Oxidant stress is an important contributor to renal dysfunction and hypertension. We have previously demonstrated that regulation of renal oxygen consumption by nitric oxide (NO) is impaired in the kidney of spontaneously hypertensive rats (SHR) due to increased superoxide production. We further explored the mechanisms of enhanced oxidant stress in the kidney of SHR. Suppression of cortical oxygen consumption by bradykinin (BK) or enalaprilat (Enal), which act through stimulation of endogenous NO, was impaired in SHR (BK: -14.1 +/- 1.2%; Enal: -15.5 +/- 1.2%) and was restored by addition of apocynin, an inhibitor of assembly of the NAD(P)H oxidase complex (BK: -21.0 +/- 0.6%; Enal: -25.<em>3</em> +/- 1.4%), suggesting this as the source of enhanced superoxide production. Addition of an angiotensin type 1 receptor blocker, losartan, also restored responsiveness to control levels (BK: -22.0 +/- 1.1%; Enal: -2<em>3</em>.6 +/- 1.<em>3</em>%), suggesting that ANG II is responsible for enhanced oxidase activity. A similar defect in responsiveness to BK and Enal could be induced in Wistar-Kyoto kidneys by ANG II and was reversed by a superoxide scavenger (tempol), apocynin or losartan. Immunoblotting of cortical samples demonstrated enhanced expression of endothelial NO synthase (eNOS 1.9x) and NAD(P)H oxidase components (gp91(phox) 1.6x and Rac-1 4.5x). Expression of <em>SOD</em>-1 and -2 were unchanged, but <em>SOD</em>-<em>3</em> was significantly decreased in SHR (0.5x). Thus NO bioavailability is impaired in SHR owing to an ANG II-mediated increase in superoxide production in association with enhanced expression of NAD(P)H oxidase components, despite increased expression of eNOS. Loss of <em>SOD</em>-<em>3</em>, an important superoxide scavenger, may also contribute to enhanced oxidant stress.

OBJECTIVE

Pancreatic stenting is an effective method to prevent post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis in high-risk patients. This retrospective study evaluated the impact of modified stent characteristics on the rate of post-ERCP pancreatitis, spontaneous stent dislodgment, and stent-related sequelae.

METHODS

A total of 228<em>3</em> patients underwent 2447 ERCPs over a 6-year period with placement of <em>3</em>-4F diameter, unflanged pancreatic stents. The indication for stenting was pancreatitis prophylaxis predominantly in suspected sphincter of Oddi dysfunction (<em>SOD</em>), pancreas divisum therapy, and precut sphincterotomy. An abdominal radiograph was obtained 10-14 days later to assess spontaneous stent passage. Post-ERCP pancreatitis was defined according to established criteria. A total of 479 patients underwent repeat ERCPs after an initial ERCP with pancreatic stent placement. The prestenting pancreatogram was then compared with follow-up studies.

RESULTS

The pancreatitis rate with <em>3</em>F, 4F, 5F, and 6F stents was 7.5%, 10.6%, 9.8%, and 14.6%, respectively (<em>3</em>F vs. 4F, 5F, 6F: P = 0.047). Spontaneous stent dislodgment was 86%, 7<em>3</em>%, 67%, and 65%, respectively (<em>3</em>F vs. 4F, 5F, 6F: P < 0.0001). The frequency of ductal changes was 24% in patients with <em>3</em>-4F stents compared with 80% with 5-6F stents. Ductal perforation from the stents occurred in <em>3</em> patients (0.1%).

CONCLUSIONS

Small diameter (<em>3</em>-4F), unflanged pancreatic stents are more effective than the traditionally used stents (5-6F) in preventing post-ERCP pancreatitis. Stent-induced ductal changes and the need for endoscopic removal are also significantly less with <em>3</em>-4F stents. The <em>3</em>F stent appears to be superior in all aspects studied. Additional studies are needed to define the ideal method to eliminate post-ERCP pancreatitis.

Oxidative stress may contribute to hypertension. The goals of this study were to determine whether extracellular superoxide dismutase (EC<em>SOD</em>) reduces arterial pressure in spontaneously hypertensive rats (SHR) and whether its heparin-binding domain (HBD), which is responsible for cellular binding, is necessary for the function of EC<em>SOD</em>. Three days after intravenous injection of an adenoviral vector expressing human EC<em>SOD</em> (AdEC<em>SOD</em>), mean arterial pressure (MAP) decreased from 165+/-4 mm Hg (mean+/-SE, n=7) to 124+/-<em>3</em> mm Hg (n=7) in adult anesthetized SHR (P<0.01) but was not altered in normotensive Wistar-Kyoto rats. Cardiac output was not changed in SHR <em>3</em> days after AdEC<em>SOD</em>. Gene transfer of EC<em>SOD</em> with deletion of the HBD (AdEC<em>SOD</em>DeltaHBD) had no effect on SHR MAP, even though plasma <em>SOD</em> activity was greater after AdEC<em>SOD</em>DeltaHBD than after AdEC<em>SOD</em>. Immunohistochemistry revealed intense staining for EC<em>SOD</em> in blood vessels and kidneys after AdEC<em>SOD</em> but not after AdEC<em>SOD</em>DeltaHBD. Impaired relaxation of the carotid artery to acetylcholine in SHR was significantly improved after AdEC<em>SOD</em>. Cumulative sodium balance in SHR was reduced by AdEC<em>SOD</em> compared with AdEC<em>SOD</em>DeltaHBD. Gene transfer of EC<em>SOD</em> also reduced MAP in conscious SHR, although the effect was not as profound as in anesthetized SHR. In summary, gene transfer of EC<em>SOD</em>, with a strict requirement for its HBD, reduces systemic vascular resistance and arterial pressure in a genetic model of hypertension. This reduction in arterial pressure may be mediated by vasomotor and/or renal mechanisms.

