Che-Hui Kuo

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.

Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain

Abstract Low concentrations of L-ascorbic acid caused release of acetylcholine from isolated synaptic vesicles (rat, guinea-pig and rabbit) in the presence of 2mM ATP, 2 mM MgCl2 and 10−5 M CaCl. The half maximum effect was obtained with about 2 to 2.5 ωM L-ascorbic acid, and the effect was inhibited by addition of 1mM EGTA. The release of noradrenaline from rat synaptic vesicles was also enhanced by L-ascorbic acid, but the concentration for half maximal stimulation was about 20 ωM, indicating that noradrenaline release was less sensitive to L-ascorbic acid than acetylcholine release. The physiological function of L-ascorbic acid in the brain is discussed in relation to release of transmitters.

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.

Molecular Cloning and Characterization of Amida, a Novel Protein Which Interacts with a Neuron-specific Immediate Early Gene Product Arc, Contains Novel Nuclear Localization Signals, and Causes Cell Death in Cultured Cells

Amida was isolated by the yeast two-hybrid system as a novel protein which associated with Arc, a non-transcriptional immediate early gene specific to the brain. Amida was confirmed to be associated with Arc in vitro and in vivo. Amida shows no homology to known proteins. Amida is ubiquitously expressed, although it is abundant in the brain. A transfection study revealed that Amida was localized in the nucleus and after 72 h the transfected cells underwent apoptosis. Furthermore, we found two nuclear localization signals and a domain needed for interacting with Arc was encompassed by two nuclear localization signals. Co-transfection experiment with Amida and Arc suggested that Amida transported Arc into the nucleus and negatively regulated Amida-induced cell death. These results indicate that Arc together with Amida may modulate cell death in the brain.

Expression and Functional Analysis of a Novel Isoform of Gicerin, an Immunoglobulin Superfamily Cell Adhesion Molecule

We have cloned a novel cDNA of gicerin, a cell adhesion molecule belonging to the immunoglobulin superfamily. Both gicerin isoforms share the same extracellular domain, which has five immunoglobulin-like loop structures and a transmembrane domain as s-gicerin, but differ in the cytoplasmic tail domain. As the newly identified form has a larger cytoplasmic domain than the previously reported form, we refer to them as l-gicerin and s-gicerin, respectively. l-gicerin is transcribed from a distinct mRNA containing an inserted sequence not found in s-gicerin mRNA which caused a frameshift for the coding region for a cytoplasmic domain. Previous studies demonstrated that gicerin showed a doublet band of 82 and 90 kDa in chicken gizzard smooth muscle. We report that the 82-kDa protein corresponds to s-gicerin and the 90-kDa protein to l-gicerin. We also found that the two gicerin isoforms are expressed differentially in the developing nervous system. Functional analysis of these gicerin isoforms in stable transfectants revealed that they had differ in their homophilic adhesion properties, as well as in heterophilic cell adhesion assayed with neurite outgrowth factor. In addition, these isoforms have neurite-promoting activity by their homophilic adhesion, but differ in their ability to promote neurite outgrowth.

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.

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.

Involvement of a single-stranded DNA binding protein, ssCRE-BP/Purα, in morphine dependence

We have purified a nuclear protein from mouse cerebella that binds to single‐stranded oligo‐DNA of cAMP response element and is modulated by morphine treatment. Isolation of the cDNA clone showed that the nuclear protein (ssCRE‐BP) was identical to Purα, a DNA binding protein for single‐stranded purine‐rich sequences that was originally isolated as a replication factor. ssCRE‐BP/Purα and mRNA were abundant in the brain. The levels of ssCRE‐BP/Purα and the transcript were not changed by chronic morphine treatment, however, the levels of an activator of ssCRE‐BP/Purα, which is necessary for the DNA binding, may be modulated by the treatment.

Ca2+/Calmodulin‐Dependent Transcriptional Activation of Neuropeptide Y Gene Induced by Membrane Depolarization: Determination of Ca2+‐ and Cyclic AMP/Phorbol 12‐Myristate 13‐Acetate‐Responsive Elements

Abstract: Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time‐dependently, without a change in β‐actin mRNA level, in NG108‐15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage‐dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin‐dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization‐induced transactivation was also blocked by CaM kinase inhibitors. The 200‐bp 5′‐upstream region (−344/−145) was localized as a Ca2+/calmodulin‐responsive element (CaMRE), which confers depolarization‐induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca‐responsive elements such as CRE, SRE, NF‐AT, or the C/EBPβ‐binding site and was separated from a 64‐bp cyclic AMP/phorbol 12‐myristate 13‐acetate‐responsive element (−144/−81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L‐type Ca channels.