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Hisatake Kondo

Publications and source records attributed to Hisatake Kondo.

At least 37 records · Page 2Linked to original sources
PubMed2005

Expression and localization of diacylglycerol kinase isozymes and enzymatic features in rat lung.

Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKalpha, -epsilon, and -zeta were detected in the lung. By immunohistochemical examination, DGKalpha and -zeta were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKalpha in the cytoplasm and DGKzeta in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKalpha and -zeta was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKepsilon was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.

Animals↗
PubMed2005

p55 protein is a member of PSD scaffold proteins in the rat brain and interacts with various PSD proteins.

p55 is a membrane-associated guanylate kinase (MAGuK) family member that consists of a single PDZ followed by SH3, HOOK and guanylate kinase (GuK or GK) domains. We investigated rat p55 (r-p55) in the brain. r-p55 mRNA was expressed widely in various tissues and in various regions of the brain. r-p55 protein was also expressed widely in various rat tissues, including brain and erythrocytes. The protein was enriched in the synaptic plasma membrane and postsynaptic density (PSD) fractions of the forebrain. An immunocytochemical study using cultured cortical neurons suggested postsynaptic localization of r-p55 protein. Pull-down assay showed that r-p55 protein interacted with r-p55 itself and various PSD proteins, such as PSD-95, SAP97, GKAP, CASK, GRIP, neuroligin, cadherin, tubulin, actin, alpha-internexin, neurofilament-L and Ca(2+)/calmodulin-dependent protein kinase II, through its PDZ, SH3, HOOK or GK domains. The interaction with PSD-95 was found to occur between the PDZ domains of PSD-95 and the HOOK and GK domains of r-p55 protein. These findings, together with the presence of r-p55 puncta in a period of early synaptogenesis, suggest that r-p55 protein functions as one of postsynaptic scaffold component in an early stage of synaptogenesis in the brain. r-p55 protein may form a basic structure, which interlinks diverse functional molecules of the PSD necessary for postsynaptic signaling and synaptic adhesion.

Amino Acid Sequence↗
PubMed2005

Importin alpha transports CaMKIV to the nucleus without utilizing importin beta.

Ca(2+)/calmodulin-dependent protein kinase type IV (CaMKIV) plays an essential role in the transcriptional activation of cAMP response element-binding protein-mediated signaling pathways. Although CaMKIV is localized predominantly in the nucleus, the molecular mechanism of the nuclear import of CaMKIV has not been elucidated. We report here that importin alpha is able to carry CaMKIV into the nucleus without the need for importin beta or any other soluble proteins in digitonin-permeabilized cells. An importin beta binding-deficient mutant (DeltaIBB) of importin alpha also carried CaMKIV into the nucleus, which strongly suggests that CaMKIV is transported in an importin beta-independent manner. While CaMKIV directly interacted with the C-terminal region of importin alpha, the CaMKIV/importin alpha complex did not form a ternary complex with importin beta, which explains the nonrequirement of importin beta for the nuclear transport of CaMKIV. The cytoplasmic microinjection of importin alpha-DeltaIBB enhanced the rate of nuclear translocation of CaMKIV in vivo. This is the first report to demonstrate definitely that mammalian importin alpha solely carries a cargo protein into the nucleus without utilizing the classical importin beta-dependent transport system.

Active Transport, Cell Nucleus↗
PubMed2005

Proper name anomia after left temporal subcortical hemorrhage.

We report here a patient with proper name anomia following subcortical hemorrhage in the left superior temporal gyrus. Despite the preserved ability to retrieve common names, the patient could not retrieve the names of people, countries, or racehorses, which he could recognize quite well. Semantic knowledge regarding people, countries, and racehorses was also preserved. In addition. the finding that phonological cueing was effective with preservation of the ability to point to photos corresponding to their names suggested that the lexicon of proper names was preserved in this patient. Thus, the output lexicon appeared to be partially disconnected from semantic knowledge. This rare and limited lesion suggested that the superior temporal gyrus plays an important role in connecting semantic knowledge and the output lexicon.

