The neuroprotective potential of cannabinoids has been examined in rats with striatal lesions caused by <em>3</em>-nitropropionic acic (<em>3</em>NP), an inhibitor of mitochondrial complex II. We used the CB1 agonist arachidonyl-2-chloroethylamide (ACEA), the CB2 agonist HU-<em>3</em>08, and cannabidiol (CBD), an antioxidant phytocannabinoid with negligible affinity for cannabinoid receptors. The administration of <em>3</em>NP reduced GABA contents and also mRNA levels for several markers of striatal GABAergic projection neurons, including proenkephalin (PENK), substance P (SP) and neuronal-specific enolase (NSE). We also found reductions in mRNA levels for superoxide dismutase-1 (<em>SOD</em>-1) and -2 (<em>SOD</em>-2), which indicated that <em>3</em>NP reduced the endogenous antioxidant defences. The administration of CBD, but not ACEA or HU-<em>3</em>08, completely reversed <em>3</em>NP-induced reductions in GABA contents and mRNA levels for SP, NSE and <em>SOD</em>-2, and partially attenuated those found in <em>SOD</em>-1 and PENK. This indicates that CBD is neuroprotective but acted preferentially on striatal neurons that project to the substantia nigra. The effects of CBD were not reversed by the CB1 receptor antagonist SR141716. The same happened with the TRPV1 receptor antagonist capsazepine, in concordance with the observation that capsaicin, a TRPV1 receptor agonist, failed to reproduce the CBD effects. The effects of CBD were also independent of adenosine signalling as they were not attenuated by the adenosine A2A receptor antagonist MSX-<em>3</em>. In summary, this study demonstrates that CBD provides neuroprotection against <em>3</em>NP-induced striatal damage, which may be relevant for Huntington's disease, a disorder characterized by the preferential loss of striatal projection neurons. This capability seems to be based exclusively on the antioxidant properties of CBD.

Superoxide anion free radical (O2-.) has been implicated in the pathogenesis of tissue injury consequent to ischemia/reperfusion in several different organs, including heart and bowel. Superoxide dismutase (<em>SOD</em>), an enzyme free radical scavenger specific for O2-., has been used successfully to protect these organs from structural damage during reoxygenation of ischemic tissue. It has been suggested that the catalytic action of xanthine oxidase in injured tissue is an important source of O2-. during reoxygenation. In order to evaluate the potential of <em>SOD</em> to protect against kidney damage resulting from transient ischemia followed by reperfusion with oxygenated blood, a model of warm renal ischemia was studied. LBNF1 rats underwent right nephrectomy and occlusion of the left renal artery for 45 minutes. Survival in the group of ischemic untreated rats (N = <em>3</em>0) was 56% at 7 days and serum creatinine was greatly elevated (p less than 0.01) in rats remaining alive over the full 7-day period. In strong contrast to these results, all of the animals treated with <em>SOD</em> before reperfusion (N = 18) were alive after 7 days similar to sham operated control rats (N = 8). Serum creatinine in the <em>SOD</em> treated rats was significantly elevated only to postoperative day <em>3</em> and thereafter returned to normal. Rats treated with inactive <em>SOD</em> (N = 4) or <em>SOD</em> before ischemia (N = 4) had decreased survival rates compared to ischemic untreated animals and prolonged elevation of serum creatinine. When the ischemia time was extended to 60 minutes, only 19% of the untreated animals (N = 16) survived at 7 days whereas nearly 60% of the <em>SOD</em>-treated animals survived (N = 19). Serum creatinine was greatly elevated during the full 7-day observation period in all surviving rats in the untreated ischemic group, whereas serum creatinine returned to normal (p less than 0.05) after 4 days in the surviving rats treated with <em>SOD</em>. To test whether the action of xanthine oxidase contributed to the kidney damage after reoxygenation, 45 min. ischemic rat kidneys were treated with allopurinol. All of the animals treated with allopurinol (N = 12) were alive at 7 days. Serum creatinine values returned to normal after the episode of ischemia and reperfusion but more slowly than after <em>SOD</em> treatment. Histologic evaluation of kidney tissue taken from animals after ischemia alone showed extensive renal tubular damage, which was essentially absent in kidneys from <em>SOD</em>-treated animals.(ABSTRACT TRUNCATED AT 400 WORDS)

