Neuroanatomical studies suggest that neuronal atrophy and destruction occur over the course of many years in neurodegenerative conditions such as schizophrenia and Alzheimer's disease. In schizophrenia, early intervention with atypical neuroleptics such as olanzapine has been shown to prevent development of some of the more serious and debilitating symptoms in many patients. The mechanisms whereby olanzapine slows or prevents symptom progression in schizophrenia remain unclear. A previous study found that olanzapine increased mRNA for the copper/zinc isoform of the superoxide dismutase enzyme (<em>SOD</em>-1). We investigated the effects of olanzapine in PC12 cells exposed to hydrogen peroxide. We measured cell viability, observed evidence of necrosis and apoptosis, checked the <em>SOD</em>-1 mRNA by Northern blot analyses, and determined <em>SOD</em>-1 enzyme activity. We found that: (1) the decrease in cell viability induced by hydrogen peroxide was attenuated in PC12 cells pretreated with olanzapine; (2) olanzapine increased <em>SOD</em> enzyme activity in PC12 cells; (<em>3</em>) inhibiting <em>SOD</em> activity with diethyldithiocarbamic acid prevented the cytoprotective actions of olanzapine; and (4) the decrease in <em>SOD</em>-1 mRNA level induced by hydrogen peroxide was blocked by pretreatment with olanzapine. These data indicate that the neuroprotective action of olanzapine includes the upregulation of <em>SOD</em>.

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-<em>3</em>beta. Long-term (<em>3</em> days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide <em>3</em>-kinase (PI<em>3</em>K) inhibitor wortmannin, but not with the superoxide dismutase (<em>SOD</em>) mimetic M40401. Long-term BMS treatment induced a PI<em>3</em>K-dependent increase in the expression and activity of catalase without affecting manganese <em>SOD</em> and copper/zinc-dependent <em>SOD</em>. Finally, the catalase inhibitor <em>3</em>-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI<em>3</em>K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.

The structure of Mn(III) superoxide dismutase (Mn(III)<em>SOD</em>) from Thermus thermophilus, a tetramer of chains 20<em>3</em> residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 20<em>3</em>) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this Mn<em>SOD</em> include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position <em>3</em>0 that distorts the helix near the first Mn ligand; and (<em>3</em>) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His8<em>3</em>, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp1<em>3</em>2A, Trp168A, Tyr18<em>3</em>A, Tyr172B, Tyr17<em>3</em>B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr<em>3</em>6 and His<em>3</em>2 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidermal levels of enzymatic and non-enzymatic antioxidants such as superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GSH-Px), vitamin E (Vit E), ubiquinol (CoQ10H2), and reduced glutathione (GSH), as well as polyunsaturated fatty acids of phospholipids (PL-PUFA), were evaluated in the affected epidermis of 15 patients with active vitiligo (AVP) and in the corresponding epidermis of 15 healthy phototype matched controls. The epidermal levels of CoQ10H2, Vit E, GSH, and CAT activity were significantly reduced in AVP and were associated with a marked increase of oxidized glutathione, whereas <em>SODs</em> and GSH-Px activities and ubiquinone concentration remained similar to control values. Antioxidant deficiency, in particular the decline of lipophilic antioxidants, i.e., CoQ10H2 and Vit E, accounts well for PL-PUFA reduction observed in vitiligo epidermis, mainly affecting C18:<em>3</em> n-<em>3</em>, C20:<em>3</em> n-6, C20:4 n-6, and C22:6 n-<em>3</em> fatty acids and suggesting the occurrence of a lipoperoxidative process. In conclusion, both an imbalance of the intracellular redox status and a significant depletion of enzymatic and non-enzymatic antioxidants feature the epidermis of AVP, and represent a fingerprint of an abnormal oxidative stress leading to epidermal cell injury.

A manganese(III) complex of biliverdin IX dimethyl ester, (MnIIIBVDME)2, was prepared and characterized by elemental analysis, UV/vis spectroscopy, cyclic voltammetry, chronocoulometry, electrospray mass spectrometry, freezing-point depression, magnetic susceptibility, and catalytic dismuting of superoxide anion (O2.-). In a dimeric conformation each trivalent manganese is bound to four pyrrolic nitrogens of one biliverdin dimethyl ester molecule and to the enolic oxygen of another molecule. This type of coordination stabilizes the +4 metal oxidation state, whereby the +<em>3</em>/+4 redox cycling of the manganese in aqueous medium was found to be at E1/2 = +0.45 V vs NHE. This potential allows the Mn(III)/Mn(IV) couple to efficiently catalyze the dismutation of O2.- with the catalytic rate constant of kcat = 5.0 x 10(7) M-1 s-1 (concentration calculated per manganese) obtained by cytochrome c assay at pH 7.8 and 25 degrees C. The fifth coordination site of the manganese is occupied by an enolic oxygen, which precludes binding of NO., thus enhancing the specificity of the metal center toward O2.-. For the same reason the (MnIIIBVDME)2 is resistant to attack by H2O2. The compound also proved to be an efficient <em>SOD</em> mimic in vivo, facilitating the aerobic growth of <em>SOD</em>-deficient Escherichia coli.