Anomia↗
PubMed2005

Functional assay of EFA6A, a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (ARF6), in dendritic formation of hippocampal neurons.

EFA6A is a guanine nucleotide exchange factor (GEF) that can activate ADP-ribosylation factor 6 (ARF6) in vitro, with prominent expression in the forebrain including the hippocampal formation. In this section, we describe the neuronal transfection method and show that the overexpression of a catalytically inactive mutant of EFA6A induces a prominent dendritic formation of the primary hippocampal neurons, suggesting the intimate involvement of EFA6A in the regulation of neuronal dendritic development. This reliable and consistent neuronal transfection method will also be applicable for the vector-based RNA interference method.

ADP-Ribosylation Factor 6↗
PubMed2005

A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins.

We cloned from the rat brain a novel gene, tanc (GenBank Accession No. AB098072), which encoded a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles (rols). The tanc gene was expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicated the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments showed that TANC protein bound PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also bound the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study showed TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins.

Age Factors↗
PubMed2005

Prominent expression and activity-dependent nuclear translocation of Ca2+/calmodulin-dependent protein kinase Idelta in hippocampal neurons.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) including CaMKI, II and IV, are thought to regulate a variety of neuronal functions. Unlike CaMKII, which is regulated by autophosphorylation, CaMKI as well as CaMKIV are activated by CaMKK. In this study, we examined the cellular and subcellular localization of CaMKIdelta, a recently identified fourth isoform of CaMKI, in the mature brain. In situ hybridization analysis demonstrated wide expression of CaMKIdelta mRNA in the adult mouse brain with prominent expression in the hippocampal pyramidal cells. FLAG-tagged CaMKIdelta was localized at the cytoplasm and neurites without nuclear immunoreactivity in approximately 80% of the transfected primary hippocampal neurons. The stimulation with either KCl depolarization or glutamate triggered the nuclear localization of FLAG-tagged CaMKIdelta by two-fold with a peak at 1 min. In contrast, the catalytically inactive mutants of CaMKIdelta remained cytoplasmic without nuclear translocation during KCl depolarization, indicating the requirement of its activation for the nuclear translocation. Furthermore, we showed that immunoprecipitated CaMKIdelta could phosphorylate cAMP response element binding protein (CREB)alphain vitro and that the over-expression of CaMKIdelta enhanced GAL4-CREB-luciferase activity in PC12 cells stimulated by KCl depolarization. Our present study provides the first evidence for the possible involvement of CaMKIdelta in nuclear functions through its nuclear translocation in response to stimuli that trigger intracellular Ca2+ influx.

Animals↗
PubMed2005

BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and palmitoylation, and interacts with alpha, but not beta, subunit of Ca/calmodulin-dependent protein kinase II.

We cloned a rat BAALC 1-6-8 isoform cDNA (GenBank Accession No. AB073318) that encoded a 22-kDa protein, and identified endogenous BAALC 1-6-8 protein in the brain. The gene was expressed widely in the frontal part of the brain, and the protein was localized to the synaptic sites and was increased in parallel with synaptogenesis. The protein interacted with the alpha, but not beta, subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIalpha). The interaction occurred between the N-terminal 35-amino-acid region of BAALC 1-6-8 protein and the C-terminal end of the regulatory domain of CaMKIIalpha, which contains alpha isoform-specific sequence. Thus, the interaction may be CaMKIIalpha-specific. We also found that BAALC 1-6-8 protein, as well as CaMKIIalpha, was localized to lipid rafts and that both myristoylation and palmitoylation of BAALC 1-6-8 N-terminal portion were required for targeting of the protein into lipid rafts. These findings suggest that BAALC 1-6-8 protein play a synaptic role at the postsynaptic lipid raft possibly through interaction with CaMKIIalpha.

