The influence of a new derivate of kisspeptin-10 - Kissorphin (KSO) on the rewarding effects of morphine in the conditioned place preference (CPP) test in male rats.

Kissorphin (KSO) is a new peptide derived from kisspeptin-10. Previous study has indicated that this peptide displays neuropeptide FF (NPFF)-like anti-opioid activity. Herein, we examined the influence of KSO (1; 3, and 10 nmol, intravenously [i.v.]), on the rewarding action of morphine (5 mg/kg, intraperitoneally [i.p.]), using the unbiased design of the conditioned place preference (CPP) paradigm in rats. To test the effect of KSO on the acquisition of morphine-induced CPP, KSO and morphine were co-injected during conditioning with no drugs treatment on the test day. To investigate the effect of KSO on the expression of morphine-induced CPP, morphine alone was given during the conditioning phase (1 × 3 days) and KSO was administered 5 min prior to the placement in the CPP apparatus on the test day. To estimate the influence of KSO on the reinstatement of morphine-induced CPP, KSO was given 5 min before a priming dose of morphine (5 mg/kg, i.p.) on the reinstatement test day. The results show that KSO inhibited the acquisition, expression and reinstatement of morphine-induced CPP. The strongest effect of KSO was observed at the dose of 10 nmol (acquisition and reinstatement) or 1 nmol (expression). KSO given alone, neither induced place preference, nor aversion. Furthermore, the morphine-modulating effects of KSO were markedly antagonized by pretreatment with RF9 (10 nmol, i.v.), the NPFF receptors selective antagonist. Thus, KSO inhibited the morphine-induced CPP mainly by involving specific activation of NPFF receptors. Overall, these data further support the anti-opioid character of KSO.

Technical note: Air compared to nitrogen as nebulizing and drying gases for electrospray ionization mass spectrometry.

In the present study we tested the application of compressed air instead of pure nitrogen as the nebulizing and drying gas, and its influence on the quality of electrospray ionization (ESI) mass spectra. The intensities of the signals corresponding to protonated molecules were significantly (twice) higher when air was used. Inspection of signal-to-noise (S/N) ratios revealed that, in both cases, sensitivity was comparable. A higher ion abundance after the application of compressed air was followed by a higher background. Another potential risk of using air in the ESI source is the possibility for sample oxidation due to the presence of oxygen. To test this, we selected five easily oxidizing compounds to verify their susceptibility to oxidation. In particular, the presence of methionine was of interest. For all the compounds studied, no oxidation was observed. Amodiaquine oxidizes spontaneously in water solutions and its oxidized form can be detected a few hours after preparation. Direct comparison of the spectra where nitrogen was used with the corresponding spectra obtained when air was applied did not show significant differences. The only distinction was slightly different patterns of adducts when air was used. The difference concerns acetonitrile, which forms higher signals when air is the nebulizing gas. It is also important that the replacement of nitrogen with air does not affect quantitative data. The prepared calibration curves also visualize an intensity twice as high (independent of concentration within tested range) of the signal where air was applied. We have used our system continuously for three months with air as the nebulizing and drying gas and have not noticed any unexpected signal deterioration caused by additional source contamination from the air. Moreover, compressed air is much cheaper and easily available using oil-free compressors or pumps.

Modulation of neuropeptide FF (NPFF) receptors influences the expression of amphetamine-induced conditioned place preference and amphetamine withdrawal anxiety-like behavior in rats.

Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24h after the last (14 days) amphetamine (2.5mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.

Start-to-end processing of two-dimensional gel electrophoretic images.

Gel electrophoresis serves as a basic analytical tool in the proteomic studies. However, processing of gel electrophoretic images is still the main bottleneck of data analysis, and there is an increasing need for the fully automated approaches. The proposed start-to-end strategy of analyzing the gel images consists of chemometric tools, which allow their effective preprocessing, automatic warping, and data modeling. The image preprocessing techniques: denoising in the wavelet domain and the penalized asymmetric least squares approach for the background estimation are proposed. Matching of images is based on fuzzy warping of features, extracted from the gel images. For the classification or calibration purpose, multivariate approaches such, as partial least squares (PLS) or kernel-PLS methods are used. Performance of the proposed strategy is demonstrated on the real set of the two-dimensional gel images.

Is the nociceptin (NOP) receptor involved in attenuation of the expression of sensitization to morphine-induced hyperlocomotion in mice?