OBJECTIVE

To explore a treatment paradigm for Leber hereditary optic neuropathy (LHON), we augmented mitochondrial antioxidant defenses to rescue cells with the G11778A mutation in mitochondrial DNA.

METHODS

Cells homoplasmic for the G11778A mutation in mitochondrial DNA were infected with an adeno-associated viral vector containing the human mitochondrial superoxide dismutase (SOD2) gene. Control cells were infected with an adeno-associated viral (AAV) vector expressing the green fluorescent protein (GFP). Two days later, the high-glucose culture medium was exchanged for a glucose-free medium containing galactose. After 1 or 2 days, cellular production of superoxide was assessed using the fluorescent probe dihydroethidium, and we used TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) staining to detect apoptotic nuclei. The effect of SOD2 on LHON cell survival was quantitated after 2 or <em>3</em> days.

RESULTS

Comparisons of AAV-SOD2-infected LHON cells relative to control cells infected with AAV-green fluorescent protein showed increased expression of mitochondrial SOD that attenuated superoxide-induced fluorescence by 26% (P = .00<em>3</em>) and suppressed TUNEL-induced fluorescence by 21% (P = .048) after 2 days of growth in galactose medium, when cell survival increased by 25% (P=.05). After <em>3</em> days in galactose medium, SOD2 increased LHON survival by 89% (P = .006) relative to controls.

CONCLUSIONS

Protection against mitochondrial oxidative stress may be useful for treatment of LHON.

CONCLUSIONS

Gene therapy with antioxidant genes may protect patients with LHON against visual loss.

OBJECTIVE

To establish how far liposomal copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD), respectively, reduce radiation-induced fibrosis (RIF), using a well-characterized pig model of RIF permitting the design of a controlled laboratory experiment.

METHODS

In this model of acute localized gamma irradiation simulating accidental overexposure in humans, three groups of five large white pigs were irradiated using a collimated 192Ir source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of subcutaneous fibrosis involving skin and skeletal muscle developed 6 months after irradiation. One experimental group of five pigs was then injected i.m. with 10 mg/10 kg b.wt. of Cu/Zn-SOD, twice a week for 3 weeks, and another experimental group of five was injected with 10 mg/10 kg b.wt. of Mn-SOD, three times a week for 3 weeks. Five irradiated control pigs were injected with physiological serum. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Block depth was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 12-14 weeks after completing SOD injections. The density, length, width, and depth of the fibrotic block, and the areas and volume of its projected cutaneous surface were compared before treatment, 1, 3, and 6 weeks thereafter, and at autopsy, 12-14 weeks after treatment ended.

RESULTS

The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. Whether they were given Cu/Zn- or Mn-SOD, significant and roughly equivalent softening and shrinking of the fibrotic block were noted in all treated animals between the first week after treatment ended and autopsy, when mean regression was 45% for length and width, 30% for depth, and 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar. This replacement of scar tissue by normal tissue in experimental animals and the 50% decrease in the linear dimensions of the scar were comparable to the results obtained in previous clinical studies and highly significant compared to the clinical and autopsy results for the control animals.

CONCLUSIONS

Our results are striking and comparable to the results obtained in our previous clinical study after liposomal Cu/Zn-SOD treatment. To our knowledge, this is the first time that two agents have been shown to reverse the radiation-induced fibrotic process in experimental animals and to permit the regeneration of normal tissue in a zone of well-established postirradiation fibrosis.

BACKGROUND

Free oxygen radicals and insufficiency of antioxidants have been implicated in the pathogenesis of hypertension. We determined the effect of edible oils on blood pressure, lipid profiles and redox status in hypertensive patients given antihypertensive therapy (nifedipine-calcium channel blocker).

