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Protein tyrosine phosphorylation in angiosperms has been implicated in various physiological processes, including seed development and germination. In conifers, the role of tyrosine phosphorylation and the mechanisms of its regulation are yet to be investigated. In this study, we examined the profile of protein tyrosine phosphorylation in Scots pine seeds at different stages of germination. We detected extensive protein tyrosine phosphorylation in extracts from Scots pine (Pinus sylvestris L.) dormant seeds. In addition, the pattern of tyrosine phosphorylation was found to change significantly during seed germination, especially at earlier stages of post-imbibition which coincides with the initiation of cell division, and during the period of intensive elongation of hypocotyls. To better understand the molecular mechanisms of phosphotyrosine signaling, we employed affinity purification and mass spectrometry for the identification of pTyr-binding proteins from the extracts of Scots pine seedlings. Using this approach, we purified two proteins of 10 and 43 kDa, which interacted specifically with pTyr-Sepharose and were identified by mass spectrometry as P. sylvestris defensin 1 (PsDef1) and aldose 1-epimerase (EC:5.1.3.3), respectively. Additionally, we demonstrated that both endogenous and recombinant PsDef1 specifically interact with pTyr-Sepharose, but not Tyr-beads. As the affinity purification approach did not reveal the presence of proteins with known pTyr binding domains (SH2, PTB and C2), we suggest that plants may have evolved a different mode of pTyr recognition, which yet remains to be uncovered., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Plants produce a variety of molecules to defend themselves from fungal pathogens. Defensins belong to the family of antimicrobial peptides that play a central role in innate immunity in all species of plants. We have previously reported the purification of antimicrobial peptides from Scots pine seedlings and the identification of some of them, including defensin, by mass spectrometry. In this study, we extend our original study on molecular cloning of Pinus sylvestris defensin 1 (PsDef1) by presenting the expression and affinity purification of recombinant defensin 1 (rPsDef1). The full-length coding sequence of PsDef1 has an open reading frame capable to encode a protein of 83 amino residues, including a signal peptide of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5,601.6 Da. We have employed pET system to express mature form of PsDef1 fussed to GST. As GST-PsDef1 fusion protein was not biologically active, we removed GST moiety from the mature defensin 1 peptide by proteolytic cleavage with Factor Xa. The resulting rPsDef1 protein exhibited strong antifungal activity against a panel of pathogenic fungi which is comparable to that of endogenous Scots pine defensin 1. In addition, rPsDef1 was used to produce specific polyclonal antibodies. Using generated antibodies, we found that the level of PsDef1 is significantly increased in Scots pine seedlings during germination and in their response to pathogenic infection with Heterobasidion annosum.

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin 1 cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins.

6-glucuronidation of morphine confers to this widely used analgesic increased potencies both in terms of receptor binding (selectivity) and of antinociceptive properties. In binding studies, 6-glucuronidation of morphine results in increased mu/kappa selectivity (comparable to DAGO). Na+ ions and Gpp(NH)p inhibit similarly the binding of morphine and M6G at the mu sites, suggesting that M6G is a mu agonist. The agonist nature of M6G was further confirmed by its antinociceptive properties: M6G (i.c.v.) was considerably more potent than morphine in the writhing (45 fold) and in the tail-flick (61 fold) tests. Furthermore, M6G induced a longer lasting analgesic effect than morphine. These data strongly suggest that M6G may significantly contribute to the pharmacological activity of morphine.

Unlabelled: To specify pharmacological interferences between droperidol and fentanyl an experimental study was carried out on mice. The analgesic kinetic was studied after the administration of the two drugs in a concentration ratio between 25/1 and 150/1., Results: Fentanyl is a potent analgesic with a short time effect. It has very good correlations between dose effect and time effect. Droperidol does not have a analgesic effect. The behaviour of the two drugs depends on the way they are administered, either simultaneously or consecutively: simultaneous administration of the two drugs increases the level and duration of analgesia: nalaxone reverses analgesia, a fentanyl injection given 5 minutes before a droperidol injection produces the same analgesia as fentanyl alone, a droperidol injection given 5 minutes before a fentanyl injection induces a level of analgesia lower than fentanyl alone. Droperidol appears to reverse the fentanyl analgesia.