Jerzy Silberring

The influence of chronic stress on multiple opioid peptide systems in the rat: pronounced effects upon dynorphin in spinal cord.

Recurrent exposure to intermittent electrical foot-shock (30 min, twice daily) for 7 days caused an increase in immunoreactive (ir) dynorphin and ir-alpha-neo-endorphin in lumbar and cervical (but not thoracic) spinal cord as measured 16 h following the final session. At this time the level of ir-Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was also increased at the lumbar level. An acute foot-shock depleted spinal cord dynorphin in chronically stressed but not in naive rats. No alterations in levels of ir-dynorphin or ir-MEAGL were seen in discrete brain tissues. In contrast to the brain, where no effects were seen, the levels of beta-endorphin increased in both lobes of the pituitary. This change, however, was not accompanied by an alteration in levels of beta-endorphin in plasma. These data show that chronic foot-shock stress selectively influences particular pools of opioid peptides, predominantly those derived from proenkephalin B in the spinal cord and from proopiomelanocortin in the anterior pituitary. It is suggested that alterations observed in the spinal cord reflect enhanced activity of the proenkephalin B system in response to chronic nociceptive stimulation.

Mass spectrometry : instrumentation, interpretation, and applications

FOREWORD. CONTRIBUTORS. PART I INSTRUMENTATION. 1 DEFINITIONS AND EXPLANATIONS (Ann Westman-Brinkmalm and Gunnar Brinkmalm). References. 2 A MASS SPECTROMETERS BUILDING BLOCKS (Ann Westman-Brinkmalm and Gunnar Brinkmalm). 2.1. Ion Sources. 2.2. Mass Analyzers. 2.3. Detectors. References. 3 TANDEM MASS SPECTROMETRY (Ann Westman-Brinkmalm and Gunnar Brinkmalm). 3.1. Tandem MS Analyzer Combinations. 3.2. Ion Activation Methods. References. 4 SEPARATION METHODS (Ann Westman-Brinkmalm, Jerzy Silberring, and Gunnar Brinkmalm). 4.1. Chromatography. 4.2. Electric-Field Driven Separations. References. PART II INTERPRETATION. 5 INTRODUCTION TO MASS SPECTRA INTERPRETATION: ORGANIC CHEMISTRY (Albert T. Lebedev). 5.1. Basic Concepts. 5.2. Inlet Systems. 5.3. Physical Bases of Mass Spectrometry. 5.4. Theoretical Rules and Approaches to Interpret Mass Spectra. 5.5. Practical Approaches to Interpret Mass Spectra. References. 6 SEQUENCING OF PEPTIDES AND PROTEINS (Marek Noga, Tomasz Dylag, and Jerzy Silberring). 6.1. Basic Concepts. 6.2. Tandem Mass Spectrometry of Peptides and Proteins. 6.3. Peptide Fragmentation Nomenclature. 6.4. Technical Aspects and Fragmentation Rules. 6.5. Why Peptide Sequencing? 6.6. De Novo Sequencing 6.7. Peptide Derivatization Prior to Fragmentation. Acknowledgments. References. Online Tutorials. 7 OPTIMIZING SENSITIVITY AND SPECIFICITY IN MASS SPECTROMETRIC PROTEOME ANALYSIS (Jan Eriksson and David Fenyo). 7.1. Quantitation. 7.2. Peptide and Protein Identification. 7.3. Success Rate and Relative Dynamic Range. 7.4. Summary. References. PART III APPLICATIONS. 8 DOPING CONTROL (Graham Trout). References. 9 OCEANOGRAPHY (R. Timothy Short, Robert H. Byrne, David Hollander, Johan Schijf, Strawn K. Toler, and Edward S. VanVleet). References. 10 OMICS APPLICATIONS (Simone Konig). 10.1. Introduction. 10.2. Genomics and Transcriptomics. 10.3. Proteomics. 10.4. Metabolomics. 11 SPACE SCIENCES (Robert Sheldon). 11.1. Introduction. 11.2. Origins. 11.3. Dynamics. 11.4. The Space MS Paradox. 11.5. A Brief History of Space MS. 11.6. GENESIS and the Future. References. 12 BIOTERRORISM (Vito G. DelVecchio and Cesar V. Mujer). 12.1. What is Bioterrorism? 12.2. Some Historical Accounts of Bioterrorism. 12.3. Geneva Protocol of 1925 and Biological Weapons Convention of 1972. 12.4. Categories of Biothreat Agents. 12.5. Challenges. 12.6. MS Identification of Biomarker Proteins. 12.7. Development of New Therapeutics and Vaccines Using Immunoproteomics. References. 13 IMAGING OF SMALL MOLECULES (Malgorzata Iwona Szynkowska). 13.1. SIMS Imaging. 13.2. Biological Applications (Cells, Tissues, and Pharmaceuticals). 13.3. Catalysis. 13.4. Forensics. 13.5. Semiconductors. 13.6. The Future. References. 14 UTILIZATION OF MASS SPECTROMETRY IN CLINICAL CHEMISTRY (Donald H. Chace). 14.1. Introduction. 14.2. Where are Mass Spectrometers Utilized in Clinical Applications? 14.3. Most Common Analytes Detected by Mass Spectrometers. 14.4. Multianalyte Detection of Clinical Biomarkers, The Real Success Story. 14.5. Quantitative Profiling. 14.6. A Clinical Example of the Use of Mass Spectrometry. 14.7. Demonstrations of Concepts of Quantification in Clinical Chemistry. 15 POLYMERS (Maurizio S. Montaudo). 15.1. Introduction. 15.2. Instrumentation, Sample Preparation, and Matrices. 15.3. Analysis of Ultrapure Polymer Samples. 15.4. Analysis of Polymer Samples in which all Chains Possess the Same Backbone. 15.5. Analysis of Polymer Mixtures with Different Backbones. 15.6. Determination of Average Molar Masses. References. 16 FORENSIC SCIENCES (Maria Kala). 16.1. Introduction. 16.2. Materials Examined and Goals of Analysis. 16.3. Sample Preparation. 16.4. Systematic Toxicological Analysis. 16.5. Quantitative Analysis. 16.6. Identification of Arsons. References. 17 NEW APPROACHES TO NEUROCHEMISTRY (Jonas Bergquist, Jerzy Silberring, and Rolf Ekman). 17.1. Introduction. 17.2. Why is there so Little Research in this Area? 17.3. Proteomics and Neurochemistry. 17.4. Conclusions. Acknowledgments. References. PART IV APPENDIX. INDEX.

