Decrease In Succinate Research Articles

Effect of a Very Low-Calorie Diet on Oxidative Stress, Inflammatory and Metabolomic Profile in Metabolically Healthy and Unhealthy Obese Subjects.

The purpose of the study was to determine the impact of weight loss through calorie restriction on metabolic profile, and inflammatory and oxidative stress parameters in metabolically healthy (MHO) and unhealthy (MUHO) obese individuals. A total of 74 subjects (34 MHO and 40 MUHO) received two cycles of a very low-calorie diet, alternating with a hypocaloric diet for 24 weeks. Biochemical, oxidative stress, and inflammatory markers, as well as serum metabolomic analysis by nuclear magnetic resonance, were performed at baseline and at the end of the intervention. After the diet, there was an improvement in insulin resistance, as well as a significant decrease in inflammatory parameters, enhancing oxidative damage, mitochondrial membrane potential, glutathione, and antioxidant capacity. This improvement was more significant in the MUHO group. The metabolomic analysis showed a healthier profile in lipoprotein profile. Lipid carbonyls also decrease at the same time as unsaturated fatty acids increase. We also display a small decrease in succinate, glycA, alanine, and BCAAs (valine and isoleucine), and a slight increase in taurine. These findings show that moderate weight reduction leads to an improvement in lipid profile and subfractions and a reduction in oxidative stress and inflammatory markers; these changes are more pronounced in the MUHO population.

Selective Targeting of Cancerous Mitochondria and Suppression of Tumor Growth Using Redox-Active Treatment Adjuvant.

Redox-active substances and their combinations, such as of quinone/ascorbate and in particular menadione/ascorbate (M/A; also named Apatone®), attract attention with their unusual ability to kill cancer cells without affecting the viability of normal cells as well as with the synergistic anticancer effect of both molecules. So far, the primary mechanism of M/A-mediated anticancer effects has not been linked to the mitochondria. The aim of our study was to clarify whether this “combination drug” affects mitochondrial functionality specifically in cancer cells. Studies were conducted on cancer cells (Jurkat, Colon26, and MCF7) and normal cells (normal lymphocytes, FHC, and MCF10A), treated with different concentrations of menadione, ascorbate, and/or their combination (2/200, 3/300, 5/500, 10/1000, and 20/2000 μM/μM of M/A). M/A exhibited highly specific and synergistic suppression on cancer cell growth but without adversely affecting the viability of normal cells at pharmacologically attainable concentrations. In M/A-treated cancer cells, the cytostatic/cytotoxic effect is accompanied by (i) extremely high production of mitochondrial superoxide (up to 15-fold over the control level), (ii) a significant decrease of mitochondrial membrane potential, (iii) a decrease of the steady-state levels of ATP, succinate, NADH, and NAD+, and (iv) a decreased expression of programed cell death ligand 1 (PD-L1)—one of the major immune checkpoints. These effects were dose dependent. The inhibition of NQO1 by dicoumarol increased mitochondrial superoxide and sensitized cancer cells to M/A. In normal cells, M/A induced relatively low and dose-independent increase of mitochondrial superoxide and mild oxidative stress, which seems to be well tolerated. These data suggest that all anticancer effects of M/A result from a specific mechanism, tightly connected to the mitochondria of cancer cells. At low/tolerable doses of M/A (1/100-3/300 μM/μM) attainable in cancer by oral and parenteral administration, M/A sensitized cancer cells to conventional anticancer drugs, exhibiting synergistic or additive cytotoxicity accompanied by impressive induction of apoptosis. Combinations of M/A with 13 anticancer drugs were investigated (ABT-737, barasertib, bleomycin, BEZ-235, bortezomib, cisplatin, everolimus, lomustine, lonafarnib, MG-132, MLN-2238, palbociclib, and PI-103). Low/tolerable doses of M/A did not induce irreversible cytotoxicity in cancer cells but did cause irreversible metabolic changes, including: (i) a decrease of succinate and NADH, (ii) depolarization of the mitochondrial membrane, and (iii) overproduction of superoxide in the mitochondria of cancer cells only. In addition, M/A suppressed tumor growth in vivo after oral administration in mice with melanoma and the drug downregulated PD-L1 in melanoma cells. Experimental data suggest a great potential for beneficial anticancer effects of M/A through increasing the sensitivity of cancer cells to conventional anticancer therapy, as well as to the immune system, while sparing normal cells. We hypothesize that M/A-mediated anticancer effects are triggered by redox cycling of both substances, specifically within dysfunctional mitochondria. M/A may also have a beneficial effect on the immune system, making cancer cells “visible” and more vulnerable to the native immune response.

