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Biomedical subjects

K Saijoh

Publications and source records attributed to K Saijoh.

49 records · Page 3Linked to original sources
PubMed1989

The interaction of aluminium with soluble protein kinase C from mouse brain.

The interaction of aluminium ion species with soluble protein kinase C, Ca2+/phospholipid-dependent protein kinase, from mouse brain has been examined in vitro. The activity of protein kinase C was increased by addition of Ca2+ displaying an EC50 value of 10.3 +/- 1.1 x 10(-6) M. The A1 species inhibited the activity with an IC50 values of 8.6 +/- 0.5 x 10(-5) M and 2.7 +/- 0.3 x 10(-5) M in the presence of 0.5 mM Ca2+ and absence of Ca2+, respectively. Concerning the EC50 for Ca2+ activation, this was increased by the A1 species in a dose-dependent manner. Moreover, the inhibition was of a non-competitive type with respect to H1 histone and of a mixed type with respect to ATP. It is likely that the inhibition was caused by 1) the blocking of Mg2+ binding to ATP, 2) the blocking of CA2+ binding to protein kinase C. Our results suggested that protein kinase C was involved in neurotoxicity of A1.

Aluminum↗
PubMed1989

Molecular cloning of cDNA for rat brain metallothionein-II and regulation of its gene expression.

A rat brain metallothionein-II (MT-II) complementary DNA (cDNA) clone was isolated from a cDNA plasmid library, which was prepared from non-treated rat brain mRNA, by a colony screening procedure using 32P-labeled synthetic oligonucleotide probes. It is deduced that the clone encodes for a protein of 61 amino acids comprising 20 cysteines, which is highly homologous to MT-IIs in other species. Northern blot analysis demonstrated major mRNA species in the brain, liver and kidneys (approximately 350 b in size), which is induced in response to dexamethasone, zinc, cadmium and mercury but not to methyl mercury. These findings confirm that MT-II genes are expressed and regulated both by steroid and heavy metals in the brain as well as in peripheral organs.

Amino Acid Sequence↗
PubMed1988

Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

The interaction of cations with activity of soluble protein kinase C from mouse brain.

The interaction of some cations with the enzymatic activity of soluble protein kinase C was determined in order to elucidate whether protein kinase C can be activated by other metal cations besides Ca2+. Protein kinase C was activated by Ca2+ and Sr2+ having EC50 values of nearly 10 microM and 200 microM, respectively. Ba2+ likewise activated protein kinase C but was less potent. Co2+, Ni2+ and Mn2+ had no activating effects on the activity in the absence of Ca2+, but was slightly reduced in the presence of Ca2+ (0.5 mM). Cations with ionic radii close to Ca2+ (0.99 A) inhibited the activity irrespective of the absence or presence of Ca2+. The order of potency is as follows: Hg2+ greater than Cd2+ approximately Cu2+ much greater than Sm3+ greater than Tb3+ greater than La3+. Pb2+ and Zn2+, which showed a high affinity to SH-groups, as well as Hg2+, Cd2+ and Cu2+, which also inhibited the activity. Thus, among the ions investigated, the alkaline-earth ions Sr2+ and Ba2+ could be substituted for Ca2+, irrespective of ionic radii. The serious environmental pollutants such as Hg2+, Cd2+ or Pb2+ impaired the activity of protein kinase C probably due to SH-blocking.

Animals↗
PubMed1988

Action of mercurials on activity of partially purified soluble protein kinase C from mice brain.

The enzymatic activity of soluble protein kinase C from mice brain was inhibited by mercuric chloride (II) (HgCl2) and organic mercurials, i.e. methyl mercury, phenyl mercury and p-chloromercuribenzoic acid (PCMB). The IC50 was 0.08 microM for HgCl2 and about 1 microM for organic mercurials. Sulfhydryl blocking reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) were less potent but nevertheless inhibited the enzymic activity of protein kinase C. The Hill coefficients of HgCl2, DTNB and NEM were close to unity whereas the values for organic mercurials were 1.3 to 1.5. The inhibition was of a non-competitive type with respect to H1 histone. 3H-PDBu binding activity was also inhibited by all of the reagents in a non-competitive manner. Mercurials apparently bind to sulfhydryl groups of protein kinase C to inhibit the enzymatic activity.

Animals↗
PubMed1985

Effect of hypoxia on uptake and acetylation of [3H]choline in brain slices from adult and newborn guinea pigs.

