BACKGROUND

Chemoresistance is a significant clinical problem in pancreatic cancer (PC) and underlying molecular mechanisms still remain to be completely understood. Here we report a novel exosome-mediated mechanism of drug-induced acquired chemoresistance in PC cells.

METHODS

Differential ultracentrifugation was performed to isolate extracellular vesicles (EVs) based on their size from vehicle- or gemcitabine-treated PC cells. Extracellular vesicles size and subtypes were determined by dynamic light scattering and marker profiling, respectively. Gene expression was examined by qRT-PCR and/or immunoblot analyses, and direct targeting of DCK by miR-155 was confirmed by dual-luciferase <em>3</em>'-UTR reporter assay. Flow cytometry was performed to examine the apoptosis indices and reactive oxygen species (ROS) levels in PC cells using specific dyes. Cell viability was determined using the WST-1 assay.

RESULTS

Conditioned media (CM) from gemcitabine-treated PC cells (Gem-CM) provided significant chemoprotection to subsequent gemcitabine toxicity and most of the chemoresistance conferred by Gem-CM resulted from its EVs fraction. Sub-fractionation grouped EVs into distinct subtypes based on size distribution and marker profiles, and exosome (Gem-Exo) was the only sub-fraction that imparted chemoresistance. Gene expression analyses demonstrated upregulation of SOD2 and CAT (ROS-detoxifying genes), and downregulation of DCK (gemcitabine-metabolising gene) in Gem-Exo-treated cells. SOD/CAT upregulation resulted, at least in part, from exosome-mediated transfer of their transcripts and they suppressed basal and gemcitabine-induced ROS production, and partly promoted chemoresistance. DCK downregulation occurred through exosome-delivered miR-155 and either the functional suppression of miR-155 or restoration of DCK led to marked abrogation of Gem-Exo-mediated chemoresistance.

CONCLUSIONS

Together, these findings establish a novel role of exosomes in mediating the acquired chemoresistance of PC.

To evaluate the role of renin-angiotensin system (RAS)-mediated oxidative stress in insulin resistance (IR), we compared the effects of the angiotensin II (ANG II) receptor blocker (ARB) valsartan and a superoxide dismutase (<em>SOD</em>) mimetic, tempol, on whole body glucose tolerance and soleus muscle insulin-stimulated glucose uptake in transgenic hypertensive TG(mREN-2)27 (Ren-2) rats. Ren-2 rats and Sprague-Dawley (SD) controls were given valsartan (<em>3</em>0 mg/kg) or tempol (1 mmol/l) in their drinking water for 21 days. IR was measured by glucose tolerance testing (1 g/kg glucose ip). IR index (AUC(glucose) x AUC(insulin)) was significantly higher in the Ren-2 animals compared with SD controls (<em>3</em>0.5 +/- 7.0 x 10(6) arbitrary units in Ren-2 vs. 10.2 +/- 2.4 x 10(6) in SD, P < 0.01). Both valsartan and tempol treatment normalized Ren-2 IR index. Compared with SD controls (100%), there was a significant increase in superoxide anion production (measured by lucigenin-enhanced chemiluminescence) in soleus muscles of Ren-2 rats (1<em>3</em><em>3</em> +/- 15%). However, superoxide production was reduced in both valsartan- and tempol-treated (85 +/- 22% and 59 +/- 12%, respectively) Ren-2 rats. Insulin (INS)-mediated 2-deoxyglucose (2-DG) uptake (%SD basal levels) was substantially lower in Ren-2 rat soleus muscle compared with SD (Ren-2 + INS = 110 +/- <em>3</em>% vs. SD + INS = 206 +/- 12%, P < 0.05). However, Ren-2 rats treated with valsartan or tempol exhibited a significant increase in insulin-mediated 2-DG uptake compared with untreated transgenic animals. Improvements in skeletal muscle insulin-dependent glucose uptake and whole body IR in rats overexpressing ANG II by ARB or <em>SOD</em> mimetic indicate that oxidative stress plays an important role in ANG II-mediated insulin resistance.

OBJECTIVE

To screen superoxide dismutase 1 (<em>SOD</em>1) on chromosome 21 as a possible candidate gene for familial keratoconus (KC).

METHODS

Total genomic DNA was extracted from the blood of 15 different KC families and 156 unaffected subjects. All five exons of the <em>SOD</em>1 gene were sequenced. For a rapid screening test, DNA was amplified by polymerase chain reaction (PCR), digested with HpyCH4 III or analyzed by radioactively end-labeled exon PCR. RNA was extracted from leukocytes and reverse transcribed to cDNA, and the PCR was amplified for splice variants. Some samples were cloned and sequenced.