An understanding of the cellular genes whose expression is altered during HSV reactivation will enable us to better understand host responses and biochemical pathways involved in the process. Furthermore, this knowledge could allow us to develop gene-targeted inhibitors to prevent viral reactivation. Mice latent with HSV-1 strain McKrae and uninfected control mice were subjected to hyperthermic stress (4<em>3</em> degrees C for 10 min) and their trigeminal ganglia (TG) collected 1 h later. Two additional groups included HSV-1 latently infected and uninfected mice not subjected to hyperthermic stress. Poly A+ mRNA was enriched from total mouse TG RNA and reverse transcribed using MMLV RT. Radioactively labeled cDNAs were analyzed by microarray analysis. A stress/toxicology array of 149 mouse genes on a nylon membrane was used. The labeled cDNAs prepared from latently infected, stressed mice demonstrated <em>3</em>-fold or greater increases in certain mRNA-early response genes (ERGs) compared to cDNAs from uninfected, stressed control mice. The ERG mRNAs that showed increases included two heat shock proteins (HSP60 and HSP40), a basic transcription factor (BTF T62), a DNA repair enzyme, two kinases [MAP kinase and a stress-induced protein kinase (SADK)], an oxidative stress-induced protein, a manganese superoxide dismutase precursor-2 (<em>SOD</em>-2), and cyclooxygenase 2 (COX-2). The gene expression in unstressed, infected TGs was similar to the gene expression in unstressed, uninfected controls. These results suggest that there is a significant difference in the ERG expression profile in latently infected TGs undergoing stress-induced reactivation compared to uninfected TGs.

Regulation of the katB catalase gene in the anaerobic bacterium Bacteroides fragilis was studied. Northern blot hybridization analyses revealed that katB was transcribed as an approximately 1.6-kb monocistronic mRNA. The levels of katB mRNA increased>> 15-fold when anaerobic, mid-logarithmic-phase cultures were exposed to O2, O2 with paraquat, or hydrogen peroxide. Under anaerobic conditions, the low levels of katB mRNA increased in a growth-dependent manner, reaching maximum expression at late logarithmic or early stationary phase, followed by a decrease in stationary phase. Under anaerobic conditions, the expression of katB mRNA was strongly repressed by glucose and to a lesser extent by xylose. However, glucose repression was completely abolished upon exposure to oxygen. The nonfermentable carbon sources fumarate, succinate, acetate, and pyruvate did not significantly affect expression. Phosphate, nitrogen, and hemin limitation did not affect the expression of katB mRNA, suggesting that the nutritional control of katB expression is restricted to carbon and energy sources and not other forms of nutrient limitation. Primer extension analysis revealed that during both oxidative stress and carbon or energy limitation, katB utilized the same promoter region but transcription initiation occurred at two different nucleotides separated by <em>3</em> or 4 bases. Interestingly, a 6-bp inverted repeat sequence present in the katB regulatory region was also observed upstream of the B. fragilis superoxide dismutase gene <em>sod</em>. It is possible that this is a recognition site for a DNA binding protein involved in the regulation of oxidative stress genes in this organism.

We tested the hypothesis that short-term exercise (STEx) training and the associated increase in pulmonary blood flow during bouts of exercise cause enhanced endothelium-dependent vasorelaxation in porcine pulmonary arteries and increased expression of endothelial cell nitric oxide synthase (eNOS) and superoxide dismutase-1 (<em>SOD</em>-1) protein. Mature, female Yucatan miniature swine exercised 1 h twice daily on a motorized treadmill for 1 wk (STEx group, n = 7); control pigs (Sed, n = 6) were kept in pens. Pulmonary arteries were isolated from the left caudal lung lobe, and vasomotor responses were determined in vitro. Arterial tissue from the distal portion of this pulmonary artery was processed for immunoblot analysis. Maximal endothelium-dependent (ACh-stimulated) relaxation was greater in STEx (71 +/- 5%) than in Sed (44 +/- 6%) arteries (P < 0.05), and endothelium-independent (sodium nitroprusside-mediated) responses did not differ. Sensitivity to ACh was not altered by STEx training. Immunoblot analysis indicated a <em>3</em>.9-fold increase in eNOS protein in pulmonary artery tissue from STEx pigs (P < 0.05) with no change in <em>SOD</em>-1 or glyceraldehyde-<em>3</em>-phosphate dehydrogenase protein levels. We conclude that STEx training enhances ACh-stimulated vasorelaxation in pulmonary arterial tissue and that this adaptation is associated with increased expression of eNOS protein.

