Progress 08/15/03 to 11/15/07
Outputs
OUTPUTS: Activities: 1. Conducted experiments proposed including characterization of retinal reductases and BCMO knock out mice. 2. Seminars were given to nutritional sciences graduate students regarding experimental process and results from proposed studies (3 seminars were given during the granting period) 3. Mentoring undergraduate students - independent research project. 4. Presentation was given to Clinical Nutrition Research Unit members regarding findings from the proposal. Products: A new mouse model, BCMO1-deficient mice were obtained from Dr. Adrian Wyss of DSM and was characterized. Dissemination: BCMO deficient mouse line will be freely available to other investigators at non-profit organization who obtain a written consent from Dr. Wyss.
PARTICIPANTS: Individuals: Jisun Paik, PD - Dr. Paik conducted the proposed studies and devoted 4.8 calendar months effort for each year Collaborators and contacts: Dr. William S. Blaner at Columbia Univeristy had been a collaborator in creating tissue specific knock out mice lacking beta-carotene 15,15' monooxygenase (BCMO). He provided total body knock out mice lacking BCMO that was obtained originally from Dr. Adrian Wyss (DSM, Switzerland). We used this mice to characterize beta-carotene absorption and metabolism. Training or professional development: Two undergraduate students had research experience by helping to conduct proposed studies and were given research credits.
Impacts
New knowledge has gained by completion of our proposed studies: 1. retinal reductase 1 is likely a downstream enzyme responsible for retinal reduction following beta-carotene cleavage. 2. Mice lacking beta-carotene 15,15' monooxygenase (BCMO-deficient mice) is a suitable model to study beta-carotene absorption. 3. Beta-carotene absorption is highly variable in animals and is dependent on multiple factors, including efficiency of cleavage in the intestine. 4. Beta-carotene 15,15' monooxygeanse is a main enzyme responsible for cleaving beta-carotene in intestine. 5. Cellular retinol binding proteins regulate flow of retinoid metabolism by channeling retinoids to specific enzymes thus, protecing cells from damage and preventing wasting of retinoids. 6. Cellular retinol binding protein type I stimulates BCMO activity while cellular retinol binding protein type II does not. In the future, we plan to explore beta-carotene absorption process in relation to other dietary
factors such as fat content, using the newly developed animal model, BCMO deficient mice.
Publications
- Fierce Y, Morais MM, Piantedosi R, Wyss A, Blaner WS and Paik J. (2008). In vitro and in vivo characterization of retinoid synthesis from beta-carotene. Arch Biochem Biophys, In press (doi:10.1016/j.abb.2008.02.010)
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Progress 10/01/04 to 09/30/05
Outputs
Progresses made in year 2. Objective 1. To examine if and how cellular retinol-binding proteins, types I and II regulate the activity of hCCE: (A) We did not detect direct physical interactions by the two proteins in year 1. However, because the presence of CRBPs in in vitro enzyme assays influenced hCCE activity, we hypothesized that interaction between hCCE and CRBPs likely occurs in the presence of substrate (beta-carotene) and/or product (retinal). To examine this possibility, we created a plasmid (hCCE/CTAP) that express hCCE fused with streptavidin binding peptide and calmodulin binding peptide when transfected into cells. We have transfected CHO cells with this plasmid and currently optimizing conditions of hCCE isolation using two-step purification method by taking advantage of the affinity of two tagged peptides for streptavidin and calmodulin. Once protein can be purified reproducibly, we will transfect Caco2, human intestinal cells with hCCE/CTAP and treat
cells with and without beta-carotene and examine presence of interacting proteins under these conditions. (B) CHO cell lines expressing hCCE and/or CRBP I or II were examined for differences in beta-carotene metabolism. These experiments did not lead to unequivocal results partly due to problems associated with beta-carotene solubilization and treatment in cells. We have tried different methods to solubilize beta-carotene but high concentrations of detergent used for solubilizing beta-carotene caused cell death. Other investigators have used water-soluble beta-carotene for this purpose and yielded reproducible results. Thus, we are negotiating terms with DSM to obtain water-soluble beta-carotene. Once this is achieved, we will resume cell culture experiments to explore influence of CRBP I or II on beta-carotene metabolism. Objective 2. To create and characterize a mouse model that can be used to study beta-carotene metabolism in humans.: (A) We have found one ES cell with
recombination events. However, Southern blot analysis showed that this cell is not a clonal cell line but a mixture of cells with and without recombination event. Using dilutional plating method, we obtained clonal cell lines that have gone through recombination event. We are in the process of creating mice with germ-line transmission of the targeting construct. (B) We also obtained a total body knock out mice from Dr. Adrian Wyss at DSM. This mice are created with conventioinal knock out strategy. We are breeding these mice to characterize phenotypes due to lack of CCE expression.
