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Mitochondrial Membrane (mitochondrial + membrane)
Kinds of Mitochondrial Membrane Terms modified by Mitochondrial Membrane Selected AbstractsCorrelation of Hippocampal Glucose Oxidation Capacity and Interictal FDG-PET in Temporal Lobe EpilepsyEPILEPSIA, Issue 2 2003Stefan Vielhaber Summary: ,Purpose: Interictal [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) demonstrates temporal hypometabolism in the epileptogenic zone of 60,90% of patients with temporal lobe epilepsy. The pathophysiology of this finding is still unknown. Several studies failed to show a correlation between hippocampal FDG-PET hypometabolism and neuronal cell loss. Because FDG is metabolized by hexokinase bound to the outer mitochondrial membrane, we correlated the glucose-oxidation capacity of hippocampal subfields obtained after surgical resection with the corresponding hippocampal presurgical FDG-PET activity. Methods: In 16 patients with electrophysiologically confirmed temporal lobe epilepsy, we used high-resolution respirometry to determine the basal and maximal glucose-oxidation rates in 400-,m-thick hippocampal subfields obtained after dissection of human hippocampal slices into the CA1 and CA3 pyramidal subfields and the dentate gyrus. Results: We observed a correlation of the FDG-PET activity with the maximal glucose-oxidation rate of the CA3 pyramidal subfields (rp = 0.7, p = 0.003) but not for the regions CA1 and dentate gyrus. In accordance with previous studies, no correlation of the FDG-PET to the neuronal cell density of CA1, CA3, and dentate gyrus was found. Conclusions: The interictal hippocampal FDG-PET hypometabolism in patients with temporal lobe epilepsy is correlated to the glucose-oxidation capacity of the CA3 hippocampal subfield as result of impaired oxidative metabolism. [source] BimEL as a possible molecular link between proteasome dysfunction and cell death induced by mutant huntingtinEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2010Rebecca Leon Abstract Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the N-terminus of the huntingtin protein. It is characterized by a selective loss of medium spiny neurons in the striatum. It has been suggested that impaired proteasome function and endoplasmic reticulum (ER) stress play important roles in mutant huntingtin (mHtt)-induced cell death. However, the molecular link involved is poorly understood. In the present study, we identified the essential role of the extra long form of Bim (Bcl-2 interacting mediator of cell death), BimEL, in mHtt-induced cell death. BimEL protein expression level was significantly increased in cell lines expressing the N-terminus of mHtt and in a mouse model of HD. Although quantitative RT-PCR analysis indicated that BimEL mRNA was increased in cells expressing mHtt, we provided evidence showing that, at the post-translational level, phosphorylation of BimEL played a more important role in regulating BimEL expression. Up-regulation of BimEL facilitated the translocation of Bax to the mitochondrial membrane, which further led to cytochrome c release and cell death. On the other hand, knocking down BimEL expression prevented mHtt-induced cell death. Taken together, these findings suggest that BimEL is a key element in regulating mHtt-induced cell death. A model depicting the role of BimEL in linking mHtt-induced ER stress and proteasome dysfunction to cell death is proposed. [source] Interaction of the alpha-helical H6 peptide from the pro-apoptotic protein tBid with cardiolipinFEBS JOURNAL, Issue 21 2009Patrice X. Petit BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices ,H6 and ,H7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c -releasing activity. In the present study, we focused on the interaction between the ,H6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the ,H6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of ,H6 into the phospholipid monolayer. The modification of two charged amino acid residues in ,H6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and ,H6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that ,H6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via ,H6. [source] Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissuesFEBS JOURNAL, Issue 13 2002Araceli Del Arco Aralar1 and citrin are members of the subfamily of calcium-binding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown. We investigated the pattern of expression of aralar1 and citrin in murine embryonic and adult tissues at the mRNA and protein levels. In situ hybridization analysis indicates that both isoforms are expressed strongly in the