ATP Synthase Expression Research Articles

Novel Antibiotics Targeting Respiratory ATP Synthesis in Gram-Positive Pathogenic Bacteria

Emergence of drug-resistant bacteria represents a high, unmet medical need, and discovery of new antibacterials acting on new bacterial targets is strongly needed. ATP synthase has been validated as an antibacterial target in Mycobacterium tuberculosis, where its activity can be specifically blocked by the diarylquinoline TMC207. However, potency of TMC207 is restricted to mycobacteria with little or no effect on the growth of other Gram-positive or Gram-negative bacteria. Here, we identify diarylquinolines with activity against key Gram-positive pathogens, significantly extending the antibacterial spectrum of the diarylquinoline class of drugs. These compounds inhibited growth of Staphylococcus aureus in planktonic state as well as in metabolically resting bacteria grown in a biofilm culture. Furthermore, time-kill experiments showed that the selected hits are rapidly bactericidal. Drug-resistant mutations were mapped to the ATP synthase enzyme, and biochemical analysis as well as drug-target interaction studies reveal ATP synthase as a target for these compounds. Moreover, knockdown of the ATP synthase expression strongly suppressed growth of S. aureus, revealing a crucial role of this target in bacterial growth and metabolism. Our data represent a proof of principle for using the diarylquinoline class of antibacterials in key Gram-positive pathogens. Our results suggest that broadening the antibacterial spectrum for this chemical class is possible without drifting off from the target. Development of the diarylquinolines class may represent a promising strategy for combating Gram-positive pathogens.

Abstract 568: Biomarkers of phenethyl isothiocyanate-mediated mammary cancer prevention in MMTV-neu mouse model

Abstract Biomarker(s) predictive of efficacy is necessary for clinical development of promising cancer chemopreventive agents because cancer incidence is too rigorous of an end point for malignancies with long latency such as breast cancer. The present study was undertaken to identify biomarker(s) associated with phenethyl isothiocyanate (PEITC)-mediated mammary cancer prevention in MMTV-neu transgenic mouse model. The PEITC is a promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress). Two-dimensional gel electrophoresis-mass spectrometry of tumor specimens from control and PEITC-fed mice (n= 3) revealed alterations in expression of several proteins, including hexokinase-1, isoform CRA_f (1.36-fold increase by PEITC administration), L-lactate dehydrogenase A chain isoform 1 (1.57-fold increase by PEITC administration), pyruvate kinase isozymes M1/M2 (1.33-fold decrease by PEITC administration), and mitochondrial ATP synthase, H+ transporting F1 complex beta subunit (1.38-fold decrease by PEITC administration). Interestingly, the PEITC-induced apoptotic cell death in cultured cancer cells is associated with inhibition of oxidative phosphorylation leading to production of reactive oxygen species. Down-regulation of mitochondrial ATP synthase protein in tumors of PEITC-fed mice was confirmed by immunohistochemistry. Expression of mitochondrial ATP synthase was reduced by about 66% in the tumors from PEITC-fed mice in comparison with control (P= 0.05 by two-sided Student's t-test, n= 6-7). In addition, plasma level of transthyretin was significantly higher in the PEITC treatment group compared with control (P= 0.04 by two-sided Student's t-test, n= 5). In conclusion, the present study not only underscores the power of proteomics in cancer chemoprevention research but also suggests that mitochondrial ATP synthase, and possibly hexokinase-1, pyruvate kinase isozymes M1/M2, and/or L-lactate dehydrogenase A chain isoform 1, may be useful biomarkers to assess the biological activity of PEITC in future clinical trials. This study was supported in part by the US PHS grants CA101753 and CA129347, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 568. doi:1538-7445.AM2012-568

Use of comparative proteomics to identify the effects of creatine pyruvate on lipid and protein metabolism in broiler chickens

Four hundred male chickens were selected to study the effects of pyruvate (Pyr), creatine pyruvate (CrPyr) and creatine (Cr) on the expression of hepatic mitochondrial and cytoplasm proteins associated with lipid and protein metabolism. Mitochondrial purification was accomplished using the two-step differential centrifugation and density gradient method, and the activities of organelle-specific marker enzymes were determined to assess the purity of the mitochondria. Proteins were extracted and fractionated by two-dimensional electrophoresis and the differential protein spots were assessed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. CrPyr reduced fatty acid accumulation by down-regulating adipose differentiation-related protein, inhibited ATP synthase expression, and reduced cholesteryl ester transfer protein (CETP) expression, thus reducing the levels of high density lipoprotein and triglycerol (TG) levels (thereby lowering fat and cholesterol deposition). CrPyr increased the expression of eukaryotic translation initiation factor (eIF) 2B, calreticulin (CRT) and eIF3a, thus promoting protein synthesis. CrPyr up-regulated the expression of fatty acid-binding proteins, CETP and apolipoprotein A-IV in cytoplasmic extracts, and these proteins accelerated the decomposition of fatty acids and TG, thus reducing fat deposition. In conclusion, CrPyr plays an important role in lipolysis and protein synthesis, and this effect was more pronounced than was the effect of Pyr and Cr.

