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Biomedical subjects

J P Reilly

Publications and source records attributed to J P Reilly.

At least 19 recordsLinked to original sources
PubMed2001

Elucidation of the initial step of oligonucleotide fragmentation in matrix-assisted laser desorption/ionization using modified nucleic acids.

To probe the mechanism of gas-phase oligonucleotide ion fragmentation, modified oligonucleotides were studied using matrix-assisted laser desorption/ionization. The oligonucleotides were of the form 5'-TTTTXTTTTT, where X was a modified nucleotide. Modifications included substitution of hydroxy, methoxy, amino, and allyl groups at the 2'-position of the deoxyribose. The modified ribose contained adenine, guanine, cytosine, or uracil bases. For comparison, we studied oligomers where X was an unmodified adenosine, guanosine, cytidine, thymidine, or uridine deoxyribonucleotide. We found a very strong dependence of the matrix-to-analyte ratio on fragmentation for these oligomers. Analysis of these modifications suggests that the initial fragmentation step in MALDI-MS involves a two-step (E1) elimination of the base.

Allyl Compounds↗
PubMed2000

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.

Chromatography, High Pressure Liquid↗
PubMed2000

Improved calibration of time-of-flight mass spectra by simplex optimization of electrostatic ion calculations.

A novel time-of-flight mass calibration method has been developed. In contrast to conventional methods, where the relationship between ion flight time and mass is an arbitrary polynomial equation, this method is based on the physics of ion motion. Parameters needed to describe the physics are numerically optimized using a simplex algorithm. Once these parameters are established, unknown masses can be determined from their times-of-flight. This calibration method gives intrinsically well-behaved results, since nonlinearities (due to extraction delay, desorption velocity, etc.) are properly taken into account in the time-of-flight calculation. The simplex method is compared to curve fitting for the analysis of time-of-flight data, and some significant advantages are demonstrated. Salient features of the method include greatly improved mass extrapolation accuracy, no loss of interpolated calibration accuracy, the ability to obtain an accurate calibration with a minimal number of calibrants, and the ability to extract unknown parameters such as desorption velocities.

Algorithms↗
PubMed2000

Observation of tetrahydrofolylpolyglutamic acid in bacteria cells by matrix-assisted laser desorption/ionization mass spectrometry.

Tetrahydrofolylpolyglutamic acid in whole bacteria cells and cell lysates is analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The speed, mass information, and tolerance to impurities of this technique make it ideal for monitoring the glutamation levels of folic acid in biological systems. Folylpolyglutamic acid is observed in a few strains of E. coli and two species of Staphylococcus bacteria. The effects of growth time, growth media, and the addition of methotrexate, a dihydrofolate reductase inhibitor, are also studied.

Antimetabolites, Antineoplastic↗
PubMed2000

Enhancing the intensities of lysine-terminated tryptic peptide ions in matrix-assisted laser desorption/ionization mass spectrometry.

Tryptic digests of three proteins are reacted with O-methylisourea in order to convert lysine residues to homoarginines. The resulting homoarginine-terminated peptides exhibit more intense MALDI mass spectral peaks than their lysine-terminated predecessors. This simple chemical reaction should therefore facilitate protein sequencing and mass mapping.

Amino Acid Sequence↗
PubMed2000

Contrast echocardiography clarifies uninterpretable wall motion in intensive care unit patients.

OBJECTIVES: The study examined the value of contrast echocardiography in the assessment of left ventricular (LV) wall motion in intensive care unit (ICU) patients. BACKGROUND: Echocardiograms done in the ICU are often suboptimal. The most common indication is the evaluation of LV wall motion and ejection fraction (EF). METHODS: Transthoracic echocardiograms were done in 70 unselected ICU patients. Wall motion was evaluated on standard echocardiography (SE), harmonic echocardiography (HE), and after intravenous (IV) contrast echocardiography (CE) using a score for each of 16 segments. A confidence score was also given for each segment with each technique (unable to judge; not sure; sure). The EF was estimated visually for each technique, and a confidence score was applied to the EF. RESULTS: Uninterpretable wall motion was present in 5.4 segments/patient on SE, 4.4 on HE (p = 0.2), and 1.1 on CE (p < 0.0001). An average of 7.8 segments were read with surety on SE, 9.2 on HE (p = 0.1), and 13.7 on CE (p < 0.0001). Ejection fraction was uninterpretable in 23% on SE, 13% on HE (p = 0.14), and 0% on CE (p = 0.002 vs. HE; p < 0.0001 vs. SE). The EF was read with surety in 56% of patients on SE, 62% on HE (p = 0.47), and 91% on CE (p < 0.0001). Thus, wall motion was seen with more confidence on CE. More importantly, the actual readings of segmental wall motion and EF significantly differed using CE. CONCLUSIONS: CE should be used in all ICU patients with suboptimal transthoracic echocardiograms.