Impacts
Beta-carotene metabolism is intimately linked to vitamin A metabolism because it is one of the precursors of vitamin A. Because animals cannot synthesize vitamin A, all vitamin A has to come from dietary sources such as beta-carotene in fruits and vegetables. We are aiming at discovering connections between beta-carotene cleavage and vitamin A metabolism by analyzing two proteins involved in this process, carotene cleavage enzyme and cellular retinol binding proteins. We are also near at developing animal models that can be used for studying beta-carotene metabolism. Once the mouse is generated, we will cross them with various Cre mice to create whole body and tissue specific knock out mice. These will be shared with other researchers who wish to study beta-carotene metabolism.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Progresses made for objective 1. To examine if and how cellular retinol-binding proteins, types I and II regulate the activity of hCCE: 1. We examined direct physical interactions between human carotene cleavage enzyme (hCCE) and cellular retinol binding proteins (CRBP), types I and II. Purified CCE was immobilized to a column packed with his-resin using histidine tag and cellular extracts containing CRBP I and II was applied to this column to interact with hCCE. Interaction was then detected on SDS gel after extensive washing and elution of his-tagged CCE from the column. Both CRBP I and II were eluted with flow through, indicating that direct interaction between these two proteins do not occur under the conditions that we employed. The possible interaction between hCCE and CRBP I was hypothesized based on the observation that increased amounts of apo-CRBP I enhance CCE activity in vitro. The enzyme activity assay included substrate (beta-carotene) and product
(all-trans-retinal) of CCE, which were not included in interaction studies. Thus, we will examine whether these components are needed for interaction of hCCE and CRBPs to occur. In addition, we are currently exploring means of detecting protein interactions in cells in culture to study whether the interactions between hCCE and CRBPs occur in vivo. 2. We generated cell lines to explore the influence of CRBP I and II on CCE. Chinese hamster ovary cells were transfected with CCE and either CRBP I or II, and were selected with G418 and zeocin. We will use these cell lines to detect changes in beta-carotene metabolism due to the presence of either CRBP I or II in these cells. These studies will answer questions regarding what factors regulate CCE activity in cells. Progresses made for objective 2. To create and characterize a mouse model that can be used to study beta-carotene metabolism in humans. This objective replaces original objective 2 - to identify short chain
dehydrogenase/reductases involved in the reduction of retinal that is generated by hCCE from beta-carotene- to avoid scientific and budgetary overlap with another grant. This was approved on May 17th, 2004. : 1. We have generated a construct to create CCE knock-out (KO) mouse and introduced this construct to embryonic stem (ES) cells. 2. We have screened ES cells for recombination events. We confirmed one positive colony. We are currently working on creating a mouse with germ-line transmission of the targeting construct. Our goal is to create tissue specific KO mice that can be used to study beta-carotene metabolism in humans. Currently, mice cannot be used to study carotenoid metabolism in humans, because rodents metabolize beta-carotene more efficiently than humans. By creating tissue specific KO mice, we are hoping to provide inexpensive animal models that can be used for this purpose.
Impacts
Identification of factors that are involved in regulation of beta-carotene metabolism may help us answer why carotenoid-rich food sources do not appear to be sufficient in overcoming vitamin A deficiency in developing countries. In addition, development of an animal model will promote studies to explore various aspects of beta-carotene metabolism to better understand regulatory mechanisms of beta-carotene conversion to vitamin A.
Publications
- No publications reported this period
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