Respiratory behaviour of a Zymomonas mobilis adhB::kan(r) mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions

Perturbation of the aerobic steady-state in a chemostat culture of the ethanol-producing bacterium Zymomonas mobilis with a small pulse of ethanol causes a burst of ethanol oxidation, although the reactant ratio of the alcohol dehydrogenase (ADH) reaction ([NADH][acetaldehyde][H(+)])/([ethanol][NAD(+)]) remains above the K(eq) value. Simultaneous catalysis of ethanol synthesis and oxidation by the two ADH isoenzymes, residing in different redox microenvironments, has been proposed previously. In the present study, this hypothesis is verified by construction of an ADH-deficient strain and by demonstration that it lacks the oxidative burst in response to perturbation of its aerobic steady-state with ethanol.     

Enrichment,isolation and characterization of fungi tolerant to 1‐ethyl‐3‐methylimidazolium acetate

Aims: This work aimed to characterize microbial tolerance to 1‐ethyl‐3‐methylimidazolium acetate ([C2mim][OAc]), an ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. Methods and Results: Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populations dominated by Aspergilli. Ionic liquid‐tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]‐tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. Conclusions: The results of the study suggest that tolerance to ionic liquids is a general property of the Aspergilli. Significance and Impact of the Study: Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.     

Parameters affecting the chemical work output of a hybrid photoelectrochemical biofuel cell.

A hybrid photoelectrochemical biofuel cell employing the photoanode architecture of a dye-sensitized solar cell has been assembled. A porphyrin dye sensitizes a TiO(2) semiconductor over the visible range to beyond 650 nm. Photoinduced charge separation at the dye-TiO(2) interface results in electron migration to a cathode, and the holes generated on surface bound dyes oxidize soluble electron mediators. The increased [Ox] : [Red] ratio of the mediator drives the solution-based enzymatic oxidation of appropriate substrates. In this report we investigate how the accumulation of anodic and cathodic products limits cell performance. The NAD(+)/NADH and benzoquinone/hydroquinone redox couples were studied as sacrificial electron donors in the absence of appropriate enzymes or substrates. Comparatively poor cell performance was observed using the benzoquinone/hydroquinone couple. This effect is explained in terms of rapid charge recombination by electron donation from the electrode to benzoquinone in solution, as compared to much less recombination with NAD(+). With the NAD(+)/NADH couple the cell performance is relatively independent of the redox poise of the anode solution, but limited by accumulation of reduction products in the cathodic compartment. Using the NAD(+)/NADH couple, the photochemical reforming of ethanol to hydrogen was demonstrated under conditions where the process would be endergonic in the dark.     

Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquids mixtures

Novozym 435-catalyzed synthesis of 6-O-lauroyl-d-glucose in ionic liquids (ILs) was investigated. The highest lipase activity was obtained in water-miscible [Bmim][TfO] which can dissolve high concentration of glucose, while the highest stability of lipase was shown in hydrophobic [Bmim][Tf(2)N]. The optimal activity and stability of lipase could be obtained in [Bmim][TfO] and [Bmim][Tf(2)N] mixture (1:1, v/v). Specifically, the activity of lipase was increased from 1.1 to 2.9 micromolmin(-1)g(-1) by using supersaturated glucose solution in this mixture, compared with reaction using saturated solution. After 5 times reuse of lipase, 86% of initial activity was remained in this mixture, while the residual activity in pure [Bmim][TfO] was 36%. Therefore, the productivity obtained by using ILs mixtures was higher than those in pure ILs.     

Thermophilic enrichment of microbial communities in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium acetate

Aims

The aim of the study was to develop an approach to enrich ionic liquid tolerant micro‐organisms that efficiently decompose lignocellulose in a thermophilic and high‐solids environment.

Methods and Results

High‐solids incubations were conducted, using compost as an inoculum source, to enrich for thermophilic communities that decompose switchgrass in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium acetate ([C2mim][OAc]). Ionic liquid levels were increased from 0 to 6% on a total weight basis incrementally. Successful enrichment of a community that decomposed lignocellulose at 55°C in the presence of 6% [C2mim][OAc] was achieved, when the [C2mim][OAc] level was increased stepwise from 2% to 4% to 5% to 6%. Pyrosequencing results revealed a shift in the community and a sharp decrease in richness, when thermophilic conditions were applied.

