OBJECTIVE

(1) To investigate the diagnostic value of some O-(2-[F]fluoroethyl)-L-tyrosine (F-18 FET) indices derived from the dynamic acquisition to differentiate low-grade gliomas from high-grade; (2) to analyze the course of tumor time-activity curves (TACs); and (<em>3</em>) to calculate the individual probability of a high-grade glioma using the logistic regression.

METHODS

Seventeen low-grade (WHO I-II) and 15 high-grade (WHO III-IV) gliomas were studied with dynamic F-18 FET PET. Regions of interests were drawn over the tumor and contralateral brain, and TACs were analyzed. We considered early standardized uptake value (SUV), middle SUV, late SUV, early-to-middle SUV tumor ratio, early-to-late SUV tumor ratio; time to peak (Tpeak), in minutes, from the beginning of the dynamic acquisition up to the maximum SUV of the tumor; and SoD (sum of the frame-to-frame differences). To assess the individual probability of high-grade, logistic regression was also used.

RESULTS

High-grade gliomas showed significantly (P < 0.0001) higher values when compared with low-grade gliomas in early SUV, early-to-middle ratio, early-to-late ratio, Tpeak, and SoD. For the grading of gliomas, the best indices were early-to-middle ratio and Tpeak providing a diagnostic accuracy of 94%. TACs analysis provided an 87% diagnostic accuracy. For individual high-grade diagnosis, the logistic regression provided 9<em>3</em>% sensitivity, 100% specificity, and 97% accuracy.

CONCLUSIONS

Early-to-middle SUV tumor ratio and Tpeak were the best indices for assessing the grading of gliomas. Since early-to-middle ratio derives from the first <em>3</em>5 minutes of the dynamic acquisition, the PET study could last half an hour instead of 1 hour. By logistic regression, it is possible to assess the individual probability of high-grade, useful for prognosis and treatment.

Aging is characterized by activation of inducible over endothelial nitric oxide synthase (iNOS and eNOS), impaired antioxidant activity and increased oxidative stress, which reduces nitric oxide bioavailability and causes endothelial dysfunction. Caloric restriction (CR) blunts oxidative stress. We investigated whether CR impacts endothelial dysfunction in aging and the underlying mechanisms. Aortas from young (YC, 6 months of age) and old (OC, 24 months of age) rats ad-libitum fed and from old rats caloric-restricted for <em>3</em>-weeks (OR, 26%) were investigated. Endothelium-dependent vasorelaxation was impaired in OC, associated with reduced eNOS and increased iNOS expression (P<0.05). Aortic nitrite was similar in OC and YC, but the contribution of calcium-independent NOS to total NOS activity was increased whereas that of calcium-dependent NOS was reduced (p≤0.000<em>3</em>). Plasma thiobarbituric acid-reactive substances (TBARS) were elevated in OC as well as aortic nitrotyrosine (P<0.05). Expression of manganese superoxide dismutase (Mn<em>SOD</em>) and total <em>SOD</em> activity were impaired in OC (P<0.05 vs. YC), whereas copper-zinc (CuZn) <em>SOD</em> expression was similar in OC and YC. CR restored endothelial dysfunction in old rats, reduced iNOS expression, total nitrite and calcium-independent NOS activity in aorta (P<0.05) without changes in eNOS expression and calcium-dependent NOS activity. Sirtuin-1 expression did not differ among groups. Plasma TBARS and aortic nitrotyrosine were reduced (P<0.05) in OR compared with OC. In OR CuZn<em>SOD</em> protein and <em>SOD</em> activity increased (P<0.05) without changes in Mn<em>SOD</em> expression. Short-term CR improves age-related endothelial dysfunction. Reversal of altered iNOS/eNOS ratio, reduced oxidative stress and increased <em>SOD</em> enzyme activity rather than enhanced NO production appear to be involved in this effect.

Autophagy is a catabolic process involved in the continuous removal of toxic protein aggregates and cellular organelles to maintain the homeostasis and functional integrity of cells. The mechanistic understanding of autophagy mediated neuroprotection during the development of neurodegenerative disorders remains elusive. Here, we investigated the potential role of rapamycin-induced activation of autophagy and PI<em>3</em>K/Akt1/mTOR/CREB pathway(s) in the neuroprotection of amyloid-beta (Aβ1-42)-insulted hippocampal neurons in rat model of Alzheimer's disease (AD) like phenotypes. A single intra-hippocampal injection of Aβ1-42 impaired redox balance and markedly induced synaptic dysfunction, neurotransmission dysfunction, and cognitive deficit, and suppressed pro-survival signaling in the adult rats. Rapamycin administration caused a significant reduction of mTOR complex 1 phosphorylation at Ser2481 and a significant increase in levels of autophagy markers such as microtubule-associated protein-1 light chain-<em>3</em> (LC<em>3</em>), beclin-1, sequestosome-1/p62, unc-51-like kinase 1 (ULK1). In addition, rapamycin induced the activation of autophagy that further activated p-PI<em>3</em>K, p-Akt1 (Ser47<em>3</em>), and p-CREB (Ser18<em>3</em>) expression in Aβ1-42-treated rats. The activated autophagy markedly reversed Aβ1-42-induced impaired redox homeostasis by decreasing the levels of prooxidants-ROS generation, intracellular Ca2+ flux and LPO, and increasing the levels of antioxidants-<em>SOD</em>, catalase, and GSH. The activated autophagy also provided significant neuroprotection against Aβ1-42-induced synaptic dysfunction by increasing the expression of synapsin-I, synaptophysin, and PSD95; and neurotransmission dysfunction by increasing the levels of CHRM2, DAD2 receptor, NMDA receptor, and AMPA receptor; and ultimately improved cognitive ability in rats. Wortmannin administration significantly reduced the expression of autophagy markers, p-PI<em>3</em>K, p-Akt1, and p-CREB, as well as the autophagy mediated neuroprotective effect. Our study demonstrate that autophagy can be an integrated part of pro-survival (PI<em>3</em>K/Akt1/mTOR/CREB) signaling and autophagic activation restores the oxidative defense mechanism(s), neurodegenerative damages, and maintains the integrity of synapse and neurotransmission in rat model of AD.

