干旱条件下大豆叶片H_2O_2代谢变化及其同抗旱性的关系

干旱条件下大豆叶片H_2O_2含量增加,AsA POD与 GR活性均表现“先上升后下降”的趋势。叶片AsA与GSH含量均随干旱时间的延长而逐渐下降,而PPOD活性则持续增加。抗旱性较强的小粒大豆品种7605在干旱条件下能维持较强的 H_2O_2清除能力,H_2O_2累积较少。     

电离辐射对内皮细胞分泌TXA2和PGI2的影响

本实验通过血管内皮细胞的体外培养,用RIA 法直接测定细胞培养液中,6-keto-PGF_(1a)和 TXB_2的含量并以此反映培养细胞合成分泌 PGI_2和 TXA_2的量。同时观察15Gyγ射线辐照前后的差别,以探索辐射损伤出血的原因。实验结果表明,在辐射后32小时中 TXA_2上升,PGI_2下降。正常组 PGI_2分泌量为 TXA_2的6倍。在血液内皮界面保持以 PGI_2为优势的平衡状态。γ-射线照射能激活内皮细胞中的环氧化酶和血栓素合成酶破坏这一平衡,使TXA_2的量增加,促使血小板在内皮细胞表面粘附聚集并释放平滑肌增殖因子,导致血管内皮细胞损伤,从而造成出血。     

BRCA1相互作用蛋白的分离及鉴定

乳腺癌易感基因(breast cancer susceptibility gene-1,BRCAl)在DNA损伤修复、细胞周期调控、染色质的稳定、基因转录激活以及细胞凋亡等方面起着重要作用。BRCAI C-末端是富含酸性氨基酸的转录激活结构域(AD),AD核心结构为两个串联的BRCT结构域(BRCTl和BRCT2)。应用酵母双杂交技术,以BRCT2为诱饵蛋白,从卵巢文库中筛选到了与BRCT2结构域相互作用蛋白FHL2(four and half LIM domains)。利用酵母交配的方法证明FHL2与BRCAlBRCT2特异结合,而不与BRCAl BRCTl、Rapl BRCT结构域结合。GST沉淀实验表明,FHL2在体外特异地与BRCT2结构域相结合;免疫共沉淀实验表明,FHL2在体内特异地与BRCT2结构域结合;FHL2可与全长BRCAl结合。BRCAl与FHL2相互作用的发现为研究BRCAl以及FHL2在肿瘤发生、发展中的作用打下了坚实的基础。     

植物细胞中蓝光诱导ROS生成的识别和亚细胞定位检测

以2’,7’-二氯二氢荧光素二乙酯(dichlorofluorescein diacetate,H2DCF-DA)为荧光探针孵育拟南芥叶表皮条,利用荧光光谱和激光共聚焦扫描显微技术,对高辐照蓝光诱导下叶肉细胞活性氧(reactive oxygen spe-cies,ROS)的生成,进行了分子识别和亚细胞定位检测。结果表明:植物细胞在蓝光诱导下,可以产生大量的ROS。过氧化氢酶清除实验表明:高辐照蓝光诱导产生的ROS,主要成分是H2O2,并且主要定位在叶绿体和细胞膜上。     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO₂浓度升高可以诱导植物叶片气孔关闭, 提高植物对高浓度CO₂的适应性。但植物如何感知CO₂浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚。利用高通量、非侵入的远红外成像技术, 建立了拟南芥(Arabidopsis thaliana)气孔对CO₂浓度变化反应相关的突变体筛选技术, 筛选出对环境CO₂浓度敏感的拟南芥突变体ecs1。遗传学分析表明, ecs1为单基因隐性突变体, 突变基因ECS1编码一个跨膜钙离子转运蛋白。与野生型拟南芥相比, 360 μL·L^–1CO₂可引起ecs1突变体叶片温度上升和气孔关闭, ecs1突变体对900 μL·L^–1CO₂长时间处理具有较强的适应性。进一步的实验表明, 360μL·L^–1CO₂即可诱导ecs1突变体叶片积累较高浓度的H₂O₂, 而900 μL·L^–1CO₂才能够诱导野生型拟南芥叶片积累H₂O₂。因此, ECS1可能参与调节高浓度CO₂诱导的拟南芥气孔关闭和H₂O₂产生, H₂O₂可能作为第二信号分子介导CO₂诱导拟南芥气孔关闭的反应。     