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (<em>SOD</em>) prior to incubation with Hx/XO. Catalase treatment, but not <em>SOD</em>, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 micromol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol <em>3</em>-kinase (PI<em>3</em>K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-l-arginine. H2O2 stimulated the phosphorylation of Akt Ser47<em>3</em> (7-fold) and Thr<em>3</em>08 (2-fold) in isolated EDL muscles. H2O2 at 600 micromol/l had no effect on ATP concentrations and did not increase the activities of either the alpha1 or alpha2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI<em>3</em>K-dependent mechanism.

Elevated oxidative stress plays a key role in diabetes-associated vascular disease. In this study, we tested the hypothesis that high glucose-induced oxidative stress was associated with changes in the expression of NADPH oxidase, superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS). Oxidative stress was assessed in cell cultures of mouse microvessel endothelial cells (MMECs) by fluorescence labelling with dihydroethidium, lucigenin-enhanced chemiluminescence and determining NADPH oxidase subunit and eNOS expression with real-time polymerase chain reaction protocol and Western blotting. Oxidative stress and expression of the NADPH oxidase subunit, p22phox, were both increased, SOD expression lowered and eNOS significantly elevated in MMECs treated with 40 mM glucose for 72 h compared to low glucose medium. Oxidative stress, p22phox mRNA, eNOS mRNA, and protein were lowered by concurrent incubation with sepiapterin. When eNOS protein expression in endothelial cells was significantly decreased by eNOS siRNA treatment, superoxide generation was significantly higher in the MMECs grown in low glucose, but reduced in those grown in high glucose for 72 h. Thus, exposure of MMECs to high glucose results in increased oxidative stress that is associated with increased eNOS and NADPH oxidase subunit expression, notably p22phox, and decreased expression of SOD.

It has been hypothesized that the formation of oxygen-derived free radicals may play an important part in ischemically induced tissue injury. Using a canine ischemic liver model, we have assessed the role of two oxygen-free radical scavengers, catalase (CAT) and superoxide dismutase (<em>SOD</em>), used alone and in combination, on the recovery of ischemic livers. Liver ischemia was induced in adult mongrel dogs by cross-clamping of the portal vein and hepatic artery for 40 min. Hepatectomy was then performed, and livers were tested in an isolated perfusion model with 500 ml of an albumin-mannitol solution at <em>3</em>7 degrees C for <em>3</em> hr. Liver function tests were performed hourly during the perfusion period. Biopsies and 99m-Tc-HIDA scans were also done at the end of perfusion. Livers in group 1 (n = 6) served as controls and were not pretreated prior to ischemic injury. Livers in group 2 (n = 6), group <em>3</em> (n = 6), and group 4 (n = 6) were pretreated, respectively, with CAT (5000 U/kg), <em>SOD</em> (5000 U/kg), and <em>SOD</em> and CAT in combination (5000 U/kg) each. The results indicated that the oxygen-derived free radical scavengers, CAT and <em>SOD</em>, were able to provide partial protection against the free radicals accumulated during ischemic damage. These studies offer some potential avenues for the protection of livers prior to and after transplantation.

Free radicals are involved in pathophysiology of ischemia/reperfusion injury (IRI). Melatonin is a potent scavenger of reactive oxygen and nitrogen species. Thus, this study was designed to elucidate its effects in a model of rat kidney transplantation. Twenty Lewis rats were randomly divided into 2 groups (n = 10 animals each). Melatonin (50 mg/kg BW) dissolved in 5 mL milk was given to one group via gavage 2 hr before left donor nephrectomy. Controls were given the same volume of milk only. Kidney grafts were then transplanted into bilaterally nephrectomized syngeneic recipients after 24 hr of cold storage in Histidine-Tryptophan-Ketoglutarate solution. Both graft function and injury were assessed after transplantation through serum levels of blood urea nitrogen (BUN), creatinine, transaminases, and lactate dehydrogenase (LDH). Biopsies were taken to evaluate tubular damage, the enzymatic activity of superoxide dismutase (<em>SOD</em>) and lipid hydroperoxide (LPO), and the expression of NF-kBp65, inducible nitric oxide synthase (iNOS), caspase-<em>3</em> as indices of oxidative stress, necrosis, and apoptosis, respectively. Melatonin improved survival (P < 0.01) while decreasing BUN, creatinine, transaminases, and LDH values up to <em>3</em>9-71% (P < 0.05). Melatonin significantly reduced the histological index for tubular damage, induced tissue enzymatic activity of <em>SOD</em> while reducing LPO. At the same time, melatonin down-regulated the expression of NF-kBp65, iNOS, and caspase-<em>3</em>. In conclusion, donor preconditioning with melatonin protected kidney donor grafts from IRI-induced renal dysfunction and tubular injury most likely through its anti-oxidative, anti-apoptotic and NF-kB inhibitory capacity.

BACKGROUND

Low-grade inflammation is a marker for cardiovascular disease (CVD). The inflammatory biomarkers C-reactive protein (CRP) and soluble urokinase plasminogen activator receptor (suPAR) independently predict CVD. We tested the hypothesis that these biomarkers reflect different aspects of the inflammation associated with CVD.

METHODS

We studied 2273 subjects without CVD. Log-transformed CRP and suPAR were included in general linear and logistic regression models to compare associations with measures of anthropometry and subclinical organ damage (SOD). Owing to interactions on body mass index (BMI) (P<0.0001), the population was stratified by gender and smoking concerning anthropometry.

RESULTS

In both genders, independent of smoking, log-CRP was positively associated with BMI (β: 0.28 to 0.40, P<0.001) and waist circumference (WC) (β: 0.27 to 0.42, P<0.001). In contrast, in smoking women and men, log-suPAR was negatively associated with BMI and WC (β: -0.09 to -0.19, P<0.05). In non-smoking women, log-suPAR was positively associated with BMI and WC (β: 0.14 and 0.16, P<0.001), whereas no associations were found in non-smoking men. No interactions were found on SOD. Adjusted for age, sex, smoking, and physical activity, log-suPAR was associated with an increased urine albumin/creatinine ratio (standardized odds ratio (95% confidence interval (CI)) for highest vs. lower quartiles: 1.36 (1.21-1.52), whereas log-CRP was not (1.10 (0.99-1.22))), and extent of atherosclerosis (standardized proportional odds ratio (95% CI) for carotid plaques 0, 1 ≤ to ≤ 3, >3: 1.31 (1.16-1.47), whereas log-CRP was not (1.00 (0.89-1.11))).

CONCLUSIONS

CRP is positively associated with anthropometric measures, whereas suPAR is linked to endothelial dysfunction and atherosclerosis.

Direct in vivo evidence for the susceptibility of human neuronal cells to dengue virus has not been reported. In this study, we demonstrated that type 2 dengue (DEN-2) virus infection induced extensive apoptosis in the human neuroblastoma cell line SK-N-SH. Phospholipase A(2) (PLA(2)) was activated by DEN-2 infection, which led to the generation of arachidonic acid (AA). Inhibition of PLA(2) activity by the PLA(2) inhibitors, AACOCF(<em>3</em>) and ONO-RS-082, diminished DEN-2 virus-induced apoptosis. In contrast, the cyclooxygenase inhibitors aspirin and indomethacin, thought to increase AA accumulation by blocking AA catabolism, enhanced apoptosis. Exogenous AA induced apoptosis in a dose-dependent manner. Superoxide anion, which is thought to be generated through the AA-activated NADPH oxidase, was increased after infection. Pretreatment with superoxide dismutase (<em>SOD</em>) protected cells against DEN-2 virus-induced apoptosis. Furthermore, generation of superoxide anion was blocked by AACOCF(<em>3</em>). In addition, the transcription factors, NF-kappaB and c-Jun, were found to be activated after DEN-2 virus infection. However, pretreatment of cells with oligodeoxynucleotides containing NF-kappaB, but not c-Jun, binding sites (transcription factor decoy) strongly prevented dengue virus-induced apoptosis. The finding that AACOCF(<em>3</em>) and <em>SOD</em> significantly block activation of NF-kappaB suggests that this activation is derived from the AA-superoxide anion pathway. Our results indicate that DEN-2 virus infection of human neuroblastoma cells triggers an apoptotic pathway through PLA(2) activation to superoxide anion generation and subsequently to NF-kappaB activation. This apoptotic effect can be either directly derived from the action of AA and superoxide anion on mitochondria or indirectly derived from the products of apoptosis-related genes activated by NF-kappaB.

OBJECTIVE

To investigate the effect of ginsenoside Rg1, an effective ingredient from ginsenoside, on 1-methyl-4-phenyl-1,2,<em>3</em>,6-tetrahydropyridine (MPTP)-induced substantia nigra neuron lesion.

METHODS

C57-BL mice were given MPTP to prepare Parkinson disease mice model. Different doses of Rg1 (5, 10, and 20 mg.kg(-1).d(-1)) or N-acetylcystein (NAC) (<em>3</em>00 mg.kg(-1).d(-1)) were given <em>3</em> d prior to MPTP in the pretreatment groups. Glutathione (GSH) level and total superoxide dismutase (T-<em>SOD</em>) activity in substantia nigra were determined by spectrophotometry. Nissl staining, tyrosine hydroxylase immunostaining, and TUNEL labeling were used to observe the damage and apoptosis of nigral neurons. Western blot analysis was used to detect the phospho-JNK and phospho-c-Jun levels in midbrain homogenates.

