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H Taguchi

Publications and source records attributed to H Taguchi.

At least 271 records · Page 15Linked to original sources
PubMed1994

Chimeric YACs were generated at unreduced rates in conditions that suppress coligation.

Chimerism is a major limitation of current YAC libraries. A method based on partially filled-in ends of restriction fragments was designed to avoid coligation as a possible source of chimeras. Model experiments using plasmid DNA as an insert showed that coligation was clearly avoided by this method. Pilot collections of YACs with an average insert size of 650kb were then constructed with and without the partial fill-in treatment. Starting from a mixture of a equal amounts of human and mouse DNA, none of 108 clones was positive by hybridization with both Alu and B2 probes, again suggesting that coligation was effectively blocked. However, 4 out of 10 clones still hybridized to 2 or more locations by FISH on chromosomes in human metaphase spreads, level similar to that in the clones made without the partial fill-in step. These results strongly suggest that chimeric clones generally arise by a mechanism independent of coligation, presumptively based on recombination.

Animals↗
PubMed1994

Monomeric chaperonin-60 and its 50-kDa fragment possess the ability to interact with non-native proteins, to suppress aggregation, and to promote protein folding.

Chaperonin-60 is usually isolated as a tetradecameric form arranged as two stacked seven-member rings, and this structure has been considered to be required for promoting protein folding. However, monomeric chaperonin-60 (cpn60m), isolated from holo-chaperonin of Thermus thermophilus, and its proteolytic 50-kDa fragment, which lacks amino-terminal 78 amino acid residues, can interact with non-native rhodanese and lactate dehydrogenase, suppress formation of aggregates, and promote productive folding under the appropriate conditions. However, different from tetradecameric chaperonin-60, folding promoted by cpn60m and the 50-kDa fragment produces lower yields of active enzymes and does not require ATP or chaperonin-10. These effects are not due to transient reassembly of cpn60m into a tetradecamer during the reaction, since immobilized cpn60m and the 50-kDa fragment, both of which can not reassemble into a tetradecamer, can still promote protein folding. An excess amount of the 50-kDa fragment shows an inhibitory effect on MgATP-triggered holochaperonin-dependent folding, indicating the 50-kDa fragment and holo-chaperonin can interact with the same species of non-native proteins. Thus, cpn60m has an intrinsic activity as a molecular chaperone and amino-terminal region of cpn60 is dispensable for this activity.

Amino Acid Sequence↗
PubMed1994

Folding intermediate binds to the bottom of bullet-shaped holo-chaperonin and is readily accessible to antibody.

Holo-chaperonin from Thermus thermophilus (Thermus holo-cpn) is a bullet-shaped particle where chaperonin-10 heptamer locates at one axial end of the cylindrical body of chaperonin-60 tetradecamer. Thermus holo-cpn promotes in-vitro folding of denatured 3-isopropylmalate dehydrogenase (IPMDH) from the same bacterium. We observed the complexes of Thermus holo-cpn and folding intermediates of IPMDH by immuno-electron microscopy after decoration by single layer labeling with anti-IPMDH IgG or by double layer labeling with anti-IPMDH IgG as first layer and antibodies against IgG as second layer. Images of the electron microscope showed that anti-IPMDH IgG was bound to the bottom end of the bullet-shaped Thermus holo-cpn. This result provides direct evidence that the folding intermediate binds to the axial end, which is opposite to the end where chaperonin-10 heptamer resides, of the cylindrical body of chaperonin-60 tetradecamer, and that bound folding intermediate in the complex is sufficiently exposed to the outside to be accessible by antibody.

3-Isopropylmalate Dehydrogenase↗
PubMed1994

Essential role of arginine 235 in the substrate-binding of Lactobacillus plantarum D-lactate dehydrogenase.

