Vitamin K2 protects against Aß42-induced neurotoxicity by activating autophagy and improving mitochondrial function in Drosophila.

OBJECTIVE: Alzheimer disease is characterized by progressive decline in cognitive function due to neurodegeneration induced by accumulation of Aß and hyperphosphorylated tau protein. This study was conducted to explore the protective effect of vitamin K2 against Aß42-induced neurotoxicity. METHODS: Alzheimer disease transgenic Drosophila model used in this study was amyloid beta with the arctic mutation expressed in neurons. Alzheimer disease flies were treated with vitamin K2 for 28 days after eclosion. Aß42 level in brain was detected by ELISA. Autophagy-related genes and NDUFS3, the core subunit of mitochondrial complex I, were examined using real-Time PCR (RT-PCR) and western blot analysis. RESULTS: Vitamin K2 improved climbing ability (P = 0.0105), prolonged lifespan (P < 0.0001) and decreased Aß42 levels (P = 0.0267), upregulated the expression of LC3 and Beclin1(P = 0.0012 and P = 0.0175, respectively), increased the conversion of LC3I to LC3II (P = 0.0206) and decreased p62 level (P =0.0115) in Alzheimer disease flies. In addition, vitamin K2 upregulated the expression of NDUFS3 (P = 0.001) and increased ATP production (P = 0.0033) in Alzheimer disease flies. CONCLUSION: It seems that vitamin K2 protect against Aß42-induced neurotoxicity by activation of autophagy and rescue mitochondrial dysfunction, which suggests that it may be a potential valuable therapeutic approach for Alzheimer disease.

Curcumin supplementation improves mitochondrial and behavioral deficits in experimental model of chronic epilepsy.

The present study was aimed to investigate the potential beneficial effect of curcumin, a polyphenol with pleiotropic properties, on mitochondrial dysfunctions, oxidative stress and cognitive deficits in a kindled model of epilepsy. Kindled epilepsy was induced in rats by administering a sub-convulsive dose of pentylenetetrazole (PTZ, 40 mg/kg body weight) every alternate day for 30 days. PTZ administered rats exhibited marked cognitive deficits assessed using active and passive avoidance tasks. This was accompanied by a significant decrease in NADH:cytochrome-c reductase (complex I) and cytochrome-c oxidase (complex IV) activities along with an increase in ROS, lipid peroxidation and protein carbonyls. The levels of glutathione also decreased in the cortex and hippocampus. Electron micrographs revealed disruption of mitochondrial membrane integrity with distorted cristae in PTZ treated animals. Histopathological examination showed pyknotic nuclei and cell loss in the hippocampus as well as in the cortex of PTZ treated animals. Curcumin administration at a dose of 100 mg/kg, p.o. throughout the treatment paradigm was able to ameliorate cognitive deficits with no significant effect on seizure score. Curcumin was able to restore the activity of mitochondrial complexes. In addition, significant reduction in ROS generation, lipid peroxidation and protein carbonyls was observed in PTZ animals supplemented with curcumin. Moreover, glutathione levels were also restored in PTZ treated rats supplemented with curcumin. Curcumin protected mitochondria from seizure induced structural alterations. Further, the curcumin supplemented PTZ rats had normal cell morphology and reduced cell loss. These results suggest that curcumin supplementation has potential to prevent mitochondrial dysfunctions and oxidative stress with improved cognitive functions in a chronic model of epilepsy.

Partial complex I deficiency due to the CNS conditional ablation of Ndufa5 results in a mild chronic encephalopathy but no increase in oxidative damage.

Deficiencies in the complex I (CI; NADH-ubiquinone oxidoreductase) of the respiratory chain are frequent causes of mitochondrial diseases and have been associated with other neurodegenerative disorders, such as Parkinson's disease. The NADH-ubiquinone oxidoreductase 1 alpha subcomplex subunit 5 (NDUFA5) is a nuclear-encoded structural subunit of CI, located in the peripheral arm. We inactivated Ndufa5 in mice by the gene-trap methodology and found that this protein is required for embryonic survival. Therefore, we have created a conditional Ndufa5 knockout (KO) allele by introducing a rescuing Ndufa5 cDNA transgene flanked by loxP sites, which was selectively ablated in neurons by the CaMKIIα-Cre. At the age of 11 months, mice with a central nervous system knockout of Ndufa5 (Ndufa5 CNS-KO) showed lethargy and loss of motor skills. In these mice cortices, the levels of NDUFA5 protein were reduced to 25% of controls. Fully assembled CI levels were also greatly reduced in cortex and CI activity in homogenates was reduced to 60% of controls. Despite the biochemical phenotype, no oxidative damage, neuronal death or gliosis were detected in the Ndufa5 CNS-KO brain at this age. These results showed that a partial defect in CI in neurons can lead to late-onset motor phenotypes without neuronal loss or oxidative damage.

Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation.

Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.

MiADMSA reverses impaired mitochondrial energy metabolism and neuronal apoptotic cell death after arsenic exposure in rats.

Arsenicosis, due to contaminated drinking water, is a serious health hazard in terms of morbidity and mortality. Arsenic induced free radicals generated are known to cause cellular apoptosis through mitochondrial driven pathway. In the present study, we investigated the effect of arsenic interactions with various complexes of the electron transport chain and attempted to evaluate if there was any complex preference of arsenic that could trigger apoptosis. We also evaluated if chelation with monoisoamyl dimercaptosuccinic acid (MiADMSA) could reverse these detrimental effects. Our results indicate that arsenic exposure induced free radical generation in rat neuronal cells, which diminished mitochondrial potential and enzyme activities of all the complexes of the electron transport chain. Moreover, these complexes showed differential responses towards arsenic. These early events along with diminished ATP levels could be co-related with the later events of cytosolic migration of cytochrome c, altered bax/bcl(2) ratio, and increased caspase 3 activity. Although MiADMSA could reverse most of these arsenic-induced altered variables to various extents, DNA damage remained unaffected. Our study for the first time demonstrates the differential effect of arsenic on the complexes leading to deficits in bioenergetics leading to apoptosis in rat brain. However, more in depth studies are warranted for better understanding of arsenic interactions with the mitochondria.

Disrupted-in-schizophrenia 1 (DISC1) plays essential roles in mitochondria in collaboration with Mitofilin.

Disrupted-in-schizophrenia 1 (DISC1) has emerged as a schizophrenia-susceptibility gene affecting various neuronal functions. In this study, we characterized Mitofilin, a mitochondrial inner membrane protein, as a mediator of the mitochondrial function of DISC1. A fraction of DISC1 was localized to the inside of mitochondria and directly interacts with Mitofilin. A reduction in DISC1 function induced mitochondrial dysfunction, evidenced by decreased mitochondrial NADH dehydrogenase activities, reduced cellular ATP contents, and perturbed mitochondrial Ca(2+) dynamics. In addition, deficiencies in DISC1 and Mitofilin induced a reduction in mitochondrial monoamine oxidase-A activity. The mitochondrial dysfunctions evoked by the deficiency of DISC1 were partially phenocopied by an overexpression of truncated DISC1 that is associated with schizophrenia in human. DISC1 deficiencies induced the ubiquitination of Mitofilin, suggesting that DISC1 is critical for the stability of Mitofilin. Finally, the mitochondrial dysfunction induced by DISC1 deficiency was partially reversed by coexpression of Mitofilin, confirming a functional link between DISC1 and Mitofilin for the normal mitochondrial function. According to these results, we propose that DISC1 plays essential roles for mitochondrial function in collaboration with a mitochondrial interacting partner, Mitofilin.

Mitochondrial tubulopathy in tenofovir disoproxil fumarate-treated rats.

OBJECTIVES: Tenofovir disoproxil fumarate (tenofovir DF) use has been associated with renal dysfunction and Fanconi syndrome. Tenofovir is taken up into renal tubules by anion transporters where high intracellular drug concentration may induce a functionally relevant depletion of mitochondrial DNA (mtDNA). We investigated if tenofovir may induce renal mtDNA depletion and respiratory chain dysfunction. METHODS: Rats (n = 8) were gavaged daily with 100 mg x kg(-1) x d(-1) of tenofovir DF or didanosine. Kidneys and livers were examined after 8 weeks of treatment. RESULTS: The tenofovir group had significantly lower body and kidney weights than rats exposed to water or didanosine. Proximal but not distal tubules were of increased diameter and contained small lipid droplets. Tubular mitochondria were enlarged, and their crystal architecture was disrupted. Tenofovir-exposed kidneys contained low mtDNA copy numbers and impaired expression of mtDNA-encoded cytochrome c oxidase (COX) I but not nucleus-encoded COX IV subunits. Histochemistry demonstrated low tubular COX and nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) activities, whereas succinate dehydrogenase activity was preserved. COX activity was preserved in the glomeruli of tenofovir-exposed rats. Didanosine did not elicit renal effects but, unlike tenofovir, depleted mtDNA in liver (by 52%). CONCLUSIONS: Tenofovir DF induces an organ-specific nephrotoxicity with mtDNA depletion and dysfunction of mtDNA-encoded respiratory chain subunits. The data do not support nephrotoxicity of didanosine.

