Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

Over-activation of p38 is implicated in many cardiovascular diseases (CVDs), including myocardial infarction, hypertrophy, heart failure, and ischemic heart disease. Numerous therapeutic interventions for CVDs have been directed toward the inhibition of the p38 mitogen-activated protein kinase activation that contributes to the detrimental effect after ischemia/reperfusion (I/R) injuries. However, the efficacy of these treatments is far from ideal, as they lack specificity and are associated with high toxicity. Previously, we demonstrated that N-acetyl cysteine (NAC) pretreatment up-regulates DUSP4 expression in endothelial cells, regulating p38 and ERK1/2 activities, and thus providing a protective effect against oxidative stress. Here, endothelial cells under hypoxia/reoxygenation (H/R) insult and isolated heart I/R injury were used to investigate the role of DUSP4 in the modulation of the p38 pathway. In rat endothelial cells, DUSP4 is time-dependently degraded by H/R (0.25±0.07-fold change of control after 2 h H/R). Its degradation is closely associated with hyperphosphorylation of p38 (2.1±0.36-fold change) and cell apoptosis, as indicated by the increase in cells immunopositive for cleaved caspase-3 (12.59±3.38%) or TUNEL labeling (29.46±3.75%). The inhibition of p38 kinase activity with 20 µM SB203580 during H/R prevents H/R-induced apoptosis, assessed via TUNEL (12.99±1.89%). Conversely, DUSP4 gene silencing in endothelial cells augments their sensitivity to H/R-induced apoptosis (45.81±5.23%). This sensitivity is diminished via the inhibition of p38 activity (total apoptotic cells drop to 17.47±1.45%). Interestingly, DUSP4 gene silencing contributes to the increase in superoxide generation from cells. Isolated Langendorff-perfused mouse hearts were subjected to global I/R injury. DUSP4^-/- hearts had significantly larger infarct size than WT. The increase in I/R-induced infarct in DUSP4^-/- mice significantly correlates with reduced functional recovery (assessed by RPP%, LVDP%, HR%, and dP/dt_max) as well as lower CF% and a higher initial LVEDP. From immunoblotting analysis, it is evident that p38 is significantly overactivated in DUSP4^-/- mice after I/R injury. The activation of cleaved caspase-3 is seen in both WT and DUSP4^-/- I/R hearts. Infusion of a p38 inhibitor prior to ischemia and during the reperfusion improves both WT and DUSP4^-/- cardiac function. Therefore, the identification of p38 kinase modulation by DUSP4 provides a novel therapeutic target for oxidant-induced diseases, especially myocardial infarction.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

pERK-dependent defective TCR-mediated activation of CD4<Superscript>+</Superscript> T cells in end-stage renal disease patients

Background

Patients with end-stage renal disease (ESRD) have an impaired immune response with a prematurely aged T-cell system. Mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK) and p38, regulate diverse cellular programs by transferring extracellular signals into an intracellular response. T cell receptor (TCR)-induced phosphorylation of ERK (pERK) may show an age-associated decline, which can be reversed by inhibiting dual specific phosphatase (DUSP) 6, a cytoplasmic phosphatase with substrate specificity to dephosphorylate pERK. The aim of this study was to assess whether ESRD affects TCR-mediated signaling and explore possibilities for intervening in ESRD-associated defective T-cell mediated immunity.

Results

An age-associated decline in TCR-induced pERK-levels was observed in the different CD4⁺ (P < 0.05), but not CD8⁺, T-cell subsets from healthy individuals (HI). Interestingly, pERK-levels of CD4⁺ T-cell subsets from young ESRD patients were in between young and elderly HI. A differentiation-associated decline in TCR-induced ERK and p38 phosphorylation was observed in T cells, although TCR-induced p38 phosphorylation was not significantly affected by age and/or ESRD. Frequencies of TCR-induced CD69-expressing CD4⁺ T cells declined with age and were positively associated with pERK. In addition, an age-associated tendency of increased expression of DUSP6 was observed in CD4⁺ T cells of HI and DUSP6 expression in young ESRD patients was similar to old HI. Inhibition of DUSP6 significantly increased TCR-induced pERK-levels of CD4⁺ T cells in young and elderly ESRD patients, and elderly HI.

Conclusions

TCR-mediated phosphorylation of ERK is affected in young ESRD patients consistent with the concept of premature immunological T cell ageing. Inhibition of DUSP6 specific for pERK might be a potential intervention enhancing T-cell mediated immunity in ESRD patients.

     

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis

The Ras/mitogen-activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual-specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal-regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium-induced colitis, as opposed to wild-type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc ^Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage-independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.     

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction  Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H₂O₂, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods  Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl⁺ cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl⁺ CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl⁺ cells. Conclusion  NAC enhances imatinib-induced apoptosis of Bcr-Abl⁺ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.     