Conclusions

A community tolerant to a thermophilic, high‐solids environment containing 6% [C2mim][OAc] was enriched from compost. Gradually increasing [C2mim][OAc] concentrations allowed the community to adapt to [C2mim][OAc].

Significance and Impact of the Study

A successful approach to enrich communities that decompose lignocellulose under thermophilic high‐solids conditions in the presence of elevated levels of [C2mim][OAc] has been developed. Communities yielded from this approach will provide resources for the discovery of enzymes and metabolic pathways relevant to biomass pretreatment and fuel production.     

C3(OH)2mim][BF4]-Au/Pt biosensor for glutamate sensing in vivo integrated with on-line microdialysis system

A new type of hydroxyl functionalized room temperature ionic liquid (RTIL), [C(3)(OH)(2)mim][BF(4)], was synthesized herein and a novel H(2)O(2) biosensor is fabricated with [C(3)(OH)(2)mim][BF(4)] as the substrate and electrodepositing bimetallic Au/Pt nanoparticles (NPs) onto the [C(3)(OH)(2)mim][BF(4)] film. The functionalization of RTIL with hydroxyl groups provided an appropriate environment for the preparation of more uniform and smaller Au/Pt NPs with the diameter of 2.5 nm±0.2 nm. Immobilized with glutamate oxidase (GlutaOx), the resulting GlutaOx-[C(3)(OH)(2)mim][BF(4)]-Au/Pt-Nafion biosensor displayed excellent electrocatalytic response to glutamate at a potential of -200 mV. An effective on-line microdialysis system, which was powered by a microdialysis pump, was set up and used for the detection of glutamate successively in the striatum of rats. The glutamate biosensor in on-line microdialysis system showed good linear range from 0.5 μM to 20.0 μM with the detection limit of 0.17 μM (S/N=3). The basal level of glutamate in the striatum of anaesthetic rats was calculated to be 3.01±0.67 μM (n=3). The application of the GlutaOx-[C(3)(OH)(2)mim][BF(4)]-Au/Pt-Nafion electrode is further demonstrated for in vivo sensing of the variation of glutamate level in the striatum when rats received intraperitoneal (i.p.) injection of 100 mM KCl and brain electrical stimulation of the subthalamic nucleus area (STN). Both of the two kinds of stimulation resulted in an increase in the extracellular concentration of glutamate. This method has proved to be sensitive and reproducible, which enables its promising application in physiology and pathology.     

Feruloyl esterase-catalysed synthesis of glycerol sinapate using ionic liquids mixtures

The ability of a feruloyl esterase (AnFaeA), either in free or immobilised (cross-linked enzyme aggregates) form, to catalyse the esterification of glycerol, a major by-product of the biodiesel industry, with sinapic acid was studied in four hexafluorophosphate anion-containing ionic liquids: ([Bmim][PF(6)], [Omim][PF(6)], [C(2)OHmim][PF(6)] and [C(5)O(2)mim][PF(6)]). Such ionic liquids are considered 'green' reaction systems. The synthetic reaction was optimised in [C(2)OHmim][PF(6)] and the highest conversion yield was 72.5+/-2.1%, while, at the same reaction conditions in [C(5)O(2)mim] [PF(6)], a similar conversion yield was obtained (76.7+/-1.5%). AnFaeA was active in its free and immobilised form, with the latter retaining a part of its synthetic activity after 5 consecutive 24h-period reaction cycles. Sinapic acid was esterified to one of the primary hydroxyl groups of glycerol and retained, after esterification, 63.1+/-0.3% and 89.5+/-1.1% of its antioxidant activity against low-density lipoprotein oxidation, when added at concentrations of 10 and 60muM, respectively, in the assay mixture.     

Protease-catalyzed esterification of amino acid in water-miscible ionic liquid

The activity of two proteases in the esterification of N-acetyl-L-phenylalanine with ethanol was examined in the water-miscible ionic liquid, 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([emim][Tf]). The activity of subtilisin was not only improved 9-fold by changing from a water-miscible organic solvent, acetonitrile, to [emim][Tf], but also was about three times greater than that in a water-immiscible organic solvent, octane. Likewise, the activity of alpha-chymotrypsin in [emim][Tf] was more effectively enhanced compared with that in a water-miscible or a water-immiscible organic solvent. The water content in [emim][Tf] affected the activity of subtilisin.     