ATP synthase ecto-α-subunit: a novel therapeutic target for breast cancer

BackgroundTreatment failure for breast cancer is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in breast cancer cells in vitro and in vivo. Identification of new targets will facilitate the developmental pace of new techniques in screening and early diagnosis. Improved abilities to predict progression and metastasis, therapeutic response and toxicity will help to increase survival of breast cancer patients.MethodsDifferential protein expression in two breast cancer cell lines, one with high and the other with low metastatic potential, was analyzed using two-dimensional liquid phase chromatographic fractionation (Proteome Lab PF 2D system) followed by matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI-TOF/MS).ResultsUp regulation of α-subunit of ATP synthase was identified in high metastatic cells compared with low metastatic cells. Immunohistochemical analysis of 168 human breast cancer specimens on tissue microarrays revealed a high frequency of ATP synthase α-subunit expression in breast cancer (94.6%) compared to normal (21.2%) and atypical hyperplasia (23%) breast tissues. Levels of ATP synthase expression levels strongly correlated with large tumor size, poor tumor differentiation and advanced tumor stages (P < 0.05). ATP synthase α-subunit over-expression was detected on the surface of a highly invasive breast cancer cell line. An antibody against the ATP synthase α-subunit inhibited proliferation, migration and invasion in these breast cancer cells but not that of a non-tumor derived breast cell line.ConclusionsOver-expression of ATP synthase α-subunit may be involved in the progression and metastasis of breast cancer, perhaps representing a potential biomarker for diagnosis, prognosis and a therapeutic target for breast cancer. This finding of this study will help us to better understand the molecular mechanism of tumor metastasis and to improve the screening, diagnosis, as well as prognosis and/or prediction of responses to therapy for breast cancer.

Cell-Free Expression and Assembly of ATP Synthase

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F1Fo-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α3β3γδɛab2c13. Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.

High insulin and saturated fatty acid exposure cause mitochondrial dysfunction via distinct mechanisms

Mitochondrial dysfunction has been associated with insulin resistance, obesity and diabetes. Both hyperinsulinaemia and dyslipidemia are hallmarks of the insulin resistant state. The purpose of this study was to examine the contributions of high insulin and saturated fatty acid exposure on mitochondrial function and gene expression in cultured myocytes. Chronic insulin treatment of myotubes reduced signalling through PI 3‐Kinase/AKT. Insulin‐resistant myocytes exhibited lower mitochondrial membrane potential and ROS production but exhibited ATP levels similar to control cells. Citrate synthase activity was increased in the insulin treated cells. The expression of a number of mitochondrial genes including COX2, COX4, ATP synthase were reduced in the chronic insulin treated cells albeit they were not significant. However, the transcriptional co‐activator PGC1alpha, and uncoupling proteins, UCP2 and UCP3 were significantly reduced in these cells. No changes were detected in Tfam or NRF‐1 transcript levels. Mitofusin2 and Porin protein levels were also not altered by chronic insulin exposure. In contrast, treatment of cells with saturated fatty acid reduced mitochondrial membrane potential, significantly increased ROS production, and increased the mRNA expression of UCP2, UCP3 and ATP synthase. These cells also displayed lower levels PGC1α and PGC1β gene expression compared to controls. Taken together these results suggest that chronic hyperinsulinemia and exposure to saturated fatty acids impair mitochondrial function by different mechanisms.

Ecto-F₁-ATPase: a moonlighting protein complex and an unexpected apoA-I receptor.

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F₁-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F₁-ATPase. Particularly, the role of the F₁-ATPase pathway(s) in HDL-cholesterol uptake and apoA-I-mediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions.

