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T Haga

Publications and source records attributed to T Haga.

At least 37 records · Page 2Linked to original sources
PubMed2000

Construction of chimeric simian and human immunodeficiency viruses that produce interleukin 12.

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for evaluating vaccine candidates against HIV-1 and for investigating the pathogenesis of HIV-1 in vivo. In addition, SHIVs are candidates for a vaccine against HIV-1 because attenuated SHIVs can induce long-lasting anti-HIV-1 Env humoral and cell-mediated immunity in monkeys without AIDS-like diseases. In this study, we inserted IL-12 genes in a nef-deleted SHIV to increase the ability of the SHIV to induce cell-mediated immunity against HIV-1. The SHIV vector was constructed by deleting the nef gene and replacing it with restriction enzyme sites. Since IL-12 consists of two subunit genes, p35 and p40, SHIVs with one or both of these genes were constructed. SHIVs with either one of the subunit genes could replicate without a deletion of the inserted gene, but SHIVs with two subunit genes replicated poorly and the inserted genes were rapidly deleted. Production of IL-12 was detected when both of the single-subunit SHIVs were coinfected. The production of IL-12 by the coinfection reached 800 pg/ml, and IL-12 was detected after serial passage in cell cultures, although this amount of IL-12 heterodimer was 150-1500 times less than that of the p40 subunits. These IL-12-producing SHIVs are candidates for a live-attenuated vaccine to induce effective cellular immunity against HIV-1.

Cell Line↗
PubMed2000

Identification and characterization of the high-affinity choline transporter.

In cholinergic neurons, high-affinity choline uptake in presynaptic terminals is the rate-limiting step in acetylcholine synthesis. Using information provided by the Caenorhabditis elegans Genome Project, we cloned a cDNA encoding the high-affinity choline transporter from C. elegans (cho-1). We subsequently used this clone to isolate the corresponding cDNA from rat (CHT1). CHT1 is not homologous to neurotransmitter transporters, but is homologous to members of the Na+-dependent glucose transporter family. Expression of CHT1 mRNA is restricted to cholinergic neurons. The characteristics of CHT1-mediated choline uptake essentially match those of high-affinity choline uptake in rat brain synaptosomes.

Amino Acid Sequence↗
PubMed2000

Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli.

The M2 muscarinic acetylcholine receptor mutant (M2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as [3H]L-quinuclidinyl benzilate ([3H]QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C. No [3H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C. The MBP-M2 mutant expressed in E. coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [(3)H]QNB with carbamylcholine and atropine. The MBP-M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gi1 in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [35S]GTP gamma S binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [(35)S]GTP gamma S binding were the same as those observed for the M2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor.

ATP-Binding Cassette Transporters↗
PubMed2000

Molecular modeling of G-protein coupled receptor kinase 2: docking and biochemical evaluation of inhibitors.

G-protein coupled receptor kinase 2 (GRK2) regulates the activity of many receptors. Because potent inhibitors of GRK2 are thus far limited to polyanionic compounds like heparin, we searched for new inhibitors with the aid of a molecular model of GRK2. We used the available crystal structure of cAMP dependent protein kinase (cAPK) as a template to construct a 3D homology model of GRK2. Known cAPK and GRK2 inhibitors were docked into the active sites of GRK2 and cAPK using DOCK v3.5. H8 docked into the hydrophobic pocket of the adenosine 5'-triphosphate (ATP) binding site of cAPK, consistent with its known competitive cAPK inhibition relative to ATP. Similarly, 3 of 4 known GRK2 inhibitors docked into the ATP binding pocket of GRK2 with good scores. Screening the Fine Chemicals Directory (FCD, containing the 3D structures of 13,000 compounds) for docking into the active sites of GRK2 identified H8 and the known GRK2 inhibitor trifluoperazine as candidates. Whereas H8 indeed inhibited light-dependent phosphorylation of rhodopsin by GRK2, but with low potency, 3 additional FCD compounds with promising GRK2 scores failed to inhibit GRK2. This result demonstrates limitations of the GRK2 model in predicting activity among diverse chemical structures. Docking suramin, an inhibitor of protein kinase C (not present in FCD) yielded a good fit into the ATP binding site of GRK2 over cAPK. Suramin did inhibit GRK2 with IC50 32 microM (pA26.39 for competitive inhibition of ATP). Suramin congeners with fewer sulfonic acid residues (NF062, NF503 [IC50 14 microM]) or representing half of the suramin molecule (NF520) also inhibited GRK2 as predicted by docking. In conclusion, suramin and analogues are lead compounds in the development of more potent and selective inhibitors of GRK2.

Animals↗
PubMed2000

Plasma levels of the chemokine RANTES in macaque monkeys infected with pathogenic and non-pathogenic SIV/HIV-1 chimeric viruses at an early stage of infection.