The advantages offered by ultra-fast (>60 kHz) magic angle spinning (MAS) rotation for the study of biological samples, notably containing paramagnetic centers are explored. It is shown that optimal conditions for performing solid-state (1<em>3</em>)C NMR under 60 kHz MAS are obtained with low-power CW (1)H decoupling, as well as after a low-power (1)H,(1<em>3</em>)C cross-polarization step at a double-quantum matching condition. Acquisition with low-power decoupling highlights the existence of rotational decoupling sidebands. The sideband intensities and the existence of first and second rotary conditions are explained in the framework of the Floquet-van Vleck theory. As a result, optimal (1<em>3</em>)C spectra of the oxidized, paramagnetic form of human copper zinc superoxide dismutase (<em>SOD</em>) can be obtained employing rf-fields which do not exceed 40 kHz during the whole experiment. This enables the removal of unwanted heating which can lead to deterioration of the sample. Furthermore, combined with the short (1)H T(1)s, this allows the repetition rate of the experiments to be shortened from <em>3</em> s to 500 ms, thus compensating for the sensitivity loss due to the smaller sample volume in a 1.<em>3</em> mm rotor. The result is that 2D (1<em>3</em>)C-(1<em>3</em>)C correlation could be acquired in about 24 h on less than 1 mg of <em>SOD</em> sample.

Our previous study demonstrated that the combination of mannosylated superoxide dismutase (Man-<em>SOD</em>) and succinylated catalase (Suc-CAT), both of which are designed to be targeted to liver nonparenchymal cells, is a promising approach to prevent the initial phase of hepatic ischemia/reperfusion injury induced by occlusion of the portal vein for <em>3</em>0 min followed by a 1-h reperfusion in mice. In this study, the preventive effects of these agents were examined on late-phase injury mediated by infiltrating neutrophils, a more severe condition than the initial one. Administration of Suc-CAT alone or with Man-<em>SOD</em> to mice undergoing hepatic ischemia/reperfusion significantly suppressed the expression of intercellular adhesion molecule-1 along the hepatic sinusoid and prevented neutrophil infiltration in the liver. Man-<em>SOD</em> and Suc-CAT also prevented the increase in plasma glutamic pyruvic transaminase and glutamic oxaloacetic transaminase activities after reperfusion lasting <em>3</em> and 6 h. Histological evaluation of liver tissues confirmed the efficacy of this treatment, suggesting that these <em>SOD</em> and catalase derivatives have the ability to suppress neutrophil-induced hepatic injury. These results demonstrate that targeted delivery of antioxidant enzymes to liver nonparenchymal cells is a promising approach to reducing the reactive oxygen species produced by Kupffer cells and neutrophils infiltrating into the tissue. Since Suc-CAT is partially taken up by hepatocytes via a catalase-specific uptake mechanism, such a fraction could also be involved in its preventive effect against the injury.