1. M4040<em>3</em> is a low molecular weight, synthetic manganese containing superoxide dismutase mimetic (<em>SOD</em>m) that removes superoxide anions (*O2-) without interfering with other reactive species known to be involved in inflammatory responses (e.g. nitric oxide, NO and peroxynitrite, ONOO-). 2. As such, M4040<em>3</em> represents an important pharmacological tool to dissect the roles of *O2- in acute and chronic inflammation. For this purpose, the pharmacological profile of M4040<em>3</em> was evaluated in carrageenan-induced pleurisy. <em>3</em>. Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by: fluid accumulation in the pleural cavity which contained a large number of neutrophils (PMNs) as well as an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx), prostaglandin E2 (PGE2), tumour necrosis factor alpha, (TNFalpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and interleukin-10 (IL-10). 4. All parameters of inflammation were attenuated by M4040<em>3</em> except for NOx, PGE2 and IL-10 which remained unaltered. Furthermore, carrageenan induced an upregulation of the adhesion molecules ICAM-1 and P-selectin, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. 5. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine and PARS was reduced by M4040<em>3</em>. 6. These results clearly indicate that *O2- plays a critical role in the development of the inflammatory response by altering key components of the inflammatory cascade. Therefore, synthetic enzymes of <em>SOD</em> such as M4040<em>3</em>, offers a novel therapeutic approach for the management of various inflammatory diseases where these radicals have been postulated to play a role.

Docosahexaenoic acid (DHA, 22:6 n-<em>3</em>), a polyunsaturated fatty acid found in fish oil, exerts cytotoxic effects on cancer cells. Although DHA was toxic toward five human cancer cell lines (MCF-7, MDA-MB-2<em>3</em>1, SiHa, Raji, and DHL-4), the lines were not uniformly sensitive. DHL-4, a bcl-2 overexpressing lymphoid line, was the most sensitive (IC50, 5.2 micromol/L) and the cervical cancer cell line, SiHa, was the most resistant (IC50,>><em>3</em>00 micromol/L). Lipid peroxidation has been cited by others as an important component of DHA toxicity, and we confirmed that vitamin E prevents the cytotoxic effects of DHA. Lipid peroxidation was greater following DHA treatment of the sensitive DHL-4 cells than in the resistant SiHa cells, as assessed by thiobarbituric acid reactive substance generation. DHL-4 cells treated with DHA for 20 hours showed a <em>3</em>.5-fold increase in thiobarbituric acid reactive substances, whereas SiHa cells showed no increase. Reverse transcription-PCR analysis detected a down-regulation of the expression of the major antioxidant enzyme, superoxide dismutase (<em>SOD</em>) 1, in DHL-4 cells but not in SiHa cells after DHA treatment. Knockdown of <em>SOD</em>1 expression in SiHa cells with small interfering RNA significantly enhanced lipid peroxidation and cytotoxicity on exposure to DHA. These results show that DHL-4 cells are highly sensitive to the cytotoxic effect of DHA and that regulation of <em>SOD</em>1 expression may play an important role in determining the sensitivity of different tumor cells to the cytotoxic effects of DHA.

Increasing evidence suggests that bone marrow derived-mesenchymal stem cells (MSCs) have neuroprotective properties and a major mechanism of action is through their capacity to secrete a diverse range of potentially neurotrophic or anti-oxidant factors. The recent discovery that MSCs secrete superoxide dismutase <em>3</em> (<em>SOD</em><em>3</em>) may help explain studies in which MSCs have a direct anti-oxidant activity that is conducive to neuroprotection in both in vivo and in vitro. <em>SOD</em><em>3</em> attenuates tissue damage and reduces inflammation and may confer neuroprotective effects against nitric oxide-mediated stress to cerebellar neurons; but, its role in relation to central nervous system inflammation and neurodegeneration has not been extensively investigated. Here we have performed a series of experiments showing that <em>SOD</em><em>3</em> secretion by human bone marrow-derived MSCs is regulated synergistically by the inflammatory cytokines TNF-alpha and IFN-gamma, rather than through direct exposure to reactive oxygen species. Furthermore, we have shown <em>SOD</em><em>3</em> secretion by MSCs is increased by activated microglial cells. We have also shown that MSCs and recombinant <em>SOD</em> are able to increase both neuronal and axonal survival in vitro against nitric oxide or microglial induced damage, with an increased MSC-induced neuroprotective effect evident in the presence of inflammatory cytokines TNF-alpha and IFN-gamma. We have shown MSCs are able to convey these neuroprotective effects through secretion of soluble factors alone and furthermore demonstrated that <em>SOD</em><em>3</em> secretion by MSCs is, at least, partially responsible for this phenomenon. <em>SOD</em><em>3</em> secretion by MSCs maybe of relevance to treatment strategies for inflammatory disease of the central nervous system.

