This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Goal: The goal of this project is to demonstrate the utility of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) as in vivo reporter of enzyme kinetics involved in the biosynthesis pathways of pyruvate. Our long-term goal is to utilize this technique for validating potential therapeutic targets that regulate protein function in vivo with the precision and control conferred by tunable gene technology, and in addition advance our understanding of basic biochemical pathways. We assume that NADH reductase is the key enzyme that regulates (rate limiting step) the NADH to NAD+ reduction. Materials and Methods: EL4 (C57BL mouse lymphoma) and A20 (BALB/c B cell lymphoma) cells are transfected with cDNA plasmid of NQO1. These cells have been tested and found negative for ectromelia virus (mousepox). The cDNA plasmid of NQO1 expresses the gene for nicotinamide adenine dinucleotide (NADH) quinone oxidoreductase 1 (NQO1). NQO1 is a ubiquitous cytosolic flavoenzyme that catalyzes two- electron reduction of various quinones, with NADH or NADPH as an electron donor. A flask of 50 million cells in 2.5 ml of RPMI 1640 medium is administered with hyperpolarized [1-13C]-pyruvate followed by data acquisition. A 3.0 Tesla Signa magnet (GE healthcare) and a dynamic nuclear polarizer (Oxford Instruments Hypersense) are utilized.
Specific Aim 1 : To transfect cell cultures with a NADH-reductase gene that over-expresses NADH. Experiment Design 1: We will transfect cells with cDNA plasmids that increases the levels of NADH in cell culture, and we will measure the chemical flux of [1-13C]-pyruvate to lactate as an indirect measure of the NADH levels. However, since hyperpolarized [1-13C]-pyruvate signal is short lived (~30s) we will prefer a regulated promoter gene of NADH-reductase that can be turned on appropriately prior to the infusion of hyperpolarized pyruvate.
Specific Aim 2 : To ectopically expressed a plasmid DNA reporter or a tunable regulated gene of NADH reductase in rat liver. Experimental Design 2: We will transfect a rat liver by hydrodynamics-based administration of plasmid DNA or by systemic delivery of conditionally regulated protein. When NADH levels are significantly higher in the liver, the hyperpolarized [1-13C]-pyruvate experiment will be performed to ascertain the conversion rates of pyruvate to lactate as an indirect measure of NADH-reductase. As a control measure, one cohort of rats will not receive the plasmid DNA or tunable form of NADH-reductase.
Showing the most recent 10 out of 446 publications
