Intracellular Redox Environment Research Articles

Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal.

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping.

Foxos Suppress Lipid Peroxidation to Promote AML Progression and Chemotherapy Resistance

Over 30% of acute myeloid leukemia (AML) patients do not respond to first-line chemotherapy, and a significant portion of patients that do initially respond subsequently relapse with resistant disease. These unsatisfactory outcomes indicate that AML cells either rapidly evolve or inherently possess mechanisms for evading standard chemotherapeutic approaches. Several studies have suggested that AML cells utilize pathways that regulate intracellular redox biology to promote chemotherapy resistance; however, the precise pathways governing resistance and redox biology in AML have yet to be fully determined.We recently discovered that the FOXO family of transcription factors, which have been traditionally considered tumor suppressor genes, actually support AML cell survival and the differentiation blockade. Specifically, we observed that the expression of FOXO1 and FOXO3 are significantly increased in approximately 40% of primary human AML samples (p<0.0001) and that short hairpin RNA (shRNA)-mediated inhibition of FOXO3 caused human AML cells to acquire characteristics of myeloid differentiation such as increased CD11b expression, cytoplasmic volume, size and granularity. Moreover, we also found that compound ablation of the FoxO family members FoxO1, FoxO3 and FoxO4 significantly extends disease latency and improves survival (p=0.0009) in a murine model of AML driven by the leukemogenic allele MLL-AF9.Previous studies have shown that, in variety of cell types, FOXOs influence the intracellular redox environment by suppressing the production of reactive oxygen species (ROS). Therefore, to determine the molecular role of FOXOs in AML, we initially focused on the impact of FOXO inhibition on AML cell redox biology. Using fluorogenic probes that detect either total intracellular ROS content (CellRox) or superoxide production (MitoSox), we found that shRNA-mediated inhibition of FOXO3 did not affect total levels of intracellular ROS or superoxides. However, using a lipid peroxidation sensor (BODIPY¨ 581/591 C11), we did observe that two distinct FOXO3-targeting shRNAs increased both homeostatic and stress-induced levels of lipid peroxides in AML cells (shFOXO3-1, p=0.0004; shFOXO3-2, p=0.0023). Consistent with this, we also found that AML cells treated with a chemical inhibitor of FOXOs (AS1842856) display increased steady-state levels of intracellular lipid peroxides (p=0.0076) as well as increased signs of differentiation (CD11b and morphological changes) and death (Annexin V staining).To elucidate the importance of lipid peroxidation in AML, we evaluated how two chemical anti-oxidants, N-acetyl-L-cysteine (NAC) and butylated hydroxyanisole (BHA), impact the anti-leukemia effects and increased lipid peroxidation mediated by FOXO inhibition. From these analyses, we have observed that BHA treatment suppresses lipid peroxide production and partly blocks AML cell death induced by shRNA-mediated FOXO3 inhibition (shFOXO3-1, p=0.0001; shFOXO3-2, p<0.0001). Interestingly, NAC treatment, which does protect healthy hematopoietic stem and progenitor cells from FOXO inhibition, is unable to reverse the anti-leukemia effects or lipid peroxidation induced by FOXO inhibition, suggesting that FOXOs may differentially regulate redox biology between normal and malignant hematopoietic progenitors.Both basic and clinical studies have shown that anthracyclines such as daunorubicin (DNR) induce lipid peroxidation; however, the role of lipid peroxidation in chemotherapy effectiveness is largely unknown. We have discovered that co-treatment of AML cells with DNR and BHA (but not NAC) completely blocks the cytotoxic effects of DNR (p<0.0001), suggesting that suppression of lipid peroxides could promote chemotherapy resistance. Consistent with this idea, we have observed that shRNA-mediated inhibition of FOXO3 enhance DNR-mediated AML cell death (p<0.0001), whereas enforced expression of FOXO3 protects human AML cells from DNR cytotoxicity (p=0.003).Collectively, these results suggest that FOXOs are critical mediators of AML progression and chemotherapy resistance by directly regulating intracellular lipid peroxide levels. DisclosuresNo relevant conflicts of interest to declare.