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit.

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.

Possible involvement of glutamic and/or aspartic acid residue(s) and requirement of mitochondrial integrity for the protective effect of creatine against inhibition of cardiac mitochondrial respiration by methylglyoxal.

We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661-669,2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3'-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine.

Dopamine toxicity involves mitochondrial complex I inhibition: implications to dopamine-related neuropsychiatric disorders.

Dopamine, which is suggested as a prominent etiological factor in several neuropsychiatric disorders such as Parkinson's disease and schizophrenia, demonstrates neurotoxic properties. In such dopamine-related diseases mitochondrial dysfunction has been reported. Dopamine oxidized metabolites were shown to inhibit the mitochondrial respiratory system both in vivo and in vitro. In the present study, we suggest an additional mechanism for dopamine toxicity, which involves mitochondrial complex I inhibition by dopamine. In human neuroblastoma SH-SY5Y cells dopamine induced a reduction in ATP concentrations, which was negatively correlated to intracellular dopamine levels (r = - 0.96, P = 0.012), and was already evident at non-toxic dopamine doses. In disrupted mitochondria dopamine inhibited complex I activity with IC50 = 11.87 +/- 1.45 microm or 8.12 +/- 0.75 microM in the presence of CoQ or ferricyanide, respectively, with no effect on complexes IV and V activities. The catechol moiety, but not the amine group, of dopamine is essential for complex I inhibition, as is indicated by comparing the inhibitory potential of functionally and structurally dopamine-related compounds. In line with the latter is the finding that chelatable FeCl2 prevented dopamine-induced inhibition of complex I. Monoamine oxidase A and B inhibitors, as well as the antioxidant butylated hydroxytoluene (BHT), did not prevent dopamine-induced inhibition, suggesting that dopamine oxidation was not involved in this process. The present study suggests that dopamine toxicity involves, or is initiated by, its interaction with the mitochondrial oxidative phosphorylation system. We further hypothesize that this interaction between dopamine and mitochondria is associated with mitochondrial dysfunction observed in dopamine-related neuropsychiatric disorders, such as schizophrenia and Parkinson's disease.

The activity of the chloroplastic Ndh complex is regulated by phosphorylation of the NDH-F subunit.

Hydrogen peroxide (H(2)O(2)) induces increases, to different degrees, in transcripts, protein levels, and activity of the Ndh complex (EC 1.6.5.3). In the present work, we have compared the effects of relatively excess light, H(2)O(2), dimethylthiourea (a scavenger of H(2)O(2)), and/or EGTA (a Ca(2+) chelator) on the activity and protein levels of the Ndh complex of barley (Hordeum vulgare cv Hassan) leaf segments. The results show the involvement of H(2)O(2) in the modulation of both the protein level and activity of the Ndh complex and the participation of Ca(2+) mainly in the activity regulation of pre-existing protein. Changes in Ndh complex activity could not be explained only by changes in Ndh protein levels, suggesting posttranslational modifications. Hence, we investigate the possible phosphorylation of the Ndh complex both in thylakoids and in the immunopurified Ndh complex using monoclonal phosphoamino acid antibodies. We demonstrate that the Ndh complex is phosphorylated in vivo at threonine residue(s) of the NDH-F polypeptide and that the level of phosphorylation is closely correlated with the Ndh complex activity. The emerging picture is that full activity of the Ndh complex is reached by phosphorylation of its NDH-F subunit in a H(2)O(2)- and Ca(2+)-mediated action.

