Reduction potential characterization of methanogen factor 390

Abstract During oxidative stress, Methanobacterium thermoautotrophicum derivatizes the two-electron carrier, coenzyme F₄₂₀, to form factor 390 (F₃₉₀). Two methods were used to estimate the reduction potential of this chromophore. Oxidative titration of reduced F₃₉₀ by potassium ferricyanide in the presence of either NADH or a redox indicator dye yielded an estimate of −320 mV for the reduction potential. A sulfite dissociation constant of 11 mM was measured which correlates to a reduction potential of −310 mV when compared to other 5-deazaflavins and nicotinamides. Thus, the F₃₉₀ reduction potential is within a useful working range for the microorganism.     

F4 - Isoprostanes as Specific Marker of Docosahexaenoic Acid Peroxidation in Alzheimer's Disease

Abstract : F₂-isoprostanes are prostaglandin-like compounds derived from free radical-catalysed peroxidation of arachidonic acid. Peroxidation of eicosapentaenoic acid produces F₃-isoprostanes, whereas peroxidation of docosahexaenoic acid would give F₄-isoprostanes. This study demonstrates the presence of esterified F₄-isoprostanes in human brain and shows that levels are elevated in certain brain cortex regions in Alzheimer's disease. Our data with Alzheimer's disease suggest that analysis of F₄-isoprostanes will provide new opportunities to study lipid peroxidation in the neurodegenerative diseases.     

Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings

Vigna cutjang Endl. cv. Pusa Barsati seedlings, subjected to increasing degrees of water stress (−0.5, −1.0, −1,5 MPa), produced an approximately proportional increase in glycolate oxidase activity, hydrogen peroxide (H₂O₂) and proline content but a decrease in catalase activity, ascorbic acid and protein content. Leaf water potential (leaf ψ) and relative water content (RWC) were also lowered with increasing stress. Pretreatment with l -cysteine and reduced glutathione (10-3 M) decreased glycolate oxidase activity, H₂O₂ content, ascorbic acid oxidase activity, proline content and also slightly improved the water status of leaves stressed (−1.0 MPa) for 2 days. Pretreatment of non-stressed seedlings with these antioxidants had little or no effect. These studies indicate that treatment with antioxidants makes the plant tolerant against water stress by modulating the endogenous levels of H₂O₂ and ascorbic acid in stressed tissue.     

Behavioral characterization of mice lacking the neurite outgrowth inhibitor Nogo-A

The membrane protein Nogo-A inhibits neurite outgrowth and regeneration in the injured central nervous system, primarily because of its expression in oligodendrocytes. Hence, deletion of Nogo-A enhances regeneration following spinal cord injury. Yet, the effects of Nogo-A deletion on general behavior and cognition have not been explored. The possibility of potential novel functions of Nogo-A beyond growth inhibition is strongly suggested by the presence of subpopulations of neurons also expressing Nogo-A – not only during development but also in adulthood. We evaluated here Nogo-A ^−/− mice in a series of general basic behavioral assays as well as functional analyses related to brain regions with notable expression levels of Nogo-A. The SHIRPA protocol did not show any major basic behavioral changes in Nogo-A ^−/− mice. Anxiety-related behavior, pain sensitivity, startle reactivity, spatial learning, and associative learning also appeared indistinguishable between Nogo-A ^−/− and control Nogo-A^+/+ mice. However, motor co-ordination and balance were enhanced in Nogo-A ^−/− mice. Spontaneous locomotor activity was also elevated in Nogo-A ^−/− mice, but this was specifically observed in the dark (active) phase of the circadian cycle. Enhanced locomotor reaction to systemic amphetamine in Nogo-A ^−/− mice further pointed to an altered dopaminergic tone in these mice. The present study is the first behavioral characterization of mice lacking Nogo-A and provides significant insights into the potential behavioral relevance of Nogo-A in the modulation of dopaminergic and motor functions.     

