Mitochondrial Supernatant Research Articles

Fabrication and evaluation of mannose decorated curcumin loaded nanostructured lipid carriers for hepatocyte targeting: In vivo hepatoprotective activity in Wistar rats

Curcumin is a well-recognized antioxidant phytoactive isolated from the rhizomes of Curcuma longa. Numerous landmark investigations have proved the antioxidant and hepatoprotective potential of curcumin. The aim of present study was to target curcumin loaded nanocarriers to hepatocytes using asialoglycoprotein receptors targeting strategy. Mannose, a water-soluble carbohydrate, was hydrophobized by anchoring stearylamine with an objective to conjugate mannose on the surface of curcumin loaded nanostructured lipid carriers for targeting asialoglycoprotein receptors on hepatocytes. Mannose conjugated stearylamine was synthesized and characterized using various analytical techniques. The synthesized targeting ligand was incorporated curcumin loaded nanostructured lipid carriers and characterized by photon correlation spectroscopy. Zeta potential measurement was used to confirm the conjugation of the synthesized ligand to the surface of drug-loaded nanostructured lipid carriers. CCl4 induced hepatotoxicity in male Wistar rats was used as an experimental animal model to evaluate the hepatoprotective potential of formulated drug encapsulated nanostructured lipid carriers. The hepatoprotective potential was assessed by measuring serum liver injury markers and oxidative stress parameters in the liver post–mitochondrial supernatant. Mannose conjugated nanostructured lipid carriers showed acceptable particle size which revealed its suitability for hepatocyte targeting. In addition to this, mannose conjugated nanocarriers revealed significantly better (p ​< ​0.05) reduction of serum liver injury markers and proinflammatory cytokines compared to the unconjugated one which confirmed hepatocytes targeting potential of the synthesized ligand. Asialoglycoprotein receptors targeting could be a landmark strategy for hepatocyte targeting. Thus, the synthesized mannose anchored stearylamine could be a promising novel targeting ligand having hepatocyte targeting potential.

INFLUENCE OF ARTESUNATE COMBINATIVE THERAPY CO-ADMINISTRATION WITH RUTIN ON INFLAMMATORY CYTOKINES AND IMMUNOGLOBULINS IN PLASMODIUM BERGHEI-INFECTED MICE.

Some antimalarial drugs are immune-modulators that impact multiple pathways of innate immunity in malarial treatment. However, information on the immunomodulatory effects of artequine and rutin in the treatment of malaria remains elusive. Twenty-five Swiss mice (18 ± 2 g) were used for this study. Twenty were infected with Plasmodium berghei (NK65). Parasitemia was confirmed, and the animals were grouped (n = 5) as follows: Group A was not infected but treated orally with vehicle. Groups B to E were infected and treated (B) orally with vehicle (10 ml/kg), (C) with 10 mg/kg artequine, (D) with 10 mg/kg of artequine supplemented with 100 mg rutin/kg, and (D) with 10 mg/kg of artequine supplemented with 200 mg rutin/kg, for 7 days. Blood was collected for hematological, inflammatory cytokines, and immunoglobulins G and M assays. Post mitochondrial supernatant fraction was used for antioxidant assays. Rutin co-administration (200 mg/kg) significantly (P < 0.001) increased platelet and neutrophil counts (P < 0.01) but significantly (P < 0.01) decreased white blood cell count and lymphocyte relative to parasitized control. Also, it significantly (P < 0.05) decreased lipid peroxidation, xanthine oxidase, and superoxide dismutase activities but significantly (P < 0.05) increased reduced glutathione and glutathione S-transferase activity. Rutin co-administration also caused a significant (P < 0.001) increase in tumor necrosis factor-alpha, interleukin-6, and immunoglobulin M levels, while interleukin-1β and immunoglobulin G decreased significantly (P < 0.001) compared with parasitized control. These results showed that rutin co-administration with artequine improved host antioxidant status and modulated the immune and inflammatory responses.