RESULTS

Pretreatments of C57-BL mice with different doses of Rg1 or NAC were found to protect against MPTP-induced substantia nigra neurons loss. Rg1 or NAC prevented GSH reduction and T-SOD activation in substantia nigra, and attenuated the phosphorylations of JNK and c-Jun following MPTP treatment.

CONCLUSIONS

The antioxidant property of Rg1 along with the blocking of JNK signaling cascade might contribute to the neuroprotective effect of ginsenoside Rg1 against MPTP.

We characterized a novel form of extracellular superoxide dismutase (ec<em>SOD</em>) in atherosclerotic vessels. Specific activity and protein expression of ec<em>SOD</em> was increased two- to threefold in apo E-deficient compared with control aortas. RNase protection assays demonstrated that the expected ec<em>SOD</em> transcript was not increased in either apo E-deficient mice or cholesterol-fed LDL receptor-deficient mice, but that a second, lower molecular weight transcript was present and became predominant as atherosclerosis progressed. Sequence analysis revealed that this novel ec<em>SOD</em> has a 10-bp deletion in the <em>3</em>' untranslated region and an asparagine to aspartic acid mutation at amino acid 21. Studies of isolated macrophages and immunohistochemistry suggested that the truncated ec<em>SOD</em> transcript was expressed by lipid-laden but not control macrophages. Recombinant wild-type and novel ec<em>SOD</em>s expressed in Sf9 cells exhibited similar <em>SOD</em> activities. These experiments show that ec<em>SOD</em> expression is increased in atherosclerotic vessels and that this is characterized by an alteration in mRNA and protein structure. Further, the source of this altered ec<em>SOD</em> is likely the lipid-laden macrophage. The enzymatic properties of this novel ec<em>SOD</em> may have important implications for the function of the lipid-laden macrophage and the atherosclerotic process.

OBJECTIVE

The role of polymorphonuclear neutrophils (PMN) was examined in local and remote organ injury after intestinal ischemia-reperfusion.

BACKGROUND

PMN have been found to mediate the local injury in low flow intestinal ischemia-reperfusion. However, in complete intestinal ischemia-reperfusion, prevention of PMN adhesion by monoclonal antibodies to CD11b and CD18 reduces remote but not local intestinal injury. The role of PMN was further investigated in this setting.

METHODS

In a rat model of 1-hour complete intestinal ischemia and 4-hour reperfusion. PMN were manipulated in the following four ways: (1) inhibition of oxygen-free radicals using manganese superoxide dismutase and catalase (<em>SOD</em>/CAT), (2) antagonism of PMN elastase using secretory leukocyte protease inhibitor (SLPI), (<em>3</em>) neutropenia using PMN antisera, and (4) inhibition of activation and adhesion using interleukin-1 receptor antagonist (IL-1ra) and tumor necrosis factor binding protein (TNFbp). Lung injury was quantified by the pulmonary permeability index, which is the ratio of bronchoalveolar lavage to blood concentration of radiolabeled bovine serum albumin, and PMN sequestration by myeloperoxidase (MPO) activity. Liver injury was estimated by PMN counts using quantitative histologic examination and by serum glutamic pyruvic transaminase (SGPT). Local injury was quantified by MPO activity and histologic grading.

RESULTS

Neutropenia reduced the pulmonary permeability 80% from 11.0 +/- 0.5 x 10(-<em>3</em>) with saline treatment to 5.50 +/- 0.12 x 10(-<em>3</em>); IL-1ra, to 5.62 +/- 0.44 x 10(-<em>3</em>); and TNFbp, to 4.<em>3</em>2 +/- 0.18 x 10(-<em>3</em>) (all p < 0.05). Pulmonary MPO rose from 0.0<em>3</em> +/- 0.01 U/g to 0.25 +/- 0.0<em>3</em> U/g (p < 0.05). This was reduced by neutropenia, 0.01 +/- 0.00<em>3</em> U/g, but not by inhibition of oxygen-free radicals or PMN elastase. IL-1ra inhibited PMN sequestration, reducing MPO to 0.12 +/- 0.01 (p < 0.05). Liver injury was 60% dependent on PMN. Ischemia-reperfusion increased SGPT from 20.<em>3</em> +/- 0.7 IU/L in the sham-treated animals to 97.0 +/- <em>3</em>.1 IU/L in the experimental animals. Neutropenia reduced this to 48.1 +/- <em>3</em>.9 IU/L; IL-1ra, to 44.7 +/- <em>3</em>.7 IU/L; <em>SOD</em>/CAT, to 64.0 +/- <em>3</em>.<em>3</em>8 IU/L; and SLPI, to 57.1 +/- <em>3</em>.4 IU/L (p < 0.05 in all cases). Local injury was severe and unaffected by manipulation of the PMN.

