KIN 406 Midterm 3 Flashcards
Redox reactions involve?
Addition (reduction) or removal (oxidation) of electrons
A pro-oxidant/oxidant is a?
oxidizing agent…accepts e_ from other molecules and it reduced itself
An antioxidant is a?
reducing agent…gives e- to another species and is oxidized itself
In a stable atom, electrons?
occupy each orbital with opposite spins to each other
What is a free radical?
an atom with an unpaired electron in an orbital
Why are free radicals so reactive?
Because of their unpaired electron
How do free raddicals become more stable?
By seeking to accept an e- from another molecule (could be a protein, a lipid, DNA, etc)
Redox reactions involving free radicals “stealing” electrons will continue until a terminal reaction is reached. What are two examples of these terminal reactions?
When radicals react with each other to form new nonradical species, or if it is quenched by an anti-oxidant to give a non-radical species.
What is a reactive oxygen species?
Compounds derived from partial chemical reductions of molecular oxygen
Are ROS always free radicals?
NO, because they don’t always contain unpaired electrons but are still highly reactive because chemical structure still wants to steal e-
What is ground state O2 like?
Has 2 unpaired electrons in different orbitals (diradical), however, it is stable because both electrons have identical spins…will not readily react with most molecules that e- in opposite spins
What can cause the reverse of the spin of one of electron to form reactive singlet oxygen (1O2)?
input of energy, such as radiation
How is the superoxid radical formed?
a one electron reduction of oxygen
What is superoxide usually converted to spontaneously because it is so reactive?
H2O2 (hydrogen peroxide)
Is H2O2 a free radical?
NO, because it has no upaired electrons, but it is a ROS
What is the functional difference between superoxide and hydrogen peroxide?
It is more stable than superoxide and is very long lived, so it can travel long distances, and it can readily pass through biological membranes.
What is H2O2 easily converted into?
A more highly reactive hydroxyl radical
What is the reaction for H2O2 –> hydroxyl radical?
Fenton reaction with a transition metal…Fe2+ + H2O2 —> Fe3+ + hydroxyl radical + stable hydroxide ion
What is nitric oxide?
a nitrogen based free radical/reactive nitrogen species
What is the reaction between nitric oxide and superoxide?
nitric oxide + superoxide —> peroxynitrire (ONOO-)
What is OONO-?
peroxynitrite…very long lived and very reactive
Why are free radicals, ROS, and RNS so bad?
can lethally damage proteins, DNA, and lipids which ultimately affect cell function
What are the 2 forms of antioxidants in the cell?
antioxidant enzymes and biological (chemical) antioxidants
What are the main antioxidants in the cell?
superoxide dismutase (SOD), catalase, gluthione peroxidase (GPx), and glutathione S-tranferase (GST)
Where are antioxidant enzymes synthesized, and what can induce them?
Synthesized in the body, and oxidant stress can induce their expression
3 forms of SOD?
Copper-Zinc (CuZnSOD), Manganese containing SOD (MnSOD), and extracellular SOD (EcSOD)
Where is CuSOD found?
In the cytosol
Where is MnSOD found?
in the mitochondrial matrix, makes up 15-20% of total SOD
What makes up 15-20% of SOD?
MnSOD
Where is extracellular SOC found?
A form of CuZnSOD found in the extracelllular fluid (plasma and interstitial fluid)
Where is MnSOD sythesized?
In the cytosol and transported to the mitochondria via Hsp70 and mitochondrial import stimulation factor, through TOMandTIM
What does SOD catalyze?
The reaction of superoxide and hydrogen to form hydrogen peroxide and oxygen (2 superoxide + 2 H+ –SOD–> H2O2 and O2
Where is catalse found?
in the mitochondria and other organelles
What does catalase do?
Dismutates hydrogen peroxide into water and oxygen (2H2O2 –catalase–> 2H2O + O2
Where is Gpx (glutathione peroxidase) found?
Mitochondria and cytosol
What does Gpx do?
