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Biomedical subjects

H Ishizaki

Publications and source records attributed to H Ishizaki.

At least 19 recordsLinked to original sources
PubMed1992

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells.

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.

Animals↗
PubMed1992

Phylogenetic relationships of the genera Arthroderma and Nannizzia inferred from mitochondrial DNA analysis.

The phylogenetic relationship of the genera Arthroderma and Nannizzia, was investigated by mitochondrial DNA analysis based on the restriction-fragment-length polymorphisms. Phylogenetic trees made on ten species. A. benhamiae, A. insingulare, A. quadrifidum, A. simii, A. vanbreuseghemii, N. fulva, N. grubyia, N. gypsea, N. incurvata and N. otae showed no definite distinctions between the genera Arthroderma and Nannizzia. These results support the conclusion of Weitzman et al. that the genera Arthroderma and Nannizzia are congeneric.

Ascomycota↗
PubMed1992

Phylogeny of the genera Trichophyton using mitochondrial DNA analysis.

Diversity of mitochondrial DNA (mtDNA) was investigated in 92 Trichophyton rubrum strains, 2 T. mentagrophytes var. mentagrophytes, 2 T. m. var. interdigitale, 2 T. m. var. goetzii, 1 T. m. var. erinacei, 2 T. quinckeanum, 2 T. schoenleinii, 1 T. tonsurans, 2 T. verrucosum var. album, 2 T. v. var. discoides, 1 T. violaceum var. violaceum, 1 Arthroderma benhamiae, and 1 A. vanbreuseghemii using endonucleases, Hae III, Msp I, Hind III, Xba I, and Bgl II. Trichophyton species were divided into 7 groups, and a phylogenetic tree was produced based on sequence divergence within mtDNA. The following results were obtained: (1) T. rubrum was divided into 2 groups Type I and Type II, and was suggested to be a complex. (2) A. benhamiae was closely related to T. m. var. erinacei. (3) T. rubrum Type II, T. tonsurans, and A. vanbreuseghemii showed identical restriction profiles, and were suggested to be closely related to each other or identical. (4) T. quinckeanum and T. schoenlenii showed identical restriction profiles, which differed slightly from those of A. vanbreuseghemii. (5) mtDNA analysis was useful in identifying pleomorphic strains.

DNA, Fungal↗
PubMed1992

Synthesis of bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by stepwise and selective formation of three disulfide bridges.

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm, Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for alpha-amino group. Three disulfide bonds were bridged step by step (A6-A11, A20-B22, and A7-B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.

Amino Acid Sequence↗
PubMed1992

Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers.

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6-A11, A7-B10, and A20-B22. An intermolecular bond of A20-B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6-A11, A7-B10, A20-B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6-A7, A11-B10, A20-B22-cystine]- and [A6-B10, A7-A11, A20-B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.

Amino Acid Sequence↗
PubMed1992

Structure and expression of bombyxin-related peptide genes of the moth Samia cynthia ricini.

From the genomic DNA of the moth Samia cynthia ricini, we cloned and characterized six clustered genes that encode precursor molecules for peptides structurally related to bombyxin, a Bombyx mori brain secretory peptide that is structurally like insulin and functionally like the prothoracicotropic hormone. The precursor molecules deduced from these genes have the domain organization of signal peptide/B-chain/C-peptide/A-chain, as in preprobombyxins and preproinsulins. The Samia bombyxin-related peptide (SBRP) genes are classified into families A and B according to their sequence homology. Two genes belonging to different families are arranged close to each other to form a pair with opposite transcriptional orientations (A-1/B-1, A-2/B-2, and A-3/B-3). None of these genes have introns, and gene B-3 has an in-frame stop codon representing a pseudogene. Four genes, A-1, A-3, B-1, and B-2, are expressed in Samia brain. Genomic Southern hybridization suggests that the Samia genome contains many other SBRP genes.

Amino Acid Sequence↗
PubMed1992

[Statistical survey of multiple primary cancers of the head and neck].