The brain exhibits regional vulnerabilities to many insults, and age itself has differential effects on neuronal populations as exemplified by the age-dependent loss of dopaminergic neurons in the nigrostriatal system. We hypothesized that oxidative damage to DNA was more likely to occur in the nigrostriatal system which undergoes significant neurochemical and functional changes with age. To test this hypothesis, oxidative damage to DNA, indicated by levels of 8-hydroxy2'-deoxyguanosine (oxo8dG), was measured in pons-medulla (PM), midbrain (MB), caudate-putamen (CP), hippocampus (HP), cerebellum (CB), and cerebral cortex (CX) at <em>3</em>, 18, and <em>3</em>4 months of age in C57/b1 mice. Steady-state levels of oxo8dG increased significantly with age in MB, CP, and CB, but not in PM, HP, or CX. Manganese superoxide dismutase (Mn<em>SOD</em>) activity decreased with age in MB, CP, and HP, but not in PM, CB, or CX. Regional activities of Cu/Zn superoxide dismutase (Cu/Zn <em>SOD</em>) and glutathione peroxidase (Glut Px) did not change significantly with age. Concomitant with the regional alterations in DNA damage, there was a significant age-dependent decline in locomotor activity, motor coordination, and striatal dopamine content especially during the interval between 18 and <em>3</em>4 months. In conclusion, oxyradical-associated damage to DNA did not accumulate uniformly across brain regions with age and was highest in brain regions that subserve spontaneous locomotor activity and motor coordination.

Oxidative stress is involved in the sarcopenia of aging muscles. On the grounds that ventilatory muscles are permanently active, and their activity may even increase with aging, we hypothesized that the levels of oxidative stress would probably be increased in the external intercostals of elderly healthy individuals. We conducted a case-control study in which reactive carbonyl groups, malondialdehyde-protein adducts, <em>3</em>-nitrotyrosine immunoreactivity, Mn-superoxide dismutase (Mn-<em>SOD</em>), and catalase were detected using immunoblotting in external intercostals and quadriceps (open muscle biopsies) obtained from 12 healthy elderly and 12 young individuals of both sexes. In elderly subjects, reactive carbonyls, malondialdehyde-protein adducts, <em>3</em>-nitrotyrosine, Mn-<em>SOD</em>, and catalase were significantly greater in the external intercostals than in the young controls. A post hoc analysis, in which men and women from both groups were analyzed separately, revealed that the external intercostals of elderly women, but not those of elderly men, showed significantly increased levels of reactive carbonyls, malondialdehyde-protein adducts, <em>3</em>-nitrotyrosine, and Mn-<em>SOD</em> compared to those of control females. This study suggests that differences in muscle activity might explain the differential pattern of oxidative stress observed in human respiratory and limb muscles with aging as well as the likely existence of a sex-related regulation of this phenomenon in these muscles.

OBJECTIVE

To investigate the chemopreventive effects of 1alpha, 25-dihydroxyvitamin D(<em>3</em>) in diethylnitrosamine induced, phenobarbital promoted rat hepatocarcinogenesis.

METHODS

The rats were randomly divided into 6 different groups (A-F). Groups A, C and E rats received a single intraperitoneal (i.p.) injection of diethylnitrosamine (DEN) at a dose of 200 mg/kg body mass in 9 g/L NaCl solution at 4 wk of age, while group B served as normal vehicle control received normal saline once. After a brief recovery of 2 wk, all the DEN treated rats were given phenobarbital (PB) at 0.5 g/L daily in the basal diet till wk 20. Group A was DEN control. Treatment of 1alpha, 25-(OH)2D<em>3</em> in group C was started 4 wk prior to DEN injection and continued thereafter till wk 20 at a dose of 0.<em>3</em> microg/100 microL propylene glycol per one single dose (os) twice a week. Group E received the treatment of 1alpha, 25-(OH)2D<em>3</em> at the same dose mentioned as above for 15 wk. The rats in group D and F received 1alpha, 25-(OH)2D<em>3</em> alone as in group C without DEN injection.