Inhibition of oxidative phosphorylation underlies the antiproliferative and proapoptotic effects of mofarotene (Ro 40-8757) in Burkitt's lymphoma cells.

In the search for retinoids active against Burkitt's lymphoma (BL), we found that the arotinoid mofarotene (Ro 40-8757) induced strong antiproliferative and apoptotic responses in most established BL cell lines as well as in primary BL cells. Ro 40-8757-induced apoptosis is associated with mitochondrial membrane depolarization, activation of caspase-3 and -9, and enhanced production of reactive oxygen species. These effects were related to a transient drop in intracellular ATP content, probably favored by a downregulation of NADH dehydrogenase subunit-1, a component of the mitochondrial respiratory chain (MRC) Complex I. Inhibition of MRC with thenoyltrifluoroacetone suppressed both the ATP recovery and apoptosis, confirming that the effects of Ro 40-8757 are mediated by changes in mitochondrial function. Compared to EBV-negative lines, EBV-carrying BLs were more resistant to Ro 40-8757-induced apoptosis. EBV infection and ectopic LMP-1 expression increased the resistance of BL cells to Ro 40-8757-induced apoptosis, probably through bcl-2 upregulation. Finally, we also show that 2-methoxyoestradiol, an inhibitor of the scavenger enzymes superoxide dismutases, enhanced Ro 40-8757-mediated apoptosis. These findings provide the rationale for evaluating the clinical efficacy of Ro 40-8757 in BL patients and suggest that the combination of Ro 40-8757 with inhibitors of scavenger enzymes may be a promising therapeutic approach for this aggressive lymphoma.

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 9: comparison of acetoxy 4-methylcoumarins and other polyphenolic acetates reveal the specificity to acetoxy drug: protein transacetylase for pyran carbonyl group in proximity to the oxygen heteroatom.

The evidences for the possible enzymatic transfer of acetyl groups (catalyzed by a transacetylase localized in microsomes) from an acetylated compound (acetoxy-4-methylcoumarins) to enzyme proteins leading to profound modulation of their catalytic activities was cited in our earlier publications in this series. The investigations on the specificity for transacetylase (TA) with respect to the number and positions of acetoxy groups on the benzenoid ring of coumarin molecule revealed that acetoxy groups in proximity to the oxygen heteroatom (at C-7 and C-8 positions) demonstrate a high degree of specificity to TA. These studies were extended to the action of TA on acetates of other polyphenols, such as flavonoids and catechin with a view to establish the importance of pyran carbonyl group for the catalytic activity. The absolute requirement of the carbonyl group in the pyran ring of the substrate for TA to function was established by the observation that TA activity was hardly discernible when catechin pentacetate and 7-acetoxy-3,4-dihydro-2,2-dimethylbenzopyran (both lacking pyran ring carbonyl group) were used as the substrates. Further, the TA activity with flavonoid acetates was remarkably lower than that with acetoxycoumarins, thus suggesting the specificity for pyran carbonyl group in proximity to the oxygen heteroatom. The biochemical properties of flavonoid acetates, such as irreversible activation of NADPH cytochrome C reductase and microsome-catalyzed aflatoxin B(1) binding to DNA in vitro were found to be in tune with their specificity to TA.

Assay-based quantitative analysis of PC12 cell differentiation.

Differentiation of PC12 cells has been quantified by measurement of neurite length. However, this procedure is not suitable for large numbers of samples, for example in 96-well tissue culture plates. For this reason, we established three simple and quantitative methods for nerve growth factor-induced differentiation of PC12 cells cultured in 96-well plates. Firstly, because neuronal markers, including neurofilament proteins and beta-tubulin isotype III, are increased during PC12 cell differentiation, we developed cell enzyme-linked immunoabsorbent assays (ELISA)-based procedures that measure the amount of these proteins. Secondly, because lactate dehydrogenase (LDH) is down-regulated and mitochondrial NADH-dehydrogenase activity is increased during PC12 cell differentiation, we established procedures to measure changes in LDH and NADH dehydrogenase. We found that the cell ELISA and cell counting assays could be used to determine the degree of PC12 cell differentiation caused by nerve growth factor, basic fibroblast growth factor and epidermal growth factor. However, neither LDH nor NADH-dehydrogenase activities changed during Thy-1 antibody-induced differentiation. These findings show that in addition to the cell ELISA procedures, the LDH and NADH-dehydrogenase procedures are useful for characterization of growth factor-induced PC12 cell differentiation.

