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Jerzy Silberring

Publications and source records attributed to Jerzy Silberring.

At least 19 recordsLinked to original sources
PubMed2006

Methods for samples preparation in proteomic research.

Sample preparation is one of the most crucial processes in proteomics research. The results of the experiment depend on the condition of the starting material. Therefore, the proper experimental model and careful sample preparation is vital to obtain significant and trustworthy results, particularly in comparative proteomics, where we are usually looking for minor differences between experimental-, and control samples. In this review we discuss problems associated with general strategies of samples preparation, and experimental demands for these processes.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

The role of neuropeptide FF (NPFF) in the expression of sensitization to hyperlocomotor effect of morphine and ethanol.

Neuropeptide FF (NPFF) has been characterized as an endogenous anti-opioid peptide because its intraventricular injection (icv) reversed morphine- and stress-induced analgesia, and precipitates withdrawal syndrome in morphine-dependent rats. The role of NPFF in other aspects of drug dependence is unknown. Therefore, the aim of this study was to determine NPFF influence on the expression of sensitization to the morphine-induced hyperlocomotion. As the opioid system plays a role in ethanol effects, the influence of NPFF on the expression of sensitization to hyperlocomotor effect of ethanol was also investigated. Our study indicated that acute administration of NPFF (5, 10, 20nmol, icv) inhibited the expression of morphine-induced sensitization at doses of 10 (P<0.05) and 20nmol (P<0.01), and also inhibited ethanol-induced sensitization at a dose of 20nmol (P<0.01). Furthermore, NPFF inhibited the acute locomotor effect of morphine (10 and 20nmol) but not that of ethanol. NPFF, given alone, did not change the locomotor activity of mice and did not disturb motor coordination of animals in the rotarod test. In conclusion, our experiments indicated that NPFF attenuated the acute morphine locomotion and the expression of sensitization to locomotion. We anticipate that NPFF may be involved in both of these effects.

Animals↗
PubMed2006

CART (85-102)-inhibition of psychostimulant-induced hyperlocomotion: importance of cyclization.

Synthetic derivative of C-terminal fragment of CART (55-102) with reduced thiol groups, [Abu(86,94)]CART (85-102)(red), given together with amphetamine (5 mg/kg, s.c.) or cocaine (15 mg/kg, s.c.), reversed hyperlocomotion induced by these drugs at a dose of 0.1 microg but not at a higher dose. In the cerebral cortex homogenate, [Abu(86,94)]CART (85-102)(red) was nonspecifically cleaved from N- and C-termini. This peptide contains two chemically blocked Cys residues, and two others in reduced form. Concomitant with cleavage, rapid cyclization occurred. The newly formed cyclic peptides were stable. The cyclic peptide [Abu(86,94)]CART (85-102)(ox) failed to inhibit amphetamine- and cocaine-induced locomotor activity. The ability to inhibit the locomotor-stimulant activity of amphetamine was retained in [Abu(86,88,94,101)]CART (85-102), in which all Cys were replaced with 2-aminobutyric acid to prevent their pairing. Disulfide bridge formation may be an interesting mechanism that prevents proteolysis of [Abu(86,94)]CART (85-102)(red) and terminates its ability to reverse amphetamine-induced hyperlocomotion.

Amphetamines↗
PubMed2006

The activity of CART peptide fragments.

Cocaine- and amphetamine-regulated transcript (CART) peptides attracted much attention after the discovery that the level of CART mRNA is increased in rat striatum after acute administration of cocaine and amphetamine. The most widely investigated sequence is CART (55-102), whose roles were confirmed in modulation of various physiological processes such as feeding, energy expenditure, stress control, endocrine secretion, and reward. However, peptides other than (55-102) may be generated from the CART precursor as well. This review describes biological activity of peptides derived from the CART precursor in vivo, and of synthetic CART fragments that have not been found in the nature. In particular, the activity of CART (85-102) is described, whose ability to exert behavioral responses was confirmed by the observed attenuation of the expression of sensitization to morphine-induced hyperlocomotion. This fragment also decreased the number of escape jumps evoked by naloxone in morphine-addicted mice after intracerebroventricular administration.