METHODS

5<em>3</em>0 patients medicated with nifedipine were divided into <em>3</em> groups (<em>3</em>56 patients-sesame oil; 87 patients-sunflower oil; 47 patients-groundnut oil) and the control group (n=40) received only the drug, nifedipine. The respective oils were supplied to the patients and instructed to use as the only edible oil for 60 days, which comes to <em>3</em>5 g of oil/day/person. Blood pressure, lipid profiles [total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG)], lipid peroxidation [thiobarbituric acid reactive substances (TBARS)], enzymatic [superoxide dismutase (<em>SOD</em>), catalase (CAT) and glutathione peroxidase (GPx)] and nonenzymatic [(vitamin C, vitamin E, beta-carotene and reduced glutathione (GSH)] in blood were measured at baseline and after 60 days of oil substitution.

RESULTS

Patients with nifedipine alone or with respective oils had significantly lowered blood pressure. TC, LDL-C and TG decreased while HDL-C elevated in sesame and sunflower oil groups. Increases of HDL-C and TG were noted in groundnut oil group. TBARS levels reduced in all the groups whereas the reduction was remarkable in sesame oil group. Activities of <em>SOD</em> elevated in the <em>3</em> oil groups whereas GPx and CAT increased only in sesame oil group. Levels of vitamin C, vitamin E, beta-carotene and GSH increased in sesame oil group whereas vitamin E and beta-carotene were elevated only in sunflower and groundnut oil groups. GSH increased in drug control group also.

CONCLUSIONS

Among the <em>3</em> oils, sesame oil offers better protection over blood pressure, lipid profiles and lipid peroxidation and increases enzymatic and nonenzymatic antioxidants.

This study determined the role of body temperature during chronic exercise on myocardial stress proteins and antioxidant enzymes as well as functional recovery after an ischemic insult. Male Sprague-Dawley rats were exercised for <em>3</em>, 6, or 9 wk in a 2<em>3</em> degrees C room (<em>3</em>WK, 6WK, and 9WK, respectively) or in a 4-8 degrees C environment with wetted fur (<em>3</em>WKC, 6WKC, and 9WKC, respectively). The colder room prevented elevations in core temperature. During weeks <em>3</em>-9 the animals ran 5 days/wk up a 6% grade at 20 m/min for 60 min. Myocardial heat shock protein 70 (HSP 70) increased 12.<em>3</em>-fold (P < 0.05) in 9WK versus sedentary (SED) rats but was unchanged in the cold-room runners. Compared with SED rats, alphaB-crystallin was 90% higher in 9WKC animals, HSP 90 was 50% higher in <em>3</em>WKC and 6WKC animals, and catalase was 2<em>3</em>% higher in <em>3</em>WK animals (P < 0.05 for all). Cytosolic superoxide dismutase increased and mitochondrial <em>SOD</em> decreased (P < 0.05) in <em>3</em>WK and 6WK rats compared with <em>3</em>WKC and 6WKC rats. Antioxidant enzymes returned to SED values in all runners by 9 wk. No differences were observed among any of the groups for glucose-regulated protein 75, heme oxygenase-1, or glutathione peroxidase. Mechanical recovery of isolated working hearts after 22.5 min of global ischemia was enhanced in 9WK (P < 0.05) but not in 9WKC rats. We conclude that exercise training results in dynamic changes in cardioprotective proteins over time which are influenced by core temperature. In addition, cardioprotection resulting from chronic exercise appears to be due to increased HSP 70.

A novel type of superoxide dismutase containing nickel as a cofactor (Ni<em>SOD</em>) has been discovered in several Streptomyces spp. The gene for Ni<em>SOD</em> (sodN) was cloned from S. coelicolor Müller using degenerate oligonucleotide probes designed from the N-terminal peptide sequence of the purified enzyme. It encodes a polypeptide of 1<em>3</em>1 amino acids (1470<em>3</em> Da), without any apparent sequence similarity to other known proteins. The N-terminus of the purified Ni<em>SOD</em> was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing. The molecular mass of the processed polypeptide was predicted to be 1<em>3</em>201 Da, in close agreement with that of the purified Ni<em>SOD</em> (1<em>3</em>.4 kDa). The transcription start site of the sodN gene was determined by S1 mapping and primer extension analysis. Ni2+ regulates the synthesis of Ni<em>SOD</em> polypeptide. S1 mapping of both 5' and <em>3</em>' ends of sodN mRNA revealed that Ni2+ increased the level of monocistronic sodN mRNA by more than ninefold without changing its half-life, thus demonstrating that Ni2+ regulates transcription. Both precursor and processed Ni<em>SOD</em> polypeptides with little <em>SOD</em> activity were produced from the cloned sodN gene in S. lividans in the absence of sufficient Ni2+; however, on addition of Ni2+, active Ni<em>SOD</em> consisting of only processed polypeptide was formed. Expression of the full-length sodN gene in E. coli produced Ni<em>SOD</em> polypeptide without any <em>SOD</em> activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N-terminal 14 amino acids from the sodN gene allowed the production of active Ni<em>SOD</em> in E. coli, indicating that N-terminal processing is required to produce active Ni<em>SOD</em>. These results reveal the unique role of nickel as a multifaceted regulator in S. coelicolor controlling sodN transcription and protein processing, as well as acting as a catalytic cofactor.