RESULTS

A heterozygous genomic 7-base deletion in intron 2 of the <em>SOD</em>1 gene was identified in two KC families (pedigrees 1 and 6). The deletion segregated within pedigree 1 and was absent in <em>3</em>12 chromosomes from normal individuals. RNA from the proband of pedigree 1 showed that in addition to the wild-type transcript, two other transcripts were expressed for the CuZn <em>SOD</em> (<em>SOD</em>1) gene: lacking entire exon 2 (LE2) and lacking entire exon 2 and entire exon <em>3</em> (LE2E<em>3</em>).

CONCLUSIONS

A unique genomic deletion within intron 2 close to the 5' splice junction of the <em>SOD</em>1 gene was identified in three patients with KC. Moreover, mRNA from one affected individual also had two transcript splice variants (LE2 and LE2E<em>3</em>) that others have shown to code for proteins lacking the active site of the <em>SOD</em>1 enzyme. Further studies should be conducted to determine whether a causal relationship exists between these two events that may increase oxidative stress and be associated with KC.

Oxidants are toxic, but at low doses they can stimulate rather than inhibit the growth of mammalian cells and play a role in the etiology of cancer and fibrosis. The effect of oxidants on cells is modulated by multiple interacting antioxidant defense systems. We have studied the individual roles and the interaction of Cu,Zn-superoxide dismutase (<em>SOD</em>) and catalase (CAT) in transfectants with human cDNAs of mouse epidermal cells JB6 clone 41. Since only moderate increases in these enzymes are physiologically meaningful, we chose the following five clones for in-depth characterization: CAT 4 and CAT 12 with 2.6-fold and 4.2-fold increased catalase activities, respectively, <em>SOD</em> 15 and <em>SOD</em> <em>3</em> with 2.<em>3</em>-fold and <em>3</em>.6-fold increased Cu,Zn-<em>SOD</em> activities, respectively, and SOCAT <em>3</em> with a <em>3</em>-fold higher catalase activity and 1.7-fold higher Cu,Zn-<em>SOD</em> activity than the parent JB6 clone 41. While the increases in enzyme activities were moderate, the human cDNAs were highly expressed in the transfectants. As demonstrated for the clone <em>SOD</em> 15, this discordance between message concentrations and enzyme activities may be due to the low stability of the human Cu,Zn-<em>SOD</em> mRNA in the mouse recipient cells. According to immunoblots the content of Mn-<em>SOD</em> was unaltered in the transfectants. While the activities of glutathione peroxidase were comparable in all strains, the concentrations of reduced glutathione (GSH) were significantly lower in <em>SOD</em> <em>3</em> and <em>SOD</em> 15. This decrease in GSH may reflect a chronic prooxidant state in these Cu,Zn-<em>SOD</em> overproducers.(ABSTRACT TRUNCATED AT 250 WORDS)

Independently, superoxide (O2-) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and O2- were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1alpha, and p5<em>3</em> were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated O2- during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1alpha, and p5<em>3</em> regulation. NO responses were restored by addition of <em>SOD</em>. The intermediates formed from the reactions of NO with O2- were non-toxic, did not form <em>3</em>-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of O2- by <em>SOD</em>, was cytotoxic at high concentrations and activated p5<em>3</em> independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of O2- with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from O2-.

BACKGROUND

An increase in oxidative stress is suggested to be intimately involved in the pathogenesis of heart failure. However, gene expression of enzymes that metabolize reactive oxygen metabolites has not been investigated in the human heart.

RESULTS

Myocardial tissue homogenates of the left ventricular wall from hearts in end-stage failure due to dilated (DCM) or ischemic (ICM) cardiomyopathy (n=12 each), as well as from nonfailing donor hearts (n=12), were analyzed for mRNA levels of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), glutathione peroxidase (GPX), and catalase by Northern blot analyses. Protein levels of MnSOD, CuZnSOD, and catalase were determined by Western blot or ELISA. MnSOD, CuZnSOD, and GPX mRNA levels were similar in all <em>3</em> groups. In contrast, catalase mRNA levels were found to be increased by 12<em>3</em>+/-2<em>3</em>% in DCM hearts and by 9<em>3</em>+/-10% in ICM hearts (P<0.01 each) compared with control hearts. Likewise, catalase protein levels were found to be increased in failing hearts (DCM by 90+/-10%, ICM by 90+/-1<em>3</em>%; P<0. 05 each) compared with control hearts. In addition, the observed upregulation of catalase mRNA and protein in failing hearts was attended by an increased catalase enzyme activity (DCM by 124+/-16%, ICM by 117+/-15%; P<0.01 each), whereas MnSOD, CuZnSOD, and GPX enzyme activity levels were unchanged in failing compared with nonfailing myocardium.