branchial arches, dermomyotome, limb and tail buds at early embryonic stages. However, citrin was more abundant in the ectodermal components of these structures whereas aralarl had a predominantly mesenchymal localization. The strong expression of citrin in the liver was acquired postnatally, whereas the characteristic expression of aralar1 in skeletal muscle was detected at E18 and that in the heart began early in development (E11) and was preferentially localized to auricular myocardium in late embryonic stages. Aralar1 was also expressed in bone marrow, T-lymphocytes and macrophages, including Kupffer cells in the liver, indicating that this is the major AGC isoform present in the hematopoietic system. Both aralar1 and citrin were expressed in fetal gut and adult stomach, ovary, testis, and pancreas, but only aralar1 is enriched in lung and insulin-secreting ,,cells. These results show that aralar1 is expressed in many more tissues than originally believed and is absent from hepatocytes, where citrin is the only AGC isoform present. This explains why citrin deficiency in humans (type II citrullinemia) only affects the liver and suggests that aralar1 may compensate for the lack of citrin in other tissues. [source] Curvature properties of novel forms of phosphatidylcholine with branched acyl chainsFEBS JOURNAL, Issue 10 2000Richard M. Epand We studied the properties of a series of phosphatidylcholine molecules with branched acyl chains. These lipids have previously been shown to have marked stimulatory effects on the side-chain cleavage activity of cytochrome P450SCC (CYP11A1), an enzyme of the inner mitochondrial membrane. The synthetic lipids used were diacyl phosphatidylcholines with the decanoyl, dodecanoyl or tetradecanoyl chain having a hexyl, octyl or decyl straight chain aliphatic branch at the 2-position. All three lipids lowered the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine, the lipids with longer acyl chains being more effective in this regard. As pure lipids all of the forms were found by X-ray diffraction to be predominantly in the hexagonal phase (HII) over the entire temperature range of 7,75 °C. The properties of the HII phase were unusual with regard to the small size of the lattice spacings and the small temperature dependence of the spacings. We used tetradecane to relieve hydrocarbon packing constraints to determine the intrinsic radius of curvature of the lipid monolayer. The elastic bending modulus was measured in the presence of tetradecane by introducing an osmotic gradient across the hexagonal phase cylinders with aqueous solutions of poly(ethylene glycol). The elastic bending modulus was found to be higher than that observed with other lipids and to increase with temperature. Both the small intrinsic radius of curvature and the high elastic bending modulus indicate that the presence of these lipids in bilayer membranes will impose a high degree of negative curvature strain. [source] Organelle-specific expression of subunit ND5 of human complex I (NADH dehydrogenase) alters cation homeostasis in Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 6 2010Wojtek Steffen Abstract The ND5 component of the respiratory complex I is a large, hydrophobic subunit encoded by the mitochondrial genome. Its bacterial homologue, the NDH-1 subunit NuoL, acts as a cation transporter in the absence of other NDH-1 subunits. Mutations in human ND5 are frequently observed in neurodegenerative diseases. Wild type and mutant variants of ND5 fused to GFP or a FLAG peptide were targeted to the endoplasmatic reticulum (ER) or the inner mitochondrial membrane of Saccharomyces cerevisiae, which lacks an endogenous complex I. The localization of ND5 fusion proteins was confirmed by microscopic analyses of S. cerevisiae cells, followed by cellular fractionation and immunostaining. The impact of the expression of ND5 fusion proteins on the growth of S. cerevisiae in the presence and absence of added salts was studied. ER-resident ND5 conferred Li+ sensitivity to S. cerevisiae, which was lost when the E145V variant of ND5 was expressed. All variants of ND5 tested led to increased resistance of S. cerevisiae at high external concentrations of Na+ or K+. The data seem to indicate that ND5 influences the salt homeostasis of S. cerevisiae independent of other complex I subunits, and paves the way for functional studies of mutations found in mitochondrially encoded complex I genes. [source] Mitochondrial preprotein translocases as dynamic molecular machinesFEMS YEAST RESEARCH, Issue 6 2006Martin Van Der Laan Abstract Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments , outer membrane, intermembrane