1. The present study was undertaken to investigate the effects of hypobaric hypoxia, equivalent to an altitude of 5500 m, on antioxidant enzymes in rats. 2. Malondialdehyde levels in serum, heart, lung, liver and kidney of hypobaric-hypoxic rats were all significantly higher than in control rats by day 21 of exposure (P < 0.05), indicating increased oxidative stress. <em>3</em>. Superoxide dismutase (<em>SOD</em>) catalyses the conversion of the superoxide anion to H2O2 and O2. The concentration of immunoreactive Mn-<em>SOD</em> in the serum of hypobaric-hypoxic rats was raised significantly from day 5 onwards, whereas in liver and lung, it had decreased significantly by day 21 (P < 0.05). 4. Glutathione peroxidase (GSH-Px) catalyses H2O2 and certain lipid peroxides. By day 21, GSH-Px activity had increased significantly in the heart and lungs, but decreased significantly in the liver (P < 0.05). 5. Catalase catalyses H2O2. Catalase activity in the liver and kidney of hypobaric-hypoxic rats was significantly decreased on day 1 (P < 0.05) though levels then recovered. 6. Mn-<em>SOD</em> mRNA in the liver of hypobaric-hypoxic rats was induced during the experiment, the effect being exceptionally marked, especially during the first <em>3</em> days of exposure to hypobaric hypoxia. 7. These results suggest that the liver may be more vulnerable than the other organs tested to oxidative stress under hypobaric hypoxia.

The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-<em>3</em>, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (<em>SOD</em>), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-<em>SOD</em>, Glut-<em>3</em>, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

The mitochondrial antioxidant manganese superoxide dismutase (Mn-<em>SOD</em>) plays a critical cytoprotective role against oxidative stress. Vascular endothelial growth factor (VEGF) was shown previously to induce expression of Mn-<em>SOD</em> in endothelial cells by a NADPH oxidase-dependent mechanism. The goal of the current study was to determine the transcriptional mechanisms underlying this phenomenon. VEGF resulted in protein kinase C-dependent phosphorylation of IkappaB and subsequent translocation of p65 NF-kappaB into the nucleus. Overexpression of constitutively active IkappaB blocked VEGF stimulation of Mn-<em>SOD</em>. In transient transfection assays, VEGF increased Mn-<em>SOD</em> promoter activity, an effect that was dependent on a second intronic NF-kappaB consensus motif. In contrast, VEGF-mediated induction of Mn-<em>SOD</em> was enhanced by the phosphatidylinositol <em>3</em>-kinase (PI<em>3</em>K) inhibitor LY294002 and by dominant negative Akt and was decreased by constitutively active Akt. Overexpression of a constitutively active (phosphorylation-resistant) form of FKHRL1 (TMFKHRL1) resulted in increased Mn-<em>SOD</em> expression, suggesting that the negative effect of PI<em>3</em>K-Akt involves attenuation of forkhead activity. In co-transfection assays, the Mn-<em>SOD</em> promoter was transactivated by TMFKHRL1. Flavoenzyme inhibitor, diphenyleneiodonium (DPI), and antisense oligonucleotides against p47phox (AS-p47phox) inhibited VEGF stimulation of IkappaB/NF-kappaB and forkhead phosphorylation, supporting a role for NADPH oxidase activity in both signaling pathways. Like VEGF, hepatocyte growth factor (HGF) activated the PI<em>3</em>K-Akt-forkhead pathway. However, HGF-PI<em>3</em>K-Akt-forkhead signaling was insensitive to diphenyleneiodonium and AS-p47phox. Moreover, HGF failed to induce phosphorylation of IkappaB/NF-kappaB or nuclear translocation of NF-kappaB and had no effect on Mn-<em>SOD</em> expression. Together, these data suggest that VEGF is uniquely coupled to Mn-<em>SOD</em> expression through growth factor-specific reactive oxygen species (ROS)-sensitive positive (protein kinase C-NF-kappaB) and negative (PI<em>3</em>K-Akt-forkhead) signaling pathways.

Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-<em>3</em>2 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 ->> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm ->> 41<em>3</em>-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or <em>3</em>-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (<em>SOD</em>) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor >> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.

