁迫下铝敏感型黑大豆SB根生理生化指标以及根中胁迫相关基因表达的变化,探讨外源CGA缓解SB根铝毒害的效果及分子机理。以不同浓度Al和CGA处理SB,筛选出CGA缓解Al毒害的最佳浓度,测定Al含量、抗氧化系统酶活性、14-3-3蛋白与H~+-ATP酶的表达、H~+泵活性。结果表明:低浓度CGA能缓解Al胁迫下黑大豆SB根伸长抑制,并促进侧根数目增加,而高浓度CGA的缓解效果下降;0.01 g·L~(-1) CGA使Al胁迫下SB根尖Al含量与MDA含量下降,促进根系柠檬酸的分泌。RTPCR和Western Bloting分析表明0.01 g·L~(-1) CGA促进Al胁迫下SB根中14-3-3b、14-3-3m、14-3-3k和GHA2基因(质膜H~+-ATP酶)的表达,抑制MATE基因的表达。同时,0.01 g·L~(-1) CGA能促进Al胁迫下质膜H~+-ATP酶蛋白磷酸化水平以及其与14-3-3蛋白结合,且能提高质膜H~+-ATP酶和H~+泵活性。因此推测外源CGA可能通过增加侧根数,增强14-3-3蛋白和质膜H~+-ATP酶基因蛋白表达水平和互作,弥补Al胁迫下MATE表达的抑制,增加柠檬酸的分泌,增强SB对铝毒害的耐受性。     

Mg~(2+)影响嵌有线粒体H~+-ATP酶脂酶体的表面电荷密度和流动性

<正> 我们实验室曾报道,用胆酸盐透析法将猪心线粒体H~+-ATP酶嵌入大豆磷脂脂质体形成脂酶体时,透析液中Mg~(2+)的存在会降低脂酶体的膜脂流动性,并明显提高重建H~+-ATP酶的活性以及对寡霉素或DCCD的敏感性,因而推论Mg~(2+)的作用很可能是通过改变膜脂的物理状态,形成了维持H~+-ATP酶较高活性的合适构象。但共确切的作用机制仍     

低温、高pH胁迫对水稻幼苗根系质膜、液泡膜ATP酶活性的影响

以耐冷性不同的两个水稻品种为材料,比较研究了幼苗根系质膜、液泡膜ATP酶对低温(8℃)及高pH(8.0)胁迫的反应。结果表明水稻根细胞质膜和液泡膜上均存在Ca3+-ATP酶,但活性远低于H+-ATP酶。耐冷品种武育粳3号经低温(8℃)处理2d,根系质膜和液泡膜H+-ATP酶、Ca2+-ATP酶活性均明显升高,至冷处理12d,H+-ATP酶、Ca2+-ATP酶活性有所下降,但仍与对照相近;而冷敏感品种汕优63经低温(8℃)处理2d,根系质膜H+-ATP酶活性略有升高,而质膜Ca2+-ATP酶以及液泡膜H+-ATP酶、Ca2+-ATP酶活性已明显下降;至冷处理12d,4种酶活性均明显低于对照。高pH胁迫使质膜和液泡膜H+-ATP酶活性下降,而使Ca2+-ATP酶活性上升。高pH胁迫会加剧低温冷害。结果表明,耐冷品种质膜、液泡膜ATP酶比冷敏感品种对低温胁迫有更强的适应能力。     

克山病是一种“心肌线粒体病(Mitochondrial Cardiomyopathy)”

亚急克病人心肌线粒体内膜电子传递链的琥珀酸氧化酶系,琥珀酸脱氢酶和细胞色素氧化酶活性明显低于对照。H~ -ATP酶的活性及其对寡霉素的敏感性都明显下降。ATP能量化后线粒体膜电位的变化也比对照明显降低。膜脂流动性低于对照。亚急克病人心肌线粒体内观察到较多的电子致密无定形物质,经电镜X射线微区等方法分析,认为这些物质不是Ca_3(PO_4)_2,而可能是一种蛋白质凝聚物。此外,心肌线粒体的硒含量远低于对照,而Ca含量明显高于对照。上述结果都反映亚急克病人心肌线粒体明显损伤。根据克山病患者心肌细胞线粒体结构与功能方面呈现的如此广泛与明显的异常,可将克山病称为“心肌线粒体病(Mitochondrial Cardiomyopathy)”。     