space (IMS), inner membrane and matrix , requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria. [source] Involvement of Ca2+ and ROS in ,-tocopheryl succinate-induced mitochondrial permeabilizationINTERNATIONAL JOURNAL OF CANCER, Issue 8 2010Vladimir Gogvadze Abstract Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox-silent analog of vitamin E, ,-tocopheryl succinate (,-TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax-mediated permeabilization of the outer mitochondrial membrane. Here we show that ,-TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T-lymphocytes. In particular, in addition to ROS production, ,-TOS stimulates rapid Ca2+ entry into the cells with subsequent accumulation of Ca2+ in mitochondria,a prerequisite step for MPT induction. Alteration of mitochondrial Ca2+ buffering capacity was observed as early as 8 hr after incubation with ,-TOS, when no activation of Bax was yet detected. Ca2+ accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca2+ uptake significantly mitigated the apoptotic response. Importantly, Ca2+ -induced mitochondrial destabilization might cooperate with Bax-mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria. [source] Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug-resistant colon carcinoma cells (LoVo) than on their wild-type counterpartsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2002Annarica Calcabrini Abstract The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H2O2 and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H2O2. Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells. © 2002 Wiley-Liss, Inc. [source] The adenine nucleotide translocase type 1 (ANT1): A new factor in mitochondrial diseaseIUBMB LIFE, Issue 9 2005J. Daniel Sharer Abstract Mitochondrial disorders of oxidative phosphorylation (OXPHOS) comprise a growing list of potentially lethal diseases caused by mutations in either mitochondrial (mtDNA) or nuclear DNA (nDNA). Two such conditions, autosomal dominant progressive external ophthalmoplegia (adPEO) and Senger's Syndrome, are associated with dysfunction of the heart and muscle-specific isoform of the adenine nucleotide translocase (ANT1), a nDNA gene product that facilitates transport of ATP and ADP across the inner mitochondrial membrane. AdPEO is a mtDNA deletion disorder broadly characterized by pathology involving the eyes, skeletal muscle, and central nervous system. In addition to ANT1, mutations in at least two other nuclear genes, twinkle and POLG, have been shown to cause mtDNA destabilization associated with adPEO. Senger's syndrome is an autosomal recessive condition characterized by congenital heart defects, abnormalities of skeletal muscle mitochondria, cataracts, and elevated circulatory levels of lactic acid. This syndrome is associated with severe depletion of ANT1, which may be the result of an as yet unidentified ANT1-specific transcriptional or translational processing error. ANT1 has also been associated with a third condition, autosomal dominant facioscapulohumeral muscular dystrophy (FSHD), an adult onset disorder characterized by variable muscle weakness in the face, feet, shoulders, and hips. FSHD patients possess specific DNA deletions on chromosome 4, which appear to cause derepression of several nearby genes, including ANT1. Early development of FSHD may involve mitochondrial dysfunction and increased oxidative stress, possibly associated with overexpression of ANT1. IUBMB Life, 57: 607-614, 2005 [source] Translocation of Proteins into MitochondriaIUBMB LIFE, Issue 6 2001Nicholas J. Hoogenraad Abstract The translocase of the outer mitochondrial membrane (TOM) is composed of receptors, a channel protein, and its modulators that function together to import proteins into mitochondria. Although the import pathway of proteins directed to the mitochondrial matrix has been well characterized, recent studies into the import pathway taken by proteins into the other submitochondrial compartments have broadened our understanding into the way the TOM machinery recognizes, interacts, and translocates proteins. [source] Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Xingshun Xu Abstract Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways. [source] Neuropathy-induced apoptosis: Protective effect of physostigmineJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2009L. Di Cesare Mannelli Abstract Traumatic, infectious, metabolic, and chemical noxa to the nervous system are the etiology of a crippling disease generally termed neuropathy. Motor disorders, altered sensibility, and pain are the pathognomonic traits. Cellular alterations induced by this