RESULTS

The comet assay showed statistically higher mean values for length to width ratios (L: W) of DNA mass and tailed cells (P<0.01; P<0.01 respectively) in DEN treated rats as compared to their normal controls. Continuous supplementation of 1alpha, 25-dihydroxyvitaminD2 showed a significant (P<0.01) decrease in L:W ratio of DNA mass tailed cells. Furthermore, 1alpha, 25-(OH)2D<em>3</em> supplementations elevated the super oxide dismutase (SOD) activity, hepatic malondialdehyde (MDA) level, reduced glutathione (GSH) and glutathione S-transferase (GST) activity (P<0.01, P<0.05, P<0.05 and P<0.05 respectively). As an endpoint marker histological changes were observed to establish the chemopreventive effects of 1alpha, 25-dihydroxyvitaminD2.

CONCLUSIONS

Supplementations of 1alpha, 25-(OH)2D<em>3</em> has a marked protection against hepatic nodulogenesis, antioxidant enzymes and DNA damages in DEN induced rat hepatocarcinogenesis promoted by phenobarbital.

A growing body of evidence indicates that carbon monoxide (CO), once perceived merely as a poisonous gas, exerts antiapoptotic and cytoprotective effects. Using a water-soluble CO-releasing molecule (CORM) tricarbonylchloro(glycinato)ruthenium(II) (CORM-<em>3</em>), we previously reported that CO induces a delayed protection against myocardial infarction similar to that observed in the late phase of ischemic preconditioning (PC). In the current study, we investigated the molecular mechanisms underlying this cardioprotective effect. The impact on apoptotic signaling pathways was first examined in the setting of ischemia/reperfusion injury. Mice were pretreated with CORM-<em>3</em> or iCORM-<em>3</em> (which does not release CO) and subjected to coronary occlusion/reperfusion 24h later. In mice that received CORM-<em>3</em>, there was a significant reduction in markers of apoptosis (cleaved lamin A, cleaved caspase-<em>3</em>, and cleaved PARP-1) after ischemia/reperfusion injury. To elucidate the mechanism of CORM-<em>3</em>-induced cardioprotection we further examined the activation of transcription factors and induction of cardioprotective and apoptosis modulating proteins. Infusion of CORM-<em>3</em> rapidly activated the stress-responsive transcription factors nuclear factor kappaB (NF-κB), signal transducers and activators of transcription (STAT)1, STAT<em>3</em>, and NF-E2-related factor-2 (Nrf2). This was followed 24h later by upregulation of cardioprotective proteins (heme oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and extracellular superoxide dismutase [Ec-<em>SOD</em>]) and antiapoptotic proteins involving both the mitochondria-mediated (Mcl-1) and the death receptor-mediated (c-FLIP(S) and c-FLIP(L)) apoptosis pathways. We conclude that CO released by CORM-<em>3</em> triggers a cardioprotective signaling cascade that recruits the transcription factors NF-κB, STAT1/<em>3</em>, and Nrf2 with a subsequent increase in cardioprotective and antiapoptotic molecules in the myocardium leading to the late PC-mimetic infarct-sparing effects. This article is part of a Special Issue entitled 'Possible Editorial'.

OBJECTIVE

Hypoxia/reoxygenation (H/R) causes oxidative stress to the cell and induces apoptotic cell death. Signal transducer and activator of transcription-<em>3</em> (Stat<em>3</em>) is one of the most important molecules involved in the initiation of liver development and regeneration, and has recently been shown to protect cells against various pathogens. In order to investigate the hepatoprotective effects of Stat<em>3</em>, we examined whether it protects against H/R-induced injury in primary hepatocytes.

METHODS

Primary cultured hepatocytes were prepared from SD rats. Adenoviruses and cytokines were added 2 days and 1h prior to the H/R insult, respectively. Hepatocytes and culture media were harvested for the assays before and after H/R insult.

RESULTS

Interleukin-6 and cardiotropin-1, which may function mainly through Stat<em>3</em> activation, protected cells from H/R-induced apoptosis. Adenoviral overexpression of the constitutively activated form of Stat<em>3</em> (Stat<em>3</em>-C) reduced H/R-induced apoptosis as well as generation of reactive oxygen species (ROS) in hepatocytes. Interestingly, Stat<em>3</em>-C induced Mn-SOD, but not Cu/Zn-SOD, both at the protein and mRNA levels. Overexpression of Mn-SOD significantly reduced H/R-induced ROS and apoptosis by inhibiting redox-sensitive activation of caspase-<em>3</em> activity.

CONCLUSIONS

Stat<em>3</em> protects hepatocytes from H/R-induced cell injury at least partly by upregulating Mn-SOD and inactivating caspase-<em>3</em>.

This study compares changes in the pro-oxidant production and buffering capacity in young and aged skeletal muscle after exposure to chronic repetitive loading (RL). The dorsiflexors from one limb of young and aged rats were loaded <em>3</em> times/week for 4.5 weeks using 80 maximal stretch-shortening contractions per session. RL increased H2O2 in tibialis anterior muscles of young and aged rats and decreased the ratio of reduced/oxidized glutathione and lipid peroxidation in aged but not young adult animals. Glutathione peroxidase (GPx) activity decreased whereas catalase activity increased with RL in muscles from young and aged rats. RL increased CuZn superoxide disumutase (<em>SOD</em>) and Mn <em>SOD</em> protein concentration and CuZn <em>SOD</em> activity in muscles from young but not aged animals. There were no changes in protein content for GPx-1 and catalase or messenger RNA for any of the enzymes studied. These data show that aging reduces the adaptive capacity of muscles to buffer increased pro-oxidants imposed by chronic RL.

