Naomasa Miki

Visinin: A novel calcium binding protein expressed in retinal cone cells

Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pI 5.1). To investigate its function, visinin cDNA was isolated from a chick retinal lambda gt11 cDNA library, using anti-visinin serum. The beta-galactosidase-visinin fusion protein was used for purifying epitope-selected antibody. The purified visinin antibody reacted only with a 24 kd protein in retinal cone cells. Visinin mRNA was expressed only in the retinal photoreceptor layer. The nucleotide sequence of the cDNA revealed that visinin has three E-F hand structures and is a Ca2+ binding protein. Visinin protein expressed in E. coli exhibited Ca2+ binding activity. These results suggest that visinin is a photoreceptor-specific Ca2+ binding protein and may be involved in phototransduction in the cone cells.

Immunohistochemical localization of chick retinal 24 kdalton protein (visinin) in various vertebrate retinae

Antiserum against a protein (24,000 daltons, visinin) of chick retina has been provided for immunohistochemical study on the localization of visinin in chick retinae during development, as well as in various vertebrate retinae. The photoreceptor cells were stained with anti-visinin serum from 7th day embryonic retinae and its intensity was gradually increased with embryonic age. In addition, visinin-like immunoreactivity was found in some kinds of amacrine and displaced amacrine cells from 11th-day embryonic retinae. When human, cat, frog and carp retinae which contain both rods and cones were examined, staining of cone cells was clearly observed in the photoreceptor cell layer, but not in the rods. Furthermore visinin-like immunoreactivity was barely detectable in the photoreceptor cells of bovine, rat and mouse retinae containing mostly rod cells. These results suggest that visinin is mainly located in the cone cells in various vertebrate retinae and is a good marker for the cone cells.

Stimulatory effect of taurine on Ca-uptake by disc membranes from photoreceptor cell outer segments

Abstract Effects of taurine and related compounds on Ca-uptake by the disc membranes prepared from dark-adapted frog retina were studied. Taurine stimulated ATP-dependent Ca-uptake and the turnover of 45Ca in the disc membranes without affecting basal activity, but it was not observed with the synaptic plasma membranes from rat brain. The stimulatory effect appears to be specific to taurine, since cysteine sulfinic acid, hypotaurine, isethionic acid, β-alanine and γ-aminobutyric acid (GABA) did not stimulate Ca-uptake. The maximal activation, observed at about 30 mM taurine, was about 3 fold, and the Km value for taurine was 10 mM. These results might suggest that taurine modifies translocation of Ca ion in the rod outer segment.

Immunohistochemical cross-reactivity and electrophoretic comigration between calbindin D-27 kDa and visinin

Calbindin D-27 kDa (previously named vitamin D-CaBP or cholecalcin) and visinin present similitude both for their purification procedure and histochemical localization. We systematically compared by histochemistry calbindin and visinin immunoreactive structures in chick and pigeon retina, in rat cerebellum and kidney and in pigeon cerebellum. The calbindin and visinin immunoreactive structures were identical except in the retina. Preabsorption of anti-visinin with purified chick or rat calbindin suppresses the labelling in every organ studied except in the photoreceptor layer of pigeon and chick retina. Such a persistence of labelling was explained by Western blotting analysis of chick-retina soluble proteins showing a pattern of 7 different proteins recognized by anti-visinin even though only one protein was recognized in rat kidney and cerebellum. Anti-visinin is thus a polyclonal antibody reacting with more than one antigen of the chick retina, one of those antigens being calbindin. Calbindin is the single antigen recognized by anti-visinin in the other tested organs. In conclusion, we present evidence that visinin is a calbindin.

Isolation of a novel retina-specific clone (MEKA cDNA) encoding a photoreceptor soluble protein

We have reported the isolation of clones which are candidates for retina-specific cDNAs. One of the cDNA clones, pCR-470, was further characterized. We found that mRNA corresponding to the pCR-470 was expressed only in the retina and encodes an unknown soluble protein whose molecular weight and pI are calculated to be 26,935 and 5.35, respectively. We designated it as a MEKA protein, because its amino acid sequence starts from M-E-K-A. It was found by in situ hybridization that MEKA mRNA was transcribed only in the photoreceptor cells and accumulated in the inner segments just like opsin mRNA. The MEKA cDNA was ligated with expression vector PEX 1, and a MEKA-fusion protein synthesized in E. coli was purified and used as an antigen. By the Western blot analysis anti-MEKA protein serum reacted with a soluble 32 kDa protein from bovine retina and 33 kDa for chick, but not with proteins from other tissues. Immunohistochemical study showed that anti-MEKA stained only the photoreceptor cells in bovine, chick, rat and mouse retinas.

