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1.
Genome Res ; 20(1): 28-35, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19923254

ABSTRACT

Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.


Subject(s)
Acetaminophen/adverse effects , Analgesics, Non-Narcotic/adverse effects , Betaine-Homocysteine S-Methyltransferase/genetics , Chemical and Drug Induced Liver Injury/genetics , Liver Failure, Acute/genetics , Vitamin U/metabolism , Acetaminophen/metabolism , Acetaminophen/pharmacokinetics , Animals , Betaine-Homocysteine S-Methyltransferase/metabolism , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Chemical and Drug Induced Liver Injury/prevention & control , Diet , Gene Expression Profiling , Liver/metabolism , Liver/pathology , Liver Failure, Acute/chemically induced , Liver Failure, Acute/pathology , Liver Failure, Acute/prevention & control , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred Strains , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Sequence Analysis, DNA
3.
Assay Drug Dev Technol ; 1(6): 823-33, 2003 Dec.
Article in English | MEDLINE | ID: mdl-15090228

ABSTRACT

Activation of cells by the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor-kappaB (NF-kappaB) via proteasomal degradation of an associated IkappaB molecule. To monitor cellular IkappaB, the protein was recombinantly expressed as a fusion protein with a novel enzymatic tag, ProLabel (PL). ProLabel is a small 5.5-kDa sequence from the amino-terminal amino acids of beta-galactosidase, possesses a simple ribbon structure, and can be fused to many proteins via the amino or carboxyl terminus. Expression of this construct allows quantitative detection of the recombinant protein in crude lysates by using a method based on beta-galactosidase enzyme fragment complementation (EFC). Transient transfection of IkappaB-PL in HeLa cells generated an EFC signal that was highly correlated with a western analysis of the protein construct. ProLabel expressed alone in the cells did not show any EFC activity, due to rapid proteolytic degradation, indicating a very low background signal from the protein tag. TNF-alpha and IL-1 treatment induced a concentration-dependent degradation of IkappaB-PL, with potency values similar to those reported using other methods. IkappaBM-PL (mutant of IkappaB-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF-alpha or IL-1, demonstrating that degradation of IkappaB-PL was specific to the NF-kappaB pathway activation. TNF-alpha and IL-1 induced maximal IkappaB-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF-alpha antibodies and the proteasome inhibitor, MG-132. The assay was amenable to HTS systems, with good precision and reproducibility. Z' values and coefficients of variance for IkappaB-PL degradation were 0.6 and <9%, respectively.


Subject(s)
I-kappa B Proteins/metabolism , Membrane Proteins/metabolism , NF-kappa B/metabolism , Protein Interaction Mapping/methods , Signal Transduction/genetics , beta-Galactosidase/genetics , Dose-Response Relationship, Drug , Genetic Complementation Test , HeLa Cells , Humans , I-kappa B Proteins/genetics , Leupeptins/pharmacology , Membrane Proteins/genetics , NF-kappa B/genetics
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