The synthesis of [3H]acetylcholine in brain slices from adult and newborn guinea pigs were suppressed by anaerobiosis. The uptake and acetylation of [3H]choline in the newborn brain were very low, compared to findings in slices of the adult brain. The most sensitive tissue to hypoxia was the striatum followed by the hippocampus. There were significant differences in the case of the frontal cortex.

Acetylation↗
PubMed1985

Influence of hypoxia on release and uptake of neurotransmitters in guinea pig striatal slices: dopamine and acetylcholine.

We studied the influence of hypoxia on the release of [3H]dopamine ([3H]DA) and [3H]acetylcholine ([3H]ACh), uptake of [3H]DA and [3H]choline and Ca2+-influx in guinea pig striatal slices. Tetrodotoxin (TTX)-sensitive and Ca2+-dependent electrically evoked release of [3H]DA was not affected by hypoxia, while spontaneous release of [3H]DA was rapidly increased. On the other hand, by hypoxia, the evoked [3H]ACh release gradually decreased and was diminished to about 45% 40 min later. Hypoxia suppressed the Vmax of [3H]DA uptake to one third and that of [3H]choline to half of the control values, but with no change in either of the Km values. Hypoxia reduced both the acetylation and the uptake of [3H]choline in slices preliminarily incubated with 3 mM or 25 mM K+ medium. Stimulation-induced Ca2+-influx was slightly suppressed and was 78.1% of the control values even after 40 min exposure to hypoxia. The Ca2+-dependent neurotransmitter release process itself appears to be well preserved against hypoxia as compared with the uptake process. Our findings imply that hypoxia could result in differential alterations of neural activity depending on the specific sensitivity of the presynaptic process of neurotransmission.

Acetylation↗
PubMed1984

Evidence for the neurotransmitter role of norepinephrine in the ventral thalamic nucleus of the guinea pig: localization, uptake and release.

The localization, uptake and release of norepinephrine (NE) by the isolated guinea pig ventral thalamic nucleus (VT) were studied to determine the possible neurotransmitter role of this catecholamine. The NE content in the VT was 356 +/- 24 ng/g wet weight and intraneuronal localization of NE was demonstrated in the VT by histofluorescence. [3H]NE accumulation into the VT slices was about 3 times that of [3H]NE (10(-7) M) in the medium 10 min after incubation. Kinetic analyses indicated 2 components of [3H]NE accumulation, one representing a high (Km1 4 X 10(-7) M and Vmax1 1.5 pmol/mg/10 min) and a low (Km2 1.8 X 10(-6) M and Vmax2 5 pmol/mg/10 min) affinity uptake system. Desmethylimipramine (10(-5) M) reduced the high affinity uptake of [3H]NE by 30% of the control value. Electrical stimulation of the slices increased the efflux of [3H]NE from tissues preloaded with [3H]NE, in a current- and frequency-dependent fashion. The release of [3H]NE induced by stimulation of up to 1 mA was inhibited by tetrodotoxin (10(-6) M) or Ca-free medium containing EGTA (10(-4) M). These findings provide strong evidence for the neurotransmitter role of NE in the VT.

Animals↗
PubMed1983

Solubilization of D2 dopamine receptor coupled to guanine nucleotide regulatory protein from bovine striatum.

D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of dopamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL-4B column chromatography as a large molecule, with a Stokes radius of approximately 90 A. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.

Animals↗
PubMedDate not supplied

Brain metallothionein gene expression and regulation.

Metallothionein (MT) gene expression in the brain has been most thoroughly studied using rodents. Although MT is considered to be a 'housekeeping' protein even in the brain, the basal MT mRNA expression level is not always high. Differences in the responses of rats and mice have made it difficult to interpret the data. Moreover, the response to inducers is not always apparent, probably because the brain is protected by the blood-brain barrier and initial responses to inducers in peripheral tissues modulate their accumulation in the brain. A relatively high content of MT protein in the brain might be sufficient to elicit minute alterations in the level of inducers. Nonetheless, regulation of MT gene expression in the brain seems to be important in e.g. maintaining the levels of trace elements and controlling redox potentials. The localization and utilization of trans elements such as MTF-I and MEP-I in the brain will provide new aspects for study. The high homology among MT isoforms with respect to nucleotide as well as amino acid sequences has made it difficult to obtain cDNA probes or antibodies capable of distinguishing MT isoforms. Thus, their cross-reactivity might make changes in MT mRNAs appear minimal when MT isoforms are differently regulated. The rapid developments in methodology permitting sensitive, rapid, high-resolution analysis could clarify the background of tissue- and cell-specific gene regulation as well as differential induction.

Amino Acids↗