Plasma levels of the chemokine RANTES were examined in monkeys infected with either a pathogenic simian and human immunodeficiency chimeric virus (SHIV) or a non-pathogenic SHIV to determine whether RANTES levels were related to the pathogenicity of the virus, the plasma viral load, or the kinetics of CD4+ T-cells. In the results no significant correlation was found between the RANTES kinetics and changes in the CD4+ T-cell numbers nor the plasma viral loads in any of the monkeys, although a transient decrease of the RANTES level was observed in the pathogenic virus-infected monkeys. At least, the plasma RANTES level can not be used as an index of the pathogenicity of the virus at the early stage of infection.

Animals↗
PubMed2000

Polymorphism of the D12S66 system in the Japanese population and its detection using degraded DNA.

The short tandem repeat (STR) polymorphism of the locus D12S66 was amplified by PCR and analyzed by denaturing polyacrylamide gel electrophoresis followed by silver staining. Among 190 DNA samples from the Japanese population, six alleles were observed. The genotypic distribution meets Hardy-Weinberg expectations, and the heterozygosity was 52.7%. When sequences of the allelic products were compared, each allelic segment was 153-173 bp in size, and contained 9 to 14 GATA tetranucleotide repeat motifs. Amplification of the locus using 27 tooth and blood stain samples as sources of degraded DNA resulted in low backgrounds and reproducible patterns, suggesting the usefulness of the application of this locus for material examination.

Adenine↗
PubMed2000

RGS domain in the amino-terminus of G protein-coupled receptor kinase 2 inhibits Gq-mediated signaling.

We have previously shown that not only G protein-coupled receptor kinase (GRK) 2, but also a catalytically inactive Lys220Trp GRK2 decreases endothelin (ET)-1-induced inositol 1,4,5-trisphosphate (IP3) formation, and demonstrated the presence of phosphorylation-independent desensitization mechanism. To clarify the role of GRK2 other than that as a kinase, we characterized an RGS (regulator of G protein signaling)-like domain in the amino-terminus of GRK2. Both GRK2(1-181) and GRK2(54-174) suppressed Ca2+ responses induced by angiotensin II (Ang II) and ET-1, and bound directly with Galphaq but not Galphas nor Galphai3 in the presence of GDP and AlF4-. These results demonstrate that GRK2 regulates Gq-mediated signaling negatively by direct interaction between its RGS domain and the transitional state of Galphaq, as well as through phosphorylation of activated receptors by its kinase domain.

Aluminum Compounds↗
PubMed1999

Functional identification and reconstitution of an odorant receptor in single olfactory neurons.

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand-receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription-coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Adenoviridae↗
PubMed1999

Control elements of muscarinic receptor gene expression.

Studies describing the structures of the M1, M2 and M4 muscarinic acetylcholine receptors (mAChR) genes and the genetic elements that control their expression are reviewed. In particular, we focus on the role of the neuron-restrictive silencer element/restriction element-1 (NRSE/RE-1) in the regulation of the M4 mAChR gene. The NRSE/RE-1 was first identified as a genetic control element that prevents the expression of the SCG-10 and type II sodium channel (NaII) genes in non-neuronal cells in culture. The NRSE/RE-1 inhibits gene expression by binding the repressor/silencer protein NRSF/REST, which is present in many non-neuronal cell lines and tissues. Our studies show that although the expression of the M4 mAChR gene is inhibited by NRSF/REST, this inhibition is not always complete. Rather, the efficiency of silencing by NRSF/REST is different in different cells. A plausible explanation for this differential silencing is that the NRSF/RE-1 interacts with distinct sets of promoter binding proteins in different types of cells. We hypothesize that modulation of NRSF/REST silencing activity by these proteins contributes to the cell-specific pattern of expression of the M4 mAChR in neuronal and non-neuronal cells. Recent studies that suggest a more complex role for the NRSE/RE-1 in regulating gene expression are also discussed.

Animals↗
PubMed1999

Gene-mutated HIV-1/SIV chimeric viruses as AIDS live attenuated vaccines for potential human use.

To develop an AIDS vaccine for human use as well as a suitable animal model for AIDS research, we constructed a series of HIV-1/SIVmac chimeric viruses (SHIVs). We successfully generated a SHIV (designated as NM-3rN) having the HIV-1 env gene, which enabled the evaluation of the efficacy of HIV-1 Env-targeted vaccines in macaque monkeys instead of chimpanzees. Two NM-3rN derivatives (NM-3 and NM-3n) induced long-term anti-virus immunities without manifesting the disease. The monkeys vaccinated with NM-3 or NM-3n became resistant to a challenge inoculation with NM-3rN. Serum from a monkey vaccinated with NM-3 neutralized not only the parental HIV-1 (NL432), but also an antigenically different HIV-1 (MN). In vivo experiments confirmed the heterologous protection against an SHIV having the HIV-1 (MN) env. In addition to specific immunity including neutralizing antibodies and cytotoxic T lymphocyte activity, nonspecific immunity such as natural killer activity is associated with this protection. These data suggest that the live vaccine has the ability to protect individuals against various types of HIVs. These SHIVs should contribute to the development of future anti-HIV-1 live vaccines in humans.