A gene with greater than 50% amino acid sequence identity to eukaryotic Cu/Zn superoxide dismutase genes (<em>sod</em>) was found at 19 map units in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). This gene was transcribed into 1.4- and 1.5-kb RNAs late in virus infection. The two late RNAs have coterminal <em>3</em>' ends but initiate from two different start points, both of which map to the central adenine of the sequence motif ATTAAG. The late gene product was found to be a low abundance 20-kDa polypeptide which was observed only upon partial purification by chloroform/ethanol extraction. Viruses with mutations in <em>sod</em> were able to replicate normally both in cell culture and in insect larvae. Viruses lacking an intact <em>sod</em> showed no evidence of a replication disadvantage even in the presence of paraquat, an inducer of superoxide anions. A viral-encoded superoxide dismutase (<em>SOD</em>) activity was not detected although endogenous insect <em>SODs</em> appeared to be induced in insect hemolymph and in other tissues following virus infection. The function of <em>sod</em> in the baculovirus infection process remains unclear.

Angiotensin-converting enzyme (ACE) inhibitors ameliorate the progression of renal disease. In combination with vitamin D receptor activators, they provide additional benefits. In the present study, uremic (U) rats were treated as follows: U+vehicle (UC), U+enalapril (UE; 25 mg/l in drinking water), U+paricalcitol (UP; 0.8 μg/kg ip, <em>3</em> × wk), or U+enalapril+paricalcitol (UEP). Despite hypertension in UP rats, proteinuria decreased by <em>3</em>2% vs. UC rats. Enalapril alone, or in combination with paricalcitol, further decreased proteinuria (≈70%). Glomerulosclerosis and interstitial infiltration increased in UC rats. Paricalcitol and enalapril inhibited this. The increase in cardiac atrial natriuretic peptide (ANP) seen in UC rats was significantly decreased by paricalcitol. Enalapril produced a more dramatic reduction in ANP. Renal oxidative stress plays a critical role in inflammation and progression of sclerosis. The marked increase in p22(phox), a subunit of NADPH oxidase, and decrease in endothelial nitric oxide synthase were inhibited in all treated groups. Cotreatment with both compounds inhibited the uremia-induced increase in proinflammatory inducible nitric oxide synthase (iNOS) and glutathione peroxidase activity better than either compound alone. Glutathione reductase was also increased in UE and UP rats vs. UC. Kidney 4-hydroxynonenal was significantly increased in the UC group compared with the normal group. Combined treatment with both compounds significantly blunted this increase, P < 0.05, while either compound alone had no effect. Additionally, the expression of Mn-<em>SOD</em> was increased and CuZn-<em>SOD</em> decreased by uremia. This was ameliorated in all treatment groups. Cotreatment with enalapril and paricalcitol had an additive effect in increasing CuZn-<em>SOD</em> expression. In conclusion, like enalapril, paricalcitol alone can improve proteinuria, glomerulosclerosis, and interstitial infiltration and reduce renal oxidative stress. The effects of paricalcitol may be amplified when an ACE inhibitor is added since cotreatment with both compounds seems to have an additive effect on ameliorating uremia-induced changes in iNOS and CuZn-<em>SOD</em> expression, peroxidase activity, and renal histomorphometry.