Alternative Splicing↗
PubMed2005

Cellular and subcellular localization of EFA6C, a third member of the EFA6 family, in adult mouse Purkinje cells.

EFA6C is a third member of the EFA6 family of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factor 6 (ARF6). In this study, we first demonstrated that EFA6C indeed activated ARF6 more selectively than ARF1 by ARF pull-down assay. In situ hybridization histochemistry revealed that EFA6C mRNA was expressed predominantly in mature Purkinje cells and the epithelial cells of the choroid plexus in contrast to the ubiquitous expression of ARF6 mRNA throughout the brain. EFA6C mRNA was already detectable in the Purkinje cells at embryonic day 13, increased progressively during post-natal development and peaked during post-natal second week. In Purkinje cells, the immunoreactivity for EFA6C was localized particularly in the post-synaptic density as well as the plasma membranes of the cell somata, dendritic shafts and spines, while the immunoreactivity in their axon terminals in the deep cerebellar nuclei was very faint. These findings suggest that EFA6C may be involved in the regulation of the membrane dynamics of the somatodendritic compartments of Purkinje cells through the activation of ARF6.

ADP-Ribosylation Factor 6↗
PubMed2004

Selective translocation of diacylglycerol kinase zeta in hippocampal neurons under transient forebrain ischemia.

The molecular mechanisms responsible for differential neuronal vulnerability to ischemic injury are incompletely understood. Previous studies have reported that the expression and activity of protein kinase C (PKC), some subtypes of which are activated by Ca(2+) and diacylglycerol (DG), are altered after ischemic insults. Therefore, DG kinase (DGK), which is responsible for controlling PKC activity through DG metabolism, may also be involved in this process. DGKzeta, which is abundantly expressed in the brain, contains a nuclear localization signal (NLS), suggesting its involvement in some nuclear processes in neuronal cells. To elucidate the functional implications of DGKzeta in ischemia, we examined detailed localization of DGKzeta in rat brain after ischemic insults. We used an ischemic model of global cerebral ischemia for 20 min by bilateral common carotid artery occlusion combined with hypotension and followed time-points of reperfusion. DGKzeta expression was evaluated by immunohistochemistry using affinity-purified anti-DGKzeta antibody. In sham-operated rats, a strong DGKzeta-immunoreactivity was observed in the nucleus of neurons in various parts of the brain. In the global ischemic model DGKzeta-immunoreactivity was reduced in intensity in the hippocampal formation and detected in the cytoplasm of CA1 pyramidal neurons throughout reperfusion time courses. Change in the subcellular localization was restricted to the pyramidal cells in CA1 and later in CA3, but not observed in other areas of hippocampus. No change was observed in the cerebral and cerebellar cortices. The present study suggests that DGKzeta might be involved in the process of selective vulnerability of hippocampal pyramidal neurons in postischemic brain.

Animals↗
PubMed2004

NIDD, a novel DHHC-containing protein, targets neuronal nitric-oxide synthase (nNOS) to the synaptic membrane through a PDZ-dependent interaction and regulates nNOS activity.

Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. Here we report the identification and characterization of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank accession number AB098078), which increases nNOS enzyme activity by targeting the nNOS to the synaptic plasma membrane in a PDZ domain-dependent manner. The deduced NIDD protein consisted of 392 amino acid residues and possessed five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif (-EDIV) at its C-terminal tail. In vitro pull-down assays suggested that the C-terminal tail region of NIDD specifically interacted with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS were enriched in synaptosome and synaptic plasma membrane fractions and were present in the lipid raft and postsynaptic density fractions in the rat brain. Co-localization of these proteins was also observed by double staining of the proteins in cultured cortical neurons. Thus, NIDD and nNOS were co-localized in the brain, although the colocalizing regions were restricted, as indicated by the distribution of their mRNA expression. Most important, co-transfection of NIDD and nNOS increased NO-producing nNOS activity. These results suggested that NIDD plays an important role in the regulation of the NO signaling pathway at postsynaptic sites through targeting of nNOS to the postsynaptic membrane.

