8-oxodG 8-oxo-7,8-dihydroguanine
Aco1p Aconitase
ADP Adenosine diphosphate
ATP Adenosine-5'-triphosphate
ALS Amyotrophic Lateral Sclerosis
AOP-1 Antioxidant protein 1
AP site Apurinic and apyrimidinic site
APS Ammonium persulfate
BER Base excision repair
bEC Bovine endothelial cells
Bp Base pairs
bSMC Bovine smooth muscle cells
ddH2O Double distilled water
D-loop Displacement-loop
DMSO Dimethyl sulfoxide
dN Deoxyribonucleotide
DNA Deoxyribonucleic acid
eNOS Endothelial NO synthase
ETC Electron transport chain
FAD Flavin adenine dinucleotide
FADU Fluorimetric analysis of DNA unwinding
FBS Fetal bovine serum
HDAC Histone deacetylase
HEK Human Embryonic Kidney cells
HMG High mobility group
HSP Heat shock proteins
hSMC Human smooth muscle cells
H-strand Heavy strand
HX Hypoxanthine
IMS Intermembrane space
iNOS Inducible NO synthase
MTA Mitochondrial theory of aging
mtDNA Mitochondrial DNA
mtNOS Mitochondrial NO synthase
mtSSB Mitochondrial single-strand binding protein NAD+ Nicotinamide adenine dinucleotide
OGG1 8-oxoguanine glycosylase
OM Outer membrane
p0 Mitochondrial DNA deficient cells
PARP Poly(ADP-ribose) polymerase
PBS Phosphate buffered saline
PHGPx Phospholipid-hydroperoxide glutathione peroxidase
Pol γ Polymerase gamma
Prx Peroxiredoxin
PTP Mitochondrial permeability transition pore
RNS Reactive nitrogen species
ROS Reactive oxygene species
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SOD1 (Cu,Zn-SOD) Copper, zinc superoxide dismutase
SOD2 (MnSOD) Manganese containing superoxide dismutase SOD3 (EC-SOD) Extracellular superoxide dismutase
TCA-cycle Tricarboxylic acid cycle
TFAM Mitochondrial transcription factor A
Trx Thioredoxin
U Unit (1 µmol/min enzyme activity)
UCP Uncoupling protein
UDG Uracil DNA glycosylase
XO Xanthine oxidase
7.2 List of figures
FIGURE 1-1: EVOLUTIONARY TRANSFER OF PROTO-MITOCHONDRIAL GENES...3
FIGURE 1-2: ELECTRON MICROGRAPH OF A MITOCHONDRION...5
FIGURE 1-3: MODELS FOR MITOCHONDRIAL MEMBRANE STRUCTURE...6
FIGURE 1-4: COMPUTER-GENERATED MODELS BASED ON SEGMENTED TOMOGRAMS OF MITOCHONDRIA...7
FIGURE 1-5: MITOCHONDRIA FUSION AND FISSION EVENTS IN LIVING CELLS...8
FIGURE 1-6: MAP OF THE HUMAN MITOCHONDRIAL GENOME...12
FIGURE 1-7: THE MINIMAL MITOCHONDRIAL REPLISOME...14
FIGURE 1-8: SKIN FIBROBLAST MTDNA NUCLEOID LABELED WITH DNA ANTIBODY...16
FIGURE 1-9: STEPS IN COMPACTION OF DNA BY TFAM...20
FIGURE 1-10: SUPEROXIDE ANION GENERATION IN MITOCHONDRIA BY THE ETC...23
FIGURE 1-11: MITOCHONDRIAL NOS IS ASSOCIATED WITH THE INNER MITOCHONDRIAL MEMBRANE...27
FIGURE 1-12: FORMATION AND INACTIVATION OF ROS AND RNS FORMATION IN MITOCHONDRIA.30 FIGURE 1-13: OXIDATION OF GUANINE...33
FIGURE 1-14: PROPOSED MECHANISM OF 8-OXODG PRODUCTION BY PEROXYNITRITE...33
FIGURE 1-15: BASE-PAIRING CONFORMATIONS OF 8-OXODG...34
FIGURE 1-16: THE MITOCHONDRIAL FREE RADICAL THEORY OF AGING...38
FIGURE 1-17: INVERSE CORRELATION BETWEEN HEART 8-OXODG IN MTDNA AND MLSP...39
FIGURE 3-1: ISOLATION PROCEDURE OF NUCLEOIDS FROM DIFFERENT CELL TYPES OR TISSUE....62
FIGURE 3-2: LAYERING OF THE STEP GRADIENT...63
FIGURE 3-3: STANDARD CURVE TO CALCULATE MTDNA CONCENTRATION FROM DNA FLUORESCENCE...64
FIGURE 3-4: PACHLT-A VECTOR AS MTDNA MODEL...71
FIGURE 3-5: PRODUCTION OF PEROXYNITRITE BY SIN-1...73
FIGURE 4-1: MITOCHONDRIA DNA WAS EXTRACTED BY THE PHENOL/CHLOROFORM METHOD FROM VARIOUS RAT TISSUE AND ANALYZED BY SDS-PAGE AND WESTERN-BLOT...75
FIGURE 4-2: NATIVE NBT-PAGE WITH MTDNA AND SOD1 AS PROTEIN STANDARD...76
