Biomedical subjects

K Hagopian

Publications and source records attributed to K Hagopian.

7 recordsLinked to original sources

PubMed2004

Ageing, oxidative stress, and mitochondrial uncoupling.

Mitochondria are a cell's single greatest source of reactive oxygen species. Reactive oxygen species are important for many life sustaining processes of cells and tissues, but they can also induce cell damage and death. If their production and levels within cells is not effectively controlled, then the detrimental effects of oxidative stress can accumulate. Oxidative stress is widely thought to underpin many ageing processes, and the oxidative stress theory of ageing is one of the most widely acknowledged theories of ageing. As well as being the major source of reactive oxygen species, mitochondria are also a major site of oxidative damage. The purpose of this review is a concise and current review of the effects of oxidative stress and ageing on mitochondrial function. Emphasis is placed upon the roles of mitochondrial proton leak, the uncoupling proteins, and the anti-ageing effects of caloric restriction.

Aging↗

PubMed1999

Preparative electrophoretic method for the purification of a hydrophobic membrane protein: subunit c of the mitochondrial ATP synthase from rat liver.

A method is described for the purification of subunit c of ATP synthase from rat liver mitochondria. After sample preparation and solvent extraction, the protein was purified to homogeneity by a single-step preparative electrophoretic procedure, using aqueous buffer and containing lithium dodecyl sulfate. The subunit is an extremely hydrophobic and insoluble protein and all solubilization attempts, using a variety of detergents, were unsuccessful except for lithium dodecyl sulfate. Buffer exchange and FPLC gel filtration removed the detergent from the purified sample, leaving the protein in a soluble form. The mammalian protein is composed of 75 amino acid residues, with a molecular mass of 7602 Da and is classified as a proteolipid. Subunit c accounts for 25 and 85% of the intralysosomal accumulation, within neurons, of storage material in juvenile and late-infantile forms of Batten's disease, respectively. This purification procedure allows access to a continuous supply of pure subunit c from a conventional source such as rat liver and preserves precious autopsy materials. The protein could be used as substrate in future proteolytic studies involving pepstatin-insensitive lysosomal proteases and for raising of more specific antibodies. The procedure could also be adapted/modified and used as a model for purifying other extremely insoluble proteins.

Animals↗

PubMed1999

Androgen-induced oxidative stress in human LNCaP prostate cancer cells is associated with multiple mitochondrial modifications.

We investigated the role of androgen-induced oxidative stress in prostate cancer using the androgen-responsive LNCaP human prostate cancer cell line exposed to a 1-nM concentration of the synthetic androgen R1881 (which correlates with serum androgen levels). Such exposure, which decreases growth rate and increases oxidative stress in LNCaP cells, induced statistically significant mitochondrial changes. A 40% increase in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, indicative of mitochondrial dehydrogenase activity, occurred 24 hr after androgen treatment. This change preceded 50-110% increases, 40-96 hr after R1881 exposure, in levels of cellular peroxides and hydroxyl radicals as measured by 2'7'-dicholorofluorescin diacetate (DCF) fluorescence. On the basis of electron microscopy measurements, R1881 treatment increased the area fraction of mitochondria per cell by approximately 100% at 72 hr. In agreement, mitochondrial mass at 96 hr, evaluated by the fluorescent dye nonyl acridine orange (NAO), was 80% higher in treated cells. R1881 exposure for 24 hr lowered the activities of electron transport system (ETS) complexes, I, II, and IV by 17-27% and ATP levels by 50%. The ETS inhibitors, rotenone and antimycin A, lowered androgen-induced DCF fluorescence readings to control levels thereby suggesting ETS involvement in androgen-induced oxidant production. Addition of alpha-tocopherol succinate abrogated R1881-induced elevations in MTT reduction. In sum, androgens may, directly or indirectly, contribute to oxidative stress in LNCaP cells by regulating mitochondrial number, activity, and oxidant production by mechanisms that are, at least in part, sensitive to an antioxidant.

Antimycin A↗

PubMed1998

Purification and characterization of phosphatidylglycerolphosphate synthase from Schizosaccharomyces pombe.

The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerolphosphate synthase; PGPS4; EC 2.7.8.5) is located in the mitochondrial inner membrane and catalyzes the committed step in the cardiolipin branch of phospholipid synthesis. Previous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this work, we report the purification to homogeneity of PGPS from S. pombe. The enzyme was solubilized from the mitochondrial membrane of S. pombe with Triton X-100. The solubilized enzyme, together with the associated detergent and intrinsic lipids, had a molecular mass of 120 kDa, as determined by gel filtration. The enzyme was further purified using salt-induced phase separation, gel filtration, and ionic exchange, hydroxylapatite, and affinity chromatographies. The procedure yielded a homogeneous protein preparation, evidenced by both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoelectric focusing under nondenaturing conditions. The purified enzyme had an apparent molecular mass of 60 kDa as determined by SDS-PAGE. The enzyme showed a strong dependence on lipid cofactors for activity in vitro. While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardiolipin. The possible physiological significance of the lipid cofactor effect is discussed. This is the first purification of a eucaryotic PGPS enzyme to date, and the first purification of a phospholipid biosynthetic enzyme from S. pombe.