Amino Acid Sequence↗
PubMed2006

Rat brain proteome in morphine dependence.

The aim of this study was to reveal potential markers associated with drug dependence, using the proteomic approach. Gels containing samples derived from morphine-treated and control animals were compared and analyzed. Inspection of protein profiles, following TCA/acetone precipitation and the use of nano-scale liquid chromatography coupled to tandem mass spectrometry, allowed for identification of eleven potential dependence markers, mainly cytoplasmic and mitochondrial enzymes, e.g. proteins that belong to GTPase and GST superfamilies, ATPase, asparaginase or proteasome subunit p27 families.

Animals↗
PubMed2006

Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells.

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10 ng/ml) and IL-6 (10 ng/ml). After 24 h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera.

Actin Depolymerizing Factors↗
PubMed2006

N-terminal H3/D3-acetylation for improved high-throughput peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer.

A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.

Acetylation↗
PubMed2006

Identification of catecholamines in the immune system by desorption/ionization on silicon.

Desorption/ionization from porous silicon dioxide (DIOSD), in combination with a standard matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrument, was used for the identification of catecholamines in the human peripheral blood lymphocytes. A routine MALDI-TOF analysis does not allow for sensitive detection of low molecular mass compounds (i.e. below 400 Da) due to the pronounced background ions arising from the matrix. Therefore, we have tested DIOSD methodology for the identification of catecholamines in the immune system. Using DIOSD, catecholamines were unambiguously identified in the cell extract of peripheral blood lymphocytes at the femtomolar level. The DIOSD extends the possible use of MALDI-TOF mass spectrometry towards small molecules that were previously detected by other methods.

Blood Chemical Analysis↗
PubMed2006

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid.

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.

Alpha-Globulins↗
PubMed2006

Fingerprinting of 3, 4-methylenedioxymethamphetamine markers by desorption/ionization on porous silicon.

Desorption/ionization on porous silicon (DIOS) is a method which extends the application range of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This technique eliminates matrix background in the low mass range; DIOS is especially advantageous in research on small organic molecules and their metabolites in biological samples. DIOS mass spectrometry was applied for 3, 4-methylenedioxymethamphetamine, (MDMA, Ecstasy) impurities identification. Trace components profiling enables the identification of by-products characteristics for the synthesis route of MDMA. Ecstasy, a synthetic psychoactive drug, is highly popular among young people, and often used as a recreational drug, most commonly used during disco parties. MDMA enhances feeling of euphoria by increasing the level of neurotransmitters such as serotonin, dopamine and norepinephrine, and causes acute behavioral and psychological effects. MDMA is almost exclusively produced illegally, primarily in Western Europe. The new method for MDMA impurities profiling has been developed to trace the origin of MDMA pills. For comparison and classification of the impurity profiles, the principal components analysis was used.

Drug Contamination↗
PubMed2006

Proteomic analysis of rat cerebral cortex, hippocampus and striatum after exposure to morphine.

Although a series of proteins in the brain have been shown to be qualitatively or quantitatively dysregulated following morphine administration, a systematic proteomic study has not been carried out so far. We therefore aimed to show the effect of morphine on protein levels in the rat brain. For this purpose rats were given a morphine base in subcutaneously placed pellets and subsequently the cerebral cortex, hippocampus and striatum were taken for proteomic studies after three days. Extracted proteins were run on two-dimensional gel electrophoresis, scanned and quantified by specific software. Proteins with significantly different levels were analysed by mass spectrometry (MALDI-TOF-TOF). Twenty-six proteins were found to be differentially expressed and were unambiguously identified. Dysregulated proteins were from several protein pathways and cascades including signaling, metabolic, protein handling, antioxidant and miscellaneous classes. These findings represent an initial approach to the generation of a 'morphinome' and may form the basis for further protein chemical studies as a valuable analytical tool. Moreover, the study reveals morphine-regulated proteins in different brain areas and indicates the pathways involved following morphine administration in the rat, the main species for pharmacological studies in the field.