FIGURE 4-3: ISOLATED RAT HEART AND BRAIN MITOCHONDRIA...77
FIGURE 4-4: WESTERN-BLOT TO CHECK PURITY OF MITOCHONDRIA SAMPLE...78
FIGURE 4-5: PREPARATION OF RAT HEART P1/S1 NUCLEOID FRACTIONS...80
FIGURE 4-6: PREPARATION OF RAT HEART P2 NUCLEOID FRACTIONS...81
FIGURE 4-7: PREPARATION OF RAT BRAIN P2 NUCLEOID FRACTIONS...82
FIGURE 4-8: BOVINE HEART NUCLEOIDS ISOLATED BY A SUCROSE DENSITY GRADIENT...83
FIGURE 4-9: BOVINE HEART NUCLEOID P2 PREPARATION...84
FIGURE 4-10: P2 FRACTIONS OF BOVINE BRAIN NUCLEOIDS...85
FIGURE 4-11: ISOLATION OF HUMAN NUCLEOIDS FROM JURKAT CELLS...87
FIGURE 4-12: TCA-CYCLE ENZYME IN JURKAT NUCLEOIDS...88
FIGURE 4-13: CONTROL DIGESTION OF MTDNA...90
FIGURE 4-14: ISOLATION OF PURE NUCLEOIDS...91
FIGURE 4-15: TCA-CYCLE ENZYME IN JURKAT P2 NUCLEOIDS...92
FIGURE 4-16: CONTROL DIGESTION OF MTDNA...93
FIGURE 4-17: ISOLATION OF RAT HEART NUCLEOID P1-FRACTIONS WITH INCREASING SALT CONCENTRATIONS...95
FIGURE 4-18: SLOT-BLOT FILTER ASSAY TO PROVE INTERACTION OF MTDNA AND RECOMBINANT SOD2 ...97
FIGURE 4-19: SLOT-BLOT FILTER ASSAY TO PROVE INTERACTION OF MTDNA AND BOVINE SOD1..99
FIGURE 4-20: IMMUNOPRECIPITATION OF BOVINE HEART NUCLEOIDS...100
FIGURE 4-21: CONFOCAL IMAGES OF HUMAN VASCULAR SMOOTH MUSCLE CELLS...102
FIGURE 4-22: LACK OF SOD2 ASSOCIATION TO NUCLEOIDS ISOLATED FROM BOVINE AORTIC ENDOTHELIAL CELLS...104
FIGURE 4-23: NUCLEOIDS ISOLATED FROM JURKAT CELLS TREATED WITH OR WITHOUT A HDAC INHIBITOR ...106
FIGURE 4-24: ELECTRON MICROGRAPH OF ISOLATED RAT HEART NUCLEOIDS...108
FIGURE 4-25: FADU-ASSAY WITH THE STANDARD PROTOCOL FOR NUCLEAR DNA... 110
FIGURE 4-26: DETECTION OF STRAND BREAKS IN PLASMID DNA BY THE MODIFIED FADU METHOD. ... 111
FIGURE 4-31: SUPEROXIDE DISMUTASES HAD NO PROTECTIVE EFFECT ON SUPEROXIDE INDUCED DNA DAMAGE...120
FIGURE 4-32: ANALYSES OF JURKAT MITOCHONDRIA FOR MTDNA STRAND BREAKS...122
FIGURE 4-33: IRRADIATED JURKAT MITOCHONDRIA SHOWED STRAND BREAKS IN A DOSE DEPENDENT MANNER...123
FIGURE 4-34: EFFECTS OF AGING IN ALDEHYDE DEHYDROGENASE OR SOD2-DEFICIENT MICE ON MITOCHONDRIAL DNA LESIONS...125
FIGURE 5-1: HIGHLY CONSERVED LYSINE RESIDUES AT THE C-TERMINUS OF SOD2...132
FIGURE 5-2: INHIBITION OF PEROXYNITRITE FORMATION INSIDE MITOCHONDRIA...139
FIGURE 5-3: MODEL TO EXPLAIN THE BIOLOGICAL SIGNIFICANCE OF SOD2 ASSOCIATION TO MTDNA ...140
FIGURE 5-4: VICIOUS CIRCLE FOR MITOCHONDRIAL SUPEROXIDE FORMATION...143
7.3 List of tables
TABLE 1-1:PROTEINS FOUND IN HUMAN MTDNA NUCLEOIDS...18
TABLE 3-1:LABORATORY EQUIPMENT...44
TABLE 3-2:PRIMARY AND SECONDARY ANTIBODIES...45
TABLE 3-3:BACTERIAL GROWTH MEDIUM...45
TABLE 3-4:GENERAL BUFFERS AND SOLUTIONS...46
TABLE 3-5:BUFFERS FOR SDS-PAGE AND WESTERN BLOT...47
TABLE 3-6:BUFFERS FOR MITOCHONDRIA AND NUCLEOID COMPLEX ISOLATION...49
TABLE 3-7:BUFFERS FOR ENZYME ACTIVITY...49
TABLE 3-8:BUFFERS FOR THE SLOT BLOT BINDING ASSAY...49
TABLE 3-9:CO-IMMUNOPRECIPITATION BUFFERS...50
TABLE 3-10:BUFFERS FOR THE FADU-ASSAY...50
TABLE 3-11:BUFFERS FOR IMMUNOFLUORESCENCE...51
TABLE 3-12:BUFFER FOR ELECTRON MICROSCOPY...51
TABLE 3-13:ANIMALS...51
TABLE 3-14:CELLS...52
TABLE 3-15:CELL CULTURE MEDIA, SUPPLEMENTS AND REAGENTS...53
TABLE 3-16:BACTERIA...54
TABLE 3-17:PLASMID...54
TABLE 3-18:ENZYMES...54
TABLE 3-19:PROTEIN MOLECULAR WEIGHT STANDARDS AND DNA MARKER...55
TABLE 3-20:KITS...55
TABLE 3-21:SOFTWARE...56
TABLE 4-1:DISTRIBUTION OF SOD AND MDH ACTIVITY IN THE VARIOUS FRACTIONS DURING NUCLEOID ISOLATION FROM JURKAT CELLS AND RAT HEART...89