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T Nakazawa

Publications and source records attributed to T Nakazawa.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus.

We previously identified two hypervariable regions [HVR1 (27 amino acids) and HVR2 (7 amino acids)] in the putative envelope glycoprotein (gp70) by comparison of the amino acid sequences of many isolates of the HCV-II genotype. To understand the functional features of these HVRs, using the polymerase chain reaction we analyzed the rate of actual sequence variability in the region including HVR1 and HVR2 of HCV isolated successively at intervals of several months from two patients with chronic C-type hepatitis. In both patients, the amino acid sequence of HVR1, but not HVR2, was found to change dramatically during the observation period (about one amino acid per month). However, no alteration of the amino acid sequence of HVR1 of HCV was observed in a patient in the acute phase of chronic hepatitis. Restriction digestion analysis of sequence diversity showed that a HCV genome with a newly introduced mutation in HVR1 often became the predominant population at the next time of examination. Alterations of amino acids in HVR1 occurred sequentially in the two patients in the chronic phase. These findings suggest that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection.

Alanine Transaminase↗
PubMed1992

Calyculin A induces contractile ring-like apparatus formation and condensation of chromosomes in unfertilized sea urchin eggs.

Calyculin A, a protein phosphatase inhibitor, induced cleavage-like morphological change in unfertilized sea urchin eggs. A contractile ring-like apparatus containing both filamentous actin and myosin was formed in the cleavage furrow. Wheat germ agglutinin receptors were also found in the same region. The eggs did not develop further after constriction of the ring. No aster-like microtubular structure was found in the calyculin A-treated eggs. The cleavage was not inhibited by the antimicrotubule drug griseofulvin. Calyculin A also increased histone H1 kinase activity and induced chromosome condensation. These changes also occurred in the presence of emetine (an inhibitor of protein synthesis) and aphidicolin (an inhibitor of DNA synthesis). It is suggested that calyculin A induced these changes in the sea urchin eggs by inhibiting the activity of protein phosphatase 1.

Actin Cytoskeleton↗
PubMed1992

Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid of Pseudomonas putida.

Transcription from the promoter of a positive regulatory gene, xylS, on the TOL plasmid of Pseudomonas putida is activated by another positive regulator, XylR, in the presence of m-xylene and is dependent on RNA polymerase containing the NtrA protein (sigma 54). Deletion analysis of the upstream region of the xylS gene revealed an upstream regulatory sequence (URS), located between 145 and 188 bp upstream from the transcription start site. The URS is active in either orientation and can be placed 3.9 kb further upstream without loss of activity. Dependence of activation on helical periodicity was observed in the region between the URS and the promoter of the xylS gene, suggesting DNA loop formation between these two sites, which are located about 100 bp apart. The expression of xylR was autogenously repressed by XylR protein. This autogenous repression is decreased in an NtrA- background, irrespective of the presence of the xylS promoter in cis, indicating that NtrA protein, or NtrA-containing RNA polymerase that is not bound to the xylS promoter, is involved in the binding of XylR protein to the URS.

Bacterial Proteins↗
PubMed1992

Marked sequence diversity in the putative envelope proteins of hepatitis C viruses.

The nucleotide sequences of cDNAs (414 base pairs) encoding parts of putative envelope proteins (gp35 and gp70) of 40 isolates of hepatitis C virus (HCV-J) derived from 30 independent plasma or liver specimens from Japanese patients (13 with chronic hepatitis, 14 with hepatocellular carcinoma and 3 hemophiliacs who had received imported clotting factors), were analyzed using the polymerase chain reaction. Approximately 29-38% of the nucleotide sequences of the HCV-J isolates examined differed from those of isolates from the United States (HCV-US). Furthermore, 12-24% and 8-17% sequence diversities were found within the isolates of HCV-J and HCV-US, respectively. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. We confirmed that two hypervariable regions (HVR1 and HVR2) were present in this amplified region, as described in our previous report (Hijikata et al., 1991a) and we found that the HVR1 regions of HCV-J and HCV-US were 27 and 21 amino acids in length, respectively, and began from the N-terminal amino acid of gp70. HVR2 was found in HCV-J, but not in HCV-US isolates, in which the corresponding region of the genome was conserved. During the analysis, plural HCV genomes were found in 6 of 30 specimens. These plural HCV genomes in a single specimen were concluded to be derived from the same HCV ancestor, because of their relative low sequence diversities (about 10% in their nucleotide sequences).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Molecular cloning of hepatitis C virus genome from a single Japanese carrier: sequence variation within the same individual and among infected individuals.

