Lower Reactive Oxygen Species Production Research Articles

Nox4 involvement in TGF-beta and SMAD3-driven induction of the epithelial-to-mesenchymal transition and migration of breast epithelial cells

The epithelial-to-mesenchymal transition (EMT) is the development of increased cell plasticity that occurs normally during wound healing and embryonic development and can be coopted for cancer invasion and metastasis. TGF-beta induces EMT but the mechanism is unclear. Our studies suggest that Nox4, a member of the NADPH oxidase (Nox) family, is a source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. We found that TGF-beta induces Nox4 expression (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells. Conversely, cells expressing a dominant-negative form of Nox4 or Nox4-targeted shRNA showed significantly lower ROS production on TGF-beta treatment. Expression of a constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein expression, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3 dependent based on the effect of constitutively active SMAD3 increasing Nox4 promoter activity, whereas dominant-negative SMAD3 or SIS3, a SMAD3-specific inhibitor, had the opposite effect. Furthermore, Nox4 knockdown, dominant-negative Nox4 or SMAD3, or SIS3 blunted TGF-beta induced wound healing and cell migration, whereas cell proliferation was not affected. Our experiments further indicate that Nox4 plays a role in TGF-beta regulation of fibronectin mRNA expression, based on the effects of dominant-negative Nox4 in reducing fibronectin mRNA in TGF-beta-treated MDA-MB-231and MCF10A cells. Collectively, these data indicate that Nox4 contributes to NADPH oxidase-dependent ROS production that may be critical for the progression of the EMT in breast epithelial cells, and thereby has therapeutic implications.

Conditional model to study the tissue- and time specific effects of nadph oxidase 2 -derived reactive oxygen species during arthritis

Background and objectivesNeutrophil cytosolic factor 1 (NCF1) is the key regulatory component of the phagocytic NADPH oxidase 2 (NOX2) complex, and mutations in this gene lead to lowered production of...

MICS-1 interacts with mitochondrial ATAD-3 and modulates lifespan in C. elegans

Caenorhabditis elegans open reading frame T21C9.1 encodes an uncharacterized protein, which is here named MICS-1 (mitochondrial scaffolding protein-1). It is predicted to be the homolog of human outer mitochondrial membrane protein 25 (OMP25 or synaptojanin-2-binding protein), which is a PDZ domain containing protein with a putative role in cellular stress response pathways. Here, we provide evidence that MICS-1 is an interacting partner of mitochondrial protein ATAD-3 (homologue of human ATAD3), which is essential for C. elegans development. We demonstrate that mics-1(RNAi) animals or mics-1 mutants display enhanced longevity with an increased mean lifespan of up to 54% compared to control animals. Of note, also atad-3(RNAi) promoted longevity, although to a lesser extend (29% compared to controls). In addition, thermal stress of mics-1 mutants induced low reactive oxygen species (ROS) production, whereas atad-3(RNAi) animals were highly sensitive to this assay, displaying drastically increased ROS levels. Further studies revealed that MICS-1 and ATAD-3 associated longevity was partially dependent on the presence of DAF-16. However, for both conditions, we also found a DAF-16 independent extension of lifespan. Finally, we observed an additional lifespan extension in mics-1 mutants when subjected to atad-3(RNAi) whereas heat induced ROS production was even aggravated under this condition. This suggests (partially) independent effects of MICS-1 and ATAD-3 on lifespan and ROS production in vivo.

Effectiveness of Transcriptional CDK Inhibitors in Targeting Non-Proliferating CD34+CD38- AML Cells,