chronic pathology include mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. Energy imbalance can compromise the maintenance of mitochondrial membrane potential, furthering the release of cytochrome C and the subsequent cleavage and activation of caspases. Chronic constriction injury (CCI) of the rat sciatic nerve is a neuropathy model able to induce a strong mitochondrial impairment with a consequent apoptotic induction. In this model, the acetylcholinesterase inhibitor physostigmine is administered at 0.125 mg/kg i.p. (twice per day) starting from the operation and for 15 days after. The cholinergic activation reduces cytosolic levels of cytochrome C, suggesting an improved stability of the mitochondrial membrane, and the expression level of the active caspase 3 fragments (19, 16 kDa) is reduced significantly with respect to saline treatment. Accordingly, physostigmine impairs caspase 3 protease activity. In fact, the target of the activated caspase 3, the 89-kDa PARP fragment, is significantly less expressed in the ligated nerve of physostigmine-treated rats, reaching levels that are comparable to those in the contralateral unligated nerve. Finally, this natural acetylcholinesterase inhibitor reduces DNA fragmentation both in the proximal and in the distal parts of the nerve. This protection correlates with the induction of XIAP. Therefore, apoptosis, central to tissue degeneration, is prevented by repeated physostigmine treatment of CCI animals. © 2009 Wiley-Liss, Inc. [source] Volatile anaesthetics depolarize neural mitochondria by inhibiton of the electron transport chainACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 5 2006R. Bains Background:, The mitochondrial membrane potential (,,m) controls the generation of adenosine triphosphate (ATP) and reactive oxygen species, and sequesteration of intracellular Ca2+[Ca2+]i. Clinical concentrations of sevoflurane affect the ,,m in neural mitochondria, but the mechanisms remain elusive. The aim of the present study was to compare the effect of isoflurane and sevoflurane on ,,m in rat pre-synaptic terminals (synaptosomes), and to investigate whether these agents affect ,,m by inhibiting the respiratory chain. Methods:, Synaptosomes were loaded with the fluorescent probes JC-1 (,,m) and Fura-2 ([Ca2+]i) and exposed to isoflurane or sevoflurane. The effect of the anaesthetics on the electron transport chain was investigated by blocking complex I and complex V. Results:, Isoflurane 1 and 2 minimum alveolar concentration (MAC) decreased the normalized JC-1 ratio from 0.92 ± 0.03 in control to 0.86 ± 0.02 and 0.81 ± 0.01, respectively, reflecting a depolarization of the mitochondrial membrane (n = 9). Isoflurane 2 MAC increased [Ca2+]i. In Ca2+ -depleted medium, isoflurane still decreased ,,m while [Ca2+]i remained unaltered. The effect of isoflurane was more pronounced than for sevoflurane. Blocking complex V of the respiratory chain enhanced the isoflurane- and sevoflurane-induced mitochondrial depolarization, whereas blocking complex I and V decreased ,,m to the same extent in control, isoflurane and sevoflurane experiments. Conclusions:, Isoflurane and sevoflurane may act as metabolic inhibitors by depolarizing pre-synaptic mitochondria through inhibition of the electron transport chain, although isoflurane seems to inhibit mitochondrial function more significantly than sevoflurane. Both agents inhibit the respiratory chain sufficiently to cause ATP synthase reversal. [source] Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulenceMOLECULAR MICROBIOLOGY, Issue 2 2010Ranadhir Dey Summary Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein-encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co-immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene-deleted parasites (Ldp27,/,) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27,/, parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis. [source] Plasma and liver carnitine status of children with chronic liver disease and cirrhosisPEDIATRICS INTERNATIONAL, Issue 4 2001Mukadder A Selimo AbstractBackground: Carnitine is an essential cofactor in the transfer of long-chain fatty acids across the inner mitochondrial membrane for oxidation. As its synthesis is performed in the liver, alterations in carnitine metabolism is expected in liver diseases, especially in cirrhosis. Methods: In this study, we investigated plasma and liver carnitine concentrations of 68 children with chronic liver disease, 36 of whom had cirrhosis as well. Carnitine level was determined by enzymatic method. Results: Plasma and liver carnitine concentrations were not correlated. Mean plasma carnitine level of cirrhotic children was significantly lower than that of the control group (P<0. 