Effects of a novel variant of the yeast \u03b3-glutamyl kinase Pro1 on its enzymatic activity and sake brewing

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

Calcitriol Reduces Adverse Effects of Diclofenac on Mitochondrial Function in Isolated Rat Heart Mitochondria.

The safety of diclofenac (DIC) use in clinical practice has been questioned because of adverse cardiovascular effects. Previous studies have indicated that DIC cause mitochondrial dysfunction and oxidative stress in heart mitochondria. The aim of this study was to investigate the protective effect of calcitriol against the mitochondrial toxicity potency of diclofenac in heart rat mitochondria. For this purpose, rat heart mitochondria were isolated with mechanical lysis and differential centrifugation. Then isolated mitochondria were pretreated with 3 different concentrations of calcitriol (2.5, 5 and 10 µM) for 5 min at 37°C, after which DIC (10 µg/ml) was added to promote deleterious effects on mitochondria. During 1 hour of incubation, using by flow cytometry and biochemical evaluations, the parameters of mitochondrial toxicity were evaluated. Our results showed that DIC (10 µg/ml) caused a significant decrease in succinate dehydrogenase (SDH) activity, mitochondrial membrane potential (MMP) collapse, and mitochondrial swelling, and a significant increase in reactive oxygen species (ROS) formation, lipid peroxidation (LP) and oxidative stress. Also, our results revealed that co-administration of calcitriol (5 and 10 µM) with diclofenac markedly ameliorates the mitochondrial toxicity effects in rat hart mitochondria. In this study, we showed that DIC impairs mitochondrial function and induces mitochondrial toxicity in rat heart isolated mitochondria, which were ameliorated by calcitriol. These findings suggest that calcitriol may be a preventive/therapeutic strategy for cardiotoxicity complications caused by DIC.

Impact of a Multistrain Probiotic Formulation with High Bifidobacterial Content on the Fecal Bacterial Community and Short-Chain Fatty Acid Levels of Healthy Adults.

The consumption of probiotic products is continually increasing, supported by growing scientific evidence of their efficacy. Considering that probiotics may primarily affect health (either positively or negatively) through gut microbiota modulation, the first aspect that should be evaluated is their impact on the intestinal microbial ecosystem. In this study, we longitudinally analyzed the bacterial taxonomic composition and organic acid levels in four fecal samples collected over the course of four weeks from 19 healthy adults who ingested one capsule a day for two weeks of a formulation containing at least 70 billion colony-forming units, consisting of 25% lactobacilli and 75% Bifidobacterium animalis subsp. lactis. We found that 16S rRNA gene profiling showed that probiotic intake only induced an increase in a single operational taxonomic unit ascribed to B. animalis, plausibly corresponding to the ingested bifidobacterial strain. Furthermore, liquid chromatography/mass spectrometry revealed a significant increase in the lactate and acetate/butyrate ratio and a trend toward a decrease in succinate following probiotic administration. The presented results indicate that the investigated probiotic formulation did not alter the intestinal bacterial ecosystem of healthy adults and suggest its potential ability to promote colonization resistance in the gut through a transient increase in fecal bifidobacteria, lactic acid, and the acetate/butyrate ratio.

Metabolic targeting of HIF-1α potentiates the therapeutic efficacy of oxaliplatin in colorectal cancer

Drug resistance is a major problem limiting the efficacy of chemotherapy in cancer treatment, and the hypoxia-induced stabilization of HIF-1α plays a role in this process. HIF-1α overexpression has been observed in a variety of human cancers, including colorectal cancer (CRC). Therefore, targeting HIF-1α is a promising strategy for overcoming chemoresistance to enhance the efficacy of chemotherapies in CRC. Here, we show that DNMT inhibitors can induce HIF-1α degradation to overcome oxaliplatin resistance and enhance anti-CRC therapy. We found that a low-toxicity DNMT inhibitor, zebularine, could downregulate HIF-1α expression and overcome hypoxia-induced oxaliplatin resistance in HCT116 cells and showed efficacy in HCT116 xenograft models and AOM/DSS-induced CRC mouse models. Zebularine could induce the degradation of HIF-1α protein through hydroxylation. LC-MS analysis showed a decrease in succinate in various CRC cells under hypoxia and in colon tissues of AOM/DSS-induced CRC mice. The decrease was reversed by zebularine. Tumor angiogenesis was also reduced by zebularine. Furthermore, zebularine potentiated the anticancer effect of oxaliplatin in AOM/DSS-induced CRC models. This finding provides a new strategy in which an increase in HIF-1α hydroxylation could overcome oxaliplatin resistance to enhance anti-CRC therapy.

Metabolic alterations in the rat cerebellum following acute middle cerebral artery occlusion, as determined by 1H NMR spectroscopy.

Supratentorial focal ischemia may reduce cerebral blood volume and cerebellar glucose metabolic rate contralateral to the region of ischemia. The present study investigated the effects of middle cerebral artery occlusion (MCAO) on cerebral metabolism in the ischemic cerebral hemisphere and the non‑ischemic cerebellum in rats 1, 3, 9 and 24h following ischemia using exvivo proton nuclear magnetic resonance (1H NMR) spectroscopy. The results demonstrated that focal ischemia induced increases in the levels of lactate and alanine, and a decrease in succinate, as early as 1h following ischemia in the left cerebral hemisphere and the right cerebellum. A continuous increase in lactate levels and decrease in creatine levels were detected in both cerebral areas 3 and 24h post‑MCAO. The most obvious difference between the two cerebral areas was that there was no statistically significant difference in N‑acetyl aspartate (NAA) levels in the right cerebellum at all time points; however, the amino acid levels of NAA in the left cerebral hemisphere were markedly decreased 3, 9 and 24h post‑MCAO. In addition, an obvious increase in glutamine was observed in the right and left cerebellum at 3, 9and 24h post‑MCAO. Furthermore, the present study demonstrated that γ‑aminobutyric acid levels were decreased at 1h in the left and right cerebellum and were evidently increased at 24h in the right cerebellum post‑MCAO. In conclusion, supratentorial ischemia has been indicated to affect the activities of the non‑ischemic contralateral cerebellum. Therefore, these results suggested that an NMR‑based metabonomic approach may be used as a potential means to elucidate cerebral and cerebellar metabolism following MCAO, which may help improve understanding regarding cerebral infarction at a molecular level. Exvivo 1HNMR analysis may be useful for the assessment of clinical biopsies.

Abstract 1497: BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers

Abstract Dysregulated mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. Perturbations in mitochondrial fluxes (i.e inhibition of electron chain complexes activity, impaired electron flow) have substantial effects on cell viability, suggesting that targeting mitochondrial function could be effective for therapeutic response in cancer. BPM31510, containing oxidized coenzyme Q10 elicits an anti-Warburg effect is currently in phase II clinical trials for solid tumors. Previously, we have demonstrated the anti-cancer properties of BPM31510 in breast and pancreatic xenograft models. Here, we examined the mechanism of action of BPM31510 in vitro. Using a multi-cancer cell panel, BPM31510 was shown to be consistently and selectively cytotoxic to cancer cells, compared to normal and non-tumorigenic controls, and sensitivity did not correlate to cell doubling time or mutational status. Treatment with BPM 31510 (EC50) in breast and pancreatic cancer cells resulted in a time- and dose-dependent decrease in mitochondrial membrane potential which preceded an increase in early and late apoptosis cells, suggesting BPM31510 initiates a mitochondrial mediated cell death pathway. Using a fluorescently labeled CoQ10 we were able to trace the subcellular location of the CoQ10, which predominantly accumulates in mitochondria and lipid droplets in a time dependent manner. Additionally, the mitochondrial enrichment of CoQ10 is accompanied by morphological changes that varied amongst the different cancer cell types. As CoQ10 is a redox molecule required for electron transfer activity between complexes, we hypothesized that disruption of Q-pool homeostasis would alter complex activity. To investigate this, Complex driven respiration was measured in cells treated with BPM31510 and compared to untreated. Alterations in mitochondrial respiration characterized by a dose-dependent decrease in succinate (Complex II) and glycerol-3-phosphate (Complex III)-fueled respiration were observed in cells treated with BPM31510, while no changes were seen in pyruvate driven respiration (Complex I), suggesting that BPM31510 specifically impairs respiration responses that are more dependent on Q-pool functionality. As impairment of the electron transport chain increases intracellular oxidative stress, we next investigated if BPM 31510 treatment increases ROS levels. After 24h treatment, BPM31510 significantly increased ROS levels in treated cancer cells compared to untreated. Furthermore, BPM31510 induced death could be in part prevented by co-treatment with antioxidants. Together, these data demonstrates BPM31510 has anti-cancer activity in multiple cancer cell types and define a unique and novel functional link between mitochondrial Q-pool disruption and the mechanism of action of BPM31510. Citation Format: Tulin Dadali, Katerina Krumova, Anne R. Diers, Pallavi Awate, Ryan Ng, Arleide Lee, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1497. doi:10.1158/1538-7445.AM2017-1497

