氯通道在葛根素注射液致溶血反应中的作用

目的：观察阻断和激活氯通道对葛根素注射液致家兔溶血反应的影响,探讨氯通道在葛根素注射液溶血反应中的作用。方法：将不同浓度的葛根素注射液（0.75、1.5、3、6、12 mg/ml）与家兔红细胞悬液共孵育6 h,使用细胞图像记录系统观测葛根素注射液是否诱导红细胞溶血,酶标仪、流式细胞仪检测红细胞溶血率,并观测激活和阻断氯通道对葛根素注射液溶血作用的影响。结果：葛根素注射液可引起家兔红细胞体外溶血,在所观察的1.5 mg/ml~12 mg/ml范围内,溶血效应呈浓度依赖性增强（n=3,P<0.01）。氯通道阻断剂Tamoxifen （20 μmol/L）、ATP （10 mmol/L）可有效抑制葛根素注射液的溶血作用（n=3~5,P<0.01）;而使用低浓度ATP （50 μmol/L）激活氯通道,则显著增强葛根素注射液的溶血作用（n=4,P<0.01）。结论：葛根素注射液的体外溶血效应呈浓度依赖性,氯通道激活与葛根素注射液诱导的溶血反应密切相关。     

高铁血红素对心肌缺血／再灌注损伤的防护作用及其机制

目的：在大鼠急性心肌缺血／再灌ii（I／R）模型上,观察高铁血红素在钙激活中性蛋白酶（calpain）介导的心肌I／R损伤中的作用。并初步探讨其可能的机制。方法：64只雄性SD大鼠随机8组（n：8）：假手术组（sham组）、（I／R）组、MDL28170＋I／R组、单纯MDL28170组、高铁血红素＋I／R组、单纯高铁血红素组、锌原卟啉Ⅸ＋高铁血红素＋I／R组、单纯锌原卟啉Ⅸ组。采用大鼠离体心脏Langendorff灌流技术,心脏I／R后,测定左室发展压（LVDP）、心肌梗死面积、冠脉流出液中的乳酸脱氢酶（LDH）释放量。检测calpain、血红素氧化酶（HO）、和半胱氨酸天冬氨酸蛋白酶3（caspase3）活性。Westernblot观察心肌钙蛋白酶抑制蛋白（calpastatin）蛋白表达。结果：①心肌I／R后,calpain、caspase3活性明显增高。calpain抑制剂MDL28170可抑制I／R诱导的LDH释放量增加,增高LVDP,缩小心肌梗死面积。②与单纯I／R组相比,大鼠预先给予高铁血红素后,心脏HO-1活性增加,calpain和caspase3活性下降。同时,LDH释放量减少,LVDP明显增高,心肌梗死面积缩小。③I／R组心肌calpastatin表达量明显低于对照组,高铁血红素组大鼠calpastatin表达量增高。HO-1的抑制剂锌原卟啉Ⅸ可取消高铁血红素对calpastain表达量的影响,并取消其心肌保护作用。结论：高铁血红素预处理可通过抑制calpain的激活,减轻大鼠心肌I／R损伤,其机制可能与增加calpastatin蛋白表达有关。     

苯丙氨酸解氨酶在诱导黄瓜幼苗抗寒性中的作用

为了探讨苯丙氨酸解氨酶(PAL)在诱导黄瓜幼苗抗寒性中的作用,采用喷施特异抑制剂(AOPP)的方法控制PAL活性,测定幼苗抗寒性的变化.结果表明： 低温可以诱导黄瓜幼苗叶片中PAL的基因表达和活性升高;喷施AOPP显著抑制了叶片中PAL活性,减少了酚类和类黄酮物质的积累.低温对黄瓜幼苗造成显著伤害,AOPP预处理加剧了低温对幼苗的损伤,幼苗抗寒性降低.与对照相比,幼苗叶片中相对电解质渗漏率和丙二醛(MDA)含量显著升高,PSII最大光化学效率(F_v/F_m)降低,光化学猝灭参数Y(NO)升高,胁迫相关基因(PR1-1a、COR47、P5CS、HSP70)的诱导表达受到抑制.低温导致黄瓜幼苗叶片中H₂O₂积累,还原型抗坏血酸(AsA)含量降低,脱氢抗坏血酸(DHA)含量升高,AsA∶DHA减小;喷施AOPP的幼苗中抗氧化酶(过氧化氢酶CAT、抗坏血酸过氧化物酶APX)活性显著低于对照,H₂O₂过量积累,AsA∶DHA更低.施用H₂O₂清除剂可以有效缓解喷施AOPP引起的低温损伤加剧,而施用CAT抑制剂的幼苗对低温胁迫更敏感.表明低温诱导了PAL活性升高,促进了苯丙烷类次生代谢产物的合成,提高了胞内抗氧化酶活性,可有效清除活性氧分子,维持AsA氧化还原状态,缓解低温引起的光损伤和氧化损伤.     

