Presentation on theme: "MODO (MODel Organisms) fellowship Tutor: Doct. Antonietta Spagnuolo Cellular and Developmental Biology Group Maria Salzano MODO meeting: 27 th of June."— Presentation transcript:
MODO (MODel Organisms) fellowship Tutor: Doct. Antonietta Spagnuolo Cellular and Developmental Biology Group Maria Salzano MODO meeting: 27 th of June 2013 Identification of thyroid hormon precursor(s) in Ciona intestinalis endostyles Satoh, 2003 Stazione Zoologica Anton Dohrn
Specific endostyle cells have been shown to concentrate radioiodine from sea water (Barrington, 1957), and to produce monoiodiotyrosine (MIT), diiodiotyrosine (DIT) and thyroxine (Roche et al., 1959); (Kennedy, 1966); (Dunn, 1974). Thyroid hormones play a critical role in amphibians [Furlow et al.,2006; Tata, 2005], urochordate [Patricolo et al., 2001], amphioxus [Paris and Laudet, 2008] and lamprey [Manzon et al., 2001] metamorphosis. Anatomical and molecular studies confirmed that a THYROID-LIKE FUNCTION lies within the ENDOSTYLES also of Urochordates and Cephalochordates. (Ogasawara et al., 1999) The link between Endostyle and Thyroid in Ciona genome, no homologus of TG gene has been found PROJECT AIM Identification of the protein(s) used as scaffold for THs biosynthesis In vertebrates TG is the protein precursor of T3 and T4
Proteins are dried and digested (pronase) T3/T4 bound to protein/s analysis Endostyles total proteins extracts Proteins are removed (ice EtOH; -20°C) free T3/T4 analysis LC analysis on column Selected Reaction Monitoring mass spectroscopy analysis D V As negative control.. Preliminary results Free T4 * in collaboration with A. Amoresano’s group
Preliminary results (2) * in collaboration with A. Amoresano’s group Proteins are dried and digested (pronase) T3/T4 bound to Protein analysis PEPTIDE IDENTIFICATION Software for identification, characterisation and quantization of proteins using mass spectrometry data.
Endostyles TE IP anti T4-Ab Sup NB 75 Kda 50 Kda 37 Kda 25 Kda Endostylus : TE Endostyles proteins Total Extract IP Anti T4-Ab: Protein Immuno – Precipitated with anti T4-Ab Sup NB : Supernatant of proteins Not Ab - Bound * in collaboration with Prof. Pucci’s group * A parallel strategy… Endostyles total proteins extracts immunoprecipitated with anti T4-Ab
Next analysis Selected Reaction Monitoring mass spectroscopy analysis blood 2) What are the effects on T4 production in endostyles?? + KClO 4 1) Is there T4? Is T4 free or bound to proteins?