Previous evidence supports the important role that oxidative stress (OxS) plays in metabolic syndrome (MetS)-related manifestations. We determined the relationship between the number of MetS components and the degree of OxS in MetS patients. In this comparative cross-sectional study from the LIPGENE cohort, a total of 91 MetS patients (4<em>3</em> men and 48 women; aged between 45 and 68 years) were divided into four groups based on the number of MetS components: subjects with 2, <em>3</em>, 4 and 5 MetS components (n=20, <em>3</em>1, 28 and 12, respectively). We measured ischemic reactive hyperemia (IRH), plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), total nitrite, lipid peroxidation products (LPO), hydrogen peroxide (H2O2), superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GPx) plasma activities. sVCAM-1, H2O2 and LPO levels were lower in subjects with 2 or <em>3</em> MetS components than subjects with 4 or 5 MetS components. IRH and total nitrite levels were higher in subjects with 2 or <em>3</em> MetS components than subjects with 4 or 5 MetS components. <em>SOD</em> and GPx activities were lower in subjects with 2 MetS components than subjects with 4 or 5 MetS components. Waist circumference, weight, age, homeostatic model assessment-β, triglycerides (TGs), high-density lipoprotein and sVCAM-1 levels were significantly correlated with <em>SOD</em> activity. MetS subjects with more MetS components may have a higher OxS level. Furthermore, association between <em>SOD</em> activity and MetS components may indicate that this variable could be the most relevant OxS biomarker in patients suffering from MetS and could be used as a predictive tool to determine the degree of the underlying OxS in MetS.

Heat shock proteins (HSP) HSP72, HSC70 and HSP25 protein levels and mRNA levels of HSP72 genes (Hsp72-1, Hsp72-2, Hsp72-<em>3</em>) and HSC70 were examined in tibialis anterior muscles from young and old rats following 4.5 weeks of heavy resistance exercise. Young (<em>3</em> months) (n=10) and old (<em>3</em>0 months) (n=9) rats were subjected to 14 sessions of electrically evoked resistance training using stretch-shortening contractions of the left limb that activated the dorsiflexor muscle group, including the tibialis anterior muscle, while the right side served as the intra-animal control. Muscle wet weight of the left tibialis anterior increased by 15.6% in young animals compared to the untrained right side, while the aged rats demonstrated no significant hypertrophy based on muscle wet weight. There were no differences in mRNA expression between the control and experimental muscles in either the old or the young animals for any of the four genes examined. On the other hand, HSP72 levels as determined by Western blots were significantly (p<0.01) higher (968.8 and 409.1%) in the trained as compared to the contralateral control muscle in young and old animals, respectively. HSP25 expression was increased significantly (p<0.01) by training in muscles of young rats (94<em>3</em>.1%) and old rats (420.<em>3</em>%). Moreover, there was no training by age interaction for HSP72, while a significant age and training by age effects were found in muscles for HSP25. There was no change in HSC70 protein expression in response to the training intervention in either age group. <em>SOD</em>-1 enzyme level increased by 66.6% in the trained muscles of the young rats, while this enzyme was <em>3</em><em>3</em>% lower in trained muscles compared to the untrained control side in old rats. Moreover, a significant (p<0.05) training by age interaction was found for <em>SOD</em>-1 enzyme levels. This study suggests that fast contracting muscles in young and old animals are capable of increasing HSP expression in response to high intensity contractile stress. Furthermore, the data are consistent with the hypothesis that higher levels of oxidative stress in muscles of old animals limit HSP levels and/or function in response to high intensity contractile stress.

Melatonin is a powerful scavenger of oxygen free radicals. In humans, melatonin is rapidly transferred from the maternal to the fetal circulation. To investigate whether or not maternal melatonin administration can protect the fetal rat brain from radical-induced damage by increasing the activities of antioxidant enzymes, we administered melatonin to pregnant rats on day 20 of gestation. Melatonin (10 mg/kg) was injected intraperitoneally at daytime (14:00 hr) and, to remove the fetuses, a laparotomy was performed at 1, 2, or <em>3</em> hr after its administration. We measured the melatonin concentration in the maternal serum and in fetal brain homogenates and determined the activities of superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GSH-Px) in fetal brain homogenates. Melatonin administration markedly increased melatonin concentrations in the maternal serum and fetal brain homogenates, with peak levels achieved 1 hr after melatonin administration (serum: 5<em>3</em>8.2+/-160.7 pM/mL; brain homogenates: 1<em>3</em>.8+/-2.8 pM/mg protein). Between 1 and <em>3</em> hr after melatonin administration, GSH-Px activity in fetal brain homogenates increased significantly (P<0.01). Similarly, <em>SOD</em> activity increased significantly between 1 and 2 hr after melatonin administration (P<0.01). These results indicate that melatonin administration to the mother increases antioxidant enzyme activities in the fetal brain and may thereby provide indirect protection against free radical injury. Thus, melatonin may potentially be useful in the treatment of neurodegenerative conditions that may involve excessive free radical production, such as fetal hypoxia and preeclampsia.