Analgesics, Opioid↗
PubMed2005

Differential effects of N-peptidyl-O-acyl hydroxylamines on dynorphin-induced antinociception in the mouse capsaicin test.

In the capsaicin test, intrathecal (i.t.) dynorphins are antinociceptive. Cysteine protease inhibitors such as p-hydroxymercuribenzoate (PHMB) given i.t. augment and prolong their activity. The effect of two novel cysteine protease inhibitors, N-peptidyl-O-acyl hydroxylamines, on the antinociception induced by i.t. administered dynorphin A or dynorphin B has been investigated. When administered i.t. 5 min before the injection of capsaicin (800 ng) into the plantar surface of the hindpaw, dynorphin A (62.5-1000 pmol) or dynorphin B (0.5-4 nmol) produced a dose-dependent and significant antinociceptive effect. The effect of dynorphin A (1 nmol) and dynorphin B (4 nmol) disappeared completely within 180 and 60 min, respectively. PHMB (2 nmol) and Boc-Tyr-Gly-NHO-Bz (BYG-Bz) (2 nmol) co-administered with dynorphin A or dynorphin B significantly prolonged antinociception induced by both. On the other hand, Z-Phe-Phe-NHO-Bz (ZFF-Bz) (1 and 2 nmol) only prolonged antinociception induced by dynorphin A. The results suggest that Z-Phe-Phe-NHO-Bz is an inhibitor of cysteine proteases preferring cleavage of dynorphin A, with less specificity towards dynorphin B in the mouse spinal cord.

Analgesics↗
PubMed2005

Proteomics and peptidomics in neuroscience. Experience of capabilities and limitations in a neurochemical laboratory.

The increasing use of proteomics has created a basis for new strategies to develop methodologies for rapid identification of protein patterns in living organisms. It has also become evident that proteomics has other potential applications than protein and peptide identification, e.g. protein characterization, with the aim of revealing their structure, function(s) and interactions of proteins. In comparative proteomics studies, the protein expression of a certain biological system is compared with another system or the same system under perturbed conditions. Global identification of proteins in neuroscience is extremely complex, owing to the limited availability of biological material and very low concentrations of the molecules. Moreover, in addition to proteins, there are number of peptides that must also be considered in global studies on the central nervous system. In this overview, we focus on and discuss problems related to the different sources of biological material and sample handling, which are part of all preparatory and analytical steps. Straightforward protocols are desirable to avoid excessive purification steps, since loss of material at each step is inevitable. We would like to merge the two worlds of proteomics/peptidomics and neuroscience, and finally we consider different practical and technical aspects, illustrated with examples from our laboratory.

Animals↗
PubMed2005

An enhanced method for peptides sequencing by N-terminal derivatization and MS.

An improved method for peptide sequencing based on acetylation/deuteroacetylation in conjunction with ESI MS is introduced. Derivatization with a 1:1 mixture of acetic anhydride and deuterated acetic anhydride incorporates a stable isotope label into the analyzed molecule. This approach has been initially applied to FAB. Using MS/MS, the technique provides a fast, highly sensitive and reliable determination of the primary structure of unknown peptides. This procedure labels N-terminal fragments formed during MS/MS analysis, resulting in a simplification and faster interpretation of the spectra. The performance of the method has been tested with several synthetic peptides and applied to an efficient sequencing of the peptide map, using a nano-scale LC coupled on-line to a tandem mass spectrometer.

Acetylation↗
PubMed2005

Proteome analysis of mouse primary astrocytes.

