Taurine reduced oxygen free radical production induced by homocysteine in electron transport chain and homocysteine inhibites taurine transporter in mitochondria membrane

AIM: To observe effects of homocysteine and antagonized effects of taurine on electronic leakage and free radical production in myocardial mitochondria. METHODS: Myocardial mitochondria of rat heart was isolated, and was broken by supersonic wave to prepare submitochondria. Recombinant of succinic acid cytochrome c reductase was prepared with mitochondria of porcine heart. They were co-incubated with homocysteine and/or taurine with various concentration. The H₂O₂ and O₂^- were determined by chemiluminescence methods. The taurine transporter of heart mitochondria and its propert, and effects of homocysteine on its function were studied with glass filter. RESULTS: Homocysteine stimulated oxygen free radical production in heart mitochondria, submitochondria, and succinic acid cytochrome c in a concentration-dependent manner. Although taurine itself did not affect oxygen free radical production, taurine did inhibit oxygen free radical production in mitochondria, submitochondria and succinic acid cytochrome c in a concentration-dependent manner. Taurine transporters of Na⁺-dependent were existed in mitochondria membrane. Homocysteine inhibited taurine transtport in mitochondria in a concentration-dependent manner. CONCLUSIONS: Taurine inhibited electronic leakage and oxygen free radical production induced by homocysteine in electron transport chain. There were taurine transporters in mitochondria membrane, and transport functions of taurine transporter were inhibited by homocysteine.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

Energy metabolism and apoptotic effect of microwave radiation on rat myocardial cells

AIM: To investigate the effect of microwave radiation at different intensities on the rat myocardium and its possible mechanism.METHODS: The rats were radiated by the intensity of 500, 1 000, 1 500 and 2 000 W/m² with 2 450 MHz microwave for 6 min. The heart tissue was collected 6 h after microwave radiation. ATP and mitochondria complex Ⅳ and Ⅴ were measured. The changes of the tissue structures were observed under transmission electron microscope. The apoptosis of the myocardial cells was detected by a cell analyzer. The protein level of cleaved caspase-3 was determined by Western blotting.RESULTS: The concentration of ATP and activity of mitochondria complex Ⅳand Ⅴ signi-ficantly decreased compared with control group in the cardiac tissues. The decreased number, morphological abnormalities such as dissolved cavitation, matrix and obvious tumefaction of mitochondria were observed under transmission electron microscope. The microwave radiation induced the apoptosis of myocardial cells in the rats. The cell apoptotic rate and the protein level of cleaved caspase-3 increased with increasing intensity of microwave radiation(P<0.05).CONCLUSION: Microwave radiation has obvious injury effect on the rat heart, which can cause cardiac energy metabolism dysregulation and cardiac myocyte apoptosis.     

Etoposide induces apoptosis via mitochondrial signaling pathway with cytochrome c release in Jurkat leukemia cells

AIM:To investigate the molecular mechanisms of apoptosis and to elucidate the apoptosis signaling pathway triggered by etoposide in Jurkat human leukemia cells. METHODS:Apoptosis was detected using annexin V-FITC and propidium iodide (PI) staining, respectively, and annexin V-FITC positive cells and hypodiploid cells were analyzed by flow cytometry. Mitochondrial membrane potential (△Ψm) was detected using 3, 3-dihexyloxycarbocyanine iodide ［DiOC6(3)］ staining and △Ψm low cells were analyzed by flow cytometry. Preparation of cytosolic extracts and isolation of mitochondria were completed by centrifugation. Western blotting analysis was used to evaluate the level of cytochrome c, caspase-3, and poly (ADP-ribose) polymerase (PARP) expression. RESULTS:Etoposide induced apoptosis showing phosphatidylserine externalization and DNA fragmentation in a time-dependent manner and the apoptosis could be inhibited by a broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Collapse of △Ψm induced by etoposide preceded DNA fragmentation and phosphatidylserine externalization. In contrast, it was not blocked by zVAD.fmk. Etoposide caused cytochrome c release from mitochondria into cytosol, subsequent activation of caspase-3 (32 kD) presented with an intermediate (20 kD) and its active product (17 kD), and cleavage of full-length PARP (116 kD) into the so-called apoptotic 85 kD fragment. CONCLUSION:Etoposide-induced Jurkat cell apoptosis is initiated through mitochondria signaling pathway with cytochrome c release into cytoplasm and caspase is the ultimate executioner of cell apoptosis.     

Serine protease HtrA2/Omi and cell death or survival

The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli.Once in the cytosol,HtrA2/Omi has been implicated in promoting cell death in caspase-dependent or caspase-independent pathway.On the other hand,HtrA2/Omi can refold and degrade misfolded proteins in the mitochondria during cell stress.     

Mdivi-1, mitochondrial fission inhibitor,impairs developmental competence and mitochondrial function of embryos and cells in pigs

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.     

Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida

Nanoparticles (NPs) of TiO₂ and ZnO are receiving increasing attention due to their widespread applications. To evaluate their toxicities to the earthworm Eisenia fetida (Savigny, 1826) in soil, artificial soil systems containing distilled water, 0.1, 0.5, 1.0 or 5.0 g kg⁻¹ of NPs were prepared and earthworms were exposed for 7 days. Contents of Zn and Ti in earthworm, activities of antioxidant enzymes, DNA damage to earthworm, activity of cellulase and damage to mitochondria of gut cells were investigated after acute toxicity test. The results from response of the antioxidant system combined with DNA damage endpoint (comet assay) indicated that TiO₂ and ZnO NPs could induce significant damage to earthworms when doses were greater than 1.0 g kg⁻¹. We found that Ti and Zn, especially Zn, were bioaccumulated, and that mitochondria were damaged at the highest dose in soil (5.0 g kg⁻¹). The activity of cellulase was significantly inhibited when organisms were exposed to 5.0 g kg⁻¹ of ZnO NPs. Our study demonstrates that both TiO₂ and ZnO NPs exert harmful effects to E. fetida when their levels are higher than 1.0 g kg⁻¹ in soil and that toxicity of ZnO NPs was higher than TiO₂.     

Effects of maternal limb ischemic preconditioning on structural and functional changes of mitochondria in fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats

AIM: To investigate the effects of maternal limb ischemic preconditioning (LIP) on the mitochondrial structures and functions of the hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats. METHODS: Pregnant rats (n=40) were randomly divided into 4 groups: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. Intrauterine ischemia model was established through the experimental design. The ultrastructure of the mitochondria in CA1 area of the hippocampus was observed. The mitochondrial membrane potential and reactive oxygen species (ROS) were measured. The content of ATP and MDA in the hippocampus tissue was detected. The activity of Mn-SOD was observed. RESULTS: Compared with sham group, the ultrastructure of mitochondria in CA1 area of the hippocampus was damaged in FD group and LIP+FD group. The mitochondrial membrane potential, the content of ATP and the activity of Mn-SOD were decreased. However, the content of ROS and MDA was increased. Compared with FD group, the ultrastructure of mitochondria in CA1 area of the hippocampus was intact in LIP+FD group. Furthermore, the reduced mitochondrial membrane potential and ATP content were inhibited. The activity of Mn-SOD was increased, but the content of ROS and MDA was decreased in LIP+FD group. CONCLUSION: Limb ischemia preconditioning inhibits the damage the mitochondria of fetal hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats.     

Proteomic study of myocardial mitochondria with ischemia/reperfusion injury and pinacidil postconditioning in isolated rat hearts

AIM: To investigate the protective effect of pinacidil postconditioning on rat myocardium suffering ischemia/reperfusion injury by mitochondrial proteomics. METHODS: Langendorff apparatus was used to establish the model of myocardial ischemia/reperfusion injury. Sprague-Dawley rats were randomly divided into 2 groups: pinacidil postconditioning group (Pina group) and ischemia/reperfusion injury group (I/R group). After 20 min of perfusion with K-H solution, the perfusion was suspended for 40-min (global ischemia) follow by 60 min of reperfusion in I/R group. In Pina group at the end of 40 min global ischemia, the isolated hearts were perfused with K-H solution containing pinacidil (50 μmol/L) for 2 min followed 58-min perfusion with regular K-H solution. Total proteins extracted from the mitochondria were applied to the two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots over 2 times were evaluated by a software. Then they were subjected to in-gel digestion, and analyzed by spectrometry. RESULTS: The expression levels of NDUFA10, NDUFS2 and NDUFV2 were elevated but those of IDHA and ECH1 were decreased in Pina group compared with I/R group. Interestingly, 2 spots in the 2-DE map were identified as ATPase subunit δ. The expression levels of one spot was elevated, while the other was decreased. CONCLUSION: Pinacidil postconditioning may decrease the degree of increased expression levels of NDUFA10, NDUFS2 and NDUFV2, promote the expression of IDHA and ECH1, and induce the phosphorylation of ATPase subunit δ, which may be related to the protective mechanism of pinacidil postconditioning.     

渗透胁迫对玉米幼苗根尖线粒体ATPase活性与结合态多胺含量的影响

[目的]探讨线粒体膜上结合态多胺与渗透胁迫的关系。[方法]以抗旱性不同的玉米(Zea maysL.)品种农大108(抗旱性较强)和掖单13(抗旱性较弱)幼苗为材料,研究在聚乙二醇(PEG)-6000渗透胁迫下,幼苗根线粒体ATPase水解活性与膜上非共价结合态亚精胺(Spd)含量的关系。[结果]渗透胁迫条件下,抗性强的农大108的ATPase活性下降的幅度明显小于抗性弱的品种掖单13,而农大108的线粒体膜上非共价结合亚精胺的上升幅度明显大于掖单13。外源Spd处理,明显促进了掖单13在胁迫条件下非共价结合Spd含量的增加,同时抑制其在胁迫条件下ATPase活性的降低,从而提高了掖单13幼苗的抗性;Spd生物合成的专一性抑制剂——甲基乙二醛-双(鸟嘌呤腙)(MGBG)处理,显著抑制了农大108在胁迫条件下膜上非共价结合Spd的增加,同时促进其在胁迫条件下ATPase活性的降低,从而明显降低了农大108幼苗的抗性。[结论]线粒体膜上的非共价结合的Spd可能通过维持ATPase活性的稳定来提高玉米幼苗的抗渗透胁迫能力。