Connexin43 Hemichannel-Mediated Regulation of Connexin43

Background

Many signaling molecules and pathways that regulate gap junctions (GJs) protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43) hemichannels were activated by cadmium (Cd²⁺), we tested this hypothesis.

Principal Findings

Incubation of Cx43-transfected LLC-PK1 cells with Cd²⁺ led to an increased expression of Cx43. This effect of Cd²⁺ was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd²⁺ on JNK activation and Cx43 expression. Indeed, Cd²⁺ induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd²⁺.

Conclusions

Collectively, our results thus indicate that Cd²⁺-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.     

CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells

The aim of this study was to characterize the role of CFTR during Cd²⁺-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr^+/+ and cftr^?/? mice were used. In cftr^+/+ cells, the application of Cd²⁺ (5 μM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl^? conductance sensitive to CFTR_inh-172. Thereafter Cd²⁺ induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl^? and TASK2 K⁺ conductances. By contrast, cftr^?/? cells exposed to Cd²⁺ were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr^+/+ cells, Cd²⁺ enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR_inh-172 or in cftr^?/? cells. Additionally, Cd²⁺ exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr^+/+ cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd²⁺ exposure by modulating the intracellular content of glutathione.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants

Industry residues, phosphate fertilisers and wastewater as a source of irrigation have considerably increased levels of heavy metals in the soil, mainly cadmium (Cd²⁺). To test the effects of a calcium (Ca²⁺) treatment on Cd²⁺ accumulation and plant tolerance to this heavy metal, plants of two citrus genotypes, Cleopatra mandarin (CM) and Carrizo citrange (CC), were watered with increasing concentrations of Cd²⁺, and phytochelatin (PC) and glutathione (GSH) content were measured. Both genotypes were able to synthesise PCs in response to heavy metal intoxication, although CM seems to be a better Cd²⁺ excluder than CC. However, data indicate that CC plants had a higher capacity for regenerating GSH than CM plants. In this context, the effects of Ca²⁺ treatment on Cd²⁺ accumulation, plant survival and PC, GSH and oxidised glutathione (GSSG) content were assessed. Data indicate that treatment with Ca²⁺ had two positive effects on citrus physiology: it reduced Cd⁺² uptake into roots and also increased GSH content (even in the absence of Cd²⁺). Overall, the data indicate that although Cd²⁺ exclusion is a powerful mechanism to avoid heavy metal build‐up into photosynthetic organs, the capacity to maintain optimum GSH levels to feed PC biosynthesis could also be an important factor in stress tolerance.     

Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

Intracellular glutathione level modulates the induction of apoptosis by Δ-prostaglandin J2

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an α, β-unsaturated carbonyl of 9-deoxy-Δ^9,12-13,14-dihydro PGD₂ (Δ¹²-PGJ₂), on the cytotoxicity of Δ¹²-PGJ₂. Δ¹²-PGJ₂ caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The Δ¹²-PGJ₂-induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by Δ¹²-PGJ₂. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of Δ¹²-PGJ₂ is due to thiol-reactivity and intracellular GSH modulates the Δ¹²-PGJ₂-induced apoptosis by regulating the accessibility of Δ¹²-PGJ₂ to target proteins containing thiol groups.     

Sulfate uptake in photosynthetic Euglena gracilis. Mechanisms of regulation and contribution to cysteine homeostasis

Background

Sulfate uptake was analyzed in photosynthetic Euglena gracilis grown in sulfate sufficient or sulfate deficient media, or under Cd²⁺ exposure or Cys overload, to determine its regulatory mechanisms and contribution to Cys homeostasis.

Results

In control and sulfate deficient or Cd²⁺-stressed cells, one high affinity and two low affinity sulfate transporters were revealed, which were partially inhibited by photophosphorylation and oxidative phosphorylation inhibitors and ionophores, as well as by chromate and molybdate; H⁺ efflux also diminished in presence of sulfate. In both sulfate deficient and Cd²⁺-exposed cells, the activity of the sulfate transporters was significantly increased. However, the content of thiol-metabolites was lower in sulfate-deficient cells, and higher in Cd²⁺-exposed cells, in comparison to control cells. In cells incubated with external Cys, sulfate uptake was strongly inhibited correlating with 5-times increased intracellular Cys. Re-supply of sulfate to sulfate deficient cells increased the Cys, γ-glutamylcysteine and GSH pools, and to Cys-overloaded cells resulted in the consumption of previously accumulated Cys. In contrast, in Cd²⁺ exposed cells none of the already elevated thiol-metabolites changed.

