Search

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) consisting of alpha and beta isoforms is highly expressed in the central nervous system and is implicated in the regulation of various Ca(2+)-dependent physiological processes. We investigated the immunohistochemical distribution of the alpha and beta isoforms of this enzyme in the rat retina, using highly specific monoclonal antibodies which recognize each isoform. Immunoblotting revealed that not only the alpha but also the beta isoform of CaM kinase II were expressed in the retina. The immunohistochemical study showed that highly alpha-immunoreactive products were localized in amacrine cells in the inner nuclear layer and displaced amacrine cells and ganglion cells in the ganglion cell layer. In addition, two well-defined bands within the inner plexiform layer were densely stained with the anti-alpha antibody. By contrast, immunoreactivity against the anti-beta antibody was very weak in the same neuronal components of the retina. beta-Immunoreactive products were homogeneously distributed throughout the inner plexiform layer and no well-defined bands were detected in this layer. Glial cells such as Muller cells were immunoreactive neither to alpha nor beta antibody. A possible co-existence of choline acetyl transferase (ChAT) within CaM kinase II alpha-immunopositive neurons was examined by evaluating adjacent sections stained with anti-CaM kinase II alpha antibody and anti-ChAT antibody, respectively. The distribution of CaM kinase II alpha immunoreactivity in the rat retina was remarkably similar to that of ChAT immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzymatic and regulatory properties of Ca2+/calmodulin-dependent protein kinase in the postsynaptic density (mPSDp CaM kinase) of the rat forebrain was compared with those of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). mPSDp CaM kinase was different from soluble CaM kinase II in terms of substrate specificity, regulatory consequences and sites of autophosphorylation. Both soluble and PSD kinases generated Ca(2+)-independent activity by autophosphorylation and Ca(2+)-independent activity almost reached the maximum during the first minute of autophosphorylation. Ca(2+)-independent activity of mPSDp CaM kinase was more stable than that of the soluble kinase under autophosphorylating conditions. Autophosphorylation of the kinases decreased the mobility of the kinases on SDS-polyacrylamide gels. The mobility shift and determination of 32P phosphate incorporation into the kinases demonstrated that there were three species in mPSDp CaM kinase alpha isoform: two active forms with and without the mobility shift (about 22 and 19%, respectively), and an inactive form (about 59%). However, there was only one species in the soluble kinase alpha isoform, which was active. The maximum incorporation of 32P phosphate into mPSDp CaM kinase alpha isoform was less than that of the soluble kinase. Tryptic peptide analysis indicated that the phosphorylation sites of mPSDp CaM kinase alpha isoform differed from those of the soluble kinase.

(1) オキアミの脂質は,その脂肪酸組成から予想されたよりも酸化の進行が遅い。(2) その酸化パターンの最も特異的なことは過酸化物の蓄積が全く認められないことである。(3) リノール酸にオキアミの脂質を添加すると過酸化物の生成が抑えられることにより,オキアミの脂質には抗酸化性物質が存在することが明らかになった。(4) トコフェロールが200～350μg/g・oil含まれているが,(3)で認めた抗酸化作用には主要な役割を果していないようである。(5) ケイ酸カラムで分画したところメタノール画分に抗酸化力が認められた。

A protein with MW approximately 350 k daltons and pI approximately 5.5, which was deleted in the dystrophic mouse (C57BL/10ScSn-mdx), was detected on two-dimensional gel electrophoresis with silver staining. Deletion of this protein was uniformly observed in the dystrophic mouse extensor digitus longus, soleus and cardiac muscle. This protein specifically reacted with the monoclonal antibody against the chemically synthesized N-terminal fragment of human dystrophin. The protein reacting with this monoclonal antibody was also detected in rabbit back-muscle, rat extensor digitus longus and human skeletal muscle at the same position as the mouse muscle protein, on the two-dimensional gel electrophoresis. Our results show that dystrophin is solubilized in 8M guanidine HCl and that the modified two-dimensional gel electrophoresis can be applied to separate dystrophin.

The inhibitory effects of 5-chloro-7-iodo-8-hydroxy-quinoline (clioquinol), methylmercuric chloride and 4-hydroxyaminoquinoline-N-oxide(4-HAQO) on DNA, RNA and protein syntheses in the neonatal rat superior cervical ganglion (SCG) were studied in relation to the action of mouse 2.5S nerve growth factor (NGF), using organ cultures. RNA and protein syntheses in SCG were stimulated approximately 3- and 2-fold, respectively, by NGF (1 microgram/ml), but the DNA synthesis was only slightly or not at all stimulated. Methylmercuric chloride and 4-HAQO dose-dependently inhibited DNA, RNA and protein syntheses, either in the presence or in the absence of NGF. On the other hand, clioquinol (up to 100 microM) slightly or not at all inhibited RNA synthesis in the absence of NGF; however, it did abolish the NGF-induced stimulation of RNA synthesis in the presence of NGF. The DNA and protein syntheses were dose-dependently inhibited by clioquinol, either in the presence or in the absence of NGF. We conclude from this study that the interaction between clioquinol and the functions of NGF raises the question of a possible toxicity of the drug on specific neurons.

To investigate molecular mechanisms involved in the neurotoxicity of clioquinol (5-chloro-7-iodo-8-hydroxyquinoline), the inhibitory effects of this drug on DNA, RNA and protein syntheses were examined, in relation to the action of nerve growth factor (2.5S NGF). We used an organ culture of neonatal rat superior cervical ganglion (SCG). Ten microM clioquinol inhibited completely DNA and protein syntheses and abolished the stimulatory effect of NGF on RNA synthesis. With regard to the chemical structure of clioquinol, hydroxylation at the 8th carbon of quinoline is essential for the inhibition of DNA, RNA and protein syntheses, and the hydrophobicity of the 8-HQ derivatives is a required property for potent inhibition. Compared with effects of EDTA, alizarine, sodium alizarine sulfate, o-phenanthroline and alpha,alpha'-dipyridyl, the loss of the NGF-induced stimulation of RNA synthesis by clioquinol does not seem to be primarily caused by its metal-chelating property. Clioquinol did not significantly alter the uptake rate of thymidine, uridine and leucine, thereby suggesting that the primary action of clioquinol on inhibition of DNA, RNA and protein syntheses does not relate to uptake of the precursor into SCG. Clioquinol did not significantly alter the degradation of 3H-uridine-labeled RNA. NGF suppressed the degradation of RNA and this suppression was overcome by clioquinol. The release of free uridine from SCG into the culture medium was enhanced by clioquinol and was partially suppressed by NGF. The inhibitory effects of clioquinol were completely prevented by bovine serum albumin (BSA), but not by NGF even at a 5-fold concentration of clioquinol.(ABSTRACT TRUNCATED AT 250 WORDS)