SCORE Program Director
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Research (during tenure)
Dr. Kuehn had an ongoing, active research program at New Mexico State University for 42 years, beginning in 1970, and ending in 2012 when he retired. His research interests centered on several topics in polyamine metabolism and roles in cellular homeostasis.
Synopsis: Polyamine oxidase (PAO) catalyzes oxidative cleavage of polyamines spermidine (spd) or spermine (spm) to produce diaminopropane (dap), H2O2, and an aminoaldehyde derivative. In plants, dap is the precursor for biosynthesis of norspermidine (nspd) and norspermine (nspm) via the enzyme, Schiff base reductase decarboxylase (SBRD). Recently, the catabolism of spd and spm by PAO has been proposed by several investigators to be a causative agent, through product H2O2 and ensuing oxidative stress, which forces animal cells into programmed cell death (apoptosis). The gene for PAO from oat seedlings was recently cloned, sequenced, and characterized in this laboratory. The availability of this newly isolated gene offers unique opportunities to gain genetic evidence for a potential role(s) of PAO and polyamine-catabolism in apoptosis in a plant model test system.
The hypothesis of this application is: (i) PAO has a causative role in apoptosis in cells through H2O2 produced by its oxidation of spd or spm, and (ii) the nspd and nspm produced from dap serve as suppressors of apoptosis through feedback inhibition of PAO and reduction in H2O2 synthesis. The specific aims of this proposal are: (1) The PAO cDNA gene sequence will be used to generate antisense PAO gene constructs ligated to a copper-inducible promoter. These constructs will be used to transform alfalfa plants with Ti-plasmid methods to analyze the consequences of controlled PAO-deficiency on apoptosis in plant tissues. (2) The cDNA gene sequence for the enzyme, SBRD, which catalyzes nspd and nspm biosynthesis from dap, will be used to generate antisense SBRD gene constructs ligated to a copper-inducible promoter. These constructs will be used to transform alfalfa plants in order to analyze the consequences of uncoupling generation of H2O2 by PAO from the biosynthesis of nspd and nspm derived from dap by SBRD.
Aims (1) and (2) are direct tests of parts (i) and (ii) of the hypothesis. (3) The PAO cDNA gene sequence from oat will be used as a gene probe to attempt the isolation of the human PAO gene from kidney and liver cDNA libraries. A characterized human PAO gene will make possible the development of a molecular biological approach to investigate the role of PAO in generating H2O2 and its alleged role to elicit animal apoptosis. (4) The cDNA gene sequence of a signal peptide for the PAO gene will be ligated to a gene coding for a green fluorescent protein (GFP). The fusion protein produced from this construction will be analyzed by fluorescent imaging techniques in tissues of alfalfa and oat plants transformed with this construction in order to identify the subcellular localization of PAO. In situ labeling of PAO by immunogold antibody reagents and electronmicroscopic analysis, will corroborate the localization studies by the GFP fusion protein technique. These results will aid in localizing the origin of events that initiate PAO-dependent apoptosis.
- G. D. Kuehn and G.C. Phillips. 2005. Role of Polyamines in Apoptosis and Other Recent Advances in Plant Polyamines. Critical Reviews in Plant Sciences 24:1-8.
- G. D. Kuehn. 2005. Bridges to American Indian Students in Community Colleges Program. Science 307:1685. [Invited Contribution to Science Next Wave at www.nextwave.org <http://www.nextwave.org/> , March 18th issue.
- Cloning, Isolation and Sequence of the Gene for Polyamine Oxidase from Avena sativa L. (Oat.) Gardner-Johnson, Yvette J., A. Dharma, N. Dong, G.C. Phillips, D. Benn. and G.D. Kuehn (2000) Plant Physiology, in review.
- Natural Polyamines and Their Biological Consequences in Mammals. Patoaka, J. and G.D. Kuehn (1999) Acta Medica, in press.
- Occurrence of uncommon polyamines in cultured tissues of maize Koc, E.C., S. Bagga, D.D. Songstad, S.R. Betz, G.D. Kuehn, and G.C. Phillips (1998) In Vitro Cellular and Developmental Biology-Plant 34: 252-255.
- Putrescine Aminopropyltransferase is Responsible for Biosynthesis of Spermidine, Spermine, and Multiple Uncommon Polyamines in Osmotic Stress-Tolerant Alfalfa (Medicago sativa L.). Bagga, S., J. Rochford, Z. Klaene, G.D. Kuehn, and G.C. Phillips (1997) Plant Physiology 114:445-454.
- Characterization of Chile Pepper Fruit Peroxidases During Ripening. Biles, C.L., G.D. Kuehn, and M.M. Wall (1997) Plant Physiology and Biochemistry 35:273-280.
- Partial Purification and Kinetic Characterization of Acyl CoA:alcohol Transacylase from Developing Jojoba Cotyledons. Garver, W.S., J.D. Kemp and G.D. Kuehn (1996) Plant Physiology: Advanced Life Sciences 13:45-49.
- A High Performance Liquid Chromatography- Based Radiometric Assay for Acyl-CoA:alcohol Transacylase from Jojoba. Garver, W.S., J.D. Kemp, and G.D. Kuehn (1995) Analytical Biochemistry 196:335-340.
- Boronic Acid Matrices for the Affinity Purification of Glycoproteins and Enzymes Hageman, J.H. and G.D. Kuehn (1992) Practical Protein Chromatographys. Methods in Molecular Biology Series (A. Kenney and S. Fowell, Eds.), The Humana Press, Inc., 11:45-71.
- Purification and Partial Characterization of Transglutaminase from Physarum polycephalum Klein, J.D., E. Guzman, and G.D. Kuehn (1992) Journal of Bacteriology 174:2599-2605.
- Evidence for the Occurrence of Polyamine Oxidase in the Dicotyledonous Plant Medicago sativa L. (Alfalfa) Bagga, S., A. Dharma, G.C. Phillips, and G.D. Kuehn (1991) Plant Cell Reports 10:550-554.
- Identification of the Large Subunit of Ribulose 1,5 bisphosphate/carboxylase/oxygenase as a Substrate for Transglutaminase in Plants. Margosiak, S.A., A. Dharma, A.P. Gonzales, D. Louie, and G.D. Kuehn (1990) Plant Physiology 92:88-96.
- Detection of Norspermidine and Norspermine in Medicago sativa L. (Alfalfa). Rodriguez-Garay, B., G.C. Phillips, and G.D. Kuehn (1989) Plant Physiology 89:525-529.