Abstract 3482Quiescent cells conserve energy and are characterised by low RNA synthesis. In contrast, cancer cells are thought to be addicted to high RNA synthesis, particularly synthesis of survival molecules. We hypothesised that quiescent cancer cells, already low in RNA, would be sensitive to apoptosis induced by transcriptional cyclin-dependent kinase (CDK) inhibitors that further deplete RNA. We cultured the CD34+CD38- KG1a cell line continuously in the presence of an mTOR inhibitor, which maintained excellent viability and enriched the cells for quiescent stem cell features including low RNA content, low metabolism, low reactive oxygen species production and decreased size. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that the basal mitochondrial apoptotic machinery is neither impaired nor improved. We treated quiescence-enriched cells for 48 hours with the nucleoside analogues ara-C, 5-azacytidine and clofarabine, the topoisomerase targeting agents daunorubicin, etoposide and irinotecan and three multikinase inhibitors with activity against transcriptional CDKs - flavopiridol, roscovotine and TG02. All of the agents used showed increased kill in the unmanipulated compared to the quiescence-enriched cells, emphasising the chemoresistant nature of quiescent cells. To put a value on the difficulty of eradicating the leukamic clone, we used the parameter 2 X IC50 (which theoretically would kill 100% cells in a totally homogeneous population). In quiescence-enriched cells, the percentage of cells killed at 2 X IC50 was roscovotine - 80%, TG02 – 72%, flavopiridol - 62%, 5-azacydidine - 61%, daunorubicin, clofarabine and irinotecan - 60%, etoposide - 58%, and for araC no IC50 for quiescence-enriched cells was reached at 20 times the IC50 of proliferating cells. The data show conclusively that, in this model, transcriptional CDK inhibitors outperformed the agents conventionally used in AML chemotherapy. We also showed that transcriptional CDK inhibitors induced serine 2 dephosphorylation of RNA polymerase II in equal measure in proliferating and quiescence- enriched cells. We have already shown that TGO2 targets primary CD34+CD38- cells in vitro (ASH 2010, abstract 1823). Here we also show that in four primary AML samples sensitive to roscovotine in vitro, the median percentage kill is greater in the CD34+CD38- subset, (which consists largely of quiescent cells and tends to contain the leukaemic stem cells,) than in the bulk cells (40.5% versus 19.5% kill at 2 micromolar roscovotine). We suggest that transcriptional CDK inhibitors may be a useful class of agent for targetting the occult quiescent CD34+CD38- cells thought to contribute to relapse in AML. Disclosures:Burrows:Tragara Pharmaceuticals: Employment.

Production and consumption of reactive oxygen species by fullerenes

Reactive oxygen species (ROS) are one of the most important intermediates in chemical, photochemical, and biological processes. To understand the environmental exposure and toxicity of fullerenes better, the production and consumption of ROS (singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals) by Buckminster fullerene (C(60) ) and fullerenol were investigated in aqueous systems. Fullerenol exhibits higher photoproduction efficiency of singlet oxygen and superoxide than aqueous suspensions of C(60) aggregates (aqu/nC(60) ), and this higher efficiency results in higher steady-state concentrations of these two ROS. Transmission electron microscopy indicates that the C(60) molecules in aqu/nC(60) are much more closely packed than the C(60) cages in fullerenol. These observations provide additional evidence that the lower ROS production efficiency of aqu/nC(60) is attributable primarily to efficient self-quenching of C(60) triplet states. Production of singlet oxygen by aqu/nC(60) is accelerated by increasing oxygen concentration and in part is sensitized by fluorescent photoproducts that accumulate during irradiation. The fullerenes react slowly with singlet oxygen (second-order rate constant <4 × 10(5) M(-1) s(-1) ), but react rapidly with hydroxyl radicals (second-order rate constants of 5.4 × 10(9) and 4 × 10(8) M(-1) s(-1) for aqu/nC(60) and fullerenol, respectively). These results show that environmental conditions, including light exposure and oxygen concentration, have the potential to impact the generation of toxic ROS by fullerenes.

