The assembly of yeast mitochondrial ATP synthase: subunit depletion in vivo suggests ordered assembly of the stalk subunits b, OSCP and d

AbstractThe abundance in vivo of each of three subunits b, OSCP and d, components of the stalk region of the yeast mitochondrial ATP synthase complex, was manipulated by a controlled depletion strategy. Western blots of whole cell lysates were used to study the effect of depletion of each of these subunits on the cellular levels of other subunits of the enzyme complex. A hierarchy of subunit stability was determined and interpreted to indicate the order of assembly of these three subunits of the stalk region. Thus, subunit b is assembled first, followed by OSCP and then by subunit d

Lack of correlation between MYCN expression and the Warburg effect in neuroblastoma cell lines

<p>Abstract</p> <p>Background</p> <p>Many cancers preferentially meet their energy requirements through the glycolytic pathway rather than via the more efficient oxidative phosphorylation pathway. It is thought that this is an important adaptation in cancer malignancy. We investigated whether use of glycolysis for energy production even in the presence of oxygen (known as the Warburg effect) varied between neuroblastoma cell lines with or without <it>MYCN </it>amplification (a key indicator of poor disease outcome in neuroblastoma).</p> <p>Methods</p> <p>We examined ATP and lactate production, oxygen consumption and mitochondrial energisation status for three neuroblastoma cell lines with varying degrees of <it>MYCN </it>amplification and MYCN expression.</p> <p>Results</p> <p>We found no correlation between MYCN expression and the Warburg effect in the cell lines investigated.</p> <p>Conclusion</p> <p>Our results suggest preferential use of glycolysis for energy production and MYCN expression may be independent markers of neuroblastoma malignancy <it>in vitro </it>if not <it>in vivo</it>.</p

Coenzyme Q10 therapy before cardiac surgery improves mitochondrial function and in vitro contractility of myocardial tissue

AbstractObjectivesPrevious clinical trials suggest that coenzyme Q10 might afford myocardial protection during cardiac surgery. We sought to measure the effect of coenzyme Q10 therapy on coenzyme Q10 levels in serum, atrial trabeculae, and mitochondria; to assess the effect of coenzyme Q10 on mitochondrial function; to test the effect of coenzyme Q10 in protecting cardiac myocardium against a standard hypoxia-reoxygentation stress in vitro; and to determine whether coenzyme Q10 therapy improves recovery of the heart after cardiac surgery.MethodsPatients undergoing elective cardiac surgery were randomized to receive oral coenzyme Q10 (300 mg/d) or placebo for 2 weeks preoperatively. Pectinate trabeculae from right atrial appendages were excised, and mitochondria were isolated and studied. Trabeculae were subjected to 30 minutes of hypoxia, and contractile recovery was measured. Postoperative cardiac function and troponin I release were assessed.ResultsPatients receiving coenzyme Q10 (n = 62) had increased coenzyme Q10 levels in serum (P = .001), atrial trabeculae (P = .0001), and isolated mitochondria (P = .0002) compared with levels seen in patients receiving placebo (n = 59). Mitochondrial respiration (adenosine diphosphate/oxygen ratio) was more efficient (P = .012), and mitochondrial malondialdehyde content was lower (P = .002) with coenzyme Q10 than with placebo. After 30 minutes of hypoxia in vitro, pectinate trabeculae isolated from patients receiving coenzyme Q10 exhibited a greater recovery of developed force compared with those in patients receiving placebo (46.3% ± 4.3% vs 64.0% ± 2.9%, P = .001). There was no between-treatment difference in preoperative or postoperative hemodynamics or in release of troponin I.ConclusionsPreoperative oral coenzyme Q10 therapy in patients undergoing cardiac surgery increases myocardial and cardiac mitochondrial coenzyme Q10 levels, improves mitochondrial efficiency, and increases myocardial tolerance to in vitro hypoxia-reoxygenation stress

Bim Links ER Stress and Apoptosis in Cells Expressing Mutant SOD1 Associated with Amyotrophic Lateral Sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1

Modulation of Cellular Hsp72 Levels in Undifferentiated and Neuron-Like SH-SY5Y Cells Determines Resistance to Staurosporine-Induced Apoptosis

Increased expression of Hsp72 accompanies differentiation of human neuroblastoma SH-SY5Y cells to neuron-like cells. By modulating cellular levels of Hsp72, we demonstrate here its anti-apoptotic activity both in undifferentiated and neuron-like cells. Thermal preconditioning (43°C for 30 min) induced Hsp72, leading to cellular protection against apoptosis induced by a subsequent treatment with staurosporine. Preconditioned staurosporine-treated cells displayed decreased Bax recruitment to mitochondria and subsequent activation, as well as reduced cytochrome c redistribution from mitochondria. The data are consistent with Hsp72 blocking apoptosis upstream of Bax recruitment to mitochondria. Neuron-like cells (with elevated Hsp72) were more resistant to staurosporine by all measured indices of apoptotic signaling. Use of stable transfectants ectopically expressing moderately elevated levels of Hsp72 revealed that such cells in the undifferentiated state showed enhanced resistance to staurosporine-induced apoptosis, which was even more robust after differentiation to neuron-like cells. Overall, the protective effects of differentiation, thermal preconditioning and ectopic Hsp72 expression were additive. The strong inverse correlation between cellular Hsp72 levels and susceptibility to apoptosis support the notion that Hsp72 acts as a significant neuroprotective factor, enabling post-mitotic neurons to withstand potentially lethal stress that induces apoptosis

Identification of a Novel Gene Product That Promotes Survival of Mycobacterium smegmatis in Macrophages

BACKGROUND: Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate. CONCLUSIONS/SIGNIFICANCE: MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria