Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-09-25
2002-11-12
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S034000, C435S039000
Reexamination Certificate
active
06479251
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention is related to the area of toxicology, mutagenesis, and carcinogenesis testing.
BACKGROUND OF THE INVENTION
Mitochondria are the major sites of energy (ATP) production in the cell. Mitochondria also perform many other cellular functions. These include respiration, heme, lipid, amino acid and nucleotide biosynthesis. Mitochondria also maintain the intracellular homeostasis of inorganic ions and initiate programmed cell death (1-5). Mitochondria contain approximately 1000 proteins. However, only 13 of these proteins are encoded by human mitochondrial DNA (mitDNA). These 13 proteins constitute the essential subunits of the electron transport system (1-5). All other mitochondrial proteins are synthesized in the cytoplasm and transported into mitochondria. Proteins destined for mitochondria contain a mitochondrial transport signal (MTS, 5).
Mitochondria are the major source of endogenous reactive oxygen species (ROS) in cells because they contain the electron transport chain that reduces oxygen to water by addition of electrons during oxidative phosphorylation. Mitochondrial respiration accounts for about 90% of cellular oxygen consumption (1-5). Therefore, each mitochondrion produces 10
7
ROS molecules/cell/day during normal oxidative phosphorylation (6-7). Human mitDNA, unlike nuclear DNA, contains no introns and has no protective histones proteins. These features of mitDNA make it more vulnerable than nuclear DNA to damage by ROS produced within the mitochondria (and by environmental agents such as ionizing radiation which produce ROS). Consistent with this finding mitDNA appears to mutate at a rate ten times higher than nuclear DNA (8). Mutations in mitDNA are involved in the pathogenesis of a variety of diseases including cancer (1, 9-10), heart disease (11), cardiomyopathies, diabetes, degenerative diseases such as Parkinson's, Alzheimer's, Huntington's disease, and other neurological disorders (1,12). Accumulation of somatic mutations of mitDNA also appears to be a constant feature of normal aging in all vertebrates thus far examined (1). The genetic changes observed in these diseases range from point mutations to deletions or insertions in the mitDNA(13-15).
There is a continuing need in the art for improved means of testing agents which may be harmful to the health of humans as well as a need for identification of agents for treating important human diseases in which mitochondrial dysfunction has been implicated.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for identifying agents which induce mitochondrial dysfunction.
It is another object of the present invention to provide a method for identifying agents which enhance mitochondrial function.
It is still another object of the invention to provide a kit for testing mitochondrial function.
These and other objects of the invention are achieved by providing one or more of the following embodiments. In one embodiment, a method is provided for identifying agents which induce mitochondrial dysfunction. Eukaryotic cells which are auxotrophic for adenine are contacted with a test agent. The eukaryotic cells are cultured on a medium comprising glucose. The fraction of the eukaryotic cells which appear white is scored. The white eukaryotic cells are tested to determine the ability to grow on a non-fermentable carbon source. The test agent is identified as a mitochondrial dysfunction inducer if the test agent increases the fraction of the eukaryotic cells (a) which become white and (b) which are unable to grow on the non-fermentable carbon source, relative to control eukaryotic cells which have not been contacted with the test agent.
According to another embodiment of the invention, a method is provided for identifying agents which enhance mitochondrial function. Eukaryotic cells which are auxotrophic for adenine and defective in mitochondrial function are contacted with a test agent. The eukaryotic cells are cultured on a medium comprising glucose. The fraction of the eukaryotic cells which appear red is scored. The red eukaryotic cells are tested to determine the ability to grow on a non-fermentable carbon source. The test agent is identified as a mitochondrial function enhancer if the test agent increases the fraction of the eukaryotic cells (a) which become red and (b) which are able to grow on the non-fermentable carbon source, relative to control eukaryotic cells which have not been contacted with the test agent.
According to still another embodiment of the invention, a kit is provided. The kit comprises a sample of eukaryotic cells which are auxotrophic for adenine; a non-fermentable medium for growth of the eukaryotic cells; a fermentable medium for cell growth of the eukaryotic cells; and instructions for testing agents for the ability to affect mitochondrial function.
The present invention thus provides the art with a simple bioassay system which can be used by toxicologists to determine the potential genetic and non-genetic hazard to mitochondria of a variety of suspected or known mutagens, toxins, or carcinogens.
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Banner & Witcoff , Ltd.
Chaudhry Mahreen
Gitomer Ralph
The Johns Hopkins University
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