This study investigated the effects of N-phenyl-2-naphthylamine, an effective allelochemical on aquatic unicellular algae Chlorella vulgaris at physiological gene transcription level. Exposure to 2.5 mg L(-1) of N-phenyl-2-naphthylamine increased the activities of the antioxidant enzymes, superoxide dismutase (<em>SOD</em>), peroxidase (POD), and catalase (CAT), which were 2.47, <em>3</em>.24, and 4.27 times higher than that of the control, however, exposure to 4.0 mg L(-1) N-phenyl-2-naphthylamine decreased the activities of these antioxidant enzymes. An increase in malondialdehyde content and a decrease in chlorophyll content following exposure to N-phenyl-2-naphthylamine suggested that the alga was severely damaged and that cell growth was greatly inhibited. Electron microscopy showed that the plasma membrane was detached from the cell wall, the nucleus was condensed, and the structure of chloroplasts was disrupted, in response to N-phenyl-2-naphthylamine exposure. Real-time PCR showed that N-phenyl-2-naphthylamine reduced the transcript abundance of psaB and psbC to <em>3</em>% and 1% of the control, respectively. These results demonstrated that N-phenyl-2-naphthylamine not only inhibited photosynthesis, but also triggered the synthesis of reactive oxygen species (ROS) to disrupt the subcellular structure of this aquatic organism.

Licorice has been used in Chinese folk medicine for the treatment of various disorders. Licorice has the biological capabilities of detoxication, antioxidation, and antiinfection. In this study, we evaluated the antihepatotoxic effect of licorice aqueous extract (LE) on the carbon tetrachloride (CCl(4))-induced liver injury in a rat model. Hepatic damage, as reveled by histology and the increased activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) activities, and decreased levels of serum total protein (TP), albumin (Alb) and globulin (G) were induced in rats by an administration of CCl(4) at <em>3</em> mL/kg b.w. (1:1 in groundnut oil). Licorice extract significantly inhibited the elevated AST, ALP and ALT activities and the decreased TP, Alb and G levels caused by CCl(4) intoxication. It also enhanced liver super oxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), Glutathione S-transferase (GST) activities and glutathione (GSH) level, reduced malondialdehyde (MDA) level. Licorice extract still markedly reverses the increased liver hydroxyproline and serum TNF-α levels induced by CCl(4) intoxication. The data of this study support a chemopreventive potential of licorice extract against liver oxidative injury.

To attain a profile of protein expression during diabetes, we applied proteomic analysis to glomeruli of 160-day-old db/db diabetic and db/m nondiabetic mice. Glomerular proteins were extracted and separated by two-dimensional gel electrophoresis to construct a proteome map. Matrix-assisted laser desorption and ionization-time of flight mass spectrometry and peptide mass fingerprinting were used to identify 190 proteins. Of 105 analyzed spots, expression of 40 proteins, including the antioxidative enzymes peroxiredoxin 1 and <em>3</em>, glutathione peroxidase 1, and <em>SOD</em>-1, was increased with diabetes, suggesting an adaptive response to oxidative stress associated with this diabetic model. However, activity of glutathione peroxidase and <em>SOD</em> was unaltered in glomeruli of diabetic mice. Expression of glyoxalase I was increased in glomeruli of diabetic mice. Because the cofactor for glyoxalase I, glutathione, is decreased in renal cortex of db/db mice, renal cortical glyoxalase I activity was measured in vitro with fixed amounts of exogenous glutathione. Glyoxalase I activity was decreased in renal cortex of db/db mice. These data indicate that diabetes-induced decreases in glyoxalase I activity are likely to be due to glutathione-dependent and -independent mechanisms and that increased expression of glyoxalase I may represent an insufficient adaptive response to increased methylglyoxal formation.

