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Relative influence of pro- and antioxidant enzymes on the cellular oxidative damage in man. - AN0401
Diet influences the activities of pro- and antioxidant
enzymes in both animals and humans. The direct consquences
of such changes on cellular oxidative damage is not clear. A
sub-cellular system will be developed based upon the
microsomal fraction ( membrane vesicles containing the
significant enzymes) isolated from various soft tissues and
human cell lines qualitatively and quantitatively engineered
with respect to key enzymes. Changes in the composition and
content of pro- and antioxidant enzymes in subcellular
fractions will be effected by a combination of the
following: a) direct addition of cytosolic enzymes;
b) induction of certain enzymes through exposure to drugs or
plants protective factors.
Data obtained from this system will form the basis for
developing a predictive model for determining the
nature/level of cellular oxidative damage in humans.
1. Purification of target enzymes.
2. Production of microsomal enzyme systems form human cell
lines expressing human membrane-bound target enzymes and
from human tissues.
3. Characterise the integrity, enzyme levels and metabolite
levels of microsomal system.
4. Set up assays to measure oxidative damage of microsomal
systems and of whole cells and use to monitor oxidative
damage. Eventual assay choice to be made in consultation
with other workers from the antioxidant programme.
5. Set up subcellular models using techniques and material
developed om 1-4.
6. Assess the relative importance and contribution of target
enzymes in subcellular model subjected to oxidative stress.
7. Develop a predictive model for determining the
level/nature of cellular oxidative damage utilising marker
enzyme activity data. Incorporate putative protective
fractions and determine their effect on oxidative damage.
8. Validate this using enzyme activity data from associated
Time-Scale and Cost
Contractor / Funded Organisations
IFR - Institute of Food Research (BBSRC)