水环境中活性氧的来源、种类及测定方法

各种环境介质和生命体中许多微观化学过程都与活性氧密切相关.本文介绍了水环境中活性氧的来源、种类和测定.它们主要包括:¹O₂(单线态氧)、O₂^-(超氧自由基)/HO₂·(氢过氧自由基)、·OH(羟基自由基)、H₂O₂、RO·(烷氧基)、ROO·(烷过氧基)和R·OH(氢过氧化物)等.其主要来源于辐射分解、热解和氧化还原法等.测定采用分子探针法、图谱法和酶法.     

杀死感染艾滋病细胞的新的治疗药

明治乳业公司开发了作用于感染艾滋病病毒(HIV)的细胞,抑制病毒产生杀死细胞的新的治疗药。开始用小鼠试验能确认安全性,最近使用艾滋病模拟动物,还未确认作用机制。新物质是结合了化学修饰剂氯高铁血红素和琥珀酰化人血清白蛋白的复合物(M-HDA)。对淋巴细胞有亲和性的琥珀酰化人血清白蛋白运送氯高铁血红素。氯高铁血红素整合到细胞内,通过某些作用杀死细胞。使用重症免疫缺陷小鼠(SCID/hu)检测体内的抗HIV效果后,计划在美国进行临床试验。明治乳业卫生科学研究所小组进行的研究是“血浆蛋白掺入到清除剂受体,以葡萄糖硫酸阻止这种作     

血红素加氧酶-1在对抗过氧化氢引起的血管低反应性中的作用

目的：研究HO-1的诱导剂是否可对抗H2O2引起的血管低反应性,并探讨其作用机制。方法：采用血管环灌流装置,观察胸主动脉环的收缩效应。结果：①SD大鼠腹腔注射高铁血红素后,主动脉HO-1活性和血中CO含量增高;同时,H2O2引起的血管收缩功能下降的现象明显改善。②KATP通道阻断剂优降糖,而非GC抑制亚甲蓝,可取消高铁血红素的抗H2O2损伤的作用。③Hemin＋H2O2组与单纯H2O2组的钙收缩曲线无明显差异。④无钙液中,高铁血红素可抑制H2O2引起的咖啡因和PE诱导的收缩幅度的下降。结论：诱导主动脉HO-1活性增加,可对抗氧化应激引起的血管收缩反应的低下,其机制可能是通过激活KATP通道,影响细胞内贮存钙的释放起作用。而与GC信号转导通路无关。     

当归水提液和醇提液体外抗脂质过氧化和红细胞溶血作用

研究当归水提液和醇提液对小鼠肝组织自发性过氧化酯质分解产物丙二醛（malondialdehyde,MDA）的生成和对红细胞膜脂质过氧化及红细胞溶血作用的影响。采用TBA比色法测定肝组织匀浆MDA生成,分光光度法测定过氧化氢诱导红细胞膜脂质过氧化和溶血。实验分为空白组、对照组、加药组。加药组分为25、50、100和200mg/mL四个浓度组。当归水提液和醇提液均在25～200mg／mL的浓度范围内,能够明显抑制小鼠肝组织匀浆自发性MDA的生成,具有抑制过氧化氢诱导红细胞膜脂质过氧化和溶血的作用,抑制效果随当归水提液和醇提液浓度的增大而逐渐增强,抑制率与药物浓度成良好的量效关系。当归水提液和醇提液具有抗脂质过氧化和红细胞溶血的作用。     