CONCLUSIONS

Increased oxidative stress in human end-stage heart failure may result in a specific upregulation of catalase gene expression as a compensatory mechanism, whereas SOD and GPX gene expression remain unaffected.

UV radiation (UVR) induces serious structural and functional alterations in human skin leading to skin aging and carcinogenesis. Reactive oxygen species are key players in UVR-mediated photodamage and induce the DNA-base-oxidized, intermediate 8-hydroxy-2'-deoxyguanosine (8-OHdG). Herein, we report the protective action of melatonin against UVR-induced 8-OHdG formation and depletion of antioxidative enzymes using ex vivo human full-thickness skin exposed to UVR in a dose (0, 100, <em>3</em>00 mJ/cm(2))- and time-dependent manner (0, 24, 48 hr post-UVR). Dynamics of depletion of antioxidative enzymes including catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (<em>SOD</em>), or 8-OHdG formation were studied by real-time PCR and immunofluorescence/immunohistochemical staining. UVR-treated skin revealed significant and immediate (0 hr <em>3</em>00 mJ/cm(2)) reduction of gene expression, and this effect intensified within 24 hr post-UVR. Simultaneous increase in 8-OHdG-positive keratinocytes occurred already after 0 hr post-UVR reaching 71% and 99% up-regulation at 100 and <em>3</em>00 mJ/cm(2), respectively (P < 0.001). Preincubation with melatonin (10(-<em>3</em>) M) led to <em>3</em>2% and 29% significant reductions in 8-OHdG-positive cells and the prevention of antioxidative enzyme gene and protein suppression. Thus, melatonin was shown to play a crucial role as a potent antioxidant and DNA protectant against UVR-induced oxidative damage in human skin.

We have reported that members of the bcl-2 gene family are expressed and gonadotropin regulated in ovarian granulosa cells during follicular maturation and atresia. Because Bcl-2, a protein that prevents apoptosis in several cell types, is reported to function as an antioxidant or free radical scavenger, the present studies were designed to investigate if oxidative stress plays a role in granulosa cell apoptosis during follicular atresia in the immature rat ovary. In the first series of experiments, the role of oxidative stress in the induction of granulosa cell apoptosis was directly tested using a defined in vitro follicle culture system. Healthy antral follicles obtained from equine CG (eCG)-primed immature (27 day old) rats were incubated in serum-free medium for 24 h in the absence or presence of FSH (100 ng/ml; a control for inhibiting apoptosis), superoxide dismutase (<em>SOD</em>; 10-1000 U/ml), ascorbic acid (0.01-1 mM; a free radical scavenger), N-acetyl-L-cysteine (25-100 mM; a free radical scavenger and stimulator of endogenous glutathione peroxidase activity), or catalase (10-1000 U/ml). Granulosa cells within follicles incubated in medium alone exhibited extensive apoptosis after 24 h of incubation, and this onset of apoptosis was blocked by treatment with FSH (29 +/- 4% of controls; P < 0.001, n = <em>3</em>). Moreover, apoptosis in follicles was also inhibited by treatment with <em>SOD</em> (44 +/- 4% of controls at 1000 U/ml; P < 0.01, n = <em>3</em>), ascorbic acid (55 +/- 9% of controls at 1 mM; P < 0.05, n = <em>3</em>), N-acetyl-L-cysteine (24 +/- 7% of controls at 100 mM; P < 0.001, n = <em>3</em>), or catalase (<em>3</em>5 +/- 6% of controls at 1000 U/ml; P < 0.001, n = <em>3</em>). In the second series of experiments, complementary DNAs corresponding to secreted (SEC-<em>SOD</em>), copper/zinc-containing (Cu/Zn-<em>SOD</em>), and manganese-containing (Mn-<em>SOD</em>) forms of rat <em>SOD</em>, rat seleno-cysteine glutathione peroxidase (GSHPx), and rat catalase were isolated and used to synthesize antisense RNA probes for Northern and slot blot analysis of changes in <em>SOD</em>, GSHPx, and catalase gene expression during follicular maturation. In vivo priming of 25-day-old female rats for 2 days with 10 IU eCG, which promoted antral follicular growth and survival, increased levels of messenger RNA encoding SEC-<em>SOD</em> (216 +/- 9% of saline-treated controls, P < 0.05, n = <em>3</em>) and Mn-<em>SOD</em> (222 +/- 14% of saline-treated controls, P < 0.05, n = <em>3</em>) vs. saline-treated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Both reactive oxygen species (ROS) and Forkhead box O (FOXO) family transcription factors are involved in the regulation of adipogenic differentiation of preadipocytes and stem cells. While FOXO has a pivotal role in maintaining cellular redox homeostasis, the interactions between ROS and FOXO during adipogenesis are not clear. Here we examined how ROS and FOXO regulate adipogenesis in human adipose-derived stem cells (hASC). The identity of isolated cells was confirmed by their surface marker expression pattern typical for human mesenchymal stem cells (positive for CD29, CD44, CD7<em>3</em>, CD90, and CD105, negative for CD45 and CD<em>3</em>1). Using a standard adipogenic cocktail consisting of insulin, dexamethasone, indomethacin, and <em>3</em>-Isobutyl-1-methylanxthine (IDII), adipogenesis was induced in hASC, which was accompanied by ROS generation. Scavenging ROS production with N-acetyl-L-cysteine or EUK-8, a catalytic mimetic of superoxide dismutase (<em>SOD</em>) and catalase, inhibited IDII-induced adipogenesis. We then mimicked IDII-induced oxidative stress through a lentiviral overexpression of Nox4 and an exogenous application of hydrogen peroxide in hASC and both manipulations significantly enhanced adipogenesis without changing the adipogenic differentiation rate. These data suggest that ROS promoted lipid accumulation in hASC undergoing adipogenesis. Antioxidant enzymes, including <em>SOD</em>2, catalase, and glutathione peroxidase were upregulated by IDII during adipogenesis, and these effects were blunted by FOXO1 silencing, which also suppressed significantly IDII-induced adipogenesis. Our findings demonstrated a balance of ROS generation and endogenous antioxidants in cells undergoing adipogenesis. Approaches targeting ROS and/or FOXO1 in adipocytes may bring new strategies to prevent and treat obesity and metabolic syndrome.