Catalyzes the reduction of hydrogen peroxide to water by using glutathione (GSH) as an electron donor…2 GSH + H2O2 –GPX–> GSSG (glutathione disulfide) + 2H2O
What does GSH donate in the reaction with Gpx?
a pair of hydrogen ions (and electrons) to H2O2
What is GSH oxidized to?
Glutathione disulfide (GSSG)
What is the reaction for reducing GSSG back to GSH?
GSSG + NADPH + H+ –GR–> 2GSH + 2 NADP+
What is the electron donor for reducing GSSG to GSH, and the enzyme that catalyzes the reaction?
NADPH, and glutathione reductase
Which enzyme has a higher affinity for H2O2, catalase or Gpx?
GPx, and it is found in both the cytosol and mitocohdrial matrix
2 classes of nonenzymatic antioxidants?
1) antioxidants such as GSH that can be synthesized by the body 2) antioxidant enzymes that cannot be synthesized by the body and must be obtained in the diet
Main vitamin antioxidants?
Vitamin E and C
What is GSH, and where is it found?
A water-soluble thiol-containing peptide that is found in high concentrations in virtually all cells. In most cells it is found ing MILLIMOLAR concentrations
What is the most important function as GSH?
to serve as a substrate for GPX
Why is the ratio of GSH:GSSG important?
high intracellular GSSG levels inactivate other important enzymes
What is the problem with cells exporting GSSG to maintain the GSH:GSSG ratio?
a loss of GSSG can reduce the ability to recycle GSH
What are GSH levels regulated by?
GSH utilization and synthesis
Where is the majority of GSH synthesized, and from what?
In the liver (90%) from the AAs cysteine, glutamate, and glycine
What is another role of GSH besides to serve as a substrate for GPX?
also important in keeping vitamins E and C (which are limited) in a reduced state
Most important fat-soluble antioxidant in the body?
vitamin E (tocopherol)
Where is tocopherol/vitamin E found?
cell membranes including inner mitochondrial membrane
How does vitamin E act as an antioxidant?
donates an electron to any lipid radical…produces a new lipid non-radical and vitamin E radical
How is a vitamin E radical reduced back to vitamin E?
gains an electron from Vitamin C OR GSH
What is the main water-soluble antioxidant found in the body?
Vitamin C/absorrbic acid
Where is vitamin C found?
in the cytosol of the cell as well as in the ECF
How does vitamin C act as an antioxidant?
Can directly act as one by donating an electron to superoxide and hydroxyl radical OR donates an electron to vitamin E radical
What is the main function of vitamin c?
to donate an electron to vitamin E radical, this forms a less reactive vitamin C radical
How can a vitamin c radical be recycled back to vitamin C?
by GSH and other means
At physiological concentrations, ROS and RNS regulate…
growth, differentiation, proliferation, and apoptosis
ROS and RNS can stimulate…
gene expression of various cytokines, transcription factors, and proteins
ROS and RNS can stimulate gene expression of various cytokines, transcription factors, and proteins by…
1) regulating kinase and phosphatase activity thereby triggering a signaling cascade through protein phosphorylation and dephosphorylation 2) regulating synthesis and breakdown of transcription factors
Transcription factors stimulates by ROS and RNS are controlled by what signalling pathway?
Nuclear Factor kB (NFkB)
Protein kinases stimulates by ROS and RNS are controlled by what signalling pathway?
Mitogen Activated Protein Kinase (MAPKinases)
Oxidant stress occurs when?
there is an imbalance between oxidant generation and antioxidant defense. Therefore, any factor than causes an increase in oxidant generation or decrease in antioxidant defense could lead to oxidative stress.