Analgesis and convulsant effects of guanidinoethylmercaptosuccinic acid (GEMSA) — A potent enkephalin convertase inhibitor

We studied behavioral effects of the intraventricularly and intrathecally administered guanidinoethylmercaptosuccinic acid (GEMSA) - a potent inhibitor of enkephalin convertase. When given intraventricularly in doses of 3 and 6 micrograms, GEMSA elicited analgesia; after doses of 12.5 and 25 micrograms the explosive motor behavior and convulsions occurred. Following the intrathecal administration of GEMSA (12.5, 25 and 50 micrograms), an increase in the tail-flick latency was observed; moreover that drug potentiated analgesic effects of the intrathecally applied Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Gly7-Leu8. All the above effects of GEMSA were significantly attenuated by naloxone. The rats subjected to chronic pain showed a weaker analgesic response to the intrathecally injected GEMSA. The 3H-GEMSA binding to enkephalin convertase in the spinal cord of these rats produced only a slight increase in KD; besides, no changes in the enzyme activity were observed. The study shows that GEMSA has a potent pharmacological action in the central nervous system. Furthermore, this effect is partly due to the influence of GEMSA on endogenous opioid peptide systems, possibly on proenkephalin A.

Kinetics of [3H]-prazosin binding to the rat cortex during aging

Kinetics (Bmax, KD, k on, k off) of [3H]-prazosin binding to the rat cortex was measured at different ages of animals (2-4, 13-15, 20-24 months old). Additionally, an agonist (phenylephrine) and an antagonist (phentolamine) inhibition of [3H]-prazosin binding was performed at two different rat ages (2-4 and 20-24 months old). The number of cortical alpha 1-adrenoceptors was significantly reduced in 20-24-month-old rats, when compared with 2-4-month ones. Association (k on) and dissociation (k off) rate constants were also reduced in the oldest group, but the dissociation constant (KD) was similar in all the age groups tested. Affinity of phenylephrine, but not of phentolamine, was reduced in the oldest group (20-24 months old). The obtained data suggest that changes in the brain alpha 1-adrenoceptors, correlated with the animal age, are connected to the number of binding sites, rate constants and the affinity of an agonist.