Neonatal rats (4--7 days old) and adult rats (approximately 80 days old) were continuously exposed to either 96--98% oxygen or air. Examination of the lungs of neonatal rats, who survived 5 days of oxygen exposure with no evidence of respiratory distress, showed significant increases in the pulmonary superoxide dismutase (<em>SOD</em>) activity (peak value: 144% of air-exposed controls), glutathione peroxidase (GP) activity (126%), glutathione reductase (GR) activity (122%), reduced glutathione (GSH) level (176%), and glucose-6-phosphate dehydrogenase activity (151%). Adult rats, most of whom succumbed within <em>3</em> days of oxygen exposure, did not show any significant increase in the activities of pulmonary <em>SOD</em>, GP, GR, and the level of GSH as compared to the air-exposed adult animals. Glucose-6-phosphate dehydrogenase was significantly elevated in the 72-hr oxygen-exposed adult rats. It is concluded that increases in the lung complement of <em>SOD</em>, GR, GP, and GSH in the neonatal rat during oxygen challenge may provide the mechanism(s) for their increased tolerance to hyperoxia-induced lung injury as compared to the adults.

In a 16-wk study, weanling Wistar rats (<em>3</em>2 males and <em>3</em>2 females) were fed a modified AIN-76 diet containing 20% fat with various (n-<em>3</em>) fatty acids. All dietary fats provided the same amount of saturates, monounsaturates, and total essential fatty acids [(n-6) + (n-<em>3</em>)]. The control diet contained lard/corn oil (L/CO). The other diets contained (n-<em>3</em>) fatty acids from linseed oil (LSO), from linseed oil + menhaden oil (LSO + MO) or from menhaden oil (MO). The (n-<em>3</em>) diets reduced total and HDL-cholesterol, particularly in rats fed the MO diet. Platelet thromboxane levels were equally depressed by the LSO and MO diets. Dietary (n-<em>3</em>) fatty acids significantly elevated docosahexaenoic acid in livers and hearts of male and female rats, with females reaching higher levels. This increase was accompanied by reduced arachidonic acid, except for hearts of females in which the major decrease was in linoleic acid. Overall, enzyme activities in the MO-fed group were decreased to the following levels (relative to the activity in the control group): heart Mn superoxide dismutase (<em>SOD</em>), 28%; liver CuZn<em>SOD</em>, 82%; aorta CuZn<em>SOD</em>, <em>3</em>2%. Greater reductions in these enzyme activities were seen in the female rats fed the MO diet compared with male rats. Lipid peroxidation, assessed by urinary, heart and liver thiobarbituric acid reactants, was increased by dietary (n-<em>3</em>) fatty acids (MO greater than LSO + MO greater than LSO greater than L/CO) and was higher in females than in males. These results indicate that enhanced lipid peroxidation occurs with the increased oxidative stress of elevated tissue (n-<em>3</em>) fatty acids accompanied by reduced <em>SOD</em> activity.

The cellular effects of carbon monoxide (CO) are produced primarily by CO binding to iron or other transition metals, which may also promote prooxidant activities of the more reactive gases, oxygen and nitric oxide. We tested the hypothesis that prooxidant effects of CO deregulate the calcium-dependent mitochondrial pore transition (MPT), which disrupts membrane potential and releases apoptogenic proteins. Rats were exposed to either CO (50 ppm) or hypobaric hypoxia (HH) for 1, <em>3</em>, or 7 days, and liver mitochondria harvested to study protein expression and sensitivity to MPT by calcium and oxidants. Both exposures induced hypoxia-sensitive protein expression: hypoxia-inducible factor 1alpha (HIF-1alpha), heme oxygenase-1 (HO-1), and manganese <em>SOD</em> (<em>SOD</em>2), but <em>SOD</em>2 induction was greater by CO than by HH, especially at 7 days. Relative to HH, CO also caused significant early mitochondrial oxidative and nitrosative stress shown by decreases in GSH/GSSG and increases in protein <em>3</em>-nitrotyrosine (<em>3</em>-NT) and protein mixed disulfide formation. This altered MPT sensitivity to calcium through an effect on the "S-site," causing loss of pore protection by adenine nucleotides. By 7 days, despite continued CO, nitrosative stress decreased and adenine nucleotide protection was restored to preexposure levels. This is the first evidence of functional mitochondrial pore stress caused by CO independently of its hypoxic effect, as well as a compensatory response exemplifying a mitochondrial phenotype shift. The implications are that cellular CO can activate or deactivate mitochondria for initiation of apoptosis in vivo.

