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Title: Study of cultured fibroblasts in vivo using NMR

Abstract

The goal was to study the compartmentation of phosphorylated glycolytic intermediates in intact Chicken Embryo Fibroblasts (CEFs) using /sup 31/P NMR at 109 MHz. A technique for maintaining functional cells at high densities in an NMR magnet is described. Signals were detected from cytoplasmic inorganic phosphate (P/sub i/), ATP, NAD, NADH, phosphorylcholine and phosphorylethanolamine. The effect of external glucose on cytoplasmic pools of phosphates was studied. When cells were perfused with glucose-free medium the rate of glycolysis decreased, the amplitudes of the ATP resonances decreased, and the P/sub i/ intensity increased. The quantity of NMR-detectable P/sub i/ produced was significantly greater than the quantity of NMR-detectable ATP which was lost. Experiments with /sup 32/P labeled P/sub i/ showed that as the concentration of glucose in the medium was increase, the amount of phosphate sequestered in the cells increased. We conclude that there is a pool of P/sub i/ which is not detected by high resolution NMR and that the size of this pool increases as the rate of glycolysis increase. Longtitudinal relaxation times of intracellular phosphates in normal, transformed, and primary CEFs were measured. The results demonstrate that relaxation times of phosphates are sensitive to structural and metabolic changes whichmore » occur when cells are grown in culture. 59 references. 31 figures.« less

Authors:
Publication Date:
Research Org.:
Lawrence Berkeley Lab., CA (USA)
OSTI Identifier:
6020082
Report Number(s):
LBL-18628
ON: DE85004604
DOE Contract Number:
AC03-76SF00098
Resource Type:
Technical Report
Resource Relation:
Other Information: Thesis
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; FIBROBLASTS; GLYCOLYSIS; GLUCOSE; BIOLOGICAL EFFECTS; CATABOLISM; ATP; CELL CULTURES; CHICKENS; CYTOPLASM; EMBRYOS; EXPERIMENTAL DATA; NAD; NUCLEAR MAGNETIC RESONANCE; PHOSPHATES; PHOSPHORUS 32; QUANTITY RATIO; ALDEHYDES; ANIMAL CELLS; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIRDS; CARBOHYDRATES; CELL CONSTITUENTS; CHEMICAL REACTIONS; COENZYMES; CONNECTIVE TISSUE CELLS; DATA; DAYS LIVING RADIOISOTOPES; DECOMPOSITION; FOWL; HEXOSES; INFORMATION; ISOTOPES; LIGHT NUCLEI; MAGNETIC RESONANCE; METABOLISM; MONOSACCHARIDES; NUCLEI; NUCLEOTIDES; NUMERICAL DATA; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; OXYGEN COMPOUNDS; PHOSPHORUS COMPOUNDS; PHOSPHORUS ISOTOPES; RADIOISOTOPES; RESONANCE; SACCHARIDES; SOMATIC CELLS; VERTEBRATES; 550501* - Metabolism- Tracer Techniques

Citation Formats

Karczmar, G.S. Study of cultured fibroblasts in vivo using NMR. United States: N. p., 1984. Web.
Karczmar, G.S. Study of cultured fibroblasts in vivo using NMR. United States.
Karczmar, G.S. Wed . "Study of cultured fibroblasts in vivo using NMR". United States. doi:.
@article{osti_6020082,
title = {Study of cultured fibroblasts in vivo using NMR},
author = {Karczmar, G.S.},
abstractNote = {The goal was to study the compartmentation of phosphorylated glycolytic intermediates in intact Chicken Embryo Fibroblasts (CEFs) using /sup 31/P NMR at 109 MHz. A technique for maintaining functional cells at high densities in an NMR magnet is described. Signals were detected from cytoplasmic inorganic phosphate (P/sub i/), ATP, NAD, NADH, phosphorylcholine and phosphorylethanolamine. The effect of external glucose on cytoplasmic pools of phosphates was studied. When cells were perfused with glucose-free medium the rate of glycolysis decreased, the amplitudes of the ATP resonances decreased, and the P/sub i/ intensity increased. The quantity of NMR-detectable P/sub i/ produced was significantly greater than the quantity of NMR-detectable ATP which was lost. Experiments with /sup 32/P labeled P/sub i/ showed that as the concentration of glucose in the medium was increase, the amount of phosphate sequestered in the cells increased. We conclude that there is a pool of P/sub i/ which is not detected by high resolution NMR and that the size of this pool increases as the rate of glycolysis increase. Longtitudinal relaxation times of intracellular phosphates in normal, transformed, and primary CEFs were measured. The results demonstrate that relaxation times of phosphates are sensitive to structural and metabolic changes which occur when cells are grown in culture. 59 references. 31 figures.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Aug 01 00:00:00 EDT 1984},
month = {Wed Aug 01 00:00:00 EDT 1984}
}

Technical Report:
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  • The goal of this thesis was to study the compartmentation of phosphorylated glycolytic intermediates in intact Chicken Embryo Fibroblasts (CEFs) using /sup 31/P NMR at 109 MHz. Because glycolysis is regulated differently in normal and virally transformed CEFs, NMR experiments were performed on both types of cells. A technique for maintaining functional cells at high densities in an NMR magnet is described. Signals were detected from cytoplasmic inorganic phosphate (P/sub i/), ATP, NAD, NADH, phosphorylcholine and phosphorylethanolamine. The effect of external glucose on cytoplasmic pools of phosphates was studied. However, experiments with /sup 32/P labelled P/sub i/ showed that asmore » the concentration of glucose in the medium was increased, the amount of phosphate sequestered in the cells increased. They conclude that there is a pool of P/sub i/ which is not detected by high resolution of NMR and that the size of this pool increases as the rate of glycolysis increases. These effects were found only in cultured cells; the data for transformed and normal cells were similar. Longitudinal relaxation times of intracellular phosphates in normal, transformed, and primary CEFs were measured.« less
  • Abstract for final report for project entitled A functional genomics approach using radiation-induced changes in gene expression to study low dose radiation effects in vitro and in vivo which has been supported by the DOE Low Dose Radiation Research Program for approximately 7 years. This project has encompassed two sequential awards, ER62683 and then ER63308, in the Gene Response Section in the Center for Cancer Research at the National Cancer Institute. The project was temporarily suspended during the relocation of the Principal Investigators laboratory to the Dept. of Genetics and Complex Diseases at Harvard School of Public Health at themore » end of 2004. Remaining support for the final year was transferred to this new site later in 2005 and was assigned the DOE Award Number ER64065. The major aims of this project have been 1) to characterize changes in gene expression in response to low-dose radiation responses; this includes responses in human cells lines, peripheral blood lymphocytes (PBL), and in vivo after human or murine exposures, as well as the effect of dose-rate on gene responses; 2) to characterize changes in gene expression that may be involved in bystander effects, such as may be mediated by cytokines and other intercellular signaling proteins; and 3) to characterize responses in transgenic mouse models with relevance to genomic stability. A variety of approaches have been used to study transcriptional events including microarray hybridization, quantitative single-probe hybridization which was developed in this laboratory, quantitative RT-PCR, and promoter microarray analysis using genomic regulatory motifs. Considering the frequent responsiveness of genes encoding cytokines and related signaling proteins that can affect cellular metabolism, initial efforts were initiated to study radiation responses at the metabolomic level and to correlate with radiation-responsive gene expression. Productivity includes twenty-four published and in press manuscripts, as well as a U.S. patent. There are several additional publications that will be submitted in 2007 that were supported in part by this program. These future publications include one manuscript on in vivo expression profiling analysis in mouse models, one manuscript on radiation responses in human cell lines, at least one on development of stress signatures in human cells, and three manuscripts on radiation metabolomics.« less
  • In this experimental investigation the polarization levels and relaxation rates for optically pumped $4He and 3He isotopes were determined. An experiment verified that simultaneous magnetic resonance of both isotopes in a common volume may be attained. Characteristics of some experimental semiconductor diode lasers were measured with a view toward replacing helium lamps as an optical pumping radiation source with diode lasers. A semiconductor diode laser was successfully used to monitor polarized 4He atoms.
  • During the summer of 2015, I participated in the DHS HS-STEM fellowship at Sandia National Laboratories (SNL, NM) under the supervision of Dr. Todd M. Alam in his Nuclear Magnetic Resonance (NMR) Spectroscopy research group. While with the group, my main project involved pursing various hydrolysis reactions with Diethyl Chlorophosphate (DECP), a surrogate for the agent Sarin (GB). Specifically, I performed different hydrolysis reactions, monitored and tracked the different phosphorous containing species using phosphorous ( 31P) NMR spectroscopy. With the data collected, I performed kinetics studies mapping the rates of DECP hydrolysis. I also used the NMR of different nucleimore » such as 1H, 13C, 17O, and 35Cl to help understand the complexity of the reactions that take place. Finally, my last task at SNL was to work with Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) NMR Spectroscopy optimizing conditions for 19F- 31P filtering NMR experiments.« less