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Biomedical subjects

R Kato

Publications and source records attributed to R Kato.

At least 109 records · Page 6Linked to original sources
PubMed1999

[Outbreak case caused by different colicin type of Shigella sonnei in a day nursery in Tokyo (1998)].

From October through December, 1998, person to person infection caused by Shigella sonnei had occurred in the day nursery in Hachioji-city Tokyo, and a total of 41 patients including 3 suspected cases and 3 carriers had been confirmed. Although one case was imported case which is one-year-old kindergarten child, the remaining was domestic case. Of patients, 33 cases (80.5%) were a three-year-old kindergarten children and their families, accounting for 20 and 13 cases, respectively. From the data of symptom onset of patients, epidemic could distinguish to the former part and the later part. The recur or reinfection, and re-detection case was observed in the later part. Clinical symptom of the patients was diarrhea (100%), fever (80%) and abdominal cramps (70%), and LVFX for adult or FOM for child was used for the therapy. In the colicin typing test and the antibiotic susceptibility test for 9 reagents, almost isolates in the former part were type 0 and susceptible, and MBC for FOM ranged from 6.25 to 100 micrograms/ml, whereas those in the later part was type 2 and resistant for the TC, and showed highly MBC for FOM with 50 or 100 micrograms/ml. However, both isolates showed same patterns in plasmid profiles and DNA fingerprints by RAPD analysis. On the other hand, the strain from imported case was also colicin 0, but it was different as regards resistant for ABPC and ST and two genetic analysis.

Adult↗
PubMed1999

Biphasic kinetics of imipramine N-oxidation in rat brain microsomes.

Imipramine (IMI) N-oxidase activity in brain microsomes from rats of both sexes was determined by high performance liquid chromatography, and compared with the results in rat liver microsomes. Brain and liver microsomal IMI N-oxidation was sensitive to thermal inactivation and had an optimal pH at around 9.0. IMI N-oxidase activity (15.54 pmol/min/mg protein) in brain microsomes was about one-hundredth that of liver microsomes (2.08 nmol/min/mg protein) at a substrate concentration of 5 mM. IMI N-oxidase activities in both brain and liver microsomes displayed biphasic kinetics that associated a low Km-low Vmax element with a high Km-high Vmax component. Furthermore, a significant sex difference was observed in Vmax values (male>female) in both phases, but Km values were similar between male and female rats, resulting in a significant sex difference (male>female) in intrinsic clearance values (Vmax/Km) of the low-Km and the high-Km phases.

Animals↗
PubMed1999

Contribution of flavin-containing monooxygenase and cytochrome P450 to imipramine N-oxidation in rat hepatic microsomes.

Enzymatic formation of desipramine (DMI) and imipramine N-oxide (IMINO) was kinetically characterized in rat liver microsomes at pH 8.5 and 7.5. The formation of IMINO was quickly suppressed by the preincubation of microsomes at 37 degrees C at pH 8.5, but the suppression was comparatively gentle at pH 7.5. In kinetic studies, the formation of DMI was monophasic at the two pH points, and a substrate inhibition was observed at pH 8.5, but not at pH 7.5. In contrast, the formation of IMINO was biphasic at both pH points, Le., the summation of a low-Km phase and a high-Km phase. Methimazole (MZ), an inhibitor of flavin-containing monooxygenase (FMO), markedly suppressed the low-Km phase of IMINO formation at both pH points. MZ also suppressed DMI formation at pH 8.5, but it elevated DMI formation at pH 7.5. SKF 525-A, an inhibitor of cytochrome P450 (CYP), markedly suppressed DMI formation at both pH points. The inhibitor suppressed IMINO formation in the high-Km phase of the biphasic kinetics at both pH points, whereas it stimulated the activity of the low-Km phase at pH 7.5. These results suggest that CYP enzyme(s) are mainly responsible for DMI formation at pH 8.5 and 7.5, and FMO enzyme(s) also are involved in IMI N-demethylation at a higher pH range in rat liver microsomes, at least in part. In the formation of IMINO, FMO is a major enzyme at both pH points, and CYP may also contribute to the N-oxide formation to some extent at pH 8.5.