The effect of endurance training on the resistance of the heart to left ventricular (LV) functional deficit and infarction after a transient regional ischemia and subsequent reperfusion was examined. Female Sprague-Dawley rats were randomly assigned to an endurance exercise training (Tr) group or a sedentary (Sed) control group. After 20 wk of training, hearts were excised, perfused, and instrumented for assessment of LV mechanical function, and the left anterior descending coronary artery was occluded to induce a transient regional ischemia (1 h) that was followed by 2 h of reperfusion. Throughout much of the regional ischemia-reperfusion protocol, coronary flow rates, diastolic function, and LV developed pressure were better preserved in hearts from Tr animals. During the regional ischemia, coronary flow to myocardium outside the ischemic zone at risk (ZAR) was maintained in Tr hearts, whereas it progressively fell in Sed hearts. On release of the coronary artery ligature, flow to the ZAR was greater in Tr than in Sed hearts. Infarct size, expressed as a percentage of the ischemic ZAR, was significantly smaller in hearts from Tr rats (24 +/- <em>3</em> vs. <em>3</em>2 +/- 2% of ZAR, P < 0.05). Mn- and CuZn-<em>SOD</em> protein expression were higher in the LV myocardium of Tr animals (P < 0.05 for both isoforms). Our data indicate that long-term exercise training leads to infarct sparing and better maintenance of coronary flow and mechanical function after ischemia-reperfusion.

Physiological levels of laminar shear stress completely abrogate apoptosis of human endothelial cells in response to a variety of stimuli and might therefore importantly contribute to endothelial integrity. We show here that the apoptosis-suppressive effects of shear stress are mediated by upregulation of Cu/Zn <em>SOD</em> and NO synthase. Shear stress-mediated inhibition of endothelial cell apoptosis in response to exogenous oxygen radicals, oxidized LDL, and tumor necrosis factor-alpha was associated with complete inhibition of caspase-<em>3</em>-like activity, the central effector arm executing the apoptotic cell death program in endothelial cells. Shear stress-dependent upregulation of Cu/Zn <em>SOD</em> and NO synthase blocks activation of the caspase cascade in response to apoptosis-inducing stimuli. These findings establish the upregulation of Cu/Zn <em>SOD</em> and NO synthase by shear stress as a central protective cellular mechanism to preserve the integrity of the endothelium after proapoptotic stimulation.

