Search Results

Prediction of Post Mortem Interval from Degradation of Endogenous Nucleotides in Human Subjects

Description: High Performance Liguid Chromatography was used to measure degradation of nucleotides in human cadavers for the purpose of prediction of post mortem interval. Endogenous nucleotides were extracted from integumentary tissue of six(6) human cadavers using six percent(6%) tricholoacetic acid. Linear regression statistical techniques were used to determine linearity of degradation of various nucleotide pools.
Date: April 1993
Creator: Williams, John Burgess
Partner: UNT Libraries

Radial Compression High Performance Liquid Chromatography as a Tool for The Measurement of Endogenous Nucleotides in Bacteria

Description: High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.
Date: August 1986
Creator: Dutta, Probir Kumar
Partner: UNT Libraries

Nucleotide Inhibition of Glyoxalase II

Description: The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" enzyme (Glo-II-i) show that a significant reduction of the affinity of the enzyme for the substrate, SLG, occurs and further suggest that only one form of the enzyme is kinetically distinguishable after "de-sensitization". Tryptophan fluorescence studies of the two enzyme preparations suggest that a subtle conformational change in the enzyme has occurred during de-sensitization. We have also observed that Glo-II-i is "resensitized" to nucleotide inhibition after incubation in the presence of a reagent that reduces disulfide bonds. The resensitized enzyme exhibits an increased KM value similar to that of the original Glo-II-s. Kinetics studies show that ATP or GTP again ...
Date: May 1999
Creator: Gillis, Glen S
Partner: UNT Libraries

Quantitation of Endogenous Nucleotide Pools in Pseudomonas aeruginosa

Description: Nucleotide pools were extracted and quantified from Pyr^+ and Pyr^- strains of P. aerucjinosa. Strains were grown in succinate minimal medium with and without pyrimidines, and nucleotides were extracted using trichloracetic acid (TCA; 6% w/v). The pyrimidine requirement was satisfied by uracil, uridine, cytosine or cytidine. Pyr^- mutants were starved for pyrimidines for two hours before nucleotide levels were measured. This starvation depleted the nucleotide pools which were restored to wild type levels by the addition of pyrimidines to the medium. When the pyrimidine analogue, 6-azauracil, known to inhibit OMP decarboxylase, was added to cultures of the wild type strain, the uridine and cytidine nucleotides were depleted to near zero. Thus, the nucleotide pool levels of Pseudomonas strains can be manipulated.
Date: August 1988
Creator: Entezampour, Mohammad
Partner: UNT Libraries

Endogenous Nucleotide Pools in Growing Cells of Azotobacter Vinelandii

Description: The objective of this investigation was to examine the changes in the nucleotide pools of Azotobacter vinelandii during the growth cycle. Endogenous ribonucleotides were extracted from A. vinelandii using trichloroacetic acid (TCA; 12% w/v). The 5' mono-, di- and triphosphates of adenine, guanine, uracil and cytosine were separated and quantified by anion-exchange high performance liquid chromatography. Results indicated that the adenylate energy charge of A. vinelandii paralleled the growth rate during exponential phase and that it declined rapidly as the stationary phase was reached. In addition, the amount of each nucleotide in A. vinelandii tended to increase in the logarithmic phase and decrease in the stationary phase in a similar manner to the energy charge.
Date: August 1987
Creator: Lee, Yick-Shun
Partner: UNT Libraries