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Liver fat represents a balance between input, secretion, and oxidation of fatty acids. As humans spend the majority of a 24-h period in a postprandial state, dietary fatty acids make an important contribution to liver fat metabolism. We compared hepatic fatty acid partitioning in healthy lean (n = 9) and abdominally obese (n = 10) males over 24 h. Volunteers received three mixed meals adjusted for basal metabolic rate. U-[sup.13]C-labeled fatty acids were incorporated into the meals, and [[sup.2][H.sub.2]]palmitate was infused intravenously to distinguish between sources of fatty acids incorporated into VLDL-TG. Immunoaffinity chromatography was used to isolate VLDL-TG of hepatic origin. Liver and whole body fatty acid oxidation was assessed by isotopic enrichment of 3-hydoxybutyrate and breath C[O.sub.2]. We found a similar contribution of dietary fatty acids to VLDL-TG in the two groups over 24 h The contribution of fatty acids from splanchnic sources was higher (P < 0.05) in the abdominally obese group. Ketogenesis occurred to a significantly greater extent in abdominally obese compared with lean males, largely due to lessened downregulation of postprandial ketogenesis (P < 0.001). The appearance of [sup.13]C in breath C[O.sub.2] was also greater (P < 0.001) in abdominally obese compared with lean men. Hepatic elongation and desaturation of palmitic acid were higher (P < 0.05) in abdominally obese than in lean males. Oxidation of dietary fatty acids and hepatic desaturation and elongation of palmitic acid occurred to a greater extent in abdominally obese men. These alterations may represent further pathways for redirection of fatty acids into export from the liver or oxidation to prevent liver fat accumulation. very low-density lipoprotein; stable isotopes; postprandial metabolism; oxidation; abdominal obesity; desaturation doi: 10.1152/ajpendo.00272.2010.

Aging presumably initiates shifts in substrate oxidation mediated in part by changes in insulin sensitivity. Similar shifts occur with cardiac hypertrophy and may contribute to contractile dysfunction. We tested the hypothesis that aging modifies substrate utilization and alters insulin sensitivity in mouse heart when provided multiple substrates. In vivo cardiac function was measured with microtipped pressure transducers in the left ventricle from control (4-6 mo) and aged (22-24 mo) mice. Cardiac function was also measured in isolated working hearts along with substrate and anaplerotic fractional contributions to the citric acid cycle (CAC) by using perfusate containing [sup.13]C-labeled free fatty acids (FFA), acetoacetate, lactate, and unlabeled glucose. Stroke volume and cardiac output were diminished in aged mice in vivo, but pressure development was preserved. Systolic and diastolic functions were maintained in aged isolated hearts. Insulin prompted an increase in systolic function in aged hearts, resulting in an increase in cardiac efficiency. FFA and ketone flux were present but were markedly impaired in aged hearts. These changes in myocardial substrate utilization corresponded to alterations in circulating lipids, thyroid hormone, and reductions in protein expression for peroxisome proliferator-activated receptor (PPAR)[alpha] and pyruvate dehydrogenase kinase (PDK)4. Insulin further suppressed FFA oxidation in the aged. Insulin stimulation of anaplerosis in control hearts was absent in the aged. The aged heart shows metabolic plasticity by accessing multiple substrates to maintain function. However, fatty acid oxidation capacity is limited. Impaired insulin-stimulated anaplerosis may contribute to elevated cardiac efficiency, but may also limit response to acute stress through depletion of CAC intermediates. diabetes; heart; efficiency; thyroid hormone; substrate oxidation doi: 10.1152/ajpheart.00931.2009.