Enkephalin convertase in the rat spinal cord

3H-Guanidinoethylmercaptosuccinic acid (3H-GEMSA) a very selective inhibitor of enkephalin convertase, binds to the crude rat spinal cord homogenates saturably, reversibly and with high affinity. Scatchard analysis revealed two classes of binding sites with KD: 4.5 nM and 215 nM. A plot of dissociation experiment was nonlinear with the T1/2: 2 min and 6 min, respectively. 3H-GEMSA binding sites are evenly distributed throughout the rat spinal cord and their high density might suggest a physiological significance of enkephalin convertase in that tissue.

iTRAQ analysis with Paul ion trap-obstacle solved.

Mass spectrometers equipped with ion trap analyzers have been significantly improved due to their high performance and wide application area accompanying the low costs of purchase. Despite several advantages, such as reasonable resolution at low cost, high sensitivity, and capability for multistage analysis, ion traps have an important drawback: low mass cutoff during tandem mass spectrometry analysis MS(n). Although the low mass cutoff associated with the ion trap does not seriously obstruct peptide identification, it may cause a serious problem in identification of small molecules (posttranslational modifications, e.g., glycan structures) and quantification of peptides with multiplexed isobaric tag reagents. The presented approach offers the possibility to use isobaric tags for relative and absolute quantification labeling (iTRAQ) for quantitative, proteomic analysis using typical, widely available ion trap devices and manufacturers software. We have performed series of analyses of standard protein labeled with isobaric tags in various concentration ratios to prove quantitative capabilities of this approach.

Highly efficient proteinase assay with chromogenic substrates and its application in a study of enzyme inhibitors

Abstract A simple and rapid method for the determination of enzyme activities with chromogenic substrates is described. Trypsin and papain were used as model proteinases and N-benzoyl- dl -arginine p-nitroanilide (BAPNA) was applied as substrate. The enzyme assay was performed on a multi-scale using 96-well microtitration plates and product release was detected with the aid of an automatic plate reader, widely used in ELISA tests. The procedure was used for electrophoretic studies of trypsin and a crude papain preparation. It was also applied for the investigation of N-peptidyl-O-acylhydroxylamine proteinase inhibitors. In comparison with commonly used procedures with chromogenic substrates, the proposed approach consumes markedly reduced amounts of all reagents. It allows an almost unlimited number of samples to be assayed in a short time and should be applicable to the detection and determination of any enzyme activitiy where chromogenic substrates are applicable.

A modified radioimmunoassay for arylsulfatase A in human serum and urine.

A radioimmunoassay was developed for the determination of arylsulfatase A (EC 3.1.6.1) in human serum and urine. An isoenzyme of arylsulfatase A purified from human urine was used as a standard antigen. The enzyme was radioiodinated with 125I using the Chloramine T method and was stable for about 4 wk. Antibody-bound enzyme was separated from free enzyme by means of a double antibody technique in the presence of polyethylene glycol (PEG). The working range of the method was 0.15-5.0 ng of arylsulfatase A per assay. The within-assay CV was about 8% for both biological fluids and the between-assay CV for serum was 14.1%. Analytical recoveries were 93.2 +/- 9.1% and 97.8 +/- 5.5% for serum and urine, respectively, and the sensitivity was 0.040 ng of arylsulfatase per assay. Serum samples of 50 healthy blood donors were assayed to establish the normal serum level of immunoreactive enzyme, which was found to be 8.3 ng/ml +/- 1.8 ng/ml of serum. Storage of frozen serum was shown to have no significant effect on results obtained using this RIA.

Desorption/ionization mass spectrometry on array of silicon micro tips

We have shown that a sample of bio-species deposited onto the spot of the array of protruded metal gated silicon tips (DIOST) may be effectively ionized and evaporated by UV laser pulsed light illumination. This phenomenon has been never observed on flat surface of silicon dioxide or TiW-Au thin film layers the materials which were used in construction of the tips array. Nature of this process is not known; the most intriguing fact is the protonization as well as negative electron ionization of bio-samples. The new DIOST platform was used for TOF MS tests, and clearly readable, low-noised mass spectrograms of LGG and dopamine were observed.