The involvement of oxidative stress in the pathogenesis of alcoholic diseases in the liver has been repeatedly confirmed. Resveratrol, a natural phytoalexin present in grape skin and red wine possesses a variety of biological activities including antioxidant. This study was conducted to evaluate whether resveratrol has a preventive effect on the main indicators of hepatic oxidative status as an expression of the cellular damage caused by free radicals, and on antioxidant defence mechanism during chronic ethanol treatment. Wistar rats were treated daily with <em>3</em>5% ethanol solution (<em>3</em> g/kg/day i.p.) during 6 weeks and fed basal diet or basal diet containing 5 g/kg resveratrol. Control rats were treated with i.p. saline and fed basal diet. Experimentally, chronic ethanol administration leads to hepatotoxicity as monitored by the increase in the level of hepatic marker enzymes and the appearance of fatty change, necrosis, fibrosis and inflammation in liver sections. Ethanol also enhanced the formation of MDA in the liver indicating an increase in lipid peroxidation, a major end-point of oxidative damage, and caused drastic alterations in antioxidant defence systems. Particularly the activities of hepatic superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GPx) and catalase (CAT) were found reduced by ethanol treatment while glutathione reductase (GR) activity was unchanged. Dietary supplementation with resveratrol during ethanol treatment inhibited hepatic lipid peroxidation and ameliorated <em>SOD</em>, GPx and CAT activities in the liver. Conclusively, we can suggest that resveratrol could have a beneficial effect in inhibiting the oxidative damage induced by chronic ethanol administration, which was proved by the experiments that we conducted on rats.

Diabetic nephropathy, the leading cause of end-stage renal disease, is characterized by a proapoptotic and prooxidative environment. The mechanisms by which lifestyle interventions, such as exercise, benefit diabetic nephropathy are unknown. We hypothesized that exercise inhibits early diabetic nephropathy via attenuation of the mitochondrial apoptotic pathway and oxidative damage. Type 2 diabetic db/db and normoglycemic wild-type mice were exercised for an hour everyday at a moderate intensity for 7 wk, following which renal function, morphology, apoptotic signaling, and oxidative stress were evaluated. Exercise reduced body weight, albuminuria, and pathological glomerular expansion in db/db mice independent of hyperglycemic status. Changes in renal morphology were also related to reduced caspase-<em>3</em> (main effector caspase in renal apoptosis), caspase-8 (main initiator caspase of the "extrinsic" pathway) activities, and TNF-alpha expression. A role for the mitochondrial apoptotic pathway was unlikely as both caspase-9 activity (initiator caspase of this pathway) and expression of regulatory proteins such as Bax and Bcl-2 were unchanged. Kidneys from db/db mice also produced higher levels of superoxides and had greater oxidative damage concurrent with downregulation of superoxide dismutase (<em>SOD</em>) 1 and <em>3</em>. Interestingly, although exercise also increased superoxides, there was also upregulation of multiple <em>SODs</em> that likely inhibited lipid (hydroperoxides) and protein (carbonyls and nitrotyrosine) oxidation in db/db kidneys. In conclusion, exercise can inhibit progression of early diabetic nephropathy independent of hyperglycemia. Reductions in caspase-<em>3</em> and caspase-8 activities, with parallel improvements in <em>SOD</em> expression and reduced oxidative damage, could underlie the beneficial effects of exercise in diabetic kidney disease.

The pathology of traumatic brain injury (TBI) is characterized by the decreased capacity of neurons to metabolize energy and sustain synaptic function, likely resulting in cognitive and emotional disorders. Based on the broad nature of the pathology, we have assessed the potential of the omega-<em>3</em> fatty acid docosahexaenoic acid (DHA) to counteract the effects of concussive injury on important aspects of neuronal function and cognition. Fluid percussion injury (FPI) or sham injury was performed, and rats were then maintained on a diet high in DHA (1.2% DHA) for 12 days. We found that DHA supplementation, which elevates brain DHA content, normalized levels of brain-derived neurotrophic factor (BDNF), synapsin I (Syn-1), cAMP-responsive element-binding protein (CREB), and calcium/calmodulin-dependent kinase II (CaMKII), and improved learning ability in FPI rats. It is known that BDNF facilitates synaptic transmission and learning ability by modulating Syn-I, CREB, and CaMKII signaling. The DHA diet also counteracted the FPI-reduced manganese superoxide dismutase (<em>SOD</em>) and Sir2 (a NAD+-dependent deacetylase). Given the involvement of <em>SOD</em> and Sir2 in promoting metabolic homeostasis, DHA may help the injured brain by providing resistance to oxidative stress. Furthermore, DHA normalized levels of calcium-independent phospholipase A2 (iPLA2) and syntaxin-<em>3</em>, which may help preserve membrane homeostasis and function after FPI. The overall results emphasize the potential of dietary DHA to counteract broad and fundamental aspects of TBI pathology that may translate into preserved cognitive capacity.

OBJECTIVE

The study was aimed to evaluate the oxidative/nitrosative stress status in prostate cancer (CaP) and benign prostatic hyperplasia (BPH).