Biocatalysis in ionic liquids: the stereoconvergent hydrolysis of trans-β-methylstyrene oxide catalyzed by soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) was shown to catalyze hydrolysis of epoxides using the ionic liquids (ILs) [bmim][PF₆], [bmim][N(Tf)₂], and [bmim][BF₄] (where bmim=1-butyl-3-methylimidazolium, PF₆=hexafluorophosphate, N(Tf)₂=bis(trifluoromethylsulfonyl)imide, and BF₄=tetrafluoroborate) as reaction medium. Reaction rates were generally comparable with those observed in buffer solution, and when the cress enzyme was used the hydrolysis of trans-β-methylstyrene oxide gave, through a stereoconvergent process, the corresponding optically active (1S,2R)-erythro-1-phenylpropane-1,2-diol.     

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed.     

Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system

In this paper, an integrated process involving the mixed ionic liquids/water two-phase system (MILWS) is proposed to improve the efficiency for enzymatic hydrolysis of penicillin G. First, hydrophilic [C4mim]BF4 (1-butyl-3-methylimidazolium tetrafluoraborate) and NaH2PO4 salt form an ionic liquids aqueous two-phase system (ILATPS), which could extract penicillin from its fermentation broth efficiently. Second, a hydrophobic [C4mim]PF6 (1-butyl-3-methylimidazolium hexafluoraphosphate) is introduced into the ionic liquids-rich phase of ILATPS containing penicillin and converses it into MILWS. Penicillin is hydrolyzed by penicillin acylase in the water phase of MILWS at pH 5. The byproduct phenylacetic acid (PAA) is partitioned into the ionic liquids mixture phase, while the intended product 6-aminopenicillanic acid (6-APA) is precipitated at this pH. In comparison with a similar butyl acetate/water system (BAWS) at pH 4, MILWS exhibits two advantages. (1) The selectivity between PAA and penicillin is greatly optimized at pH 5 by varying the mole ratio of [C4mim]PF6/[C4mim]BF4 in MILWS, whereas in BAWS the unalterable nature of the organic solvent restricts the optimized pH for maximum selectivity between PAA and penicillin at pH 4. (2) The pH for 6-APA precipitation in BAWS is 4, whereas it shifts to pH 5 in MILWS due to the complexation between negatively charged 6-APA and the cationic surface of the ionic liquids micelle. As a result, the removal of the two products from the enzyme sphere at relatively high pH is permitted in MILWS, which is beneficial for enzymatic activity and stability in comparison with the acidic pH 4 environment in BAWS.     

Effect of High Physiological Temperatures on NAD+ Content of Green Leaf Mitochondria (Apparent Inhibition of Glycine Oxidation)

We observed a rapid decline in the rate of glycine oxidation by purified pea (Pisum sativum L.) leaf mitochondria preincubated at 40[deg]C for 2 min. In contrast, exogenous NADH and succinate oxidations were not affected by the heat treatment. We first demonstrated that the inhibition of glycine oxidation was not attributable to a direct effect of high temperatures on glycine decarboxylase/serine hydroxymethyltransferase. We observed that (a) addition of NAD+ to the incubation medium resulted in a resumption of glycine-dependent O2 uptake by intact mitochondria, (b) addition of NAD+ to the suspending medium prevented the decline in the rate of glycine-dependent O2 consumption by pea leaf mitochondria incubated at 40[deg]C, (c) NAD+ concentration in the matrix space collapses within only 5 min of warm temperature treatment, and (d) mitochondria treated with the NAD+ analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3[prime]-NAD+ retained high rates of glycine-dependent O2 uptake after preincubation at 40[deg]C. Therefore, we conclude that the massive and rapid efflux of NAD+, leading to the apparent inhibition of glycine oxidation, occurs through the specific NAD+ carrier present in the inner membrane of plant mitochondria. Finally, our data provide further evidence that NAD+ is not firmly bound to the inner membrane.     

