Epidemiology of nosocomial infections at Fukuoka University Hospital.
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Bacterial Infections↗
Biomedical subjects
T Furuichi
Publications and source records attributed to T Furuichi.
[Postnatal development of the cerebellar circuit and gene expression].
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Actins↗
Inositol 1,4,5-trisphosphate receptors are strongly expressed in the nervous system, pharynx, intestine, gonad and excretory cell of Caenorhabditis elegans and are encoded by a single gene (itr-1).
Inositol 1,4,5-trisphosphate (InsP3) activates receptors (InsP3Rs) that mediate intracellular Ca(2+ )release, thereby modulating intracellular calcium signals and regulating important aspects of cellular physiology and gene expression. To further our understanding of InsP3Rs we have characterised InsP3Rs and the InsP3R gene, itr-1, from the model organism Caenorhabditis elegans. cDNAs encoding InsP3Rs were cloned enabling us to: (a) identify three putative transcription start sites that result in alternative mRNA 5' ends: (b) detect alternative splicing at three sites and: (c) determine the full genomic organisation of the itr-1 gene. The InsP3R protein (ITR-1) is approximately 42 % identical with known InsP3Rs and possesses conserved structural features. When the putative InsP3 binding domain was expressed in Escherichia coli, specific binding of InsP3 was detected. Using antibodies against ITR-1 we detected a protein of 220 kDa in C. elegans membranes. These antibodies and itr-1::GFP (green fluorescent protein) reporter constructs were used to determine the expression pattern of itr-1 in C. elegans. Strong expression was observed in the intestine, pharynx, nerve ring, excretory cell and gonad. These results demonstrate the high degree of structural and functional conservation of InsP3Rs from nematodes to mammals and the utility of C. elegans as a system for studies on InsP3R mediated signalling.
Amino Acid Sequence↗
Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner.
The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.
Amino Acid Sequence↗
Microinjection of Ca2+ store-enriched microsome fractions to dividing newt eggs induces extra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2+ release.
The cleavage signal transferred to the future cleavage cortex during anaphase has been proposed as "cleavage stimulus," but no signal has proved to induce cleavage furrows. The local Ca2+ transient along the cleavage furrow has been reported, but the Ca2+ source has remained unknown. To address these questions, we studied functions of Ca2+ stores in dividing newt eggs and found that microinjection of the Ca2+ store-enriched microsome fraction to the dividing newt egg induced a local extra-cleavage furrow at the injection site in 64-67% of the injected newt eggs while coinjection with inositol 1,4, 5-trisphosphate receptor (IP(3)R) antagonists heparin or anti-type 1-IP(3)R antibody clearly suppressed this induction (5 and 11% in induction rates, respectively). Injection of cerebellar microsomes from the type 1-IP(3)R-deficient mice induced extracleavage furrows albeit at a low rate (19%). Our observations strongly suggest that Ca2+ stores with IP(3)R induce and position a cleavage furrow via IP(3)-induced Ca2+ release (IICR) as Ca(2+)-releasing machinery and putative cleavage stimulus itself.
Anaphase↗
Cupidin, an isoform of Homer/Vesl, interacts with the actin cytoskeleton and activated rho family small GTPases and is expressed in developing mouse cerebellar granule cells.
A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Delta11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin-mGluR1-actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent manner in vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.
Actins↗
Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.
Our previous studies have demonstrated that calmodulin binds to IP3R type I (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real-time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3-induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of k(a) = 4.46 x 10(2) and > 10(4) M(-1) s(-1) k(d) = 1.44 x 10(-2) and 1.17 x 10(-1) s(-1). The apparent dissociation constant was calculated to be 27.3 microM. The IP3-induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3-induced Ca2+ release by Ca2+.
Animals↗
Real time analysis of interaction between inositol 1,4, 5-trisphosphate receptor type I and its ligand.
Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger that releases intracellular Ca(2+) by binding to its specific receptor, inositol 1,4,5-trisphosphate receptor (IP(3)R), in a wide range of cellular processes. We report here large-scale expression and purification of N-terminal 604 amino acids of IP(3)R type 1 (T604) expressed in E. coli, which contains the ligand binding domain. Surface plasmon resonance biosensor studies showed that purified T604 could bind to its ligands with binding specificity identical to that of full-length native IP(3)R type 1. Kinetic parameters of T604 for IP(3) consisted of a fast association rate constant (K(ass) = 1.2 x 10(6) M(-1) s(-1)) and a rapid dissociation rate constant (k(diss) = 1 s(-1)), and the equilibrium dissociation constant was determined to be 336 nM, at 150 mM NaCl and pH 7.4. However, association and dissociation patterns depended on the pH level and ionic strength. These results pave the way toward detail analysis of structure-function analysis of the ligand binding domain of IP(3)R type 1 for its ligands.
Amino Acid Sequence↗
High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli.
Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3.
Amino Acid Substitution↗
Inositol 1,4,5-trisphosphate receptor down-regulation is activated directly by inositol 1,4,5-trisphosphate binding. Studies with binding-defective mutant receptors.
Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate (InsP3) receptors and, thus, lead to InsP3 receptor down-regulation. To gain insight into this process, we examined whether or not InsP3 receptor degradation is a direct consequence of InsP3 binding by analyzing the down-regulation of exogenous wild-type and binding-defective mutant InsP3 receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP3 binding appears to mediate down-regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP3-induced Ca2+ release in permeabilized cells, presumably by increasing the number of InsP3-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobilization and InsP3 formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down-regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SH-SY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP3 binding directly activates InsP3 receptor degradation. Because InsP3 binding induces a conformational change in the InsP3 receptor, these data suggest that this change provides the signal for accelerated proteolysis.
Amino Acid Sequence↗
Trypsinized cerebellar inositol 1,4,5-trisphosphate receptor. Structural and functional coupling of cleaved ligand binding and channel domains.
The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a tetrameric intracellular inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ release channel (calculated molecular mass = approximately 313 kDa/subunit). We studied structural and functional coupling in this protein complex by limited (controlled) trypsinization of membrane fractions from mouse cerebellum, the predominant site for IP3R1. Mouse IP3R1 (mIP3R1) was trypsinized into five major fragments (I-V) that were positioned on the entire mIP3R1 sequence by immuno-probing with 11 site-specific antibodies and by micro-sequencing of the N termini. Four fragments I-IV were derived from the N-terminal cytoplasmic region where the IP3-binding region extended over two fragments I (40/37 kDa) and II (64 kDa). The C-terminal fragment V (91 kDa) included the membrane-spanning channel region. All five fragments were pelleted by centrifugation as were membrane proteins. Furthermore, after solubilizing with 1% Triton X-100, all were co-immunoprecipitated with the C terminus-specific monoclonal antibody that recognized only the fragment V. These data suggested that the native mIP3R1-channel is an assembly of four subunits, each of which is constituted by non-covalent interactions of five major, well folded structural components I-V that are not susceptible to attack by mild trypsinolysis. Ca2+ release experiments further revealed that even the completely fragmented mIP3R1 retained significant IP3-induced Ca2+ release activity. These data suggest that structural coupling among five split components conducts functional coupling for IP3-induced Ca2+ release, despite the loss of peptide linkages. We propose structural-functional coupling in the mIP3R1, that is neighboring coupling between components I and II for IP3 binding and long-distant coupling between the IP3 binding region and the channel region (component V) beyond trypsinized gaps for ligand gating.
Amino Acid Sequence↗
Cooperative formation of the ligand-binding site of the inositol 1,4, 5-trisphosphate receptor by two separable domains.
Limited trypsin digestion of mouse cerebellar membrane fractions leads to fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316-327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity. This is despite the fact that the IP3-binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core. Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathione S-transferase (GST) fusion proteins, which were respectively composed of residues 1-343 and 341-604, almost corresponding to two split binding components, and separately expressed in Escherichia coli. The GST-fused residues 1-343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341-604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor. Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared. We suggest that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity.
Animals↗
Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor.
The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.
Animals↗
Transcriptional regulation of mouse type 1 inositol 1,4,5-trisphosphate receptor gene by NeuroD-related factor.
The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a Ca2+ channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box-I element, which is located -334 relative to the transcription initiation site of the mouse IP3R1 gene and includes an E-box consensus sequence, is involved in the up-regulation of such IP3R1 gene expression. Furthermore, the previous study also indicated that some CNS-related basic helix-loop-helix (bHLH) factors bind to the box-I and activate IP3R1 gene expression. In this study, we demonstrated that one of the CNS-related bHLH factors, neuronal differentiation factor (NeuroD)-related factor (NDRF), specifically bound to the box-I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP3R1 gene expression. In situ hybridization of adult mouse brain revealed that IP3R1 and NDRF mRNA were co-expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue-specific regulation of IP3R1 gene expression in the CNS.
Animals↗
Effects of long-term use of benzodiazepines on gait and standing balance in the elderly.
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Accidental Falls↗
Cloning and characterization of two genes encoding dihydroxyacetone kinase from Schizosaccharomyces pombe IFO 0354.
We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354. The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively. Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products. A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose. A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S. pombe 972h-.
Amino Acid Sequence↗
T-cell-receptor signalling in inositol 1,4,5-trisphosphate receptor (IP3R) type-1-deficient mice: is IP3R type 1 essential for T-cell-receptor signalling?
Stimulation of T-cells via the T-cell receptor (TCR) complex is accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i). Recently, it was reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of inositol 1,4,5-trisphosphate receptor (IP3R) type 1 (IP3R1), failed to demonstrate increased [Ca2+]i or interleukin-2 production after TCR stimulation and was also resistant to apoptotic stimuli. This cell line lacked IP3R1 expression, but expressed the type-2 and -3 receptors, IP3R2 and IP3R3 respectively [Jayaraman, Ondriasova, Ondrias, Harnick and Marks (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 6007-6011, and Jayaraman and Marks (1997) Mol. Cell. Biol. 17, 3005-3012]. The authors concluded that IP3R1 is essential for TCR signalling and suggested that Ca2+ release via IP3R1 is a critical mediator of apoptosis. To establish whether a loss of IP3R1 function in T-cells occurred in vivo and in vitro, we investigated Ca2+ signalling after TCR stimulation and the properties of T-cells using IP3R1-deficient (IP3R1-/-) mice. As IP3R1-/- mice die at weaning, we transplanted bone marrow cells of IP3R1-/- mice into irradiated wild-type mice. Western blot analysis showed that the recipient IP3R1-containing (IP3R1+/+) lymphocytes were replaced by the donor IP3R1-/- lymphocytes after transplantation and that expression of IP3R2 and IP3R3 was unaltered. In contrast with the previous reports, T-cells lacking IP3R1 were able to mobilize Ca2+ from intracellular Ca2+ stores after stimulation via the TCR. We observed no significant differences between IP3R1+/+ and IP3R1-/- T-cells in terms of the number of thymocytes and splenocytes, the proportion of the T-cell phenotype, proliferative response to anti-CD3 monoclonal antibody (mAb) stimulation and cell viability. Therefore IP3R1 is not essential for T-cell development and function.
Animals↗
Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal epithelium of submandibular gland.
In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some "pioneer cells," rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.
Animals↗