In the present study we investigated whether synthetic agonists of the nociceptin (NOP) receptors, Ro 64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one] and Ro 65-6570 (8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one), influence the expression of sensitization to the locomotor stimulant effect of morphine in mice. Sensitization was produced by five repeated administrations of morphine (10 mg/kg, s.c.) at 3-day intervals. Seven days after the last morphine injection, Ro 64-6198 (1 and 3 mg/kg, i.p.) and Ro 65-6570 (3 and 6 mg/kg, i.p.) were given immediately before the challenge dose of morphine (10 mg/kg, s.c.). Both substances inhibited the expression of sensitization to the locomotor stimulant action of morphine. However, the selective NOP receptor antagonist, [Nphe1]NC(1-13)NH2 (30 nmol, i.c.v.) did not reverse the inhibitory effect of the Ro-compounds. Therefore, our results suggest that the NOP receptor may not be critical for the influence of Ro-compounds on the morphine-induced sensitization, or the observed effect may be attributed to one functional subset of this receptor, stimulation of which is not blocked by [Nphe1]NC(1-13)NH2.

Morphinome--proteome of the nervous system after morphine treatment.

Proteome is a natural consequence of the post-genome era when the HUGO project (Human Genome Organization) has almost been completed. Here, a specifically aimed proteome in drug dependence--morphinome, is described, including tasks, strategies and pitfalls of the methodology.

Clinical mass spectrometry in neuroscience. Proteomics and peptidomics.

In this review we discuss the merits and drawbacks with the use of proteomic and peptidomic strategies for identification of proteins and peptides in their multidimensional interactions in complex biological processes. The progress in proteomics and peptidomics during the last years offer us new challenges to study changes in the protein and peptide synthesis. These strategies also offer new tools to follow post-translational modifications and other disturbed chemical processes that may be indicative of pathophysiological alteration(s). Furthermore these techniques can contribute to improvements in the diagnosis and therapy of neurodegenerative diseases, such as Alzheimer's disease, and psychiatric diseases, as depression and post traumatic stress disorders. We also consider different practical aspects of the applications of mass spectrometry in clinical neuroscience, illustrated by example from our laboratories. The new proteomic and peptidomic strategies will further enable the progress for clinical neuroscience research.

Orphanin FQ/nociceptin but not Ro 65-6570 inhibits the expression of cocaine-induced conditioned place preference.

The present study investigated the effect of orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like 1 (ORL-1) receptor on the expression of cocaine-induced conditioned place preference (CPP) in rats. To extend this study, the new non-peptidic compound Ro 65-6570 (8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one), with agonist activity at ORL-1 receptors, was examined. The influence of both compounds on cocaine-induced hyperactivity was also studied. Our experiments indicated that intracerebroventricular (i.c.v.) injection of OFQ/N, at doses of 10 and 20 microg/rat, significantly suppressed the expression of cocaine-induced place preference. Ro 65-6570 (3 and 6 mg/kg, i.p.) did not change the effect of cocaine, although its acute injection in control rats significantly increased the time spent in the drug-associated compartment of the CPP apparatus. The substances exhibited opposite effects on cocaine-induced hyperactivity (OFQ/N suppressed it but Ro 65-6570 increased it). Our results suggest that the effect of OFQ/N on the expression of cocaine-induced CPP may be a result of its influence on dopamine (DA) neurotransmission in mesolimbic structures. Ro 65-6570 does not share this effect with OFQ/N.

Dynorphin A inhibits nociceptin-converting enzyme from the rat spinal cord.

Cysteine proteinase found in the spinal cord of rat, called nociceptin-converting enzyme (NCE), is competitively inhibited by dynorphin A and its fragment des-[Tyr(1)]-DYN A. This proteinase converts orphanin FQ/nociceptin (OFQ/N) to two major fragments: OFQ/N(1-11) and further OFQ/N(1-6) with analgesic properties. Dynorphin A at the concentration of 10 microM increases K(M) from 15.0 to 55.9 microM. The calculated K(i) for this interaction was estimated at 3.7 microM. This observation may suggest an interaction between opioid and nociceptive systems which may be affected by the balance between opioid and antiopioid systems. This balance between particular OFQ/N sequences that are derived from the same precursor and regulated by proteinases may play an important role in pain. Interestingly, dynorphin B does not reveal a similar action on the NCE.

Supramolecular assistance to regioselectivity in the reactions of chlorocyclophosphazenes with sodium oxyanions: macrocyclic effect and anion dependence.