0001). While there was no difference between liver carnitine concentrations of children with chronic liver disease and cirrhosis (P>0.05), mean plasma level of cirrhotics were lower (P<0.05). Plasma carnitine was correlated with albumin, triglyceride and gamma glutamyl transpeptidase (GGT) in patients with chronic liver disease (P<0.05). Liver carnitine was correlated with GGT in cirrhotic patients (P<0.005). Children with malnutrition had higher plasma and liver carnitine levels (P<0.05). The highest plasma and liver carnitine levels were detected in children with biliary atresia and criptogenic cirrhosis, respectively. Both the lowest plasma and liver carnitine levels were detected in Wilson's disease. Conclusion: Children with cirrhosis have low plasma carnitine concentrations. This finding is prominent in children with Wilson's disease. As carnitine is an essential factor in lipid metabolism, the carnitine supplementation for patients with cirrhosis in childhood, especially with Wilson's disease, seems to be mandatory. [source] The Binding Characteristics and Intracellular Localization of Temoporfin (mTHPC) in Myeloid Leukemia Cells: Phototoxicity and Mitochondrial Damage,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2000J. Y. Chen ABSTRACT The state of aggregation of the photosensitizer meso -tetrahydroxyphenylchlorin (mTHPC) in both cell free and intracellular environment was elucidated by comparing its absorption and excitation spectra. In methanol, mTHPC existed as monomers and strongly fluoresced. In aqueous solutions such as phosphate-buffered saline (PBS), mTHPC formed nonfluorescent aggregates. Some portion of mTHPC monomerized in the presence of 10% fetal calf serum PBS. In murine myeloid leukemia M1 and WEHI-3B (JCS) cells, cytoplasmic mTHPC were monomeric. By using organelle-specific fluorescent probes, it was found that mTHPC localized preferentially at the mitochondria and the perinuclear region. Photodynamic treatment of mTHPC-sensitized leukemia cells caused rapid appearance of the apoptogenic protein cytochrome c in the cytosol. Results from flow cytometric analysis showed that the release of cytochrome c was especially pronounced in JCS cells, and well correlated with the extent of apoptotic cell death as reported earlier. Electron microscopy revealed the loss of integrity of the mitochondrial membrane and the appearance of chromatin condensation as early as 1 h after light irradiation. We conclude that rapid release of cytochrome c from photodamaged mitochondria is responsible for the mTHPC-induced apoptosis in the myeloid leukemia JCS and M1 cells. [source] Disorders of carnitine transport and the carnitine cycle,AMERICAN JOURNAL OF MEDICAL GENETICS, Issue 2 2006Nicola Longo Abstract Carnitine plays an essential role in the transfer of long-chain fatty acids across the inner mitochondrial membrane. This transfer requires enzymes and transporters that accumulate carnitine within the cell (OCTN2 carnitine transporter), conjugate it with long chain fatty acids (carnitine palmitoyl transferase 1, CPT1), transfer the acylcarnitine across the inner plasma membrane (carnitine-acylcarnitine translocase, CACT), and conjugate the fatty acid back to Coenzyme A for subsequent beta oxidation (carnitine palmitoyl transferase 2, CPT2). Deficiency of the OCTN2 carnitine transporter causes primary carnitine deficiency, characterized by increased losses of carnitine in the urine and decreased carnitine accumulation in tissues. Patients can present with hypoketotic hypoglycemia and hepatic encephalopathy, or with skeletal and cardiac myopathy. This disease responds to carnitine supplementation. Defects in the liver isoform of CPT1 present with recurrent attacks of fasting hypoketotic hypoglycemia. The heart and the muscle, which express a genetically distinct form of CPT1, are usually unaffected. These patients can have elevated levels of plasma carnitine. CACT deficiency presents in most cases in the neonatal period with hypoglycemia, hyperammonemia, and cardiomyopathy with arrhythmia leading to cardiac arrest. Plasma carnitine levels are extremely low. Deficiency of CPT2 present more frequently in adults with rhabdomyolysis triggered by prolonged exercise. More severe variants of CPT2 deficiency present in the neonatal period similarly to CACT deficiency associated or not with multiple congenital anomalies. Treatment for deficiency of CPT1, CPT2, and CACT consists in a low-fat diet supplemented with medium chain triglycerides that can be metabolized by mitochondria independently from carnitine, carnitine