The incidence and factors associated with multiple primary cancer were surveyed and analyzed from April 1978 through December 1990 in our clinic. Fifty-nine cases with multiple primary cancers were selected for study from among 579 cases with malignant head and neck region tumors. The cancers involved the larynx in 17 cases (8.4%), oral cavity in 14 (19.4%), oropharynx in 13 (41.9%), hypopharynx in 7 (8.0%) and nasal cavity or paranasal sinus in 7 (8.0%). Twenty-one cases (35.6%) arose synchronously within one year. From our observations, heavy drinking and smoking were most strongly suspected to be risk factors for induction of malignancy. Most multiple primary cancers in the head and neck region were observed to coexistent in a "so called multicentric zone" consisting of the oral area, pharyngeal area, larynx, esophagus, stomach, and tracheobronchial tree. To avoid overlooking concealed cancer in the multicentric zone, upper gastro-intestinal endoscopy, bronchial endoscopy, chest X-ray and sputum examination should be performed for screening of patients with head and neck malignancies.

Adult↗
PubMed1992

[A case of transfusion-associated GVHD after total laryngectomy].

We report a case of fatal transfusion-associated graft versus host disease (GVHD) that developed in a patient with laryngeal cancer. After a 39.6Gy irradiation dose, total laryngectomy with right radical neck dissection was performed. The postoperative course was uneventful. However, seven days after blood transfusion, high fever (38.5 degrees C) suddenly appeared. On the ninth day, watery diarrhea and facial erythema were observed. On the 12th day, liver disturbance and pancytopenia developed. The patient died on the 16th day because of overwhelming sepsis. Transfusion-associated GVHD has a mortality rate of more than 90%. Therefore, the most important procedure for preventing GVHD is the use of irradiated blood products. Furthermore unnecessary blood transfusion should be avoided.

Graft vs Host Disease↗
PubMed1992

Relevance of the Tullio phenomenon in assessing perilymphatic leak in vertiginous patients.

The Tullio phenomenon was studied in 57 control subjects, in 7 patients with confirmed PLF and in 149 patients with different types of inner ear pathology. The subjects were exposed to low-frequency sound (LFS) with sound pressure levels ranging from 130 to 132 dB and at frequencies of 25, 50, and 63 Hz. The duration of each stimulation was 30 seconds. The postural responses were measured using a force platform and analyzed with a computer. In control subjects the LFS stimulation somewhat improved the postural stability. All patients with PLF showed significantly altered postural stability during LFS exposure. Also about 20 percent of patients with labyrinthine vertigo, patients with Menièré's disease, patients with chronic middle ear disease (with vertigo) had pathologic response to LFS. None of the patients with pure sensorineural hearing loss without vertigo responded to LFS. Post-urography with LFS loading is a new, promising tool in detection of PLF, but the specificity of the test is still unsatisfactory.

Adolescent↗
PubMed1991

Phylogeny and molecular epidemiology of Sporothrix schenckii in Japan.

Mitochondrial DNA(mtDNA) diversity was investigated in 257 clinical isolants of Sporothrix schenckii obtained from 4 districts in Japan. S. schenckii was classified into 10 types based on Hae III restriction profiles. Phylogeny of types constructed by the method of Fitch and Margoliash [1] on the estimated sequence divergence within mtDNA using the methods of Nei and Li [2], showed that S. schenckii are grouped into 2 clusters, one group consisting of types 1, 2 and 3, and the other group consisting of the other seven types. In addition, types 1, 2, and 5 were correlated with their geographic origin, whereas type 4 was present throughout Japan.

DNA, Fungal↗
PubMed1991

Metabolism of azoxymethane, methylazoxymethanol and N-nitrosodimethylamine by cytochrome P450IIE1.

The metabolism of azoxymethane (AOM), methylazoxymethanol (MAM) and N-nitrosodimethylamine (NDMA) by liver microsomes from acetone-induced rats as well as by a reconstituted system containing purified cytochrome P450IIE1 was examined. The products consisted of MAM from AOM; methanol and formic acid from MAM; and methylamine, formaldehyde, methanol, methylphosphate and formic acid from NDMA. Compared to liver microsomes from untreated rats, the metabolic activity of acetone-induced microsomes was approximately 4 times higher for all three carcinogens. Using the reconstituted system, the enzyme activities (nmol substrate metabolized/nmol P450/min) for AOM, MAM and NDMA were 2.88 +/- 1.14, 2.87 +/- 0.59 and 9.47 +/- 2.24 respectively. Incubations carried out in the presence of a monoclonal antibody to cytochrome P450IIE1 resulted in a 85-90% inhibition of all three reactions in this system. These results provide conclusive evidence that AOM, MAM and NDMA are metabolized by the same form of rat liver cytochrome P450. In addition, the stoichiometry of NDMA products formed in these reactions indicates that denitrosation, a presumed detoxication process, and alpha-hydroxylation, an activation reaction, are also catalyzed by the same cytochrome P450 isozyme.