Sialylated human milk oligosaccharides prevent intestinal inflammation by inhibiting toll like receptor 4/NLRP3 inflammasome pathway in necrotizing enterocolitis rats

BackgroundNecrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates and has very limited therapeutic options. Sialylated human milk oligosaccharides (SHMOs) improve pathological changes in experimental NEC models. The objectives of this study were to investigate the involvement of NLRP3 inflammasome in NEC pathology and to explore the effects of SHMOs on toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/NLRP3 inflammatory pathway in experimental NEC.MethodsThe intestinal-tissue segments were collected from NEC infants, NLRP3 and caspase-1 positive cell were examined by immunohistochemistry. Newborn rats were hand-fed with formula containing or non-containing SHMOs (1500 mg/L) and exposed to hypoxia/cold stress to induce experimental NEC. The NEC pathological scores were evaluated; ileum protein expression of membrane TLR4 (mTLR4), inhibitor κB-α (IκB-α), NF-κB p65 subunit and phospho-NF-κB p65, as well as NLRP3 and caspase-1 were analyzed; ileum concentrations of interleukin-1β, interleukin-6, tumor necrosis factor-α (TNF-α) were also measured. Human colon epithelial Caco-2 cells were pre-treated with or without SHMOs and stimulated with TLR4 activator, lipopolysaccharide. Cell viabilities, mitochondrial membrane potential and supernatant matrix metalloprotease 2 (MMP-2) activities were analyzed.ResultsIncreased frequencies of NLRP3 and caspase-1 positive cells were found in the lamina propria of damaged intestinal area of NEC neonates. SHMOs supplementation reduced NEC incidence and pathological damage scores of rats challenged with hypoxia/cold stress. Accumulation of interleukin-1β, interleukin-6 and TNF-α in NEC group were attenuated in SHMOs + NEC group. Protein expression of mTLR4, NLRP3 and caspase-1 were elevated, cytoplasmic IκB-α were reduced, nuclear phospho-NF-κB p65 were increased in the ileum of NEC rats. SHMOs supplementation ameliorated the elevation of mTLR4, NLRP3 and caspase-1, restored IκB-α in the cytoplasmic fraction and reduced phospho-NF-κB p65 in the nuclear fraction in the ileum of NEC rats. SHMOs pre-treatment improved Caco-2 cell viability, mitigated loss of mitochondrial membrane potential and modulated MMP-2 activities in the presence of lipopolysaccharide in-vitro.ConclusionsThis study provided clinical evidence of involvement of NLRP3 inflammasome in NEC pathology, and demonstrated the protective actions of SHMOs might be owing to the suppression of TLR4/NF-κB/NLRP3-mediated inflammation in NEC.

Demonstration of exogenous nuclear histone H3 binding to mitochondria and subsequent cytochrome c release in cauliflower

The life cycle of a cell is partly regulated by the programmed cell death (PCD) process. From development to demise, a cell's PCD process must respond to external signals and internal factors mediated by mitochondria. Previous studies show that the release of histones into the cytosol caused by DNA damage or loss of nuclear integrity is correlated with apoptosis in mammalian cells. These released histones bind to mitochondria and permeabilize its inner and outer membranes, which causes the release of cytochrome c into the cytosol that leads to caspase activation and the demise of the cell. Owing to the high conservation of histones, we hypothesize that histone-mediated cytochrome c release from mitochondria may be conserved across a wide range of eukaryotes. We investigated this histone-mitochondrial interaction in cauliflower using density-gradient purified mitochondria and exogenous histones from a crude histone fraction, then added the exogenous histone fractions to the purified cauliflower mitochondria and analyzed the mitochondrial pellets and supernatants by immunoblotting against cytochrome c and H3. Our data clearly shows that histone-enriched fractions elicited cytochrome c release from mitochondria, and that mitochondria bind exogenous histone H3.

Localization and distribution of connexin 43 in mitochondria of cervical cancer HeLa cells

Objective To study the distribution of connexin 43 (Cx43) in cervical cancer HeLa cells, and to verify the localization of Cx43 in mitochondria. Methods HeLa cells were segregated into cytoplasm, cell nucleus, mitochondria and supernatant after segregation by using the method of homogenate and centrifuge. Immunoelectron microscope was used to observe the morphology of mitochondria and the localization as well as the distribution of Cx43 in HeLa cells. Voltage-dependent anion channel 1 (VDAC1) was used to confirm the localization of mitochondria. Immunofluorescence was used to costain HeLa cells with Cx43 and mitochondrial marker VDAC1 to verify mitochondria localization of Cx43 in cervical cancer HeLa cells. Then Western blot was used to quantify the expression of Cx43 in fractions (cytoplasmic fraction, nuclear, mitochondria and post mitochondrial supernatant). Mitochondrial markers including VDAC1 and cytochrome c oxidase Ⅳ (COX Ⅳ) were used to confirm mitochondria. Plasma membrane marker (LHR) was used to confirm plasma membrane. Results Immunoelectron microscope confirmed that the normal mitochondria or cystic swollen one could be seen in the complete HeLa cells and the detached HeLa cells mitochondria, with the presence of Cx43 and VDAC1 in detached mitochondria. Immunofluorescence showed Cx43 colocalized with VDAC1. There was a significant difference in the Cx43 expressions of the subcellular structure in the HeLa cells [cytoplasm (1.23±0.11), cell nucleus (0.39±0.09), mitochondria (3.67±0.59), supernatant after segregation (0.16±0.06); F = 84.17, P < 0.05]. It also showed that the relative amount of Cx43 in mitochondria was enriched. Conclusions Cx43 is enriched in mitochondria in cervical cancer HeLa cells. Therefore, Cx43 in mitochondria might be a potential target in diagnosis, therapy and prognosis of cervical cancer. Key words: HeLa cells; Connexin 43; Mitochondria; Localization; Distribution

