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K Fukunaga

Publications and source records attributed to K Fukunaga.

At least 181 records · Page 10Linked to original sources
PubMed1994

Activation of Ca2+/calmodulin-dependent protein kinase II and phosphorylation of intermediate filament proteins by stimulation of glutamate receptors in cultured rat cortical astrocytes.

We investigated the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) via stimulation of glutamate receptors and subsequent phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) in cultured rat cortical astrocytes. The indirect immunofluorescence analysis with the anti-CaM kinase II antibody revealed that the enzyme was detected diffusely in the cytoplasm and more intensely in the nucleus. Glutamate elevated the Ca(2+)-independent activity of CaM kinase II through autophosphorylation, and this response was blocked by both DL-2-amino-3-phosphonopropionate and 6-cyano-7-nitroquinoxaline-2,3-dione, but not by D-2-amino-5-phosphonovalerate. In the experiments using 32P-labeled astrocytes, the phosphorylation of vimentin and GFAP as well as autophosphorylation of CaM kinase II were found to be stimulated after the exposure to glutamate. It was concluded by two-dimensional phosphopeptide analysis that the increased phosphorylation of vimentin and GFAP observed in intact cells were due to the activation of CaM kinase II by glutamate. These results suggest that glutamate can activate CaM kinase II through stimulation of both the metabotropic and non-N-methyl-D-aspartate receptors, and that the concomitant phosphorylation of vimentin and GFAP may in turn regulate the functions of intermediate filament proteins in intact astrocytes.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

Excitatory interactions between glutamate receptors and protein kinases.

One of the most active areas of neurobiology research concerns mechanisms involved in paradigms of synaptic plasticity. A popular model for cellular learning and memory is long term potentiation (LTP) in hippocampus. LTP requires postsynaptic influx of Ca2+ which triggers multiple biochemical pathways resulting in pre- and postsynaptic mechanisms enhancing long term synaptic efficiency. This article focuses on an acute postsynaptic mechanism that can enhance responsiveness of glutamate receptors. Evidence is presented that calcium/calmodulin-dependent protein kinase II, the major postsynaptic density protein at excitatory glutaminergic synapses, can phosphorylate glutamate receptors and enhance ion current flowing through them.

Amino Acid Sequence↗
PubMed1994

Cellular localization of type II Ca2+/calmodulin-dependent protein kinase in the rat basal ganglia and intrastriatal grafts derived from fetal striatal primordia, in comparison with that of Ca2+/calmodulin-regulated protein phosphatase, calcineurin.

We investigated immunohistochemically the cellular localization of multifunctional type II Ca2+/calmodulin-dependent protein kinase in the rat basal ganglia and intrastriatal grafts derived from fetal striatal primordia, in comparison with that of calcineurin, a reliable marker for striatal medium-sized spinous neurons. The type II Ca2+/calmodulin-dependent protein kinase-positive neurons were of medium size, with a mean diameter of 16.1 +/- microns (average +/- S.D., n = 72, range 13.6-18.3 microns) and comprised approximately 70% of the total neuronal population in the striatum. Light microscopy showed that the type II Ca2+/calmodulin-dependent protein kinase-positive cells had round, triangular or polygonal cell bodies with relatively little cytoplasm. Analysis of serial sections showed that type II Ca2+/calmodulin-dependent protein kinase and calcineurin immunoreactivities were co-localized in the striatal neurons examined with a similar distribution pattern. Type II Ca2+/calmodulin-dependent protein kinase-positive cells were always immunoreactive for calcineurin and cells negative for type II Ca2+/calmodulin-dependent protein kinase showed no apparent calcineurin immunoreactivity. Type II Ca2+/calmodulin-dependent protein kinase-positive nerve fibers in the globus pallidus and substantia nigra almost disappeared following striatal ischemic injury produced by transient middle cerebral artery occlusion and cerebral hemitransection, respectively, suggesting that these immunopositive fibers were striatal projections. Thus, most type II Ca2+/calmodulin-dependent protein kinase-positive neurons in the rat striatum are considered to be of the medium-sized spinous type. Type II Ca2+/calmodulin-dependent protein kinase or calcineurin immunoreactivity was also observed in a large number of neurons in transplants derived from fetal striatal primordia grafted into striatal ischemic lesions. In addition, type II Ca2+/calmodulin-dependent protein kinase- or calcineurin-immunoreactive nerve fibers appeared in the deafferented globus pallidus of the host rats, suggesting that the striatopallidal pathway was reformed by striatal projection neurons of the transplants. This finding may also indicate that Ca2+/calmodulin-regulated enzymes are useful for tracing striatal projection fibers as endogenous marker proteins.