One of the histopathologic hallmarks of early diabetic retinopathy is the selective loss of pericytes. Evidences suggest that the pericyte loss in vivo is mediated by apoptosis. However, the underlying cause of pericyte apoptosis is not fully understood. This study investigated the effect of advanced glycation end products (AGEs) on apoptotic cell death in bovine retinal pericytes (BRPs). After incubation of BRPs with 0.47, 1.88, 7.5, <em>3</em>0 microM of AGE-bovine serum albumin (BSA) for 4 days, we assayed the pericytes apoptosis by FACS (fluorescence activated cell sorting), and further measured the signaling pathway involved. The results showed that AGE-BSA could induce significantly the apoptosis of BRPs in a dose-dependent manner compared with controls, associated with an increase in intracellular malondialdehyde level and caspase-<em>3</em> activity; a decrease in intracellular catalase, <em>SOD</em> activities and Bcl-2/Bax ratio. <em>SOD</em> and selective caspase-<em>3</em> inhibitor Z-DEVD-fmk can inhibit pericyte apoptosis induced by AGE-BSA. These data suggest that the pericyte loss in diabetic retinopathy involves an apoptotic process, and that elevated AGE observed in diabetes may cause apoptosis in BRPs through an oxidative stress mechanism. The decreased Bcl-2/Bax ratio and activation of caspase-<em>3</em> are associated with apoptotic process.

Disorder of physiological signaling functions of reactive oxygen species (ROS) superoxide and hydrogen peroxide and reactive nitrogen species (RNS) nitric oxide and peroxynitrite is an important feature of diabetes mellitus type 1 and type 2. It is now known that hyperglycemic conditions of cells are associated with the enhanced levels of ROS mainly generated by mitochondria and NADPH oxidase. It has been established that ROS stimulate many enzymatic cascades under normal physiological conditions, but hyperglycemia causes ROS overproduction and the deregulation of ROS signaling pathways initiating the development of diabetes mellitus. On the other hand the deregulation of RNS signaling leads basically to a decrease in NO formation with subsequent damaging disorders. In the present work we will consider the pathological changes of ROS and RNS signaling in enzyme/gene regulated processes catalyzed by protein kinases C and B (Akt/B), phosphatidylinositol <em>3</em>'-kinase (PI<em>3</em>-kinase), extracellular signal-regulated kinase 1/2 (ERK1/2), and some others. Furthermore we will discuss a particularly important role of several ROS-regulated genes and adapter proteins such as the p66shc, FOXO<em>3</em>a, and Sirt2. The effects of low and high ROS levels in diabetes will be also considered. Thus the regulation of damaging ROS levels in diabetes by antioxidants and free radical scavengers must be one of promising treatment of this disease, however, because of the inability of traditional antioxidative vitamin E and C to interact with superoxide and hydrogen peroxide, new free radical scavengers such as flavonoids, quinones and synthetic mimetics of superoxide dismutase (<em>SOD</em>) should be intensively studied.

The human <em>SOD</em>1(G9<em>3</em>A) transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS). In that time, a great many insights into the toxicity of mutant <em>SOD</em>1 have been gained using this and other mutant <em>SOD</em> transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (<em>3</em>-4 months) is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) <em>SOD</em>1(G9<em>3</em>A) transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved.