水分胁迫下丛枝菌根真菌对红橘叶片活性氧代谢的影响

研究了水分胁迫下接种地表球囊霉（Glomus versiforme (Karsten) Berch）对红橘(Citrus tangerine Hort. ex Tanaka)叶片活性氧代谢的影响.结果表明：水分胁迫显著抑制了地表球囊霉对红橘根系的侵染,抑制率为33％.在正常供水和水分胁迫下,接种地表球囊霉处理的红橘叶片磷含量显著增加,与未接种处理相比,分别增加了45％和27%,丙二醛（MDA）和H₂O₂含量分别降低了25％、21％和16％、16％.正常供水和水分胁迫下接种地表球囊霉增强了叶片超氧化物岐化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性;提高了可溶性蛋白质、还原型抗坏血酸（ASC）和总抗坏血酸(TASC)含量.水分胁迫下接种处理显著降低了叶片超氧阴离子自由基（O₂^-_·）含量,与正常供水相比降低了31％.表明菌根化红橘植株的抗旱性增强.     

金樱子多糖的抗氧化作用

目的：探讨金樱子多糖(PRL)体外抗氧化作用。方法：邻苯三酚自氧化法测定PRL清除超氧阴离子自由基效果;比色法测定PRL对羟自由基诱导红细胞溶血、脂质过氧化反应的影响。结果：PRL能显著清除超氧阴离子自由基、押制羟自由基对细胞膜的破坏而引起的溶血和脂质过氧化产物的形成。结论：PRL具有显著的抗氧化作用。     

Hemin-mediated Hemolysis in Erythrocytes： Effects of Ascorbic Acid and Glutathione

In the present work,we investigated the effect of ascorbic acid and glutathione on hemolysisinduced by hemin in erythrocytes.Ascorbic acid not only enhanced hemolysis,but also induced formationof thiobarbituric acid-reactive substances in the presence of hemin.It has been shown that glutathioneinhibits hemin-induced hemolysis by mediating hemin degradation.Erythrocytes depleted of glutathionebecame very sensitive to oxidative stress induced by hemin and ascorbic acid.H_2O_2 was involved in hemin-mediated hemolysis in the presence of ascorbic acid.However,a combination of glutathione and ascorbicacid was more effective in inhibiting hemolysis induced by hemin than glutathione alone.Extracellular andintracellular ascorbic acid exhibited a similar effect on hemin-induced hemolysis or inhibition of hemin-induced hemolysis by glutathione.The current study indicates that ascorbic acid might function as anantioxidant or prooxidant in hemin-mediated hemolysis,depending on whether glutathione is available.     

Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1^–/–; Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1^–/– mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1^–/– erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 ± 3.2 in 4.1^–/– vs. 9.8 ± 1.3 mmol/10¹³ cell x h in control mice), with an abnormal dependence on osmolality (EC₅₀ = 417 ± 42 in 4.1^–/– vs. 460 ± 35 mosmol/kgH₂O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered (K_0.5 = 489.7 ± 0.7 vs. 537.0 ± 0.56 nM in control mice), the V_max of the H-induced Na/H exchange activity was markedly elevated in 4.1^–/– erythrocytes (V_max 91.47 ± 7.2 compared with 46.52 ± 5.4 mmol/10¹³ cell x h in control mice). Na/H exchange activation by okadaic acid was absent in 4.1^–/– erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically downregulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of 4.1^–/– erythrocytes. spherocytosis; cell Na; Na/H exchange     