PIP2信号与PIP2的再合成

磷脂酰肌醇-4,5-二磷酸（phosphatidylinositol-4,5-bisphosphate,PIP2）是一种分布在细胞膜内侧面的微量磷脂。虽然含量很低,但PIP2在细胞信号转导以及膜蛋白功能调节等方面却起着十分重要的作用。细胞膜中PIP2的含量水平呈动态平衡,在其代谢调节改变时,PIP2局部浓度的变化可影响特定蛋白的功能。该文就近二十年来针对PIP2信号和PIP2代谢调节相关的研究作一综述。     

过氧化氢—植物体内的一种信号分子

本文概述了H2O2的产生机制,总结了H2O2介导植物生理生化变化及相关基因的表达,提出了H2O2是植物体内的一种信号分子。     

TXA2/PGI2与心血管疾病

血栓素(Thromboxane,TXA2)和前列环素(Prostacyclin,PGI2)均为花生四烯酸的代谢物,是前列腺素(Prostaglandins,PGs)中生物活性最强的一对。在正常情况下,二者在体内保持一定的平衡,相互拮抗、相互协调,共同维持血液循环畅通,与心血管疾病关系密切。本文即就其生物特性及与心血管病的关系等进行综述,对人们全面认识TXA2/PGI2具有一定的参考价值。     

Hypertension augments ethanol-induced depression of cell shortening and intracellular Ca(2+) transients in adult rat ventricular myocytes.

Ethanol, a risk factor for myocardial dysfunction, depresses myocardial contraction. This study was to determine whether ethanol-induced myocardial depression is affected by hypertension. Mechanical properties of ventricular myocytes isolated from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were evaluated using a video edge-detection system. Myocytes were electrically stimulated to contract at 0.5 Hz. Contractile properties analyzed include peak twitch amplitude (PTA), time-to-PTA (TPS), time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes. Acute ethanol exposure (80-640 mg/dl) caused a concentration-dependent inhibition of PTA and DeltaFFI in both WKY and SHR myocytes. The extent of maximal inhibition of PTA and FFI was significantly greater in SHRs (53.7 and 38.9%) compared to the WKY group (21.0 and 25.4%). Ethanol did not affect TPS but shortened TR(90) and slowed +/-dL/dt at high concentration ranges. Interestingly, the augmented ethanol-induced inhibition of cell shortening in hypertension was greatly attenuated by Ca(2+) channel opener BayK 8644 (1 microM). These results suggest that ethanol-induced myocardial depression may be augmented in hypertension, possibly due to mechanism(s) involving sarcolemmal Ca(2+) channels.     

Salvia miltiorrhiza attenuates the changes in contraction and intracellular calcium induced by anoxia and reoxygenation in rat cardiomyocytes

The aim of the present study is to investigate the effect of Salvia miltiorrhiza (SM) on contraction and the intracellular calcium of isolated ventricular myocytes during normoxia or anoxia and reoxygenation using a video tracking system and spectrofluorometry. Cardiac ventricular myocytes were isolated enzymatically by collagenase and exposed to 5 min of anoxia followed by 10 min of reoxygenation. SM (1-9 g/L) depressed both contraction and the [Ca(2+)](i) transient in a dose-dependent manner. SM did not affect the diastolic calcium level and the sarcolemmal Ca(2+) channel of myocytes but decreased the caffeine-induced calcium release. During anoxia, the +/-dL/dtmax, amplitudes of contraction (dL) of cell contraction and [Ca(2+)](i) transients were decreased, while the diastolic calcium level was increased. None of the parameters returned to the pre-anoxia level during reoxygenaton. However, SM (3 g/L) did attenuate the changes in cell contraction and intracellular calcium induced by anoxia and reoxygenation. It is concluded that SM has different effects on normoxic and anoxic cardiomyocytes. The SM-induced reduction of changes in contraction and intracellular calcium induced by anoxia/reoxygenation indicates that SM may be beneficial for cardiac tissue in recovery of mechanical function and intracellular calcium homeostasis.     