Substitutions of the conserved Arg-235 with Lys and Gln induced drastic decreases in the catalytic efficiency of Lactobacillus plantarum D-lactate dehydrogenase (D-LDH). Both the mutant enzymes showed a marked resistance to 2,3-butanedione, by which the wild-type enzyme is rapidly inactivated unless NADH and oxamate are present. The pKa of the catalytic His was markedly shifted to the alkaline side by the Arg-to-Gln substitution, while it was not significantly shifted by the Arg to Lys substitution. The Arg-to-Lys replacement, by which the catalytic efficiency was less damaged, also induced decreases in kcat/Km for alternative substrates, such as 2-ketobutyrate, by approximately 10,000-fold, virtually the same level as in the case of pyruvate. Although both the wild-type and mutant enzymes exhibited lower kcat/Km for the alternative substrates than that for pyruvate, in the case of the mutant enzyme, the decrease in kcat/Km for the alternative substrates was mostly due to a decrease in kcat, while it was caused mainly by an increase in Km in the wild-type enzyme, suggesting that the mutant enzyme tends to form a nonproductive enzyme-substrate complex, in particular with more unfavorable substrates. The pH-dependence of the kinetic constants also indicated that there is a nonproductive binding that does not require the protonated or deprotonated form of the catalytic His residue. These results strongly suggest that Arg-235 plays an essential role in the tight and correct binding of substrate to the binding site of D-LDH, as in the case of Arg-171 in L-LDH.

Arginine↗
PubMed1994

Detection and characterization of antibodies to bacterial heat-shock protein 60 in sera of patients with primary biliary cirrhosis.

The enzyme-linked immunosorbent assay (ELISA) with bacterial heat-shock protein 60 (HSP60) purified from Yersinia enterocolitica (Ye) revealed that the antibodies directed against YeHSP60 existed in sera of patients with primary biliary cirrhosis (PBC). To characterize the epitope specificity of the antibodies in patients, the epitope mapping of HSP60 by means of the antibodies was performed. The results have suggested that the epitope recognized with anti-HSP60 antibodies in PBC relates to the amino acid sequence of YeHSP60 molecule as follows: DLGQAKRVVINKDTTIIIDGVGDEAAIQGRLAQIRQQIEEATSDYDKEK.

Amino Acid Sequence↗
PubMed1994

Severe neurological abnormalities associated with a mutation in the zinc-finger domain in a group A xeroderma pigmentosum patient.

All the reported Japanese patients with group A xeroderma pigmentosum (XP) have two or three mutations at codon 116 in exon 3, codon 228 in exon 6, and the splicing acceptor site of intron 3 of XP group A complementing (XPAC) gene. A homozygote (XP39OS) with a nonsense mutation at codon 228 has less severe neurological abnormalities than patients with the splicing mutation at the acceptor site of intron 3. As homozygotes for the nonsense mutation at codon 116, which truncates a carboxyl-terminal site of XPAC protein at an early part of its zinc-finger domain, have not been reported previously, the possible severity of associated neurological abnormalities was not known. We report a group A XP patient, XP18OS, who had neurological abnormalities which were more severe than those in patients homozygous for the splicing mutation. The polymerase chain reaction product from exon 3 of the patient's XPAC gene was digested completely into three fragments by MseI restriction endonuclease. Thus, the patient was homozygous for the mutation at codon 116.

Base Sequence↗
PubMed1994

ATP-sensitive K+ channels mediate dilatation of cerebral arterioles during hypoxia.

We tested the hypothesis that dilatation of cerebral arterioles during hypoxia is mediated by activation of ATP-sensitive K+ channels. The diameter of pial arterioles was measured through a closed cranial window in anesthetized rabbits. Topical application of aprikalim (10(-6) mol/L), a direct activator of ATP-sensitive K+ channels, dilated pial arterioles by 18 +/- 3% (mean +/- SEM). Glibenclamide (10(-6) mol/L), an inhibitor of ATP-sensitive K+ channels, virtually abolished aprikalim-induced vasodilatation. When arterial PO2 was reduced from 129 +/- 3 to 25 +/- 1 mm Hg, the diameter of cerebral arterioles increased by 66 +/- 9% (P < .05). Glibenclamide inhibited dilatation of pial arterioles during hypoxia by 46 +/- 5% (P < .05). In contrast, vasodilatation in response to sodium nitroprusside was not altered by glibenclamide. Topical application of adenosine (10(-4) mol/L) increased arteriolar diameter by 21 +/- 4%. Glibenclamide did not affect adenosine-induced vasodilatation. These findings suggest that dilatation of cerebral arterioles in response to hypoxia is mediated, in part, by activation of ATP-sensitive K+ channels.

