Characterization of Endoproteolytic Processing of Dynorphins by Proprotein Convertases using Mouse Spinal Cord S9 Fractions and Mass Spectrometry

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous Big Dynorphin (BDyn) and Dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild type (WT), PC1-/+ and PC2-/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments Dynorphin 1-19, Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7 and C-terminal processing of Dyn A generates Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even partial inhibition of PC1 or PC2 may impair the endogenous opioid system.

Concentrations of perfluorinated carboxylic acids (PFCAs) and sulfonic acids (PFSAs) in liver samples of different marine mammals (1984-2009)

Temporal variations in concentrations of perfluorinated carboxylic acids (PFCAs) and sulfonic acids (PFSAs), including perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) structural isomers, were examined in livers of pilot whale (Globicephala melas), ringed seal (Phoca hispida), minke whale (Balaenoptera acutorostrata), harbor porpoise (Phocoena phocoena), hooded seal (Cystophora cristata), Atlantic white-sided dolphin (Lagenorhynchus acutus) and in muscle tissue of fin whales (Balaenoptera physalus). The sampling spanned over 20 years (1984-2009) and covered a large geographical area of the North Atlantic and West Greenland. Liver and muscle samples were homogenized, extracted with acetonitrile, cleaned up using hexane and solid phase extraction (SPE), and analyzed by liquid chromatography with negative electrospray tandem mass spectrometry (LC-MS/MS). In general, the levels of the long-chained PFCAs (C9-C12) increased whereas the levels of PFOS remained steady over the studied period. The PFOS isomer pattern in pilot whale liver was relatively constant over the sampling years. However, in ringed seals there seemed to be a decrease in linear PFOS (L-PFOS) with time, going from 91% in 1984 to 83% in 2006.

Performance tests for the evaluation of computerized gas chromatography/mass spectrometry equipment and laboratories /

"April 1980"--Cover.

The determination of uranium isotope abundances and reactor fuel burnup by mass spectrometry /

April 17, 1963--page 11.

Investigation of solid surfaces by field emission microscopy and mass spectrometry; final report.

"Contract no. AF 49 (638)-748. Division file 30-4. AFOSR-2273."

Method of analysis and quality-assurance practices for determination of pesticides in water by solid-phase extraction and capillary-column gas chromatography/mass spectrometry at the U.S. Geological Survey California District Organic Chemistry Laboratory, 1996-99 /

Mode of access: Internet.

Measurement of free drug and clinical end-point by high-performance liquid chromatography-mass spectrometry: Applications and implications for pharmacokinetic and pharmacodynamic studies

Free drug measurement and pharmacodymanic markers provide the opportunity for a better understanding of drug efficacy and toxicity. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) is a powerful analytical technique that could facilitate the measurement of free drug and these markers. Currently, there are very few published methods for the determination of free drug concentrations by HPLC-MS. The development of atmospheric pressure ionisation sources, together with on-line microdialysis or on-line equilibrium dialysis and column switching techniques have reduced sample run times and increased assay efficiency. The availability of such methods will aid in drug development and the clinical use of certain drugs, including anti-convulsants, anti-arrhythmics, immunosuppressants, local anaesthetics, anti-fungals and protease inhibitors. The history of free drug measurement and an overview of the current HPLC-MS applications for these drugs are discussed. Immunosuppressant drugs are used as an example for the application of HPLC-MS in the measurement of drug pharmacodynamics. Potential biomarkers of immunosuppression that could be measured by HPLC-MS include purine nucleoside/nucleotides, drug-protein complexes and phosphorylated peptides. At the proteomic level, two-dimensional gel electrophoresis combined with matrix-assisted laser desorption/ionisation time-of-flight (TOF) MS is a powerful tool for identifying proteins involved in the response to inflammatory mediators. (C) 2003 Elsevier Science B.V. All rights reserved.

Effect of atorvastatin on cyclosporine pharmacokinetics in liver transplant recipients

BACKGROUND: The development of hyperlipidemia after liver transplant is frequently treated with hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) such as atorvastatin. As atorvastatin and the primary immunosuppressant drug, cyclosporine, are metabolized by the same pathway, there is the potential for an interaction. OBJECTIVE: To determine the effect of atorvastatin on cyclosporine pharmacokinetics in liver transplant recipients. METHODS: Six stable, long-term adult liver transplant recipients from a single center who developed posttransplant dyslipidemia were recruited to participate in a 14-day, open-label study of atorvastatin 10 mg/d coadministered with standard posttransplant immunosuppression using constant oral doses-of cyclosporine and corticosteroids. A 10-point pharmacokinetic profile was performed prior to and on day 14 after commencement of atorvastatin therapy. Cyclosporine concentrations were measured by HPLC-electrospray-tandem mass spectrometry. The AUC was calculated by the linear trapezoidal rule, with other parameters determined by visual inspection. RESULTS: Atorvastatin coadministration increased the cyclosporine AUC by 9% (range 0-20.6%; 3018 vs 3290 ng(.)h/mL; p = 0.04). No significant change was evident for other cyclosporine pharmacokinetic parameters. Total cholesterol and low-density lipoprotein cholesterol levels were significantly lower on day 14 than at baseline (p < 0.02). One patient developed a twofold increase in transaminases after 2 weeks of atorvastatin therapy, but no other clinical or biochemical adverse events were recorded. CONCLUSIONS: Atorvastatin coadministration increases the cyclosporine AUC by approximately 10% in stable liver transplant recipients. This change in systemic exposure to cyclosporine is of questionable clinical significance. Atorvastatin is effective in reducing cholesterol levels in liver transplant recipients.

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: Investigations of the cyclotide kalata B1 in biological fluids

A rapid method has been developed for the quantification of the prototypic cyclotide kalata B I in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity (R-2 > 0.99) in the concentration range 0.05-10 mu g/mL with a limit of detection of 0.05 mu g/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological samples without the need for extensive extraction procedures. (c) 2005 Elsevier B.V. All rights reserved.