supplements, and avoidance of fasting and sustained exercise. © 2006 Wiley-Liss, Inc. [source] Proteomic and transcriptomic study on the action of a cytotoxic saponin (Polyphyllin D): Induction of endoplasmic reticulum stress and mitochondria-mediated apoptotic pathwaysPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2008Fung-Ming Siu Abstract Polyphyllin D (PD) is a potent cytotoxic saponin found in Paris polyphylla. In the present study, bioinformatic, proteomic and transcriptomic analyses were performed to study the mechanisms of action of PD on human nonsmall cell lung cancer (NSCLC) cell line (NCI-H460). Using a gene expression-based bioinformatic tool (connectivity map), PD was identified as a potential ER stress inducer. Our proteomic and transcriptomic analyses revealed that PD treatment led to upregulation of typical ER stress-related proteins/genes including glucose-regulated protein 78 (BiP/GRP78) and protein disulfide isomerase (PDI). In particular, elevated expression of C/EBP homologous transcription factor (chop) and activation of caspase-4 occurred at early time point (8,h) of PD treatment, signifying an initial ER stress-mediated apoptosis. Induction of tumor suppressor p53, disruption of mitochondrial membrane, activation of caspase-9 and caspase-3 were detected upon prolonged PD treatment. Collectively, these data revealed that PD induced the cytotoxic effect through a mechanism initiated by ER stress followed by mitochondrial apoptotic pathway. The ability of activating two major pathways of apoptosis makes PD an attractive drug lead for anticancer therapeutics. [source] Cell death: regulation by the Bcl-2 protein familyPSYCHOGERIATRICS, Issue 2006Yoshihide TSUJIMOTO Abstract An increase in mitochondrial membrane permeability is central to cell death including apoptosis and necrosis. During apoptosis, permeabilization of outer mitochondrial membrane leads to the release of several apoptogenic factors, such as cytochrome c and Smac/Diablo, into the cytoplasm that activate downstream death programs, including apoptotic proteases called caspases, although the detailed mechanism of outer mitochondrial membrane permeabilization remains elusive. Although the mitochondrial membrane permeability transition (MPT), resulting in ,, loss, mitochondrial swelling and rupture of the outer membrane has initially been proposed as a general mechanism for apoptotic permeabilization of outer mitochondrial membrane, the recent studies with cyclophilin D-deficient mice indicate that MPT regulates some forms of necrotic death, but not apoptotic death, and that MPT is involved in ischemia,reperfusion injury in heart and brain. Anti-apoptotic proteins, Bcl-2 and Bcl-xL, efficiently block not only apoptotic mitochondrial permeabilization but also MPT. The present paper focuses on the mechanisms by which Bcl-2 family members control the permeability of mitochondrial membrane during apoptosis and necrosis. [source] Prohibitin regulates TGF-, induced apoptosis as a downstream effector of smad-dependent and -independent signalingTHE PROSTATE, Issue 1 2010Brian Zhu Abstract BACKGROUND Prohibitin (PHB), a protein located on the inner mitochondrial membrane and nuclei, is an intracellular effector of transforming growth factor-, (TGF-,) signaling in prostate cancer cells. This study investigated the involvement of PHB in the apoptosis and survival outcomes of human prostate cancer cell to TGF-,. shRNA PHB loss of function in prostate cancer cells led to enhanced apoptotic response to TGF-, via Smad-dependent mechanism. METHOD TGF-, activation of Raf-Erk intracellular signaling, led to PHB phosphorylation, decreased inner mitochondrial permeability, and increased cell survival. Calcein-based immunofluorescence studies revealed the functional involvement of PHB in maintaining inner mitochondrial membrane permeability as an integral component of TGF-, induced apoptosis in prostate cancer cells. RESULTS These finding indicates that induction of TGF-, apoptosis is mediated by Smad-dependent and Smad-independent signaling (MAPK) converging at PHB as a downstream effector regulating inner mitochondrial permeability. Putative PHB associated proteins were identified by subjecting TGF-, treated cells to immunoprecipitation with anti-PHB, and mass spectrometry. A screen for the kinase specific phosphorylation sites of PHB revealed three protein kinase (PKC) binding sites. CONCLUSION Our results demonstrate that TGF-, led to upregulation of the PKC inhibitor 14-3-3 protein and promoted its association with PHB, while PHB association with PKC-,, was inhibited by the MEK1 inhibitor, documenting a critical interdependence