Animals↗
PubMed1999

Distribution of JC virus DNA in peripheral blood lymphocytes of hematological disease cases.

OBJECTIVE: The distribution of JC virus DNA in peripheral blood was surveyed by the polymerase chain reaction using the late genes as markers. RESULTS: Six out of 52 cases of hematological diseases and one systemic lupus erythematosus case out of 17 cases were positive for JCV DNA. After separation into B and T lymphocytes by a cell sorter, JCV DNA was found in both cell types prepared from adult T cell leukemia and PML patients. CONCLUSION: Only 1 or 2 copies of JCV genome were calculated to exist in a cell based on the time course analysis of PCR. Only B lymphocytes and glial brain cells are known to produce nuclear factors which support the growth of the virus. The result that B lymphocytes contained a copy number of JCV genome similar to T lymphocytes suggests that there is some barrier to viral growth in susceptible B lymphocytes, and that the growth of JCV is different from that of other virulent viruses.

Aged↗
PubMed1999

[Validity analysis of Sapporo Medical University-sexual function questionnaire].

BACKGROUND: We evaluated the validity of the Sapporo Medical University-sexual function questionnaire, comparing the response to each question of patients having sexual dysfunction with those of normal volunteers as controls. PATIENTS AND METHODS: Responses from 335 patients with sexual dysfunction and 490 normal volunteers aged from 20 to 39 years old were evaluated. We compared mean scores of each question for patients having sexual dysfunction with those for controls. Discriminant analysis was used for evaluating which questions contributed more strongly to discriminating patients having the disease from controls. The analysis was also used for validating the questionnaire. RESULTS: Mean scores for patients with sexual dysfunction were significantly lower than those for controls in all questions. Nine of the 11 questions were statistically useful to discriminate these two groups with discriminant analysis. The analysis also revealed that questions about frequency of erection, rigidity of the erectile penis and duration of erection highly contributed to discriminate these two groups. The discriminant analysis achieved high sensitivity and specificity for classifying the groups. CONCLUSION: These results suggest that the use of the Sapporo Medical University-sexual function questionnaire is valid for discriminating patients with sexual dysfunction from subjects with normal sexual function.

Adult↗
PubMed1998

In vitro reconstitution of human B-cell ontogeny: from CD34(+) multipotent progenitors to Ig-secreting cells.

We describe a long-term, in vitro culture system initiated with CD34(+) or CD34(+)CD38(-) umbilical cord blood hematopoietic progenitors that supports normal human B-lineage development, including the production of mature Ig-secreting B cells. In the first stage (human B-progenitor long-term culture [HB-LTC]), CD34(+) hematopoietic progenitors are cultured on the murine stromal cell line, S17, leading to the sustained production of large numbers of CD10(+), CD19(+) early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrogate light chain), cytoplasmic mu chain, and surface IgM expression were used to characterize the CD19(+) B progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B cells, and IgM+, IgD-/+ immature B cells. The limited expansion of IgM+ B cells and the immature surface phenotype of this population (IgM+, IgD+, CD10(+), CD38(+)) suggested that HB-LTC conditions were unable to provide appropriate signals for further differentiation. A second culture stage was used to determine if these immature B cells were functionally competent. Purified CD19(+) cells were transferred onto fibroblasts expressing human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell proliferation, modulation of the IgM+ cell surface phenotype to one consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated using two-stage cultures established with highly purified multipotent CD34(+)CD38(-) hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transition points in human B ontogeny, beginning with commitment to the B lineage and leading to development and activation of mature B cells.

Animals↗
PubMed1998

The novel substrate recognition mechanism utilized by aspartate aminotransferase of the extreme thermophile Thermus thermophilus HB8.