白介素—2对心肌细胞[Ca^2＋]i的作用及其信号转导途径

为研究白介素－2（interleukin-2,IL-2）对心肌细胞内钙浓度（[Ca^2 ]i）的影响及其信号转导途径,实验采用酶解法分离成年大鼠心室肌细胞,以Fura-2/AM为钙探针,用细胞内双波长钙荧光系统检测细胞[Ca^2 ]i的变化。结果发现：（1）IL－2（0.5-200U/ml）浓度依赖性地降低单个心室肌细胞内钙态,IL－2（200U／ml）对咖啡因诱导的肌浆网内储钙的释放无影响;（2）纳洛酮(naloxone,Nal)(10^-8mol/L）和nor-binaltorphimine(nor-BNI,10^-8mol/L）可阻断IL－2对心肌细胞钙瞬态的作用,而纳曲吲哚（naltrindole,NTI）（10^-6mol/L）不能阻断此作用;（3）κ阿片受体激动剂U50488H（10^-6mol/L）降低心肌细胞钙瞬态,nor-BNI(10^-8mol/L）可阻断此作用;（4）5mg/L百日咳毒素（PTX）预处理可取消IL－2降低心肌细胞钙瞬态的作用,而酪氨酸激酶抑制剂genistein(10^-4mol/L）不能取消IL－2的作用;（5）U73122预处理可阻断IL－2的作用。研究结果表明,IL－2降低心肌细胞钙瞬态的作用,是通过心肌细胞上κ阿片受体介导的,其下游途径包括PTX敏感的G蛋白和磷脂酶C。     

Acetaldehyde depresses myocardial contraction and cardiac myocyte shortening in spontaneously hypertensive rats: role of intracellular Ca2+.

Acetaldehyde (ACA), the major metabolite of ethanol, exerts both stimulatory and depressive actions on myocardial tissue. We have recently shown that ACA depresses myocardial contraction, cardiac myocyte shortening and intracellular Ca2+ transients in normal rat heart. The purpose of the present study was to determine the influence of hypertension on ACA-induced myocardial actions. Mechanical properties of left ventricular papillary muscles and ventricular myocytes isolated from both 25-week-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were evaluated using force-transducer and video edge-detection, respectively. Papillary muscles and cardiac myocytes were electrically stimulated to contract at 0.5 Hz. Contractile properties analyzed include: peak tension development (PTD), peak twitch amplitude (PTA), time-to-PTD/PTA (TPT/TPS), time-to-90% relaxation/relengthening (RT90/TR90) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT/+/-dL/dt). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity (FFI) changes. ACA (1-30 mM) depressed PTD without affecting other mechanical indices in both WKY and SHR myocardium, with maximal inhibition of 64 and 69%, respectively. SHR myocytes exhibited increased cell dimension, baseline PTA and resting intracellular Ca2+ levels, compared to WKY counterparts. ACA (0.03-30 mM) depressed PTA without affecting TPT, TR90 and +/-dL/dt. The maximal inhibitions were 31 and 36% in WKY and SHR groups, respectively. Interestingly, ACA exerted a biphasic effect on FFI, displaying potentiation at lower doses (<3 mM) and inhibition at higher doses (>3 mM). The maximal increase in FFI changes were 19 and 22% at 0.3 mM and the maximal decreases were 37 and 29% at 30 mM ACA, in WKY and SHR myocytes, respectively. Neither resting intracellular Ca2+ levels (FFI) nor fluorescence decay time (FDT) were affected by ACA. The increase in FFI was attenuated by propranolol (1 microM), whereas the decrease in FFI was reversed by BayK 8644 (1 microM). These results suggest that hypertension does not appear to alter ACA-induced myocardial depression. The mechanism underlying ACA-induced myocardial actions may involve increased beta-adrenergic activity at low doses and reduced Ca2+ entry and/or release at high doses.     

