Glutathione-mediated Antioxidative Mechanisms in Sunflower (<Emphasis Type="Italic">Helianthus Annuus</Emphasis> L.) Cells in Response to Cadmium Stress

Cadmium (Cd) homeostasis and detoxification in sunflower (Helianthus annuus L.) cells differing in Cd sensitivity/tolerance were studied by analyzing the glutathione-mediated antioxidant mechanism vis-à-vis phytochelatin biosynthesis in vitro. Calluses exposed to Cd-shock/-acclimatization (150μM) were assayed for oxidative stress, reduced glutathione (GSH), glutathione disulfide (GSSG), phytochelatins (PCs) and reactive oxygen species (ROS). Although Cd did not induce any oxidative stress in Cd-tolerant callus (TCd), it generated oxidative stress in Cd-shock callus (SCd) both in terms of lipid peroxidation and protein oxidation. GSH/GSSG ratio remained similar to control values in the cadmium-acclimatized calluses. However, after acute treatment, there was a decline in both GSH and GSSG levels in SCd with concomitant reduction in the GSH/GSSG ratio. Analysis of PCs was performed using HPLC and mass spectrometry methods. PC concentration in TCd were approximately twice those that in SCd, showing in both cases a 1:2:1 relative proportion for PC n = 2 (PC2): PC n = 3 (PC3): PC n = 4 (PC4). Calluses growing in the presence of Cd developed an increased resistance to paraquat oxidative stress generation. These results indicated that PCs synthesis was an important mechanism for Cd detoxification in sunflower calluses, but the capacity to grow in the presence of Cd is related to the tissues ability to maintain high intracellular levels of GSH.     

Inhibitory Effect of Ascorbic Acid on the Proliferation and Invasion of Hepatoma Cells in Culture

Effect of ascorbic acid (AsA) on the proliferation and invasion of rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cells and by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells, respectively. AsA suppressed the invasion of AH109A cells in a dose-dependent manner at concentrations of 62.5–500 μM, while it inhibited the proliferation of the cells at higher concentrations of 250 and 500 μM. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. AsA suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with AsA, HX and XO or with AsA and hydrogen peroxide. Furthermore, AsA reduced the intracellular peroxide levels in AH109A cells. These results suggest that the antioxidative property of AsA may be involved in its anti-invasive action on hepatoma cells.     

Lung Ischemia: A Model for Endothelial Mechanotransduction

Endothelial cells in vivo are constantly exposed to shear associated with blood flow and altered shear stress elicits cellular responses (mechanotransduction). This review describes the role of shear sensors and signal transducers in these events. The major focus is the response to removal of shear as occurs when blood flow is compromised (i.e., ischemia). Pulmonary ischemia studied with the isolated murine lung or flow adapted pulmonary microvascular endothelial cells in vitro results in endothelial generation of reactive oxygen species (ROS) and NO. The response requires caveolae and is initiated by endothelial cell depolarization via K_ATP channel closure followed by activation of NADPH oxidase (NOX2) and NO synthase (eNOS), signaling through MAP kinases, and endothelial cell proliferation. These physiological mediators can promote vasodilation and angiogenesis as compensation for decreased tissue perfusion.     

High-dose vitamin C supplementation increases skeletal muscle vitamin C concentration and SVCT2 transporter expression but does not alter redox status in healthy males

Antioxidant vitamin C (VC) supplementation is of potential clinical benefit to individuals with skeletal muscle oxidative stress. However, there is a paucity of data reporting on the bioavailability of high-dose oral VC in human skeletal muscle. We aimed to establish the time course of accumulation of VC in skeletal muscle and plasma during high-dose VC supplementation in healthy individuals. Concurrently we investigated the effects of VC supplementation on expression levels of the key skeletal muscle VC transporter sodium-dependent vitamin C transporter 2 (SVCT2) and intramuscular redox and mitochondrial measures. Eight healthy males completed a randomized placebo-controlled, crossover trial involving supplementation with ascorbic acid (2×500 mg/day) over 42 days. Participants underwent muscle and blood sampling on days 0, 1, 7, and 42 during each treatment. VC supplementation significantly increased skeletal muscle VC concentration after 7 days, which was maintained at 42 days (VC 3.0±0.2 (mean±SEM) to 3.9±0.4 mg/100 g wet weight (ww) versus placebo 3.1±0.3 to 2.9±0.2 mg/100 g ww, p=0.001). Plasma VC increased after 1 day, which was maintained at 42 days (VC 61.0±6.1 to 111.5±10.4 µmol/L versus placebo 60.7±5.3 to 59.2±4.8 µmol/L, p<0.001). VC supplementation significantly increased skeletal muscle SVCT2 protein expression (main treatment effect p=0.006) but did not alter skeletal muscle redox measures or citrate synthase activity. A main finding of our study was that 7 days of high-dose VC supplementation was required to significantly increase skeletal muscle vitamin C concentration in healthy males. Our findings implicate regular high-dose vitamin C supplementation as a means to safely increase skeletal muscle vitamin C concentration without impairing intramuscular ascorbic acid transport, antioxidant concentrations, or citrate synthase activity.     

