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Biomedical subjects

H Taguchi

Publications and source records attributed to H Taguchi.

At least 235 records · Page 13Linked to original sources
PubMed1996

Induction and epitope analysis of Helicobacter pylori heat shock protein.

Induction of heat shock proteins (HSPs) was analyzed in Helicobacter pylori strains. With heat shock at 42 degrees C, a synthesized 60 kDa-HSP (HSP60) was detected on autoradiography. The expression of HSP60 on the cell surface of H. pylori was examined by flow cytometric analysis. All strains used in this study expressed HSP60 on the cell surface, although the intensity differed among the strains, depending on culture conditions. The reactivity of a monoclonal antibody (mAb), 3C8, directed against bacterial HSP60, with HSP60 derived from ten strains of H. pylori and with human gastric carcinoma cell HSP60 was examined by immunoblot analysis. An epitope that reacted with the mAb was detected in the HSP60 of H. pylori and on the surface of human gastric carcinoma cells.

Antibodies, Monoclonal↗
PubMed1996

Vascular expression of inducible nitric oxide synthase is associated with activation of Ca(++)-dependent K+ channels.

We tested the hypothesis that expression of inducible nitric oxide synthase (NO-synthase) in response to endotoxin (lipopolysaccharide) produces activation of potassium channels. Contraction of the rat thoracic aorta in response to phenylephrine was measured in vitro after treatment in vivo for 15 hr with vehicle (control) or lipopolysaccharide (10 mg/kg i.p.). Impaired contraction in response to phenylephrine was used as an index of inducible NO-synthase expression, and activation of potassium channels was examined with specific inhibitors. Contraction in response to 10(-5) M phenylephrine (expressed as a percentage of contraction in response to 85 mM KCI) was markedly impaired in lipopolysaccharide-treated rats, compared with control (15 +/- 5% vs. 131 +/- 10%, P < .05, mean +/- S.E.). Expression of inducible NO-synthase mRNA in the vessel wall in lipopolysaccharide-treated rats was confirmed using reverse transcription-polymerase chain reaction. Contraction of the aorta in lipopolysaccharide-treated rats was restored to normal by 0.3 mM aminoguanidine (an inhibitor of inducible NO-synthase). Contraction of the aorta in response to phenylephrine, which was inhibited by lipopolysaccharide, was not affected by glibenclamide (an inhibitor of ATP-sensitive potassium channels) but was increased 2-fold (P < .05) by iberiotoxin (50 nM), an inhibitor of Ca(+2)-dependent potassium channels. Relaxation of the aorta in response to sodium nitroprusside, an exogenous donor of nitric oxide, and 8-bromo-cyclic GMP was also inhibited by iberiotoxin. These findings suggest that nitric oxide produced by vascular expression of inducible NO-synthase activates calcium-dependent potassium channels and that this mechanism may contribute to impaired vasoconstrictor responses during sepsis.

Animals↗
PubMed1995

Finding of a homarine-synthesizing enzyme in turban shell and some properties of the enzyme.

A homarine-synthesizing enzyme was found for the first time in cell-free extract from turban shell (Batillus cornutus) and the enzyme was purified 36.2-fold and characterized. Properties of the enzyme were as follows: substrates were picolinic acid (pyridine-2-carboxylic acid) and S-adenosyl-L-methionine; optimum temperature for the enzymic reaction was 25 degrees C; optimum pH for the enzymic reaction was 6.3; and the Km values for picolinic acid and S-adenosyl-L-methionine were calculated at 317 and 14.5 microM, respectively. Among pyridine carboxylic acids, only picolinic acid was methylated with S-adenosyl-L-methionine by this enzyme. The molecular weight of the enzyme was estimated to be 70,800. The enzyme activity was inhibited by heavy metal ions, S-adenosyl-L-homocysteine, adenosine, homocysteine, and sinefungin. Homarine, which is an osmotic pressure regulator, morphogen, etc.; is enzymatically synthesized by the methylation of picolinic acid with S-adenosyl-L-methionine and the enzyme activity may be controlled by S-adenosyl-L-homocysteine (reaction product) and its related compounds.

Adenosine↗
PubMed1995

Kinetic analysis of interactions between GroEL and reduced alpha-lactalbumin. Effect of GroES and nucleotides.

The real-time analysis of the association and dissociation of chaperonin with respect to its substrate protein was carried out using the BIAcore system. We immobilized alpha-lactalbumin (LA) as a substrate protein on the sensor chip and the GroEL solution was passed over it. Whereas GroEL did not bind to the immobilized native LA, it associated with the immobilized Ca(2+)-depleted, disulfide bond-reduced form of LA (rLA) rapidly (kon = 1.96 x 10(5) M-1 S-1) and dissociated extremely slowly (koff = 2.08 x 10(-4) S-1), giving a low dissociation constant (KD = 1.06 nM). MgATP greatly accelerated the dissociation (koff = 0.15 +/- 0.02 S-1). The KD value remained almost unchanged when GroES and/or 10 microM ADP was included in the GroEL solution. However, when 1 mM ADP was included, the KD value of GroEL increased by 2 orders of magnitude solely due to the change in koff. When GroES and 1 mM ADP were included, no interaction with rLA was detected due to changes in both kon and koff. These results indicate that GroEL/ES has high and low affinity ADP binding sites and that occupation of the low affinity sites by ADP was responsible for the loss of ability to interact with the substrate protein. The effect of excess GroES on the preformed GroEL.rLA and GroEL/ES.rLA complexes was also examined. With increasing GroES, the dissociation of GroEL and GroEL/ES from rLA was accelerated, and thus the possibility is suggested that the substrate protein and GroES compete for the same site on GroEL.

Adenine Nucleotides↗
PubMed1995

Design of noncovalent trypsin inhibitor based on the X-ray crystal structure of the complex.

The inhibitory mechanism of trans-4-aminomethylcyclohexanecarbonyl-L-phenyl-alanine-4-carbo xymethylanilide (1), a noncovalent serine protease inhibitor synthesized based on previous structure-activity studies, was clarified based on the X-ray crystal structure of the complex (2.2 A resolution, R = 0.175), where the amino group of the aminomethylcyclohexane moiety was bifurcately hydrogen-bonded to the carboxyl oxygens of Asp 189 side group (specificity pocket), and the hydrogen bonds of the cyclohexanecarbonyl oxygen to NHs of Gly 193 and Ser 195 residues (oxyanion hole) and of Phe NH to Ser 195 O gamma atom (catalytic triad) were observed. In contrast, the Phe benzene moiety and terminal carboxymethylanilide of 1 were not well located on the electron density map, suggesting the conformational freedom of these P1' and P2' sites at the binding pocket. Based on these insights, trans-4-aminomethylcyclohexanecarbonyl-4-nitro-L-phenylalanine-4-+ ++benzoylanilide (2) was designed, in which the P1' and P2' sites were modified so as to effectively interact with the amino acid residues of trypsin binding pocket via hydrogen bonding and van der Waals interactions, respectively. Consequently, 2 showed 40 times higher inhibitory activity against trypsin than 1.

Crystallization↗
PubMed1995

Assignment of the 49-kDa (PRIM1) and 58-kDa (PRIM2A and PRIM2B) subunit genes of the human DNA primase to chromosome bands 1q44 and 6p11.1-p12.

DNA primase is an essential replication protein that catalyzes the synthesis of oligoribonucleotide primers. DNA primase, consisting of two subunits (p49 and p58), plays a key role in both the initiation of DNA replication and the synthesis of Okazaki fragments for lagging strand synthesis. We mapped the locations of human chromosomes of the genes coding for both subunits [p49 (PRIM1) and p58 (PRIM2)] by PCR amplification using DNAs of a panel of somatic hybrids, to chromosomes 1 and 6, respectively. The PRIM1 gene was mapped to 1q44, and two PRIM2 loci (PRIM2A and PRIM2B) were detected at 6p11.1-p12 by fluorescence in situ hybridization using several genomic DNA probes.

Animals↗
PubMed1995

Equatorial split of holo-chaperonin from Thermus thermophilus by ATP and K+.

Holo-chaperonin molecule from Thermus thermophilus is a bullet-shaped particle whose cylinder part and round top are composed of two stacked rings of the cpn60 heptamer and a single ring of the cpn10 heptamer, respectively. We found that it splits at the plane between two cpn60 rings into two halves under physiological conditions, that is, in the presence of ATP (but not AMP-PNP, ADP) + K+ (but not Na+) at 60 degrees C. This equatorial split could be functionally important although it has not been considered in any current mechanistic model of chaperonin functioning.

Adenosine Triphosphate↗
PubMed1995

Chaperonin releases the substrate protein in a form with tendency to aggregate and ability to rebind to chaperonin.

To know whether the protein released from chaperonin GroEL/ES is in a form committed to the native state or still an aggregatable non-native one, two experiments were carried out. Dilution of the [GroEL-substrate protein] binary complex prior to ATP addition significantly improved the yield of folding, suggesting that the released protein has a tendency to aggregate. When N-ethylmaleimide treated GroEL, which can form the binary complex but not release the bound protein, was added to the binary complex prior to ATP addition, productive folding was severely inhibited, indicating that the protein released from GroEL/ES can bind to N-ethylmaleimide treated chaperonin. These data favor the 'reservoir' or 'reversion' model, in which GroEL/ES acts as a buffer of folding intermediate or mediates reversion of a misfolded protein to a less folded primitive form, rather than the 'marsupium' model in which folding of the substrate protein proceeds in chaperonin.