Role of genetics in resistance to type 1 diabetes

A single nucleotide polymorphism in the mitochondrial gene encoding NADH dehydrogenase subunit 2 (mt-ND2) has been associated with reduced incidence of human type 1 diabetes (T1D). We identified the orthologue of this mitochondrial single nucleotide polymorphism in mouse and using NOD mouse models linked this genetic polymorphism to T1D resistance. The mechanism how this single nucleotide polymorphism affects the development of diabetes is studied using mouse models and beta cell lines. The impact of this single nucleotide polymorphism on mitochondrial function and resistance to reactive oxygen species was assessed. For these studies we measured oxygen consumption by isolated mitochondria under different doses of nitric oxide. In addition, alloxan sensitivity of beta cell lines was tested using the MTT method to measure cell survival. mt-Nd2a is associated with protection against mouse T1D and alloxan-induced diabetes. Mice with mt-Nd2a exhibited resistance to transfer of diabetes by single clone of diabetogenic CD4+ or CD8+ T cells. Beta cell line with mt-Nd2a resist in vitro attack of diabetogenic CD8+ cytotoxic T cells, as well as free radicals generated by alloxan; isolated mitochondria with mt-Nd2a showed lower reactive oxygen species production and were more resistant to nitric oxide. mt-Nd2a protects against T1D in mouse models. The protection is at beta cell level and is associated with resistance against reactive oxygen species-mediated damage and death.

Monascin from red mold dioscorea as a novel antidiabetic and antioxidative stress agent in rats and Caenorhabditis elegans

Monascin is a major yellow compound from red mold dioscorea. We investigated monascin to test whether this compound acts as an antidiabetic and antioxidative stress agent in diabetic rats and Caenorhabditis elegans. The mechanisms by which monascin exerts its action in vivo were also examined. Streptozotocin (STZ)-induced diabetic rats were given monascin at 30mg/kg/day and sacrificed after 8weeks. Blood glucose and serum insulin, triglyceride, total cholesterol, and high-density lipoprotein and antioxidative enzymes in the pancreas of rats were measured. In addition, monascin was evaluated for stress resistance and potential associated mechanisms in C. elegans. Throughout the 8-week experimental period, significantly lowered blood glucose, serum triglyceride, and total cholesterol and higher high-density lipoprotein levels were observed in monascin-treated rats. Monascin-treated rats showed higher serum insulin level, lower reactive oxygen species production, and higher activities of glutathione peroxidase, superoxide dismutase, and catalase in the pancreas compared to diabetic control rats. In addition, monascin significantly induced the hepatic mRNA levels of FOXO3a, FOXO1, MnSOD, and catalase in STZ-induced diabetic rats. Monascin-treated C. elegans showed an increased survival rate during oxidative stress and heat stress treatments compared to untreated controls. Moreover, monascin extended the life span under high-glucose conditions and enhanced expression of small heat shock protein (sHSP-16.2), superoxide dismutase (SOD-3), and glutathione S-transferase (GST-4) in C. elegans. Finally, we showed that monascin affected the subcellular distribution of the FOXO transcription factor DAF-16, whereas it was unable to enhance oxidative stress resistance in the daf-16 deletion mutant in C. elegans. Mechanistic studies in rats and C. elegans suggest that the protective effects of monascin are mediated via regulation of the FOXO/DAF-16-dependent insulin signaling pathway by inducing the expression of stress response/antioxidant genes, thereby enhancing oxidative stress resistance.

Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system

Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition. A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively. Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0-0.05 RLU; normal (NRP), 0.05-0.1 RLU; high (HRP), 0.1-0.4 RLU; very high (VHRP) 0.4-2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples. This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production.

Acquisition of Chemoresistance in Gliomas Is Associated with Increased Mitochondrial Coupling and Decreased ROS Production

Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches.

Peripheral Blood Neutrophil Granulocytes from Patients with Head and Neck Squamous Cell Carcinoma Functionally Differ from Their Counterparts in Healthy Donors

Solid tumors such as head and neck squamous cell carcinoma (HNSCC) display an intense interaction between tumoral factors and the immune system. Functional modulation of tumor-infiltrating and peripheral blood immune cells plays an important role during tumor progression. In this pilot study we compared biological functions of polymorphonuclear granulocytes (PMN) from the peripheral blood of HNSCC patients and healthy subjects. PMN were simultaneously isolated from the peripheral blood of HNSCC patients and healthy donors for functional analysis (apoptosis, production of reactive oxygen species (ROS), cytokine release and immunophenotyping). PMN from HNSCC patients showed a significantly lower inducible production of ROS (P = 0.02) and reduced spontaneous apoptosis (P= 0.008) compared with PMN from healthy donors. Under standard culture conditions, there was no significant difference regarding the release of inflammatory cytokines between PMN from HNSCC patients and PMN from healthy donors. Confirming previous observations, serum concentrations of PMN-related cytokines were significantly higher in the peripheral blood of HNSCC patients than in that of controls. Importantly, immunophenotyping revealed an increased number of immature PMN in PMN fractions isolated from HNSCC patients. Peripheral blood PMN from HNSCC patients and healthy donors show distinct functional differences. The presence of increased numbers of immature stages of PMN in HNSCC patients may partly contribute to the changes observed. After recruitment to and infiltration of the tumor, PMN may be further modulated in the local tumor microenvironment. This pilot study justifies functional analyses of myeloid cells in larger cohorts of patients with HNSCC.