溶酶体膜H^＋—ATP酶的细胞化学观察

本实验在以p-NPP为底物、氯化铈为捕捉剂、Tricine-KOH为缓冲液的中性最适环境中温育大鼠肝组织,进行肝细胞、血窦内皮细胞和枯否氏细胞内溶酶体膜上H~+-ATP酶定位的细胞化学研究。温育反应形成的磷酸铈反应产物电子密度高、颗粒细、分布均匀、非特异性反应产物少、重复性好。对照实验证实,溶酶体膜上的H~+-ATP酶是对哇巴因耐受的,对NEM敏感的囊泡膜H~+-ATP酶。     

The Role of Mitochondria in T-2 Toxin-Induced Human Chondrocytes Apoptosis

T-2 toxin, a mycotoxin produced by Fusarium species, has been shown to cause diverse toxic effects in animals and is also a possible pathogenic factor of Kashin–Beck disease (KBD). The role of mitochondria in KBD is recognized in our recent research. The aim of this study was to evaluate the role of mitochondria in T-2 toxin-induced human chondrocytes apoptosis to understand the pathogenesis of KBD. T-2 toxin decreased chondrocytes viabilities in concentration- and time-dependent manners. Exposure to T-2 toxin can reduce activities of mitochondrial complexes III, IV and V, ΔΨm and the cellular ATP, while intracellular ROS increased following treatment with T-2 toxin. Furthermore, mitochondrial cytochrome c release, caspase-9 and 3 activation and chondrocytes apoptosis were also obviously observed. Interestingly, Selenium (Se) can partly block T-2 toxin -induced mitochondria dysfunction, oxidative damage and chondrocytes apoptosis. These results suggest that the effect of T-2 toxin on human chondrocytes apoptosis may be mediated by a mitochondrial pathway, which is highly consistent with the chondrocytes changes in KBD.     

Is there a plasma membrane-located anion-sensitive ATPase? IV. Distribution of the enzyme in rat pancreas.

The intracellular localization of anion-sensitive Mg2+-ATPase in rat pancreas was studied by differential centrifugation, density gradient centrifugation and by the use of inhibitors of mitochondrial Mg2+-ATPase. The anion-sensitive MG2+-ATPase appears to be localized almost exclusively in a mitochondrial (15 min, 15 000 times g) fraction which shows two peaks after density gradient centrifugation. Both peaks coincide with the highest levels of cytochrome c oxidase activity, but not with alkaline phosphatase, (Na+ plus K+)-ATPase and leucine aminopeptidase activities or RNA. They appear to be equal sensitive to inhibition by oligomycin, aurovertin D and the rat liver mitochondrial inhibitor protein, at least when 1 mM EDTA is present in the isolation media. We conclude that no significant plasma membrane-located anion-sensitive Mg2+-ATPase activity is present in rat pancreas.     

T-2 Toxin Contamination in Grains and Selenium Concentration in Drinking Water and Grains in Kaschin–Beck Disease Endemic Areas of Qinghai Province

It has been strongly suggested that two factors are involved in the development of Kaschin?CBeck Disease (KBD), namely grains contamination with T-2 toxin and selenium deficiency. So our team undertook a survey about grains and drinking water in three rural KBD endemic villages and one non-KBD village in Qinghai Province. The level of T-2 toxin contamination in 364 grain samples was assayed using an ELISA kit. The selenium concentration in these grains and 15 drinking water samples from three KBD endemic villages were determined using the 2,3-diaminonaphthalene fluorometric assay. The results revealed that the level of T-2 toxin contamination in the samples from three KBD endemic villages was relatively high with an average level of 78.91?ng/g in wheat and 47.47?ng/g in flour. The T-2 toxin level in samples from the non-KBD village (12.23?ng/g) was significantly lower than that of local grains from the three KBD endemic villages. The average selenium content in wheat and flour from KBD areas was 0.0045 and 0.0067???g/g, respectively. The selenium concentration in local grain samples was significantly lower than that in samples from the non-KBD village (0.0604???g/g). In addition, the selenium concentration in drinking water from three KBD endemic villages was also low (0.156???g/L). These results support a potential role of T-2 toxin contamination and selenium deficiency in KBD. Compared with non-KBD endemic areas, health hazards in grains and in the environment of KBD endemic areas were observed.     

Cross-reconstitution of isolated F1-ATPase from potato tuber mitochondria with F1-depleted beef heart and yeast submitochondrial particles

Cross-reconstitution of isolated potato mitochondrial F1-ATPase with F1-depleted beef heart and yeast submitochondrial particles is reported. Potato F1 binds to the heterologous membrane and confers oligomycin sensitivity on the ATPase activity of the reconstituted system. Binding of F1 is promoted by the presence of Mg2+ with the maximal stimulatory effect at 20 mM. Mg2+ increase the sensitivity to oligomycin of the reconstituted system consisting of potato F1 and yeast membranes, however, they do not influence oligomycin sensitivity of potato F1 and beef heart membranes.     