Adult↗
PubMed2000

The yeast heat shock transcription factor changes conformation in response to superoxide and temperature.

In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide.

Amino Acid Sequence↗
PubMed1999

The action of N-terminal acetyltransferases on yeast ribosomal proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to determine the state of N-terminal acetylation of 68 ribosomal proteins from a normal strain of Saccharomyces cerevisiae and from the ard1-Delta, nat3-Delta, and mak3-Delta mutants (), each lacking a catalytic subunit of three different N-terminal acetyltransferases. A total 30 of the of 68 ribosomal proteins were N-terminal-acetylated, and 24 of these (80%) were NatA substrates, unacetylated in solely the ard1-Delta mutant and having mainly Ac-Ser- termini and a few with Ac-Ala- or Ac-Thr- termini. Only 4 (13%) were NatB substrates, unacetylated in solely the nat3-Delta mutant, and having Ac-Met-Asp- or Ac-Met-Glu- termini. No NatC substrates were uncovered, e.g. unacetylated in solely mak3-Delta mutants, consistent with finding that none of the ribosomal proteins had Ac-Met-Ile-, Ac-Met-Leu-, or Ac-Met-Phe- termini. Interestingly, two new types of the unusual NatD substrates were uncovered, having either Ac-Ser-Asp-Phe- or Ac-Ser-Asp-Ala- termini that were unacetylated in the ard1-Delta mutant, and only partially acetylated in the mak3-Delta mutant and, for one case, also only partially in the nat3-Delta mutant. We suggest that the acetylation of NatD substrates requires not only Ard1p and Nat1p, but also auxiliary factors that are acetylated by the Mak3p and Nat3p N-terminal acetyltransferases.

Acetylation↗
PubMed1999

Safety profile of the proton-pump inhibitors.

The adverse effect profile of proton-pump inhibitors is presented. The proton-pump inhibitors are a well-tolerated class of drugs. The most common adverse events of headache, diarrhea, and nausea have been reported in fewer than 5% of patients treated with lansoprazole or omeprazole. The frequency of these adverse events with the two proton-pump inhibitors is comparable to that of placebo and histamine H2-receptor antagonists. Few clinically important interactions have been observed between proton-pump inhibitors and other drugs metabolized by the cytochrome P-450 system. The interaction potential should be considered when drugs with a narrow therapeutic window, such as phenytoin, warfarin, and theophylline, are used concomitantly with proton-pump inhibitors. Theoretical concerns about the consequences of chronic administration of proton-pump inhibitors, such as the impact of sustained hypergastrinemia on gastric morphology and the development of atrophic gastritis, have been dismissed. While increased gastrin levels are observed among patients taking proton-pump inhibitors, for the majority they remain within the normal range. After long-term use of the drugs, patients do not appear to be at increased risk of atrophic gastritis or gastric cancer. Helicobacter pylori infection, rather than acid suppression, may be the more important factor for the development of atrophic gastritis. Bacterial overgrowth and altered nutrient absorption resulting from sustained hypochlorhydria induced by chronic administration of proton-pump inhibitors have not been realized as clinical concerns. Not only are proton-pump inhibitors well tolerated during short-term administration, but there also do not appear to be clinically important adverse sequelae associated with their long-term use.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1999

Toward a simple, expedient, and complete analysis of human hemoglobin by MALDI-TOFMS.

MALDI mass spectrometry is explored as a method for hemoglobin characterization. To simplify and expedite the analysis, hemoglobin is obtained without purification directly from whole human blood. The use of trypsinactivated bioreactive MALDI probes is evaluated as a means to further reduce the analysis time from hours to minutes. Moreover, variations of the MALDI matrix preparation facilitate detection of the problematic tryptic peptides alpha T12, alpha T13, and beta T12. The results reveal that MALDI-based methods are easily implemented, are rapid, and allow detection of traditionally elusive tryptic peptides.

Hemoglobins↗
PubMed1999

Monitoring the growth of a bacteria culture by MALDI-MS of whole cells.

