The central focus of the research described in this proposal is an understanding of the mechanisms which regulate the expression of lactase- phlorizin hydrolase (L-Ph) during the development of the mammalian small intestine. This enzyme is solely responsible for the hydrolysis of lactose, the primary carbohydrate in milk, and is essential for survival of mammalian neonates; adult animals and most human adults are considered to be lactase-deficient. The laboratory of the principal investigator has recently isolated and partially sequenced a 2.3 kb cDNA encoding a portion of rat L-Ph. On the basis of recent data obtained using probes derived from this clone, it now seems clear that, in addition to the expected high levels of L-Ph mRNA in suckling animals, significant quantities also persist in adult enterocytes. In combination with data obtained during the previous grant period demonstrating hydrolysis of glycolipids by L-Ph and the continued presence of total L-Ph activity in adult small intestine, these observations suggest a more complex function of the L-Ph subsequent to the suckling period than previously understood. These findings alter the interpretation of all previous studies of the development of the L-Ph which relied exclusively on measurements of specific activity. In order to elucidate the mechanisms regulating developmental L-Ph expression studies have been designed to: 1) determine the temporal pattern of L-Ph gene expression during development in the rat total small intestine by measuring concurrently L-Ph mRNA, L-Ph enzyme activity and L-Ph protein content; 2) characterize the cellular basis for alteration sin L-Ph activity and content using in situ hybridization and immunohistochemistry; 3) define regional differences in L-Ph enzyme activity and protein content in relation to L-Ph mRNA levels along the length of the small intestine, and examine the mechanisms by which L-Ph gene expression is influenced by hormonal status; 4) isolate a full-length cDNA encoding L-Ph; 5) elucidate the structural requirements for correct processing and intracellular targeting of L-Ph using transfection of the full length cDNA and specific constructs or deletions; and 6) identify the active sites in L-Ph and their structural features by peptide mapping and use these data to guide deletional analysis using expression cDNAs. Taken together, these patterns, derived in rats which are considered to be lactase deficient in adulthood, should be representative of the developmental changes occurring in other mammals and in the intestine of lactase-deficient humans.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Tufts University
United States
Zip Code
Boudreau, Francois; Rings, Edmond H H M; van Wering, Herbert M et al. (2002) Hepatocyte nuclear factor-1 alpha, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription. Implication for the developmental regulation of the sucrase-isomaltase gene. J Biol Chem 277:31909-17
Krasinski, S D; Van Wering, H M; Tannemaat, M R et al. (2001) Differential activation of intestinal gene promoters: functional interactions between GATA-5 and HNF-1 alpha. Am J Physiol Gastrointest Liver Physiol 281:G69-84
Li, W; Wang, J; Coluccio, L M et al. (2000) Brush border myosin I (BBMI): a basally localized transcript in human jejunal enterocytes. J Histochem Cytochem 48:89-94
Montgomery, R K; Mulberg, A E; Grand, R J (1999) Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology 116:702-31
Estrada, G; Krasinski, S D; Grand, R J et al. (1998) Defective intracellular processing of lactase-phlorizin hydrolase protein in rats prenatally exposed to ethanol. Alcohol Clin Exp Res 22:1177-83
Lee, M F; Krasinski, S D (1998) Human adult-onset lactase decline: an update. Nutr Rev 56:1-8
Barth, J A; Li, W; Krasinski, S D et al. (1998) Asymmetrical localization of mRNAs in enterocytes of human jejunum. J Histochem Cytochem 46:335-43
Li, W; Krasinski, S D; Verhave, M et al. (1998) Three distinct messenger RNA distribution patterns in human jejunal enterocytes. Gastroenterology 115:86-92
Montgomery, R K; Rings, E H; Thompson, J F et al. (1997) Increased C/EBP in fetal rat small intestine precedes initiation of differentiation marker mRNA synthesis. Am J Physiol 272:G534-44
Lee, M F; Russell, R M; Montgomery, R K et al. (1997) Total intestinal lactase and sucrase activities are reduced in aged rats. J Nutr 127:1382-7

Showing the most recent 10 out of 29 publications