Gentamicin (GM) is an antibiotic whose clinical use is limited by its nephrotoxicity. Experimental evidences suggest a role of reactive oxygen species in GM-induced nephrotoxicity. Therefore, we investigated if aged garlic extract (AGE), an antioxidant, has a protective role in this experimental model. Four groups of male Wistar rats were studied: 1) Control (CT), injected subcutaneously (s.c.) and intraperitoneally (i.p.) with saline, 2) GM, treated s.c. with GM (70 mg/kg/12 hours/4 days), <em>3</em>) AGE, treated i.p with AGE (1.2 mL/kg/12 hours/6 days), and 4) GM + AGE treated with GM and AGE. The treatment with AGE started two days before the first dose of GM (GM + AGE group) or saline (AGE group). Animals were sacrificed on day 5, and blood, urine, and kidneys were obtained. Nephrotoxicity was made evident by: 1) the increase in blood urea nitrogen and plasma creatinine, 2) the decrease in plasma glutathione peroxidase (GPx) activity and the urinary increase in N-acetyl-beta-D-glucosaminidase activity and total protein, and <em>3</em>) necrosis of proximal tubular cells. These alterations were prevented or ameliorated by AGE treatment. Furthermore, AGE prevented the GM-induced increase in the renal levels of oxidative stress markers: nitrotyrosine and protein carbonyl groups and the decrease in manganese superoxide dismutase (Mn-<em>SOD</em>), GPx, and glutathione reductase (GR) activities. The protective effect of AGE was associated with the decrease in the oxidative stress and the preservation of Mn-<em>SOD</em>, GPx, and GR activities in renal cortex. These data suggest that AGE may be a useful agent for the prevention of GM-nephrotoxicity.

Formulations of antioxidant enzymes, superoxide dismutase 1 (<em>SOD</em>1, also known as Cu/Zn <em>SOD</em>) and catalase were prepared by electrostatic coupling of enzymes with cationic block copolymers, polyethyleneimine-poly(ethylene glycol) or poly(L-lysine)-poly(ethylene glycol), followed by covalent cross-linking to stabilize nanoparticles (NPs). Different cross-linking strategies (using glutaraldehyde, bis-(sulfosuccinimidyl)suberate sodium salt or 1-Ethyl-<em>3</em>-[<em>3</em>-dimethylaminopropyl]carbodiimide hydrochloride with N-hydroxysulfosuccinimide) and reaction conditions (pH and polycation/protein charge ratio) were investigated that allowed immobilizing active enzymes in cross-linked NPs, termed "nanozymes." Bienzyme NPs, containing both <em>SOD</em>1 and catalase were also formulated. Formation of complexes was confirmed using denaturing gel electrophoresis and western blotting; physicochemical characterization was conducted using dynamic light scattering and atomic force microscopy. In vivo studies of (125)I-labeled <em>SOD</em>1-containing nanozymes in mice demonstrated their increased stability in both blood and brain and increased accumulation in brain tissues, in comparison with non-cross-linked complexes and native <em>SOD</em>1. Future studies will evaluate the potential of these formulations for delivery of antioxidant enzymes to the central nervous system to attenuate oxidative stress associated with neurological diseases.

UNASSIGNED

Formulations of antioxidant enzyme complexes were demonstrated along with their increased stability in both blood and brain and increased accumulation in CNS tissue. Future studies will evaluate the potential of these formulations for antioxidant enzyme deliver to the CNS to attenuate oxidative stress in neurodegenerative diseases.

Traditional Chinese medicine uses a systemic treatment approach, targeting multiple etiological factors simultaneously. Danhong injection (DHI), a very popular Chinese medicine injection, is reported to be effective for many cardiovascular conditions. The primary active ingredients of DHI, and their systemic and interrelated mechanism have not been evaluated in an established myocardial ischemia/reperfusion (MI/R) model. We identified the main active constituents in DHI, including hydroxysafflor yellow A (A), salvianolic acid B (B), and danshensu (C), by HPLC fingerprint analysis and assessed their effect on MI/R rats and cardiomyocytes. These <em>3</em> compounds and DHI all decreased the levels of IL-1, TNF- α , and MDA, increased those of IL-10 and <em>SOD</em> activity in vivo and in vitro, and had antiapoptotic effects, as shown by flow cytometric analysis and TUNEL assay. Moreover, these compounds increased phosphorylation of Akt and ERK1/2 in cardiomyocytes. Interestingly, we found compound A exerted a more prominent anti-inflammatory effect than B and C, by decreasing NF- κ B levels; compound B had more powerful antioxidative capacity than A and C, by increasing Nrf2 expression; compound C had stronger antiapoptotic ability than A and B, by lowering caspase-<em>3</em> activity. Our results elucidate the mechanisms by which DHI protects against MI/R induced injury.

