effect of superoxide dismutase/catalase mimetics on lifespan and oxidative stress in housefly
DESCRIPTION
Effect of Superoxide Dismutase/Catalase Mimetics on Lifespan and Oxidative Stress in Housefly. Bayne, Sohal. Hypothesis. SOD/catalase mimetics extend lifespan in houseflies. Materials. EUK-8 and EUK-134 Houseflies (musca domestica) 200/cage/group. Method s. Divide flies into groups: - PowerPoint PPT PresentationTRANSCRIPT
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Effect of Superoxide Dismutase/Catalase Mimetics
on Lifespan and Oxidative Stress in Housefly
Bayne, Sohal
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Hypothesis
• SOD/catalase mimetics extend lifespan in houseflies.
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Materials
• EUK-8 and EUK-134
• Houseflies (musca domestica) 200/cage/group
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Methods
• Divide flies into groups:– controls– low dose– medium dose– high dose
• Administer mimetics at various concentrations via drinking water
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Observe
• effects on lifespan
• amounts of protein carbonyls under different conditions:– normoxic– hyperoxic (100% ambient oxygen at 10-
12 days for 48 hrs)
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EUK-8 under normoxic conditions:
• flies drank less water
• shortened lifespan 67% at high doses
• no effect at low doses
• no effect on protein carbonyl content
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EUK-8 under hyperoxic conditions:
• shortened lifespan 13%
• no effect on protein carbonyl content
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EUK-134 under normoxic conditions:
• shortened lifespan at high doses
• no effect at low doses
• no effect on protein carbonyl content
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Conclusions
• The effects of SOD/catalase mimetics appear to be species specific.
• SOD/catalase mimetics do not extend lifespan in house flies.– (contrast with results in worms)
• SOD/catalase mimetics do not affect protein carbonyl levels in house flies.
• SOD/catalase mimetics do shorten lifespan during oxidation stress.– (possibly due to toxicity or its opposite, low intake)
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Effects of caloric restriction on mitochondrial reactive oxygen
species production and bioenergetics: reversal by insulin
Lambert, A.J. and Merry, B.J.
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• Putting it all together: CR, insulin, mitochondria, and free radicals
• Study the effects of CR and insulin on mitochondrial reactive oxygen species production.
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Background
• Caloric restriction extends mean and maximum lifespan in model organisms.
• CR delays the onset and incidence of age-related diseases.
• CR ameliorates the age-related decline in DNA repair and protein turnover.
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• The exact mechanisms of how CR causes these effects are not known.
• Attenuation of the rate of accrual of tissue oxidative damage by decreased generation of free radicals is a plausible explanation.
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• Mitochondria are the main producers of free radicals.
• Mitochondrial DNA is particularly susceptible to free radical damage.
• Mitochondria from CR animals show reduced rates of free-radical generation.
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Methods
• Male Brown Norway rats, housed singly
• Intake of food was limited such that body weights were maintained at 55% of the age-matched controls (fully-fed rats).
• Insulin miniosmotic pumps implanted subcutaneously.
• Give insulin (0.55 microL/hr) to group of adult rats continuously for 2 weeks.
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• Measure production of free radicals, specifically hydrogen peroxide (H2O2).
• Measure activity in specific components of mitochondrial energetic pathways (proton gradient, substrate oxidation, other).
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Results
• Plasma insulin levels were significantly lower in CR than in control rats.
• Hydrogen peroxide production rate significantly lower in CR (0.25 nmol/min/mg) than in fully-fed rats (0.60 nmol/min/mg)
• Decrease in hydrogen peroxide production rate was partially reversed (0.40 nmol/min/mg) by 2 weeks of 0.55 microL/hr insulin treatment of CR rats.
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Conclusions
• CR decreases insulin levels and decreases free-radical levels in the mitochondria
• Increasing insulin levels counteract the reduction of free-radical levels in the mitochondria
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Q and Extension of Life Span in Worms
Larsen, Clarke
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Background
• Coenzyme Q (ubiquinone) is sold in drug stores as an anti-oxidant.
• Many people take Q as a life-extension supplement.
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• Coenzyme Q is a carrier of electrons in the mitochondrial Electron Transport Chain.
• Electron transport in complexes I & III create a proton gradient across the inner membrane.
• This is coupled to the synthesis of ATP by complex V (Fo/F1 ATPase).
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Q functions:
• antioxidant (scavenges electrons)
• prooxidant (generates superoxide)
• a redox-active component of plasma-membrane electron transport
• uridine synthesis
• a cofactor for proton-pumping activity in uncoupling proteins in mitochondria.
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Q6, Q7, Q8, Q9, and Q10
• Coenzyme Q can have a variable length side chain, with typically 6 to 10 subunits, hence Q6, Q7, Q8, Q9, and Q10.
• Different species tend to produce Q with a particular length side chain – Q10 in human– Q9 in worm– Q8 in bacteria
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Q mutants in worms
• Clk-1 mutants in worms lack endogenous Q9 – relies instead on Q8 from bacterial diet.
• Clk-1 mutants live twice as long as wildtype worms.
• The missing clk-1 gene encodes a di-iron carbolxylate enzyme:– responsible for final hydroxylase step in Q
synthesis
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• A test to see if dietary Q alters lifespan of worms.– Normal diet is OP50 (Q8 replete E.coli
bacteria)– Q-less diet is GDI (Q-less E.coli bacteria)
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Experiment 1
• Wild worms switched to Q-less diet during larval stage 4– avoids developmental interference
• Wildtype lifespan extended 59%.