In anaerobic enrichment cultures for phototrophic nitrite-oxidizing bacteria from different freshwater sites, two different cell types, i.e. non-motile cocci and motile, rod-shaped bacteria, always outnumbered all other bacteria. Most-probable-number (MPN) dilution series with samples from two freshwater sites yielded only low numbers ([less than or equal to] 3x[10.sup.3] [cm.sup.-3]) of phototrophic nitrite oxidizers. Slightly higher numbers (about [10.sup.4] [cm.sup.-3]) were found in activated sewage sludge. Anaerobic phototrophic oxidation of nitrite was studied with two different isolates, the phototrophic sulfur bacterium strain KS1 and the purple nonsulfur bacterium strain LQ17, both of which were isolated from activated sludge collected from the municipal sewage treatment plant in Konstanz, Germany. Strain KS1 converted 1 mM nitrite stoichiometrically to nitrate with concomitant formation of cell matter within 2-3 days, whereas strain LQ17 oxidized only up to 60 % of the given nitrite to nitrate within several months with the concomitant formation of cell biomass. Nitrite oxidation to nitrate was strictly light-dependent and required the presence of molybdenum in the medium. Nitrite was oxidized in both the presence and absence of oxygen. Nitrite inhibited growth at concentrations higher than 2 mM. Hydroxylamine and hydrazine were found to be toxic to the phototrophs in the range 5-50 mM and did not stimulate phototrophic growth. Based on morphology, substrate-utilization pattern, in vivo absorption spectra, and 16S rRNA gene sequence similarity, strain KS1 was assigned to the genus Thiocapsa and strain LQ17 to the genus Rhodopseudomonas. Also, Thiocapsa roseopersicina strains DSM 217 and DSM 221 were found to oxidize nitrite to nitrate with concomitant growth. We conclude that the ability to use nitrite phototrophically as electron donor is widespread in nature, but low MPN counts indicate that its contribution to nitrite oxidation in the studied habitats is rather limited. DOI 10.1099/rnic.0.036004-0

Carbon 13 nuclear magnetic resonance (NMR) isotopomer analysis was used to measure the rates of oxidation of long-chain fatty acids, ketones, and pyruvate to determine the minimum pyruvate concentration ([pyruvate]) needed to suppress oxidation of these alternative substrates. Substrate mixtures were chosen to represent either the fed or fasted state. At physiological [pyruvate], fatty acids and ketones supplied the overwhelming majority of acetyl-CoA. Under conditions mimicking the fed state, 3 mM pyruvate provided ~80% of acetylCoA, but under fasting conditions 6 mM pyruvate contributed only 33% of acetyl-CoA. Higher [pyruvate], 10-25 mM, was associated with transient reduced cardiac output, but overall hemodynamic performance was unchanged after equilibration. These observations suggested that 3-6 mM pyruvate in the coronary arteries would be an appropriate target for studies with hyperpolarized [1-[sup.13]C]pyruvate. However, the metabolic products of 3 mM hyperpolarized [1-[sup.13]C]pyruvate could not be detected in the isolated heart during perfusion with a physiological mixture of substrates including 3% albumin. In the presence of albumin even at high concentrations of pyruvate, 20 mM, hyperpolarized [H.sup.13]C[O.sup.-.sub.3] could be detected only in the absence of competing substrates. Highly purified albumin (but not albumin from plasma) substantially reduced the longitudinal relaxation time of [1-[sup.13]C]pyruvate. In conclusion, studies of cardiac metabolism using hyperpolarized [1-[sup.13]C]pyruvate are sensitive to the effects of competing substrates on pyruvate oxidation. myocardium; pyruvate; dynamic nuclear polarization; substrate oxidation; isolated rat heart; carbon-13; nuclear magnetic resonance doi: 10.1152/ajpheart.00656.2009.

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis., With the ubiquitous use of fertilizers in agriculture, nitrate (N[O.sub.3.sup.-]) and nitrite (N[O.sub.2.sup.-]) have become major electron acceptors in freshwater environments (1). The feedback of eutrophication on the atmospheric methane [...]

d-Limonene kept in a refrigerator for 6 years (old limonene) showed unusually high anti-Candida activity as compared with that of fresh d-limonene. Fresh d-limonene exposed to solar radiation showed potent antifungal activity in parallel to the exposure time. Compositional analysis using GC/MS revealed that a similar composition of the oxidised products between old limonene and fresh limonene exposed to sun light. Fresh d-limonene kept in the vapour state in the dark for 10 days showed extensive oxidation and potent anti-Candida activity.These results suggested that care must be taken on the degree of oxidation before measurement of antimicrobial activity of d-limonene and essential oils containing d-limonene. Key words: d-limonene, anti-Candida activity, air-oxidation, filament formation

Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Ru61a and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the [alpha]/[beta]-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1-H-3-hydroxy-4-oxoquinaldine substrate, its N-acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical [alpha]/[beta]-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The 'oxyanion hole' of the [alpha]/[beta]-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions. oxygenase | oxygen chemistry | structural enzymology www.pn as.org/cgi/doi/10.1073/pnas.0909033107

Sorensen LP, Gormsen LC, Nielsen S. VLDL-TG kinetics: a dual isotope study for quantifying VLDL-TG pool size, production rates, and fractional oxidation in humans. Am J Physiol Endocrinol Metab 297: E1324-E1330, 2009. First published September 22, 2009; doi: 10. 1152/ajpendo.00366.2009.--Very-low-density lipoproteins (VLDLs) are large, complex particles containing both surface proteins (e.g., ApoB100) and core lipids, e.g., cholesterol and triglycerides (TG). Whereas ApoB100 kinetics have been thoroughly studied, accurate measurement of VLDL-TG kinetics have proven difficult due to either complex mathematics or laborious procedures. The present study was therefore designed to measure VLDL-TG kinetics by dual isotope ex vivo labeled VLDL-TG tracers and well-established kinetics equations (bolus injection or the primed continuous infusion). Ten healthy Caucasian men [age, 23 [+ or -] 3 yr old (mean [+ or -] SD); body mass index, 24.7 [+ or -] 1.3 kg/[m.sup.2]] were included in the study. VLDL-TG rate of appearance (Ra) was measured using a dual-tracer technique ([9,[10.sup.-3]H]-labeled VLDL-TG and [1-[sup.14]C]- labeled VLDL-TG) to allow comparison of various bolus decay curve fits with the Ra obtained by the primed continuous infusion (PCI; considered the gold standard). In addition, VLDL-TG fatty acid oxidation was measured as [sup.14]C[O.sub.2] in exhaled breath, using the hyamine trapping technique. Following a bolus injection, tracer decay was better described by a biexponential than a monoexponential fit ([r.sup.2] = 0.99 [+ or -] 0.01 vs. 0.97 [+ or -] 0.04, respectively, P = 0.01). VLDL-TG Ra calculated using the PCI correlated significantly with the biexponential fit ([rho] = 0.62, P < 0.05), whereas this was not the case for the monoexponential fit ([rho] = -0.18, P = not significant). VLDL-TG Ra using the best fit of the bolus injection method (biexponential) was less than values obtained by the constant infusion technique [biexponential, 34.3 (range, 27.1-69.6) vs. PCI, 44.4 (range, 33.0-72.7), P < 0.05]. Fractional oxidation of VLDL-TG was 37.2 [+ or -] 8.8% at 240 min corresponding to 198.8 [+ or -] 55.9 kcal/day or 10.6 [+ or -] 3.3% of resting energy expenditure (REE). Our data demonstrate that VLDL-TG Ra measured by a biexponential fit to a bolus decay curve correlates well with VLDL-TG Ra measured by a primed continuous infusion, and therefore that a 'second' peripheral VLDL-TG compartment with rapid exchange of TG exists. VLDL-TG volume of distribution is therefore greater than previously anticipated. Finally our data supports that VLDL-TG contributes quantitatively to REE. lipoproteins; tracer kinetics; methods; margination doi: 10.1152/ajpendo.00366.2009

In enteric bacteria, the cellular response to oxidative stress is activated by oxidation of the iron-sulfur clusters in SoxR, which then induces transcription of soxS, turning on a battery of defense genes. Here we demonstrate both in vitro and in cells that activation of SoxR can occur in a DNA-mediated reaction with guanine radicals, an early genomic signal of oxidative stress, serving as the oxidant. SoxR in its reduced form is found to inhibit guanine damage by repairing guanine radicals. Moreover, cells treated with a DNA-binding photooxidant, which generates guanine radicals, promotes the expression of soxS. In vitro, this photooxidant, tethered to DNA 80 bp from the soxS promoter, induces transcription by activating SoxR upon irradiation. Thus, transcription can be activated from a distance through DNA-mediated charge transport. This chemistry offers a general strategy for DNA-mediated signaling of oxidative stress. DNA charge transport | guanine radicals | oxidative stress

