Isolation and Structure of a New Victoxinine Derivative Produced by Helminthosporium victoriae

The structures of alkyl radicals generated in several methyl esters of fatty acids by irradiation with UV light were studied by the spin trapping technique. A spin trap, deuterated nitrosodurene, traps alkyl radicals in both saturated and unsaturated esters at the ambient temperature. The trapped radicals and their hyperfine splitting constants from several esters were as follows: pentadienyl radicals (a_N= 13.8 ~ 14.0 G, a_H = 5.9 ~ 6.0 G) from methyl linoleate, linolenate and docosahexaenoate; allyl radicals (a_N = 13.9 G, a_H = 6.8 G) and α-carbon radicals (a_N = 13.3 G, a_H = 10.0 G) from methyl oleate and elaidate; α-carbon radicals (a_N = 13.3 ~ 13.4 G, a_H = 9.6 ~ 10.0 G) and secondary alkyl radicals (a_N = 13.9 G, a_H = 6.8 ~ 7.2 G) from saturated esters.     

高通量低功率激光照射诱导肿瘤细胞凋亡的分子机制研究

低功率激光照射（low—power laser irradiation,LPLI）能够引起广泛的促细胞增殖、分化等生物刺激效应。基于这些效应,低功率激光治疗已经成为一种临床上广泛应用的有效的激光理疗手段。从2005年开始,邢达小组开始对LPu在较高激光通量（剂量）时的肿瘤细胞杀伤效应进行初步探讨。研究发现,高通量低功率激光照射（high fluencelow—power laser irradiation,HF—LPLI）通过激活内源光受体来触发线粒体氧应激,进而激活线粒体凋亡通路。该研究工作加深了对LPLI生物刺激效应分子机制的了解,为低功率激光治疗在临床应用时激光剂量的合理选择提供重要理论参考依据。与此同时,基于HF—LPLI有效杀死肿瘤细胞的效应,HF—LPLI可以作为一种潜在的、有效的临床肿瘤治疗手段。     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

Flow cytometric analysis of mitochondria from CA1 and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation

Mitochondria are important in the pathophysiology of several neurodegenerative diseases, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes and release of apoptogenic proteins are involved in these processes. Following brain insults, cell death often occurs in discrete regions of the brain, such as the subregions of the hippocampus. To analyse mitochondrial structure and function in such subregions, only small amounts of mitochondria are available. We developed a protocol for flow cytometric analysis of very small samples of isolated brain mitochondria, and analysed mitochondrial swelling and formation of ROS in mitochondria from the CA1 and CA3 regions of the hippocampus. Calcium-induced mitochondrial swelling was measured, and fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide) and to measure production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). We found that formation of ROS and mitochondrial permeability transition pore activation were higher in mitochondria from the CA1 than from the CA3 region, and propose that differences in mitochondrial properties partly underlie the selective vulnerability of the CA1 region to brain insults. We also conclude that flow cytometry is a useful tool to analyse the role of mitochondria in cell death processes.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Abstract

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H₂O₂) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H₂O₂-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H₂O₂-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H₂O₂, and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca^{2 +}) chelator, overexpression of Homer 1a had no significant effects on H₂O₂-induced oxidative stress. These results suggest that Homer 1a has protective effects against H₂O₂-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca^{2 +} homeostasis.     

The role of mitochondria-derived reactive oxygen species in the pathogenesis of non-steroidal anti-inflammatory drug-induced small intestinal injury

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) have been implemented in clinical settings for a long time for their anti-inflammatory effects. With the number of NSAID users increasing, gastroenterological physicians and researchers have worked hard to prevent and treat NSAID-induced gastric mucosal injury, an effort that has for the large part being successful. However, the struggle against NSAID-induced mucosal damage has taken on a new urgency due to the discovery of NSAID-induced small intestinal mucosal injury. Although the main mechanism by which NSAIDs induce small intestinal mucosal injury has been thought to depend on the inhibitory effect of NSAIDs on cyclooxygenase (COX) activity, recent studies have revealed the importance of mitochondria-derived reactive oxygen species (ROS) production, which occurs independently of COX-inhibition. ROS production is an especially important factor in the increase of small intestinal epithelial cell permeability, an early stage in the process of small intestinal mucosal injury. By clarifying the precise mechanism, together with its clinical features using novel endoscopy, effective strategies for preventing NSAID-induced small intestinal damage, especially targeting mitochondria-derived ROS production, may be developed.     