Aspartate aminotransferase (AspAT) is a unique enzyme that can react with two types of substrate with quite different properties, acidic substrates, such as aspartate and glutamate, and neutral substrates, although the catalytic group Lys-258 acts on both types of substrate. The dynamic properties of the substrate-binding site are indispensable to the interaction with hydrophobic substrates (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63). AspATs from various organisms are classified into two subgroups, Ia and Ib. The former includes AspATs from Escherichia coli and higher eukaryotes, whereas the latter includes those from Thermus thermophilus and many prokaryotes. The AspATs belonging to subgroup Ia each have an Arg-292 residue, which interacts with the distal carboxyl groups of dicarboxylic (acidic) substrates, but the functionally similar residue of subgroup Ib AspATs has not been identified. In view of the x-ray crystallographic structure of T. thermophilus AspAT, we expected Lys-109 to be this residue in the subgroup Ib AspATs and constructed K109V and K109S mutants. Replacing Lys-109 with Val or Ser resulted in loss of activity toward acidic substrates but increased that toward the neutral substrate, alanine, considerably. These results indicate that Lys-109 is a major determinant of the acidic substrate specificity of subgroup Ib AspATs. Kinetic analysis of the interactions with neutral substrates indicated that T. thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E. coli AspAT. A flexible active site in the rigid T. thermophilus AspAT molecule may explain its high activity even at room temperature.

Amino Acid Sequence↗
PubMed1998

Quick identification and localization of CpG islands in large genomic fragments by partial digestion with Hpa II and Hha I.

More than 50% of mammalian genes are associated with CpG islands and thus they serve as a good gene marker. We have devised a simple method to scan large pieces of native or cloned genomic DNA for CpG islands. The method is based on the presence of multiple Hpa II and Hha I sites in CpG islands, at a frequency 30 times higher than in the rest of the genome. The steps include complete digestion of DNA with a rare-cutting restriction endonuclease (to produce large fragments with defined ends), partial digestion with Hpa II and Hha I, and subsequent Southern hybridization with an end probe. This identifies a CpG island as a cluster of sub-bands and, based on their electrophoretic mobility, one can immediately locate the island relative to the ends. For many vectors, universal probes flanking the cloning site are available, enabling the simultaneous analysis of a large number of samples. We demonstrated the usefulness of the method by analyzing known CpG islands in native genomic DNA and lambda, cosmid and P1 clones, and by isolating two novel transcribed islands from anonymous cosmid clones. Our method is quick, inexpensive, and can detect CpG islands with few or even no rare-cutter sites.

Animals↗
PubMed1998

Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament.

Self-assembly of RecA protein in solution and on single-stranded DNA exerts a significant effect on the catalytic activities of this protein. To manipulate the self-association reaction, we examined the effects of various salts on the self-association of RecA from Thermus thermophilus (ttRecA) by circular dichroism spectroscopy and gel-filtration analysis. We showed that the self-association of ttRecA strongly depends on the kind and concentration of the salt, as well as on the protein concentration. Chaotropic ions were especially useful for obtaining RecA in its hexameric and monomeric states. On the basis of these observations, we were able to regulate the oligomeric states of ttRecA and we then examined the activity of RecA in various oligomeric states. Monomeric ttRecA bound to ssDNA and formed a nucleoprotein filament, which showed ssDNA-dependent ATPase activity. These results suggest that the monomeric form of RecA is an intermediate in filament formation on ssDNA.

Chromatography, Gel↗
PubMed1998

Domain organization and functional analysis of Thermus thermophilus MutS protein.

MutS protein binds to DNA and specifically recognizes mismatched or small looped out heteroduplex DNA. In order to elucidate its structure-function relationships, the domain structure of Thermus thermophilus MutS protein was studied by performing denaturation experiments and limited proteolysis. The former suggested that T. thermophilus MutS consists of at least three domains with estimated stabilities of 12.3, 22.9 and 30.7 kcal/mol and the latter revealed that it consists of four domains: A1 (N-terminus to residue 130), A2 (131-274), B (275-570) and C (571 to C-terminus). A gel retardation assay indicated that T.thermophilus MutS interacts non-specifically with double-stranded (ds), but not single-stranded DNA. Among the proteolytic fragments, the B domain bound to dsDNA. On the basis of these results we have proposed the domain organization of T. thermophilus MutS and putative roles of these domains.

Adenosine Triphosphatases↗
PubMed1998

Crystallization and preliminary X-ray characterization of aspartate aminotransferase from an extreme thermophile, Thermus thermophilus HB8.