CONCLUSIONS

These data suggest that PMN and their products mediate most of the lung, part of the liver, and none of the local gut injury after intestinal ischemia-reperfusion.

OBJECTIVE

Recently, various transgenic and knock-out mouse models have become available for studying the pathogenesis of diabetic retinopathy. At the same time, diabetes-induced retinal changes in the wild-type mice remain poorly characterised. The present study compared retinal biochemical changes in rats and mice with similar (6-week) durations of streptozotocin-induced diabetes.

METHODS

The experiments were performed on Wistar rats and C57Bl6/J mice. Retinal glucose, sorbitol, fructose, lactate, pyruvate, glutamate, alpha-ketoglutarate and ammonia were measured spectrofluorometrically by enzymatic methods. Vascular endothelial growth factor (VEGF) protein was assessed by ELISA, and poly(ADP-ribosyl)ation by immunohistochemistry and western blot analysis. Free mitochondrial and cytosolic NAD(+)/NADH ratios were calculated from the glutamate and lactate dehydrogenase systems.

RESULTS

Retinal glucose concentrations were similarly increased in diabetic rats and mice, vs controls. Diabetic rats manifested approximately 26- and 5-fold accumulation of retinal sorbitol and fructose, respectively, whereas elevation of both metabolites in diabetic mice was quite modest. Correspondingly, diabetic rats had (1) increased retinal malondialdehyde plus 4-hydroxyalkenal concentrations, (2) reduced superoxide dismutase (<em>SOD</em>), glutathione peroxidase, glutathione reductase and glutathione transferase activities, (<em>3</em>) slightly increased poly(ADP-ribose) immunoreactivity and poly(ADP-ribosyl)ated protein abundance, and (4) VEGF protein overexpression. Diabetic mice lacked these changes. <em>SOD</em> activity was 21-fold higher in murine than in rat retinas (the difference increased to 54-fold under diabetic conditions), whereas other antioxidative enzyme activities were <em>3</em>- to 10-fold lower. With the exception of catalase, the key antioxidant defence enzyme activities were increased, rather than reduced, in diabetic mice. Diabetic rats had decreased free mitochondrial and cytosolic NAD(+)/NADH ratios, consistent with retinal hypoxia, whereas both ratios remained in the normal range in diabetic mice.

CONCLUSIONS

Mice with short-term streptozotocin-induced diabetes lack many biochemical changes that are clearly manifest in the retina of streptozotocin-diabetic rats. This should be considered when selecting animal models for studying early retinal pathology associated with diabetes.

BACKGROUND

Endothelium-dependent vasodilation is impaired in large conduit vessels in diabetes mellitus. Oxygen radicals contribute to the impaired endothelium-dependent vasodilation. We tested the hypothesis that stimulated endothelium-dependent vasodilation is reduced in renal afferent arterioles in diabetes and is caused by an increase in vascular superoxide (O2(-)).

METHODS

Renal afferent arterioles from normal and insulin-treated alloxan-diabetic rabbits were microdissected and microperfused in vitro for the study of luminal diameter responses to acetylcholine (Ach; 10(-11) to 10(-6) mol/L). The blood glucose concentration of insulin-treated alloxan-diabetic rabbits was elevated fourfold compared with normal rabbits (<em>3</em>19 +/- 2<em>3</em> vs. 79 +/- 6 mg/dL, P < 0.001).

RESULTS

In norepinephrine (NE)-preconstricted afferent arterioles of normal rabbits, Ach significantly (P < 0.001) increased luminal diameter by 165 +/- 44%. The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (10(-4) mol/L) blocked this Ach-induced vasodilation. In marked contrast, in NE-preconstricted arterioles of diabetic rabbits, Ach significantly (P < 0.01) decreased luminal diameter by 41 +/- 11%. Pretreatment of diabetic afferent arterioles with the superoxide dismutase (<em>SOD</em>) mimetic tempol (10(-<em>3</em>) mol/L) restored a vasodilator response to Ach. In NE-preconstricted diabetic afferent arterioles treated with tempol, Ach significantly (P < 0.001) increased luminal diameter by 25 +/- 6%.

CONCLUSIONS

Ach-induced afferent arteriolar vasodilation is dependent on nitric oxide and is impaired in diabetes. O2(-) contributes to the impaired Ach-induced vasodilation in renal afferent arterioles in diabetes.

OBJECTIVE

Experimental studies provide evidence of an association between ischemic stroke and increased oxidative stress, but data in humans are still limited and controversial. The purpose of this study was to investigate the time course of plasma antioxidant changes in ischemic stroke patients.

METHODS

Plasma antioxidants, including water-soluble (vitamin C and uric acid) and lipid-soluble (vitamins A and E) compounds as well as antioxidant enzyme activities in plasma (superoxide dismutase [<em>SOD</em>] and glutathione peroxidase) and erythrocytes (<em>SOD</em>), were measured by high-performance liquid chromatography (antioxidant vitamins) and by spectrophotometry (antioxidant enzymes) in <em>3</em>8 subjects (25 men and 1<em>3</em> women aged 77.2+/-7.9 years) with acute ischemic stroke of recent onset (<24 hours) on admission, after 6 and 24 hours, and on days <em>3</em>, 5, and 7. Antioxidant levels in patients on admission were compared with those of age- and sex-matched controls.