Adenosine↗
PubMed1994

Carboxypeptidase Taq, a thermostable zinc enzyme, from Thermus aquaticus YT-1: molecular cloning, sequencing, and expression of the encoding gene in Escherichia coli.

The gene for carboxypeptidase Taq, a thermostable metallo-carboxypeptidase from Thermus aquaticus YT-1, was cloned and sequenced. The gene comprised an open reading frame of 1,536 base pairs with a GTG initiation codon and a TGA termination codon, which encodes a protein of 56,210 Da consisting of 511 amino acid residues. The GTG initiation codon of the gene was replaced with ATG by site-directed mutagenesis, and then the gene was expressed in Escherichia coli. The enzyme purified from E. coli cells showed the same properties as those of carboxypeptidase Taq prepared from T. aquaticus cells. Analysis for metal ions bound to the enzyme found that one molecule of the enzyme contains one tightly bound zinc ion. Comparison of the entire sequence showed that the enzyme has no obvious sequence similarity to any other metallo-peptidases. However, a His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent endopeptidases and aminopeptidases, was found at positions 276 to 280 of the enzyme. These findings suggest that carboxypeptidase Taq is a novel type of zinc-dependent metallocarboxypeptidase.

Amino Acid Sequence↗
PubMed1994

Glyceryl-ether monooxygenase (EC 1.14.16.5): nature of the glyceryl-ether lipid substrates in aqueous buffer.

The kinetics of inhibition of glyceryl-ether monooxygenase with 6-methyl-5,6,7,8-tetrahydropterin as cofactor (saturating) by the detergent Mega-10 with batyl alcohol and (RS)-3-(1-hexadecyloxy)-2-hydroxypropane-1-phosphocholine as substrates exhibited noncompetitive inhibition with apparent Ki values of 1.74 +/- 0.37 mM and 185 +/- 23 microM, respectively. Inhibition by octadecan-1-ol (with batyl alcohol as substrate in the presence of 2.3 mM Mega-10) was competitive with an apparent Ki value of 765 +/- 80 microM. The critical micelle concentration values of various solutions of Mega-10 were determined and used to show that the active ether-lipid substrates were in micelle form (for a water soluble substrate) or mixed micelle form (for substrates solubilised with Mega-10).

Animals↗
PubMed1994

[Screening for thyroid dysfunction in adults residing in Hokkaido Japan: in relation to urinary iodide concentration and thyroid autoantibodies].

The prevalence of thyroid dysfunction and its relation to thyroid autoantibodies (TAA) and urinary iodide concentration (UI) was studied in apparently healthy adults in Sapporo (n = 4110) (Sapporo group), and in five coastal areas of Hokkaido (n = 1061) (coastal group) which produce iodine-rich seaweed (kelp). The frequency of above normal UI (high UI) in the morning urinary samples of coastal group was 10.8%, significantly higher than that of Sapporo group (6.4%) (p < 0.001). Frequency of positive TAA in both groups were similar. In Sapporo group TAA was positive in 6.4% of males and 13.8% of females with an age-related increase. The overall prevalence of hyperthyroidism (TSH < 0.15 mU/L) in coastal group (0.6%) was similar to that in Sapporo group (1.1%), while that of hypothyroidism (TSH > 5.0 mU/L) in coastal group (3.8%) was significantly higher than that in Sapporo group (1.3%) (P < 0.001). The frequency of high UI correlated significantly with that of hypothyroidism with negative TAA (r = 0.829, P < 0.05), but not with positive TAA, or with that of hyperthyroidism. Hypothyroidism was more prevalent in TAA negative subjects with high UI than with normal UI. Moreover, serum TSH and thyroglobulin levels were higher and free T4 level was lower in former than in latter group. These results indicate that 1) the prevalence of TAA negative hypothyroidism in iodine sufficient areas may be associated with the amount of iodine ingested, 2) this hypothyroidism is more prevalent and marked in subjects consuming further excess amounts of iodine, and 3) excessive intake of iodine should be considered an etiology of hypothyroidism in addition to chronic thyroiditis in these areas.