Chloroplast ultrastructure in two Crassulacean species and an F1 hybrid with differing biomass δ13C values

Abstract. The chloroplasts of two species of the Crassulaceae and their F₁ hybrid were compared by electron microscopy. The two species had contrasting leaf tissue δ¹³C values of −25°/_∞ ( Sedum greggii ) and −13°/_∞ ( Cremnophila linguifolia ), and the F₁ hybrid had a value of − 18°/_∞. S. greggii had a mean of 8.9 thylakoids per granum in contrast to C. linguifolia which had a mean of only 3.8 thylakoids per granum. The F₁ hybrid had a mean of 6.4 thylakoids per granum. Crystaloids were observed in S. greggii and the hybrid but not in C. linguifolia     

Ascorbic acid influences the development and immunocompetence of larval Heliothis virescens

Ascorbic acid, known to be a free radical scavenger and vital to insect development, is important in larval resistance to baculovirus infection. We sequentially elevated the ascorbic acid content in an ascorbic acid-depleted diet and evaluated the effect on larval Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) development and immunocompetence. Dietary ascorbic acid levels lower than 0.7 g l⁻¹ slowed the growth rate of larvae, reduced pupal weights significantly, and severely inhibited adult emergence. Larvae developing on ascorbic acid-free diet experienced far higher levels of mortality following per os infection with virus. Additionally, viral infection in larvae fed an ascorbic acid-free diet, as monitored by epifluorescence microscopy, showed signs of infection much earlier than larvae fed control levels of ascorbic acid. These results demonstrate an indirect correlation between the level of ascorbic acid in the food stream of larval H. virescens and the susceptibility of the insect to baculoviral infection.     

Seizure-induced formation of isofurans: novel products of lipid peroxidation whose formation is positively modulated by oxygen tension

We have previously shown that seizures induce the formation of F₂-isoprostanes (F₂-IsoPs), one of the most reliable indices of oxidative stress in vivo . Isofurans (IsoFs) are novel products of lipid peroxidation whose formation is favored by high oxygen tensions. In contrast, high oxygen tensions suppress the formation of F₂-IsoPs. The present study determined seizure-induced formation of IsoFs and its relationship with cellular oxygen levels (pO₂). Status epilepticus (SE) resulted in F₂-IsoP and IsoF formation, with overlapping but distinct time courses in hippocampal subregions. IsoF, but not F₂-IsoP formation coincided with mitochondrial oxidative stress. SE resulted in a transient decrease in hippocampal pO₂ measured by in vivo electron paramagnetic resonance oximetry suggesting an early phase of seizure-induced hypoxia. Seizure-induced F₂-IsoP formation coincided with the peak hypoxia phase, whereas IsoF formation coincided with the 'reoxygenation' phase. These results demonstrate seizure-induced increase in IsoF formation and its correlation with changes in hippocampal pO₂ and mitochondrial dysfunction.     

Influence of some environmental variables on the ascorbic acid status of mullet, Mugil cephalus L., tissues. I. Effect of salinity, capture-stress, and temperature

The effects of capture stress, exposure to a hypo-osmotic environment and elevated water temperatures on the ascorbic acid (AsA) content of several mullet, Mugil cephalus , tissues were examined. All the treatments significantly altered tissue AsA levels, but the pattern of AsA fluctuations varied. Gill AsA concentrations increased two fold after exposure to a hypo-osmotic medium (salinity changed from 30‰ to 5‰), whereas AsA content in this tissue declined after capture. Both treatments depleted AsA reserves in the kidney. AsA concentrations in the brain increased after exposure to low salinity and elevated water temperatures, but were unaffected by capture stress. None of the treatments caused long term alteration of hepatic AsA reserves. Ascorbic acid inhibited oubain-sensitive Na⁺, K⁺-ATPase activity of gill tissue in vitro . The results suggest an involvement of AsA in osmo- or ion-regulatory functions of teleosts gills, salinity and thermal adaptation mechanisms in neural tissue, and the response of renal tissue to adverse environmental stimuli.     