Comparative studies of the naphtholysis of crown-bearing tetrachlorocyclotriphosphazene 2 and its acyclic analogue, nongem-diethoxytetrachlorocyclotriphosphazene 5 (which was synthesized and isolated in the pure cis form for the purpose of this study), reveal a significant macrocyclic effect of the substituent on both the kinetics and regiochemistry of chlorine substitution in the N(3)P(3) ring. Whereas substitution of 5 with sodium naphthoxide provided moderate yields of the respective sterically and electronically favored nongem-naphthoxydiethoxy derivative as the major product, isolated as the mixture of cis and trans isomers 8 + 9, the corresponding reaction of crown substrate 2 resulted in highly regioselective formation of the gem-to-macrocycle substituted mononaphthoxy PNP-crown derivative 11a. The importance of the PNP-crown related effects for the regio- and stereocontrol of the substitution of chlorine atoms in the N(3)P(3) ring is discussed in terms of supramolecular sodium cation-assisted interactions between the ring oxy substituent(s) and incoming oxyanions. To approach the problem of the anion-dependence of the regioselectivity, a comparison has been drawn between the reactions of the PNP-crown substrate 2 with sodium arylates derived from beta-naphthol (10a) and phenol (10b), carried out with equimolar or 100% excess sodium oxyanion with respect to 2, and the corresponding reactions of 2 with sodium monoenolates derived from the beta-dicarbonyl compounds: acetylacetone (10c) and ethyl acetylacetate (10d). The effectiveness of supramolecularly assisted transition state stabilization is found to be dependent not only on the dimensional complementarity between the cation and the macrocycle but also on the properties of the four interacting ionic centers according to the HSAB principle and on the nature of the electronic interaction of the PNP-crown substituent with the N(3)P(3) ring, the latter determining the extent of regiocontrol to either one or two macrocyclic chloride functions. The increase of the extent of regioselectivity of gem-substitution at the PNP-macrocycle when passing from arylate to beta-dicarbonyl enolate oxy anions is related to the observed trends in the (31)P NMR chemical shifts and refers to the electronic structure of the oxy substituent.

Structure-property relationships of a tetrapyrrolidinyl PNP-lariat ether and its complexes with potassium, sodium, and silver cations.

Spectral properties of the tetrapyrrolidinyl PNP-lariat ether, L, and its complexes with K+, Na+, and Ag+ were investigated. Crystal structures of L and its complex with potassium iodide [KL]I were determined. Protonation constants of the ligand and formation constants of its complexes with Ag+, K+, and Na+ in aqueous solutions were determined. 1H NMR, 31P NMR, FTIR, and ESIMS spectra as well as potentiometric measurements indicate that complexation of Ag+ involves participation of the polyether oxygen donors and the endocyclic nitrogen atom of the cyclophosphazene ring. On the other hand, for complexation of K+ and Na+, only polyether oxygen atoms are involved. The latter conclusion is confirmed in the solid-state structure of the [KL]I complex.

C-Terminal glycine is crucial for hyperalgesic activity of nociceptin/orphanin FQ-(1-6).

A C-terminal analog of the hexapeptide orphanin FQ/nociceptin-(1-6), [Ala(6)]-orphanin FQ/nociceptin-(1-6), and a pentapeptide orphanin FQ/nociceptin-(1-5) were tested in vivo for their analgesic/hyperalgesic activity in the hot-plate test with rats. Replacement of the C-terminal glycine by L-alanine (Phe-Gly-Gly-Phe-Thr-Ala) in orphanin FQ/nociceptin-(1-6) abolished the hyperalgesic potency of native orphanin FQ/nociceptin-(1-6) (Phe-Gly-Gly-Phe-Thr-Gly), but analgesic activity was retained and was diminished by naloxone. Removal of the C-terminal amino acid (glycine or alanine) from orphanin FQ/nociceptin-(1-6) caused a significant loss of analgesic activity. It is anticipated that glycine plays a crucial role in the biphasic activity of orphanin FQ/nociceptin-(1-6). This may suggest the existence of a mechanism for terminating the biological action of orphanin FQ/nociceptin.

Antinociceptive effect produced by intracerebroventricularly administered dynorphin A is potentiated by p-hydroxymercuribenzoate or phosphoramidon in the mouse formalin test.

The antinociceptive effects of intracerebroventricularly (i.c.v.) administered dynorphin A, an endogenous agonist for kappa-opioid receptors, in combination with various protease inhibitors were examined using the mouse formalin test in order to clarify the nature of the proteases involved in the degradation of dynorphin A in the mouse brain. When administered i.c.v. 15 min before the injection of 2% formalin solution into the dorsal surface of a hindpaw, 1-4 nmol dynorphin A produced a dose-dependent reduction of the nociceptive behavioral response consisting of licking and biting of the injected paw during both the first (0-5 min) and second (10-30 min) phases. When co-administered with p-hydroxymercuribenzoate (PHMB), a cysteine protease inhibitor, dynorphin A at the subthreshold dose of 0.5 nmol significantly produced an antinociceptive effect during the second phase. This effect was significantly antagonized by nor-binaltorphimine, a selective kappa-opioid receptor antagonist, but not by naltrindole, a selective delta-opioid receptor antagonist. At the same dose of 0.5 nmol, dynorphin A in combination with phosphoramidon, an endopeptidase 24.11 inhibitor, produced a significant antinociceptive effect during both phases. The antinociceptive effect was significantly antagonized by naltrindole, but not by nor-binaltorphimine. Phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, bestatin, a general aminopeptidase inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, were all inactive. The degradation of dynorphin A by mouse brain extracts in vitro was significantly inhibited only by the cysteine protease inhibitors PHMB and N-ethylmaleimide, but not by PMSF, phosphoramidon, bestatin or captopril. The present results indicate that cysteine proteases as well as endopeptidase 24.11 are involved in two steps in the degradation of dynorphin A in the mouse brain, and that phosphoramidon inhibits the degradation of intermediary delta-opioid receptor active fragments enkephalins which are formed from dynorphin A.

Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I.

Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile2-Pro3 Pro3-Pro4 and Val6-Pro7 replaced by their tetrazole analogues Ile2-psi[CN4]-Ala3', Pro3-psi[CN4]-Ala4 and Val6-psi[CN4]-Ala7 were synthesized by the solid phase peptide synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation test (LPT).

Hematological and antifungal properties of temporin A and a cecropin A-temporin A hybrid.

Temporin A (TA) and a cecropin A-temporin A hybrid peptide (CATA) were synthesized and assayed for their hemolytic, anticoagulant, and antifungal properties. CATA retains significant antifungal activity, is less hemolytic than TA, and inhibits blood coagulation. These results recommend further studies of the biological activities of CATA.

Determination of nociceptin-orphanin FQ metabolites by capillary LC-MS.

Nociceptin-orphanin FQ (OFQ/N) is a newly discovered peptide involved in pain transmission. The method is described to identify metabolic pathway of this neuropeptide in the spinal cord of rats using capillary size-exclusion liquid chromatography coupled to electrospray ionization mass spectrometry. The applied technique is rapid and selective, and allows for simultaneous measurement and quantitation of several fragments in the incubation mixture.

Antibacterial activities of temporin A analogs.

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and -resistant Staphylococcus aureus as well as vancomycin-resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N-terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all-D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non-chiral interactions with membrane lipids.

Orphanin FQ/nociceptin inhibits morphine withdrawal.

The influence of orphanin FQ/nociceptin (OFQ/N) on the morphine-withdrawal symptom was investigated. Withdrawal syndrome was induced in the morphine-dependent rats by an intraperitoneal (i.p.) injection of 2 mg/kg naloxone hydrochloride--an opioid receptors antagonist. Wet-dog shakes were used as a measure of the abstinence syndrome. Intraventricular injections of OFQ/N (5-20 microg/animal) caused significant inhibition of the withdrawal signs at doses between 15-20 microg, in the morphine-dependent rats. OFQ/N alone did not change behavior of the morphine-dependent animals. The obtained results indicate that OFQ/N can inhibit the morphine withdrawal symptoms induced by naloxone.

Antimicrobial peptides derived from heme-containing proteins: hemocidins.

Deprived of heme and partially unfolded hemoglobin, myoglobin and cytochrome c display microbicidal activity against a broad spectrum of microorganisms with half maximal lethal dose estimated at micromolar concentrations. The intact proteins were ineffective. Antibacterial activity of these apohemoproteins was also sustained after digestion to approximately 50 amino acids long peptides but further fragmentation abolished microbicidal properties. The most active fragment of apomyoglobin (corresponding to 56-131 region) showed a pronounced effect on the E. coli membrane permeabilization and its action was sensitive to salt as well as to divalent cations concentrations. The membrane-directed effect was specific toward bacteria but no lipopolysaccharide binding properties were observed. No hemolytic properties, even at high peptide concentrations were found; however, a slight but dose-independent cytotoxic effect was observed on fibroblasts and hepatoma cells. The presented data suggest a 'carpet-like' mechanism of the membrane-directed activity and may result from exceptional abilities of hemoprotein-derived peptides to form alpha-helical structures. We postulate that the antimicrobial peptides obtained from the heme-containing proteins should be named hemocidins, in contrast to, e.g., hemorphins displaying opioid-like activity.

Electrospray ionization tandem mass spectrometry for poly(propylene oxide) starting and end group analysis

Electrospray ionization tandem mass spectrometry was applied to study poly(propylene oxide) obtained in the presence of potassium hydride. It was found that the polyether chains possess different starting groups, i.e. those situated at the beginning of the polymer backbone. These were isopropoxy, hydroxy, methoxy and allyloxy groups when the polymerization was stopped by the addition of methyl iodide. Each macromolecule was terminated by the methoxy group in this case. Copyright 1999 John Wiley & Sons, Ltd.