György Orosz

Genetic study of glutathione accumulation during cold hardening in wheat

Abstract. The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots of wheat (Triticum aestivum L.) cv. Cheyenne (Ch, frost-tolerant) and cv. Chinese Spring (CS, moderately frost-sensitive), in a T. spelta L. accession (Tsp, frost-sensitive) and in chro- mosome substitution lines CS (Ch 5A) and CS (Tsp 5A). The fast induction of total glutathione accumulation was detected during the first 3 d of hardening in the shoots, especially in the frost-tolerant Ch and CS (Ch 5A). This observation was corroborated by the study of de novo GSH synthesis using [35S]sulfate. In Ch and CS (Ch 5A) the total cysteine, γ-glutamylcysteine (precursors of GSH), hydroxymethylglutathione and GSH contents were greater during the 51-d treatment than in the sensitive genotypes. After 35 d hardening, when the maximum frost tolerance was observed, greater ratios of reduced to oxidised hydroxymethylglutathione and glutathione were detected in Ch and CS (Ch 5A) compared to the sensitive genotypes. A correspondingly greater glutathione reductase (EC 1.6.4.2) activity was also found in Ch and CS (Ch 5A). It can be assumed that chromosome 5A of wheat has an influence on GSH accumulation and on the ratio of reduced to oxidised glutathione as part of a complex regulatory function during hardening. Consequently, GSH may contribute to the enhancement of frost tolerance in wheat.

New strategy for the synthesis of large peptides as applied to the C-terminal cysteine-rich 41 amino acid fragment of the mouse agouti protein

Abstract In this new convergent peptide synthetic approach the advantages of the Fmoc and Boc techniques are combined. Boc-protected peptide fragments prepared on 2-chlorotrityl resin by Fmoc-technique were coupled sequentially in solution. As an example, the C-terminal (91–131) part of the agouti protein comprising all the 5 disulfide bridges in the original molecule has been synthesized by this method.

Nociceptin antagonism: probing the receptor by N-acetyl oligopeptides

In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra- and pentapeptides selective for the kappa-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [(35)S]GTPgammaS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (K(e)) of 2.44 nM, and no agonist effect at 10 microM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the delta-opioid receptor agonist [D-Ala(2),D-Leu(5)]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [(3)H]nociceptin-NH(2) binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [(35)S]GTPgammaS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N- and the C-terminal region of the molecule.

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91–131) (AP91–131) at the melanocortin type-1 receptor (MC1-R) were assessed using B16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91–131 was about 3-fold less potent than the natural agonist α-melanocyte-stimulating hormone (α-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-α-MSH (Ki 6.5±0.8 nmol/l). α-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP91–131; the IC50 values for AP91–131 in the two assay systems were 91±22 nM and 95±15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91–131 with IC50 values of 9.6±1.8 nM and 5.0±2.4 nM, respectively. This indicates inverse agonist activity of AP91–131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91–131 in the adenylate cyclase and melanin assays. On the other hand, AP91–131 inhibited cell growth similar to α-MSH (IC50 11.0±2.1 nM; maximal inhibition 1.8-fold higher than that of α-MSH). Furthermore, MC1-R was down-regulated by AP91–131 with about the same potency and time-course as with α-MSH. These results demonstrate that AP91–131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different α-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.

Simple and efficient synthesis of 2-chlorotritylchloride resin

Abstract The laboratory synthesis of 2-chlorotritylchloride polystyrene resin was described. Polystyrene was acylated with 2-chlorobenzoyl chloride, reacted with phenyl lithium and finally converted to trityl chloride in the presence of dimethyl sulfoxide and trimethylsilyl chloride. A process for the regeneration of the resin is described.