Amino Acid Sequence↗
PubMed2004

Phenotype of palmitic acid transport and of signalling in alveolar type II cells from E/H-FABP double-knockout mice: contribution of caveolin-1 and PPARgamma.

Based on the assumption that fatty-acid-binding proteins (FABPs) of the epidermal-type (E-FABP) and heart-type (H-FABP) in murine alveolar type II (TII) cells mediate the synthesis of dipalmitoyl phosphatidylcholine (DPPC), the main surfactant phospholipid, we analysed TII cells isolated from wild-type (wt) and E/H-FABP double-knockout (double-ko) mice. Application of labelled palmitic acid to these cells revealed a drop in uptake, beta-oxidation, and incorporation into neutral lipids and total phosphatidylcholine (PC) of TII cells from double-ko mice. Whereas incorporation of labelled palmitic acid into DPPC remained unchanged, degradation studies demonstrated a substantial shift in DPPC synthesis from de novo to reacylation. In addition, increased expression of mRNAs and proteins of caveolin-1 and PPARgamma, and an increase of the mRNA encoding fatty acid translocase (FAT) was observed in the double-ko phenotype. As caveolin-1 interacted with PPARgamma, we assumed that FAT, caveolin-1, and PPARgamma form a signalling chain for fatty acid or drug. Consequently, PPARgamma-selective pioglitazone was added to the diet of double-ko mice. We found that further activation of PPARgamma could 'heal' the E/H-FABP double-ko effect in these TII cells as transport and utilisation of labelled palmitic acid restored a wt phenocopy. This indicated that E-FABP and/or H-FABP are involved in the mediation of DPPC synthesis in wt TII cells.

Animals↗
PubMed2004

Cloning and characterization of diacylglycerol kinase iota splice variants in rat brain.

Diacylglycerol kinase (DGK) catalyzes phosphorylation of a second messenger diacylglycerol (DG) to phosphatidic acid in cellular signal transduction. Previous studies have revealed that DGK consists of a family of isozymes including our rat clones. In this study we isolated from rat brain cDNA library the cDNA clones for a rat homologue of DGKiota (rDGKiota-1) that contains two zinc finger-like sequences, the highly conserved DGK catalytic domain, a bipartite nuclear localization signal, and four ankyrin repeats at the carboxyl terminus. In addition, we found novel splice variants, which contain either insertion 1 (71 bp) or insertion 2 (19 bp) or both in the carboxyl-terminal portion. Each of the insertions causes a frameshift, and the resultant premature stop codons produce two truncated forms (termed rDGKiota-2 and -iota-3), the former lacking the ankyrin repeats at the carboxyl terminus and the latter lacking a part of the catalytic domain and the ankyrin repeats. Truncation of the carboxyl-terminal portion clearly exerts effects on the detergent solubility and enzymatic activity of the splice variants, although all three variants showed similar cytoplasmic localization in cDNA-transfected cultured neurons despite the continued presence of the nuclear localization signal sequence. Immunoblot analysis using anti-rDGKiota antibody raised against the common amino-terminal portion clearly shows that these rDGKiota variants are indeed expressed in the brain. These results suggest that the carboxyl-terminal truncated forms of rDGKiota-2 and -iota-3 that exhibit reduced enzymatic activities might show a dominant negative effect against the intact rDGKiota-1, and that the modulation of signal transduction by the splice variants may play some roles in the physiologic and/or pathologic conditions of neurons.

Alternative Splicing↗
PubMed2004

Somatodendritic localization of the mRNA for EFA6A, a guanine nucleotide exchange protein for ARF6, in rat hippocampus and its involvement in dendritic formation.

