Folate is a water-soluble vitamin that is essential for nucleic acid and amino acid metabolism. Folate absorption requires a two-stage process of hydrolysis of dietary pteroylpolyglutamates and binding and transport of the pteroylmonoglutamate derivative at the brush-border surface of the intestine. The overall objective of the proposed research is to further understanding of the process of folate absorption by study in the pig of the molecular properties and tissue expression of two intestinal folate-interactive proteins: jejunal brush-border folate hydrolase (BBFH) and folate-binding protein (FBP).
Specific aims i nclude: a) to develop cDNA and antibody probes for both proteins; b) to determine the full-length cDNA for both proteins; and c) to study the tissue expression of both proteins. Methods include use of oligonucleotide probes for BBFH and a gift full-length cDNA for a human FBP to isolate specific clones for BBFH and FBP from a pig jejunal cDNA library. Antibodies will be developed from purified proteins, fusion proteins, and synthetic peptides, and will be used to confirm oligonucleotide screens and for tissue expression studies. Sequencing of full-length cDNAs for each protein will be followed by homology comparisons. Tissue studies will utilize Northern blots and in-situ hybridization for transcripts, and immunoprecipitation, rocket immunoelectrophoresis, immunoblots, and immunohistochemistry to determine molecular sizes, amounts, and cellular distribution of each protein in the intestine and other tissues. Data from These studies will be used to develop a subsequent proposal to study the regulation of protein synthesis in animal models, thus providing a framework for study of the regulation of folate absorption in human tissue from patients with different disorders of folate absorption and deficiency.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK045301-01
Application #
3246819
Study Section
Nutrition Study Section (NTN)
Project Start
1992-09-30
Project End
1995-09-29
Budget Start
1992-09-30
Budget End
1993-09-29
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California Davis
Department
Type
Schools of Medicine
DUNS #
094878337
City
Davis
State
CA
Country
United States
Zip Code
95618
Halsted, Charles H; Villanueva, Jesus A; Devlin, Angela M et al. (2002) Interactions of ethanol and folate deficiency in development of alcoholic liver disease in the micropig. Trans Am Clin Climatol Assoc 113:151-62; discussion 162-3
Halsted, Charles H; Villanueva, Jesus A; Devlin, Angela M et al. (2002) Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig. Proc Natl Acad Sci U S A 99:10072-7
Halsted, Charles H; Villanueva, Jesus A; Devlin, Angela M (2002) Folate deficiency, methionine metabolism, and alcoholic liver disease. Alcohol 27:169-72
Villanueva, J A; Devlin, A M; Halsted, C H (2001) Reduced folate carrier: tissue distribution and effects of chronic ethanol intake in the micropig. Alcohol Clin Exp Res 25:415-20
Devlin, A M; Ling, E H; Peerson, J M et al. (2000) Glutamate carboxypeptidase II: a polymorphism associated with lower levels of serum folate and hyperhomocysteinemia. Hum Mol Genet 9:2837-44
Halsted, C H; Ling, E H; Luthi-Carter, R et al. (1998) Folylpoly-gamma-glutamate carboxypeptidase from pig jejunum. Molecular characterization and relation to glutamate carboxypeptidase II. J Biol Chem 273:20417-24
Villanueva, J; Ling, E H; Chandler, C J et al. (1998) Membrane and tissue distribution of folate binding protein in pig. Am J Physiol 275:R1503-10
Van Hoozen, C M; Ling, E H; Halsted, C H (1996) Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum. Biochem J 319 ( Pt 3):725-9