外源海藻糖对小麦幼苗耐盐性的影响

以盐敏感小麦品种鲁麦15为材料,分别用完全Hoagland营养液、150mmol／L NaCl和150mmol／L NaCl 10mmol／L海藻糖处理小麦幼苗,测定小麦幼苗生长、离子含量、根系质膜H^ -ATPase、SOD活性、MDA含量等指标,旨在探讨外源海藻糖在抗盐性中的作用。结果表明：外源海藻糖可明显缓解盐胁迫对小麦幼苗生长的抑制作用;明显提高NaCl胁迫条件下小麦幼苗叶片中K^ 的含量,降低Na^ 的含量,降低其Na^ ／K^ ;提高NaCl胁迫条件下小麦幼苗SOD活性,降低MDA的含量,降低细胞质膜透性,缓解根系质膜H^ -ATPase活性抑制。以上结果表叫外源海藻糖可能通过增加活性氧清除能力、缓解质膜伤害、维持胞质离子稳态提高植物抗盐性。     

The effect of T-2 toxin on active sodium transport across frog skin in the presence of ADH and amphotericin B

1. The effect of T-2 toxin on active sodium transport across frog skin both in the presence and in the absence of stimulants of sodium transport, such as Amphotericin B and ADH, was studied using the short circuit current technique with the following results. 2. T-2 toxin produces inhibition of active sodium transport in a dose-response correlation. 3. This effect is irreversible since the washing out of the tissue does not restore its functionality. This indicates that the micotoxin may cross the cellular membrane and act on the internal site. 4. ADH partially removes the inhibitory effect of T-2 toxin. 5. The increase of the sodium pool in the cell as determined by Amphotericin B does not reverse the inhibitory effect of T-2 toxin. 6. The biological significance of these data is discussed in regard to the possible effect of T-2 toxin on Na+, K+-ATPase activity either directly or by a reduction in the metabolic supply of substrates, or by a modified stoichiometry of the pump reaction.     

胡杨液泡膜H+-ATPase的部分纯化及其耐盐性研究

为了阐明液泡膜H^ -ATPase在盐胁迫下的作用和适应性机制,对悬浮培养的胡杨细胞在50mmol／L盐浓度下处理10d,结果表明液泡膜H^ -ATPase、焦磷酸酶的水解活性、质子泵活性增加。将通过差速离心和不连续蔗糖密度梯度离心富积的液泡膜微囊先由脱氧胆酸钠(DOC)和n-辛基-β-D-葡萄糖(OG)分步破膜抽提,经蔗糖密度梯度离心分离,部分纯化的酶含V型H^ -ATPase的主要亚基。     

Cholera toxin blocks glucagon-mediated inhibition of the liver plasma membrane (Ca2+-Mg2+)-ATPase

We have previously shown that liver plasma membrane (Ca2+-Mg2+)-ATPase activity is inhibited by glucagon. To investigate the possible involvement of a GTP-binding (G) protein in this regulation, we have examined the effects of pertussis toxin and cholera toxin on inhibition of (Ca2+-Mg2+)-ATPase by glucagon. Treatment of liver plasma membranes with pertussis toxin did not affect the sensitivity of (Ca2+-Mg2+)-ATPase to the hormone. In contrast, treatment of plasma membranes or prior injection of animals with cholera toxin prevented inhibition of the (Ca2+-Mg2+)-ATPase by glucagon. Even though adenylate cyclase activity was increased by cholera toxin treatment, addition of cyclic AMP did not mimic the effect of cholera toxin in blocking glucagon-mediated inhibition of (Ca2+-Mg2+)-ATPase activity. These data suggest that a cholera toxin-sensitive protein, perhaps Gs or a Gs-like protein, is involved in the regulation of liver (Ca2+-Mg2+)-ATPase activity. The results emphasize the possible role of Gs-like proteins in regulation of enzymes other than adenylate cyclase and suggest that the study of (Ca2+-Mg2+)-ATPase may provide a useful enzymatic system to examine such regulation.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes.

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of (Mg2+ + Na+ + K+)-ATPase than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for (Mg2+ + Na+ +k+)-ATPase and 5'-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome c reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADPH cytochrome c reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome c reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.