Leptin modulates the negative inotropic effect of interleukin-1β in cardiac myocytes

Interleukin-1beta (IL-1beta) is a potent negative inotrope implicated in the functional abnormalities of heart failure. Because the adipokine, leptin, protects against some of the cardiovascular effects of endotoxin, we hypothesized that leptin may modulate the cardiosuppressive effects of IL-1beta in isolated cardiomyocytes. Ventricular cardiac myocytes isolated from adult male Sprague Dawley rats were analyzed simultaneously for electrically stimulated contractility and calcium transients following 30 min exposure to IL-1beta (10 ng/ml) with or without 60 min pretreatment with leptin (25 ng/ml). IL-1beta decreased cell shortening, depressed maximal velocities of shortening and relengthening, and prolonged the time to 90% relaxation. The change in fura2-AM fluorescence ratio amplitude (Delta[Ca(2+)]) was significantly depressed and the time to return to baseline [Ca(2+)] was prolonged. The negative inotropic effects of IL-1beta were blocked by the neutral sphingomyelinase inhibitor Manumycin A (5 microM) or the ceramidase inhibitor N-oleoyl ethanolamine (1 microM). Prior exposure of myocytes to leptin blocked IL-1beta-induced cardiosuppression in conjunction with a blunting of IL-1beta stimulated ceramide accumulation. These data suggest that leptin may modulate IL-1beta signaling through the sphingolipid signaling pathway in cardiomyocytes.     

Functional effects of C-type natriuretic peptide and nitric oxide are attenuated in hypertrophic myocytes from pressure-overloaded mouse hearts

Increases in the myocardial level of cGMP usually exert negative inotropic effects in the mammalian hearts. We tested the hypothesis that the negative functional effects caused by nitric oxide (NO) or C-type natriuretic peptide (CNP) through cGMP would be blunted in hypertrophied cardiac myocytes. Contractile function, guanylyl cyclase activity, cGMP-dependent protein phosphorylation, and calcium transients were assessed in ventricular myocytes from aortic stenosis-induced hypertrophic and age-matched control mice. Basal percentage shortening was similar in control and hypertrophic myocytes. S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor, 10(-6) and 10(-5) M) or CNP (10(-8) and 10(-7) M) reduced percentage shortening in both groups, but their effects were blunted in hypertrophic myocytes. Maximal rates of shortening and relaxation were depressed at the basal level, and both reagents had attenuated effects in hypertrophy. Similar results were also found after treatment with guanylin and carbon monoxide, other stimulators of particulate, and soluble guanylyl cyclase, respectively. Guanylyl cyclase activity was not significantly changed in hypertrophy. Addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (an inhibitor of cGMP-dependent protein kinase, 5 x 10(-6) M) blocked SNAP or the effect of CNP in control mice but not in hypertrophy, indicating the cGMP-dependent kinase (PKG) may not mediate the actions of cGMP induced by NO or CNP in the hypertrophic state. Calcium transients after SNAP or CNP were not significantly changed in hypertrophy. These results suggest that in hypertrophied mice, diminished effects of NO or CNP on ventricular myocyte contraction are not due to changes in guanylyl cyclase activity. The data also indicated that PKG-mediated pathways were diminished in hypertrophied myocardium, contributing to blunted effects.     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

Leptin-induced suppression of cardiomyocyte contraction is amplified by ceramide

Uncorrected obesity is often accompanied by ventricular contractile dysfunction, elevation of the lipotoxic mediator ceramide and the obesity gene product leptin. Both ceramide and leptin participate in the regulation of cardiac function and are speculated to play roles in obesity-related cardiac dysfunctions. The purpose of this study was to examine the effect of ceramide on leptin-elicited cardiac contractile response. Adult rat left ventricular myocytes were incubated for 24 h with low (5 nM) or high (50 nM) concentration of leptin in the absence or presence of the active ceramide analog C2-dihydroceramide (25 microM). Contractile and intracellular Ca2+ properties were evaluated using an IonOptix MyoCam system including peak shortening (PS), maximal velocity of shortening/relengthening (+/-dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise (Delta[Ca2+]) and intracellular Ca2+ decay. While ceramide did not elicit any effect on cell mechanics and intracellular Ca2+ transients, it sensitized leptin-induced effects on myocyte shortening and intracellular Ca2+ transients. In the absence of ceramide, 5 nM leptin had no effect on cell mechanics while 50 nM depressed PS, +/-dL/dt, Delta[Ca2+] and prolonged TR90. With ceramide co-incubation, 5 nM leptin depressed PS, +/-dL/dt, Delta[Ca2+] and prolonged TR90 whereas 50 nM leptin-elicited effects on PS, +/-dL/dt, Delta[Ca2+] and TR90 were significantly potentiated in addition to slowing intracellular Ca2+ decay. In summary, our data demonstrated that ceramide sensitizes cardiac depressive effects of leptin and may contribute to hyperleptinemia-related cardiac contractile dysfunction.     