Regulation of MMP-1 expression in response to hypoxia is dependent on the intracellular redox status of metastatic bladder cancer cells

High steady-state reactive oxygen species (ROS) production has been implicated with metastatic disease progression. We provide new evidence that this increased intracellular ROS milieu uniquely predisposes metastatic tumor cells to hypoxia-mediated regulation of the matrix metalloproteinase MMP-1. Using a cell culture metastatic progression model we previously reported that steady-state intracellular H2O2 levels are elevated in highly metastatic 253J-BV bladder cancer cells compared to their non-metastatic 253J parental cells. 253J-BV cells display higher basal MMP-1 expression, which is further enhanced under hypoxic conditions (1% O2). This hypoxia-mediated MMP-1 increase was not observed in the non-metastatic 253J cells. Hypoxia-induced MMP-1 increases are accompanied by the stabilization of hypoxia-inducible transcription factors (HIFs)-1α and HIF-2α, and a rise in intracellular ROS in metastatic 253J-BV cells. RNA interference studies show that hypoxia-mediated MMP-1 expression is primarily dependent on the presence of HIF-2α. Further, hypoxia promotes migration and spheroid outgrowth of only the metastatic 253J-BV cells and not the parental 253J cells. The observed HIF stabilization, MMP-1 expression and migration under hypoxia are dependent on increases in intracellular ROS, as these effects are attenuated by treatment with the antioxidant N-acetyl-L-cysteine. These data show that ROS play an important role in hypoxia-mediated MMP-1 expression and that an elevated intracellular redox environment, as observed in metastasis, predisposes tumor cells to an enhanced hypoxic response. It further supports the notion that metastatic tumor cells are uniquely able to utilize intracellular increases in ROS to drive pro-metastatic signaling events and highlights the important interplay between ROS and hypoxia in malignancy.

Further characterization of the role of the mitochondrial high-mobility group box protein in the intracellular redox environment of Aspergillus nidulans.

HmbB, a predominantly mitochondrial high-mobility group box (HMGB) protein, of Aspergillus nidulans affects diverse biological activities, such as sterigmatocystin production, the maintenance of mitochondrial DNA copy number, germination of asexual and sexual spores, and protection against oxidative stress agents. We hypothesized that the latter correlates with an unbalanced intracellular redox state, in which case, a not yet fully characterized physiological function could be attributed to this mitochondrial HMGB protein. Here, we studied the intracellular redox environment and oxidative stress tolerance in hmbB+ and hmbBΔ strains under normal and oxidative stress conditions by measuring glutathione redox couple, intracellular reactive oxygen species (ROS) content and ROS-protecting enzyme activities. Our results revealed that the intracellular redox environment is different in hmbBΔ conidia and mycelia from that of hmbB+, and shed light on the seemingly contradictory difference in the tolerance of hmbBΔ mycelia to diamide and menadione oxidative stressors.

Ethanol Attenuates Histiotrophic Nutrition Pathways and Alters the Intracellular Redox Environment and Thiol Proteome during Rat Organogenesis.

Ethanol (EtOH) is a reactive oxygen-generating teratogen involved in the etiology of structural and functional developmental defects. Embryonic nutrition, redox environment, and changes in the thiol proteome following EtOH exposures (1.56.0 mg/ml) were studied in rat whole embryo culture. Glutathione (GSH) and cysteine (Cys) concentrations with their respective intracellular redox potentials (Eh) were determined using high-performance liquid chromatography. EtOH reduced GSH and Cys concentrations in embryo (EMB) and visceral yolk sac (VYS) tissues, and also in yolk sac and amniotic fluids. These changes produced greater oxidation as indicated by increasingly positive Eh values. EtOH reduced histiotrophic nutrition pathway activities as measured by the clearance of fluorescin isothiocyanate (FITC)-albumin from culture media. A significant decrease in total FITC clearance was observed at all concentrations, reaching approximately 50% at the highest dose. EtOH-induced changes to the thiol proteome were measured in EMBs and VYSs using isotope-coded affinity tags. Decreased concentrations for specific proteins from cytoskeletal dynamics and endocytosis pathways (α-actinin, α-tubulin, cubilin, and actin-related protein 2); nuclear translocation (Ran and RanBP1); and maintenance of receptor-mediated endocytosis (cubilin) were observed. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis also identified a decrease in ribosomal proteins in both EMB and VYS. Results show that EtOH interferes with nutrient uptake to reduce availability of amino acids and micronutrients required by the conceptus. Intracellular antioxidants such as GSH and Cys are depleted following EtOH and Eh values increase. Thiol proteome analysis in the EMB and VYS show selectively altered actin/cytoskeleton, endocytosis, ribosome biogenesis and function, nuclear transport, and stress-related responses.