A hepatitis C virus (HCV) genome was isolated and sequenced from a single Japanese patient with chronic non-A, non-B hepatitis. The genome (HCV-JT), which was constructed with 23 cDNA clones, consisted of 9436 nucleotides with a long open reading frame which could encode a sequence of 3010 amino acid residues. To study the sequence variation of the HCV genome in an individual, we analyzed another sequence of the HCV genome (HCV-JT') constructed with different cDNA clones derived from the same patient. The nucleotide variation between HCV-JT and -JT' was less than 1%, and was distributed throughout the genome except in the 5' non-coding region, where no variation was observed. The diversity was higher (1.6%) in the putative envelope protein region than in other regions. The nucleotide and deduced amino acid sequences of HCV-JT showed homologies of about 91 and 95%, respectively, with those of other Japanese HCV isolates. The nucleotide diversity was high in the gp 70 region (corresponding to the NS 1 region of flaviviruses) and low in the 5' non-coding and p22 (putative core protein) regions. A similar pattern of distribution of nucleotide changes was observed on comparison of HCV-JT with an American isolate HCV-US, where the homologies in nucleotide and amino acid sequences were about 79 and 85%, respectively. Base transversions contributed about 50% of the total base exchanges between the Japanese and American HCV sequences, but only 20% or less of those among Japanese HCV or among American HCV sequences. Thus, the Japanese and American HCVs are genetically distinguishable, supporting our earlier prediction that these two HCVs could be classified as different subtypes.

Amino Acid Sequence↗
PubMed1992

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.

Algorithms↗
PubMed1992

Cytopathic effects of Helicobacter pylori on cultured mammalian cells.

Cytopathic effects of broth-culture filtrates from eight clinical isolates and one reference strain of Helicobacter pylori on three cultured mammalian cell lines were investigated. All the strains, including NCTC 11637, produced cytotoxic factors that caused intracellular vacuolation on these cell lines. AGS and SflEp cells were more sensitive than HEp-2 cells. To examine the role of urease in the cytotoxic effect, a urease-negative mutant was produced. Filtrates from both wild-type and mutant strains produced similar vacuolation on SflEp cells in the absence of urea, suggesting that H. pylori produces a cytotoxic substance other than urease. In contrast, ammonia alone, or jack bean urease with urea, also induced rounding and detachment of SflEp cells, whereas ammonium salts induced the production of small vacuoles. The combination of the broth filtrate of the wild-type strain and urea induced vacuolation followed by rounding and detachment of SflEp cells. Evidence is presented that the latter changes are due to ammonia produced during incubation. Nevertheless, the amounts produced were less than that needed to induce cytopathic effects by itself. These results suggest that the cytotoxic substance induces intracellular vacuolation, and that the vacuolated cells are more susceptible to killing by ammonia. Thus both the cytotoxic substance and urease may contribute to the lethal cytotoxicity of H. pylori in vitro.

Ammonia↗
PubMed1992

Evaluation of airway smooth muscle contractions in vitro by high-frequency ultrasonic imaging.

To visualize ASM contraction in vitro, we measured changes in cross-sectional area and inner circumference of isolated porcine and human bronchi in response to acetylcholine or carbamylcholine chloride (Carbachol) using high-frequency ultrasound. A mechanical ultrasonic catheter (20 MHz; diameter, 1.7 mm; echo element, 1 mm2) demonstrated three histologic layers (mucosa, cartilage and adventitia) and allowed measurement of dose-dependent contraction of porcine bronchi over time. Acetylcholine (10(-4)M) significantly decreased the cross-sectional area by 17 +/- 2.85 percent but did not change the circumference. The mean initial value for area was 0.6 +/- 0.08 cm2 and for circumference was 3.0 +/- 0.24 cm (n = 5). The EC50 of acetylcholine was 1.5 x 10(-8) M. Carbachol (10(-4)M)-induced contractions of human bronchi were also observed under the same conditions. In conclusion, high-frequency ultrasonic imaging can be used to study the morphology of ASM contractions in vitro, providing valuable information on the natural dynamics of ASM.

Acetylcholine↗
PubMed1992

Behavioral study on the gracile axonal dystrophy (GAD) mutant mouse.