A mitochondrial protein increases glycolytic flux

The purpose of this study was to determine the role of mitochondria in postmortem muscle metabolism. Isolated mitochondria were incorporated into a reaction buffer that mimics postmortem glycolysis with or without mitochondrial electron transport chain inhibitors. Addition of mitochondria lowered pH values at 240 and 1440min regardless of inhibitors. Reduction in pH was accompanied by enhanced glycogen degradation and lactate accumulation. To explore the mechanism responsible for this exaggerated metabolism, mitochondrial preparations were mechanically disrupted and centrifuged. Resulting supernatants and pellets each were added to the in vitro model. Mitochondrial supernatants produced similar effects as those including intact mitochondria. To narrow further our target of investigation, mitochondrial supernatants were deproteinized with perchloric acid. The effect of mitochondrial supernatant was lost after perchloric acid treatment. These data indicate that a mitochondrial-based protein is capable of increasing glycolytic flux in an in vitro model and may partially explain acid meat development in highly oxidative AMPKγ3R200Q mutated pigs.

Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Treated with Single and Combinatorial Herbal Formulations.

Background/Aim: The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Materials and Methods: Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Results: Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was with in the range of 66.30±0.71-133.00±0.63 mg/dL compared to the untreated hyperglycemic rat group=368.20±0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/GSSG ratios were significantly (p<0.05) increased when compared with treated HyGR. Conclusion: Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Antioxidant Status of Liver and Kidney Homogenates from Hyperglycemic Rats Administered with Single and Combinatorial Herbal Formulations

Aims: The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Materials and Methods: Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Results: Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was within the range of 66.30 ± 0.71133.00 ± 0.63 mg/dL as against the untreated hyperglycemic rat group=368.20 ± 0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/ GSSG ratios were significantly ( p<0.05) increased when compared with treated HyGR. Conclusion: Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

Glycemic Indices/Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Treated with Single and Combinatorial Herbal Formulations

The present study ascertained the comparative capacities of single and combinatorial herbal formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats (HyGR) following 30 days treatment. Phytochemical composition of the herbal extracts and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione (GSH) concentration. Comparatively, flavonoids were the most abundant phytochemical present in the four herbal extracts. FPGC of treated HyGR was within the range of 66.30 ± 0.71 - 133.00 ± 0.63 mg/dL as against the untreated hyperglycemic rat group=368.20 ± 0.72 mg/dL (p<0.05). At the end of the experimental time, treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/GSSG ratios were significantly (p<0.05) increased when compared with treated HyGR. Combinatorial herbal formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on renal and hepatic antioxidants against reactive oxygen species.

English

Pathogenesis of several chronic liver diseases has been attributed to overwhelmed antioxidant protective system against reactive oxygen species (ROS). The present study ascertained the capacity of short-term administration of ethanolic extract of Allium sativa to neutralize ROS and ameliorate hyperlipidemia. Hyperlipidemia was induced in rats by single intra-peritoneal injection of CCl4 (dosage = 2.0 mL/kg), followed by treatment with ethanolic extract of A. sativa (dosage: 200 and 400 mg/kg) at a regular interval of 16 for 64 h. Blood samples were drawn from the rats at t = 0 h and t = 76 h, that is, 12 h after the end of 64 h treatment with CCl4/A. sativa extract, to ascertain for hepatic function and serum lipid profile (SLP). In addition, liver post mitochondrial supernatant (PMS) fraction was measured for oxidative stress indicators: lipid peroxidation (LPOx), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and reduced glutathione (GSH). On the average, short-term administration of ethanolic extract of A. sativa caused reduction of SLP in the following magnitude: total cholesterol (TC) = 19.48%, triacylglycerol (TAG) = 48.59%, VLDL-C = 48.57%, LDL-C = 19.49% and increase in HDL-C = 32.43%. Also, improvement in oxidative stress indicators gave SOD = 10.20%, GPx = 30.92%, CAT = 18.18%, LPOx = 35.92% and GSH = 51.09%. Although the administration of A. sativa extract to the rats did not restore full therapeutic benefits within the experimental time (t = 76 h), the capacity of the plant extract to ameliorate oxidative stress and hyperlipidemia in the animals was fairly at par with the standard hepatic drug-hepaticum. &nbsp; Key words: Allium sativa, hepatocyte, hyperlipidemia, lipid profile, oxidative stress.