Type 1 diabetes is associated with a unique form of cardiomyopathy that is present without atherosclerosis. Redox imbalance and/or changes in vascular endothelial growth factor (VEGF) expression have been associated with diabetes-related cardiomyopathy. However, the mechanisms of these changes and their interrelationships remain unclear. Using a murine type 1 diabetes model, we tested the hypothesis that alterations in cardiac performance are associated with decreased cardiac microvascular prevalence, as well as downregulation of VEGF isoforms. We also investigated oxidative stress as a contributor to regulate individual VEGF isoforms and microvascular rarefaction. Significant and rapid hyperglycemia was observed at 1 wk post-streptozotocin (STZ) and persisted throughout the 5-wk study. Left ventricular (LV) fractional shortening was reduced at week 1 and 5 post-STZ insult relative to age-matched controls. We also observed the early reduction in E/A ratio at 1 wk. Immunostaining for CD31 and digital image analysis demonstrated a 35% reduction in microvessels/myocardial area, indicative of rarefaction, which was highly correlated with fractional shortening. Furthermore, a significant increase in the prevalence of protein 3-nitrotyrosine was observed in the diabetic cardiac tissue, which was inversely associated with microvascular rarefaction. The expressions of three VEGF isoforms were significantly reduced to different extents. The reduction of VEGF164 was associated with GSSG accumulation. These data demonstrate that the mouse model of STZ-induced diabetes has hallmark features observed in humans with respect to nonischemic systolic and diastolic performance and microvascular rarefaction, which are associated with changes in VEGF isoform expression and redox imbalance in the myocardium. microvascular density; vascular endothelial growth factor; cardiomyopathy; oxidants

To sustain growth, the catabolic formation of the redox equivalent NADPH must be balanced with the anabolic demand. The mechanisms that ensure such network-wide balancing, however, are presently not understood. Based on [sup.13]C-detected intracellular fluxes, metabolite concentrations, and cofactor specificities for all relevant central metabolic enzymes, we have quantified catabolic NADPH production in Agrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, Paracoccus versutus, Pseudomonas fluorescens, Rhodobacter sphaeroides, Sinorhizobium meliloti, and Zymomonas mobilis. For six species, the estimated NADPH production from glucose catabolism exceeded the requirements for biomass synthesis. Exceptions were P. fluorescens, with balanced rates, and E. coli, with insufficient catabolic production, in which about one-third of the NADPH is supplied via the membrane-bound transhydrogenase PntAB. P. versutus and B. subtilis were the only species that appear to rely on transhydrogenases for balancing NADPH overproduction during growth on glucose. In the other four species, the main but not exclusive redox-balancing mechanism appears to be the dual cofactor specificities of several catabolic enzymes and/or the existence of isoenzymes with distinct cofactor specificities, in particular glucose 6-phosphate dehydrogenase. An unexpected key finding for all species, except E. coli and B. subtilis, was the lack of cofactor specificity in the oxidative pentose phosphate pathway, which contrasts with the textbook view of the pentose phosphate pathway dehydrogenases as being [NADP.sup.+] dependent.

The twin-arginine translocase (Tat) system is used by many bacteria to move proteins across the cytoplasmic membrane. Tat substrates are prefolded and contain a conserved SRRxFLK twin-arginine (RR) motif at their N termini. Many Tat substrates in Escherichia coli are cofactor-containing redox enzymes that have specific chaperones called redox enzyme maturation proteins (REMPs). Here we characterized the interactions between 10 REMPs and 15 RR peptides of known and predicted Tat-specific redox enzyme subunits. A combination of in vitro and in vivo experiments demonstrated that some REMPs were specific to a redox enzyme(s) of similar function, whereas others were less specific and bound peptides of unrelated enzymes. Results from Biacore surface plasmon resonance (SPR) and bacterial two-hybrid experiments identified interactions in addition to those found in far-Western experiments, suggesting that conformational freedom and/or other cellular factors may be required. Furthermore, we show that the interaction of the two prevents both from being proteolytically degraded in vivo, and kinetic data from SPR show up to 10-fold-tighter binding to the expected RR substrate when multiple binding partners existed. Investigations using full-length sequences of the RR proteins showed that the mature portion for some redox enzyme subunits is required for detection of the interactions. Sequence alignments among the REMPs and RR peptides indicated that homology between the REMPs and the hydrophobic regions following the RR motifs in the peptides correlates to cross-recognition.