RESULTS

Mean antioxidant levels and activities in patients on admission were lower than those of controls and showed a gradual increase over time. Patients with the worst early outcome (death or functional decline) had higher vitamin A and uric acid plasma levels and lower vitamin C levels and erythrocyte SOD activity than those who remained functionally stable.

CONCLUSIONS

These results suggest that the majority of antioxidants are reduced immediately after an acute ischemic stroke, possibly as a consequence of increased oxidative stress. A specific antioxidant profile is associated with a poor early outcome.

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (<em>SOD</em>) (viz. Mn-<em>SOD</em> or CuZn-<em>SOD</em>, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (<em>3</em>0 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of <em>SOD</em> isoforms caused a 2- and <em>3</em>.5-fold elevation in CuZn-<em>SOD</em> and Mn-<em>SOD</em> activities in the cytoplasm and mitochondria, respectively, and <em>3</em>-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-<em>SOD</em> attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-<em>SOD</em> expression similarly prevented PC-12 apoptosis, this was associated with increases in <em>SOD</em> activity but not GSH, indicating that cytoprotection by Mn-<em>SOD</em> overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-<em>SOD</em> overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-<em>3</em> activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-<em>SOD</em> overexpression and is independent of cellular GSH levels per se.

The increasing use of carbon nanotubes (CNTs) in biomedical applications underlines the importance of its potential toxic effects to human health. In the present study, we first exposed PC12 cells, a commonly used in vitro model for neurotoxicity study, to two kinds of commercially available single-walled carbon nanotubes (SWCNTs), to investigate the effect of SWCNTs on nervous system in vitro. The decrease of PC12 cells viability was time and dose-dependent with exposure to SWCNTs demonstrated by <em>3</em>-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release and morphological observation. Flow cytometry analysis showed that the PC12 cells' cycle was arrested in the G2/M phase, and their apoptotic rate induced by SWCNTs was dose-dependent. Further studies revealed SWCNTs decreased mitochondrial membrane potential (MMP), induced the formation of reactive oxygen species (ROS) and increased the level of lipid peroxide and decreased the activities of superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GSH-Px), catalase (CAT) and the content of glutathione (GSH) in a time and dose-dependent manner. These findings reveal that SWCNTs may induce oxidative stress to nervous system in vivo, causing the occurrence of diseases related to cellular injuries of neuronal cells, such as neurodegenerative disorders, and demonstrating the necessity of further research in vivo.

Basic leucine zipper proteins (bZIPs) are transcription factors that bind abscisic acid (ABA)-responsive elements (ABREs) and enable plants to withstand adverse environmental conditions. In the present study, a novel bZIP gene, ThbZIP1 was cloned from Tamarix hispida. Expression studies in T. hispida showed differential regulation of ThbZIP1 in response to treatment with NaCl, polyethylene glycol (PEG) 6000, NaHCO(<em>3</em>), and CdCl(2), suggesting that ThbZIP1 is involved in abiotic stress responses. To identify the physiological responses mediated by ThbZIP1, transgenic tobacco plants overexpressing exogenous ThbZIP1 were generated. Various physiological parameters related to salt stress were measured and compared between transgenic and wild type (WT) plants. Our results indicate that overexpression of ThbZIP1 can enhance the activity of both peroxidase (POD) and superoxide dismutase (<em>SOD</em>), and increase the content of soluble sugars and soluble proteins under salt stress conditions. These results suggest that ThbZIP1 contributes to salt tolerance by mediating signaling through multiple physiological pathways. Furthermore, ThbZIP1 confers stress tolerance to plants by enhancing reactive oxygen species (ROS) scavenging, facilitating the accumulation of compatible osmolytes, and inducing and/or enhancing the biosynthesis of soluble proteins.

In order to assess the role of the antioxidative defense system against salt treatment, the activities of some antioxidative enzymes and levels of antioxidants were monitored in a true mangrove, Bruguiera parviflora, subjected to varying levels of NaCl under hydroponic culture. In the leaves of B. parviflora, salt treatment preferentially enhanced the content of H2O2 as well as the activity of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (<em>SOD</em>), whereas it induced the decrease of total ascorbate and glutathione (GSH+GSSG) content as well as catalase (CAT) activity. Analysis of isoforms of antioxidative enzymes by native PAGE and activity staining revealed that leaves of B. parviflora had one isoform each of Mn-<em>SOD</em> and Cu/Zn-<em>SOD</em> and three isoforms of Fe-<em>SOD</em>. Expression of Mn-<em>SOD</em> and Fe-<em>SOD</em>-2 was preferentially elevated by NaCl. Similarly, out of the six isoforms of GPX, the GPX-1, 2, <em>3</em> and 6 were enhanced by salt treatment but the levels of GPX-4 and -5 changed minimally as compared to those of a control. Activity staining gel revealed only one prominent isoform of APX and two isoforms of GR (GR-1 and GR-2), all of these isoforms increased upon salt exposure. Four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced, suggesting differential down regulation of CAT isoforms by NaCl. The concentrations of malondialdehyde (MDA), a product of lipid peroxidation, remained unchanged in leaves of the plant treated with different concentrations of NaCl. This suggests that plants are protected against activated oxygen species by the elevated levels of certain antioxidative enzymes, thus avoiding lipid peroxidation during salt exposure. The differential changes in the levels of the isoforms due to NaCl treatment may be useful as markers for recognizing salt tolerance in mangroves.