Germ-line mitochondria exhibit suppressed respiratory activity to support their accurate transmission to the next generation

Mitochondria are accurately transmitted to the next generation through a female germ cell in most animals. Mitochondria produce most ATP, accompanied by the generation of reactive oxygen species (ROS). A specialized mechanism should be necessary for inherited mitochondria to escape from impairments of mtDNA by ROS. Inherited mitochondria are named germ-line mitochondria, in contrast with somatic ones. We hypothesized that germ-line mitochondria are distinct from somatic ones. The protein profiles of germ-line and somatic mitochondria were compared, using oocytes at two different stages in Xenopus laevis. Some subunits of ATP synthase were at a low level in germ-line mitochondria, which was confirmed immunologically. Ultrastructural histochemistry using 3,3′-diaminobenzidine (DAB) showed that cytochrome c oxidase (COX) activity of germ-line mitochondria was also at a low level. Mitochondria in one oocyte were segregated into germ-line mitochondria and somatic mitochondria, during growth from stage I to VI oocytes. Respiratory activity represented by ATP synthase expression and COX activity was shown to be low during most of the long gametogenetic period. We propose that germ-line mitochondria that exhibit suppressed respiration alleviate production of ROS and enable transmission of accurate mtDNA from generation to generation.

Comparative proteomic analysis of Typha angustifolia leaf under chromium, cadmium and lead stress

The present study investigated Typha angustifolia leaf proteome in response to Cr, Cd and Pb stress. T. angustifolia of 90 (D90) and 130d (D130) old plants were subjected to 1mM Cr, Cd and Pb and samples were collected 30d after treatment. 2-DE coupled with MS (mass spectrometry) was used to analyze and identify Cr, Cd and Pb-responsive proteins. More than 1600 protein spots were reproducibly detected on each gel, wherein 44, 46, 66 and 33, 26, 62 spots in D90 and D130 samples were differentially expressed by Cr, Cd, Pb over the control, respectively. Of these differentially expressed proteins, 3, 1, 8 overlapped in D90 and D130; while 5, 8, 5 with regulation factors above 3 in one of D90 or D130 samples. Total of 22 and 4 up- and down-regulated proteins were identified using MS and data bank analysis. Cr-induced expression of ATP synthase, RuBisCO small subunit and coproporphyrinogen III oxidase; Cd-induced RuBisCO large subunit; Pb up-regulated carbohydrate metabolic pathway enzymes of fructokinase, and improved RuBisCO activase and large subunit, Mg-protoporphyrin IX chelatase. Contrarily, elF4F was inhibited by Cr/Pb, chloroplast FtsZ-like protein and GF14omega impeded by Cd and Pb, respectively.

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction.

Vital function of PRELI and essential requirement of its LEA motif

Proteins containing the late embryogenesis abundant (LEA) motif comprise a conserved family, postulated to act as cell protectors. However, their function and mechanisms of action remain unclear. Here we show that PRELI, a mammalian LEA-containing homolog of yeast Ups1p, can associate with dynamin-like GTPase Optic Atrophy-1 (OPA1) and contribute to the maintenance of mitochondrial morphology. Accordingly, PRELI can uphold mitochondrial membrane potential (ΔΨm) and enhance respiratory chain (RC) function, shown by its capacity to induce complex-I/NADH dehydrogenase and ATP synthase expression, increase oxygen consumption and reduce reactive oxygen species (ROS) production. PRELI can also inhibit cell death induced by STS, TNF-α or UV irradiation. Moreover, in vitro and in vivo dominant-negative overexpression of mutant PRELI/LEA− (lacking the LEA motif) and transient in vitro PRELI-specific knockdown can render lymphocytes vulnerable to apoptosis, cause mouse embryo lethality and revert the resistance of lymphoma cells to induced death. Collectively, these data support the long-presumed notion of LEA protein-dependent mechanisms of cytoprotection and suggest that PRELI interacts with OPA1 to maintain mitochondria structures intact, sustain balanced ion−/proton+ gradients, promote oxidative phosphorylation reactions, regulate pro- and antiapoptotic protein traffic and enable cell responses to induced death. These findings may help to understand how bioenergetics is mechanistically connected with cell survival cues.

Effect of dietary crude protein level on visceral organ mass, cellularity, and the protein expression of ATP synthase, Na+/K+-ATPase, proliferating cell nuclear antigen and ubiquitin in feedlot steers

