Progress 09/01/03 to 08/31/09
Outputs
OUTPUTS: The goal of this project was to identify proteins that were involved in zinc metabolism and transport. The primary methods used for this research was two-dimensional gel electrophoresis and mass spectrometry, two well-established proteomic techniques. Cellular proteins that were identified as responsive to zinc included calcium binding proteins, molecular chaperones, signal transduction proteins, proteins/enzymes linked to energy metabolism, nuclear or DNA/RNA binding proteins, and structural proteins. A similar array of proteins in the hippocampus was associated with dietary zinc deficiency and caloric restriction in the rat brain. Livers from dogs afflicted with the lethal acrodermatitis genetic defect were analyzed using proteomic techniques. Thirteen proteins were differentially expressed in the affected animals, indicating that the disease affects numerous cellular physiologic functions (this was the first report of proteomic analysis of the lethal acrodermatitis mutation). Rat skeletal muscle (soleus) was also analyzed. The muscle was from a dietary zinc deficiency/supplementation study. Four muscle proteins were significantly reduced in the rats consuming a marginally zinc deficient diet. The results were consistent with the observations of others that zinc deficiency reduces muscle function. Zinc nanoparticles are increasingly being used in commercial products, yet their impact on the environment has not been addressed fully. The techniques developed in my lab were used to identify proteomic changes in bacteria grown in zinc nanoparticles. Zinc nanoparticle exposure reduced several proteins involved in cellular metabolic processes and increased a number of proteins involved in protein synthesis. PARTICIPANTS: Arthur Grider - PI; Michael Mouat - Research Associate; Angus Scrimgeour - collaborator (USARIEM, Military Nutrition Division); Nadine Kabengi - collaborator (University of Kentucky Plant and Soil Sciences Depart.); Margret Casal - colaborator (University of Pennsylvania School of Veterinary Medicine) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The economic and social impact of these findings are associated with determining the adequate level of zinc intake for optimal human health. The proteins that have been identified thus far are involved in numerous cellular reactions necessary for maintaining normal cellular function, and their altered expression during inadequate zinc intake may affect such processes as cellular senescence, apoptosis, oncogenesis, and protection against oxidative damage.
Publications
- Grider, A., Kabengi, N. J., Bertsch, P. M., Neal, A. L. (2009) Bacterial protein expression is altered by treatment with zinc nanoparticles. #922.3 FASEB.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: It is the goal of this project to identify proteins that are involved in zinc metabolism and transport. Analysis of the proteome of serum, small intestine and kidney from bull terrier dogs affected by lethal acrodermatitis was performed this year. The proteomic techniques used included immobilized pH gradient two dimensional gel electrophoresis (IPG 2DE) for cytosolic proteins, and 16-BAC cationic 2DE for separation of membrane proteins. Over 200 separations were performed this year. Serum samples were first fractionated through Cibachron blue to remove albumin, then the proteins separated by IPG 2DE. Kidney membrane proteins from lipid rafts fractions were separated by 16-BAC 2DE. Differentially expressed proteins from the serum and kidney were analyzed by mass spectrometry. Work is ongoing to analyze the lipid raft proteome of rat liver, following the consumption of a normal and marginally zinc deficient diet. The 2DE for these samples is being repeated to confirm the observed differential expression patterns, as well as to improve separations using updated 2DE methods. No mass spectrometry has been performed at this time on the differentially expressed protein spots from these samples. PARTICIPANTS: Arthur Grider is the PI of the project. Chris Dowd helped with the protein extraction methods and 2DE separations as a research associate. Angus Scrimgeour Ph.D. from the Military Nutrition Division U.S. Army Research Institute of Environmental Medicine supplied the immobilized pH gradient isoelectric focusing cell, supplies, and tissues. Margret Casal, Ph.D., Veterinary Hospital of the University of Pennsylvania, provided serum and kidney samples from dogs with lethal acrodermatitis. Jenny Hwang is a M.S. graduate student undergoing training in proteomic techniques. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: The loss of the MALDI mass spectrometer power supply, problems with its replacement, and loss of personnel within the mass spectrometer facility handling my samples has caused a delay in the identification of excise differentially expressed protein spots.