METHODS

<em>3</em>12 men from two different populations were included: 16<em>3</em> men from Macedonia (7<em>3</em> CaP patients, 67 BPH patients and 2<em>3</em> control subjects) and 149 men from Turkey (<em>3</em>4 prostate cancer patients, 100 BPH patients and 15 control subjects). We measured erythrocyte malondialdehyde (MDA) levels, erythrocyte activities of superoxide dismutase (CuZn-<em>SOD</em>), glutathione peroxidase (GPX) and catalase (CAT); plasma nitrite/nitrate (NO(2)(-)/NO(<em>3</em>)(-)), cGMP and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels.

RESULTS

A similar pattern of alteration in the oxidative/nitrosative stress-related parameters was found in both, Macedonian and Turkish studied samples: higher MDA concentrations with lower GPX and CuZn-<em>SOD</em> activities in CaP patients versus controls and BPH groups. The CAT activity was decreased in the CaP patients versus controls in the Turkish studied sample. Furthermore, CaP patients had increased plasma NO(2)(-)/NO(<em>3</em>)(-) and cGMP levels versus controls and BPH groups in both studied samples.

CONCLUSIONS

This study has confirmed an imbalance in the oxidative stress/antioxidant status and revealed an altered nitrosative status in prostate cancer patients.

Alternatives to the canonical insulin-stimulated pathway for glucose uptake are exercise- and exogenous reactive oxygen species (ROS)-stimulated glucose uptake. We proposed a model wherein mechanical loading, i.e. stretch, stimulates production of ROS to activate AMP-activated kinase (AMPK) to increase glucose uptake. Immunoblotting was used to measure protein phosphorylation; the fluorochrome probe 2'7'-dichlorofluorescin diacetate was used to measure cytosolic oxidant activity and 2-deoxy-d[1,2-(<em>3</em>)H]glucose was used to measure glucose uptake. The current studies demonstrate that stretch increases ROS, AMPKalpha phosphorylation and glucose transport in murine extensor digitorum longus (EDL) muscle (+121%, +164% and +184%, respectively; P < 0.05). We also demonstrate that stretch-induced glucose uptake persists in transgenic mice expressing an inactive form of the AMPKalpha2 catalytic subunit in skeletal muscle (+17<em>3</em>%; P < 0.05). MnTBAP, a superoxide dismutase (<em>SOD</em>) mimetic, N-acteyl cysteine (NAC), a non-specific antioxidant, ebselen, a glutathione mimetic, or combined <em>SOD</em> plus catalase (ROS-selective scavengers) all decrease stretch-stimulated glucose uptake (P < 0.05) without changing basal uptake (P>> 0.16). We also demonstrate that stretch-stimulated glucose uptake persists in the presence of the phosphatidylinositol <em>3</em>-kinase (PI<em>3</em>-K) inhibitors wortmannin and LY294001 (P < 0.05) but is diminished by the p<em>3</em>8-MAPK inhibitors SB20<em>3</em>580 and A<em>3</em>04000 (P>> 0.99). These data indicate that stretch-stimulated glucose uptake in skeletal muscle is mediated by a ROS- and p<em>3</em>8 MAPK-dependent mechanism that appears to be AMPKalpha2- and PI<em>3</em>-K-independent.

The role of superoxide radical formation in the pathogenesis of perinatal hypoxic-ischemic injury was examined using transgenic (Tg) mice expressing three times normal amounts of copper/zinc-superoxide dismutase (CuZn/<em>SOD</em>). Fourteen litters of postnatal d 7 strain 218/<em>3</em> mice were subjected to right common carotid artery ligation followed by 90 min of hypoxia in an 8% oxygen/humidified chamber maintained at <em>3</em>7 degrees C. Both Tg mice (n = <em>3</em>2) and their nontransgenic (nTg) littermates (n = <em>3</em>0) survived the injury equally. Evaluation of infarcted brain areas measured by video image analysis of three coronal brain sections through the anterior hippocampus from each animal revealed that the Tg animals suffered brain infarction more frequently than did nTg mice. Blinded histologic scoring of cerebral cortex and striatum 5 d after injury revealed that Tg mice were more likely to have higher histologic severity scores than their nTg littermates (p = 0.046<em>3</em>, Mann-Whitney U test). These findings suggest that brain injury in perinatal hypoxia-ischemia may be mediated in part by free radical formation from excessive hydrogen peroxide or nitric oxide production.