AIDS Vaccines↗
PubMed1999

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.

Animals↗
PubMed1999

Dynamin and rab5 regulate GRK2-dependent internalization of dopamine D2 receptors.

Dopamine D2 receptors (D2Rs; short form, which is one of the alternative splicing variants) expressed in COS-7 cells are internalized in an agonist-dependent manner only when G protein-coupled receptor kinase 2 (GRK2) is coexpressed [Ito, K., Haga, T., Lameh, J. & Sadée, W., (1999) Eur. J. Biochem. 260, 112-119]. We have examined the effects of coexpression of dynamin, a small molecular mass GTP-binding protein, rab5A, and their mutants on the internalization of D2Rs in the presence of both dopamine (10 or 100 microM) and GRK2. The rate and extent of D2R internalization was increased or decreased by coexpression of dynamin I or a dominant-negative form of dynamin I (dynamin I K44E), respectively. The effects of coexpressing these two dynamins were more prominent at 10 microM dopamine than at 100 microM. In the presence of 10 microM dopamine, internalization of D2R was completely suppressed when dynamin I K44E was coexpressed, and the half-life (t 1/2) of D2R internalization decreased relative to cells not expressing dynamin from 82 to 29 min when dynamin I was coexpressed. Internalization of D2Rs was facilitated or suppressed by coexpression of a constitutively active form of rab5A (rab5A Q79L) or a dominant-negative form of rab5A (rab5A S34N), respectively. The t 1/2 of D2R internalization at 10 microM dopamine decreased from 82 to 16 min in cells coexpressing rab5A Q79L. The effect of coexpression of rab5A S34N was more apparent at 100 microM dopamine than at 10 microM; the t 1/2 of D2R internalization at 100 microM dopamine increased from 20 to 56 min and the proportion of internalized D2Rs after 120 min decreased from 53 to 28%. These results indicate that the internalization of D2Rs is dependent on the action of dynamin as well as GRK2, and is regulated by the action of rab5A.

Animals↗
PubMed1999

Characterization of the carboxyl terminal-truncated endothelin B receptor coexpressed with G protein-coupled receptor kinase 2.

The role of phosphorylation of the C-terminal tail of endothelin B receptor (ETBR) in agonist-induced desensitization was investigated, using a mutant lacking C-terminal 40 amino acids (delta 40 ETBR). In cells expressing the wild type or delta 40 ETBR, ET-1 caused rapid desensitization of calcium responses. The wild type ETBR was phosphorylated by biotinylated ET-1, and the phosphorylation was markedly enhanced by coexpression with G protein-coupled receptor kinase 2 (GRK2). However, delta 40 ETBR was not phosphorylated regardless of coexpression with GRK2. On the other hand, ET-1-induced IP3 formation in these cells was decreased by coexpression with GRK2 or catalytically inactive Lys220Arg GRK2 to the similar extent. The present study demonstrates the presence of phosphorylation-independent desensitization mechanism in delta 40 ETBR and suggests that GRK2 might play a role other than that as a kinase.

Animals↗
PubMed1999

Alterations of muscarinic acetylcholine receptor subtypes in diffuse lewy body disease: relation to Alzheimer's disease.