Astrocytes play a role in energy metabolism, neuronal homeostasis and release of neuronal growth factors and several neurotransmitters. They also relate to a variety of brain diseases and contribute to restore brain dysfunction. Although current research has revealed several roles for astrocytes, knowledge on astrocytic protein expression is limited and a systematic and comprehensive proteome study of astrocytes has not been reported so far. We applied a proteomics technique based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 301 spots corresponding to 191 individual proteins in primary mouse astrocytes. The identified proteins were from antioxidant, chaperone, cytoskeleton, nucleic acid binding, signaling, proteasomal, hypothetical and miscellaneous proteins. A reference database is provided and proteins were identified in astrocytes specifically and unambiguously for the first time. A reliable analytical tool independent of antibody availability and specificity along with tentative astrocytic marker proteins is described.

Animals↗
PubMed2004

Solution conformational study of nociceptin an(d its 1-13 and 1-11 fragments using circular dichroism and two-dimensional NMR in conjunction with theoretical conformational analysis.

Conformational studies of nociceptin (NC-NH2), its fully active fragment, NC(1-13)-NH2, and two significantly less potent fragments, NC(1-13)-OH and NC(1-11)-OH, were conducted in water and TFE solutions by the employment of circular dichroism, and in DMSO-d6 by 2DNMR spectroscopy in conjunction with theoretical conformational analysis. The conformations of all thepeptides studied were calculated taking two approaches. The first assumes multiconformational equilibrium of the peptide studied, which is characterized by a set of conformations (and their statistical weight values)obtained from a global conformational analysis using three methods: the electrostatically driven Monte-Carlo (EDMC) with the ECEPP/3 force field, the simulated annealing (SA) protocols in the AMBER and CHARMM force fields. The second approach incorporates the interproton distance and dihedral angle constraints into the starting conformation. Calculations were performed using the distance geometry and SA protocol in the CHARMM force field implemented in the X-PLOR program. The CD experiments indicated that for the active peptides, hydrophobic solvents induced a significantly higher (compared with those remaining)content order, probably a helical structure. Unfortunately, as a result of the conformational flexibility of thepeptides, the analysis of conformations obtained with both approaches and different force fields did not alllow the selection of any structural elements of the NC peptides that might be connected with their bioactivity. The only common element found in most conformations of the active peptides was a helical character of fragment 8-13, which allowed the side chains of basic amino acid residues to be exposed to the outside of the molecule and probably to interact with the ORL1 receptor.

Animals↗
PubMed2004

Influence of nociceptin(1-17) fragments and its tyrosine-substituted derivative on morphine-withdrawal signs in rats.

Our previous studies demonstrated that endogenous ligand of nociceptin (NOP) receptor, nociceptin(1-17) (also known as orphanin FQ), inhibits morphine-withdrawal syndrome measured as wet dog shakes in rats [Life Sci. 66 (2000) PL119]. This peptide is metabolized in the spinal cord, both in vitro and in vivo, to shorter fragments, including nociceptin(1-11) and nociceptin(1-6). These fragments, formed after cleavage by endogenous peptidase, are behaviorally active and modulate nociception in a bi-phasic process [Peptides 20 (1999) 239]. As these peptides induced transient naloxone-reversible analgesia in behavioral tests [Peptides 20 (1999) 239], in the present study we tested the influence of nociceptin(1-11) (10 and 20 microg) and nociceptin(1-6) (10, 20 and 40 microg) on the morphine-withdrawal syndrome in rats. Furthermore, the modified fragment of nociceptin(1-6) with an opioid-message domain achieved by replacement of Phe1 with Tyr was tested. Morphine-withdrawal syndrome was precipitated by the i.p. injection of naloxone hydrochloride (2 mg/kg), 72 h after implantation of morphine pellets. The wet-dog shakes were chosen for statistical analyses of the abstinence signs. The results show that nociceptin(1-11) and (1-6) attenuate this morphine-withdrawal symptom. The replacement of Phe1 with Tyr in nociceptin(1-6) fragment did not potentiate the influence of nociceptin(1-6) on wet dog shakes precipitated by naloxone in morphine-dependent rats.

Animals↗