Brain is susceptible to oxidative stress and it is associated with age-related brain dysfunction. Previously, we have pointed out a dramatic decrease of glutathione levels in the rat brain after acetaminophen (APAP) oral administration overdose. Silymarin (SM) is a mixture of bioactive flavonolignans isolated from Silybum marianum (L.) Gaertn., employed usually in the treatment of alcoholic liver disease and as anti-hepatotoxic agent in humans. In this study, we have evaluated the effect of SM on enzymatic and non enzymatic antioxidant defensive systems in rat brain after APAP-induced damage. Male albino Wistar rats were treated with SM (200 mg/kg/die orally) for three days, or with APAP single oral administration (<em>3</em> g/kg) or with SM (200 mg/kg/die orally) for <em>3</em> days and APAP single oral administration (<em>3</em> g/kg) at third day. Successively the following parameters were measured: reduced and oxidized glutathione (GSH and GSSG), ascorbic acid (AA), enzymatic activity variations of superoxide dismutase (<em>SOD</em>) and malondialdehyde levels (MDA). Our results showed a significant decrease of GSH levels, AA levels and <em>SOD</em> activity and an increase of MDA and GSSG levels after APAP administration. After SM administration GSH and AA significantly increase and <em>SOD</em> activity was significantly enhanced. In the SM+APAP group, GSH values significantly increase and the others parameters remained unchanged respect to control values. These results suggest that SM may to protect the SNC by oxidative damage for its ability to prevent lipid peroxidation and replenishing the GSH levels.

The oxidative metabolism of catecholamines produce quinones which react with oxygen to produce superoxide anions (O2-.) and H2O2. The catecholamines, however, are important under stress conditions but may have damaging effects due to the generation of reactive oxygen species (ROS) and formation of oxidation products. ROS are involved as causative factors in many diseases, therefore, the generation of ROS by catecholamines may also contribute to this process. Isoproterenol (ISO) was administered to rats in two doses so as to evaluate their beta-adrenergic and toxicological actions in terms of lipid peroxidation (LPO) and the changes in the antioxidant enzymes such as superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione-S-transferase (GST) and glutathione (GSH) content in heart, liver and kidney. ISO treatment caused LPO in tissues, however, the heart initially showed decreased LPO. This is attributed to the condition of hypertrophy by which the heart can protect itself to a limited extent against oxidative stress. The second dose of ISO, administered 24 h after the first treatment, showed toxic effects resulting in a higher increase in LPO. The increased <em>SOD</em> activity in tissues <em>3</em> days after a dose of ISO suggests that the ROS may induce <em>SOD</em> activity to dismutate O2-. However, increased amounts of O2-., inhibited <em>SOD</em> activity at <em>3</em> and 6 h with recovery towards control values at 12 h of a second dose of ISO treatment. CAT activity in tissues increased at 6 h of a second dose of ISO treatment. The elevated <em>SOD</em> and CAT enzymes in tissues indicate a response due to increased ROS. The increase in GSH content in the heart, liver and kidney at day 2 of ISO treatment and 12 h after the second dose of ISO may also neutralise oxidative stress. The inhibition in GST activity in tissues was observed probably due to increased ROS generation, however, GST activity partially recovered by 12 h after the second dose of ISO, in an attempt to counteract oxidative stress. The result shows that ISO induced oxidative stress and the increase of the antioxidant system in tissues may attenuate oxidative stress. It is suggested that ROS generation in the oxidation of catecholamines may be partially counteracted by the antioxidant system in tissues.