Under conditions of oxidative stress, biomolecules will be ________ in tissues and organs
oxidized
Oxidized biomolecules can be?
proteins, lipids, and nucleic acids
What are the important consequences of oxidizing proteins, lipids, and DNA?
proteins = make a variety of active molecules and structures, lipids = make up organelles and cell membranes, nucleic acids = make up DNA (change code = change proteins that are made = change cell function)
Why is mtDNA particularly sensitive to oxidative stress?
close proximity to ETC, low non-coding area, lack protective histones, insufficient repair mechanisms
Why is it important to know what is causing oxidative stress?
to make a treatment of antioxidants that can better target the stress
Main cellular sources of ROS/RNS?
mitochondria, NADPH oxidase, xanthine oxidase, myeloperoxidase, nitric oxide synthase
What precentage of the total O2 used by mitochondria is incompletely reduced to superoxide?
2-3%…although this only represents a small fraction of the total O2 utilized this can be a significant source of ROS in metabolically active tissues
How is superoxide generated?
during mitochondrial respiration E- passed down the ETC are accepted by O2 to form H2O in a final step at Complex IV. Normally, O2 undergoes 4e- reduction; however, some e- leak out of the ETC and incompletely reduce O2 to superoxide
Superoxide is considered a : a) free radical e b) ROS c) RNS d) both and b e) a.b.c
d) free radical and ROS
The antioxidant that is responsible for the breakdown of H2O2 to H2O : a0 EcSOD b) MnSOD c) CuZnSOD d) glutathione reductase e) catalase
e) catalase
The major sources of the superoxide production in the ETC are?
Complex I and Complex III
The greater the flux of e- through the ETC the greater the…
superoxide generation
Where do the electrons leaked from Complex I go?
Tend to leak towards the matrix
When the e- from Complex 1 lead into the matrix, what do they do?
They can either cause damage to mtDNA or proteins in the matrix OR be taken up by antioxidants
Where do the leaked e- from Complex III go?
either into the intermembrane space or the matrix
E- from complex 3 that are leaked into the IMS, do what?
The superoxide can leave the IMS via an anion channel and go into the cytosol OR become H2O2 from GPx and leave into the cytosol
Decreased mitochondrial content and function does what to mitochondrial ROS generation?
increases it
Increased mitochondrial content and function does what to mitochondrial ROS generation?
decreases it
Why is there decreased ROS and free radical generation from the mitochondria with increased mitochondrial content?
The metabolic load is distributed amongst more mitochondria, therefore, the flow of e- is slower, so there is less e- leak.
Where is nicotinamide adenine dinucleotide phosphate oxidase found?
in membranes (plasma, SR, vacoules)
How many subunits is NADPH oxidase?
6
What does NADPH oxidase do?
produces superoxide by catalyzing the transfer of one electron from reduced NADH or NADPH to oxygen
What cells is NADPH oxidase found in?
immune cells (neutrophils and macrophages), smooth muscle, and skeletal muscle
What does NADPH oxidase do in immune cells?
Functions in host defense by killing microbes by causing free radical damage
Where is myeloperoxidase found?
lysozymes of macrophages and neutrophils
What does myeloperoxidase do?
Involves in host defense. Uses H2O2 to oxidize L-tyrosine to produces.
Where is xanthine oxidase found?
cystolic enzyme
What does xanthine oxidase do?
Produces superoxide and H2O2 from the oxidation of xanthine and hypoxanthine . Participates in cell signaling and can act as an NADH oxidase to help maintain reducing equivalents such as NAD+
What does nitric oxide synthase do?
Catalyzes the oxidation of L-arginine to L-citrulline to release nitric oxide free radical
3 main isoforms of nitric oxide synthase?
neuronal NOS (nNOS), indiucble NOS (iNOS), endothelial NOS (eNOS)
Where is neuronal NOS found?
primarily found in skeletal muscle where it is localized to the sarcolemma region and associated with dystrophin
Where is indicible NOS found?
Primary found in macrophages and aid in host defense
Where is endothelial NOS founf?
Primarily found in endothelial cells and aids in vasodilation
Antioxidant levels in different muscle types from most to least?
Highest in TypeI, then mixed, then Type II
What type of mithochondria have a higher rate of respiration and a lower rate of ROS generation?