Hyperglycemia-linked oxidative stress and/or consequent endoplasmic reticulum (ER) stress are the causative factors of pathogenesis of diabetic retinopathy. Dietary bioactive components which mitigate oxidative stress may serve as potential chemopreventive agents to prevent or slow down the disease progression. Wolfberry is a traditional Asian fruit consumed for years to prevent aging eye diseases in Asian countries. Here we report that dietary wolfberry ameliorated mouse retinal abnormality at the early stage of type 2 diabetes in db/db mice. Male mice at six weeks of age were fed the control diet with or without 1% (kcal) wolfberry for eight weeks. Dietary wolfberry restored the thickness of the whole retina, in particular the inner nuclear layer and photoreceptor layer, and the integrity of the retinal pigment epithelia (RPE), and the ganglion cell number in db/db mice. Western blotting of whole retinal cell lysates revealed that addition of wolfberry lowered expression of ER stress biomarkers binding immunoglobulin protein (BiP), protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and caspase-12, and restored AMP-activated protein kinase (AMPK), thioredoxin, Mn superoxide dismutase (Mn <em>SOD</em>) and forkhead O transcription factor <em>3</em> α (FOXO<em>3</em>α) activities. To determine if our observations were due to the high contents of zeaxanthin and lutein in wolfberry, additional studies using these carotenoids were conducted. Using the human adult diploid RPE cell line ARPE-19, we demonstrated that both zeaxanthin and lutein could mimic the wolfberry preventive effect on activation of AMPK, thioredoxin, Mn <em>SOD</em>, FOXO<em>3</em>α activities, normalize cellular reactive oxygen species and attenuate ER stress in ARPE-19 cells exposed to a high glucose challenge. The zeaxanthin preventive effect was abolished by small interfering RNA knockdown of AMPKα. These results suggested that AMPK activation appeared to play a key role in upregulated expression of thioredoxin and Mn <em>SOD</em>, and mitigation of cellular oxidative stress and/or ER stress by wolfberry and zeaxanthin and/or lutein. Taken together, dietary wolfberry on retinal protection in diabetic mice is, at least partially, due to zeaxanthin and/or lutein.

Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (<em>SOD</em>) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine <em>3</em>4 of human Mn<em>SOD</em> was exclusively nitrated to <em>3</em>-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated Mn<em>SOD</em> has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of <em>SOD</em>. Since phosphorylation and nitration of <em>SOD</em> have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of <em>SOD</em>, focusing particularly on the iron misincorporation of n<em>SOD</em>, as a part of post-translational modifications.

Recently, we reported that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) contribute to aberrant responses in pulmonary resistance arteries (PRAs) of piglets exposed to <em>3</em> days of hypoxia (Am J Physiol Lung Cell Mol Physiol 295: L881-L888, 2008). An objective of the present study was to determine whether NOX-derived ROS also contribute to altered PRA responses at a more advanced stage of pulmonary hypertension, after 10 days of hypoxia. We further wished to advance knowledge about the specific NOX and antioxidant enzymes that are altered at early and later stages of pulmonary hypertension. Piglets were raised in room air (control) or hypoxia for <em>3</em> or 10 days. Using a cannulated artery technique, we found that treatments with agents that inhibit NOX (apocynin) or remove ROS [an <em>SOD</em> mimetic (M4040<em>3</em>) + polyethylene glycol-catalase] diminished responses to ACh in PRAs from piglets exposed to 10 days of hypoxia. Western blot analysis showed an increase in expression of NOX1 and the membrane fraction of p67phox. Expression of NOX4, <em>SOD</em>2, and catalase were unchanged, whereas expression of <em>SOD</em>1 was reduced, in arteries from piglets raised in hypoxia for <em>3</em> or 10 days. Markers of oxidant stress, F(2)-isoprostanes, measured by gas chromatography-mass spectrometry, were increased in PRAs from piglets raised in hypoxia for <em>3</em> days, but not 10 days. We conclude that ROS derived from some, but not all, NOX family members, as well as alterations in the antioxidant enzyme <em>SOD</em>1, contribute to aberrant PRA responses at an early and a more progressive stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