Inositol Polyphosphate 5-Phosphatase7 Regulates the Production of Reactive Oxygen Species and Salt Tolerance in Arabidopsis

Plants possess remarkable ability to adapt to adverse environmental conditions. The adaptation process involves the removal of many molecules from organelles, especially membranes, and replacing them with new ones. The process is mediated by an intracellular vesicle-trafficking system regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Although PtdIns comprise a fraction of membrane lipids, they function as major regulators of stress signaling. We analyzed the role of PtdIns 5-phosphatases (5PTases) in plant salt tolerance. The Arabidopsis (Arabidopsis thaliana) genome contains 15 At5PTases. We analyzed salt sensitivity in nine At5ptase mutants and identified one (At5ptase7) that showed increased sensitivity, which was improved by overexpression. At5ptase7 mutants demonstrated reduced production of reactive oxygen species (ROS). Supplementation of mutants with exogenous PtdIns dephosphorylated at the D5' position restored ROS production, while PtdIns(4,5)P(2), PtdIns(3,5)P(2), or PtdIns(3,4,5)P(3) were ineffective. Compromised salt tolerance was also observed in mutant NADPH Oxidase, in agreement with the low ROS production and salt sensitivity of PtdIns 3-kinase mutants and with the inhibition of NADPH oxidase activity in wild-type plants. Localization of green fluorescent protein-labeled At5PTase7 occurred in the plasma membrane and nucleus, places that coincided with ROS production. Analysis of salt-responsive gene expression showed that mutants failed to induce the RD29A and RD22 genes, which contain several ROS-dependent elements in their promoters. Inhibition of ROS production by diphenylene iodonium suppressed gene induction. In summary, our results show a nonredundant function of At5PTase7 in salt stress response by regulating ROS production and gene expression.

Manipulating RuBisCO accumulation in the green alga, Chlamydomonas reinhardtii

The nuclear factor, Maturation/stability of RbcL (MRL1), regulates the accumulation of the chloroplast rbcL gene transcript in Chlamydomonas reinhardtii by stabilising the mRNA via its 5' UTR. An absence of MRL1 in algal mrl1 mutants leads to a complete absence of RuBisCO large subunit protein and thus a lack of accumulation of the RuBisCO holoenzyme. By complementing mrl1 mutants by random transformation of the nuclear genome with the MRL1 cDNA, different levels of rbcL transcript accumulate. We also observe that RuBisCO Large Subunit accumulation is perturbed. Complemented strains accumulating as little as 15% RuBisCO protein can grow phototrophically while RuBisCO in this range is limiting for phototrophic growth. We also observe that photosynthetic activity, here measured by the quantum yield of PSII, appears to be a determinant for phototrophic growth. In some strains that accumulate less RuBisCO, a strong production of reactive oxygen species is detected. In the absence of RuBisCO, oxygen possibly acts as the PSI terminal electron acceptor. These results show that random transformation of MRL1 into mrl1 mutants can change RuBisCO accumulation allowing a range of phototrophic growth phenotypes. Furthermore, this technique allows for the isolation of strains with low RuBisCO, within the range of acceptable photosynthetic growth and reasonably low ROS production. MRL1 is thus a potential tool for applications to divert electrons away from photosynthetic carbon metabolism towards alternative pathways.

High insulin and saturated fatty acid exposure cause mitochondrial dysfunction via distinct mechanisms

Mitochondrial dysfunction has been associated with insulin resistance, obesity and diabetes. Both hyperinsulinaemia and dyslipidemia are hallmarks of the insulin resistant state. The purpose of this study was to examine the contributions of high insulin and saturated fatty acid exposure on mitochondrial function and gene expression in cultured myocytes. Chronic insulin treatment of myotubes reduced signalling through PI 3‐Kinase/AKT. Insulin‐resistant myocytes exhibited lower mitochondrial membrane potential and ROS production but exhibited ATP levels similar to control cells. Citrate synthase activity was increased in the insulin treated cells. The expression of a number of mitochondrial genes including COX2, COX4, ATP synthase were reduced in the chronic insulin treated cells albeit they were not significant. However, the transcriptional co‐activator PGC1alpha, and uncoupling proteins, UCP2 and UCP3 were significantly reduced in these cells. No changes were detected in Tfam or NRF‐1 transcript levels. Mitofusin2 and Porin protein levels were also not altered by chronic insulin exposure. In contrast, treatment of cells with saturated fatty acid reduced mitochondrial membrane potential, significantly increased ROS production, and increased the mRNA expression of UCP2, UCP3 and ATP synthase. These cells also displayed lower levels PGC1α and PGC1β gene expression compared to controls. Taken together these results suggest that chronic hyperinsulinemia and exposure to saturated fatty acids impair mitochondrial function by different mechanisms.

Epigallocatechin Gallate Elicits Contractions of the Isolated Aorta of the Aged Spontaneously Hypertensive Rat

The present study examined the effect of the green tea catechin epigallocatechin gallate (EGCG) on endothelium-dependent responses in the aorta of 36-week-old spontaneously hypertensive rats (SHR). Isometric tension was measured in isolated aortic rings. The release of prostanoid end products was determined using enzyme immunoassay kits and the intracellular reactive oxygen species (ROS) concentration using confocal microscopy. EGCG did not improve endothelium-dependent relaxations evoked by acetylcholine, except in the presence of indomethacin. EGCG did not inhibit endothelium-dependent contractions induced by acetylcholine or ATP. At 10(-6) M and higher concentrations, EGCG caused increases in tension in the SHR aorta. The EGCG-induced contractions were accompanied by an increased production of ROS. The amount of prostanoid end products was increased significantly by EGCG, indicating that their production followed the activation of cyclooxygenase (COX). These prostanoids in turn stimulated thromboxane-prostanoid (TP) receptors and caused contractions. EGCG induced significantly smaller contractions in aortae of normotensive Wistar-Kyoto rats (WKY), accompanied with a lower production of ROS and a lesser release of prostanoids. These observations suggest that EGCG-induced contractions occur more readily in blood vessels of hypertensive than normotensive animals. The present findings indicate that the increased oxidative stress in the ageing hypertensive animals contributes to the loss of the beneficial effects and the enhancement of the adverse effects of EGCG.

Xenotransplantation of Mitochondrial Electron Transfer Enzyme, Ndi1, in Myocardial Reperfusion Injury

A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury.

Age-dependent regulation of skeletal muscle mitochondria by the thrombospondin-1 receptor CD47

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O 2 ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1–CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.

Ethylene perception inhibitor 1-MCP decreases oxidative damage of leaves through enhanced antioxidant defense mechanisms in soybean plants grown under high temperature stress