Cations, Divalent↗

PubMed1997

The role of pyruvate dehydrogenase, phosphofructo-1-kinase and acetyl-CoA carboxylase in the regulation of fatty acid synthesis in the lactating rat mammary gland during the starved to re-fed transition.

Re-feeding 24-h-starved lactating rats resulted in a rapid (within 0.5 h) restoration of glucose uptake by the mammary gland and a slower (within 3 h) restoration of fatty acid synthesis. The rapid reactivation of glucose uptake (82% of fed value within 0.5 h of re-feeding) correlated with a rapid reactivation of 6-phosphofructo-1-kinase (6-PF-1-K) and glycolysis (as determined by a 97% decrease in the [fructose-6-phosphate]/[fructose-1,6-bisphosphate] ratio). This could not be fully explained by a fall (29%) in the tissue concentration of its allosteric inhibitor, citrate. The delayed reactivation of pyruvate dehydrogenase (PDH) correlated very closely with the delayed reactivation of fatty acid synthesis and explained the continued output of pyruvate and lactate within the first 0.5 h of re-feeding. PDH reactivation preceded the reactivation of acetyl-CoA carboxylase (ACC), which did not occur significantly until 1.5 h of re-feeding. ACC reactivation correlated with a decrease in the tissue concentration of citrate and a second late phase of 6-PF-1-K activation. It is clear that the important regulatory steps 6-PF-1-K, PDH and ACC, are reactivated asynchronously in the lactating mammary gland in response to re-feeding starved rats and that PDH is more important than ACC in the regulation of fatty acid synthesis.

Acetyl-CoA Carboxylase↗

PubMed1995

Late-infantile Batten disease: purification of the subunit c of the mitochondrial ATP synthase from storage material.

The accumulation of subunit c of the mitochondrial ATP synthase in late-infantile neuronal lipofuscinosis (LINCL) and juvenile neuronal lipofuscinosis (JNCL) is well documented. The purification of the subunit from diverse sources has been reported previously, although not from the brain of Batten disease patients. This proteolipid has now been purified from late-infantile Batten disease brain. The procedures used were an original combination of the conventional solubilisation, differential centrifugation, organic solvent extractions, preparative gel electrophoresis, and FPLC. Gel filtration of the purified protein indicated molecular mass equal to or greater than 2 x 10(6) Da; however, electrophoresis of this pure protein suggested a molecular mass of approximately 3,500 Da, which is a characteristic of subunit c. The pure protein may be solubilised in aqueous buffer containing < 1% lithium dodecyl sulphate (LDS). The protein binds dicyclohexylcarbodiimide (DCCD) and shows immunoreactivity to antibodies raised against ovine storage bodies.

Blotting, Western↗

PubMed1991

Regulation of fatty acid synthesis in lactating rat mammary gland in the fed to starved transition: asynchronous control of pyruvate dehydrogenase, phosphofructokinase and acetyl-CoA carboxylase.

1. Withdrawal of food from lactating rats produced a rapid and dramatic decrease in the uptake of glucose by the mammary gland and an inhibition of the rate of fatty acid synthesis that could not be explained alone by decreased substrate supply to the tissue. 2. Within the first 6 hr starvation, fatty acid synthesis and pyruvate dehydrogenase activity were inhibited by 87 and 80%, respectively, but acetyl-CoA carboxylase activity did not change significantly. 3. Between 6 and 24 hr starvation, total and expressed activities of acetyl-CoA carboxylase decreased by 62 and 55%, respectively. 4. The ratio of fructose-6-phosphate/fructose-1,6-bisphosphate concentration in mammary tissue increased 9-fold during the first 6 hr starvation, indicating an inhibition of 6-phosphofructo-1-kinase. However, the major inhibition of this enzyme occurred between 6 and 24 hr starvation when this metabolite ratio increased a further 160-fold in parallel with increased tissue citrate concentration. 5. The increase in citrate concentration between 6 and 24 hr starvation correlated with acetyl-CoA carboxylase inactivation and ketone body accumulation in the mammary gland. 6. This study confirms the asynchronous control of three important regulatory steps in the pathway of glucose utilization and fatty acid synthesis in the lactating rat mammary gland.

Acetyl-CoA Carboxylase↗