Adult↗
PubMed1993

Truncated GroEL monomer has the ability to promote folding of rhodanese without GroES and ATP.

Similar to chaperonins from other sources, intact chaperonin from Escherichia coli (GroEL) exists as a tetradecamer, and the ability to promote folding of other proteins has been considered to be dependent on this oligomeric structure. However, the peptide fragments of GroEL of molecular size 34-50 kDa, which are produced by limited proteolysis of monomeric GroEL and are unable to assemble into an oligomer, retain the ability to promote folding of rhodanese even though the yield of productive folding is lower than the intact GroEL/GroES/ATP system. This promotion by truncated GroEL obeys rapid kinetics and does not require GroES and ATP.

Adenosine Triphosphate↗
PubMed1993

Histidine 296 is essential for the catalysis in Lactobacillus plantarum D-lactate dehydrogenase.

Two His residues, His-205 and His-296, in Lactobacillus plantarum D-lactate dehydrogenase are highly conserved in the D-isomer-specific 2-hydroxyacid dehydrogenase family, suggesting that they are candidates for the catalytic His in the enzyme. The substitution of His-296 with Tyr by means of site-directed mutagenesis induced a drastic decrease in the reaction rate, while a His-205 to Tyr substitution induced no large change in the catalytic properties. In pyruvate reduction, the Tyr-296 enzyme showed a slightly increased Km below pH 6.0 but no significant pH dependence above pH 6.0, where the wild-type enzyme showed an increased Km value. In D-lactate oxidation, in contrast, the Tyr-296 enzyme showed a greatly increased Km value for D-lactate and strong pH dependence. An additional substitution of His-296 with Gln induced more complete loss of the catalytic activity. In contrast to the Tyr-296 enzyme, the Gln-296 enzyme showed a greatly increased Km value and a strong pH-dependent reaction rate even in pyruvate reduction. Unlike the wild-type or His-205 enzyme, both the Tyr-296 and Gln-296 enzymes showed significant resistance against diethyl pyrocarbonate. These results clearly indicate that His-296 is essential for the catalysis by D-lactate dehydrogenase, as in the case of His-195 in L-lactate dehydrogenase.

Amino Acid Sequence↗
PubMed1993

A chaperonin from a thermophilic bacterium, Thermus thermophilus.

Unlike Escherichia coli chaperonins, a chaperonin (cpn) from a thermophilic bacterium, Thermus thermophilus, consisting of homologues to GroEL (cpn 60) and GroES (cpn 10) is co-purified as a large complex. Thermus chaperonin shows a bullet-like shape in the side view seen by electron microscopy, and antibody against cpn 10 binds only to the round side of the bullet. We conclude that a single cpn 60-heptamer ring with two stripes stacks into two layers and a cpn 10 oligomer binds to one side of the layers. The purified Thermus chaperonin contains endogenously bound ADP, and incubation with ATP causes a partial dissociation of chaperonin into cpn 60 monomers and a cpn 10 heptamer. The effect of Thermus chaperonin on protein refolding upon dilution from guanidine HC1 is different at three temperature ranges. At high temperatures above 55 degrees C, where the native proteins are stable but their spontaneous foldings fail, the chaperonin induces productive folding in an ATP-dependent manner. At middle temperatures (25-55 degrees C) where spontaneous foldings of the enzymes occur, the chaperonin slows down the rate of folding without changing the final yield of productive folding. At lower temperatures below 25 degrees C where spontaneous foldings also occur, the chaperonin arrests the folding even in the presence of ATP. When a solution of relatively heat labile protein is incubated at high temperatures, and then residual activity of the protein is measured at its optimal temperature after incubation with ATP, the temperature that causes irreversible heat denaturation of the protein is elevated about 10 degrees C by inclusion of Thermus chaperonin in the solution.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Isopropylmalate Dehydrogenase↗
PubMed1993