Ethylene is a stress hormone involved in early senescence and abscission of vegetative and reproductive organs under stress conditions. Ethylene perception inhibitors can minimize the impact of ethylene-mediated stress. The effects of high temperature (HT) stress during flowering on ethylene production rate in leaf, flower and pod and the effects of ethylene inhibitor on ethylene production rate, oxidative damage and physiology of soybean are not understood. We hypothesize that HT stress induces ethylene production, which causes premature leaf senescence and flower and pod abscission, and that application of the ethylene perception inhibitor 1-Methyl cyclopropene (1-MCP) can minimize HT stress induced ethylene response in soybean. The objectives of this study were to (1) determine whether ethylene is produced in HT stress; (2) quantify the effects of HT stress and 1-MCP application on oxidative injury; and (3) evaluate the efficacy of 1-MCP at minimizing HT-stress-induced leaf senescence and flower abscission. Soybean plants were exposed to HT (38/28°C) or optimum temperature (OT; 28/18°C) for 14d at flowering stage (R2). Plants at each temperature were treated with 1-MCP (1μgL−1) gas for 5h or left untreated (control). High temperature stress increased rate of ethylene production in leaves, flowers and pods, production of reactive oxygen species (ROS), membrane damage, and total soluble carbohydrate content in leaves and decreased photosynthetic rate, sucrose content, Fv/Fm ratio and antioxidant enzyme activities compared with OT. Foliar spray of 1-MCP decreased rate of ethylene production and ROS and leaf senescence traits but enhanced antioxidant enzyme activities (e.g. superoxide dismutase and catalase). In conclusion, HT stress increased ethylene production rates, caused oxidative damage, decreased antioxidant enzyme activity, caused premature leaf senescence, increased flower abscission and decreased pod set percentage. Application of 1-MCP lowered ethylene and ROS production, enhanced antioxidant enzyme activity, increased membrane stability, delayed leaf senescence, decreased flower abscission and increased pod set percentage. The beneficial effects of 1-MCP were greater under HT stress compared to OT in terms of decreased ethylene production, decreased ROS production, increased antioxidant protection, decreased flower abscission and increased pod set percentage.

Isolated Mitochondria from Skeletal Muscle Show Exaggerated Impairments with Aging Compared to Mitochondria from Permeabilized Myofibers

PURPOSE: Mitochondria regulate cellular bioenergetics and apoptosis and have been implicated in the pathogenesis of several diseases. However, it is unclear if age-related loss of muscle mass, known as sarcopenia, is associated with abnormal mitochondrial function. Two technically different approaches have mainly been used to measure mitochondrial function: isolated mitochondria (IsoMitos) and permeabilized myofibers (PermMyo) but the utility of these measures in the context of sarcopenia has not been previously assessed. We hypothesized that isolated mitochondria from skeletal muscle would show more subtle impairments with aging compared to permeabilized myofibers, due to selective harvest of the healthiest mitochondria during isolation procedures. METHODS: Using young adult (YA) and senescent (SEN) rats, we prepared IsoMitos and PermMyo from the mixed region of gastrocnemius muscle. Oxidative capacity, mitochondrial reactive oxygen species (ROS) production, and calcium retention capacity (CRC) were measured from both preparations. CRC was used as an indicator of sensitivity to permeability transition pore (PTP) opening, a physiological trigger for apoptosis. Between-group differences were analyzed using T-tests. RESULTS: Muscle mass was 38% lower in SEN gastrocnemius muscle (1.28±0.08 g; mean±SE) compared to YA (2.05±0.09 g). In PermMyo, no age differences were detected in oxidative capacity, ROS production, or CRC, except for a 30% shorter time to opening of the PTP in SEN compared to YA (p<0.05). In contrast, IsoMitos from SEN had a 65% lower oxidative capacity, 58% lower ROS production, 39% lower CRC, and 48% shorter time to PTP opening (all p<0.05). CONCLUSIONS: Contrary to our hypothesis, mitochondria from aged muscles show functional impairments that are exaggerated in IsoMitos preparations compared to PermMyo. The data suggest mitochondrial isolation reveals vulnerabilities in aged mitochondria that are not manifest under conditions where mitochondrial network structure is preserved. Care should be taken in interpreting data on aged muscle from isolated mitochondrial preparations.

Green light radiation effects on free radicals inhibition in cellular and chemical systems

Free radicals generation is inhibited through green light (GL) irradiation in cellular systems and in chemical reactions. Standard melanocyte cultures were UV-irradiated and the induced cellular reactive oxygen species (ROS) were quantified by the fluorescence technique. The same cell cultures, previously protected by a 24 h GL exposure, displayed a significantly lower ROS production. A simple chemical reaction is subsequently chosen, in which the production of free radicals is well defined. Paraffin wax and mineral oil were GL irradiated during thermal degradation and the oxidation products checked by chemiluminescence [CL] and Fourier transform infrared spectra [FT-IR]. The same clear inhibition of the radical oxidation of alkanes is recorded. A quantum chemistry modeling of these results is performed and a mechanism involving a new type of Rydberg macromolecular systems with implications for biology and medicine is suggested.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.