Impact of initial lipid content and oxygen supply on alcoholic fermentation in champagne-like musts

Available nitrogen, lipids, or oxygen are nutrients with major impact on the kinetics of winemaking fermentation. Assimilable nitrogen is usually the growth-limiting nutrient which availability determines the fermentation rate and therefore the fermentation duration. In some particular cases, as in Champagne, grape musts have high available nitrogen content and low turbidity, i.e., below 50 Nephelometric Turbidity Unit (NTU). In the case of low turbidity, the availability of lipids, particularly phytosterols, becomes limiting. In this situation, control of oxygenation, which is necessary for lipid synthesis by yeast, is particularly crucial during fermentation.To mimic and understand these situations, a synthetic medium simulating the average composition of a Champagne must was used. This medium contained phytosterol (mainly β-sitosterol) concentrations ranging from 0 to 8mg/L corresponding to turbidity between 10 and 90 NTU. Population reached during the stationary phase and the maximum fermentation rate are conditioned by the initial phytosterol concentration determining the amount of nitrogen consumption. An early loss of viability was observed when the lipid concentrations were very low. For example, the viability continuously decreased during the stationary phase to a final value of 50% for an initial phytosterol concentration of 1mg/L. In some fermentations, 10mg/L oxygen were added at the end of the growth phase to combine the effects of initial content of phytosterols in the musts and the de novo synthesis of ergosterol and unsaturated fatty acids induced by oxygen addition. Effect of oxygen supply on the fermentation kinetics was particularly significant for media with low phytosterol contents. For example, the maximum fermentation rate was increased by 1.4-fold and the fermentation time was 70h shorter with oxygen addition in the medium containing 2mg/L of phytosterols. As a consequence of the oxygen supply, for the media containing 3, 5 and 8mg/L of phytosterols, the assimilable nitrogen was completely exhausted and the fermentation kinetics, as well as the final populations and viabilities (greater than 90%), were identical for the 3 conditions. The impacts of the lipid content and additional oxygen on acetate, glycerol and succinate synthesis were also studied. The phytosterols decreased the acetate and increased the succinate synthesis, and oxygenation resulted in a decrease in succinate formation.This work highlights the similarities and differences between the effects of lipids and oxygen on fermentation kinetics and yeast metabolism. This research highlights the need for an optimal combined management of lipid content in the must via turbidity and oxygenation, particularly in nitrogen-rich musts.

Differential Metabolic Rearrangements after Cold Storage Are Correlated with Chilling Injury Resistance of Peach Fruits.

Reconfiguration of the metabolome is a key component involved in the acclimation to cold in plants; however, few studies have been devoted to the analysis of the overall metabolite changes after cold storage of fruits prior to consumption. Here, metabolite profiling of six peach varieties with differential susceptibility to develop mealiness, a chilling-injury (CI) symptom, was performed. According to metabolic content at harvest; after cold treatment; and after ripening, either following cold treatment or not; peach fruits clustered in distinct groups, depending on harvest-time, cold treatment, and ripening state. Both common and distinct metabolic responses among the six varieties were found; common changes including dramatic galactinol and raffinose rise; GABA, Asp, and Phe increase; and 2-oxo-glutarate and succinate decrease. Raffinose content after long cold treatment quantitatively correlated to the degree of mealiness resistance of the different peach varieties; and thus, raffinose emerges as a candidate biomarker of this CI disorder. Xylose increase after cold treatment was found only in the susceptible genotypes, indicating a particular cell wall reconfiguration of these varieties while being cold-stored. Overall, results indicate that peach fruit differential metabolic rearrangements due to cold treatment, rather than differential metabolic priming before cold, are better related with CI resistance. The plasticity of peach fruit metabolism renders it possible to induce a diverse metabolite array after cold, which is successful, in some genotypes, to avoid CI.