Ascorbic acid as negative effector of the peroxidase-catalyzed degradation of indole-3-acetic acid

Ascorbic acid is a strong inhibitor of indole-3-acetic oxidation catalyzed by commercial horse-radish peroxidase. In the presence of excess ascorbic acid, the indole-acetic acid oxidation catalysis is apparently blocked. The activity of peroxidase for indoleacetic acid at pH 3.7 and 33°C, in the presence of 2,4-dichlorophenol and MnCl₂ as promotors was measured by polarographic technique. The K_m was 0.27 m M and the maximum velocity was 1.02 mmol O₂ (mg protein)⁻¹ min⁻¹. Dixon plots lead to an apparent K_i of 1.25 (μ M for ascorbic acid and the inhibition was apparently competitive. Ascorbic acid, besides appearing to be a strong inhibitor of the IAA oxidase activity of peroxidase, seemed to protect IAA from total degradation. Addition of more than 5 μ M ascorbic acid produced both an exponential increase in the lag time before the onset of reaction and, at the end, an oxidation protection of 26 μ M IAA when 111 μ M IAA was present at the stawrt. The possibility of ascorbic acid-IAA auxin from endogenous oxidation in plants, is proposed.     

Dural Mast Cells: Source of Contaminating Histamine in Analyses of Mouse Brain Histamine Levels

Abstract: Histamine levels were determined in mouse brains from WBB6F₁- +/+ (mast cell normal) and WBB6F₁- W/W^v (mast cell-deficient) mice whose brains were dissected immediately after decapitation or after freezing the severed heads in liquid nitrogen for 10 s. In WBB6F₁-+/+ mice, brains obtained from frozen heads contained significantly higher levels of histamine than those obtained from unfrozen heads. The converse was found in brains obtained from the WBB6F₁- W/W^v mice. When CF-1 mice (which also contain brain-associated mast cells) were treated as described above, results very similar to those found with the WBB6F₁- +/+ mice were obtained. Further, the high levels of histamine found in CF-1 mice whose brains had been frozen in situ were accompanied by an extensive degranulation of mast cells in the dura mater of these mice. Because of this degranulation of mast cells, and the fact that increased levels of brain histamine were not found in mast cell-deficient mice, it is concluded that dural mast cells are the likely source of the artifactually higher levels of histamine seen in brains frozen in situ.     

Manipulation of fatty acid amide hydrolase functional activity alters sensitivity and dependence to ethanol

The aim of this study was to examine the role of fatty acid amide hydrolase (FAAH) on ethanol sensitivity, preference, and dependence. The deletion of FAAH gene or the inhibition of FAAH by carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) (0.1 mg/kg) markedly increased the preference for ethanol. The study further reveals that URB597 specifically acts through FAAH and that cannabinoid-1 (CB₁) receptor is critical for N -arachidonoyl ethanolamide (AEA) mediated ethanol-reinforced behavior as revealed by lack of URB597 effect in both FAAH and CB₁−/− mice compared with vehicle-treated −/− mice. The FAAH −/− mice displayed a lower sensitivity to hypothermic and sedative effects to acute ethanol challenge. The FAAH −/− mice also exhibited a reduction in the severity of handling-induced convulsions following withdrawal from chronic ethanol exposure. The CB₁ receptor and proenkephalin gene expressions, and CB₁ receptor and μ-opioid (MO) receptor-mediated G-protein activation were found to be significantly lower in the caudate-putamen, nucleus accumbens core and shell of FAAH −/− than +/+ mice. Interestingly, the MO receptor-stimulated G-protein signaling was greater in the striatum of FAAH −/− than +/+ mice following voluntary ethanol consumption. These findings suggest that an elevation in the AEA content and its action on the limbic CB₁ receptor and MO receptor might contribute to ethanol-reinforced behavior. Treatment with drugs that decrease AEA tone might prove useful in reducing excessive ethanol consumption.     

Calcium taste preferences: genetic analysis and genome screen of C57BL/6J x PWK/PhJ hybrid mice

To characterize the genetic basis of voluntary calcium consumption, we tested C57BL/6J mice (B6; with low avidity for calcium), PWK/PhJ mice (PWK; with high avidity for calcium) and their F₁ and F₂ hybrids. All mice received a series of 96-h two-bottle preference tests with a choice between water and the following: 50 m m CaCl₂, 50 m m calcium lactate, 50 m m MgCl₂, 100 m m KCl, 100 m m NH₄Cl, 100 m m NaCl, 5 m m citric acid, 30 μ m quinine hydrochloride and 2 m m saccharin. Most frequency distributions of the parental and F₁ but not F₂ groups were normally distributed, and there were few sex differences. Reciprocal cross analysis showed that B6 × PWK F₁ mice had a non-specific elevation of fluid intake relative to PWK × B6 F₁ mice. In the F₂ mice, trait correlations were clustered among the divalent salts and the monovalent chlorides. A genome screen involving 116 markers showed 30 quantitative trait loci (QTLs), of which six involved consumption of calcium chloride or lactate. The results show pleiotropic controls of calcium and magnesium consumption that are distinct from those controlling consumption of monovalent chlorides or exemplars of the primary taste qualities.     