New opioid affinity labels containing maleoyl moiety

Opioid receptor binding properties and pharmacological profiles of novel peptides containing maleoyl function were determined in order to develop new affinity labels. Based on the enkephalin structure peptide ligands were synthesized and tested. Both in in vitro receptor binding experiments and pharmacological studies, all ligands showed agonist character with relatively high affinity (Ki values in the nanomolar range) and good to moderate selectivity. Replacement of Gly2 in the enkephalin frame with D-Ala led to higher affinities with a small decrease in selectivity. The longer peptide chains resulted in compounds with high percentage (up to 86%) of irreversible binding. The selectivity pattern of the ligands is in good agreement with the data obtained from the pharmacological assays (guinea pig ileum and mouse vas deferens bioassays). The newly synthesized peptides could be used in further studies in order to determine more detailed characteristics of the ligand-receptor interaction.

Irreversible labelling of the opioid receptors by a melphalan-substituted [Met5]enkephalin-Arg-Phe derivative

[Met5]enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe) was modified with the methyl esther of melphalan (Mel; 4-bis(2-chloroethyl)amino-L-phenylalanine) and the resulting compounds were studied for their opioid binding properties in guinea pig and rat brain membranes. Three new peptides, with a substitution of a single amino acid, were synthesized (Mel-Gly-Gly-Phe-Met-Arg-Phe, Tyr-Gly-Gly-Mel-Met-Arg-Phe and Tyr-Gly-Gly-Phe-Met-Arg-Mel). In the rat brain, none of these ligands displayed any type specificity, whereas in guinea pig brain membranes the C-terminally modified peptide, Tyr-Gly-Gly-Phe-Met-Arg-Mel ([Mel7]peptide), displayed a kappa-binding profile and was a weak kappa-opioid-receptor agonist in isolated guinea pig ileum. The effect of sodium ions on [Mel7]peptide competition against [3H]naloxone binding indicated a weak agonist nature of the compound. When guinea pig brain membranes were preincubated with 1-10 microM of [Mel7]peptide, an apparently irreversible inhibition of [3H]naloxone ligand binding was observed. These results suggest that the heptapeptide containing melphalan at the C-terminus can be used as a relatively high-affinity irreversible label for the kappa-opioid receptor.

Synergism Between Agouti Protein Fragment 91–131 and Melanin Concentrating Hormone

Murine agouti protein (AP) is a 131-amino acid cysteine-rich peptide responsible for the induction of phaeomelanin production in melanocytes [1]. Overexpression and ubiquitous secretion of AP in mice carrying the lethal yellow Ay mutation leads to a bright yellow coat and obesity [1]. AP antagonizes α-melanocyte-stimulating hormone (α-MSH) at the melanocortin-1 receptor (MC1-R) and hence inhibits MSH-induced cAMP formation and melanin synthesis. AP was also shown to exhibit some MSH-agonist effects (e.g. receptor down-regulation) and to display the characteristics of an inverse agonist [2]. The finding that the C-terminal cysteine-rich fragment of AP is sufficient for a-MSH antagonism and the observation that AP evokes biological effects independent from α-MSH antagonism have prompted us to study the biological characteristics of a C-terminal AP fragment in assays typical for both types of action of AP. We have selected the mouse AP91_131 fragment which does not contain Val83, a crucial residue for the bioactivity of native AP. Also, we were interested in whether melanin-concentrating hormone (MCH), a physiological antagonist of a-MSH and inducer of food intake [3], exerts any synergistic effects on AP91_131 action. In a previous report, the strategy of the chemical synthesis of AP91_131 had been described [4]. Here we report the biological data obtained with this AP fragment. The salient finding is that the C-terminal part of AP mediates both antagonist and agonist activities, and that MCH potentiates the antagonist activity of AP91_131.

Novel, receptor-type selective tetra- and pentapeptide opioid antagonists, related to a natural sequence found in snake venom

The BOC-protected Tyr-Lys-Lys-Trp-Trp-NH 2 peptide, the original pentapeptide sequence found in the venom of Philippine cobra, and its partially modified derivatives showed significant μ opioid receptor antagonist activity in both the mouse vas deferens and guinea-pig ileum bioassays. Neither the N-protected, nor the de-blocked peptides had any appreciable agonist activity. No antagonist activity was detected after the removal of the N-terminal protecting group. The Tyr can be replaced by Phe with minor loss of activity. Omitting one lysine from the sequence (BOC-Tyr-Lys-Trp-Trp-NH 2 ) gave an antagonist structure with increased κ selectivity