Abstract 208: BPM 31510-induced alteration in Complex II activity is functionally linked to cell death activation pathway in a preclinical model of triple-negative breast cancer

Abstract Although only 15-20% of total breast cancer diagnoses are of the triple-negative breast cancer (TNBC) subtype, they account for a significant portion of the mortality rate due to their more aggressive phenotype and a high risk of reoccurrence. Metabolic rewiring supports breast cancer progression and metastasis, particularly in ER-negative and triple-negative (TNBC) breast tumors. Thus, we examined the effects of BPM 31510, a metabolic-modulating agent in clinical trials for solid tumors, in in vitro and in vivo ER-negative and TNBC models. BPM 31510 EC50/EC>90 values were determined for a panel of the breast cancer cell lines and compared to non-tumorigenic MCF12A cells in vitro, and the MDA-MB231 and SkBr-3, TNBC and ER-negative models respectively, were found to be the most sensitive to BPM 31510. Treatment with BPM 31510 (EC50 and EC>90 doses) resulted in a time- and dose-dependent decrease the viable cell population (PI- and Annexin V-negative) and a concomitant increase in cells in early and late apoptosis (PI-negative and PI-positive Annexin V-positive cells, respectively), suggesting that BPM 31510 activates regulated cell death pathways. Consistent with the in vitro data, MDA-MB231 tumor-bearing mice had smaller tumors after 30 days of treatment with BPM 31510 and increased cleaved caspase 3 staining in resected tumors. In vitro, BPM 31510-dependent breast cancer cell death was preceded by mitochondrial membrane potential depolarization (TMRE flow cytometry) and alterations in mitochondrial respiration characterized by a consistent, dose-dependent decrease in succinate (Complex II)-fueled respiration with more varied responses to BPM 31510 in cells provided the Complex I substrates (pyruvate or palmitoyl carnitine). To investigate the role of Complex II in BPM 31510-mediated cell death, pharmacological inhibitors of the dicarboxylate site (malonate) and Qp site (atpenin A5) of Complex II were used in combination with BPM 31510 to assess the resultant effects on cell death in MDA-MB231 cells. Co-treatment with malonate significantly attenuated BPM 31510-mediated cell death while atpenin A5 did not affect BPM 31510-induced cell death, indicating succinate oxidation at the dicarboxylate site of Complex II is required, in part, for induction of cell death by BPM 31510. Together, these data demonstrate BPM 31510 has a potent anti-cancer activity in preclinical breast cancer models and define a functional link between Complex II activity and the mechanism of action for BPM 31510. Citation Format: Tulin Dadali, Anne R. Diers, Arleide Lee, Ezer Benaim, Joaquin J. Jimenez, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM 31510-induced alteration in Complex II activity is functionally linked to cell death activation pathway in a preclinical model of triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 208.

Metabolic profiling of mussel larvae: effect of handling and culture conditions

Gas chromatography mass spectroscopy was applied to characterize the metabolic profiles of hatchery-reared mussel (Perna canaliculus) larvae before and after a prolonged handling and water exchange process, and to investigate the effect of culture conditions. A decrease in succinate and an increase in alanine were observed after the water exchange, which indicated alterations in energy production and osmotic balance. However, these variations were subtle and it is unlikely that the water exchange practice had any lasting negative effects on larval physiology and performance. Multivariate pattern recognition tools (hierarchical clustering, principal component analysis and projection to latent squares discriminant analysis) were used to assess metabolite variations in larvae reared in low-density static and high-density flow through systems and to construct a culture condition classification model. Twelve metabolites contributed most towards the model, which indicated differences in energy, protein and lipid metabolism. The clear group separations were not represented by observable variations in morphological traits. This suggests that growth performance is metabolically buffered through an adaptive physiological mechanism to provide similar developmental characteristics under these conditions.