Age-Related Regional Changes in Hydroxyl Radical Stress and Antioxidants in Gerbil Brain

Abstract— The levels of hydroxyl radicals and oxidized GSH have been examined as indices of oxidative stress in young (3 months), middle-aged (15 months), and old (20–24 months) gerbil brain hippocampus, cortex, and striaturn. The hydroxyl radical stress was estimated by measuring the salicylate hydroxyl radical trapping products 2,5-and 2,3-dihydroxybenzoic acid. The stress was significantly higher in all three brain regions in middle-aged and old gerbils versus young animals (66.0%). Regional comparisons showed that the stress was significantly higher in cortex than in either the hippocampus or striatum of the middle-aged and old gerbils (32.0%). The ratio of oxidized to total GSH also increased progressively in middle-aged and old animals in all three brain regions (p < 0.05, 41.1%), further indicating a general age-related increase in oxidative stress. Parallel to this age-related increase in oxidative stress, a significant, albeit slight (8%), decrease in neuronal number in hippocampal CA1 region was observed in both the middle-aged and old animals. Possible differences in antioxidant levels were also examined. Total GSH levels were similar across age groups (variance <12%). However, the regional comparison showed that it was highest in striatum in all age groups. The levels of a-tocopherol (vitamin E) were significantly higher in the middle-aged and old animals in all three regions (70.4%). Vitamin E was highest in the hippocampus and the differences between the hippocampus and the cortex and striatum increased with age. Although of a lesser magnitude, significant increases in hippocampal total ascorbic acid level were also noted with age (p < 0.05, 10%). Ascorbic acid was the most regionally specific of the three antioxidants examined, with hippocampus > cortex > striatum for all age groups. The difference in ascorbic acid level between hippocampus and cortex also increased with age (64.4%). The results suggest that the general age-related, regionally specific increases in oxidative stress stimulate the accumulation of antioxidants. It is interesting that the hippocampus, which is selectively vulnerable to various insults such as ischemia, epilepsy, and insulin-induced hypoglycemia, exhibits the greatest age-related increase in vitamin E and ascorbic acid, perhaps reflective of a greater impact of the progressive increase in baseline oxidative stress.     

Exaggerated emotional behavior in mice heterozygous null for the sodium channel Scn8a (Nav1.6)

The Scn8a gene encodes the α-subunit of Na_v1.6, a neuronal voltage-gated sodium channel. Mice homozygous for mutations in the Scn8a gene exhibit motor impairments. Recently, we described a human family with a heterozygous protein truncation mutation in SCN8A . Rather than motor impairment, neuropsychological abnormalities were more common, suggesting a role for Scn8a in a more diverse range of behaviors. Here, we characterize mice heterozygous for a null mutation of Scn8a ( Scn8a^+/− mice) in a number of behavioral paradigms. We show that Scn8a^+/− mice exhibit greater conditioned freezing in the Pavlovian fear conditioning paradigm but no apparent abnormalities in other learning and memory paradigms including the Morris water maze and conditioned taste avoidance paradigm. Furthermore, we find that Scn8a^+/− mice exhibit more pronounced avoidance of well-lit, open environments as well as more stress-induced coping behavior. Together, these data suggest that Scn8a plays a critical role in emotional behavior in mice. Although the behavioral phenotype observed in the Scn8a^+/− mice only partially models the abnormalities in the human family, we anticipate that the Scn8a^+/− mice will serve as a valuable tool for understanding the biological basis of emotion and the human diseases in which abnormal emotional behavior is a primary component.     