IMF mitochondria
What explains the lower rate of ROS generation in IMF mitochondria?
different levels of MnSOD but possibly other antioxidants AND higher levels of cytochrome c
What effect do ROS have on satellite cells?
Satellite cells incubated with H2O2 show reduced divisions in culture
DNA isolated from skeletal muscle of SOD deficient animals have _____________
increased oxidative damage
What does aging do to ROS and free radical generation and oxidative stress?
It increases them
What does aging do to cellular antioxidant defenses?
decreases them
Skeletal muscle from aged animals and humans usually shows an _______ in antioxidant enzyme activity.
increase in SOD, catalase, GPx, GST, and GR
Why is there an increase in antioxidant enzymes with age?
Very responsive to change in the oxidative environment so antioxidant enzymes increases with the increased ROS generation with age, but oxidative stress still occurs because more ROS than antioxidant enzymes
Are GSH, vitamin C, and vitamin E levels affected by age?
No
What happens to malondialdehyde , a marker of oxidative damage to lipids, with age?
it is increased
Basal mitochondrial H2O2 release is _________ in mitochondrial isolated from aged skeletal muscle
elevated
Older adults that are displaying greater fiber type changes (+40% vs -40% type II fibers) changes in mixed muscle (vastus lateralis) have ________ levels of lipid peroxidation and __________ levels of antioxidants.
higher levels of lipis\d peroxidation and lower levels of antioxidants
ETC complex deficiencies that occur in muscle during aging result in?
Increased ROS generation at the mitochondria and increased muscle damage
What happens to antioxidant levels and ROS generation in reponse to disuse?
Antioxidant enzyme levels decrease, protein oxidation levels increase, and ROS generation increase
Denervation does what to MnSOD levels in SS and IMF mitochondria?
decreases them
What does the decreased levels of MnSOD do RR ROS production, but not IMF ROS producction?
increase SS ROS
What happens to force generation when an antioxidant is taken with exercise?
It decreases
Why does optimal force generation decrease with an antioxidant?
Antioxidants cause a lack of ROS. ROS are important in generating optimal force production such as cross bridge formation.
To maintain optimal muscle force generation, what must be maintained?
An optimal cellular redox status may influence
What does regular exercise do to muscle redox status?
Elevates the level of several antioxidants (positive adaptation) and basal ROS goneration is reduced in muscle (positive adaptation)
What happens to exercise-indices signaling and antioxidant adaptation if ROS generation is blocked?
No activation of p38, ERK, NFkB (MAPK pathways) by ROS, thish inhibits the exercise-induced upregulation of these pathways which causes a decrease in antioxidant enzymes.
What does oral vitamin intake do to exercise-induced antioxidant adaptations for normal exercise?
prevents it
What happens to mitochondrial biogenesis with decreased ROS signaling from increased antioxidants?
Mitochondrial biogeneis is decreased (less PCG1-alpha and PPAR)
What are the 2 types of cell death?
necrosis and apoptosis
What are the characteristics of necrosis?
accidental form of death, usually occurs during disease or acute injury only, not part of normal development, results in rapid cellular swelling and rupture of the cell membrane, leads to major inflammatory response due to the release of intracellular material into the surrounding environment
What are the characteristic of apoptosis?
Highly conserved cell death, important part of normal tissue development and homeostasis, increased or decreased during numerous diseases. Active process regulated by various intracellular signals. Results in chromatin condensation, cell shrinking, membrane blebbing. Cell membrane remains intact, little to no inflammation
In regards to the cell membrane, how are apoptosis and necrosis different
In apoptosis, the membrane stays intact, whereas in necrosis, it blows apart
What is cell blebbing?
When the cell packages its intracellular contents into packages that express ligands that recruit phagocytes to the bleb to destroy it
What is the hallmark of apoptotic cell death?
fragmented DNA. There is also a breakdown of important structural, regulatory, and repair proteins.
Stimuli for necrosis vs. apoptosis?
