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K Mikoshiba

Publications and source records attributed to K Mikoshiba.

At least 19 recordsLinked to original sources
PubMed2001

A proposed mechanism for the potentiation of cAMP-mediated acid secretion by carbachol.

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [(14)C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca(2+) and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 microM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase due to release from the Ca(2+) store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca(2+) release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca(2+) store and the receptor is maintained by actin microfilaments.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Role of the conserved WHXL motif in the C terminus of synaptotagmin in synaptic vesicle docking.

Synaptotagmin (Syt) I, an abundant synaptic vesicle protein, consists of one transmembrane region, two C2 domains, and a short C terminus. This protein is essential for both synaptic vesicle exocytosis and endocytosis via its C2 domains. Although the short C terminus is highly conserved among the Syt family and across species, little is known about the exact role of the conserved C terminus of Syt I. In this paper, we report a function of the Syt I C terminus in synaptic vesicle docking at the active zones. Presynaptic injection of a peptide corresponding to the C-terminal 21 amino acids of Syt I (named Syt-C) into the squid giant synapse blocked synaptic transmission without affecting the presynaptic action potential or the presynaptic Ca(2+) currents. The same procedure repeated with a mutant C-terminal peptide (Syt-CM) had no effect on synaptic transmission. Repetitive presynaptic stimulation with Syt-C produced a rapid decrease in the amplitude of the postsynaptic potentials as the synaptic block progressed, indicating that the peptide interferes with the docking step rather than the fusion step of synaptic vesicles. Electron microscopy of the synapses injected with the Syt-C peptide showed a marked decrease in the number of docked synaptic vesicles at the active zones, as compared with controls. These results indicate that Syt I is a multifunctional protein that is involved in at least three steps of synaptic vesicle cycle: docking, fusion, and reuptake of synaptic vesicles.

Action Potentials↗
PubMed2000

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1).

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.

Amino Acid Sequence↗
PubMed2000

Expression of synaptotagmin I or II promotes neurite outgrowth in PC12 cells.

Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, was suggested to be involved in neurite outgrowth of chick dorsal root ganglion (DRG) neurons, based on introduction of the antibody against the C2A domain into cells via mechanical lesions. Recently, however, the functional block antibody against the C2A domain was shown to block axonal repair processes, which raised a question as to whether Syt I is indeed involved in neurite outgrowth. In this study, we expressed Syt I or II in PC12 cells and found that these expression did indeed promote neurite outgrowth, as compared to control cells. We further showed that expression of the phospholipid binding activity-deficient mutant of Syt II (Delta180-183) had little effect on the neurite outgrowth of PC12 cells. These results indicate the Ca(2+)/phospholipid binding site of Syt I or II to be essential for neurite outgrowth.

Animals↗
PubMed2000

Calcium stores regulate the polarity and input specificity of synaptic modification.

Activity-induced synaptic modification is essential for the development and plasticity of the nervous system. Repetitive correlated activation of pre- and postsynaptic neurons can induce persistent enhancement or decrement of synaptic efficacy, commonly referred to as long-term potentiation or depression (LTP or LTD). An important unresolved issue is whether and to what extent LTP and LTD are restricted to the activated synapses. Here we show that, in the CA1 region of the hippocampus, reduction of postsynaptic calcium influx by partial blockade of NMDA (N-methyl-D-aspartate) receptors results in a conversion of LTP to LTD and a loss of input specificity normally associated with LTP, with LTD appearing at heterosynaptic inputs. The induction of LTD at homo- and heterosynaptic sites requires functional ryanodine receptors and inositol triphosphate (InsP3) receptors, respectively. Functional blockade or genetic deletion of type 1 InsP3 receptors led to a conversion of LTD to LTP and elimination of heterosynaptic LTD, whereas blocking ryanodine receptors eliminated only homosynaptic LTD. Thus, postsynaptic Ca2+, deriving from Ca2+ influx and differential release of Ca2+ from internal stores through ryanodine and InsP3 receptors, regulates both the polarity and input specificity of activity-induced synaptic modification.

Animals↗
PubMed2000

Molecular properties of Zic proteins as transcriptional regulators and their relationship to GLI proteins.

Zic family genes encode zinc finger proteins, which play important roles in vertebrate development. The zinc finger domains are highly conserved between Zic proteins and show a notable homology to those of Gli family proteins. In this study, we investigated the functional properties of Zic proteins and their relationship to the GLI proteins. We first established an optimal binding sequence for Zic1, Zic2, and Zic3 proteins by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence was almost identical to the GLI binding sequence. However, the binding affinity was lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins was less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activated a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins were cotransfected into cultured cells, Zic proteins enhanced or suppressed sequence-dependent, GLI-mediated transactivation depending on cell type. Taken together, these results suggest that Zic proteins may act as transcriptional coactivators and that their function may be modulated by the GLI proteins and possibly by other cell type-specific cofactors.

Base Sequence↗
PubMed2000

Drosophila AD3 mutation of synaptotagmin impairs calcium-dependent self-oligomerization activity.