between the MEK-ERK signaling and prohibitin phosphorylation. These findings suggest a dual role for PHB as a downstream determinant of the cellular response to TGF-, via Smad-dependent pathway (apoptosis) and MAPK intracellular signaling (survival). Prostate 70: 17,26, 2010. © 2009 Wiley-Liss, Inc. [source] Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotidesACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010Matthias Haffke The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg2+ and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix ,1 in full-length ABCB6. [source] Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Mrs2, a magnesium ion transporter from yeast inner mitochondrial membraneACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Muhammad Bashir Khan Mrs2 transporters are distantly related to the major bacterial Mg2+ transporter CorA and to Alr1, which is found in the plasma membranes of lower eukaryotes. Common features of all Mrs2 proteins are the presence of an N-terminal soluble domain followed by two adjacent transmembrane helices (TM1 and TM2) near the C-terminus and of the highly conserved F/Y-G-M-N sequence motif at the end of TM1. The inner mitochondrial domain of the Mrs2 from Saccharomyces cerevisae was overexpressed, purified and crystallized in two different crystal forms corresponding to an orthorhombic and a hexagonal space group. The crystals diffracted X-rays to 1.83 and 4.16,Å resolution, respectively. Matthews volume calculations suggested the presence of one molecule per asymmetric unit in the orthorhombic crystal form and of five or six molecules per asymmetric unit in the hexagonal crystal form. The phase problem was solved for the orthorhombic form by a single-wavelength anomalous dispersion experiment exploiting the sulfur anomalous signal. [source] Differential effects of redox-cycling and arylating quinones on trans-plasma membrane electron transportBIOFACTORS, Issue 3 2008An S. Tan Abstract Cytotoxicity of quinones has been attributed to free radical generation and to arylation of cellular nucleophiles. For redox-cycling quinones, cell injury is associated with mitochondrial permeability transition, whereas arylating quinones directly depolarise the mitochondrial membrane and deplete ATP. Like mitochondrial electron transport, plasma membrane electron transport (PMET), plays a multifaceted role in cellular redox homeostasis but the effects of quinones on PMET are unknown. Here we investigate the effects of redox-cycling 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), arylating 1,4-benzoquinone (BQ) and mixed mechanism 2-methyl-1,4-naphthoquinone (MNQ) on PMET, viability and growth of P815 mouse mastocytoma cells. BQ and MNQ rapidly and extensively inhibited PMET as determined by WST-1 /mPMS reduction (IC50 3.5-5 ,Mat 30 min) whereas the effects of DMNQ were less pronounced. In contrast, MTT reduction (cytosolic NADH dehydrogenase activity over 30 min) was weakly inhibited by BQ (IC50 20 ,M) but not by MNQ or DMNQ and cell viability was unaffected. Inhibition of WST-1/mPMS reduction by BQ and MNQ but not DMNQ was fully reversed by NAC. Treatment with DMNQ, MNQ and to a lesser extent BQ inhibited cell proliferation as determined by MTT reduction at 48 h. The effects of BQ and MNQ were reversed by NAC through covalent bonding to BQ and MNQ, but not DMNQ. These results show that arylating quinones are more potent inhibitors of PMET than pure redox-cycling quinones, but that redox-cycling quinones are more cytotoxic. [source] The novel 2Fe,2S outer mitochondrial protein mitoNEET displays conformational flexibility in its N-terminal cytoplasmic tethering domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Andrea R. Conlan A primary role for mitochondrial dysfunction is indicated in the pathogenesis of insulin resistance. A widely used drug for the treatment of type 2 diabetes is pioglitazone, a member of the thiazolidinedione class of molecules. MitoNEET, a 2Fe,2S outer mitochondrial membrane protein, binds pioglitazone [Colca et al. (2004), Am. J. Physiol. Endocrinol. Metab.286, E252,E260]. The soluble domain of the human mitoNEET protein has been expressed C-terminal to the superfolder green fluorescent protein and the mitoNEET protein has been isolated. Comparison of the crystal structure of mitoNEET isolated from cleavage of the fusion protein (1.4,Å resolution, R factor = 20.2%) with other solved structures shows that the CDGSH domains are superimposable, indicating proper assembly of mitoNEET. Furthermore, there is considerable flexibility in the position of the cytoplasmic tethering arms, resulting in two different conformations