Clotrimazole enhances lysis of human erythrocytes induced by t-BHP

Clotrimazole (CLT) is an antifungal and antimalarial agent also effective as a Gardos channel inhibitor. In addition, CLT possesses antitumor properties. Recent data provide evidence that CLT forms a complex with heme (hemin), which produces a more potent lytic effect than heme alone. This study addressed the effect of CLT on the lysis of normal human erythrocytes induced by tert-butyl hydroperoxide (t-BHP). For the first time, it was shown that 10 μM CLT significantly enhanced the lytic effect of t-BHP on erythrocytes in both Ca²⁺-containing and Ca²⁺-free media, suggesting that the effect is not related to Gardos channels. CLT did not affect the rate of free radical generation, the kinetics of GSH degradation, methemoglobin formation and TBARS generation; therefore, we concluded that CLT does not cause additional oxidative damage to erythrocytes treated with t-BHP. It is tempted to speculate that CLT enhances t-BHP-induced changes in erythrocyte volume and lysis largely by forming a complex with hemin released during hemoglobin oxidation in erythrocytes: the CLT–hemin complex destabilizes the cell membrane more potently than hemin alone. If so, the effect of CLT on cell membrane damage during free-radical oxidation may be used to increase the efficacy of antitumor therapy.     

Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes

Accelerated phospholipid catabolism occurs early after the onsetof myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipaseA₂(PLA₂) activity in isolatedventricular myocytes, resulting in increased lysophospholipid andarachidonic acid production, contributing to arrhythmogenesis inischemic heart disease. The majority of ventricular myocyte arachidonicacid was found in plasmalogen phospholipids. Hypoxia increasedmembrane-associated,Ca²⁺-independent,plasmalogen-selective PLA₂activity, resulting in increased arachidonic acid release andlysoplasmenylcholine production. Pretreatment with the specificCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked hypoxia-induced increases inPLA₂ activity, arachidonic acidrelease, and lysoplasmenylcholine production. Lysoplasmenylcholineproduced action potential derangements, including shortening of actionpotential duration, and induced early and delayed afterdepolarizationsin normoxic myocytes. The electrophysiological alterations induced bylysoplasmenylcholine would likely contribute to the initiation ofarrhythmogenesis in the ischemic heart.

     

Hydroxyl Radicals and a Thylakoid-Bound Endopeptidase are Involved in Light- and Oxygen-Induced Proteolysis in Oat Chloroplasts

The hypothesis that light- and oxygen-induced proteolysis inchloroplasts is mediated by active oxygen species was examined.In order to determine whether or not H₂O₂ and/or ^{dot}OH radicalsare involved in these degradative processes we compared thedegradation of proteins in isolated oat chloroplasts exposedto white light at 80 W m^-2 with that in chloroplasts incubatedin darkness in the absence or presence of H₂O₂ or a ^{dot}OH-generatingsystem composed by ascorbic acid, FeCl₃ and H₂O₂ (Asc-Fe-H₂O₂).Light enhanced the rate of degradation of at least 18 polypeptides,while proteolysis was almost negligible in darkness in the abscenceof additives. H₂O₂ had a very small effect. However, Asc-Fe-H₂O₂-treatedchloroplasts in darkness showed a pattern of protein degradationalmost identical to that observed in the light. A thylakoid-boundendopeptidase (EP), the activity of which increased under photooxidativeenvironmental conditions and treatment with an ^{dot}OH-generatingsystem, was partially purified and characterized as a serinetypeprotease. Treatments with inhibitors of serine-type proteaseprevented both light- and Asc- Fe-H₂O₂-induced proteolysis.EP was more active against both soluble and membranous proteinsthat had been pretreated with Asc-Fe-H₂O₂ than against untreatedproteins. It is proposed that a high dose of light irradiationpromotes proteolysis by increasing the formation of ^{dot}OH,which may modify proteins such that they become more susceptibleto EP-catalyzed hydrolysis. ¹Fisiología Vegetal, Dept. de Biología Vegetal,Universidad de Alcalá de Henares, Present address: 28871Alcalá de Henares (Madrid), España.     

Decreased cation channel activity and blunted channel-dependent eryptosis in neonatal erythrocytes

Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca²⁺-permeable cation channels with subsequent entry of Ca²⁺ and stimulation of Ca²⁺-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl^– removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca²⁺ activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E₂ (PGE₂) formation, cation channel activity, Ca²⁺ entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl^– in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE_2, and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca²⁺ leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes. annexin V; osmotic cell shrinkage; calcium; apoptosis     