Genetic analysis of a Drosophila synaptotagmin (Syt) I mutant (AD3) has revealed that Tyr-334 within the C2B domain is essential for efficient Ca(2+)-dependent neurotransmitter release. However, little is known as to why a missense mutation (Tyr-334-Asn) disrupts the function of the C2B domain at the molecular level. Here, we present evidence that a Tyr-312 to Asn substitution in mouse Syt II, which corresponds to the Drosophila AD3 mutation, completely impairs Ca(2+)-dependent self-oligomerization activity mediated by the C2B domain but allows partial interaction with wild-type proteins in a Ca(2+)-dependent manner. This observation is consistent with the fact that the AD3 allele is homozygous lethal but complements another mutant phenotype. We also showed that the Ca(2+)-dependent C2B self-oligomerization is inhibited by inositol 1,3,4, 5-tetrakisphosphate, a potent inhibitor of neurotransmitter release. All of these findings strongly support the idea that self-oligomerization of Syt I or II is essential for neurotransmitter release in vivo.

Amino Acid Sequence↗
PubMed2000

Galphas family G proteins activate IP(3)-Ca(2+) signaling via gbetagamma and transduce ventralizing signals in Xenopus.

During early embryonic development, IP(3)-Ca(2+) signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, we examined effects of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies showed direct inhibition of their targets, their effects on redirection of the ventral mesoderm to a dorsal fate varied. Anti-Galphas/olf antibody showed strong induction of dorsal fate, anti-Galphai/o/t/z antibody did so weakly, and anti-Galphaq/11 antibody was without effect. Injection of betaARK, a Gbetagamma inhibitor, mimicked the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration which inhibited Galphas-coupled cAMP increase did not do so. The activation of Galphas-coupled receptor gave rise to Ca(2+) transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetagamma, which then stimulates PLCbeta and then the IP(3)-Ca(2+) system. This signaling pathway may play a crucial role in transducing ventral signals.

Amino Acid Sequence↗
PubMed2000

Doc2gamma, a third isoform of double C2 protein, lacking calcium-dependent phospholipid binding activity.

The Doc2 (double C2) family consists of two isoforms (Doc2alpha and Doc2beta) characterized by an N-terminal Munc13-1 interacting domain (Mid) and two C2 domains that interact with Ca(2+) and phospholipid at the C-terminus. This Ca(2+)-binding property is thought to be important to the regulation of neurotransmitter release. In this paper, we report a third isoform of mouse Doc2, named Doc2gamma. Doc2gamma also contains a putative Mid domain and two C2 domains, and it is 45.6 and 43.2% identical to mouse Doc2alpha and Doc2beta, respectively, at the amino acid level. In contrast to the other Doc2 isoforms, the C2 domains of Doc2gamma impair Ca(2+)-dependent phospholipid binding activity. The highest expression of Doc2gamma mRNA was found in the heart, but occurs ubiquitously, the same as Doc2beta. These findings indicate that Doc2gamma may also function as an effector for Munc13-1 and that it may be involved in the regulation of vesicular trafficking.

Amino Acid Sequence↗
PubMed2000

Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII.

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, showed both Ca(2+)-dependent (via the C2 domain) and -independent (via the NH(2)-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca(2+)-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca(2+)-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca(2+)-dependent self-oligomerization at the cytoplasmic domain with EC(50) values of about 150 micrometer Ca(2+). In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca(2+)-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca(2+)-dependent manner without being tethered at the NH(2)-terminal domain, whereas Ca(2+)-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH(2)-terminal domain.

Animals↗
PubMed2000

Combination of biomolecular interaction analysis and mass spectrometric amino acid sequencing.

We describe an approach for the combination of biomolecular interaction analysis (BIA) and electrospray tandem mass spectrometry (ESI/MS/MS) to obtain sequence information on the affinity-bound proteins on the sensor chip of BIA. The procedure is illustrated with stable and unstable interactions of recombinant proteins, i.e., histidine-tagged protein-Ni2+/NTA and 1,4,5-inositol trisphosphate receptor-ligand interactions. The E. coli lysates expressing the recombinant proteins were passed through the sensor chips, and biomolecular interactions were monitored in real time. The molecules detected on the sensor chip were digested by delivering proteolytic enzyme to the sensing flow cells. The resulting on-chip digested peptide mixture at the mid- to low-femtomole level was recovered on a microcapillary reversed-phase precolumn by an on-line system and analyzed using HPLC-MS/MS. In both cases, unambiguous sequence information on the recombinant proteins isolated on the sensor chip was obtained from only a single run of analysis. The combined BIA-MS/MS may prove to be a general and versatile system to discover novel biomolecular interactions and to analyze protein complexes.

Calcium Channels↗
PubMed2000

Membrane topogenesis of a type I signal-anchor protein, mouse synaptotagmin II, on the endoplasmic reticulum.