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.     

Bacillus coagulans tolerance to 1‐ethyl‐3‐methylimidazolium‐based ionic liquids in aqueous and solid‐state thermophilic culture

The use of ionic liquids (ILs) to disrupt the recalcitrant structure of lignocellulose and make polysaccharides accessible to hydrolytic enzymes is an emerging technology for biomass pretreatment in lignocellulosic biofuel production. Despite efforts to reclaim and recycle IL from pretreated biomass, residual IL can be inhibitory to microorganisms used for downstream fermentation. As a result, pathways for IL tolerance are needed to improve the activity of fermentative organisms in the presence of IL. In this study, microbial communities from compost were cultured under high‐solids and thermophilic conditions in the presence of 1‐ethyl‐3‐methylimidazolium‐based ILs to enrich for IL‐tolerant microorganisms. A strain of Bacillus coagulans isolated from an IL‐tolerant community was grown in liquid and solid‐state culture in the presence of the ILs 1‐ethyl‐3‐methylimidazolium acetate ([C2mim][OAc]) or 1‐ethyl‐3‐methylimidazolium chloride ([C2mim][Cl]) to gauge IL tolerance. Viability and respiration varied with the concentration of IL applied and the type of IL used. B. coagulans maintained growth and respiration in the presence of 4 wt% IL, a concentration similar to that present on IL‐pretreated biomass. In the presence of both [C2mim][OAc] and [C2mim][Cl] in liquid culture, B. coagulans grew at a rate approximately half that observed in the absence of IL. However, in solid‐state culture, the bacteria were significantly more tolerant to [C2mim][Cl] compared with [C2mim][OAc]. B. coagulans tolerance to IL under industrially relevant conditions makes it a promising bacterium for understanding mechanisms of IL tolerance and discovering IL tolerance pathways for use in other microorganisms, particularly those used in bioconversion of IL‐pretreated plant biomass. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:311–316, 2014     

A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus.

An NAD+-dependent alcohol dehydrogenase (ADH) was purified to homogeneity from an aerobic strain of Bacillus stearothermophilus, DSM 2334 (ADH 2334), and compared with the ADH from B. stearothermophilus NCA 1503 (ADH 1503). When an antibody raised against ADH 2334 was used, no cross-reactivity with ADH 1503 was observed on Western blots; by means of an enzyme-linked-immunoabsorbent-assay ('e.l.i.s.a.') procedure, it was found that ADH 1503 had less than 6% of the antigenic activity of ADH 2334. Amino acid analyses detected very small differences in composition, equivalent to about 40 sequence changes, between the two enzymes. The new enzyme has the same six-amino-acid N-terminal sequence as ADH 1503. ADH 2334, but not ADH 1503, is reactive towards methanol; both enzymes can oxidize ethanol, propan-1-ol, butan-1-ol and butan-2-ol. The new enzyme has a distinctive pH optimum at pH 5.5-6 and has significantly lower KEthanolm and kEthanolcat. values than those of ADH 1503. From steady-state kinetic parameters of the reaction with ethanol, propan-1-ol and butan-1-ol, it was shown that ADH 2334 has an ordered mechanism in both directions, with NAD+ being the compulsory first substrate in alcohol oxidation and NADH release being the rate-limiting step. ADH 1503 has an ordered addition of NAD+ and alcohol, but NADH release is not rate-limiting.     

Peroxidase biocatalysis in water-soluble ionic liquids: activity,kinetic and thermal stability

Abstract

The activity and stability of commercial peroxidase was investigated in the presence of five 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) with either bromide or chloride anions: [C_xmim][X]. The peroxidase activity and stability were better for the shorter alkyl chain lengths of the ILs and peroxidase was more stable in the presence of the bromide anion, rather than chloride. The thermal inactivation profile was studied from 45 to 60 °C in [C₄mim][Cl] and [C₄mim][Br]. The activation energy was also determined. Kinetic analysis of the enzyme in the presence of the [C₄mim][Br] or control (buffer solution) showed that the KM value increased 5-fold and Vm decreased 13-fold in the presence of the IL. The increase in KM indicates that this IL can reduce the binding affinity between substrate and enzyme.     