OBJECTIVES: Dementia associated with Lewy bodies in cortical and subcortical areas is classified as dementia of the non-Alzheimer type and termed diffuse Lewy body disease (DLBD). The generic term "dementia with Lewy bodies (DLB)" was proposed in the international workshop on Lewy body dementia to include the similar disorders presenting Lewy bodies. In DLB, a lower level of choline acetyltransferase (ChAT) activity in the neocortex was found compared with that in Alzheimer's disease. The purpose of the present study was to determine the total amount of muscarinic acetylcholine receptors (mAChRs) and relative proportion of each subtype (m1-m4) of mAChRs in the frontal and temporal cortex of seven DLBD and 11 Alzheimer's disease necropsied brains. METHODS: A [(3)H]quinuclidinyl benzilate (QNB) binding assay and an immunoprecipitation assay using subtype-specific antibodies were performed. Each antibody was raised against fusion proteins containing peptides corresponding to the third intracellular (i3) loops of the respective mAChR subtype. RESULTS: The total amounts of mAChRs were significantly lower in the preparations of temporal cortices from DLBD and Alzheimer's disease than in those from dead controls (seven cases). In both diseases, the proportion of the m3 receptor in the frontal cortex was significantly increased and that of the m4 receptor in the temporal cortex was significantly decreased compared with the control specimens. The proportions of the m1 and m2 subtypes were significantly different in the temporal cortex. The proportion of the m1 receptor was significantly greater in the DLBD brains, whereas that of the m2 receptor was significantly greater in the Alzheimer's disease brains than in the controls. CONCLUSIONS: The m1 receptor is the major subtype in the cerebral cortex, and m2 is known to be present at presynaptic terminals. The higher proportions of m1 in DLBD and m2 in Alzheimer's disease suggest that the manner of degeneration in the cholinergic system is different between the diseases. It is hypothesised that a severe depletion of presynaptic cholinergic projective neurons causes the upregulation of m1 receptor in the temporal cortex in DLBD.

Aged↗
PubMed1999

Sequence variations of the CST2 gene related to the polymorphism of salivary cystatin SA.

The CST2 locus has two polymorphic alleles, CST2*1 and CST2*2, which produce cystatin proteins SAI and SA2, respectively (Shintani et al., 1994). The purpose of this study was to define nucleotide sequence variations of the protein-coding region of the two alleles. The variations were investigated by direct sequencing of amplified DNA from individuals with different CST2 phenotypes. The sequence of three exons obtained from DNA of the CST2 1 phenotype was found to be identical to the published sequence of the CST2 gene (Saitoh et al., 1987), whereas two-point mutations were found in the sequence obtained from DNA of the CST2 2 phenotype. One of the mutations was a G --> A transition in exon 2, resulting in loss of a commonly occurring AciI restriction site. This mutation resulted in a Gly59 --> Asp59 substitution in the protein. The other mutation was an A --> T transversion in exon 3, resulting in the generation of a SfaNI restriction site. This mutation also produced a Glu120 --> Asp120 substitution in the protein. PCR-RFLP assay with AciI and SfaNI restriction enzymes revealed that the two-point mutations were always correlated with cystatin SA polymorphism. The difference in the electrophoretic positions of the two proteins, SA1 and SA2, in a basic gel and in an isoelectric focusing gel agreed with the expected mobilities of the proteins with the SA2 variant at a more anodal position. The CST2*2 allele is a unique allele, which shows amino acid substitution in one of the most conserved regions responsible for cysteine proteinase inhibitory activity.

Alleles↗
PubMed1999

[Catheter drainage of late cardiac tamponade guided by computed tomography].

Delayed cardiac tamponade is an unusual but serious complication of cardiac surgery. Echocardiography and computed tomography (CT) are well established methods for the detection of pericardial effusions. Catheter insertion guided by CT has been used to accomplish non operative drainage of symptomatic postoperative pericardial effusion in seven cases. These patients were grouped into four types according to distribution of the fluid. General pericardial effusion around the heart is classified as type 1, effusion adjacent to the right side of the heart as type 2 and left side as type 3, effusion localized only at the apex as type 4. CT imaging is useful not only to localize and assess the size of the effusions, but also to select the way of catheter insertion. As the fluid might be trapped in compartments, for instance right-sided or left sided type, investigation of the pericardial spaces is important in planning a catheter pericardiocentesis.

Aged↗
PubMed1998

GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin.

Tubulin was found to bind to a glutathione S-transferase fusion protein containing the carboxy-terminal domain of GTP-binding-protein-coupled receptor kinase 2 (GRK2) (residues 467-689), which is known to contain a pleckstrin homology site and to bind GTP-binding protein betagamma subunits. The binding of tubulin to the fusion protein was not affected by GTP-binding protein betagamma subunits, indicating that tubulin and betagamma subunits bind GRK2 independently. Western-blotting analysis with anti-GRK2 Ig indicated that GRK2 was copurified with tubulin through the polymerization-depolymerization procedure. Tubulin was phosphorylated by GRK2, in contrast with the facts that the known substrates of GRK2 are restricted to activated forms of GTP-binding-protein-coupled receptors and that tubulin is a poor substrate for most kinases. GRK2 did not phosphorylate microtubule-associated proteins (MAPs), under conditions where MAPs were well phosphorylated by endogenous kinases copurified with tubulin. The Km for tubulin was estimated to be 3 microM, and 1.3 mol phosphate/tubulin dimer was incorporated. The phosphorylation of tubulin was stimulated by betagamma subunits and agonist-bound muscarinic receptors. Phosphorylated tubulin could be polymerized into microtubules, and polymerized tubulin was also phosphorylated by GRK2.

Animals↗