Manganese superoxide dismutase (Mn-<em>SOD</em>) is induced in ischemic hearts 24 h after ischemic preconditioning, when tolerance to ischemia is acquired. We examined the relationship between Mn-<em>SOD</em> induction and the protective effect of preconditioning using cultured rat cardiac myocytes. Exposure of cardiac myocytes to brief hypoxia (1 h) decreased creatine kinase release induced by sustained hypoxia (<em>3</em> h) that follows when the sustained hypoxia was applied 24 h after hypoxic preconditioning (57% of that in cells without preconditioning). The activity and content of Mn-<em>SOD</em> in cardiac myocytes were increased 24 h after hypoxic preconditioning (activity, 170%; content, 1<em>3</em>9% compared with cells without preconditioning) coincidentally with the acquisition of tolerance to hypoxia. Mn-<em>SOD</em> mRNA was also increased 20-40 min after preconditioning. Antisense oligodeoxyribonucleotides corresponding to the initiation site of Mn-<em>SOD</em> translation inhibited the increases in the Mn-<em>SOD</em> content and activity and abolished the expected decrease in creatine kinase release induced by sustained hypoxia after 24 h of hypoxic preconditioning. Sense oligodeoxyribonucleotides did not abolish either Mn-<em>SOD</em> induction or tolerance to hypoxia. These results suggest that the induction of Mn-<em>SOD</em> in myocytes by preconditioning plays a pivotal role in the acquisition of tolerance to ischemia at a later phase (24 h) of ischemic preconditioning.

In recent years, the exploration and development of the effective methods of treatment and prevention to algal blooms, especially Microcystis aeruginosa blooms has been an important issue in the field of water environment protection. Allelochemicals (natural plant toxins) are considered promising sources of algicides to control algal blooms. The objective of this study is to determine the inhibitory effects and potential mechanisms of a well-known allelochemical gramine (N,N-dimethyl-<em>3</em>-amino-methylindole) on bloom-forming cyanobacterium M. aeruginosa. The results showed that this indole alkaloid effectively inhibited the growth of M. aeruginosa. The effective concentration causing a 50% inhibition at <em>3</em> d (EC(50, <em>3</em> d)) increased with the initial algal density (IAD) increasing. When IAD increased from 5x10(4) to 5x10(5)cellsmL(-1), the values of EC(50, <em>3</em> d) increased from 0.5 to 2.1mgL(-1). In the cells of M. aeruginosa, gramine caused an obvious increase in the level of reactive oxygen species (ROS). The lipid-peroxidation product malondialdehyde (MDA) increased significantly in gramine-treated cells. The effects of gramine on enzymatic and non-enzymatic antioxidants were in different manners. The activity of superoxide dismutase (<em>SOD</em>) was decreased after gramine exposure. The catalase (CAT) activity was increased after 4h but decreased from 60h. Both the contents and the regeneration rates of ascorbic acid (AsA) and reduced glutathione (GSH) were increased after 4h of exposure to gramine. However, only GSH content was still increased after 40h of exposure. These results suggested that the activation of antioxidants in M. aeruginosa played an important role to resist the stress from gramine at initial time, the inactivation of <em>SOD</em> is crucial to the growth inhibition of M. aeruginosa by gramine, and the phytotoxicity of gramine on M. aeruginosa may be due to oxidative damage via oxidation of ROS.

The macula densa detects changes in NaCl concentration in tubular fluid and transmits a feedback signal, known as tubuloglomerular feedback (TGF), which helps to control glomerular afferent arteriole resistance. We and other investigators have reported that synthesis of NO in the macula densa inhibits TGF. NO can be scavenged by superoxide (O(-)(2)) to form peroxynitrite, effectively reducing the bioavailability of NO; there is growing evidence that O(-)(2) regulates vascular tone in the kidney. We hypothesized that O(-)(2) produced in the macula densa enhances TGF and this effect acts only in an autocrine manner within the cells of the macula densa. Afferent arterioles and attached macula densas from Sprague-Dawley rats were simultaneously microperfused in vitro and TGF response examined before and after perfusing the tubular lumen, bath, or vascular lumen with a superoxide scavenger. The macula densa was perfused with solutions containing either 5 mmol/L Na(+) and <em>3</em> mmol/L Cl(-) (low NaCl) or 80 mmol/L Na(+) and 77 mmol/L Cl(-) (high NaCl) while keeping pressure in the afferent arteriole constant at 60 mm Hg. When 10(-4) M Tempol, a stable membrane-permeant superoxide dismutase (<em>SOD</em>) mimetic, was added to the tubular lumen, it inhibited TGF by 56% (before Tempol: TGF, <em>3</em>.2 +/- 0.<em>3</em> microm; after Tempol: TGF, 1.4 +/- 0.2 microm; n=6; P<0.05, control versus Tempol). Adding Tempol to the bath inhibited TGF by 48% (before Tempol: TGF, 2.5 +/- 0.25 microm; after Tempol: TGF, 1.<em>3</em> +/- 0.18 microm; n=6; P<0.05). However, adding Tempol to the vessel lumen did not change TGF response significantly (before Tempol: TGF, 2.7 +/- 0.<em>3</em>7 microm; after Tempol: TGF, <em>3</em>.2 +/- 0.25 microm; n=7; P=0.25). When <em>3</em>00 U/mL of the enzyme <em>SOD</em>, which is not membrane-permeant, was added to either the tubular lumen or bath, it had no effect on TGF response. Finally, to determine whether the effect of O(-)(2) in the macula densa is mediated by its scavenging of NO, 7-nitroindazole (7-NI) was added to the macula densa to inhibit neuronal nitric oxide synthase (nNOS). In the presence of 7-NI, Tempol had no effect (7-NI only: TGF, <em>3</em>.0 +/- 0.4 microm; 7-NI plus Tempol: TGF, 2.8 +/- 0.5 microm; n=6; P=0.<em>3</em>4<em>3</em>). Our findings suggest that (1) reducing O(-)(2) increases the bioavailability of NO, which inhibits TGF, (2) both O(-)(2) and NO act within the macula densa, and (<em>3</em>) O(-)(2) appears to have no effect on its own.