Regulation of Serotonin Release in the Frontal Cortex and Ventral Hippocampus of Homozygous Mice Lacking 5-HT1B Receptors: In Vivo Microdialysis Studies

Abstract: To assess the involvement of the serotonin receptor subtype 5-HT_1B as terminal autoreceptor regulating 5-HT release in mice, we compared basal values and potassium-evoked changes of extracellular 5-HT levels obtained by in vivo microdialysis in two serotoninergic terminal projection areas of conscious wild-type mice with those measured in homozygous mutant mice lacking the gene encoding the 5-HT_1B receptor. In the frontal cortex and ventral hippocampus, basal and K⁺-evoked 5-HT release did not differ between the two strains of mice studied. The infusion via reverse microdialysis of the selective 5-HT_1B receptor agonist CP-93,129 (500 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −44%) and ventral hippocampus (by −32%) of wild-type mice but had no effect in mutants. In a similar manner, the mixed 5-HT_1B-5-HT_1D receptor agonist sumatriptan (800 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −46%) of wild-type mice but had no effect in mutants. These results demonstrated that 5-HT_1B knockout mice are not as sensitive to full (CP-93,129) and mixed (sumatriptan) 5-HT_1B receptor agonists as are wild-type mice. These data provide in vivo evidence that, in mice, 5-HT_1B, but not 5-HT_1D, autoreceptors inhibit 5-HT release at nerve terminals located in the frontal cortex and ventral hippocampus.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Aged endothelial nitric oxide synthase knockout mice exhibit higher mortality concomitant with impaired open-field habituation and alterations in forebrain neurotransmitter levels

Endothelial nitric oxide synthase (eNOS) has been implicated in various brain and peripheral pathologies such as renal failure, heart failure or stroke. Consequently, the mortality rate of aged eNOS knockout mice (eNOS^–/–) was higher than that of age-matched (18–22 months old) controls. Only seven of the original 14 eNOS^–/– animals that participated in the study reached the age of 18 months or older, whereas no control mice died during this life span. In order to assess the behavioral and neurochemical consequences of chronic eNOS deficiency we examined whether the surviving aged eNOS^–/– mice showed changes in terms of motor, emotional, exploratory and neurochemical parameters. Aged eNOS^–/– mice showed reduced exploratory activity in the open-field with no habituation observable neither within sessions nor after repeated exposures. Pole test performance of eNOS^–/– mice was comparable to controls. In the elevated plus-maze eNOS^–/– mice did not differ from controls in terms of time spent in and entries into arms, but showed less locomotion on the open arms. The most prominent neurochemical alterations in the forebrains of aged eNOS^–/– mice were: (a) increased acetylcholine levels in the neostriatum; (b) decreased noradrenaline concentrations in the ventral striatum; and (c) lower serotonin levels in the frontal cortex and ventral striatum. The present findings suggest that mice which survived chronic eNOS-deficiency into old age, show some behavioral and neurochemical phenotypes distinct from adult eNOS^–/– mice.     

OXIDATIVE STRESS AND ASCORBIC ACID CONTENTS INPARMOTREMA RETICULATUMANDPARMELIA SULCATATHALLI

Paraquat-treatment ofParmotrema reticulatumand high light levels in the habitat ofParmelia sulcatacan cause oxidative stress, which promotes an increase in ascorbic acid contents of thalli. Ascorbic acid was measured by an HPLC method. InP. reticulatumthe increase was more pronounced in summer than in other seasons. InP. sulcata, thalli growing in sun-exposed habitats showed a higher ascorbic acid content than shade-growing specimens.     

Rat Frontal Cortex β1-Adrenoceptors Are Activated by the β3-Adrenoceptor Agonists SR 58611A and SR 58878A but Not by BRL 37344 or ICI 215,001

Abstract: SR 58611A, a selective agonist of gut and brown adipose tissue β₃-adrenoceptors (β₃ARs), has been reported to have antidepressant-like activity in rodents by indicating brain β₃ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine- or (−)-isoproterenol-stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by β₁AR antagonist CGP 20712A but not by β₂AR antagonist ICI 118,551. In addition, PCR analysis did not reveal β₃AR mRNA, and no specific β₃AR binding sites were detected by [³H]CGP 12177 in rat frontal cortex. When down-regulation of the β₁AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (−)-isoproterenol increased the cyclic AMP production less markedly. Our findings indicate that β₃ARs are absent in the adult rat frontal cortex, and that various β₃AR agonists differently affect the frontal cortex β₁ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex β₁ARs.