Impacts
The economic and social impact of the findings are associated with determining the adequate level of zinc intake for optimal health. Although the analysis of the differentially expressed proteins is incomplete, the results from this past year indicate that the lethal acrodermatitis mutation in bull terriers significantly alters protein expression in the kidney and serum. The protein differences compared to control dogs may provide for the identification of biomarkers for the disease, as well as biomarkers for altered zinc metabolism in otherwise normal human individuals. The improved proteomic methods that are employed increase the level of confidence that the differences observed in protein expression are real and not artifacts of the separation procedures. The proteins that have been identified thus far are involved in numerous cellular reactions necessary for maintaining normal cellular function, and their altered expression during inadequate zinc intake may affect such processes as cellular senescence, apoptosis, oncogenesis, and protection against oxidative damage.
Publications
- Grider, A. & Casal, M.L. (2008) Differential analysis of the serum proteome from bull terrier pups with lethal acrodermatitis. [Abstract] FASEB Journal, 22, 697.13.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: It is the goal of this project to identify proteins that are involved in zinc metabolism and transport. The primary method used by this lab for protein identification is two-dimensional gel electrophoresis, which allows the investigator to visualize the cellular or tissue proteome that is affected by changes in zinc nutriture. This past year this technique was used to identify zinc-dependent changes in protein expression in rat liver and small intestine. A new two-dimensional gel electrophoresis technique has been developed in the lab that utilizes denaturing cationic detergent (16-BAC cationic electrophoresis) in the first dimension. The second dimension utilizes denaturing SDS-PAGE as before. This technique was applied to the separation of membrane proteins to determine whether zinc-induced changes in the expression of these proteins occurred. A few changes have been observed, but not identified yet by mass spectrometry. In collaboration with investigators from the University of Pennsylvania Veterinary Medicine, tissues (liver, kidney, and small intestine) and serum from bull terriers afflicted with the lethal acrodermatitis (a putative zinc deficiency disease) were also analyzed by both isoelectric focusing and 16-BAC two-dimensional gel electrophoresis. The proteome from the affected tissues exhibits differential protein expression compared to unaffected tissue. The differentially expressed proteins have not been fully identified by mass spectrometry, however. PARTICIPANTS: Arthur Grider - PI Michael Mouat - Research Associate
Impacts
The economic and social impact of the findings are associated with determining the adequate level of zinc intake for optimal health. Proteins that have been identified thus far are involved in numerous cellular reactions necessary for maintaining normal cellular function, and their altered expression during inadequate zinc intake may affect such processes as cellular senescence, apoptosis, oncogenesis, and protection against oxidative damage.
Publications
- Grider, A., Mouat, M.F., Mauldin, E.A., & Casal, M.L. (2007) Analysis of the liver soluble proteome from bull terriers affected with inherited lethal acrodermatitis. Molecular Genetics and Metabolism 92:249-257.
- Grider, A., Mouat, M.F., & Scrimgeour, A.G. (2007) Consumption of a moderately Zn-deficient and Zn-supplemented diet affects soluble protein expression in rat soleus muscle. Journal of Nutritional Biochemistry 18:753-759.
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Progress 01/01/06 to 12/31/06
Outputs
It is the goal of this project to identify proteins that are involved in zinc metabolism and transport. The primary method used by this lab for protein identification is two-dimensional gel electrophoresis, which allows the investigator to visualize the cellular or tissue proteome that is affected by changes in zinc nutriture. Previously, several proteins from the rat hippocampus were shown to be differentially expressed as a function of zinc nutriture. This past year we identified soluble proteins from the rat soleus muscle that responded to zinc nutriture. An abstract was presented to the FASEB Experimental Biology Meeting in San Francisco April 1-5, 2006. The full report has been accepted for publication in the Journal of Nutritional Biochemistry. We will continue to use this proteomic technique to identify proteins in other tissues that are affected by zinc nutriture with the goal of developing a more complete understanding of the role zinc plays in maintaining
optimal human health.
Impacts
The economic and social impact of the findings are associated with determining the adequate level of zinc intake for optimal health. Proteins that have been identified thus far are involved in numerous cellular reactions necessary for maintaining normal cellular function, and their altered expression during inadequate zinc intake may affect such processes as cellular senescence, apoptosis, oncogenesis, and protection against oxidative damage.