Necrosis - pathological, usually a consequence of irreversible cell injury or disease, “cell homicide.” Apoptosis - physiological and sometime pahtological, active process that is genetically controlled by a series of biochemical and molecular events, “cell suicide”
Histology of necrosis vs. apoptosis?
Necrosis: typically affects a larger number of cells, cell swelling, loss of membrane integrity. Apoptosis: typically affects a smaller number of cells, cell shrinkage, cell breaks down into membrane-bound fragments (apoptotic bodies) aka blebbing
DNA breakdown in necrosis versus apoptosis?
Necrosis = random, diffuse fragmentation. Apoptosis = orderly nuclear condensation and fragmentation
Tissue reaction in necrosis versus apoptosis?
Necrosis = inflammation with secondary injury to surrounding unaffected tissues. Apoptosis = little to no inflammation or secondary tissue injury, cell membrane signals allow for elimination by phagocytes
What is the most popular researched biological topic currently, and why?
apoptosis because it affects almost every cell in the body, and it is in normal and diseased cells
Proliferation > cell death =
accumulation of cells…cancer, autoimmune disorders
Proliferation = cell death =
homeostasis
Cell death > proliferation =
cell loss…Alzheimer’s, cardiac and muscle diseases
Inhibition of cell death leads to?
Cell accumulations…undesirable tissue growth and in this case, an accumulation of immune cells and a variety of immune disorders
What would happen to the size of the spleen and lymph nodes in a bax-/- and bak -/- mouse?
undesirable growth of these organs
Promotion of cell death leads to?
Cell loss…this leads to an undesirable tissue atrophy, retarded development, decreased immune function, and premature death
What happens to the size of the spleen and lymph nodes in Bcl-2-/- mice?
Smaller in size and smaller kidney and spleen
Where does the name caspase come from?
Cysteine ASPartic acid proteASES (caspases)
How many caspases are there?
14 have been identified
How are caspases found?
in an inactive (pro-form) and active form
2 main classes of caspases?
initiator and effector
What are the initiator caspases?
Caspase 2,8,9,12
What are the effector caspases?
Caspase 3,6,7
What do caspases do?
Cleave numerous cellular substrates that ultimately result in apoptosis…actin, poly(ADP)ribose polymerase (DNA repair enzyme), lamins (nuclear structure proteins)
How did BCl-2s get their names?
first identified as a gene that alters growth and cell death in B-cell Lymphoma (Bcl-2)
How many Bcl-2 proteins are there in the family?
25
What is the role of the Bcl-2 family?
function to inhibit or promote cell death in a variety of tissues
Where do Bcl-2 family proteins act?
on various membranes (mitochondrial, ER/SR, and nuclear)
2 main classes in the BCl-2 family?
Anti-apoptotic and pro-apoptotic
Pro-apoptotic proteins of the Bcl-2 family?
Bax, Bid, Bak
Anti-apoptotic proteins of the Bcl-2 family?
Bcl-2, Bcl-xl
What is used as a measure of a cell’s susceptibility to apoptosis?
The ratio and pro to anti-apoptotic proteins
What are some inducers of apoptosis?
high levels of free radicals, ROS, RNS. Chemotherapeutic drugs. High levels of stress hormones (glucocorticoids, catecholamines), high cystolic calcium levels. UV radiation, ethanol, growth factor withdrawal, nutrient deprivation.
Inhibitors of apoptosis?
Antioxidants (superoxide dismutase, gluthione, catalase, vitamin C, vitamin E). Various grwoth factors (IGF-I and GH)
What is the death receptor pathway?
Cytokines FasL or TNF-alpha bind to their receptors on the sarcolemma. This activates the transmembrane the Fas associated death domain, which cleaves procaspase-8 into active caspase 8. Caspase 8 cleaves procaspase 3 into caspase 3, which then begins to dismantle to cell by cleaving its substrates.
Which of the following is false in respect to muscle during aging? a) ETC deficiencies occur b) elevated lipid peroxidation c) increased catalase levels d) higher mito. ROS generation e) none of the above
e) none of the above…they are all true