To examine the response of rice to salt stress, changes in protein expression were analyzed using a proteomic approach. To investigate dose- and time-dependent responses, rice seedlings were exposed to 50, 100 and 150 mM NaCl for 6 to 48 h. Proteins were extracted from leaf sheath and separated by two-dimensional polyacrylamide gel electrophoresis. Eight proteins showed 1- to <em>3</em>-fold up-regulation in leaf sheath, in response to 50 mM NaCl for 24 h. Among these, three proteins were unidentified (LSY081, LSY262 and LSY<em>3</em>6<em>3</em>) while five proteins were identified as fructose bisphosphate aldolases, photosystem II (PSII) oxygen evolving complex protein, oxygen evolving enhancer protein 2 (OEE2) and superoxide dismutase (<em>SOD</em>). The maximum expression levels of seven proteins were at 24 h. Their expression declined after 48 h of 50 mM NaCl treatment. In contrast, <em>SOD</em> maintained its elevated expression throughout these conditions. The increased expression of proteins seen in the 50 mM NaCl treatment group was less pronounced in the groups receiving 100 or 150 mM NaCl for 24 h. The expression of <em>SOD</em> was a common response to cold, drought, salt and abscisic acid (ABA) stresses while the expression of LSY081, LSY<em>3</em>6<em>3</em> and OEE2 was enhanced by salt and ABA stresses. LSY262 was expressed in leaf sheath and root, while fructose bisphosphate aldolases, PSII oxygen evolving complex protein and OEE2 were expressed in leaf sheath and leaf blade. LSY<em>3</em>6<em>3</em> was expressed in leaf sheath but was below the level of detection in leaf blade and root. These results indicate that specific proteins expressed in specific regions of rice show a coordinated response to salt stress.

BACKGROUND

Generation of extracellular, cytotoxic superoxide anion (O2-) by polymorphonuclear neutrophils (PMNs) contributes to an unbridled inflammatory response that can precipitate multiple organ failure (MOF). Release of O2- is markedly enhanced when activated PMNs have been previously "primed" by inflammatory mediators, such as those expressed after trauma. We therefore hypothesized that PMN priming occurs as an integral part of the early inflammatory response to trauma.

METHODS

PMNs were obtained from 17 high-risk patients with torso trauma at <em>3</em>, 6, 12, 24, 48, and 72 hours after injury, as well as from 10 healthy donors, and the in vitro release of O2- was quantitated with a kinetic, superoxide dismutase (<em>SOD</em>)-inhibitable cytochrome c reduction assay. PMN O2- release was measured in the presence and absence of 1 mumol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) and after priming and activation with 20 nmol/L platelet-activating factor (PAF) and 1 mumol/L fMLP, respectively.

RESULTS

In vitro PMN O2- release was used to determine whether postinjury PMNs were (1) activated in vivo, (2) primed in vivo, or (<em>3</em>) primable in vitro. Unstimulated PMNs from trauma patients spontaneously expressed modest amounts of O2- in vitro from 6 to 48 hours after injury, suggesting endogenous activation. Also, fMLP-activated PMNs collected between <em>3</em> and 24 hours after injury expressed more O2- than controls (p < or = 0.02), indicating in vivo, trauma-related priming. Furthermore, postinjury PMNs were maximally primed in vivo (i.e., in vitro exposure to PAF before fMLP activation failed to significantly enhance O2- release) as compared to PMNs treated with fMLP.

CONCLUSIONS

These data indicate that major torso trauma (first hit) primes and activates PMNs within <em>3</em> to 6 hours after injury. Consequently, we postulate that postinjury priming of PMNs may create an early vulnerable window during which a second hit (e.g., a secondary operation or delayed hemorrhage) activates exuberant PMN O2- release, rendering the injured patient at high risk for MOF.

The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase (SOD could not be detected in the retina of Sod1(-/-) mice. In addition to the age-related macular degeneration-like phenotypes previously reported, Sod1(-/-) mice also present progressive retinal degeneration. Our results indicate that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated retinal degeneration.

Oxidative stress is widely recognized as a key mediator of degenerative processes in Parkinson's disease (PD). Recently, we demonstrated that the dopaminergic toxin MPP+ initiates oxidative stress to cause caspase-<em>3</em>-dependent apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this study, we determined the source of reactive oxygen species (ROS) produced during MPP+-induced apoptotic cell death. In addition to mitochondria, plasma membrane NADPH oxidase is considered a major producer of ROS inside the cell. Here, we show that N27 neuronal cells express key NADPH oxidase subunits gp91phox and p67phox. We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000microM), apocynin (100-1000microM), and diphenylene iodonium (DPI, <em>3</em>-<em>3</em>0microM), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked <em>3</em>00microM MPP+-induced ROS production for over 45min in N27 cells, in a dose-dependent manner. Further treatment with DPI, apocynin, and <em>SOD</em> also blocked MPP+-induced ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin, or DPI for 24h significantly suppressed MPP+-induced cytotoxic cell death. Similarly, co-treatment with these inhibitors also significantly attenuated MPP+-induced increases in caspase-<em>3</em> enzymatic activity. Furthermore, quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and apocynin rescue N27 cells from MPP+-induced apoptotic cell death. Together, these results indicate for the first time that intracellular ROS generated by NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key determinant of MPP+-mediated dopaminergic degeneration in in vitro models of dopaminergic degeneration. This study supports a critical role of NADPH oxidase in the oxidative damage in PD; targeting this enzyme may lead to novel therapies for PD.