• Lack of Q8 extends lifespan.
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Experiment 2
• Wild type fed Q-less diet from egg to old age.
• A small fraction died earlier than normal.
• Surviving worms lived longer than normal
• Had reduced medial lifespan compared to worms switched at L4.
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Experiment 2 Conclusions
• Q is beneficial in early development.
• Q contributes to short lifespan.
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Q vs. daf genes
• The daf-2 gene in worms regulates both:– development– lifespan.
• The longevity pathway includes 2 genes:– daf-2– age-1
• that extend lifespan when mutated.
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Suppression Analysis
• Used to determine the gene products required for a specific phenotype
• Example:– The longevity phenotype of daf-2 is
considered to be suppressed by daf-16 because the double mutant daf-16/daf-2 is short lived
• Thus, daf-2 mutants longevity requires wildtype daf-16 activity.
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Analysis 1
• Suppression tests were performed on the Age phenotype with these gene products:
• daf-16• daf-12.• On a Q-replete diet, daf-16 and daf-12 mutants
live shorter than wildtype.• On a Q-less diet they live longer than wildtype.• Neither mutation suppressed the lifespan
extension produced by the Q-less diet.• Therefore, these genes are not required for
longevity.
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Analysis 2
• Effects of Q-less diet on longlived daf-2 mutants:– e1370– m41.
• Both had longer median and max lifespans on Q-less compared to Q-replete diet.
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Analysis 3
• In C.elegans, daf-2 (e1370) is the longest lived when that mutation is combined with a mutation in either:
• daf-12
• or
• clk-1.
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• The daf-12/ daf-2 double mutant had no extended lifespan on either diet.
• The clk-1/ daf-2 double mutant had extended lifespans on a Q-less diet.
• The longevity mechanism here is additive:long-lived mutant + Q-less diet =even more
longevity
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• The combination of mechanisms that reduce ROS generation and increase ROS scavenging result in decrease in total cellular ROS and apparently allows for an extended lifespan.
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Silencing of Ubiquinone Biosynthesis Genes Extends
Lifespan of Worms
Asencio, Rodrigues-aguilera et al
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Aims
• Identify genes that synthesize Coenzyme Q in C. elegans
• Determine their effect on lifespan
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This study observes
• 1. the effect of double-stranded RNA (dsRNA) as it interferes (RNAi) with gene sequences that are homologous to those of Q6 biosynthesis in saccharomyces cerevisiae during Q9 biosynthesis and Q8 intake.
• 2. the respiratory chain properties in mitochondrial of silenced worms and some aspects of phenotype (especially lifespan)
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Results
• Using dsRNA interference, 8 genes were identified that participate in Q9 biosynthesis in worms.
• RNA interference (RNAi) of Q9 biosynthesis genes extends lifespan.
• Worms treated with RNAi produce less superoxide anions (30-50% less).
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Conclusions
• At least 8 genes participate in Q9 biosynthesis.• Silencing the genes results in:
– lowered Q9 levels– lower superoxide production (in ETC)– extended lifespan– less damage to macromolecules in mitochondria.
• Findings support the endogenous oxidative stress hypothesis.
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Small Molecule Activators of Sirtuins Extend Yeast Lifespan
Howitz, Bitterman, et al.
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Background
• In budding yeast, CR extends lifespan by increased activity of Sir2 gene.
• Other members of this sirtuin (NAD+ dependent protein deacetlylases) family are:– Sir2 in yeast (extends lifespan)– Sir2.1 in worms (extends lifespan)– Sirt1 in human (promotes cell survival).
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• Sirtuins are thought to be part of an evolutionarilly conserved longevity pathway because:– they occur in rodents, flies, worms,
yeast and humans– appear to promote survival by inducing a
response to stress (drought etc.)
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Compounds that affect Sir
• resveratrol (found in red wine) activated Sirt1 2fold.
• quercetin (protein kinase inhibitor) activated Sirt1 5fold.
• piceatannol (protein kinase inhibitor) activated Sirt1 8fold.
• STACs (14 small molecule sirtuin activators) activated Sirt1 2fold.
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Resveratrol
• In yeast, resveratrol mimics CR by:– stimulating Sir2 (by not inhibiting it)– increasing DNA stability– extending lifespan by 70%
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Stabilizing DNA
• Sir2 probably extends lifespan of yeast by stabilizing repetitive DNA.
• Recombination of ribosomal RNA (rRNA) can generate an extrachromosomal molecule.
• This error is replicated to toxic levels in old cells.• Resveratrol in combination with Sir2 reduce the
frequency of rRNA recombination 60%.• Suggests aging in yeast caused by genomic
instability – not gene dysregulation.
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• Preliminary experiments indicate resveratrol extends the lifespan of mulit-cell animals (flies and worms)
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resveratrol in humans
• lowers the Michaelis Constant of Sirt1 for both:– the acetylated substrate– and NAD+.
• is associated with health benefits including the mitigation of:– neurodegeneration– carcinogenesis– atherosclerosis.
• increases cell survival by stimulating Sirt1-dependent deacetylation of gene p53.– protect cultured human cells from radiation
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• Glucose restricted yeast had no extension of longevity when treated with resveratrol.
• Resveratrol had no effect on lifespan of Sir2 null mutants.
• Indicates that resveratrol probably acts through CR pathway.