We have probed the time evolution of a growing bacteria culture by extracting samples periodically and performing matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) on whole cells. The mass spectra generated by this method contain tens of peaks in the 3-11-kDa mass range. Cultures of E. coli strain K-12 were grown in two types of containers and at two nutrient concentrations and sampled periodically from 6 to 84 h after inoculation. The relative intensities of several of the stronger peaks vary quite dramatically as a function of time. These temporal characteristics must be taken into account when MALDI-MS is applied to identify bacteria. The results also suggest that MALDI-MS can be used to follow the aging of a bacteria culture.

Escherichia coli↗
PubMed1999

Observation of Escherichia coli ribosomal proteins and their posttranslational modifications by mass spectrometry.

Ribosomes from the K-12 strain of Escherichia coli were analyzed with good sensitivity and high mass accuracy using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fifty-five of the 56 subunit proteins were observable. Mass spectral peak locations were consistent with previously reported post-translational modifications involving N-terminal methionine loss, methylation, thiomethylation, and acetylation for all but one case. The speed and accuracy of mass spectrometry make it a good candidate for phylogenetic studies of ribosomes and the observation of posttranslational modifications in other organisms.

Escherichia coli↗
PubMed1998

Three-dimensional ion mobility/TOFMS analysis of electrosprayed biomolecules.

An ion mobility/mass spectrometry technique has been developed to record mass-resolved ion mobility distributions for multiple ions simultaneously. The approach involves a new instrument that couples an electrospray ion source to an injected-ion drift tube/time-of-flight mass spectrometer. Individual components in a mixture of ions are separated by mobility differences in a drift tube and subsequently dispersed by mass-to-charge ratios in a time-of-flight instrument. Flight times in the mass spectrometer are much shorter than residence times in the drift tube, making it possible to record mass-resolved ion mobilities for all ions simultaneously. The result is a three-dimensional spectrum that contains collision cross section, mass-to-charge, and ion abundance information. The instrument and data acquisition system are described. Examples of combined ion mobility/time-of-flight data are presented for distributions of electrosprayed bradykinin and ubiquitin ions.

Bradykinin↗
PubMed1998

Detection of the bacteriological sex factor in E. coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is employed to detect the presence of the bacteriological sex factor, a trait that allows some bacteria to transfer genetic information to others by means of conjugation. The principal advantage of the method is its speed. Twenty different strains of E. coli were analyzed and the results were consistent with previously known genetic information. The sex factor could be transferred from one strain to another and the outcome verified by mass spectrometry.

Escherichia coli↗
PubMed1998

Fingerprint matching of E. coli strains with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of whole cells using a modified correlation approach.

We have developed a mathematical algorithm to compare and distinguish matrix-assisted laser desorption/ionization (MALDI) mass spectra of whole bacteria cells. This fingerprint matching technique eliminates the subjectivity involved in visually comparing two spectra to determine whether they match and it provides a quantitative measure of spectral similarity. Using it, we have distinguished twenty five different strains of a single bacteria species, E. coli. Cells are grown in culture, samples are prepared, and MALDI-TOF mass spectra are recorded for each strain. Pairs of spectra are compared by a modified cross-correlation procedure. This modified approach increases the sensitivity of correlation analysis to small spectral differences. The technique can be fine-tuned by varying the number of intervals into which spectra are divided.

DNA Fingerprinting↗
PubMed1998

Gas-phase separations of protease digests.

A mixture of peptides from a complete tryptic digest of ubiquitin has been analyzed by ion mobility/time-of-flight mass spectrometry techniques. All components of the mixture were electrosprayed and ions were separated in the gas phase based on differences in their mobilities through helium before being dispersed into a time-of-flight mass spectrometer for mass-to-charge analysis. The data show that ions separate into families primarily according to differences in their charge states and, to a lesser extent, differences in conformation. This approach reduces spectral congestion typically associated with electrosprayed mixtures and provides charge assignments for mass-to-charge ratio data. Gas-phase separations of ions appear to provide a new physical basis for characterizing components of biological mixtures.

Endopeptidases↗
PubMed1998

Maximum pulsed electromagnetic field limits based on peripheral nerve stimulation: application to IEEE/ANSI C95.1 electromagnetic field standards.

This communication proposes a rationale for maximum pulsed magnetic field limits in the electromagnetic-field standards of IEEE/ANSI C95.1. The peak limits, intended to protect against peripheral nerve excitation by pulsed fields, are adapted from existing standards for patient exposure in magnetic resonance imaging (MRI) examinations.

Electric Stimulation↗