Free radical scavenging potential of in vitro raised and greenhouse acclimatized plants of Artemisia amygdalina

AimArtemisia amygdalina Decne. (Asteraceae) is a critically endangered and endemic herb of Kashmir Himalayan sub-alpine region and Pakistan. Scientific research throughout the world has evidence to support the tremendous medicinal utility of the genus Artemisia. The natural resources of medicinal plants are being reduced day by day. This study provides the alternative way for medicinal resource utilization and conservation of A. amygdalina. MethodsIn vitro-raised plants and greenhouse acclimatized plants were obtained by culturing wild explants on Murashige and Skoog's medium. Plant extracts were obtained and subjected to different antioxidant assays: DPPH assay, riboflavin photo-oxidation assay, deoxy ribose assay, ferric thiocyanate assay, thiobarbituric acid assay, post mitochondrial supernatant assay and DNA damage on agarose gel. ResultsIn vitro grown plants, as well as those acclimatized in the greenhouse reveals antioxidant activity against hydroxyl, superoxide, and lipid peroxyl radicals. ConclusionThis preliminary study revealed the free radical scavenging potential of tissue culture-raised plant extracts of A. amydalina.

Ethylmalonyl-CoA Decarboxylase, a New Enzyme Involved in Metabolite Proofreading

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.

Antioxidant potential and radioprotective effect of soy isoflavone against gamma irradiation induced oxidative stress

The in vitro antioxidant potential and in vivo radioprotective ability of soy isoflavones was studied. Male Wistar rats were orally administered with soybean isoflavones (60mg/kg) for 21days followed by gamma irradiation exposure. Survival studies in rats exposed at 10Gy and endogenous spleen colony forming unit assay (CFU) at 6.0Gy were performed in order to find radioprotective and immunomodulatory nature of the compound. The rat liver post mitochondrial supernatant and erythrocytes were used to measure lipid peroxidation (LPO) and glutathione (GSH) content along with various antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) after gamma irradiation exposure at 2.0Gy. Pretreatment with soy isoflavone, prior to gamma irradiation resulted in the increased survival rate of the animals as compared to irradiated group. CFU counts in the isoflavone treated group followed by gamma irradiation at 6 Gy were significantly high as compared to control and the irradiated group, showing immunomodulatory nature of the isoflavones. Pretreatment with isoflavones also significantly reduced the LPO, enhanced the activity of antioxidant enzymes and improved haematological and histological parameters. The present study suggests that supplementation with isoflavone has potent antioxidant activity and act as probable radioprotector against gamma radiation induced oxidative damage.

Abstract B79: Multidrug resistance protein 1 (MRP-1) is expressed in the mitochondria of normal and cancer cells and may play a role in intrinsic drug resistance of ESFT cells.

Abstract Overexpression of plasma membrane MRP-1 can lead to multidrug resistance. In this study we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues and investigate the functional significance of mitochondrial MRP-1 with a view to identifying a new therapeutic starategy. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Protein extracts were de-glycosylated using the Protein Deglycosylation Mix (NEB). Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased by retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-PI labelling of cells. Mitochondrial membranes were solubilised by incubation with increasing concentrations of the detergent digitonin and MRP-1 localisation within the mitochondria evaluated by Western blot; protein markers of the mitochondrial outer and inner membranes, the matrix and intermembrane space were used to confirm localisation within the mitochondria. For the first time we have detected MRP-1 in the mitochondria of untreated cancer and normal cells and tissues by Western blot and confirmed these findings by immunofluorescence and confocal microscopy. The size of the MRP-1 protein in the mitochondria of cancer and normal cells was between 150–250 kDa, reflecting differences in glycosylation. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than plasma membrane MRP-1 (11–22%; p&amp;lt;0.001). Specific overexpression of MRP-1 in TC-32 cells by retroviral infection induced a preferential increase in mitochondrial MRP-1 (150%), resulting in an 85% increase in MRP-1 efflux activity (p&amp;lt;0.0001), suggesting selective targeting to this organelle. This was associated with an increase in the IC50 of the MRP-1 substrates vincristine (4.5nM to 14.7nM; p&amp;lt;0.001) and etoposide (101.7nM to 121.6nM (p&amp;lt;0.001) but not the non MRP-1 substrate actinomycin D (0.5nM). Treatment with a non-lethal concentration of doxorubicin (0.85nM, 8h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. The glycosylation of MRP-1 and the efflux of calcein-F from whole mitochondria are consistent with localisation of MRP-1 to the outer mitochondrial membrane. This was confirmed by the increased expression of MRP-1 protein in the mitochondrial supernatant after treatment with digitonin and centrifugation. The expression pattern of MRP-1 was similar to that of porin, an outer mitochondrial membrane marker. Analysis of the MRP-1 sequence (using Predator and MitoProtII) failed to reveal published mitochondrial targeting sequences, and so we are currently investigating the mechanism of MRP-1 transport to the mitochondria to identify a druggable therapeutic target. MRP-1 expressed in the outer mitochondrial membrane has increased efflux activity compared to plasma membrane MRP-1, and so represents an exciting new therapeutic target where historically conventional MRP-1 inhibitor strategies targeting plasma membrane MRP-1 have had limited clinical success. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B79.