Typical 2-Cys peroxiredoxins (Prxs) are ubiquitous peroxidases that are involved in peroxide scavenging and/or the regulation of peroxide signaling in eukaryotes. Despite their prevalence, very few Prxs have been reliably characterized in terms of their substrate specificity profile and redox potential even though these values are important for gaining insight into physiological function. Here, we present such studies focusing on Salmonella typhimurium alkyl hydroperoxide reductase C component (StAhpC), an enzyme that has proven to be an excellent prototype of this largest and most widespread class of Prxs that includes mammalian Prx I-Prx IV. The catalytic efficiencies of StAhpC ([k.sub.cat]/[K.sub.m]) are >[10.sup.7] [M.sup.-1] x [s.sup.-1] for inorganic and primary hydroperoxide substrates and [approximately equal to] 100-fold less for tertiary hydroperoxides, with the difference being exclusively caused by changes in [K.sub.m]. The oxidative inactivation of AhpC through reaction with a second molecule of peroxide shows parallel substrate specificity. The midpoint reduction potential of StAhpC is determined to be -178 [+ or -] 0.4 mV, a value much higher than most other thiol-based redox proteins. The relevance of these results for our understanding of Prx and the physiological role of StAhpC is discussed. antioxidants | peroxidases | redox regulation | redox-active disulfide

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on [C.sub.2]-[C.sub.9] alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of [beta]-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of [O.sub.2] when lactate-grown, BMOrepressed cells were washed free of lactate and incubated in N[H.sub.4]CI-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low [O.sub.2] (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O.sub.2], CuS[O.sub.4] (0.5 [micro]M) repressed 1-butanol-dependent induction of [beta]-galactosidase activity. Under oxic conditions (20% [O.sub.2] [vol/vol]), significantly higher concentrations of CuS[O.sub.4] (2 [micro]M) were required for almost complete repression of induction in lactate-grown cells. A combination of the [Cu.sup.2+] reducing agent Na ascorbate (100 [micro]M) and CuS[O.sub.4] (0.5 [micro]M) repressed the induction of [beta]-galactosidase activity under oxic conditions to the same extent that 0.5 [micro]M CuS[O.sub.4] alone repressed it under anoxic conditions. Under oxic conditions, 2 [micro]M CuS[O.sub.4] repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuS[O.sub.4] repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

The opportunistic pathogen Pseudomonas aeruginosa produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with P. aeruginosa infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by P. aeruginosa PAl4 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NA[D.sup.+] in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in P. aeruginosa PAl4, indicating that pyocyanin may also influence the intraceilular redox state by decreasing carbon flux through central metabolic pathways.

Yeast cytokinesis entails a rejuvenation process by which the aged mother cell generates daughter cells enjoying full replicative potential. Here we show that this process includes a precipitous reduction in the levels of reactive oxygen species in the progeny immediately after completion of cytokinesis. The reduction in hydrogen peroxide is the result of a Sir2p and actin cytoskeleton-dependent segregation of the cytosolic catalase Ctt1p such that the daughter cell receives a higher load of undamaged and active Ctt1p than the progenitor cell. Such spatial quality control provides the daughter cells with a superior capacity to combat external oxidative stress and delays self-inflicted oxidative damage to their cellular proteins. aging | catalase | damage segregation | rejuvenation