Calcium (Ca2+) may be involved in plant tolerance to heat stress by regulating antioxidant metabolism or/and water relations. This study was designed to examine whether external Ca2+ treatment would improve heat tolerance in two C(<em>3</em>), cool-season grass species, tall fescue (Festuca arundinacea L.) and Kentucky bluegrass (Poa pratensis L.), and to determine the physiological mechanisms of Ca2+ effects on grass tolerance to heat stress. Grasses were treated with CaCl(2) (10 mM) or H(2)O by foliar application and then exposed to heat stress (<em>3</em>5/<em>3</em>0 degrees C) in growth chambers. Some of the Ca2+ -untreated plants were maintained at 20/15 degrees C as the temperature control. Heat stress reduced grass quality, relative water content (RWC), and chlorophyll (Chl) content of leaves in both species, but Ca2+ treatment increased all three factors under heat stress. The Ca2+ concentration in cell saps increased with heat stress and with external Ca2+ treatment in both species. Osmotic potential increased with heat stress, but external Ca2+ treatment had no effect. Osmotic adjustment increased during short-term heat stress, but then decreased with a prolonged period of stress; it was not influenced by Ca2+ treatment. The activity of superoxide dismutase (<em>SOD</em>) in both species increased transiently at 12 d of heat stress and then remained at a level similar to that of the control. External Ca2+ treatment had no effect on <em>SOD</em> activity. The activities of catalase (CAT), ascorbate peroxidase (AP), and glutathione reductase (GR) of both species decreased during heat stress. Plants treated with Ca2+ under heat stress had higher CAT, GR and AP activities than untreated plants. Lesser amounts of malondialdehyde (MDA) accumulated in Ca2+ -treated plants than in untreated plants during extended periods of heat stress. The results suggested that exogenous Ca2+ treatment enhanced heat tolerance in both tall fescue and Kentucky bluegrass. This enhancement was related to the maintenance of antioxidant activities and a decrease in membrane lipid peroxidation, but not to the regulation of osmotic potential and osmotic adjustment.

BACKGROUND

Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings.

METHODS

Male Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70-80% VO2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx-) were measured. Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting.

RESULTS

High caloric diet increased triglycerides concentration (SDD: 216 +/- 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 +/- 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 +/- 0.1 and TRD: 1.24 +/- 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 +/- 0.1 and SDD: 2.57 +/- 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 +/- 6; TR: 140 +/- 8; SDD: 156 +/- 8 and TRD 153 +/- 9 mg/dl). Neither high caloric diet nor RT modified NOx- levels (SD: 27 +/- 4; TR: 28 +/- 6; SDD: 27 +/- 3 and TRD: 30 +/- 2 microM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels.

CONCLUSIONS

The improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation.

Normalization of data, by choosing the appropriate reference genes (RGs), is fundamental for obtaining reliable results in reverse transcription-quantitative PCR (RT-qPCR). In this study, we assessed Actinidia deliciosa leaves inoculated with two doses of Pseudomonas syringae pv. actinidiae during a period of 1<em>3</em> days for the expression profile of nine candidate RGs. Their expression stability was calculated using four algorithms: geNorm, NormFinder, BestKeeper and the deltaCt method. Glyceraldehyde-<em>3</em>-phosphate dehydrogenase (GAPDH) and protein phosphatase 2A (PP2A) were the most stable genes, while β-tubulin and 7s-globulin were the less stable. Expression analysis of three target genes, chosen for RGs validation, encoding the reactive oxygen species scavenging enzymes ascorbate peroxidase (APX), superoxide dismutase (<em>SOD</em>) and catalase (CAT) indicated that a combination of stable RGs, such as GAPDH and PP2A, can lead to an accurate quantification of the expression levels of such target genes. The APX level varied during the experiment time course and according to the inoculum doses, whereas both <em>SOD</em> and CAT resulted down-regulated during the first four days, and up-regulated afterwards, irrespective of inoculum dose. These results can be useful for better elucidating the molecular interaction in the A. deliciosa/P. s. pv. actinidiae pathosystem and for RGs selection in bacteria-plant pathosystems.

Berberine (BBR) is a well-known anti-diabetic herbal medicine in Asia due to its beneficial effects on insulin sensitivity, glucose metabolism and glycolysis. Here, we identified the critical role of phosphatidylinositol <em>3</em>-kinase (PI<em>3</em>K)/Akt involved BBR cellular defense mechanisms and first revealed the novel effect of BBR on nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2)/heme oxygenase (HO)-1 induction in NSC<em>3</em>4 motor neuron-like cells. BBR (0.1-10 nM) led to increasing insulin receptor expression, Akt phosphorylation and enhanced oxidant-sensitive Nrf2/HO-1 induction, which were blocked by a PI<em>3</em>K inhibitor, LY294002. In H(2)O(2)-treated cells, BBR significantly attenuated ROS production and increased cell viability, antioxidant defense (GSH and <em>SOD</em>) and oxidant-sensitive proteins (HO-1 and Nrf2), which also were blocked by LY294002. Furthermore, BBR improved mitochondrial function by increasing mitochondrial membrane potential and decreasing the oxygen consumption rate. BBR-induced anti-apoptotic function was demonstrated by increasing anti-apoptotic protein Bcl-2 and survival of motor neuron protein (SMN) and by decreasing apoptotic proteins (cytochrome c, Bax and caspase). These results suggest that BBR, which is active at nanomolar concentration, is a potential neuroprotective agent via PI<em>3</em>K/Akt-dependent cytoprotective and antioxidant pathways.

