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WP6: Cytoskeletal proteins – one year in.

WP6: Cytoskeletal proteins – one year in. Susan M. Trigwell and Paul T. Lynch, University of Derby, U.K. Alan J. Hargreaves and Philip L.R. Bonner, Nottingham Trent University, U.K.

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WP6: Cytoskeletal proteins – one year in.

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  1. WP6: Cytoskeletal proteins – one year in. Susan M. Trigwelland Paul T. Lynch, University of Derby, U.K. Alan J. Hargreaves and Philip L.R. Bonner, Nottingham Trent University, U.K.

  2. Earlier collaborative work showed link between cytoskeletal protein modification and cryopreservation pretreatment Work of other groups has linked the stability of microtubules and actin filaments with post-thaw recovery Why look at cytoskeletal proteins?

  3. Develop methods to correlate post-thaw tissue regrowth with: cytoskeletal protein modification and stability transglutaminase activity Year One objectives:

  4. Garlic (Allium sativum L.) stem discs Olive (Olea europaea L.) somatic embryos Olive (Olea europaea L.) shoot tips Tissue types

  5. In vitro transglutaminase assays

  6. Transglutaminase assay development Biotin-cadaverine incorporation assay

  7. Biotin-cadaverine incorporation assay COLOUR REACTION -cadaverine extravidin- HRPO detection -cadaverine TG N, N-dimethyl casein substrate

  8. Effect of garlic extract on GPL TG biotin-cadaverine incorporating activity

  9. Effect of olive extract on GPL TG biotin-cadaverine incorporating activity

  10. Small-scale semi-purification of protein extracts

  11. Biotin-cadaverine incorporation assay of semi-purified garlic extract

  12. Biotin-cadaverine incorporation assay of semi-purified olive extract

  13. GPL TG activity inhibited by crude garlic extract biotin-cadaverine incorporation assay crude olive extract crude or semi-purified garlic extract Summary of biotin-cadaverine incorporation assay optimisation

  14. Transglutaminase assay development Peptide crosslinking assay

  15. In vitro transglutaminase assays

  16. Peptide crosslinking assay -TVQQEL -TVQQEL COLOUR REACTION extravidin- HRPO detection TG EDC-modified casein substrate

  17. Effect of garlic extract on GPL TG peptide crosslinking activity

  18. Effect of olive extract on GPL TG peptide crosslinking activity

  19. Small-scale semi-purification of protein extracts

  20. Peptide crosslinking assay of semi-purified garlic extract

  21. Peptide crosslinking assay of semi-purified olive extract

  22. GPL TG peptide crosslinking activity inhibited by crude garlic extract peptide crosslinking assay crude or semi-purified olive extract crude or semi-purified garlic extract Summary of peptide crosslinking assay optimisation

  23. Immunoprobing of garlic extract ‘dots’ with antibody recognising the ‘crosslink’. This allows in vivo TG activity occurring during cryopreservation to be quantified. Alternative assay of TG activity in garlic stem-disc extracts Y anti-mouse Ig-AP Y anti-CL

  24. Plant species under investigation: - apple - almond Transglutaminase activity measured by: - [3H]-putrescine incorporation - in vivo TG assay of Prof. S. Beninati Contribution of Partner 5 to the WP6 workpackage

  25. Develop methods to correlate post-thaw tissue regrowth with: transglutaminase activity cytoskeletal protein modification and stability Year One objectives:

  26. Pretreatment of garlic stem-discs for analysis

  27. Garlic stem-disc cryopreservation procedure

  28. Different pretreatments of garlicstem-discs * 0.09M

  29. Probing in vivo cytoskeletal protein modification

  30. Cytoskeletal protein modification in treated garlic stem-disc extracts

  31. 250 250 160 160 105 105 75 75 50 50 Immunoprobed blots of treated garlic stem-disc extracts. T1 T2 T3 T4 M T1 T2 T3 T4 M anti-tyrosinated tubulin anti-a tubulin

  32. 250 250 160 160 105 105 75 75 50 50 Immunoprobed blots of treated garlic stem-disc extracts. T1 T2 T3 T4 M T1 T2 T3 T4 M anti-acetylated tubulin anti-transglutaminase- mediated crosslink

  33. no encapsulation: - increase in proteolysis of a-tubulin - additional transglutaminase-mediated crosslinked proteins no sucrose pretreatment: - increase in high molecular weight (transglutaminase-crosslinked?) tyrosinated and acetylated proteins Summary of correlation between garlic stem-disc pretreatment and protein modification

  34. Develop methods to correlate post-thaw tissue regrowth with: transglutaminase activity - by biotin-cadaverine incorporation assay - by peptide crosslinking assay - by immunoprobing of protein blots - by dot blot Attainment of Year One objectives:

  35. Develop methods to correlate post-thaw tissue regrowth with: cytoskeletal protein modification and stability - by immunoprobing of protein blots - by immunofluorescent probing of fixed tissue Attainment of Year One objectives:

  36. Complete method development Analyse extracts from each stage of the cryopreservation procedure of garlic stem-disc, olive somatic embryo and olive shoot-tip explants Integrate treated apple and almond explant extracts from P5 (Dr. Carmine Damiano) into the assays Plans for Year Two

  37. WP1 – water thermal behaviour WP3 – sugars WP4 – membrane components WP7 – oxidative stress Contribution of P3 to other work packages

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