Animals↗
PubMed1994

Glycolipids isolated from Aplysia kurodai can activate cyclic adenosine 3',5'-monophosphate-dependent protein kinase from rat brain.

Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonoglycosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGal beta 1-->3GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2](2-aminoethylphosphonyl-->6)Glc beta 1-->4Glc beta 1-->1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 microM. Activation of cAMP-kinase was maximal with 250 microM SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.

Animals↗
PubMed1994

Aluminium beta-cyclodextrin sulphate as a stabilizer and sustained-release carrier for basic fibroblast growth factor.

The water-insoluble aluminium salt of beta-cyclodextrin sulphate (Al.beta-CyD-Sul) was used as a stabilizer and sustained-release carrier for recombinant human basic fibroblast growth factor (bFGF). An adsorbate of bFGF with Al.beta-CyD-Sul was prepared by incubating the protein with a suspension of Al.beta-CyD-Sul in water. The mitogenic activity of bFGF released from the adsorbate, as indicated by the proliferation of kidney cells of baby hamster (BHK-21), was almost comparable with that of the intact bFGF. Al.beta-CyD-Sul significantly protected bFGF from proteolytic degradation by pepsin and alpha-chymotrypsin, compared with the water-soluble sodium salt. The in-vitro release of bFGF from the adsorbate was sustained in proportion to a rise in the ratio of Al.beta-CyD-Sul to the protein in the adsorbate. Of the bFGF preparations evaluated, the adsorbate of bFGF with Al.beta-CyD-Sul, when given subcutaneously to the rat, showed the most prominent increase in the formation of granulation tissues, due to the stabilization and slow-release of the mitogen. The limited data presented here suggest that the adsorbate of bFGF with Al.beta-CyD-Sul has a potent therapeutic efficacy for wound healing, and may be applicable to oral protein formulations for the treatment of intestinal mucosal erosions.

Animals↗
PubMed1993

Highly sensitive high-performance liquid chromatography for the measurement of malondialdehyde in biological samples.

A highly sensitive method for the measurement of malondialdehyde as thiobarbituric acid-malondialdehyde complex by reversed-phase high-performance liquid chromatography with fluorescence detection in biological samples is described. As samples, 20 microliters of rat plasma or 10% (w/v) liver homogenate mixed with 2.0% thiobarbituric acid in 2 M sodium acetate buffer containing 0.05% butyl hydroxytoluene (pH 3.5) were heated at 95 degrees C for 45 min to give the complex. The complex, extracted with n-butanol, was chromatographed on a system equipped with a reversed-phase column, and the eluted peak was monitored with a fluorescence detector (excitation wavelength 515 nm, emission wavelength 553 nm). The mobile phase was a acetonitrile-water (2:8, v/v) under isocratic conditions at ambient temperature, and a single analysis was done in ca. 4 min. The minimum detection level for malondialdehyde was as low as 0.05 pmol. The n-butanol extract was stable at least for 3 days. The simple mobile phase, the extremely sensitive detection limit, and the stability of the complex make this system applicable to routine clinical analysis with a small amount of tissue or biopsy sample.

Animals↗
PubMed1993

Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase II.

Among the molecular mechanisms that have been proposed to contribute to long-term potentiation in hippocampus are the activation and autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Here we report that high, but not low frequency stimulation applied to two groups of CA1 afferents resulted in a long lasting increase in the Ca(2+)-independent and total activities of the enzyme as well as an increase in the ratio of Ca(2+)-independent to total activity. The effect was obtained using two different CaM kinase II substrates, it was observed in hippocampal slices and in hippocampal organotypic cultures, and it could be blocked by preincubation of slices with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoate. Treatment of slices with calyculin A, a phosphatase inhibitor, modified the activity of the enzyme, but long term potentiation could still be induced and a further increase in Ca(2+)-independent CaM kinase II activity still observed.

