Partial cloning of neuronal nitric oxide synthase (nNOS) cDNA and regional distribution of nNOS mRNA in the central nervous system of the Nile tilapia Oreochromis niloticus.

A constitutive NOS complementary DNA (cDNA) was partially cloned by RT-PCR from the brain of a teleost, the Nile tilapia (Oreochromis niloticus), using degenerate primers against conserved regions of NOS. The predicted 206-long amino acid sequence showed a high degree of identity with other vertebrate neuronal NOS (nNOS) protein sequences. In addition, phylogenetic analysis revealed that Nile tilapia NOS clustered with other known nNOS. Using the coupled reaction of semi-quantitative RT-PCR and Southern blotting, the basal tissue expression pattern of the cloned nNOS gene was investigated in discrete areas of the central nervous system (CNS) and in the heart and skeletal muscle tissue. As revealed, expression of nNOS transcripts was detected in all the CNS regions examined, whereas nNOS gene was not expressed in the heart and skeletal muscle. The distribution pattern of nNOS gene expression showed the highest expression levels in the forebrain followed by the optic tectum, the brainstem and the spinal cord, whereas scarce expression was detected in the cerebellum. Cellular expression of nNOS mRNA was analyzed in the CNS by means of in situ hybridization. According to the RT-PCR results, most nNOS mRNA expressing neurons are localized in the telencephalon and diencephalon, whereas in the mesencephalic optic tectum, the brainstem and the spinal cord, nNOS mRNA expressing neurons are relatively more scattered. A very low hybridization signal was detected in the cerebellar cortex. These results suggest that NO is involved in numerous brain functions in teleosts

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized''. From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at similar to 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases