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T Haga

Publications and source records attributed to T Haga.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cloning and expression of a cDNA encoding a new neurocalcin isoform (neurocalcin alpha) from bovine brain.

Neurocalcin (NC), a neuron-specific EF-hand Ca2+-binding protein, purified from bovine brain [Terasawa, Nakano, Kobayashi and Hidaka (1992) J. Biol. Chem. 267, 19596-19599] contains multiple isoforms. We previously cloned NCdelta from bovine brain and showed high expression in neuronal tissues [Okazaki, Watanabe, Ando, Hagiwara, Terasawa and Hidaka (1992) Biochem. Biophys. Res. Commun. 185, 147-153]. We report here the molecular cloning and expression of a cDNA encoding bovine brain NCalpha. The translated bovine protein is 191 amino acids long and shares 69.1% of its amino acid sequence with NCdelta. Recombinant NCalpha migrates as a single 23 kDa band and exhibits a Ca2+-dependent mobility shift on SDS/PAGE. Analysis of fluorescence emission spectra showed the Ca2+-induced peak at 337 nm. Interestingly, the mobility shift and the fluorescence intensity at 337 nm were larger for NCalpha than for NCdelta. In Ca2+-overlay experiments, however, the apparent affinity of NCalpha for 45Ca2+ was similar to that of NCdelta. Immunohistochemical analysis revealed NCalpha expression in the granular layer of the rat cerebellar cortex whereas NCdelta was found in the Purkinje cell layer. In the rat olfactory bulb, NCalpha was located in external tufted cells, and NCdelta was found in the periglomerular cells. These data demonstrate that NC isoforms differ in their tissue distribution and conformational changes induced by Ca2+ binding. Thus differential regulation of the two NC isoforms may be involved in control of neuron function.

Amino Acid Sequence↗
PubMed1998

Design of beamline optics for EUVL.

The design of front-end collimating optics for extreme-ultraviolet lithography (EUVL) is reported. For EUVL, collimating optics consisting of a concave toroidal mirror and a convex toroidal mirror can achieve shorter optical path lengths than collimating optics consisting of two concave toroidal mirrors. Collimating optics consisting of a concave toroidal mirror and a convex toroidal mirror are discussed. The design of collimating optics for EUVL beamlines based on ray-tracing studies is described.

Journal Article↗
PubMed1998

Soft X-ray multilayer beam splitters.

A semitransparent Mo/Si multilayer beam splitter with a completely self-standing active area (10 x 10 mm) and a flatness of 1.1 nm (r.m.s.) was fabricated. The influence of the roughness of the membrane substrate on the reflectivity of a beam splitter was investigated for different materials and deposition schemes. Precise control of multilayer stress to give a slightly tensile state not only enables the fabrication of a large and flat reflection surface, but also makes it possible to etch away the supporting membrane and obtain a completely self-standing structure. The performance evaluation using synchrotron radiation revealed that the fabricated beam splitter works as a one-to-one beam splitter whose reflectivity and transmittance are both 27% (s-polarization, 45 degrees, lambda = 13.4 nm).

Journal Article↗
PubMed1998

Design and fabrication of highly heat-resistant Mo/Si multilayer soft X-ray mirrors with interleaved barrier layers.

Introducing interleaved carbon barrier layers improves the heat-resistance of Mo/Si multilayers. The soft X-ray reflectivities of the multilayers were calculated, and the effects of heating on both the reflectivities and layer structures of Mo/Si multilayers with and without barrier layers were investigated using X-ray diffraction and transmission electron microscopy. The results show that, for applications using intense soft X-ray beams, Mo/Si multilayers with interleaved carbon barrier layers are better mirrors than Mo/Si multilayers because they have much better heat resistance and almost the same soft X-ray reflectivity as the Mo/Si multilayers.

Journal Article↗
PubMed1998

Role of receptor kinase in short-term desensitization of cardiac muscarinic K+ channels expressed in Chinese hamster ovary cells.

1. The cardiac muscarinic receptor-K+ channel system was reconstructed in Chinese hamster ovary (CHO) cells by transfecting the cells with the various components of the system. The activity of the muscarinic K+ channel was measured with the cell-attached configuration of the patch clamp technique. 2. In CHO cells transfected with the channel (Kir3.1/Kir3.4), receptor (hm2) and receptor kinase (GRK2), on exposure to agonist, there was a decline in channel activity as a result of desensitization, similar to that in atrial cells. 3. Whereas the desensitization was almost abolished by not transfecting with the receptor kinase or by transfecting with a mutant receptor lacking phosphorylation sites, it was only reduced (by approximately 39%) by transfecting with a mutant receptor kinase with little/kinase activity. 4. These results suggest that the receptor kinase is responsible for desensitization of the muscarinic K+ channel and that this involves phosphorylation-dependent and -independent mechanisms.

