Recently we have demonstrated the presence of calbindin D-28K both on the surface and in the core of aragonite but not calcite crystals in the P.sicula otoconia (Piscopo et al., 2004). The otoconia of frog consist of aragonite and calcite; while the saccule, lagena and endolimphatic sac contain aragonite crystals, the utricle contains calcite crystals. Immunoblot assays with monoclonal anti-calbindin D28K antibodies were performed on proteins extracted from utricle, saccule and lagena and endolimphatic sac in order to determine if also in frog this protein is present only in aragonite crystals. Moreover, calcite and aragonite otoconia were analyzed by CLSM using the same antibodies both after weak and strong decalcification. Besides the presence and distribution of calbindin in the sensorial epithelium of saccule, utricle, lagena epithelium and in the endolimphatic sac were also studied. After weak decalcification (24-48 h in dH2O at 37 °C), fluorescence on the surface of the crystal is evident; while in those decalcified with 1N HCl for 2-3 min at room temperature, fluorescence is found in the central portion of the otoconia but only in the aragonite crystals. These data were confirmed by immunoblotting. Immunoreactivity was observed only in the sample containing the proteins extracted from otoconia of saccule and lagena. Moreover, CB-D28K immunoreactivity was present in all the structures of the inner ear unless in the saccule. Our observations confirm that calbindin is a component of aragonite but not calcite crystals and probably participates in the formation of otoconia and to their following phases of growth. These data can be correlated to the more specific function of calcareous reserve that aragonitic otoconia carry out in many classes of vertebrates. Thus, it is possible to hypothesize that calbindin can be involved both in the process of mineralization of otoconia and in their decalcification in the case otoconia is functioning as calcareous reserve.

Localization of Calbindin-D28K in the inner ear of the frog Rana esculenta.

AVALLONE, BICE;BALSAMO, GIUSEPPE;PISCOPO, MARINA;MARMO, FRANCESCO
2006

Abstract

Recently we have demonstrated the presence of calbindin D-28K both on the surface and in the core of aragonite but not calcite crystals in the P.sicula otoconia (Piscopo et al., 2004). The otoconia of frog consist of aragonite and calcite; while the saccule, lagena and endolimphatic sac contain aragonite crystals, the utricle contains calcite crystals. Immunoblot assays with monoclonal anti-calbindin D28K antibodies were performed on proteins extracted from utricle, saccule and lagena and endolimphatic sac in order to determine if also in frog this protein is present only in aragonite crystals. Moreover, calcite and aragonite otoconia were analyzed by CLSM using the same antibodies both after weak and strong decalcification. Besides the presence and distribution of calbindin in the sensorial epithelium of saccule, utricle, lagena epithelium and in the endolimphatic sac were also studied. After weak decalcification (24-48 h in dH2O at 37 °C), fluorescence on the surface of the crystal is evident; while in those decalcified with 1N HCl for 2-3 min at room temperature, fluorescence is found in the central portion of the otoconia but only in the aragonite crystals. These data were confirmed by immunoblotting. Immunoreactivity was observed only in the sample containing the proteins extracted from otoconia of saccule and lagena. Moreover, CB-D28K immunoreactivity was present in all the structures of the inner ear unless in the saccule. Our observations confirm that calbindin is a component of aragonite but not calcite crystals and probably participates in the formation of otoconia and to their following phases of growth. These data can be correlated to the more specific function of calcareous reserve that aragonitic otoconia carry out in many classes of vertebrates. Thus, it is possible to hypothesize that calbindin can be involved both in the process of mineralization of otoconia and in their decalcification in the case otoconia is functioning as calcareous reserve.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/307692
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