Wei Cui1, Gang Liu, Ruiwen Liang. 1. Department of Parasitology, Weifang Medical College, Weifang 261042, China.
Abstract
OBJECTIVE: To demonstrate the molecular expression of carbonic anhydrase IV (CA IV) in rabbit corneal endothelium. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using cultured and fresh rabbit corneal endothelial total RNA and specific primers for CA IV. The RT-PCR product was subcloned and sequenced. Immunoblotting and indirect immunofluorescence staining were performed to detect protein expression and distribution of CA IV using fresh and cultured rabbit corneal endothelium and rat anti-CA IV polyclonal antibody. RESULTS: RT-PCR screening gave positive bands at the predicted size for CA IV from fresh and cultured rabbit corneal endothelium. Sequencing further confirmed the identity of CA IV in corneal endothelium. Immunoblotting analysis showed a single band at 52 kDa for freshly isolated and cultured endothelial cells. Indirect immunofluorescence staining revealed an apparent positive staining in cultured endothelial cells. CONCLUSION: Carbonic anhydrase IV is expressed in rabbit corneal endothelium, which could contribute to the transendothelial HCO(3)(-) flux that is necessary to maintain corneal hydration and transparency.
OBJECTIVE: To demonstrate the molecular expression of carbonic anhydrase IV (CA IV) in rabbit corneal endothelium. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using cultured and fresh rabbit corneal endothelial total RNA and specific primers for CA IV. The RT-PCR product was subcloned and sequenced. Immunoblotting and indirect immunofluorescence staining were performed to detect protein expression and distribution of CA IV using fresh and cultured rabbit corneal endothelium and rat anti-CA IV polyclonal antibody. RESULTS: RT-PCR screening gave positive bands at the predicted size for CA IV from fresh and cultured rabbit corneal endothelium. Sequencing further confirmed the identity of CA IV in corneal endothelium. Immunoblotting analysis showed a single band at 52 kDa for freshly isolated and cultured endothelial cells. Indirect immunofluorescence staining revealed an apparent positive staining in cultured endothelial cells. CONCLUSION:Carbonic anhydrase IV is expressed in rabbit corneal endothelium, which could contribute to the transendothelial HCO(3)(-) flux that is necessary to maintain corneal hydration and transparency.