Gastrodin (GAS), an active constituent of the Chinese herbal medicine Tianma, has anti-oxidant and anti-inflammation activities but its protective effect to the prevention of neurotoxicity induced by ischemic stroke is unclear. In the present study, middle cerebral artery occlusion (MCAO) was used to establish a mice ischemic stroke model. Infarct volume ratio and neurobehavioral score were evaluated, Nissl staining was performed and the expression of cleaved Caspase <em>3</em>, Bax and B cell lymphoma 2 (Bcl-2) were assessed at 24 h or 7 days after reperfusion. In addition, the total superoxide dismutase (<em>SOD</em>) activity and malondialdehyde (MDA) content, as well as the expression of Nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), <em>SOD</em>1, phospho-Akt and total Akt and TNF-α and IL-1β in the ischemic hemispheres were also observed at 6 h after reperfusion to assess oxidative stress and inflammatory changes after GAS treatment. It was found that GAS, especially at high dose (100 mg/kg) reduced tested neuronal injury and neurobehavioral deficient in MCAO mice. Enhanced expression of cleaved Caspase <em>3</em> and Bax and decreased expression of Bcl-2 by MCAO were also reversed by GAS. Moreover, GAS treatment decreased the MDA content and the expression of TNF-α and IL-1β, and increased amount of <em>SOD</em> activity and the expression of HO-1 and <em>SOD</em>1 in GAS-treated ischemic brain. Furthermore, GAS significantly increased Akt phosphorylation and Nrf2 expression. These results support the neuroprotective effects of GAS, and the activation of Akt/Nrf2 pathway may play a critical role in the pharmacological action of GAS.

BACKGROUND

Ginger rhizomes are used traditionally for management of different gastrointestinal disturbances. Several studies proved that the rhizome possesses diverse biological activities such as cytotoxic, antioxidant, and anti-inflammatory effects. Recently, interest in ginger for treatment of chronic inflammatory conditions has been renewed.

OBJECTIVE

The purpose of the present study is to evaluate the potential role of ginger extract [GE] in modulating the extent and severity of ulcerative colitis (UC), a chronically recurrent inflammatory bowel disease of unknown origin.

METHODS

Male Wistar rats received <em>3</em> different doses of GE, sulfasalazine, or vehicle for <em>3</em> consecutive days before induction of UC by intra-rectal acetic acid administration, and continued further for 7 days after the induction. The colonic mucosal injury was assessed by macroscopic scoring, and histological examination. Furthermore, the mucosal content of malondialdehyde (MDA), protein carbonyl (PCO), and reduced glutathione (GSH) with the catalase (CAT) and superoxide dismutase (<em>SOD</em>) activity, were appraised as parameters of the redox state. Acute inflammatory response was determined by measuring myeloperoxidase (MPO), tumor necrosis factor (TNF-alpha), and prostaglandin E2 (PGE2).

RESULTS

All parameters were altered in ulcerated rats, and improved in animals receiving GE, an effect that was comparable to that of the standard sulfasalazine, especially at the highest dose level. Colonic mucosal injury parallels with the histological and biochemical evaluations.

CONCLUSIONS

Results showed a valuable effect of ginger extract against acetic acid-induced ulcerative colitis possibly by its antioxidant and anti-inflammatory properties.

OBJECTIVE

To investigate the mechanisms underlying the protective effects of quercetin-rutinoside (rutin) and its aglycone quercetin against CCl(4)-induced liver damage in mice.

METHODS

BALB/cN mice were intraperitoneally administered rutin (10, 50, and 150 mg/kg) or quercetin (50 mg/kg) once daily for 5 consecutive days, followed by the intraperitoneal injection of CCl(4) in olive oil (2 mL/kg, 10% v/v). The animals were sacrificed 24 h later. Blood was collected for measuring the activities of ALT and AST, and the liver was excised for assessing Cu/Zn superoxide dismutase (SOD) activity, GSH and protein concentrations and also for immunoblotting. Portions of the livers were used for histology and immunohistochemistry.