in the crystal structure. This flexibility affords multiple orientations on the outer mitochondrial membrane. [source] Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 geneBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Shin Sik Choi Abstract It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein. © 2006 Wiley Periodicals, Inc. [source] Preliminary X-ray crystallographic studies of yeast mitochondrial protein Tom70pACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2006Yunkun Wu Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tom70p is an important TOM-complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) was crystallized. The crystals diffract to 3.2,Å using a synchrotron X-ray source and belong to space group P21, with unit-cell parameters a = 44.89, b = 168.78, c = 83.41,Å, , = 90.00, , = 102.74, , = 90.00°. There are two Tom70p molecules in one asymmetric unit, which corresponds to a solvent content of approximately 51%. Structure determination by MAD methods is under way. [source] The synthetic furanonaphthoquinone induces growth arrest, apoptosis and differentiation in a variety of leukaemias and multiple myeloma cellsBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2005Julian C. Desmond Summary 2-Methyl-naphtho[2,3- b]furan-4,9-dione (FNQ3), a synthetic analogue of the quinone kigelinone, has demonstrated a real potential for use in the treatment of a variety of solid tumours. Unlike other quinones, such as mitomycin-C and adriamycin, the cytotoxicity of FNQ3 is often 10- to 14-fold more potent towards the tumour cells than their normal counterparts. We report, for the first time, that the drug had activity against a broad spectrum of leukaemias and multiple myeloma cells. It decreased the growth of acute myeloid leukaemia (AML) and multiple myeloma cell lines in a dose-dependent fashion (50% inhibitory concentration ,1·25 ,g/ml against most of the leukaemia cell lines). This dose apparently initiated mitochondrial collapse as measured by depolarisation of the mitochondrial membrane. FNQ3 potentiated the differentiation of HL-60 myeloid cells in the presence of either 1,, 25(OH)2 dihydroxyvitamin D3 [1,,25(OH)2D3] or all- trans -retinoic acid (ATRA). FNQ3 inhibited the proliferation of primary AML cells while inducing apoptosis. Eleven of 14 (79%) AML marrow samples had a prominent decrease in their clonogenic growth when cultured in the presence of the drug. In summary, this drug has growth inhibitory, apoptotic and differentiative effects against myeloid leukaemias and multiple myeloma cells. FNQ3 may represent a new therapeutic approach to these malignancies. [source] Interference of MI-D, a new mesoionic compound, on artificial and native membranesCELL BIOCHEMISTRY AND FUNCTION, Issue 1 2002Silvia M.S.C. Cadena Abstract MI-D (4-phenyl-5-(4-nitrocinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride), a new mesoionic compound, decreased the rate of swelling induced by valinomycin-K+, as well as induced swelling in the presence of nigericin-K+. Shrinkage was also affected, suggesting interference with the inner mitochondrial membrane, which would affect both fluidity and elasticity. Fluorescence polarization of DPH and DPH-PA, probing the core and outer regions respectively, of the DMPC and native membranes, indicated that MI-D shifts the midpoint of phase transition to higher values and orders of the fluid phase. These alterations in membrane fluidity are thus related to MI-D effects on the energy-linked functions of mitochondria. Copyright © 2001 John Wiley & Sons, Ltd. [source] Structural Consideration of Mammalian D -Aspartyl EndopeptidaseCHEMISTRY & BIODIVERSITY, Issue 6 2010Tadatoshi Kinouchi Abstract D -Aspartyl endopeptidase (DAEP) is a specific protease for D -aspartic acid (D -Asp)-containing protein, which has been implicated in the pathogenesis of age-related and misfolding diseases such as Alzheimer's disease. Therefore, DAEP would serve as a defensive system against the noxious D -Asp-containing protein. However, it is unclear how DAEP exerts its unique enzymatic function, since its higher-order structure remains quite unsolved. In this study, we analyzed the conformation of purified DAEP from the mitochondrial membrane of mouse by atomic force microscopy the advantage of which is its ability to study biological macromolecules and even living organisms in an ambient air environment. DAEP formed a ring-like structure with a diameter of ca. 40,nm. Our data suggest that DAEP topologically belongs to the AAA+ protease family such as proteasome, Lon, and mitochondrial membrane-bound i-/m-AAA protease. [source] | ||||||||||||||||||||||||||||