Computational Analysis of Reactive Oxygen Species Generation by Mitochondria Resulting from Substrate Manipulation

Mitochondria tightly couple the transport of electrons and proton pumping with oxidative phosphorylation to synthesize ATP. However, such energetic processes can “leak” electrons to oxygen forming reactive oxygen species (ROS). Increased ROS associated with oxidative stress has been implicated in numerous pathological conditions. To better understand the complex system regulating ROS, we present a mechanistic mitochondrial model that includes the electron transport chain, tricarboxylic acid cycle kinetics, mitochondrial calcium, sodium and proton handling, membrane ion transport processes that is coupled to oxidative phosphorylation, generation of ROS by complexes I and III, and matrix and extramitochondrial antioxidant defenses. Consistent with earlier study we found that in heart mitochondria respiring in presence of complex-II linked substrate succinate there is an increased ROS production with increasing membrane potential. Studies from experiments suggest that this relationship between ROS production and ΔѰm due to substrate modification is attributed to the process of Reverse Electron Transport. However, the model suggests that ROS generation with increasing membrane potential in presence of succinate is not because of reverse electron flow, but occurs because of the fact that as the mitochondrial membrane potential increases, it becomes harder for the protons to be pumped out to the p-side, thereby decreasing proton pumpingflux, and the most favorable pathway for the electrons is to move forward and cause the single electron reduction of oxygen to produce more ROS. Furthermore the mechanism of malonate induced reduction in ROS production is not because of decrease in reverse electron flow due to decrease in succinate, but occurs owing to the fact that since succinate oxidation decreases, there is tendency of electrons to flow across the respiratory chain to maintain the diminishing ΔѰm rather than leaking them to produce ROS.

Antioxidant function of alternative oxidase in mitochondria of winter wheat during cold hardening

The activity of alternative oxidase (AOX) and generation of reactive oxygen species (ROS) in mitochondria of winter wheat Triticum aestivum L. isolated from seedlings subjected to one (7-day exposure to 2–3°C) and two (7-day exposure to 2–3°C and 2-day exposure to −2°C) phases of a cold hardening has been studied. The antioxidant role of AOX in the first phase of the cold hardening has been determined using inhibitors of respiratory chain. Exposure to low temperature was shown to lead to inhibition of cytochrome pathway in mitochondria, increase of ROS production, and switching of the electron transport to the alternative pathway. Decrease in succinate- and antimycin A-induced ROS generation was found during two phases of cold hardening. This fact may point out to functioning of uncoupling proteins under these conditions. Thus, antioxidant function of AOX during the first phase of cold hardening may be an important component of the cold adaptation mechanism in winter crops. The data suggest that ROS and free fatty acids may be signal molecules regulating the activity of two energy-dissipation systems (AOX and uncoupling proteins).

Metabolic responses in gills of Manila clam Ruditapes philippinarum exposed to copper using NMR-based metabolomics

Copper is an important heavy metal contaminant with high ecological risk in the Bohai Sea. In this study, the metabolic responses in the bioindicator, Manila clam ( Ruditapes philippinarum), to the environmentally relevant copper exposures were characterized using NMR-based metabolomics. The significant metabolic changes corresponding to copper exposures were related to osmolytes, intermediates of the Krebs cycle and amino acids, such as the increase in homarine, branched chain amino acids and decrease in succinate, alanine and dimethylamine in the copper-exposed clam gills during 96 h exposure period. Overall, Cu may lead to the disturbances in osmotic regulation and energy metabolism in clams during 96 h experimental period. These results demonstrate that NMR-based metabolomics is applicable for the discovery of metabolic biomarkers which could be used to elucidate the toxicological mechanisms of marine heavy metal contaminants.

Furoxan-, alkylnitrate-derivatives and related compounds as anti-trypanosomatid agents: Mechanism of action studies

A series of over a hundred furoxans, alkylnitrates and related compounds were studied as growth inhibitors of the two major kinetoplastids of Latin America, Trypanosoma cruziand Leishmania spp., in in vitro assays. The most active compounds showed 50% inhibitory doses of the same order of that of Nifurtimox and Miltefosine, reference drugs used to treat Chagas Disease and Leishmaniasis respectively. Among the studied compounds derivative 4, presenting excellent inhibitory activity against the tryposmastigote and amastigote forms of T. cruzi, has emerged as a lead compound. Mechanism of action seems to involve mitochondrial dehydrogenases as a distinct effect with respect to Nifurtimox. Excreted metabolites, studied by NMR, showed a significant decrease in succinate, confirming the observed effect on the mitochrondrial dehydrogenases.