The aim of present study was to evaluate the protective effects of dexmedetomidine (DEX) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and investigate its possible mechanisms mediated by HMGB1. In vivo, pulmonary pathology observation and myeloperoxidase (MPO) activity were also examined to evaluate the protective effect of DEX in the lungs. Tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid (BALF), serum and lung tissues LPS-induced rats were detected. The oxidative indices including superoxide dismutase (<em>SOD</em>), Malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in serum were also determined. Additionally, nitric oxide (NO), TNF-α, IL-6 and IL-1β, MDA, <em>SOD</em> and GSH-Px in the supernatants of LPS-induced BEAS-2B cells were measured. Furthermore, we detected the protein expression of high mobility group box-1 protein (HMGB1), Toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), inhibitor of NF-κB (IκBα), p-IκBα, nuclear factor kappa-B (NF-κB), p-NF-κB, phosphatidylinositol <em>3</em>'-kinase (PI<em>3</em>K), p-PI<em>3</em>K, protein kinase B (Akt), p-Akt, mammalian target of rapamycin (mTOR) and p-mTOR in LPS-induced ALI rats and LPS-induced BEAS-2B cells. Immunohistochemical and immunofluorescence analyses of HMGB1 in lung tissues or BEAS-2B cells were also conducted to evaluate the mechanisms of DEX. DEX effectively attenuated pulmonary pathology, and ameliorated the levels of MPO, <em>SOD</em>, MDA, GSH-Px, TNF-α, IL-6, IL-1β and NO in LPS-stimulated rats and BEAS-2B cells. Additionally, treatment with DEX inhibited the expression of HMGB1, TLR4, MyD88, p-IκB, p-NF-κB, p-PI<em>3</em>K, p-Akt and p-mTOR in vivo and in vitro. Immunohistochemical and immunofluorescence analyses also showed that DEX suppressed HMGB1 levels in lung sections and BEAS-2B cells. Treatment with glycyrrhizin, an inhibitor of HMGB1, confirmed that HMGB1 was involved in the mechanism of DEX on LPS-induced ALI. The transfection of HGMB1 siRNA also confirmed these findings in vitro. In conclusion, the present study showed that DEX exerted a protective effect on LPS-induced ALI rats likely through the HMGB1-mediated TLR4/NF-κB and PI<em>3</em>K/Akt/mTOR pathways.

Chronic systemic exposure of D-galactose to mice, rats, and Drosophila causes the acceleration of senescence and has been used as an aging model. However, the underlying mechanism is as yet unclear. To investigate the mechanisms of neurodegeneration in this model, we studied cognitive function, hippocampal neuronal apoptosis and neurogenesis, and peripheral oxidative stress biomarkers and also the protective effects of the antioxidant R-alpha-lipoic acid. Chronic systemic exposure of mice to D-galactose (100 mg/kg, s.c., 7 weeks) induced a spatial memory deficit, an increase in cell karyopyknosis, apoptosis, and caspase-<em>3</em> protein levels in hippocampal neurons, a decrease in the number of new neurons in the subgranular zone in the dentate gyrus, a reduction of migration of neural progenitor cells, and an increase in death of newly formed neurons in the granular cell layer. The D-galactose exposure also induced an increase in peripheral oxidative stress, including an increase in malondialdehyde and decreases in total antioxidative capabilities (T-AOC), total superoxide dismutase (T-<em>SOD</em>), and glutathione peroxidase (GSH-Px) activities. A concomitant treatment with lipoic acid ameliorated cognitive dysfunction and neurodegeneration in the hippocampus and also reduced peripheral oxidative damage by decreasing malondialdehyde and increasing T-AOC and T-<em>SOD</em>, without an effect on GSH-Px. These findings suggest that chronic D-galactose exposure induces neurodegeneration by enhancing caspase-mediated apoptosis and inhibiting neurogenesis and neuron migration, as well as increasing oxidative damage. In addition, D-galactose-induced toxicity in mice is a useful model for studying the mechanisms of neurodegeneration and neuroprotective drugs and agents.

Several theories have been advanced to explain the mechanisms by which varicocele impairs male fertility. These theories include scrotal hyperthermia, retrograde flow of adrenal or renal metabolites, Leydig cell dysfunction and hypoxia. Varicocele is reported to be associated with elevated reactive oxygen species (ROS) production in spermatozoa and diminished seminal plasma antioxidant activity. The aim of this study was to investigate whether surgical correction of varicocele might reduce ROS or increase the antioxidant capacity of seminal plasma from infertile patients with varicocele. The study group consisted of 68 infertile males, selected from patients scheduled for varicocelectomy at Cairo University Hospital during the year 1999. Seminal plasma levels of two ROS [malondialdehyde (MDA), hydrogen peroxide (H2O2)] and one ROS radical [nitric oxide (NO)] were estimated as well as six antioxidants [superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPx), vitamin C (Vit C), vitamin E (Vit E), albumin) on the day prior to varicocelectomy. For comparison, the same parameters were measured again <em>3</em> and 6 months post-operatively. A statistically significant reduction in the <em>3</em> month post-operative levels of MDA, H2O2 and NO was observed when compared with the pre-operative values. A further significant reduction took place during the following <em>3</em> months. Four of the six antioxidants tested (<em>SOD</em>, CAT, GPx, and Vit C) showed a significant increase in seminal levels when comparing <em>3</em>-month post-operative with pre-operative values. A further significant increase of the four antioxidant levels took place during the following <em>3</em> months. No significant change in the level of seminal plasma albumen took place during the first <em>3</em> months after varicocelectomy, however, a significant increase was noted during the next <em>3</em> months. In contrast to other antioxidants, seminal plasma levels of Vit E showed a significant decrease when comparing <em>3</em>-month post-operative with pre-operative values. A further significant decrease took place during the following <em>3</em> months. It is concluded that varicocelectomy reduces ROS levels and increases antioxidant activity of seminal plasma from infertile men with varicocele.