Amyloid beta-peptide effects on synaptosomes from apolipoprotein E-deficient mice

Apolipoprotein E (apoE) is present in the brain and may contribute to neurophysiologic or neuropathologic events, depending on environmental and genetic influences. Recent studies indicate a role for apoE in synaptic plasticity and maintenance of synaptic membrane symmetry, suggesting that apoE may be involved in regulating synaptic homeostasis. In the present study, cerebrocortical synaptosomes were prepared from transgenic mice lacking apoE (apoE KO) to analyze the possible contribution of apoE toward maintaining homeostasis in synaptosomes. Synaptosomal preparations from apoE KO and wild-type mice exhibited similar basal levels of reactive oxygen species, mitochondrial function, and caspase activity; however, following application of amyloid beta-peptide [Abeta(1-40)], apoE KO synaptosomes displayed increased levels of oxidative stress, mitochondrial dysfunction, and caspase activation compared with synaptosomes from wild-type mice. Synaptosomal membranes from apoE KO mice were more fluid than wild-type synaptosomes and contained higher levels of thiobarbituric acid-reactive substances, consistent with elevated levels of lipid peroxidation occurring in the synapses of apoE KO mice. Together, these data are consistent with a role for apoE in maintaining homeostasis by attenuating oxidative stress, caspase activation, and mitochondrial homeostasis in synapses.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25–25 µg/ml) for an hour then incubated with H₂O₂ (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H₂O₂. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H₂O₂ only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.     

Proteome changes in the myocardium of experimental chronic diabetes and hypertension: role of PPARα in the associated hypertrophy

Diabetes with or without the presence of hypertension damages the heart. However, there is currently a lack of information about these associated pathologies and the alteration of linked proteins. For these reasons, we were interested in the potential synergistic interaction of diabetes and hypertension in the heart, focusing on the proteome characterization of the pathological phenotypes and the associated hypertrophic response. We treated normotensive and spontaneously hypertensive (SHR) rats with either streptozotocin or vehicle. After 22 weeks, type-I diabetic (DM1), SHR, SHR/DM1 and control left-ventricles were studied using proteomic approaches. Proteomics revealed that long-term DM1, SHR and SHR/DM1 rats exhibited 24, 53 and 53 altered proteins in the myocardia, respectively. DM1 myocardium showed over-expression of apoptotic and cytoskeleton proteins, and down-regulation of anti-apoptotic and mitochondrial metabolic enzymes. In both SHR and SHR/DM1 these changes were exacerbated and free fatty-acid (FFA) ß-oxidation enzymes were additionally decreased. Furthermore, SHR/DM1 hearts exhibited a misbalance of specific pro-hypertrophic, anti-apoptotic and mitochondrial ATP-carrier factors, which could cause additional damage. Differential proteins were validated and then clustered into different biological pathways using bioinformatics. These studies suggested the implication of FFA-nuclear receptors and hypertrophic factors in these pathologies. Although key ß-oxidation enzymes were not stimulated in DM1 and hypertensive hearts, peroxisome proliferator-activated receptors-α (PPARα) were potentially activated for other responses. In this regard, PPARα stimulation reduced hypertrophy and pro-hypertrophic factors such as annexin-V in high-glucose and angiotensin-II induced cardiomyocytes. Thus, activation of PPARα could reflect a compensatory response to the metabolic-shifted, apoptotic and hypertrophic status of the hypertensive-diabetic cardiomyopathy.     

Sustained delivery of exogenous melatonin influences biomarkers of oxidative stress and total antioxidant capacity in summer-stressed anestrous water buffalo (Bubalus bubalis)

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (μmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes.     

镉胁迫对拟南芥的毒害作用及自噬现象的观测

镉离子(Cd2+)具有强植物毒性,可抑制植物生长,甚至导致植物死亡。为了研究重金属镉对拟南芥的毒害作用,采用叶绿素荧光技术、流式细胞技术、激光共聚焦技术及半定量RT-PCR技术,检测光合参数的变化、活性氧(reactive oxygen species,ROS)的累积、自噬的发生,以及病原相关蛋白(pathogenesis-related protein,PR)基因表达的变化。实验结果显示,随着50μmol/L CdCl2处理时间的延长,ROS和Cd2+在细胞中大量积累。而在镉胁迫的初期,会观察到自噬的发生及PR基因表达的变化。说明植物受到外界Cd2+作用的初期,会通过自噬及增强PR基因表达来抵抗外界胁迫。但随着处理时间的延长,植物细胞内累积了大量的ROS和Cd2+,当植物不足以通过自噬途径抵抗胁迫时,就会导致生长受阻,最终对光合系统造成损伤。