314 - Transcriptional Regulation of MMP-1 by Hypoxia Is Dependent on the Intracellular Redox Environment of Metastatic Bladder Cancer Cells

314 - Transcriptional Regulation of MMP-1 by Hypoxia Is Dependent on the Intracellular Redox Environment of Metastatic Bladder Cancer Cells

Loss of the thioredoxin reductase Trr1 suppresses the genomic instability of peroxiredoxin tsa1 mutants.

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a CanR mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the CanR mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.

G.P.198: Selenoprotein N and oxidative stress are novel regulators of myogenesis and muscle cell stemness

The pathophysiology of SEPN1-related myopathy (SEPN1-RM), due to inherited defects of Selenoprotein N (SelN, encoded by <i>SEPN1</i>), remains poorly understood. Previously we reported an increase in intracellular oxidant activity and protein carbonylation in human muscle cells devoid of SelN, suggesting a key role of SelN in antioxidant protection. In parallel, <i>sepn1</i>−<i>/</i>− mice have been shown to have defective muscle repair and depletion of the satellite cell population after repeated injury. To clarify the so-far unknown underlying mechanisms, we quantified <i>SEPN1</i> expression and ROS levels in different populations of muscle stem cells from <i>sepn1</i>−<i>/</i>− mice. We demonstrated a direct correlation between SelN expression, ROS content and degree of stemness. Oxidant activity and <i>SEPN1</i> expression were significantly higher in PICs and SCA+ cells than in the more committed fibroblasts and satellite cells. Furthermore, cells from SEPN1-RM patients devoid of SelN, muscles from the <i>sepn1</i>−<i>/</i>− mice and C2C12 myoblasts knocked-down for <i>SEPN1</i> showed perturbations in the level and subcellular localisation of myogenic and cell cycle exit factors. Congruently, we observed an accelerated initiation of differentiation in C2C12 knocked-down for <i>SEPN1</i>. We further showed that this is, at least in part, due to the role of SelN in regulating redox homeostasis, including modulation of expression of specific antioxidant genes. These results show that SelN has an important role in maintaining the balance between self-renewal and differentiation of muscle stem cells, thus representing a novel link between the intracellular and tissue redox environment and myogenesis. We show for the first time that these mechanisms can target preferentially specific stem cell populations, and that different types of muscle stem cells are maintained in different redox states, suggesting that close control of redox homeostasis is important for maintenance of their stemness and self-renewal.

External cys/cySS redox state modification controls the intracellular redox state and neurodegeneration via Akt in aging and Alzheimer's disease mouse model neurons.

The extracellular redox environment of cells is mainly set by the redox couple cysteine/cystine (cys/cySS) while intracellular redox is buffered by reduced/oxidized glutathione (GSH/GSSG), but controlled by NAD(P)H/NAD(P). With aging, the extracellular redox environment shifts in the oxidized direction beyond middle-age. Since aging is the primary risk factor in Alzheimer's disease (AD), here our aim was to determine if a reduced extracellular cys/cySS redox potential of cultured primary mouse neurons changes the intracellular redox environment, affects pAkt levels, and protects against neuron loss. A reductive shift in cys/cySS in the extracellular medium of neuron cultures from young (4 month) and old (21 month) neurons from non-transgenic) and triple transgenic AD-like mice (3xTg-AD) caused an increase in intracellular NAD(P)H and GSH levels along with lower reactive oxygen species levels. Importantly, the imposed reductive shift decreased neuron death markedly in the 21 month neurons of both genotypes. Moreover, a reduced cys/cySS redox state increased the pAkt/Akt ratio in 21 month aging and AD-like neurons that positively correlated with a decreased neuron loss. Our findings demonstrate that manipulating the extracellular redox environment toward a more reduced redox potential is neuroprotective in both aging and AD-like neurons and may be a powerful and pragmatic therapeutic tool in aging and age-related diseases like AD.

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.

Expression of serum toll-like receptor 9 and oxidative damage markers in benign and malignant breast diseases.