We investigated motor function and pain sensation in the gracile axonal dystrophy (GAD) mutant mouse, using the tail-flick test and the rotarod test. GAD (gad/gad) and normal sib mice (gad/+ or +/+) were used between 5 and 11 weeks of age, during which time the behavioral signs of GAD mice shifted from sensory ataxia (about 4 to 8 weeks of age) to paresis (after about 9 weeks of age). In the tail-flick test, significant shortening of latency was observed at 6 and 8 weeks of age in female GAD mice, in comparison with normal female mice. This may be related to dysfunction or degeneration of axons in the fasiculus gracilis, whose collaterals are thought to control the transmission of nociceptive information. In the rotarod test, a cumulative chi 2 test showed significant reduction in the performance times of GAD mice beginning at 5 and 6 weeks of age in males and females, respectively, indicating that the rotarod test can detect the development of motor incoordination as early as these ages. The performance times of GAD mice dropped sharply from 9 weeks of age onwards, and this is believed to reflect the progression of paresis. The rotarod test therefore appears to be a good method of quantifying behavioral changes in GAD mice and to be applicable both to objective selection of GAD mice before 8 weeks of age and to evaluation of drugs to treat ataxia or paresis.

Age Factors↗
PubMed1992

Hypersensitivity pneumonitis induced by Shiitake mushroom spores.

Hypersensitivity pneumonitis due to the inhalation of Shiitake mushroom spores was demonstrated in a 38-year-old woman. Symptoms of cough, nausea and malaise, and clinical findings of cyanosis, bibasilar crackles, reduced lung volumes, hypoxemia, leukocytosis, elevated ESR, positive C-reactive protein, and bilateral diffuse reticulonodular shadows on chest roentgenogram improved after the patient was removed from exposure. Alveolitis was demonstrated by transbronchial lung biopsy, as well as an increase in lymphocytes in bronchoalveolar lavage. Serum precipitins and specific IgG antibodies to an extract of Shiitake mushroom spores, but not to other common molds or mushroom body, were detected in serum. Provocative inhalation test with the extract of mushroom spores caused the same clinical symptoms and signs as experienced in the workroom. This is the first report of typical hypersensitivity pneumonitis induced by Shiitake mushroom spores. Mushroom spores as well as thermophilic actinomycetes must be considered a causative agents for mushroom worker's lung.

Adult↗
PubMed1992

dNTP pool imbalance induced endonuclease: 5-fluorodeoxyuridine induced DNA double strand break in mouse FM3A cells and the mechanism of cell death.

The mechanism of intracellular deoxyribonucleotide triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A cells was studied. When the cells were treated with 1 microM 5-fluorodeoxyuridine (FdUrd), the imbalance of the cellular dNTP pool was induced. The imbalance was followed by DNA double stranded breaks and subsequent cell death. The endonuclease toward double stranded DNA has been found in a fraction of FdUrd treated cell lysate, and isolated using column chromatography. SDS-polyacrylamide gel electrophoresis showed a major protein species of approximate 45 kDa. The endonuclease was revealed, using electrophoretic separation in SDS-polyacrylamide gels containing DNA, by incubating the gels in buffer to remove SDS and to allow renaturation and enzyme activity.

Animals↗
PubMed1992

Effect of histamine antagonists and agonists on IgE production in mice.

This study was undertaken to investigate the effect of histamine, its receptor antagonists and agonists on IgE antibody production in mice. BALB/c mice were immunized intraperitoneally with 1 mg alum plus 30 micrograms of Ag90, the antigen of Japanese occupational asthma. Histamine receptor antagonists were administered before the immunization and simultaneous injection of histamine. The mice were bled 14 days after immunization and anti-Ag90 IgE antibody was obtained. Titers of the antisera were measured by passive cutaneous anaphylaxis reaction in Sprague-Dawley rats. Treatment with histamine only did not affect the level of specific IgE antibody. Administration of H1 antagonist or H2 agonist suppressed IgE production significantly. In contrast, treatment with H2 antagonist or H1 agonist augmented the IgE antibody production. Injection of H1 + H2 antagonists had no effect on the antibody production. These results suggest that histamine suppressed specific IgE production via H2 receptors and enhanced it through H1 receptors in the induction phase of the system.

Animals↗
PubMed1992

[Superoxide dismutase activity in the broncho-alveolar lavage fluid in late asthmatic response model in guinea pigs].

In order to investigate the mechanism of the late asthmatic responses (LAR), LAR model in guinea pigs was made by inhalation of a large quantity of egg albumin antigen. Serial changes of inflammatory cells and superoxide dismutase activities in broncho-alveolar lavage fluids obtained from these animals were observed. In this model, increase in inflammatory cells, such as neutrophils, was observed in the broncho-alveolar lavage fluid during the late asthmatic responses of the attack and superoxide dismutase activity increased in the broncho-alveolar lavage fluid in parallel with the increase in inflammatory cells. These results suggest that change in superoxide dismutase activity in the broncho-alveolar lavage fluid may be involved in the onset of the late asthmatic responses.