Novel FMN-containing rotenone-insensitive NADH dehydrogenase from Trypanosoma brucei mitochondria: isolation and characterization.

A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.

Effects of deoxycholic acid and its epimers on lipid peroxidation in isolated rat hepatocytes.

We studied the effects of deoxycholic acid and its three epimers with beta-hydroxyl groups (3alpha,12beta-, 3beta,12alpha-, and 3beta,12beta-dihydroxy-5beta-cholan-24-oic acids), which were hydrophilic and less cytotoxic, on lipid peroxidation to elucidate the relationship between structural features of bile acids and their effect on lipid peroxidation. Taurodeoxycholate markedly increased the production of thiobarbituric acid-reactive substances, end products of lipid peroxidation, in isolated rat hepatocytes, whereas epimers of taurodeoxycholate did not. Deoxycholic acid inhibited mitochondrial NADH dehydrogenase and NADH:ferricytochrome c oxidoreductase activities, leading to free radical generation, whereas epimers of deoxycholic acid had no effect on mitochondrial enzymes. These findings suggested that hydrophobic bile acids cause lipid peroxidation by impairment of mitochondrial function, leading to the generation of free radicals; and epimerization of alpha-hydroxyl groups in the steroid nucleus to beta-hydroxyl groups results in a decrease of the toxic effects of deoxycholic acid on lipid peroxidation.

[The effect of reaferon on the level of catecholamines and mitochondrial enzymes in the ischemic kidney]. / Vliianie reaferona na uroven' katekholaminov i fermentov mitokhondrii v ishemicheskoi pochke.

The effect of reaferon (introduced at a dose of 1 x 10(6) IU/kg over six days prior to ischemia induction) on the levels of catecholamines and the activity of succinate dehydrogenase and NADH-dehydrogenase in various structures of kidney was studied in experiments on white rats. The ischemia was modeled by 90-min ligation of renal vessels. Reaferon retained the luminescence of catecholamines in all renal structures 24 h after circulation was restored on the level of intact kidney, except for the nerve trunks where the luminescence intensity decreased by 40%. Preliminary introduction of reaferon stimulated restoration of the enzymatic activity in the Krebs cycle and mitochondrial respiratory chain after 24- and 48-h revascularization, respectively.

[The action of the neurotoxin AF64A on the reactions of neuroticized rats]. / Deistvie neirotoksina AF64A na reaktsii nevrotizirovannykh krys.

The effects of intracerebroventricular administration of ethylholine aziridinum ion (AF64A) were studied in neuroticized male Wistar rats. The cholinotoxin was bilaterally injected in the dose of 3 nmol. AF64A produced a significant decrease in arterial pressure and activity of respiratory enzymes succinate dehydrogenase and NADH-dehydrogenase in hippocampus and motor cortex. Increase in the local blood flow in the hippocampus and motor cortex had a compensatory character.

Mechanisms for isoniazid action and resistance.

Isoniazid is the most widely used antituberculosis drug. Genetic studies in Mycobacterium smegmatis identified the inhA-encoded, NADH-dependent enoyl acyl carrier protein reductase as the primary target for this drug. A reactive form of isoniazid inhibits InhA by reacting with the NAD(H) cofactor bound to the enzyme active site forming a covalent adduct (isonicotinic acyl NADH) that is apt to bind with high affinity. Resistance can occur by increased expression of InhA or by mutations that lower the enzyme's affinity to NADH. Both of these resistance mechanisms are observed in 30% of clinical tuberculosis isolates. Mutation in katG, which encodes catalase peroxidase, is the most common source for resistance. Another mechanism for isoniazid resistance, in M. smegmatis, occurs by defects in NADH dehydrogenase (Ndh) of the respiratory chain. Genetic data indicated that ndh mutations confer resistance by lowering the rate of NADH oxidation and increasing the intracellular NADH/NAD+ ratio. An increased amount of NADH may prevent formation of isonicotinic acyl NADH or may promote displacement of the isonicotinic acyl NADH from InhA. While our studies have identified this mechanism in M. smegmatis, results reported in early literature lead us to believe that it can occur in Mycobacterium tuberculosis.