Chaperonin from Thermus thermophilus can protect several enzymes from irreversible heat denaturation by capturing denaturation intermediate.

Chaperonin isolated from Thermus thermophilus is stable up to 80 degrees C. Taking advantage of this heat stability, we have studied the effects of chaperonin on heat denaturation of several relatively heat labile enzymes. When the enzymes are incubated at their denaturating temperatures, the presence of T. thermophilus chaperonin in the solution has little effect on the rate of apparent inactivation of the enzyme. However, this inactivation is not irreversible since most activity is recovered when the solution is shifted to the second incubation at a moderate temperature with concomitant addition of MgATP. When the chaperonin is omitted from the solution, no recovery is observed. Recovery of the activity is also dependent on MgATP in the second incubation and 50% of recovery is attained at 5 microM MgATP. When the chaperonin is added after starting the incubation at a denaturing temperature, recovery of the activity becomes poorer as the delay of chaperonin addition increases. The critical temperature of the incubation at which irreversible denaturation occurs to the enzymes is elevated 8-15 degrees C by inclusion of T. thermophilus chaperonin in the solution. The heat stability of captured proteins by the chaperonin, assessed as retention of the ability to resume productive folding under optimal conditions, is measured more accurately using chemically produced folding intermediate-chaperonin complexes. The ability is lost at about 78 degrees C being irrespective of variable heat stability of individual enzymes. These results indicate that during heat denaturation proteins assume a common structure which is recognizable by the chaperonin. Once a protein with this structure is captured by T. thermophilus chaperonin, it retains the ability to resume productive folding even after exposure to the otherwise denaturing high temperature. Its heat stability seems to be limited solely by heat stability of chaperonin.

Adenosine Triphosphate↗
PubMed1993

Central nervous system involvement of adult T-cell leukemia-lymphoma with multinucleated giant cells in the brain, skin, and kidney.

BACKGROUND: A case of central nervous system (CNS) involvement in a patient with adult T-cell leukemia-lymphoma (ATLL) with multinucleated giant cells (MNGC) is presented. METHODS: A 48-year-old woman with human T-lymphotropic virus type I (HTLV-I) antibody titer had multiple focal brain symptoms and skin eruptions without lymphadenopathy, hepatosplenomegaly, or increased abnormal lymphocytes in the peripheral blood. No spastic paraparesis of the lower limbs was found. The encephalopathy was progressive, and she died 5 months later despite repeated intrathecal administration of methotrexate, cytosine arabinoside, and prednisolone and monthly systemic chemotherapy with doxorubicin, cyclophosphamide, vincristine, and prednisolone. RESULTS: Postmortem examinations identified unusual ATLL lesions composed of marked infiltrations of atypical mononuclear cells and bizarre MNGC with histiocytic granulomatous reactions in the leptomeninges, brain tissues along the Virchow-Robin spaces, skin, and kidney. Immunohistochemical stains confirmed the T-cell nature of such mononuclear cells and partially T-cell and partially macrophage nature of the MNGC, although no evidence of HTLV-I expression was found. CONCLUSIONS: ATLL presenting with CNS symptoms is rare. It was assumed that the direct cytopathic effects of HTLV-I were responsible for the formation of the MNGC after considering the similarity with MNGC in encephalopathy caused by the human immunodeficiency virus.

Brain↗