Publications
- Grider, A., Mouat, M.F., Scrimgeour, A.G. (2007) Consumption of a moderately Zn-deficient and Zn-supplemented diet affects soluble protein expression in rat soleus muscle. Journal of Nutritional Biochemistry (in press).
- Grider, A., Mouat, M.F. (2006) Protein expression in skeletal muscle is influenced by changes in dietary zinc. FASEB Journal 20:A626.
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Progress 01/01/05 to 12/31/05
Outputs
The goal of this project is to identify and characterize proteins involved in zinc metabolism and transport. This is essential for a more complete understanding of zinc bioavailability in normal human nutrition, and in understanding the role zinc plays in human health. Proteomic techniques are currently being employed to determine the effects of changes in zinc nutriture on protein expression at the cellular and organismal level. The 2DPAGE techniques for separating cytosolic and membrane proteins are being developed. One abstract was presented at the FASEB Experimental Biology Meeting in San Diego Apr. 2-6, 2005. This study showed that the mutation in bull terriers, lethal acrodermatitis, affects protein expression in cultured fibroblasts, though we were unable to obtain mass spectrometry data needed to identify the proteins. Another study was published that identified proteins in a colon carcinoma cell line that are affected by quercetin treatment. The proteins that
exhibited differential expression were related the cell's survival strategies that mitigate tumorigenesis. As an antioxidant, we are now set to compare the effects of quercetin with those of zinc, also considered important for protection against free radical damage.
Impacts
The primary impact of these findings is that proteins were identified have not been previously linked to zinc nutriture. Several of the proteins are linked to apoptosis besides being involved in normal cellular physiology. These proteins may be associated with protecting the cell from the detrimental effects of zinc deficiency.
Publications
- Grider, A, Mouat, M.F., Casal, M.L. (2005) Proteomic profile of lethal acrodermatitis in Bull Terriers. FASEB Journal 19:A457.
- Mouat, M.F., Kolli, K., Orlando, R., Hargrove, J.L., Grider, A. (2005)The effects of quercetin on SW480 colon carcinoma cells: a proteomic study. Nutrition Journal 4:11-17.
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Progress 01/01/04 to 12/31/04
Outputs
Zinc (Zn) is an essential nutrient that is necessary for a wide range of cellular processes. It is essential for the structural and functional integrity of cells, and plays a pivotal role in the control of gene expression. The effect of zinc deficiency on protein expression in a colon adenocarcinoma (Caco-2) cell-culture model was studied using two-dimensional gel electrophoresis (2DGE) and mass spectrometry (MS). Caco-2 cells were grown on membrane inserts in minimal essential medium (MEM) supplemented with 20% fetal bovine serum (FBS) for 20 days. At day 14, the cells were maintained in either fresh normal MEM (12 uM Zn) or medium containing 20% dialyzed FBS (0.35 uM Zn) for 7 days. The total cell lysates (2 mg) were separated using a large format 2DGE system. The gels were stained with 0.1% Coomassie Blue R-250. Following destaining, the gels were analyzed by Phoretix 2D Advanced software (v6.01). Approximately 400 spots were detected between the two gel
preparations. The differentially expressed proteins were excised, digested with trypsin, and analyzed by mass spectrometry. Forty-seven proteins exhibited differential expression. The proteins were identified by MS followed by database searches. The proteins that responded to zinc status in Caco-2 cells could be sorted into 8 functional categories. Identified groups included calcium binding (3), chaperones (3), signal transduction (4), energy (9), nuclear or DNA/RNA binding (6), channel (1), structural (8), and serum (4).
Impacts
The primary impact of these findings is that proteins were identified have not been previously linked to zinc nutriture. Several of the proteins are linked to apoptosis besides being involved in normal cellular physiology. These proteins may be associated with protecting the cell from the detrimental effects of zinc deficiency.
Publications
- Berelc, E, Mouat, M.F., Grider, A. (2004) Protein expression in Caco-2 cells is affected by zinc deficiency. FASEB J 18:A529.
- Grider, A. 2004. Proteomics: Tools for nutrition research in the postgenomic era. In, Genomics and Proteomics in Nutrition. C.D. Berdanier and N. Moustaid-Moussa, eds. Marcel Dekker, Inc. New York. Pp. 375-394
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