The intracellular redox environment plays an important role in the maintenance of proper cellular homeostasis and functions. Disturbances in redox equilibrium of cells result in pro-inflammatory conditions, and these inflammatory conditions can induce carcinogenesis or increase the malignant potential of the tumor. Oxidative stress or tissue damage can trigger toll-like receptor (TLR) family of receptors that are involved in altering the innate immune system. The present study was aimed at evaluating the level of oxidative damage markers in breast diseases by measuring the 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyl (PC), malondialdehyde (MDA), and total antioxidant status (TAS) alterations in relation to expression of TLR-9. A significant increase in the level of oxidative damage markers was observed in breast carcinoma patients in comparison to benign and normal controls, which was accompanied by a significant decrease in TAS and expression of TLR-9 concentrations. 8-OHdG, PC, and MDA were negatively correlated with expression of TLR-9 and TAS levels. Altered levels of biomarkers of oxidative stress and TLR-9 among the malignant, benign, and controls suggest a correlation of oxidative stress and TLR signaling in the progression of disease in breast carcinoma patients. Receiver operating characteristic analysis depicts that expression of TLR-9 is a good indicator for distinguishing cancer patients from benign and normal controls. High accuracy, specificity, and sensitivity of oxidative stress markers and expression of TLR-9 can be used as discriminatory marker/s for efficient diagnosis.

20S proteasome activity is modified via S-glutathionylation based on intracellular redox status of the yeast Saccharomyces cerevisiae: Implications for the degradation of oxidized proteins

Protein S-glutathionylation is a post-translational modification that controls many cellular pathways. Recently, we demonstrated that the α5-subunit of the 20S proteasome is S-glutathionylated in yeast cells grown to the stationary phase in rich medium containing glucose, stimulating 20S core gate opening and increasing the degradation of oxidized proteins. In the present study, we evaluated the correlation between proteasomal S-glutathionylation and the intracellular redox status. The redox status was controlled by growing yeast cells in distinct carbon sources which induced respiratory (glycerol/ethanol) or fermentative (glucose) metabolism. Cells grown under glycerol/ethanol displayed higher reductive power when compared to cells grown under glucose. When purified from cells grown in glucose, 20S proteasome α5-subunit exhibited an intense anti-glutathione labeling. A higher frequency of the open catalytic chamber gate was observed in the S-glutathionylated preparations as demonstrated by transmission electron microscopy. Therefore, cells that had been grown in glucose displayed an increased ability to degrade oxidized proteins. The results of the present study suggest that 20S proteasomal S-glutathionylation is a relevant adaptive response to oxidative stress that is capable to sense the intracellular redox environment, leading to the removal of oxidized proteins via a process that is not dependent upon ubiquitylation and ATP consumption.

The succinated proteome

The post-translational modifications (PTMs) of cysteine residues include oxidation, S-glutathionylation, S-nitrosylation, and succination, all of which modify protein function or turnover in response to a changing intracellular redox environment. Succination is a chemical modification of cysteine in proteins by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in 3T3 adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes in mice. Increased succination of proteins is also detected in the kidney of a fumarase deficient conditional knock-out mouse which develops renal cysts. A wide range of proteins are subject to succination, including enzymes, adipokines, cytoskeletal proteins, and ER chaperones with functional cysteine residues. There is also some overlap between succinated and glutathionylated proteins, suggesting that the same low pKa thiols are targeted by both. Succination of adipocyte proteins in diabetes increases as a result of nutrient excess derived mitochondrial stress and this is inhibited by uncouplers, which discharge the mitochondrial membrane potential (ΔΨm) and relieve the electron transport chain. 2SC therefore serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes, and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic PTM of proteins by proteomics approaches.

Manganese Superoxide Dismutase Regulates a Redox Cycle Within the Cell Cycle

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded and mitochondria-matrix-localized oxidation-reduction (redox) enzyme that regulates cellular redox homeostasis. Cellular redox processes are known to regulate proliferative and quiescent growth states. Therefore, MnSOD and mitochondria-generated reactive oxygen species (ROS) are believed to be critical regulators of quiescent cells' entry into the cell cycle and exit from the proliferative cycle back to the quiescent state. Recent evidence suggests that the intracellular redox environment fluctuates during the cell cycle, shifting toward a more oxidized status during mitosis. MnSOD activity is higher in G0/G1 cells compared with S, G2 and M phases. After cell division, MnSOD activity increases in the G1 phase of the daughter generation. The periodic fluctuation in MnSOD activity during the cell cycle inversely correlates with cellular superoxide levels as well as glucose and oxygen consumption. Based on an inverse correlation between MnSOD activity and glucose consumption during the cell cycle, it is proposed that MnSOD is a central molecular player for the "Warburg effect." In general, loss of MnSOD activity results in aberrant proliferation. A better understanding of the MnSOD and mitochondrial ROS-dependent cell cycle processes may lead to novel approaches to overcome aberrant proliferation. Since ROS have both deleterious (pathological) and beneficial (physiological) effects, it is proposed that "eustress" should be used when discussing ROS processes that regulate normal physiological functions, while "oxidative stress" should be used to discuss the deleterious effects of ROS.

