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J R Thayer

Publications and source records attributed to J R Thayer.

10 recordsLinked to original sources
PubMed2005

Control of oligonucleotide retention on a pH-stabilized strong anion exchange column.

Strong anion exchange columns are preferred for oligonucleotide analyses due to their ability to effectively control secondary structure and poly(G) interactions. Methacrylate-based anion exchange phases minimize hydrophobic interactions with oligonucleotides, but they also tend to hydrolyze under alkaline conditions. In this article, we report the use of an anion exchange column prepared from a new class of methacrylate monomers designed to improve hydrolytic stability. This column is used to show predictable adjustment of oligonucleotide retention by eluent pH and composition. Features of the new column include (i) large, predictable, pH-dependent retention shifts (varying with specific changes in 5' or 3' terminal bases with NaCl-based eluents); (ii) reduced retention when solvent is added to NaCl-based eluents; and (iii) suppression of much of the column's hydrophobic interactions when CH3CN is used with NaClO4-based eluents at a neutral pH (i.e., this eluent system separates oligonucleotides primarily in order of their length). These observations will aid the development of elution conditions for both size-dependent and base sequence-dependent (or base composition-dependent) separations.

Chromatography, Ion Exchange↗
PubMed1998

Improvements to in-line desalting of oligosaccharides separated by high-pH anion exchange chromatography with pulsed amperometric detection.

High-pH anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD) (1) is routinely used to separate neutral and charged oligosaccharides differing by branch, linkage, and positional isomerism. Oligosaccharides are eluted in 0.1 M NaOH with gradients of sodium acetate (up to 0.25 M). Analyses of HPAEC/PAD-purified oligosaccharides generally require neutralization and removal of eluent salts. To facilitate the process, we designed and produced a cation-exchange system to remove sodium ions (Na+) from the eluent after oligosaccharide detection [the Carbohydrate Membrane Desalter (CMD), with a volatile regenerant]. Exchange of >99.5% of eluent Na+ for hydronium ions (H3O+) within the CMD generates dilute acetic acid (removable by vacuum evaporation). The exchange process desalts up to 0.35 M Na+ at 1.0 ml/min. Oligosaccharides collected after on-line desalting, evaporated and resuspended in their original volume of deionized water contained < or = 350 muM residual Na+ when the eluting sodium concentration was 300 mM. This represents a desalting efficiency of >99.8%. Recovery of neutral and sialylated oligosaccharides under these conditions ranged from 75 to 100%. With the CMD system and postcollection evaporation, HPAEC/PAD can purify oligosaccharides ready for further characterization. As a proof test, oligosaccharides from a human monoclonal antibody were separated by HPAEC/PAD, desalted with the CMD system, dried, and analyzed by matrix-assisted laser desorption-ionization, time-of-flight mass spectrometry.

Acetates↗
PubMed1982

Gut reactions of radioactive nitrite after intratracheal administration in mice.

Intratracheal administration to mice of radioactive nitrite labeled with nitrogen-13 (13NO2-) (half-life, 9.96 minutes) in dosages that do not cause pharmacological perturbation reveals that oxidative and reductive reactions occur in different organs. Oxidation of 13NO2- to radioactive nitrate (13NO3-) predominates in the blood and liver. Reduction of 13NO2- occurs in those mice that harbor intestinal microflora; this reduction does not occur in germ-free mice. The intestinal reduction products include ammonium, glutamate, glutamine, and urea. With a detection limit of about 0.01 percent of the instilled nitrogen-13, no labeled nitrosamines were detected within 30 minutes. Reduced nitrogen-13 is transported out of the intensive into the circulatory system and appears in the urine along with 13NO3-. The biological half-period for 13NO2- destruction is about 7 minutes, and both oxidation and reduction products are formed.

Animals↗
PubMed1982

Active transport of benzoate in Pseudomonas putida.

Benzoate uptake in Pseudomonas putida is mediated by an active transport system capable of accumulating benzoate against a 150-fold concentration gradient when subsequent metabolism is blocked by mutation. Initial benzoate transport rates are inhibited by CCCP, sodium azide, arsenate and DCCD. Uptake is stimulated by including a respirable carbon source during preincubation of the bacteria. The initial uptake rate and the ATP pool levels are not correlated and no periplasmic components were found to bind benzoate. These observations indicate that benzoate uptake is energized by the membrane potential, rather than by ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1982

Kinetic evaluation, using 13N, reveals two assimilatory nitrate transport systems in Klebsiella pneumoniae.

A kinetic evaluation of initial rates of nitrate transport at concentrations between 1 microM and 1 mM indicated the presence of two transport processes. Analysis of the contribution of each process to the total activity permitted the determination of kinetic constants (Km) of 4.9 microM and 4.2 mM for the high-and low-affinity systems, respectively. The ratio of the maximal velocity of the high-affinity system to that of an apparent low-affinity system was about 0.3. Both systems were inhibited by the presence of NH4+ in the transport assay. Growth in the presence of equimolar NO3- and NH4+ repressed the synthesis of both systems when compared with growth in NO3- alone.

Biological Transport↗
PubMed1976

Characterization of a benzoate permease mutant of Pseudomonas putida.

A spontaneous mutant of Pseudomonas putida (PRS 2017) has been isolated which is incapable of growth on benzoate, does not induce the enzymes of the catechol branch of the beta-ketoadipate pathway when grown in the presence of benzoate, cannot accumulate radioactively labeled benzoate, yet grows well with mandelate as sole source of carbon and energy. This strain apparently lacks a benzoate permease, which in the wild type shows a Km of about 0.1 mM for benzoate, is inducible, and is not under the control of the regulatory system which governs the induction of the enzymes of the catechol branch of the beta-ketoadapate pathway. The lesion in PRS2017 is apparently single site and maps near other genes governing benzoate dissimilation.

Adipates↗