Animals↗
PubMed1992

[Inhibitory effect of amlexanox on asthmatic attacks in an aspirin sensitive asthmatic].

The anti-allergic action of amlexanox is reported to be associated with inhibition of the release of LTC4, LTD4 and histamine, and with antagonistic activity on leukotrienes. The patient was a 18-year-old female who had suffered from bronchial asthma since the age of fifteen. We diagnosed the case as aspirin sensitive asthma because of development of a severe asthmatic attack with syncope after oral administration of an analgesic for treatment of a common cold at the age of seventeen. She also had sensitivity to toothpaste. Three-minute aerosol inhalation challenge with 0.1, 1 and 10% solution of sulpyrine was performed as a stepwise increment at 20 minutes intervals. This inhalation test was positive and it evoked simultaneous increases of LTC4, LTD4 and histamine in the peripheral blood. Since the commencement of oral administration of 150 mg per day of amlexanox, the patient has had no attacks for about 8 months. In the second inhalation test, premedication with amlexanox elevated the threshold of sulpyrine and inhibited the release of LTC4, LTD4 and histamine. We report that amlexanox was of use to control asthmatic attacks in this aspirin-sensitive asthmatic patient.

Adolescent↗
PubMed1992

[Blood coagulation, fibrinolysis and various values of Lansbury's activity index in rheumatoid arthritis patients].

To clarify pathophysiological significance of blood coagulation and fibrinolysis in rheumatoid arthritis (RA) patients with various values of Lansbury's activity index, we analysed by means of the urokinase activated platelet rich plasma thromboelastography. In RA patients, coagulation time (k) and reaction time (r)+k were shorter, maximal amplitude (ma) was larger and lysis time (LT) was longer comparing with those in healthy control. Especially LT and ma showed marked differences between RA patients and control subjects. The mild RA patients whose Lansbury's activity indexes were 0-19% had significant differences from healthy control subjects, in order of LT less than ma less than r+less than k, the probability values were smaller. Significant coefficients of positive correlations with Lansbury's activity index were higher in order of LT greater than LT/k greater than ma x LT. These findings suggested that RA patients had fibrin formation, which had close relations with maximal amplitude (ma) and lysis time (LT) which had positive correlation with Lansbury's activity index.

Activities of Daily Living↗
PubMed1992

[Evaluation of allergen-specific IgE antibody and total IgE with a new IgE detection system named FAST: fluorescence allergosorbent test].

The measurement of IgE antibodies and total IgE was performed with FAST (fluorescene allergosrobent test) system using 171 serum samples obtained from normal donors and 789 serum samples obtained from patients with various allergic diseases. These were collected by departments of internal medicine, pediatrics, otorhinolaryngology and dermatology at 17 institutes in Japan. In addition to a comparative study with RAST, skin tests and provocation tests were also performed to establish a clinical diagnosis. Simultaneous measurements were made and an excellent correlation with RAST was observed with a concordance rate of 85.7% and correlation coefficient of 0.848. The specificity determined in the normal serum samples was 95.5% by RAST and 93.1% by FAST. The sensitivity determined in the samples, of which etiological allergens were identified clinically, was 82.3% by RAST and 85.3% by FAST. Among them, the sensitivity of skin test and provocation test were 92.4% and 94.7%, respectively. The concordance rate with clinical diagnosis was as high as 89.7% for both RAST and FAST. The normal upper limit of total IgE by cumulative 95% value was calculated to be 250 IU/ml for adult subjects. These results indicated the clinical usefulness of the FAST system in evaluating IgE antibodies and total IgE.

Allergens↗
PubMed1992

[Analysis of blood coagulation and fibrinolysis in rheumatoid arthritis using the urokinase activated thromboelastography].

The urokinase activated thromboelastography is useful for the analysis of blood coagulation and fibrinolysis in rheumatoid arthritis (RA). In 43 RA patients without extraarticular symptoms (mean year old +/- SD = 50.3 +/- 10.9) and 20 healthy control subjects (mean year old +/- SD = 49.5 +/- 13.8, which is not significant), we have examined reaction time (r), coagulation time (k), r+k, maximal amplitude (ma), lysis time (LT) of the urokinase (final density = 85U/ml) activated platelet rich plasma thromboelastography. In RA patients r, k, r+k are shorter, ma is larger, LT is longer than in control subjects. Especially, k, ma and LT are remarkably statistically significant. these findings suggest that most of RA patients have hypercoagulability and/or prolongation of lysis time simultaneously, k, ma and LT are pathophysiologically important.

Adult↗