Concurrent regulation of the transcription factors Nrf2 and ATF4 mediates the enhancement of glutathione levels by the flavonoid fisetin

Glutathione (GSH) and GSH-associated metabolism provide the major line of defense for the protection of cells from various forms of toxic stress. GSH also plays a key role in regulating the intracellular redox environment. Thus, maintenance of GSH levels is developing into an important therapeutic objective for the treatment of a variety of diseases. Among the transcription factors that play critical roles in GSH metabolism are NF-E2-related factor 2 (Nrf2) and activating transcription factor 4 (ATF4). Thus, compounds that can upregulate these transcription factors may be particularly useful as treatment options through their effects on GSH metabolism. We previously showed that the flavonoid fisetin not only increases basal levels of GSH but also maintains GSH levels under oxidative stress conditions. However, the mechanisms underlying these effects have remained unknown until now. Here we show that fisetin rapidly increases the levels of both Nrf2 and ATF4 as well as Nrf2- and ATF4-dependent gene transcription via distinct mechanisms. Although fisetin greatly increases the stability of both Nrf2 and ATF4, only the effect on ATF4 is dependent on protein kinase activity. Using siRNA we found that ATF4, but not Nrf2, is important for fisetin's ability to increase GSH levels under basal conditions whereas both ATF4 and Nrf2 appear to cooperate to increase GSH levels under oxidative stress conditions. Based upon these results, we hypothesize that compounds able to increase GSH levels via multiple mechanisms, such as fisetin, will be particularly effective for maintaining GSH levels under a variety of different stresses.

Surface Enhanced Raman Scattering Traceable and Glutathione Responsive Nanocarrier for the Intracellular Drug Delivery

A surface enhanced Raman scattering (SERS) traceable nanocarrier is presented through a simple strategy for the intracellular redox environment triggered drug delivery. Basically, the nanocarrier has a core-shell structure, with the Raman molecule tagged Au@Ag nanorods as the SERS active core and mesoporous silica (MS) as the drug containing shell. In the presented system, the locations of nanocarriers can be tracked by SERS signals while those of drugs can be monitored through their fluorescence, allowing the simultaneous investigation of the intracellular distribution of both the nanocarriers and the drugs. To endow the nanocarrier with the glutathione (GSH) responsive behavior, disulfide, which can be cleaved by GSH, is used to directly attach drug molecules to the MS. Compared with other disulfide based drug delivery strategies, this is a quite simple and efficient method. The experimental results confirmed that the drug release can be triggered by the stimuli. Moreover, after the cellular uptake of the nanocarriers, a gradual drug release from the nanocarriers was observed by monitoring both the fluorescence of the drug molecules and the SERS signals of the nanocarriers. Considering its stimuli-responsive properties, this kind of nanocarrier would have great potential in improving the efficacy of cancer chemotherapy by avoiding premature drug leakage. More importantly, this SERS based tracking method of the nanocarrier would be more powerful than that based only on the fluorescence of the drug in the studies of drug release dynamic processes.

Identification of GR and TrxR systems in Setaria cervi: Purification and characterization of glutathione reductase

The glutathione reductase (GR) and thioredoxin reductase (TrxR) are important enzymes of the redox system that aid parasites to maintain an adequate intracellular redox environment. In the present study, the enzyme activity of GR and TrxR was investigated in Setaria cervi (S. cervi). Significant activity of both enzymes was detected in the somatic extract of adult and microfilariae stages of S. cervi. Both GR and TrxR were separated by partial purification using ammonium sulfate fractionation and DEAE ion exchange chromatography suggesting the presence of both glutathione and thioredoxin systems in S. cervi. The enzyme glutathione reductase (ScGR) was purified to homogeneity using affinity and ion exchange chromatography that resulted in 90 fold purification with a yield of 11.54%. The specific activity of the ScGR was 643U/mg that migrated as a single band on SDS-PAGE. The subunit molecular mass was determined to be ~50kDa while the optimum pH and temperature were found to be 7.0 and 35°C respectively. The activation energy (Ea) was calculated from the slope of Arrhenius plot as 16.29±1.40kcal/mol. The Km and Vmax were determined to be 0.27±0.045mM; 30.30±1.30U/ml with NADPH and 0.59±0.060mM; 4.16±0.095U/ml with GSSG respectively. DHBA, a specific inhibitor for GR has completely inhibited the enzyme activity at 1μM concentration. The inhibition of ScGR activity with NAI (IC50 0.71mM), NEM (IC50 0.50mM) and DEPC (IC50 0.27mM) suggested the presence of tyrosine, cysteine and histidine residues at its active site. Further studies on characterization and understanding of these antioxidant enzymes may lead to designing of an effective drug against lymphatic filariasis.