RESULTS

Pretreatment with rutin and, to a lesser extent, with quercetin significantly reduced the activity of plasma transaminases and improved the histological signs of acute liver damage in CCl(4)-intoxicated mice. Quercetin prevented the decrease in Cu/Zn SOD activity in CCl(4)-intoxicated mice more potently than rutin. However, it was less effective in the suppression of nitrotyrosine formation. Quercetin and, to a lesser extent, rutin attenuated the inflammation in the liver by down-regulating the CCl(4)-induced activation of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α) and cyclooxygenase (COX-2). The expression of inducible nitric oxide synthase (iNOS) was more potently suppressed by rutin than by quercetin. Treatment with both flavonoids significantly increased NF-E2-related factor 2 (Nrf2) and heme oxygenase (HO-1) expression in injured livers, although quercetin was less effective than rutin at an equivalent dose. Quercetin more potently suppressed the expression of transforming growth factor-β1 (TGF-β1) than rutin.

CONCLUSIONS

Rutin exerts stronger protection against nitrosative stress and hepatocellular damage but has weaker antioxidant and anti-inflammatory activities and antifibrotic potential than quercetin, which may be attributed to the presence of a rutinoside moiety in position 3 of the C ring.

Salinity negatively affects plant growth and causes significant crop yield losses world-wide. Maize is an economically important cereal crop affected by high salinity. In this study, maize seedlings were subjected to 75 mM and 150 mM NaCl, to emulate high soil salinity. Roots, mature leaves (basal leaf-pair 1,2) and young leaves (distal leaf-pair <em>3</em>,4) were harvested after <em>3</em> weeks of sowing. Roots showed the highest reduction in biomass, followed by mature and young leaves in the salt-stressed plants. Concomitant with the pattern of growth reduction, roots accumulated the highest levels of Na(+) followed by mature and young leaves. High salinity induced oxidative stress in the roots and mature leaves, but to a lesser extent in younger leaves. The younger leaves showed increased electrolyte leakage (EL), malondialdehyde (MDA), and hydrogen peroxide (H2O2) concentrations only at 150 mM NaCl. Total antioxidant capacity (TAC) and polyphenol content increased with the increase in salinity levels in roots and mature leaves, but showed no changes in the young leaves. Under salinity stress, reduced ascorbate (ASC) and glutathione (GSH) content increased in roots, while total tocopherol levels increased specifically in the shoot tissues. Similarly, redox changes estimated by the ratio of redox couples (ASC/total ascorbate and GSH/total glutathione) showed significant decreases in the roots. Activities of enzymatic antioxidants, catalase (CAT, EC 1.11.1.6) and dehydroascorbate reductase (DHAR, EC 1.8.5.1), increased in all organs of salt-treated plants, while superoxide dismutase (<em>SOD</em>, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione-s-transferase (GST, EC 2.5.1.18) and glutathione reductase (GR, EC 1.6.4.2) increased specifically in the roots. Overall, these results suggest that Na(+) is retained and detoxified mainly in roots, and less stress impact is observed in mature and younger leaves. This study also indicates a possible role of ROS in the systemic signaling from roots to leaves, allowing leaves to activate their defense mechanisms for better protection against salt stress.

The seedlings of wheat were grown in the presence of CdCl2 (500 or 1000 μM Cd), were applied with 500 μM of indole-<em>3</em>-acetic acid (IAA) or 500 μM salicylic acid (SA) as seed soaking and were sampled at 56 days after sowing. The plants exposed to Cd exhibited a substantial decline in growth, pigment content, relative water content (RWC) activities of superoxide dismutase (<em>SOD</em>), catalase (CAT) and peroxidase (POX) and leaf structure. However, pretreatment with IAA or SA mitigated the stress generated by Cd and markedly improved the aforesaid parameters. The Cd increased proline content, electrolyte leakage and plant Cd content. However, the IAA or SA treatment attenuated the adverse effects of Cd on these attributes. The results showed that pretreatment with IAA or SA enhanced the antioxidant defense activities in Cd stressed wheat, thus alleviating Cd induced oxidative damage and enhancing Cd tolerance and leaf anatomy.