Curcumin inhibits TNFα-induced lectin-like oxidised LDL receptor-1 (LOX-1) expression and suppresses the inflammatory response in human umbilical vein endothelial cells (HUVECs) by an antioxidant mechanism

In this study, the anti-oxidative activities of 70% ethanol extract from Curcuma aromatica Salisb. (CAS) and curcumin (CUR) were studied. The CAS extracts and CUR were both found to have a potent scavenging activity against the reactive species tested, as well as an inhibitory effect on LDL oxidation. Cultured human umbilical vein endothelial cells (HUVECs) were stimulated with tumour necrosis factor α (TNFα), expression of intracellular reactive oxygen species (ROS), nitric oxide (NO), endothelial nitric oxide synthase (eNOS), lectin-like oxidised LDL receptor-1 (LOX-1), adhesion molecules, inhibitory kappa Bα (IκBα) and nuclear factor kappa B (NFκB) were measured. In HUVECs stimulated with TNFα, CUR significantly suppressed expression of the intracellular ROS, LOX-1 and adhesion molecules, degradation of IκBα and translocation of NFκB, while inducing production of NO by phosphorylation of eNOS (p <0.05). In conclusion, CAS and CUR may modulate lipoprotein composition and attenuate oxidative stress by elevated antioxidant processes.     

Redox regulation of cellular functions

Maintenance of normal intracellular redox status plays an important role in such processes as DNA synthesis, gene expression, enzymatic activity, and others. In addition, it is clear that changes in the redox status of intracellular content and individual molecules, resulting from stress or intrinsic cellular activity, are involved in the regulation of different processes in cells. Small changes in intracellular levels of reactive oxygen species participate in intracellular signaling. Thiol-containing molecules, such as glutathione, thioredoxins, glutaredoxins, and peroxiredoxins, also play an important role in maintaining redox homeostasis and redox regulation. This review attempts to summarize the current knowledge about redox regulation in different cell types.     

Abnormalities of mitochondrial functioning can partly explain the metabolic disorders encountered in sarcopenic gastrocnemius

Aging triggers several abnormalities in muscle glycolytic fibers including increased proteolysis, reactive oxygen species (ROS) production and apoptosis. Since the mitochondria are the main site of substrate oxidation, ROS production and programmed cell death, we tried to know whether the cellular disorders encountered in sarcopenia are due to abnormal mitochondrial functioning. Gastrocnemius mitochondria were extracted from adult (6 months) and aged (21 months) male Wistar rats. Respiration parameters, opening of the permeability transition pore and ROS production, with either glutamate (amino acid metabolism) or pyruvate (glucose metabolism) as a respiration substrate, were evaluated at different matrix calcium concentrations. Pyruvate dehydrogenase and respiratory complex activities as well as their contents measured by Western blotting analysis were determined. Furthermore, the fatty acid profile of mitochondrial phospholipids was also measured. At physiological calcium concentration, state III respiration rate was lowered by aging in pyruvate conditions (-22%), but not with glutamate. The reduction of pyruvate oxidation resulted from a calcium-dependent inactivation of the pyruvate dehydrogenase system and could provide for the well-known proteolysis encountered during sarcopenia. Matrix calcium loading and aging increased ROS production. They also reduced the oxidative phosphorylation. This was associated with lower calcium retention capacities, suggesting that sarcopenic fibers are more prone to programmed cell death. Aging was also associated with a reduced mitochondrial superoxide dismutase activity, which does not intervene in toxic ROS overproduction but could explain the lower calcium retention capacities. Despite a lower content, cytochrome c oxidase displayed an increased activity associated with an increased n-6/n-3 polyunsaturated fatty acid ratio of mitochondrial phospholipids. In conclusion, we propose that mitochondria obtained from aged muscle fibers display several functional abnormalities explaining the increased proteolysis, ROS overproduction and vulnerability to apoptosis exhibited by sarcopenic muscle. These changes appear to be related to modifications of the fatty acid profile of mitochondrial lipids.     