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1G93A motor neurons.

Inhibition of Iron Induced Lipid Peroxidation and Antioxidant Activity of Indian Spices and Acacia In Vitro

The spices used in the Indian foods such as Star anise (Illicium verum), Bay leaves (Cinnamomum zeylanicum) and Cobra's saffron (Mesua ferrea), and Acacia (Acacia catechu), which have medicinal value, were used as test samples, to find their effect on in vitro lipid peroxidation (LPO). Rat liver post mitochondrial supernatant (PMS) in Tris HCl buffer, pH 7.4 was incubated for 0 and 1 h, with various test extracts in three different oxidant systems. The results show that addition of test samples to FeCl(3) medium at 0 h significantly stop the initiation of the LPO. However, the propagation phase of LPO was inhibited by Cobra's saffron and Acacia and not by Star anise and Bay leaves. The test samples also showed strong reducing power and superoxide radical scavenging activity. Cobra's saffron and Acacia showed the highest antioxidant activity, probably due to the higher polyphenol content as compared to other test samples.

Impact of phorate on malate dehydrogenases, lactate dehydrogenase and proteins of epigeic, anecic and endogeic earthworms

The effects of an organophosphate pesticide phorate on cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH), lactate dehydrogenase (LDH), supernatant and mitochondrial proteins of an epigeic ( Perionyx sansibaricus), anecic ( Lampito mauritii) and endogeic ( Metaphire posthuma) earthworms were studied. The treatment of different concentrations (20, 40, 80 and 160 ppm) of phorate for 16 days gradually decreased the specific activities of cMDH, mMDH and LDH as well as cytoplasmic and mitochondrial protein contents. This showed the inhibitory effect of phorate on metabolic enzymes and proteins in tropical earthworms. The inhibition was dose- and time-dependent. The inhibitory response in mitochondrial enzyme (mMDH) and protein was somewhat earlier and more as compared to the inhibitory effect of phorate on cytoplasmic enzymes (cMDH, LDH) and protein. This indicates a greater interference of phorate in cellular respiration of earthworms. The phorate related decreases in enzyme and protein profiles were about 60% and 58% in P. sansibaricus, 54% and 49% in L. mauritii and 47% and 42% in M. posthuma, respectively. It reflects phorate-induced substantial decline in protein synthesis and aerobic and anaerobic capacity of earthworms. The maximum effect of phorate was on epigeic earthworm followed by anecic and endogeic species. The present findings suggest the differential sensitivity of different earthworm species in enzymatic and protein responses to phorate and the sensitivity was associated with the ecophysiological categories of earthworms.

Identification and Characterization of AIFsh2, a Mitochondrial Apoptosis-inducing Factor (AIF) Isoform with NADH Oxidase Activity

Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.

SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans. Because adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat. We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane. Caloric restriction activates SIRT3 expression in both white and brown adipose. Additionally, cold exposure up-regulates SIRT3 expression in brown fat, whereas elevated climate temperature reduces the expression. Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1alpha, UCP1, and a series of mitochondria-related genes. Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action. Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression. This inhibitive effect can be abolished by the coexpression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action. In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species production while increasing cellular respiration. Finally, SIRT3, along with genes related to mitochondrial function, is down-regulated in the brown adipose tissue of several genetically obese mice. In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.

Aep3p Stabilizes the Mitochondrial Bicistronic mRNA Encoding Subunits 6 and 8 of the H+-translocating ATP Synthase of Saccharomyces cerevisiae

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to approximately 35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.