Twenty-four steers [initial body weight (BW) = 510 ± 4.9 kg] predominately of Angus breeding were used to determine the effect of dietary crude protein (CP) level [8.5, 10.7, 12.3 or 14.5%, dry matter (DM) basis; high-moisture-corn-based diets] on visceral mass, cellularity, and protein expression of ATP synthase, Na+/K+-ATPase, proliferating cell nuclear antigen (PCNA) and ubiquitin. Steers were on dietary treatment for 28 d. Kidney, liver, and reticulorumen weights (g) increased linearly (P &lt; 0.05) with increased dietary CP. Lung weight (g; g kg-1 BW) linearly increased (P &lt; 0.05) with increased CP. Ruminal and small intestinal DNA concentration, and the ratios of RNA:DNA and protein:DNA in small intestine were affected quadratically by increased dietary CP (P &lt; 0.05). Hepatic ATP synthase expression was affected quadratically with an increase when dietary CP increased from 8.5 to 10.7%, and a decrease when dietary CP increased from 10.7 to 14.5% (P &lt; 0.05). Renal ATP synthase expression decreased linearly (P &lt; 0.05) and small intestine mucosal Na+/K+-ATPase expression increased linearly (P = 0.05) with increased CP. These results indicate that increasing dietary CP increases liver, kidney, lung, and rumen masses, and differentially influences expression of proteins influencing energy utilization and efficiency in liver, kidney, and small intestine.Key words: Dietary crude protein, visceral organ mass, cellular energy metabolism, steer

Effect of dietary corn silage inclusion on visceral organ mass, cellularity, and the protein expression of ATP synthase, Na+/K+-ATPase, proliferating cell nuclear antigen and ubiquitin in feedlot steers

Twenty-four steers (initial body weight = 535 ± 5.0 kg) predominately of Angus breeding were used to determine the effect of dietary corn silage inclusion [20, 40, 60, or 80% of dry matter (DM)] on visceral mass, cellularity, and the protein expression of ATP synthase, Na+/K+-ATPase, proliferating cell nuclear antigen (PCNA) and ubiquitin. Steers were fed at similar energy levels (2.1 × NEm requirement). There were no significant treatment effects on specific visceral organ weights. Hepatic Na+/K+-ATPase expression linearly increased (P = 0.01) and ruminal Na+/K+-ATPase expression linearly decreased (P = 0.01) with increasing corn silage inclusion. Hepatic PCNA expression was quadratically affected (P = 0.05) with a decrease when corn silage inclusion increased from 20 to 60%, and an increase when corn silage inclusion increased from 60 to 80%. Renal ATP synthase (P = 0.02) and ubiquitin expression (P = 0.01) were quadratically affected in a similar pattern with an increase when corn silage inclusion increased from 20 to 60%, and a decrease when corn silage inclusion increased from 60 to 80%. These results indicate that different dietary corn silage inclusions, at similar dietary energy intake, may alter rumen, liver, and kidney energy expenditure, at least in part, through changes in specific metabolism rather than mass. Key words: Corn silage inclusion, visceral organ mass, cellular energy metabolism, steer

Isolation and characterization of a N,N′-dicyclohexylcarbodiimide-resistant mutant of Methanothermobacter thermautotrophicus with alterations to the ATP synthesis machinery

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant toward the ATP-synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 micromol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A(1) and A(o) subcomplexes of the A(1)A(o)-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A(1)A(o) ATP synthase. The results imply that DCCD resistance is a consequence of increased A(1)A(o) ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.

Effect of dry matter intake on visceral organ mass, cellularity, and the protein expression of ATP synthase, Na+/K+-ATPase, proliferating cell nuclear antigen and ubiquitin in feedlot steers

Twenty-four steers [467 ± 7.2 kg initial body weight (BW)] predominately of Angus breeding were used to determine the effect of dry matter intake (1.25, 1.50, 1.75, and 2.00% of BW) on visceral mass, cellularity, and the protein expression of ATP synthase, Na+/K+-ATPase, proliferating cell nuclear antigen (PCNA) and ubiquitin. There were linear increases (P ≤ 0.05) in weights of total viscera, total digestive tract, liver, kidney, heart, lung, spleen, rumen, and abomasum with increasing dry matter intake (DMI). Protein concentration decreased linearly (P &lt; 0.05) in small intestinal mucosa as DMI increased. PCNA expression increased linearly (P &lt; 0.01) in liver as DMI increased. PCNA expression was affected quadratically (P &lt; 0.05) in pancreas and small intestinal mucosa with an increase when DMI increased from 1.25 to 1.75% of BW, and a decrease when DMI increased from 1.75 to 2% of BW. ATP synthase, Na+/K+-ATPase, and ubiquitin expression in pancreas and ubiquitin expression in small intestinal mucosa increased linearly (P &lt; 0.05) as DMI increased. These results indicate that increasing DMI increases the mass of visceral organs and carcass and influences expression of proteins influencing energy utilization and efficiency in pancreas, small intestine, liver, and sternomandibularis muscle.Key words: Dry matter intake, visceral organ mass, cellular energy metabolism, steer

Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits

Heterologous expression of cDNA library in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis, with cDNA sizes of 2–5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that 75% (61) are full-length genes, which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform Arabidopsis plants. Among the 7,000 mutants, one was found to be a dwarf due to the expression of an ATP synthase, and another vegetative mutant did not produce flowers even after 7 months. The technique was validated by reintroducing the tomato ribosomal protein L9 gene and can be used in any other plant species as a gene discovery tool.

Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage

A myopathy characterized by mitochondrial pathology and oxidative stress is present in patients with peripheral arterial disease (PAD). Patients with PAD differ in disease severity, mode of presentation, and presence of comorbid conditions. In this study, we used a mouse model of hindlimb ischemia to isolate and directly investigate the effects of chronic inflow arterial occlusion on skeletal muscle microanatomy, mitochondrial function and expression, and oxidative stress. Hindlimb ischemia was induced by staged ligation/division of the common femoral and iliac arteries in C57BL/6 mice, and muscles were harvested 12 wk later. Muscle microanatomy was examined by bright-field microscopy, and mitochondrial content was determined as citrate synthase activity in muscle homogenates and ATP synthase expression by fluorescence microscopy. Electron transport chain (ETC) complexes I through IV were analyzed individually by respirometry. Oxidative stress was assessed as total protein carbonyls and 4-hydroxy-2-nonenal (HNE) adducts and altered expression and activity of manganese superoxide dismutase (MnSOD). Ischemic muscle exhibited histological features of myopathy and increased mitochondrial content compared with control muscle. Complex-dependent respiration was significantly reduced for ETC complexes I, III, and IV in ischemic muscle. Protein carbonyls, HNE adducts, and MnSOD expression were significantly increased in ischemic muscle. MnSOD activity was not significantly changed, suggesting MnSOD inactivation. Using a mouse model, we have demonstrated for the first time that inflow arterial occlusion alone, i.e., in the absence of other comorbid conditions, causes myopathy with mitochondrial dysfunction and increased oxidative stress, recapitulating the muscle pathology of PAD patients.

Niacin inhibits surface expression of ATP synthase β chain in HepG2 cells: implications for raising HDL

Niacin is an effective agent for raising HDL, but its cellular target sites are largely unknown. We examined effects of niacin on the surface expression of ATP synthase beta chain, a newly described HDL/apolipoprotein A-I (apoA-I) receptor for HDL endocytosis, in HepG2 cells. A significant amount of immunodetectable beta chain was observed on the surface of HepG2 cells, which was competitively displaced by apoA-I. Niacin treatment reduced the surface expression of beta chain in HepG2 cells by approximately 27%, and decreased (125)I-labeled HDL uptake up to approximately 35%. However, nicotinamide, a niacin metabolite that does not have clinical lipid effects, exhibited weaker inhibition on the beta chain cell surface expression, and failed to show inhibitory action on (125)I-labeled HDL uptake. Furthermore, anti-beta chain antibody significantly reduced (125)I-labeled HDL uptake and abolished the inhibitory effect of niacin. Niacin did not change beta chain mRNA expression. These data suggest that niacin inhibits cell surface expression of the ATP synthase beta chain, leading to reduced hepatic removal of HDL protein, thus implicating a potential cellular target for niacin action to raise HDL.

Oxidative stress disturbs energy metabolism of mitochondria in ethanol-induced gastric mucosa injury

To study the role of mitochondrial energy disorder in the pathogenesis of ethanol-induced gastric mucosa injury. Wistar rats were used in this study. A gastric mucosal injury model was established by giving the rats alcohol. Gross and microscopic appearance of gastric mucosa and ultrastructure of mitochondria were evaluated. Malondiadehyde (MDA) in gastric mucosa was measured with thiobarbituric acid. Expression of ATP synthase (ATPase) subunits 6 and 8 in mitochondrial DNA (mtDNA) was determined by reverse transcription polymerase chain reaction (RT-PCR). The gastric mucosal lesion index was correlated with the MDA content in gastric mucosa. As the concentration of ethanol was elevated and the exposure time to ethanol was extended, the content of MDA in gastric mucosa increased and the extent of damage aggravated. The ultrastructure of mitochondria was positively related to the ethanol concentration and exposure time. The expression of mtDNA ATPase subunits 6 and 8 mRNA declined with the increasing MDA content in gastric mucosa after gavage with ethanol. Ethanol-induced gastric mucosa injury is related to oxidative stress, which disturbs energy metabolism of mitochondria and plays a critical role in the pathogenesis of ethanol-induced gastric mucosa injury.