Redox environment is an intracellular factor to operate distinct pathways for aggregation of Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS). Misfolding and aggregation of mutant SOD1 proteins are a pathological hallmark of SOD1-related fALS cases; however, the molecular mechanism of SOD1 aggregation remains controversial. Here, I have used E. coli as a model organism and shown multiple distinct pathways of SOD1 aggregation that are dependent upon its thiol-disulfide status. Overexpression of fALS-mutant SOD1s in the cytoplasm of E. coli BL21 and SHuffleTM, where redox environment is reducing and oxidizing, respectively, resulted in the formation of insoluble aggregates with notable differences; a disulfide bond of SOD1 was completely reduced in BL21 or abnormally formed between SOD1 molecules in SHuffleTM. Depending upon intracellular redox environment, therefore, mutant SOD1 is considered to misfold/aggregate through distinct pathways, which would be relevant in description of the pathological heterogeneity of SOD1-related fALS cases.

High-fat load: mechanism(s) of insulin resistance in skeletal muscle.

Skeletal muscle from sedentary obese patients is characterized by depressed electron transport activity, reduced expression of genes required for oxidative metabolism, altered mitochondrial morphology and lower overall mitochondrial content. These findings imply that obesity, or more likely the metabolic imbalance that causes obesity, leads to a progressive decline in mitochondrial function, eventually culminating in mitochondrial dissolution or mitoptosis. A decrease in the sensitivity of skeletal muscle to insulin represents one of the earliest maladies associated with high dietary fat intake and weight gain. Considerable evidence has accumulated to suggest that the cytosolic ectopic accumulation of fatty acid metabolites, including diacylglycerol and ceramides, underlies the development of insulin resistance in skeletal muscle. However, an alternative mechanism has recently been evolving, which places the etiology of insulin resistance in the context of cellular/mitochondrial bioenergetics and redox systems biology. Overnutrition, particularly from high-fat diets, generates fuel overload within the mitochondria, resulting in the accumulation of partially oxidized acylcarnitines, increased mitochondrial hydrogen peroxide (H2O2) emission and a shift to a more oxidized intracellular redox environment. Blocking H2O2 emission prevents the shift in redox environment and preserves insulin sensitivity, providing evidence that the mitochondrial respiratory system is able to sense and respond to cellular metabolic imbalance. Mitochondrial H2O2 emission is a major regulator of protein redox state, as well as the overall cellular redox environment, raising the intriguing possibility that elevated H2O2 emission from nutrient overload may represent the underlying basis for the development of insulin resistance due to disruption of normal redox control mechanisms regulating protein function, including the insulin signaling and glucose transport processes.

The Good and Bad Effects of Cysteine S-nitrosylation and Tyrosine Nitration upon Insulin Exocytosis: A Balancing Act

As understanding of the mechanisms driving and regulating insulin secretion from pancreatic beta cells grows, there is increasing and compelling evidence that nitric oxide (•NO) and other closely-related reactive nitrogen species (RNS) play important roles in this exocytic process. •NO and associated RNS, in particular peroxynitrite, possess the capability to effect signals across both intracellular and extracellular compartments in rapid fashion, affording extraordinary signaling potential. It is well established that nitric oxide signals through activation of guanylate cyclase-mediated production of cyclic GMP. The intricate intracellular redox environment, however, lends credence to the possibility that •NO and peroxynitrite could interact with a wider variety of biological targets, with two leading mechanisms involving 1) Snitrosylation of cysteine, and 2) nitration of tyrosine residues comprised within a variety of proteins. Efforts aimed at delineating the specific roles of •NO and peroxynitrite in regulated insulin secretion indicate that a highly-complex and nuanced system exists, with evidence that •NO and peroxynitrite can contribute in both positive and negative regulatory ways in beta cells. Furthermore, the ultimate biochemical outcome within beta cells, whether to compensate and recover from a given stress, or not, is likely a summation of contributory signals and redox status. Such seeming regulatory dichotomy provides ample opportunity for these mechanisms to serve both physiological and pathophysiologic roles in onset and progression of diabetes. This review focuses attention upon recent accumulating evidence pointing to roles for nitric oxide induced post-translational modifications in the normal regulation as well as the dysfunction of beta cell insulin exocytosis.