OBJECTIVE

This study investigated the protective effect of vitamin D analog paricalcitol combined with angiotensin-converting enzyme inhibitor (enalapril) on aortic oxidative injury in atherosclerotic mice.

METHODS

Female mice were treated for 16 weeks as follows: (1) ApoE deficient + vehicle, (2) ApoE deficient + paricalcitol (200 ng <em>3</em> times a week), (<em>3</em>) ApoE deficient + enalapril (<em>3</em>0 mg/l in drinking water), (4) ApoE deficient + paricalcitol + enalapril, and (5) wild-type controls.

RESULTS

ApoE-deficient mice developed hypertension which was prevented by enalapril or enalapril + paricalcitol treatment but not by paricalcitol treatment. Histology showed atherosclerotic plaque in the aorta of ApoE-deficient mice which was prevented by paricalcitol, enalapril, and paricalcitol + enalapril treatments. Aortic malondialdehyde levels, NADPH oxidase subunit p22(phox), manganese-superoxide dismutase (Mn-SOD), inducible nitric oxide synthase, monocyte chemoattaractant protein-1, tumor necrosis factor (TNF)-α, and cyclooxygenase-2 protein expressions increased, whereas glutathione levels, CuZn-SOD, and endothelial protein expressions decreased in ApoE-deficient mice compared to controls. Treatment with paricalcitol and enalapril alone or in combination protected the inflammatory and oxidative endothelial injury of the aorta in atherosclerotic mice.

CONCLUSIONS

Combination therapy affords greater protection against aortic inflammatory and oxidative injury in atherosclerosis than monotherapy.

BACKGROUND

Contrast media (CM)-induced nephropathy (CIN) is an acute deterioration of renal function following administration of CM mediated to a large extent by the increased production of ROS within the kidney. Aim of this study was to evaluate whether a novel isoform of a recombinant Manganese SOD (rMnSOD) could provide an effective protection against CIN; this molecule shares the same ability of physiological SODs in scavenging reactive oxygen species (ROS) but, due to its peculiar properties, enters inside the cells after its administration.

METHODS

We studied the effects rMnSOD on oxidative damage in a rat model of CIN in uninephrectomized rats, that were randomly assigned to 3 experimental Groups: Group CON, control rats treated with the vehicle of CM, Group HCM, rats treated with CM and Group SOD, rats treated with CM and rMnSOD.

RESULTS

In normal rats, pretreatment with rMnSOD, reduced renal superoxide anion production, induced by the activation of NAPDH oxidase, by 84 % (p < 0.001). In rats of Group HCM, ROS production was almost doubled compared to rat of Group CON (p < 0.01) but returned to normal values in rats of Group SOD, where a significant increase of SOD activity was detected (+16 % vs HCM, p < 0.05). Administration of CM determined a striking fall of GFR in rats of Group HCM (-70 %, p < 0.001 vs CON), greatly blunted in Group SOD (-28 % vs CON, p < 0.01); this was associated with a lower presence of both tubular necrosis and intratubular casts in SOD-treated rats (both p < 0.01 vs Group HCM).

CONCLUSIONS

Our data indicate that rMnSOD is able to reduce renal oxidative stress, thus preventing the reduction of GFR and the renal histologic damage that follows CM administration.

BACKGROUND

Estimation of HIV incidence rates is important for timing interventions, planning prevention studies, and monitoring the epidemic. This requires accurate estimation of the "recency period" (also known as the "window period") between seroconversion and achievement of specific detectable levels of anti-HIV antibody titers, such as the standardized optical density (SOD) in the early phase of HIV-1 infection.

METHODS

To obtain a better understanding of interpatient variation of the recency period, prospective measurements of antiviral antibody titers in the early phase of HIV-1 subtype C infection were quantified by Vironostika-LS. Time of seroconversion was estimated by Fiebig staging.

RESULTS

The profiles of SOD values during the first year of infection commonly showed slow initial increase followed by a more rapid increase, although in some patients, SOD values increased rapidly soon after seroconversion. Using an SOD cutoff of 1.0, the average duration of the recency period in subtype C infection in the local epidemic in Botswana was estimated to be 151 days (95% confidence interval: 130 to 172 days) post seroconversion. The recency period was significantly associated (P = 0.007) with the level of viral replication during the first 2-3 months post seroconversion. Reduction of SOD values after initiation of antiretroviral therapy (ART) was a dominant pattern in antiretroviral drug (ARV)-treated subjects.

CONCLUSIONS

Our data suggest that HIV incidence estimation based on sensitive/less sensitive enzyme immunoassay cross-sectional testing could be potentially improved by incorporation of viral load levels at the time of detection of a recent infection.