Animals↗
PubMed1998

Internalization and down-regulation of human muscarinic acetylcholine receptor m2 subtypes. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2.

Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [3H]N-methylscopolamine binding sites from the cell surface, and transfer of [3H]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [3H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in the presence of 10(-4) M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10(-6) M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1/2 = 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but down-regulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loop-independent pathways.

Animals↗
PubMed1998

Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization.

Desensitization of human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [35S]GTPgammaS binding activity in membrane preparations after pre-treatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity was found to decrease to 64% following pretreatment of cells with 10 microM carbamylcholine for 30 min, and under the same conditions 51-59% of hm2 receptors were sequestered/internalized as assessed by decreases in the [3H]N-methylscopolamine binding activity on the cell surface. A similar reduction in the carbamylcholine-stimulated [35S]GTPgammaS binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm2 receptors. When the cells were pretreated with 10 microM carbamylcholine in the presence of 0.32 M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/internalization of hm2 receptors was observed, and the extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity did not change. These results indicate that desensitization of hm2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.

Animals↗
PubMed1998

A new approach to AIDS research and prevention: the use of gene-mutated HIV-1/SIV chimeric viruses for anti-HIV-1 live-attenuated vaccines.

The lack of a suitable animal model is a major obstacle to developing anti-HIV-1 vaccines. We successfully generated an SIVmac/HIV-1 chimeric virus (SHIV) (designated as NM-3rN) that contains the HIV-1 env gene and is infectious to macaque monkeys. Challenging the vaccinated macaque monkeys with NM-3rN, we developed an evaluation system for anti-HIV-1 Env-targeted vaccines. For the purpose of making the vaccine, a series of gene-mutated SHIVs were constructed. The monkeys vaccinated with these SHIVs had long-term anti-virus immunities without manifesting the disease, and became resistant to a challenge inoculation with NM-3rN. The sera from a monkey showed that, after the vaccination, the neutralizing antibodies not only against the parental HIV-1 but also against an antigenically different HIV-1 were raised. In vivo experiments confirmed that the vaccinated monkeys were protected from the challenge inoculum of an antigenically different SHIV-MN. Vaccination of monkeys with the attenuated SHIVs showed that further gene-deletion of the SHIV resulted in less immunogenicity. Nevertheless, the attenuated SHIVs had a vaccine effect against the challenge inoculation. In addition to specific immunities including neutralizing antibodies and cytotoxic T cells, a more complicated immune mechanism induced by live vaccine appears to play a role in this protection. Our data suggest that the live vaccine can induce strong and wide-range immunity against HIV-1. These SHIVs should contribute to understanding the pathogenicity of AIDS and to the development of future anti-HIV-1 live vaccines for humans.

AIDS Vaccines↗
PubMed1998

Effects of an agonist, allosteric modulator, and antagonist on guanosine-gamma-[35S]thiotriphosphate binding to liposomes with varying muscarinic receptor/Go protein stoichiometry.

We investigated whether alcuronium, an allosteric modulator of muscarinic acetylcholine receptors, can induce receptor-mediated activation of Go proteins in liposomal membranes incorporating purified M2 receptors and Go proteins and whether its action is affected by the receptor/Go protein (R/Go) ratio. The binding of guanosine-gamma-[35S]thiotriphosphate ([35S]GTPgammaS) served as the indicator of G protein activation. It was stimulated by empty receptors at high receptor densities, and the dose-response curve was shifted to the left by the agonist carbachol and to the right by the antagonist atropine. At an R/Go ratio of 300:100, the rate of [35S]GTPgammaS binding was the same in the presence or absence of 0. 1 mM carbachol. Alcuronium increased the binding of [35S]GTPgammaS at R/Go ratios of <3:100 and diminished it at R/Go ratios of >10:100, similar to previous observations on intact cells expressing muscarinic receptors at different densities. The apparent biphasicity of alcuronium action indicates that the allosteric modulator has at least two effects on muscarinic receptor/G protein interaction but its mechanistic basis is unclear. The "active state" of muscarinic receptors induced by alcuronium probably is different from that induced by carbachol. Changes in the densities of receptors and Go proteins had little effect on the kinetics of [35S]GTPgammaS binding and on receptor affinity for carbachol, provided the R/Go ratio was kept constant. This suggests that the receptors and G proteins are located in microdomains in which their concentrations remain constant, despite variations in the amounts of lipidic membranes in the system.

Alcuronium↗
PubMed1998

Sequestration of human muscarinic acetylcholine receptor hm1-hm5 subtypes: effect of G protein-coupled receptor kinases GRK2, GRK4, GRK5 and GRK6.

Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4 delta, GRK5 and GRK6 on sequestration of human m1-m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4 delta, GRK5 and GRK6 did not facilitate sequestration of m1-m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4 delta, GRK5 and GRK6. However, coexpression of GRK4 delta, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4 delta, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.

Animals↗
PubMed1997

Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail.

A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H] palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein-coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein beta gamma subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate Gi2 and G(o). However, the rate of [35S] GTP gamma S binding to Gi2 was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42-42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

Animals↗
PubMed1997

Expression of the rat m4 muscarinic acetylcholine receptor gene is regulated by the neuron-restrictive silencer element/repressor element 1.

Neuronal cell-specific expression of the rat m4 muscarinic acetylcholine receptor (mAChR) is regulated by a silencer element. A likely mediator of this silencing is the neuron-restrictive silencer element/repressor element 1 (NRSE/RE1), which is present 837 base pairs (bp) upstream from the transcription initiation site of the m4 mAChR gene (Wood, I. C., Roopra, A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940; Mieda, M., Haga, T., and Saffen, D. W. (1996) J. Biol. Chem. 271, 5177-5182). In the present study, we examined whether this putative NRSE/RE1 functions as a silencer. Transient expression assays using m4 mAChR promoter/luciferase expression vectors showed that the m4 NRSE/RE1 is necessary and sufficient to repress m4 promoter activity in non-neuronal L6 cells. m4 promoter activity was only partially repressed, however, in neuronal NG108-15 cells exogenously expressing the neuronal-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST). By contrast, the promoter activity of the type II sodium channel (NaII) gene was nearly completely repressed in NRSF/REST-expressing NG108-15 cells. Experiments with expression vectors containing chimeric promoters revealed that the NRSE/RE1 elements derived from both the m4 and NaII genes are independently sufficient to silence NaII gene promoter activity, but only partially repress m4 mAChR gene promoter activity in NRSF/REST-expressing NG108-15 cells. Thus, the repression activity of NRSF/REST depends upon the species of promoter to which it is linked. Gel-shift assays showed that the NRSF/REST is the only protein that binds to a 92-bp segment from the m4 mAChR promoter containing NRSE/RE1. This and the fact that m4 promoter activity was completely repressed in L6 cells suggest that the proteins that bind to the m4 constitutive promoter may be different from those in NG108-15 cells. Deletion analysis of the m4 constitutive promoter revealed that a 90-bp segment immediately upstream from the transcription initiation site contains significant promoter activity. Gel-shift assays revealed that several proteins in nuclear extracts prepared from L6 and NG108-15 cells bind to this 90-bp segment and that some of these proteins are L6 or NG108-15 cell-specific. These data support the idea that the repression activity of NRSF/REST depends upon the species of promoter to which it is linked and upon the proteins that bind to those promoters.

Animals↗
PubMed1997

Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.

Animals↗
PubMed1996

Interactions between wild-type and mutant prion proteins modulate neurodegeneration in transgenic mice.

Transgenic mice overexpressing approximately eightfold the mouse (Mo) prion protein (PrP) gene carrying the P102L mutation of GSS developed neurodegeneration between 150 and 300 days of age, while controls expressing the wild-type MoPrP-A transgene at the same level remained healthy. Mice overexpressing the wild-type MoPrP-A transgene were highly susceptible to inoculated mouse prions, exhibiting abbreviated scrapie incubation times of 45 days. After crossing the mutant transgene onto a null (Prnp 0/0) background, the resulting Tg(MoPrP-P101L)Prnp 0/0 mice displayed a highly synchronous onset of illness at 145 days of age, which was shortened to 85 days upon breeding to homozygosity for the transgene array. Besides occasional PrP plaques and modest spongiform degeneration, Tg(MoPrP-P101L) mice suffered from a myopathy and a peripheral neuropathy. Disruption of the wild-type MoPrP gene increased the number of PrP plaques and the severity of spongiform degeneration. Brain extracts prepared from spontaneously ill transgenic mice transmitted disease to Tg196/Prnp 0/0 mice, expressing low levels of the mutant transgene. Our results demonstrate that the presence of wild-type PrP genes, the level of PrP transgene expression, and the sequence of the transgene can profoundly modify experimental prion disease.

Animals↗
PubMed1996

Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation.

In rod photoreceptor cells, Ca2+-bound recoverin associates with disk membranes and inhibits light-dependent phosphorylation of rhodopsin. However, the functional significance of Ca2+-induced membrane association of recoverin has not been fully evaluated. We found that Ca2+-bound recoverin forms a complex with rhodopsin kinase preferentially at the membrane surface. Addition of increasing amounts of membranes promoted the membrane association of recoverin, and remarkably suppressed rhodopsin kinase activity. It was concluded that the Ca2+-recoverin-rhodopsin kinase complex is stabilized by membrane association, leading to effective suppression of the kinase activity.

Animals↗