短期CO2加富对凤梨光合作用及生长发育的影响

 研究了CO₂加富对丹尼斯凤梨（Guzmania`Denise’）和吉利凤梨（Guzmania `Cherry’）叶片光合速率、植株生长、开花和光合相关酶活性的 影响。结果表明,处理30 d期间,处理(600±40)、(900±40) μmol CO₂&;#8226;mol^-1的净光合速率分别比同期对照增加了6.24%～31.91%和11.92%～ 41.48%;CO₂加富下促进了叶片中可溶性糖和淀粉的积累, 蒸腾速率和气孔导度下降,Rubisco活性增加,乙醇酸氧化酶活性则明显下降。(600 ±40)μmol CO₂&;#8226;mol^-1处理下的株高、叶面积分别比同期对照下增加了6.94%～14.63%和1.66%～7. 06%,而处理(900±40) μmol CO₂&;#8226;mol^-1下 分别增加了9.71%～20.85%和2.87%～11.62%;CO₂加富下促进了干重和鲜重的积累。此外,CO₂加富提前了吉利凤梨的花期。     

Lidocaine: An inhibitor in the free‐radical‐induced hemolysis of erythrocytes

Lidocaine was reported to protect erythrocytes from hemolysis induced by 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH). Since AAPH‐induced hemolysis was a convenient in vitro experimental system to mimic erythrocytes undergoing peroxyl radicals attack, the aim of this work was to investigate the antioxidant effect of lidocaine on AAPH‐induced hemolysis by chemical kinetics. As a result, one molecule of lidocaine can only trap 0.37 radical, much lower than melatonin. Meanwhile, lidocaine cannot protect erythrocytes from hemolysis induced by hemin, which the mechanism of hemolysis was due to the erythrocyte membrane destroyed by hemin. Accordingly, lidocaine protected erythrocytes by scavenging radicals preferentially rather than by stabilizing membrane. Moreover, the interactions of lidocaine with two radical species, including 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) radical cation (ABTS^+?) and 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH), indicated that lidocaine can reduce ABTS^+? with 260 µM as the 50% inhibition concentration (IC₅₀) and cannot react with DPPH. Thus, lidocaine served as a reductant rather than a hydrogen donor to interact with radicals. Finally, the quantum calculation proved that, compared with the melatonin radical, the stabilization of N‐centered radical of lidocaine was higher than the amide‐type N‐centered radical but lower than the indole‐type N‐centered radical in melatonin. These results provided basic information for lidocaine to be an antiradical drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:81–86, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20267     

Isolation and Identification of Sodium 2-Propenyl Thiosulfate from Boiled Garlic (Allium sativum) That Oxidizes Canine Erythrocytes

Sodium 2-propenyl thiosulfate was identified in boiled garlic (Allium sativum). When canine erythrocytes were incubated with sodium 2-propenyl thiosulfate, the methemoglobin concentration and Heinz body percentage in erythrocytes were both increased, indicating that the compound induced oxidative damage in canine erythrocytes. It seems that this compound is one of the causative agents of garlic-induced hemolysis in dogs.     

碱胁迫对黑麦草幼苗根系活性氧代谢和渗透溶质积累的影响

为了探讨牧草对碱胁迫的耐受程度,采用营养液砂培方法,研究了不同浓度NaHCO3（0、50、100、150和200 mmol·L^-1）胁迫对黑麦草幼苗根系生长、活性氧代谢和渗透溶质积累的影响。结果表明：NaHCO3胁迫显著抑制黑麦草幼苗根系的生长,其抑制程度随胁迫浓度提高而增强,黑麦草可耐受的最高NaHCO₃浓度约为150 mmol·L^-1。随着NaHCO₃胁迫浓度的增加,黑麦草根中超氧阴离子(O^-·₂)、过氧化氢(H₂O₂)和丙二醛(MDA)含量明显上升,超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)含量显著下降,过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性及抗坏血酸(ASA)含量先升后降。黑麦草根中Na⁺含量随NaHCO₃浓度增大而增加,K⁺含量和K⁺/Na⁺比降低,可溶性糖含量先升后降,脯氨酸含量则先降后升,游离氨基酸含量呈先升后降再升高变化。表明碱胁迫导致的活性氧代谢失调和Na⁺、K⁺失衡及积累有机溶质进行渗透调节时更多能量的消耗可能是黑麦草根系生长受抑的重要因素。