Adenosine Triphosphate↗
PubMed1995

Isolation and characterization of the HC8 subunit gene of the human proteasome.

For study of the molecular basis of regulation of proteasome gene expression, we isolated the gene encoding the alpha-type HC8 subunit of the human proteasome. About 2.3 kb of the 5' flanking region of this gene was tested for promoter function by chloramphenicol acetyltransferase assay. This analysis revealed that CAAT and TATA boxes, but not a GC box, are essential for its promoter activity. These results differed from previous findings that the genes for the alpha-type HC3 and beta-type HC5 subunits of the human proteasome have a TATA-less promoter and that two or three GC boxes function as the promoter sequences (Tamura, T. et al. (1994) J. Mol. Biol. 244, 1117-1124). We mapped the HC8 gene at q23 on human chromosome 14, which differs from the chromosomal locations of nine other proteasomal subunit genes mapped so far.

Animals↗
PubMed1995

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23.

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.

Base Sequence↗
PubMed1995

Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8.

The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced. The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins. The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells. The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E. coli. T.th cpn60 was expressed as a tetradecameric form, like GroEL of E. coli. Since chaperonin from T. thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before. T.th cpn60 tetradecamer tended to dissociate into monomers during storage. T.th cpn10 expressed in E. coli was purified as a stable oligomer, most likely a heptamer. The activity as holo-chaperonin was reconstituted by mixing both subunits. T.th cpn60 tetradecamer itself arrested refolding of other proteins. The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography. Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin.

3-Isopropylmalate Dehydrogenase↗
PubMed1995

The primary cytotoxicity in ultraviolet-a-irradiated riboflavin solution is derived from hydrogen peroxide.

The cytotoxic action of near-ultraviolet (UVA) radiation on cultured mammalian cells is dependent upon oxygen, suggesting that reactive oxygen species are involved in the cellular action of the radiation. Flavins are thought to be an important chromophore for photo-induced skin injury. Irradiation of riboflavin with UVA radiation is known to produce singlet oxygen, superoxide anions, and triplet-state riboflavin radicals, which, however, are immediately quenched by many constituents of the human skin. If the chemical produces a long-lived reactive oxygen species, hydrogen peroxide (H2O2), after UVA radiation, its deleterious effect is not limited to its generation site. Thus, we investigated whether H2O2, is produced in UVA-irradiated riboflavin solution and whether it plays an important role in the cytotoxic action of the solution. The solution showed a marked cytotoxic effect when placed on human fibroblasts, and cytotoxicity was retained in the solution for at least 40 min after radiation. Most of the toxicity appeared to be derived from H2O2 produced in the solution, because the solution lost its cytotoxicity as a result of catalase treatment, and the resultant restoration of survival was almost complete. Under our conditions, two molecules of riboflavin were calculated to produce one molecule of H2O2 after UVA radiation.

Catalase↗
PubMed1995

Flow cytometric analysis using lipophilic dye PKH-2 for adhesion of Vibrio cholerae to Intestine 407 cells.

A comparative study of indirect and direct flow cytometric analysis for adherence of Vibrio cholerae to Intestine 407 cells was performed. The direct flow cytometric analysis employed the lipophilic dye PKH-2. It was concluded that direct flow cytometry using the lipophilic dye PKH-2 is useful and convenient for analyzing bacterium-host cell interactions, since it does not require any specific antibody as the first antibody.

Bacterial Adhesion↗
PubMed1995

Chronological difference in walking impairment among Japanese group A xeroderma pigmentosum (XP-A) patients with various combinations of mutation sites.

Almost all Japanese group A xeroderma pigmentosum (XP-A) patients have nonsense and/or nonsense codon-leading mutations in the XP group A (XPA) gene, and develop neurological abnormalities. Walking ability is one of the most important neuromuscular functions of the patients, because it determines their daily activities. We studied the correlation between the various combinations of mutations found by PCR-RFLP in Japanese XP-A patients and their chronological walking impairment. We classified these patients into six groups. Group I: A patient who was homozygous for the mutation at codon 116 in exon 3 (Type 1 mutation) could never walk unaided. Group III: Typical patients who were homozygous for the mutation at intron 3 (Type 2 mutation) could walk unaided till 7-16 years of age. Group V: Patients who were compound heterozygous for Type 2 mutation and for the mutation at codon 228 in exon 6 (Type 3 mutation) began to develop some walking difficulty at 5-13 years of age and became unable to walk at 25-28 years of age. Group VI: A patient who was homozygous for Type 3 mutation could walk unaided without any difficulty till the age of 21. The walking ability of group II and IV patients is not known yet.

Adolescent↗