Animals↗
PubMed1993

Cellular colocalization of calcium/calmodulin-dependent protein kinase II and calcineurin in the rat cerebral cortex and hippocampus.

An immunoperoxidase technique was used to locate multifunctional Ca2+/calmodulin-regulated protein phosphatase (calcineurin) and kinase (CaM-kinase II) in the rat cerebral cortex and hippocampus. Immunoreactivities for both enzymes were highly concentrated in the brain regions, where pyramidal-shaped neurons revealed strong immunoreactivities in their perikarya and dendrites. Serial thin section analysis using the polyethylene glycol embedding procedure disclosed that the cellular distribution of calcineurin immunolabelling in the cerebral cortex and hippocampus was similar to that of CaM-kinase II. The present findings suggest that the phosphatase and kinase may interact with each other in such neuronal subsets.

Animals↗
PubMed1993

Dephosphorylation of autophosphorylated Ca2+/calmodulin-dependent protein kinase II by protein phosphatase 2C.

It has been demonstrated that okadaic acid-insensitive protein phosphatases are involved in dephosphorylation of autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in rat cerebellar granule cells (Fukunaga, K., Rich, D. P., and Soderling, T. R. (1989) J. Biol. Chem. 264, 21830-21836). In the present study, recombinant rat protein phosphatase 2C (PrP-2C) expressed in Escherichia coli could dephosphorylate both Thr286/287 and Thr305/306 phosphorylation sites of CaM kinase II, which are responsible for the generation of Ca(2+)-independent activity and the inhibition of the total activity, respectively. The dephosphorylation of Thr286/287 and Thr305/306 was accomplished within 15 min at 0 degrees C and totally dependent on Mg2+. Phosphopeptide mapping of the CNBr-cleaved 32P-labeled CaM kinase II revealed that PrP-2C was relatively specific for dephosphorylation of Thr286/287 and Thr305/306 in the autophosphorylated CaM kinase II. These results suggest that PrP-2C has a role in the regulation of the Ca(2+)-independent activity of CaM kinase II in the neural cells.

Animals↗
PubMed1993

Protein kinase C inhibitors prevent impairment of endothelium-dependent relaxation by oxidatively modified LDL.

The mechanism(s) of inhibition of endothelium-dependent relaxation (EDR) by oxidized low-density lipoprotein (Ox-LDL) was examined in isolated porcine coronary arteries and rabbit aortas. Incubation with Ox-LDL but not native LDL caused the inhibition of thrombin- or acetylcholine-induced EDR, whereas A23187-induced EDR was preserved after incubation with Ox-LDL. Lysophosphatidylcholine (lysoPC), which was abundant in Ox-LDL and was found to be transferred from Ox-LDL to endothelial cells, also caused the inhibition of EDR in response to thrombin or acetylcholine but not to A23187. Ox-LDL depleted of lysoPC, which was prepared by phospholipase B, failed to inhibit the vasorelaxation. Coincubation with staurosporine or calphostin C, potent inhibitors of protein kinase C, attenuated the EDR inhibition by Ox-LDL or lysoPC. Phorbol 12-myristate 13-acetate, a specific protein kinase C activator, caused the EDR inhibition, and its effect was attenuated by staurosporine or calphostin C. Furthermore, lysoPC was capable of activating protein kinase C purified from cultured porcine endothelial cells. In conclusion, protein kinase C activation plays a role in the inhibition of surface receptor-mediated EDR by Ox-LDL, and lysoPC transferred from Ox-LDL to endothelial cells may be involved in the activation of protein kinase C.

Animals↗
PubMed1993

[The role of Ca2+/calmodulin-dependent protein kinase II in the cellular signal transduction].