Characterization of Chick Gizzard Extract That Promotes Neurite Outgrowth in Cultured Ciliary Neurons

Abstract: Chicken gizzard extract contains a macromolecule(s) that promotes the neurite outgrowth of dissociated neurons from the ciliary ganglia (CG) of chick embryos. The factor in gizzard extract was partially purified and estimated to be about 12S (M.W. 200,000‐300,000) on sucrose density gradient centrifugation. The neurite outgrowth of CG neurons by the factor strictly depends on the embryonal age. The maximal neurite outgrowth was observed when CG neurons were dissociated from the embryos younger than 10 days. After that time the response of CG neurons to the factor rapidly declined and was almost lost at day 14. The amount of factor in the gizzard began to increase rapidly from 12‐day‐old embryo and reached the maximal level at day 16, and thereafter a fairly steady level was maintained. When CG neurons were co‐cultured with rat myotubes, the ratio of muscle cells with synaptic responses (miniature end‐plate potentials) was significantly higher in the presence of gizzard factor than its absence. The results suggest that this factor acts as an external signal on CG neurons to form synaptic connections in vivo.

Molecular Cloning of cDNA to mRNA for a Cerebellar Spot 35 Protein

Abstract: The nucleotide sequence of mRNA for rat cerebellar spot 35 protein, a Ca‐binding protein, was determined from recombinant complementary DNA (cDNA) clones. The sequence was composed of 1, 714 base pairs (bp) which included the 783 bp of the complete coding region, the 130 bp of the 5′‐noncoding region, and the 801 bp of the 3‐noncoding region containing a polyadenylation signal. In addition, a polyadenylic acid [poly(A)] tail was also found. Because the size of spot 35 mRNA was estimated to be about 1,900 bases by Northern blot analysis, the longest insert was verified to contain a nearly full‐length cDNA sequence including the poly(A) tail. The amino acid sequence of the protein deduced from the nucleotide sequence contains 261 amino acids and at least five Ca‐binding domains. There was a high homology in the amino acid sequences (79%) and the nucleotide sequences (77%) between spot 35 protein and chick intestinal Ca‐binding protein (28K).

An immunohistochemical study on the river lamprey retina

The localization of structures immunoreactive to various polypeptides and proteins in the retina of the river lamprey (Lampetra japonica) was investigated by means of an indirect immunohistofluorescence method. In the majority of frozen sections, a subpopulation of amacrine cells showed the immunoreactivity (IR) to one of the examined antisera against corticotropin-releasing factor, glucagon, neuropeptide Y, somatostatin, visinin and 5-hydroxytryptamine, respectively. A few fibers in the inner plexiform layer were immunoreactive to cholecystokinin or substance P antiserum. The visinin-like IR was also found in two types of bipolar cells. The IR was positive in Müller cells against glutamine synthetase but not to glial fibrillary acidic protein. In some flat-mounted preparations, glucagon- and serotonin-like reactive amacrine cells and visinin-like immunoreactive bipolar cells (distally located) could be observed. The results obtained suggest that in the lamprey retina the cytoplasmic property of some cells is similar to, but that of others is different from, vertebrate retinal cells.

Presence of retina-specific proteins in the lamprey pineal complex.

The pineal complex of river lamprey reacted with the antisera raised against retina specific proteins including bovine opsin, chick visinin and frog light-sensitive cyclic GMP phosphodiesterase (PDE). Immunoreactive materials stained with anti-opsin were evenly located at the outer segment of photoreceptor cells in the pineal organ and also found in the parapineal organ. Although anti-visinin stained the pineal and parapineal photoreceptor cells, the immunopositive photoreceptor cells were observed only at the lateral portion and not at the medial portion of the pineal organ. No immunoreactive materials were found in the pineal complex by the anti-PDE, whereas the anti-PDE reacted with photoreceptor cells of the retinal tissue. The data suggest that the pineal and parapineal retinas of lamprey contain opsin- and visinin-like proteins with different distribution in their photoreceptor cell layer as found in the lamprey retinal tissue.

Analysis of a distinctive protein in chick retina during development

Soluble proteins from the chick retina were analyzed at various developmental stages by SDS-polyacrylamide gel electrophoresis. A peptide of about 24,000 daltons (24 Kd protein) appeared in the 14-day embryo and gradually increased with embryonic age, maintaining a fairly steady level after hatching. Polypeptides which correspond to actin and tubulin, however, remained almost unchanged during development. The 24 Kd protein was not detected in the cerebrum, tectum, pigment epithelium or vitreous body at any age. To characterize this protein, it was partially purified by gel filtration and ion exchange column chromatography, and its isoelectric point was measured. It was focused in a diffuse spot at about pH 5.5. In the bovine retina, a protein was observed at 24,000 daltons on SDS-polyacrylamide gel, but its isoelectric point was more basic than that of chick retina. It is suggested that the 24 Kd protein is one of the distinctive proteins that increase in concentration during the chick retinal development, and would be closely associated with retinal functions.