Conclusions

(i) Sulfate transport is an energy-dependent process; (ii) sulfate transporters are over-expressed under sulfate deficiency or Cd²⁺ stress and their activity can be inhibited by high internal Cys; and (iii) sulfate uptake exerts homeostatic control of the Cys pool.     

Intracellular glutathione level modulates the induction of apoptosis by Δ-prostaglandin J2

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an α, β-unsaturated carbonyl of 9-deoxy-Δ^9,12-13,14-dihydro PGD₂ (Δ¹²-PGJ₂), on the cytotoxicity of Δ¹²-PGJ₂. Δ¹²-PGJ₂ caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The Δ¹²-PGJ₂-induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by Δ¹²-PGJ₂. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of Δ¹²-PGJ₂ is due to thiol-reactivity and intracellular GSH modulates the Δ¹²-PGJ₂-induced apoptosis by regulating the accessibility of Δ¹²-PGJ₂ to target proteins containing thiol groups.     

Endogenous nitric oxide activation protects against cigarette smoking induced apoptosis in endothelial cells

Cigarette-induced endothelial dysfunction could be an early mediator of atherosclerosis. In this study, we explored the mechanisms of cigarette smoke extract (CSE)-induced human aortic endothelial cells (HAEC) apoptosis. We found that 10-65% of HAECs underwent apoptotic changes when HAECs were exposed to 0.001-0.02 cigarette equivalent unit of CSE for 4 h. CSE activated the caspases-3 and 8, the p38 MAP kinase and stress activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK). Specific inhibitors of p38 MAP or SAPK/JNK reduced CSE-induced caspase activation. We further showed that eNOS pre-activation by L-arginine reduced endothelial apoptosis from 65% to 5%; and eNOS inhibition by N-omega-nitro-L-arginine methyl ester accentuated CSE-induced endothelial apoptosis. We suggest that appropriate endogenous NO production may be an important protective mechanism against smoking-induced endothelial damage.     

Protective Effects of Quercetin Against Cadmium Chloride-Induced Oxidative Injury in Goat Sperm and Zygotes

Quercetin, a plant-derived flavonoid, is frequently used as an antioxidant for efficient anti-oxidative capacity. However, whether quercetin has protective effects on goat sperm and preimplantation embryos against Cd²⁺-induced oxidative injury is still unclear. So, we researched the influence of quercetin on goat sperm and zygotes respectively under the oxidative stress induced by Cd²⁺. In our study, quercetin decreased the malonaldehyde (MDA) and reactive oxygen species (ROS) levels caused by Cd²⁺ in goat sperm (p?<?0.05), which facilitated sperm characteristics including motility, survival rates, membrane integrity, and mitochondria activity during storage in vitro and subsequent embryo development (p?<?0.05). Moreover, in goat zygotes, quercetin decreased peroxidation products including ROS, MDA, and carbonyl through preserving or maintaining mitochondrial function, gene expression, and anti-oxidative products such as glutathione peroxidase, superoxide dismutase, and catalase, which ameliorated subsequent embryo development and embryo quality (p?<?0.05). Taken together, these results suggest that quercetin protects both goat sperm and preimplantation embryos from Cd²⁺-induced oxidative stress.     

Stress response in two strains of the aquatic hyphomycete <Emphasis Type="BoldItalic">Heliscus lugdunensis</Emphasis> after exposure to cadmium and copper ions

Biochemical responses to cadmium (Cd²⁺) and copper (Cu²⁺) exposure were compared in two strains of the aquatic hyphomycete (AQH) Heliscus lugdunensis. One strain (H4-2-4) had been isolated from a heavy metal polluted site, the other (H8-2-1) from a moderately polluted habitat. Conidia of the two strains differed in shape and size. Intracellular accumulation of Cd²⁺ and Cu²⁺ was lower in H4-2-4 than in H8-2-1. Both␣strains synthesized significantly more glutathione (GSH), cysteine (Cys) and γ-glutamylcysteine (γ-EC) in the presence of 25 and 50 μM Cd²⁺, but quantities and rates of synthesis were different. In H4-2-4, exposure to 50 μM Cd²⁺ increased GSH levels to 262% of the control; in H8-2-1 it increased to 156%. Mycelia of the two strains were analysed for peroxidase, dehydroascorbate reductase, glutathione reductase and glucose-6-phosphate dehydrogenase. With Cd²⁺ exposure, peroxidase activity increased in both strains. Cu²⁺ stress increased dehydroascorbate reductase activity in H4-2-4 but not in H8-2-1. Dehydroascorbate reductase and glucose-6-phosphate dehydrogenase activities progressively declined in the presence of Cd²⁺, indicating a correlation with Cd²⁺ accumulation in both strains. Cd²⁺ and Cu²⁺ exposure decreased glutathione reductase activity.