Synaptotagmin II is a type I signal-anchor protein, in which the NH(2)-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

Amino Acid Sequence↗
PubMed2000

Reelin molecules assemble together to form a large protein complex, which is inhibited by the function-blocking CR-50 antibody.

Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both in vitro and in vivo. The Reelin-Reelin interaction clearly is inhibited by the function-blocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment.

Animals↗
PubMed2000

Inhibition of Fas-mediated fulminant hepatitis in CrmA gene-transfected mice.

Hyperimmune response via Fas/Fas-ligand and perforin/granzyme pathways may be essential in pathogenesis of virus-induced fulminant hepatitis. CrmA inhibits activation of caspases and granzyme B, suggesting it may block these pathways. We investigated whether CrmA expression would inhibit Fas-associated lethal hepatitis in mice. We successfully generated AxCALNLCrmA, a recombinant adenovirus expressing CrmA gene with a Cre-mediated switching cassette. We increased CrmA expression level in the liver transfected with AxCALNLCrmA (10(9) pfu) by increasing administration dose (10(7)-10(9) pfu) of AxCANCre, a recombinant, adenovirus-expressing Cre gene. Injection of anti-Fas antibody into the control mice rapidly led to animal death due to massive liver apoptosis, while the apoptosis was dramatically reduced in the CrmA-expressed mice. The animal survival increased with an increase of CrmA expression. The formation of active caspase-3 was markedly inhibited in the crmA-transfected hepatocytes in vitro. These results suggest that crmA is an effective gene that can inhibit immune-related liver apoptosis.

Adenoviridae↗
PubMed2000

Impaired learning with enhanced hippocampal long-term potentiation in PTPdelta-deficient mice.

Protein tyrosine phosphatase delta (PTPdelta) is a receptor-type PTP expressed in the specialized regions of the brain including the hippocampal CA2 and CA3, B lymphocytes and thymic medulla. To elucidate the physiological roles of PTPdelta, PTPdelta-deficient mice were produced by gene targeting. It was found that PTPdelta-deficient mice were semi-lethal due to insufficient food intake. They also exhibited learning impairment in the Morris water maze, reinforced T-maze and radial arm maze tasks. Interestingly, although the histology of the hippocampus appeared normal, the magnitudes of long-term potentiation (LTP) induced at hippocampal CA1 and CA3 synapses were significantly enhanced in PTPdelta-deficient mice, with augmented paired-pulse facilitation in the CA1 region. Thus, it was shown that PTPdelta plays important roles in regulating hippocampal LTP and learning processes, and that hippocampal LTP does not necessarily positively correlate with spatial learning ability. To our knowledge, this is the first report of a specific PTP involved in the regulation of synaptic plasticity or in the processes regulating learning and memory.

Animals↗
PubMed2000

Identification of the C-terminal activation domain of the NeuroD-related factor (NDRF).

NeuroD-related factor (NDRF) is a basic helix-loop-helix (bHLH) protein whose expression is restricted to the central nervous system, and is considered to be responsible for maintenance of differentiated neurons as well as neurogenesis. NDRF structurally resembles NeuroD in the bHLH region and can induce neurogenesis ectopically in ectodermal cells of the Xenopus embryo. In this study, we delineated the functional domains of NDRF. Using GAL4/NDRF fusion proteins, we identified the C-terminal activation domain (C-AD) in NDRF between amino acid positions 294 and 383. This region was highly homologous to one part of the activation domain of NeuroD. We further investigated the transactivational function of C-AD in the mouse type 1 inositol 1,4,5-trisphosphate receptor promoter, which has an NDRF site. Truncation of C-AD resulted in reduction of the activation function, whereas the DNA-binding specificity was not affected. These results suggest that C-AD has a stimulatory function in the mammalian nervous system.

Amino Acid Sequence↗
PubMed2000

A 5' segment of the mouse Zic1 gene contains a region specific enhancer for dorsal hindbrain and spinal cord.

Zic1 encodes a zinc finger protein, which is required for the development of the dorsal neural tissue. The gene is a mammalian homologue of the Drosophila odd-paired. We examined the regulatory elements in the 5' flanking region of the Zic1 gene as an initial step to understanding how the Zic1 expression is restricted to the dorsal neural tissue. When a 2.9-kb fragment of the 5' flanking segment of the mouse Zic1 gene was linked to the E. coli beta-galactosidase gene, the enzyme was consistently expressed in the dorsal half of the embryonic spinal cord and in the vestibulocochlear nucleus in all four transgenic mouse lines. The transgene expression mimics the Zic1 expression with respect to the region where it occurs. But this is not so for the neuronal cell types. This suggests that the segment contains a region-specific enhancer. In vivo and in vitro deletion analyses indicated that there are essential regions between -2.0 and -0.9 kb and within the proximal 0.9 kb. The distal element is necessary for the transgene expression in the embryonic dorsal spinal cord whereas the adult vestibulocochlear nucleus expression is regulated by both elements. In these regions, there are sequences similar to the binding sequences for potential regulatory proteins.

5' Untranslated Regions↗