Apoptosis induction by the natural product cancer chemopreventive agent deguelin is mediated through the inhibition of mitochondrial bioenergetics

Deguelin exhibits chemopreventive properties in animal carcinogenesis models. The mechanism underpinning the chemopreventive effects of deguelin has not been fully elucidated. However, it has been suggested that this agent reduces ornithine decarboxylase activity, and perhaps the activity of other signaling intermediates associated with tumorigenesis, by inhibiting mitochondrial bioenergetics. We sought to determine if deguelin could trigger apoptosis by inhibiting mitochondrial bioenergetics. Therefore, we compared and contrasted the effects of deguelin on cells from two human cutaneous squamous cell carcinoma cell lines (parental cells) and their respiration-deficient clones lacking mitochondrial DNA (rho0). While deguelin promoted marked apoptosis in the parental cells in a dose- and time-dependent manner, it failed to do so in the rho0 clones. Furthermore, short-term exposure to deguelin diminished oxygen consumption by the parental cells and promoted mitochondrial permeability transition as evidenced by the dissipation of mitochondrial inner transmembrane potential, reactive oxygen species production, cardiolipin peroxidation, caspase activation, and mitochondrial swelling. Mitochondrial permeability transition was not observed in the rho0 clones exposed to deguelin. These results demonstrate that deguelin induces apoptosis in skin cancer cells by inhibiting mitochondrial bioenergetics and provide a novel mechanism for the putative anticancer activity of this agent.     

Hydrogen peroxide and hydroxyl radicals mediate palmitate-induced cytotoxicity to hepatoma cells: relation to mitochondrial permeability transition

We studied the toxicological responses of a human hepatoblastoma cell line (HepG2/C3A) to various free fatty acids (FFA) in order to identify the relation between reactive oxygen species (ROS) production and mitochondrial permeability transition (MPT). Exposure to the saturated FFA, palmitate, led to a time-dependent ROS production and hydrogen peroxide release as well as a loss of mitochondrial potential. The cytotoxicity of palmitate was significantly reduced by treating with scavengers of hydrogen peroxide, hydroxyl radical and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN). Superoxide dismutase (SOD) mimics, nitric oxide scavenger, and inhibitor of de novo ceramide synthesis had no effect on the toxicity. MPT-inhibitor, cyclosporine, prevented the loss of mitochondrial potential but did not reduce the cytotoxicity. In contrast, inhibiting mitochondrial complexes I and III reduced the early potential loss and the cytotoxicity. These results suggest that palmitate-cytotoxicity to hepatoma cells is mediated through the production of H₂O₂ and *OH and independent of MPT.     

Hydrogen peroxide and hydroxyl radicals mediate palmitate-induced cytotoxicity to hepatoma cells: Relation to mitochondrial permeability transition

We studied the toxicological responses of a human hepatoblastoma cell line (HepG2/C3A) to various free fatty acids (FFA) in order to identify the relation between reactive oxygen species (ROS) production and mitochondrial permeability transition (MPT). Exposure to the saturated FFA, palmitate, led to a time-dependent ROS production and hydrogen peroxide release as well as a loss of mitochondrial potential. The cytotoxicity of palmitate was significantly reduced by treating with scavengers of hydrogen peroxide, hydroxyl radical and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN). Superoxide dismutase (SOD) mimics, nitric oxide scavenger, and inhibitor of de novo ceramide synthesis had no effect on the toxicity. MPT-inhibitor, cyclosporine, prevented the loss of mitochondrial potential but did not reduce the cytotoxicity. In contrast, inhibiting mitochondrial complexes I and III reduced the early potential loss and the cytotoxicity. These results suggest that palmitate-cytotoxicity to hepatoma cells is mediated through the production of H₂O₂ and *OH and independent of MPT.