EFA6A is a guanine nucleotide exchange protein (GEP) that can specifically activate ADP-ribosylation factor 6 (ARF6) in vitro. A recent study has demonstrated that ARF6 is involved in the dendritic formation of developing hippocampal neurons [Hernandez-Deviez et al. (2002) Nature Neurosci., 5, 623-624]. This study examined a potential role for EFA6A in hippocampal development in Wistar rats. Our results provided definitive evidence for somatodendritic localization of EFA6A mRNA in both cultured and in vivo hippocampal neurons by nonradioactive in situ hybridization. During postnatal development, EFA6A mRNA was dramatically increased and its dendritic localization was most evident between P7 and P14. In contrast, ARF6 mRNA was confined to the neuronal layers of the hippocampus throughout development. In addition, the overexpression of a GEP-defective mutant of EFA6A enhanced the dendritic formation of the primary hippocampal neurons. The present findings suggest that EFA6A is intimately involved in the regulation of the dendritic development of hippocampal neurons.

ADP-Ribosylation Factor 6↗
PubMed2004

Brain-specific potential guanine nucleotide exchange factor for Arf, synArfGEF (Po), is localized to postsynaptic density.

We cloned from a rat brain cDNA library a novel cDNA and named it a potential synaptic guanine nucleotide exchange factor (GEF) for Arf (synArfGEF (Po)) (GenBank Accession no. AB057643) based on its domain structure and localization. The cloned gene was 7410 bases long with a 3585-bp coding sequence encoding a protein of 1194 amino acids. The deduced protein contained a coiled-coil structure in the N-terminal portion followed by Sec7 and Plekstrin homology (PH) domains. Thus, the protein was a member of the Sec7 family of proteins, GEFs. Conservation of the ADP-ribosylation factor (Arf)-binding sequence suggested that the protein was a GEF for Arf. The gene was expressed specifically in the brain, where it exhibited region-specific expression. The protein was highly enriched in the postsynaptic density (PSD) fraction prepared from the rat forebrain. Uniquely, the protein interacted with PSD-95, SAP97 and Homer/Vesl 1/PSD-Zip45 via its C-terminal PDZ-binding motif and co-localized with these proteins in cultured cortical neurons. These results supported its localization in the PSD. The postsynaptic localization was also supported by immunohistochemical examination of the rat brain. The mRNA for the synArfGEF was also localized to dendrites, as well as somas, of neuronal cells. Thus, both the mRNA and the protein were localized in the postsynaptic compartments. These results suggest a postsynaptic role of synArfGEF in the brain.

ADP-Ribosylation Factors↗
PubMed2004

Differential localization of brain-type and epidermal-type fatty acid binding proteins in the adrenal gland of mice.

In immuno-light and -electron microscopy, brain-type fatty acid binding protein (B-FABP) is localized in the sustentacular cells enclosing the chromaffin cells in the adrenal medulla. This represents another new feature commonly shared by the sustentacular cells and ganglionic satellite cells, the latter of which has already been reported to localize this molecule, and suggests a common feature in lipid metabolism shared by the two cells enclosing peripheral neurons and paraneurons. On the other hand, epidermal-type fatty acid binding protein (E-FABP) is localized in two discrete cells in the adrenal gland: the one is a subpopulation of intra-adrenal macrophages which are intensely immunoreactive for F4/80, a marker of macrophages, and are rich in pleomorphic lysosomes. Because of their direct apposition to adjacent cortical endocrine cells and medullary chromaffin cells, the macrophages may be involved not only in phagocytosis of degenerating adrenal cells but also in exertion of some yet unknown effects on the endocrine function of the cortical and medullary cells via humoral factors such as cytokines which have recently been known to be secreted by macrophages. The other is a population of cells having scanty perikaryal cytoplasm poor in organneles and several thinny extended processes in the cortex and exhibiting weak immunoreactivity for E-FABP. The possible natures of these cells immunoreactive for E-FABP are discussed in view of a subpopulation of endothelial cells or the dendritic cells of antigen-presenting property.

Adrenal Cortex↗