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Specific Detection of Type III Iodothyronine Deiodinase Protein in Chicken Cerebellar Purkinje Cells

Laboratory of Comparative Endocrinology, K. U. Leuven, Zoological Institute (C.H.J.V., K.V., T.S., E.R.K., S.V.d.G., V.M.D.), B-3000 Leuven, Belgium; Laboratory of Endocrinology, Academic Medical Center (O.B., B.Z.D.), 1105 AZ Amsterdam, The Netherlands; and Department of Cellular and Molecular Physiology, University of Massachusetts Medical Center (J.L.B.), Worcester, Massachusetts 01655

Address all correspondence and requests for reprints to: Dr. Carla H. J. Verhoelst, Laboratory of Comparative Endocrinology, Zoological Institute, K. U. Leuven, Naamsestraat 61, B-3000 Leuven, Belgium. E-mail: . carla.verhoelst{at}bio.kuleuven.ac.be

Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T₃ concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T₃ levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western blot analysis, a band of the expected molecular weight (30 kDa) could be detected in both tissues. Using a full-length ³²P-labeled type III deiodinase cRNA probe, we identified a single mRNA species in the cerebellum that was of the exact same size as the hepatic control mRNA (±2.4 kb). RT-PCR, followed by subcloning and sequence analysis, confirmed the expression of D3 mRNA in the chicken cerebellum. In this study we provide the first evidence of the presence of the D3 protein in a neuronal cell type, namely Purkinje cells, by means of immunocytochemical staining. We were able to detect a protein fragment corresponding to the expected molecular mass (30 kDa) for type III deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot analysis confirmed the presence of D3 mRNA in the cerebellum.

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