Acetone↗
PubMed1991

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cytochrome P450IIB1 in a reconstituted system.

Several previous studies have suggested that cytochrome P450IIB1 is involved in the bioactivation of the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rats as well as in mouse lung microsomes. The present investigation was undertaken to study the metabolism of NNK by purified cytochrome P450IIB1 in a reconstituted system. The metabolites 4-hydroxy-4-(3-pyridyl) butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl) butyric acid (keto acid), 4-oxo-4-(3-pyridyl) butanol (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide) and 4-oxo-4-(3-pyridyl)-1-butanol (keto alcohol) were quantitated by HPLC. The results showed that, in addition to alpha-hydroxylations, cytochrome P450IIB1 also catalyzed the formation of NNK-N-oxide efficiently, and to a certain extent, the conversion of NNK primary hydroxylation metabolites (keto aldehyde and keto alcohol) to secondary metabolites (keto acid and hydroxy acid). Cytochrome b5 at a ratio of 1:1 or 2:1 to P450IIB1 had no significant effect on the metabolic activities and profiles of NNK. The apparent Km values for the formation of keto aldehyde, NNK-N-oxide and keto alcohol were respectively 191.2, 131.4 and 318.0 microM with corresponding apparent Vmax values of 89.7, 295.5 and 333.3 pmol/min/nmol P450, indicating that hydroxylation at the alpha-methyl position is preferred over the alpha-methylene position. Measurement of formaldehyde, a product derived from the alpha-methyl hydroxylation, was developed as a convenient method to study NNK metabolism. Thiourea activated cytochrome P450IIB1-catalyzed NNK metabolism significantly. Phenethyl isothiocyanate, an inhibitor of NNK-induced lung carcinogenesis, inhibited P450IIB1-catalyzed NNK demethylation in a concentration-dependent manner. This work demonstrates that purified P450IIB1 can catalyze the conversion of NNK to most of its oxidative metabolites.

Animals↗
PubMed1991

Tullio phenomenon and postural stability: experimental study in normal subjects and patients with vertigo.

The effect of low-frequency sound (LFS) on postural stability was studied in 55 healthy volunteers and in 152 patients with vertigo due to different types of inner ear disease. The sound pressure levels ranged from 130 to 132 dB and were given at frequencies of 25, 50, and 63 Hz. The duration of each stimulation lasted 30 seconds. The postural responses were measured with a force platform. The LFS stimulation improved the postural stability of the healthy subjects through the alerting response. We did not observe any difference in the body sway according to whether the LFS was delivered with monaural or binaural stimulation. Twenty-six percent of the patients with Meniere's disease, 40% with chronic otitis media with vertigo, and 26% with vertigo of peripheral origin experienced significant postural instability at least at two of the three test frequencies during stimulation with LFS. The results demonstrate that in subjects with different types of inner ear disease the vestibular system starts to respond to LFS. The activation of vestibulospinal responses by LFS seems to be an expression of the Tullio phenomenon.

Acoustic Stimulation↗
PubMed1991

Postural stability on moving platform oscillating at high frequencies. Effect of vestibular lesion.

The effect of linear perturbation at a frequency range of 8 to 24 Hz on postural stability was studied in 7 patients with unilateral vestibular function loss. Force platform technique was used with automatic analysis resulting in the sway velocity of the center point of force. In all patients the support surface movement produced increased sway velocity which was significantly greater than in the controls. Tests at lower frequencies (8 and 10 Hz) produced higher sway velocities than tests at higher frequencies (12, 14, 16 and 24 Hz). The results indicate that the vestibular system controls postural reflexes at a substantially higher frequency range than hitherto suggested.

Adult↗
PubMed1991

Repeatability and effect of instruction of body sway.

The effect of instruction and repeatability of posturography measurement was studied in 24 healthy volunteers. In an instruction, "stable" (inverted pendular model) and "relaxed" (segmental oscillating model) positions were studied. In addition, the effect of habituation adaptation, and day-to-day variation were investigated. Sway velocity and average and maximum displacement during vibration perturbated stance were analysed. In individual responses, no statistically significant day-to-day variation could be observed and the individual variation was small. In the "relaxed" condition, the instability was greater when maximum displacement was analysed. No significant changes in sway velocity and average displacement were observed. The results demonstrated remarkable stability of normal postural control mechanism.

Adult↗