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on changes of the activities of major antioxidant enzymes, superoxide anion accumulation (O2-), ascorbate, total glutathione (TG), malondialdehyde (MDA) content and ginsenoside accumulation were investigated in ginseng roots (Panax ginseng L.) in 4 l (working volume) air lift bioreactors. Single treatment of 200 microM MJ and SA to P. ginseng roots enhanced ginsenoside accumulation compared to the control and harvested <em>3</em>, 5, 7 and 9 days after treatment. MJ and SA treatment induced an oxidative stress in P. ginseng roots, as shown by an increase in lipid peroxidation due to rise in O2- accumulation. Activity of superoxide dismutase (<em>SOD</em>) was inhibited in MJ-treated roots, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), <em>SOD</em>, guaiacol peroxidase (G-POD), glutathione peroxidase (GPx) and glutathione reductase (GR) were induced in SA-treated roots. A strong decrease in the activity of catalase (CAT) was obtained in both MJ- and SA-treated roots. Activities of ascorbate peroxidase (APX) and glutathione S transferase (GST) were higher in MJ than SA while the contents of reduced ascorbate (ASC), redox state (ASC/(ASC+DHA)) and TG were higher in SA- than MJ-treated roots while oxidized ascorbate (DHA) decreased in both cases. The result of these analyses suggests that roots are better protected against the O2- stress, thus mitigating MJ and SA stress. The information obtained in this work is useful for efficient large-scale production of ginsenoside by plant-root cultures.

Oxidative stress and apoptosis are important factors in the etiology of renal ischemia-reperfusion (I/R) injury. The present study tested the hypothesis that the cell-permeant <em>SOD</em> mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) protects the kidney from I/R-mediated oxidative stress and apoptosis in vivo. Male Sprague-Dawley rats (175-220 g) underwent renal I/R by bilateral clamping of the renal arteries for 45 min followed by reperfusion for 24 h. To examine the role of reactive oxygen species (ROS) in renal I/R injury, a subset of animals were treated with either saline vehicle (I/R Veh) or MnTMPyP (I/R Mn) (5 mg/kg ip) <em>3</em>0 min before and 6 h after surgery. MnTMPyP significantly attenuated the I/R-mediated increase in serum creatinine levels and decreased tubular epithelial cell damage following I/R. MnTMPyP also decreased TNF-alpha levels, gp(91phox), and lipid peroxidation after I/R. Furthermore, MnTMPyP inhibited the I/R-mediated increase in apoptosis and caspase-<em>3</em> activation. Interestingly, although MnTMPyP did not increase expression of the antiapoptotic protein Bcl-2, it decreased the expression of the proapoptotic genes Bax and FasL. These results suggest that MnTMPyP is effective in reducing apoptosis associated with renal I/R injury and that multiple signaling mechanisms are involved in ROS-mediated cell death following renal I/R injury.

Various studies have demonstrated longevity effects of flavonoids, a major sub-group of plant polyphenolic compounds, in Caenorhabditis elegans. To better understand their structure-activity relationship in vivo we have used a comparative approach by exposing C. elegans to the structurally related flavonoids myricetin, quercetin, kaempferol and naringenin, and assessed their impact on lifespan and on putative modes of action. The bioavailability of the tested flavonoids was demonstrated by high-performance liquid chromatography with diode-array detection (HPLC/DAD) and a 2-aminoethyl diphenyl borate-based in vivo approach. While all flavonols increased lifespan in wild-type, only myricetin elongated the mev-1(kn1) lifespan, suggesting that the flavonols antioxidant action alone is not sufficient for longevity. Structural prerequisites of high antioxidant action in vitro were also essential to reduce the reactive oxygen species (ROS) load in vivo in C. elegans and were tested in isolated mouse muscle mitochondria. Since the insulin/IGF-like signaling (IIS) cascade is a key regulator of lifespan, all compounds were tested for the ability to cause nuclear translocation of the FOXO transcription factor DAF-16 and changes in target gene expression. An increased DAF-16 translocation and <em>sod</em>-<em>3</em> promoter activity were observed with all flavonoids but was independent of their ROS scavenging capability and their effects on lifespan.

1. Erythrocyte antioxidant systems--superoxide dismutase (<em>SOD</em>), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and glutathione reductase (GR)--were discussed in relation to life-spans in some mammalian species. 2. The erythrocyte life-span of different mammals was found to be correlated with the levels of <em>SOD</em>, GSH-Px and GSH. <em>3</em>. Data reviewed indicates that the erythrocyte life-span of each species is governed by both the oxygen radical formation and the efficiency of intrinsic antioxidant systems.