Analysis of tyrosine phosphorylation and phosphotyrosine-binding proteins in germinating seeds from Scots pine

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.

Human monstrosity: rape, ambiguity and performance in Rosemary’s Baby (Roman Polanski, 1968)

The narrative of Rosemary’s Baby hinges on a central hesitation between pregnancy induced madness and the existence of Satanism. Accordingly, the monstrous element is embodied in both the real and the supernatural: Rosemary’s husband Guy (John Cassavetes) is responsible for her victimisation through rape in either explanation. However, I will argue that the inherent ambiguity of the plot makes it difficult to place him as such a figure typical to the archetypal horror binaries of normality/monster, human/inhuman. By displacing generic convention the film complicates the issue of monstrosity, whilst simultaneously offering the possibility for the depiction of female experience of marriage to be at the centre of the narrative, for the real to be possibly of more significance than the supernatural. Previous writing has tended to concentrate on Rosemary and her pregnancy, so through detailed consideration of Cassavetes’ performance and its placement in the mise-en-scène this focus on Guy aims to demonstrate that he changes almost as much as Rosemary does. The chapter will focus on the film’s depiction of rape, during Rosemary’s nightmare and after it, in order to demonstrate how the notion of performance reveals Guy’s monstrousness and the difficulties this represents in our engagement with him.