Both Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and protein kinase C (PKC) have been implicated as possible candidates for contributing to the induction of long-term potentiation (LTP) in the hippocampus. The induction of LTP in the CA1 region of the hippocampus, an event which requires postsynaptic Ca2+ influx through NMDA-type glutamate receptors, is blocked by calmodulin antagonists and inhibitors of CaM kinase II and PKC. In the present study, we describe the activation characteristics of CaM kinase II and PKC through the stimulation of glutamate receptors and regulation of the phosphorylation of substrates for CaM kinase II in the hippocampus. In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of CaM kinase II through autophosphorylation, and this response was blocked by specific antagonists of the NMDA receptor. In addition, glutamate stimulated the translocation of PKC from the cytosol to the membrane fraction through the metabotropic glutamate receptor. In the experiments with 32P-labeled cells, the phosphorylation of microtubule-associated protein 2 (MAP2) and synapsin I was stimulated by the exposure to glutamate. Finally, we demonstrated that high, but not low, frequency stimulation applied to two groups of CA1 afferents in the slices resulted in the induction of LTP with concomitant long-lasting increases in the Ca(2+)-independent and total CaM kinase II activities as well as the autophosphorylation. It could be blocked by preincubation of the slices with NMDA-receptor antagonist. These results suggest that glutamate can activate CaM kinase II through NMDA receptors in the induction of LTP and in turn stimulates the phosphorylation of target proteins such as MAP2 and synapsin I.

Animals↗
PubMed1993

Lymphoplasmacytic lymphoma in a stallion.

Lymphoplasmacytic lymphoma found in a 6-year-old Anglo-Arabian stallion was investigated histologically, immunohistochemically and ultrastructurally. The animal showed a large mediastinal mass and generalized lymph node involvement. The neoplastic cells were in various differentiation stages of small lymphocyte, centrocyte, centroblast, immunoblast and plasma cell. Some neoplastic cells showed positive cytoplasmic reactivity for mu and lambda chains. There were well developed rough endoplasmic reticulum (RER) and Golgi complexes in plasmacytoid cells, and slightly developed RER or a few long strands of RER in medium-sized to large lymphoid cells. These findings suggest that this neoplasm is of B-cell origin.

Animals↗
PubMed1993

Effects of casein and soy protein on accumulation of cholesterol and dolichol in rat liver.

A diet containing 15% (w/w) fat and 20% (w/w) of either casein (CAS) or soy protein (SOY) was fed to 4-week-old rats for a period of 18 months. The effects of these dietary proteins on the accumulation of cholesterol and dolichol in livers were studied. After 1 month, the amount of liver cholesterol was about 5 mg/g of liver. After an additional 5 months of feeding, there was a slight decrease in cholesterol per gram of liver (3.6 mg/g of liver in CAS-fed rats and 2.6 mg/g of liver in SOY-fed rats). However, after 18 months, there were a remarkable increase (7.5 mg/g of liver) in CAS-fed rats and only a slight increase in SOY-fed rats. The proportions of liver cholesterol ester in rats fed the CAS diet were 60-70% of the total cholesterol during the experimental period, but in the case of the SOY diet, only rats fed the diet for 1 month showed a high level, 70%, of cholesterol ester. The amounts of liver dolichol in rats fed the CAS and SOY diets after feeding for 18 months were 60 micrograms and 47 micrograms of liver, respectively. There was a 1.5-fold increase in both diets for a period of 18 months. The proportions of liver dolichyl fatty ester in rats fed the CAS diet were 35-40% of the total dolichol during the experimental period, but in the case of the SOY diet, only rats fed the diet for 1 month showed a high level, 36%, of dolichyl fatty ester. The proportions of dolichol ester in rats fed the SOY diet were 25-30% after 6 and 18 months of feeding. These observations indicated that the SOY diet depresses the accumulation of both liver dolichol and cholesterol.

Animals↗
PubMed1992

Activation of Ca2+/calmodulin-dependent protein kinase II by stimulation with bradykinin in neuroblastoma x glioma hybrid NG108-15 cells.

To elucidate the mechanisms of the intracellular signal transduction elicited with bradykinin in NG108-15 neuroblastoma x glioma hybrid cells, we examined the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) by bradykinin stimulation. When the extract of NG108-15 cells was immunoprecipitated with the affinity-purified antibody to brain CaM kinase II, a 50-kDa protein in the immunoprecipitate mainly became autophosphorylated in a Ca2+/calmodulin-dependent manner. The results suggest that the 50-kDa protein is the subunit of CaM kinase II in NG108-15 cells. The Ca2+/calmodulin-independent activity (autonomous activity) of the enzyme increased twice within 10 s by stimulation with 1 microM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases: the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 microM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca2+ with 1 mM EGTA or by the pretreatment with 1 microM nifedipine. Stimulation of 32P-labeled NG108-15 cells with 1 microM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. These results suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells.

Bradykinin↗
PubMed1992

Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.

In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.

Animals↗