Metabolomics by Proton Nuclear Magnetic Resonance Spectroscopy of the Response to Chloroethylnitrosourea Reveals Drug Efficacy and Tumor Adaptive Metabolic Pathways

Metabolomics of tumors may allow discovery of tumor biomarkers and metabolic therapeutic targets. Metabolomics by two-dimensional proton high-resolution magic angle spinning nuclear magnetic resonance spectroscopy was applied to investigate metabolite disorders following treatment by chloroethylnitrosourea of murine B16 melanoma (n = 33) and 3LL pulmonary carcinoma (n = 31) in vivo. Treated tumors of both types resumed growth after a delay. Nitrosoureas provoke DNA damage but the metabolic consequences of genotoxic stress are little known yet. Although some differences were observed in the metabolite profile of untreated tumor types, the prominent metabolic features of the response to nitrosourea were common to both. During the growth inhibition phase, there was an accumulation of glucose (more than x10; P < 0.05), glutamine (x3 to 4; P < 0.01), and aspartate (x2 to 5; P < 0.01). This response testified to nucleoside de novo synthesis down-regulation and drug efficacy. However, this phase also involved the increase in alanine (P < 0.001 in B16 melanoma), the decrease in succinate (P < 0.001), and the accumulation of serine-derived metabolites (glycine, phosphoethanolamine, and formate; P < 0.01). This response witnessed the activation of pathways implicated in energy production and resumption of nucleotide de novo synthesis, thus metabolic pathways of DNA repair and adaptation to treatment. During the growth recovery phase, it remained polyunsaturated fatty acid accumulation (x1.5 to 2; P < 0.05) and reduced utilization of glucose compared with glutamine (P < 0.05), a metabolic fingerprint of adaptation. Thus, this study provides the proof of principle that metabolomics of tumor response to an anticancer agent may help discover metabolic pathways of drug efficacy and adaptation to treatment.

Metabolic Engineering of Corynebacterium glutamicum for Fuel Ethanol Production under Oxygen-Deprivation Conditions

The central metabolic pathway of Corynebacterium glutamicum was engineered to produce ethanol. A recombinant strain which expressed the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) was constructed. Both genes placed under the control of the C. glutamicum ldhA promoter were expressed at high levels in C. glutamicum, resulting, under oxygen-deprivation conditions, in a significant yield ofethanol from glucose in a process characterized by the absence of cellular growth. Addition of pyruvate in trace amounts to the reaction mixture induced a 2-fold increase in the ethanol production rate. A similar effect was observed when acetaldehyde was added. Disruption of the lactate dehydrogenase (ldhA) gene led to a 3-fold higher ethanol yield than wild type, with no lactate production. Moreover, inactivation of the phosphoenolpyruvate carboxylase (ppc) and ldhA genes revealed a significant amount of ethanol production and a dramatic decrease in succinate without any lactate production, when pyruvate was added. Since the reaction occurred in the absence of cell growth, the ethanol volumetric productivity increased in proportion to cell density of ethanologenic C. glutamicum in a process under oxygen-deprivation conditions. These observations corroborate the view that intracellular NADH concentrations in C. glutamicum are correlated to oxygen-deprived metabolic flows.

Succinate:quinol oxidoreductases in the cyanobacterium synechocystis sp. strain PCC 6803: presence and function in metabolism and electron transport.

The open reading frames sll1625 and sll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp. strain PCC 6803. When the organic acid content in the Deltasll1625 and Deltasll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Deltasll1625 Deltasll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.

1H NMR investigation of toxic effects of lindane and paraquat on Hep 3B and Hep G2 human hepatoma cell lines.

Lindane and paraquat induce biochemical changes in the liver. In order to specify their molecular impact at the cellular level, a 300 MHz 1H NMR investigation of hepatoma cell lines Hep 3B and Hep G2 responses was performed. Cells were exposed over 24 h to 50 mg/L lindane (0.178 mM) or to 100 mg/L (0.389 mM) paraquat concentrations. The main observation following exposure to lindane was a decrease in betaine methyl groups (3.26 ppm) which could be related to the steatosis reported by some authors. Specifically, in Hep G2 cells with this pesticide, the glycine peak (3.56 ppm) was lowered, thus confirming that the glycine synthesis pathway involving methionine, choline, and betaine was disturbed by lindane. Moreover, in this hepatoma cell line, the p-chlorobenzoate ion could be detected as a doublet at 7.55 ppm. In Hep 3B cells, paraquat increased betaine and methionine levels, suggesting disturbance in glycine biosynthesis. Possibilities of cellular uptake were considered, and the presence of this herbicide in cells was revealed by spectrophotometric and NMR measurements after chlorhydric hydrolysis, suggesting interaction with cellular components. The impact of paraquat on Hep G2 cells appeared to be located on mitochondrial function, as indicated by the observed decrease in succinate and pyruvate levels.