NO underlies the muscarinic receptor-mediated inhibition of If in early embryonic heart cells.

BACKGROUND/AIMS: Early embryonic cardiomyocytes beat spontaneously. The hyperpolarization-activated cyclic-nucleotide-modulated current (I(f)) appears to be involved in its modulation as it is highly expressed at this stage. The spontaneous beating of early embryonic heart cells is slowed by acetylcholine (ACh), and our earlier studies identified a key role for nitric oxide (NO) in the regulation of the voltage dependent L-type Ca(2+) current (I(Ca,L)). The aim of the present study was to clarify whether and via which signalling pathway(s) I(f) is regulated upon muscarinic receptor activation in early embryonic (E9.5 to E11.5) cardiomyocytes. METHODS: The whole-cell patch clamp technique in combination with pharmacology and/or knock out mouse models was used to investigate the regulation of I(f). RESULTS: We found that the ACh analogue carbachol (CCh, 10 micromol) led in the majority of cells (68%, n=50) to a significant depression of I(f) by 16.3+/-1.4% (n=34, p<0.01, voltage steps from -35 mV to -110 mV). This cholinergic inhibition was mediated by the NO/cGMP signalling pathway as it was largely reversed by superfusion with the non selective nitric oxide synthase (NOS) inhibitor N(G)-Methyl-L-arginine acetate salt (L-NMMA, 1 mmol), the inhibitor of the soluble guanylyl cyclase (sGC) 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ, 100 micromol) and a selective inhibitor of the phosphodiesterase (PDE) type 2 Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 micromol). Analysis of the muscarinic signalling in embryonic cardiomyocytes harvested from NOS2 (-/-) and NOS3 (-/-) mice revealed that the NOS3 isoform was entirely responsible for the muscarinic receptor-induced NO production. CONCLUSIONS: Muscarinic receptor stimulation depresses I(f) by generating NO via the NOS3 and the cGMP/PDE type 2 signalling pathway in early embryonic cardiomyocytes. This suggests that NO is a key signalling molecule involved in the regulation of chronotropy of early embryonic heart cells.     

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one induces cell cycle arrest and apoptosis in HeLa cells by preventing microtubule polymerization

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is known as a specific inhibitor of soluble guanylyl cyclase (sGC). Previously, however, ODQ was reported to induce cell death via sGC-dependent and sGC-independent means in a variety of cell types. The aim of this study was to investigate the mechanism by which ODQ induces cell death in HeLa cells.Treatment of HeLa cells with ODQ induced a concentration-dependent decrease in cell viability over the range from 10 to 100 μM. DNA fragmentation and fluorescence-activated cell sorting analysis using annexin V and propidium iodide staining revealed that ODQ triggered apoptosis at concentrations of 50 and 100 μM within 24 to 48 h. The addition of 8-Br-cGMP in the presence of ODQ failed to rescue HeLa cells from death, suggesting that the inhibition of sGC was not responsible for the pro-apoptotic action of ODQ. ODQ arrested the cell cycle at the G2/M phase and caused disassembly of the microtubule network. This process was reversed by dithiothreitol. In addition, ODQ was shown to inhibit the polymerization of purified tubulin, and this was also prevented by dithiothreitol. These results indicate that ODQ inhibits microtubule assembly by direct oxidation of tubulin, induces cell cycle arrest at the G2/M phase, and triggers apoptosis in HeLa cells.