The time-course of the effects of ethanol on the redox and phosphorylation states of rat liver

1. The time-course of the effects of ethanol administration on the metabolite concentrations, redox states and phosphorylation state was studied in the freeze-clamped liver of starved rats. The response was found to vary with the time after ethanol administration. 2. Administration of ethanol caused an immediate decrease in the [NAD(+)]/[NADH] ratio of both cytoplasm and mitochondria, which persisted over the 30min studied. 3. The free cytoplasmic [NADP(+)]/[NADPH] ratio in liver decreases immediately after ethanol administration but returns nearly to control values after 15min. 4. The cytoplasmic [ATP]/[ADP][HPO(4) (2-)] ratio is elevated 15min after ethanol administration in the starved rat. 5. The rapid and large changes in most metabolite concentrations measured appeared to result from the maintenance of near-equilibrium in a wide interlinked network. 6. Differences between fed and starved rats 15min after ethanol administration were slight.     

Mouse alcohol dehydrogenase 4: kinetic mechanism, substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37 degrees C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD(+) is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K(m) for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

A relation between (NAD+)/(NADH) potentials and glucose utilization in rat brain slices.

Changes in several parameters involved in the control of metabolism were correlated with changes in glucose utilization in rat brain slices incubated under conditions which reduced glucose oxidation by 40 to 70%. The parameters included: the concentrations of ATP, ADP, AMP, and the adenylate energy charge; the cytoplasmic oxidation-reduction state ([NAD+]/[NADH]), determined from the [pyruvate]/[lactate] equilibrium; the mitochondrial oxidation-reduction state, determined from the [NH4+] ]2-oxoglutarate]/[glutamate] Equilibrium; the cytoplasmic and mitochondrial oxidation-reduction potentials (in volts), calculated from the respective [NAD+]/ [NADH] ratios using the Nernst equation; and the difference between the cytoplasmic and mitochondrial [NAD+]/[NADH] potentials. The conversion of [3, 4-14C] glucose to 14CO2 and of [U-14C] glucose to acetylcholine and to lipids, proteins, and nucleic acids by the brain slices were also determined. The values obtained by subtracting the mitochondrial from the cytoplasmic [NAD+1/[NADH] potentials correlated more closely with glucose utilization than did other parameters, under the conditions studied. For the synthesis of acetylcholine, the correlation coefficient was 0.96, and for the production of 14CO2 from [3, 4-14C] glucose it was 0.82.     

Liposomal NAD(+) prevents diminished O(2) consumption by immunostimulated Caco-2 cells

Accumulating data support the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O(2), a phenomenon that has been termed "cytopathic hypoxia." We sought to use an in vitro "reductionist" model system using cultured cells stimulated with proinflammatory cytokines to test the hypothesis that cytopathic hypoxia is mediated, at least in part, by depletion of intracellular levels of NAD(+)/NADH secondary to activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We measured O(2) consumption by Caco-2 enterocytes growing on microcarrier beads after cells were incubated for 24 h under control conditions or with cytomix, a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma. Immunostimulated cells consumed O(2) at about one-half the rate of control cells, but this effect was largely prevented if any one of the following pharmacological agents was present during the period of incubation with cytomix: 4,5-dihydroxy-1,3-benzene disulfonic acid, a superoxide radical anion scavenger; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger; 5,10,15,20- tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III], a peroxynitrite (ONOO(-)) decomposition catalyst; urate, an ONOO(-) scavenger; 3-aminobenzamide, a PARP inhibitor; or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide HCl, a chemically dissimilar and more potent PARP inhibitor. The decrease in O(2) uptake induced by cytomix was associated with decreased cellular levels of NAD(+)/NADH. The decrease in cellular NAD(+)/NADH content and the decrease in O(2) uptake induced by cytomix were completely abrogated if liposome-encapsulated NAD(+) was added to the cultures during immunostimulation. Empty liposomes also increased O(2) uptake by immunostimulated Caco-2 cells, but much less effectively than liposomes containing NAD(+). These data are consistent with the view that enterocytes exposed to proinflammatory cytokines consume less O(2) due to NAD(+)/NADH depletion secondary to activation of PARP by ONOO(-) or other oxidants.