<em>3</em>-Hydroxyanthranilic acid (<em>3</em>-HAA), a metabolite of L-tryptophan, accumulates in monocyte-derived cells (THP-1), but not in other cell lines tested (MRC-9, H4, U<em>3</em>7<em>3</em>MG, Wil-NS), following immune stimulation that induces indoleamine-2,<em>3</em>-dioxygenase (IDO), a rate-limiting enzyme in the L-tryptophan kynurenine pathway. We examined whether metabolites of the L-tryptophan-kynurenine pathway act to induce apoptosis in monocytes/macrophages. Of the L-tryptophan metabolites tested, only <em>3</em>-HAA at a concentration of 200 micromol/L was found to induce apoptosis in THP-1 and U9<em>3</em>7 cells. The addition of ferrous or manganese ions further enhanced apoptosis and free radical formation by <em>3</em>-HAA in these two types of cells. The apoptotic response induced by <em>3</em>-HAA was significantly attenuated by the addition of antioxidant, alpha-tocopherol or Trolox (a water-soluble analogue of vitamin E), and the xanthine oxidase inhibitor, allopurinol. In addition, the <em>3</em>-HAA-induced apoptotic response was slightly attenuated by catalase, but not by superoxide dismutase (<em>SOD</em>), indicating that generation of hydrogen peroxide is involved in this response. Interferon-gamma (IFN-gamma), an inducer of IDO, potently induced apoptosis in THP-1 cells, but not in U9<em>3</em>7 cells, in the presence of ferrous or manganese ions. This different susceptibility to apoptosis inducer between THP-1 and U9<em>3</em>7 cells may depend on the capacity of the cells for <em>3</em>-HAA synthesis following IDO induction by IFN-gamma. Furthermore, apoptosis was suppressed by cycloheximide in THP-1 cells, suggesting that newly synthesized proteins may be essential for apoptotic events. These results suggest that <em>3</em>-HAA induces apoptosis in monocytes/macrophages under inflammatory or other pathophysiological conditions.

Spilanthes acmella Murr. (Compositae) has been used as a traditional medicine for toothache, rheumatism and fever. Its extracts had been shown to exhibit vasorelaxant and antioxidant activities. Herein, its antimicrobial, antioxidant and cytotoxic activities were evaluated. Agar dilution method assays against 27 strains of microorganisms were performed. Results showed that fractions from the chloroform and methanol extracts inhibited the growth of many tested organisms, e.g. Corynebacterium diphtheriae NCTC 10<em>3</em>56 with minimum inhibitory concentration (MIC) of 64-256 mg/mL and Bacillus subtilis ATCC 66<em>3</em><em>3</em> with MIC of 128-256 mg/mL. The tested fractions all exhibited antioxidant properties in both DPPH and <em>SOD</em> assays. Potent radical scavenging activity was observed in the DPPH assay. No cytotoxic effects of the extracts against KB and HuCCA-1 cell lines were evident. Bioassay-guided isolation resulted in a diverse group of bioactive compounds such as phenolics [vanillic acid (2), trans-ferulic acid (5) and trans-isoferulic acid (6)], coumarin (scopoletin, 4) and triterpenoids like <em>3</em>-acetylaleuritolic acid (1), b-sitostenone (<em>3</em>), stigmasterol and stigmasteryl-<em>3</em>-O-b-D-glucopyranosides, in addition to a mixture of stigmasteryl-and b-sitosteryl-<em>3</em>-O-b-D-glucopyranosides. The compounds 1-6 represent bioactive metabolites of S. acmella Murr. that were never previously reported. Our findings demonstrate for the first time the potential benefits of this medicinal plant as a rich source of high therapeutic value compounds for medicines, cosmetics, supplements and as a health food.

The expression or activity of manganese superoxide dismutase (Mn-<em>SOD</em>) is reduced in a variety of malignant tumors and Mn-<em>SOD</em> may act as a new type of tumor suppressor gene. On the other hand, increased expression of Mn-<em>SOD</em> can diminish the cytotoxic effects of several anticancer modalities, including tumor necrosis factor alpha, ionizing radiation, certain chemotherapeutic agents and hyperthermia. Although Mn-<em>SOD</em> expression and its role in various cancers are intensely studied, little is known about its function in gastrointestinal carcinomas. To examine the expression level and significance of Mn-<em>SOD</em> in gastrointestinal carcinomas, Mn-<em>SOD</em> mRNA expression was examined in 5<em>3</em> gastric carcinoma and <em>3</em>8 colorectal carcinoma by reverse transcription-polymerase chain reaction and was compared with those in the corresponding normal mucosal tissues. The tumor/normal (T/N) ratio was calculated and the data were clinicopathologically analyzed. The average T/N ratios of Mn-<em>SOD</em> mRNA expression in gastric and colorectal carcinomas were 2.19 and <em>3</em>. 72, respectively. Clinicopathologic analyses revealed positive correlation between the Mn-<em>SOD</em> expression level and venous invasion in both gastric and colorectal carcinomas (p<0.05 and p<0.05, respectively). Furthermore, the colorectal carcinoma with lymph node metastasis showed significantly higher Mn-<em>SOD</em> expression than those without it (p<0.05). Our results suggest that Mn-<em>SOD</em> mRNA overexpression can occur in gastric and colorectal carcinomas and may be related to increased aggressiveness.