Ischemic preconditioning (IPC) induces distinctive changes in mitochondrial bioenergetics during warm (37[degrees]C) ischemia and improves function and tissue viability on reperfusion. We examined whether IPC before 2 h of hypothermic (27[degrees]C) ischemia affords additive cardioprotection and improves mitochondrial redox balance assessed by mitochondrial NADH and ftavin adenine dinucleotide (FAD) autofluorescence in intact hearts. A mediating role of ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel opening was investigated. NADH and FAD fluorescence was measured in the left ventricular wall of guinea pig isolated hearts assigned to five groups of eight animals each: hypothermia alone, hypothermia with ischemia, IPC with cold ischemia, 5-hydroxydecanoic acid (5-HD) alone, and 5-HD with IPC and cold ischemia. IPC consisted of two 5-min periods of warm global ischemia spaced 5 min apart and 15 min of reperfusion before 2 h of ischemia at 27[degrees]C and 2 h of warm reperfusion. The [K.sub.ATP] channel inhibitor 5-HD was perfused from 5 min before until 5 min after IPC. IPC before 2 h of ischemia at 27[degrees]C led to better recovery of function and less tissue damage on reperfusion than did 27[degrees]C ischemia alone. These improvements were preceded by attenuated increases in NADH and decreases in FAD during cold ischemia and the reverse changes during warm reperfusion. 5-HD blocked each of these changes induced by IPC. This study indicates that IPC induces additive cardioprotection with mild hypothermic ischemia by improving mitochondrial bioenergetics during and after ischemia. Because effects of IPC on subsequent changes in NADH and FAD were inhibited by 5-HD, this suggests that mitochondrial [K.sub.ATP] channel opening plays a substantial role in improving mitochondrial bioenergetics throughout mild hypothermic ischemia and reperfusion. nicotinamide adenine dinucleotide; flavin adenine dinucleotide; ATP-sensitive potassium channels; 5-hydroxydecanoic acid

Phosphorylation of eukaryotic translation initiation factor 2[alpha] (elF2[alpha]) on serine 51 is effected by specific stress-activated protein kinases. elF2[alpha] phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting elF2[alpha] dephosphorylation, however, GADD34 mutant cells retain significant elF2[alpha]-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates elF2[alpha]. RNAi of this gene, which we named constitutive repressor of elF2[alpha] phosphorylation (CReP, or PPP1R15B), repressed the constitutive elF2[alpha] directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of elF2[alpha] dephosphorylation by targeting the CReP-protein-phosphatase-1 complex may be used to access the salubrious qualities of the ISR.

Byline: T. B. Shvets-Teneta-Gurii (1), G. I. Troshin (1), M. R. Novikova (1), N. M. Khonicheva (1), O. A. Shostak (1), I. V. Borovskaya (1) Keywords: rat; cerebral cortex; sleep-waking cycle; oxidative-reductive state Abstract: Freely mobile mongrel male rats weighing 300--350 g were used for studies of changes in the oxidative-reductive (redox) state of brain tissue during cycles of waking, slow-wave sleep, and paradoxical sleep, by recording the potential of the oxidative-reductive state of brain tissue with platinum electrodes implanted into the cerebral cortex ad hippocampus. Electromyograms were also recorded from the cervical muscles, and overall movement activity was also recorded. A common platinum reference electrode was implanted into the nasal bones. These experiments showed that in rats, episodes of waking and paradoxical sleep occurred on the background of increases in the oxidation-reduction potential state of brain tissue at a series of brain points, which we termed 'metabolically active.' Transitions from waking and paradoxical sleep to slow-wave sleep were accompanied by decreases in the potential of the redox state. The magnitude of changes in the tissue redox state varied up to 100 mV. It is suggested that transitions from waking and paradoxical sleep to slow-wave sleep are accompanied by dynamic changes in the balance of brain tissue energy metabolism between the main energy sources. Oxidative phosphorylation dominates in waking and paradoxical sleep, while aerobic glycolysis dominates slow-wave sleep. We suggest that this latter should be interpreted as a decrease in the potential of the tissue redox state and the formation within the tissue of oscillations during slow-wave sleep. Formation of oscillations is typical for acceleration of glycolytic processes. Recently published data suggest that the major compartment or aerobic glycolysis is the astroglia. Author Affiliation: (1) Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow Article History: Registration Date: 12/10/2004

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobaciteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of FurA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.