Recombinant aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8, has been crystallized in two different crystal forms. The crystals of both forms are orthorhombic and belong to space group P212121 with cell dimensions a = 124.3, b = 113.6 and c = 61.6 A for form I and a = 197.3, b = 109.7 and c = 80.3 A for form II. The crystals of form I and II diffract to 2.1 and 2.5 A resolution, respectively, on a conventional laboratory rotating-anode source. Two heavy-atom derivatives have been identified for form I.

Aspartate Aminotransferases↗
PubMed1998

Prognostic and Therapeutic Implications of the MIB-1 Labeling Index in Breast Cancer.

BACKGROUND: Assessment of tumor proliferative activity is considered to be the most powerful prognostic factor aside from axillary lymph node status. The purpose of this study is to assess the clinical value of measurement of proliferative activity using the MIB-1 labeling index in patients with breast cancer. METHODS: Surgical specimens from 36 patients with benign breast disorders and146 patients with breast cancer were investigated. The MIB-1 labeling index wasdetermined on the specimens stained by immunohistochemical methods as much as possible. Clinical factors associated with the MIB-1 labeling index were reviewed. RESULTS: The MIB-1 labeling index for non-proliferative disorders, proliferative disorders, and breast cancer was 3.4 +/-1.9%, 8.9 +/-6.2% and 20+/-12%, respectively. The MIB-1 labeling index and tumor size, lymph node metastasis status, and clinical stage according to the TNM classification correlated significantly. Survival rate was inversely correlated with the MIB-1 labeling index. No patientwith an MIB-1 labeling index of less than 10% had lymph node metastases, and all are alive without recurrence. Patients with an MIB-1 labeling index of over 30% had an extremely poor prognosis. CONCLUSION: The MIB-1 labeling index is very useful for predicting both either extremely good or extremely poor prognosis, and axillary lymph node metastasis

Journal Article↗
PubMed1998

A methylation imprint mark in the mouse imprinted gene Grf1/Cdc25Mm locus shares a common feature with the U2afbp-rs gene: an association with a short tandem repeat and a hypermethylated region.

We identified a sperm-specific methylation imprint mark (Site II) associated with a short tandem repeat sequence and a site/region methylated in both gametes (Site I) in the Grf1 locus on mouse chromosome 9, which shared a common feature with the U2afbp-rs gene. Sites or regions of gamete-specific methylation in imprinted genes are strong candidates for carrying information regarding the parental origin of alleles. The gamete-specific methylation pattern of Sites I and II was conserved after fertilization, but attained the somatic cell pattern by the blastocyst stage. In primordial germ cells, Site I was methylated, but Site II was unmethylated in both male and female embryos, suggesting that the sperm-specific methylation imprint mark in Site II was established during spermatogenesis. These common features in methylation imprint regions may be a clue to identifying regions carrying primary information for the imprinting regulation.

Animals↗
PubMed1998

Genetic polymorphisms in human liver phenol sulfotransferases involved in the bioactivation of N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines.

Three related forms of phenol sulfotransferase (PSULT), thermostable ST1A2 (SULT1A2hum) and ST1A3 (SULT1A1hum) and a thermolabile TL-PST (SULT1A3hum), are known to exist in human livers. Thermostable forms, whose activities are polymorphically distributed, have been shown to mediate the bioactivation of carcinogenic N-hydroxy arylamines and heterocyclic amines. To clarify the nature of the sulfation polymorphism, the study compared the expressed levels of ST1A2, ST1A3 and TL-PST mRNAs in human livers by the method of reverse-transcriptase polymerase chain reaction (RT-PCR), utilizing HindIII, BamHI and SnaBI sites which were unique to the above PSULT cDNAs, respectively. Of the PCR products derived from human liver (n = 26), 43-89, < 1-29 and < 1-21% showed the restriction pattern characteristic for ST1A3, ST1A2 and TL-PST cDNAs, respectively, thus indicating that ST1A3 mRNA is the major transcript. Hepatic p-nitrophenol and dopamine sulfation rates ranged from 440-2670 and < 5-460 pmol/min per mg protein in the 26 individuals, respectively. The observed differences in the ST1A3 and TL-PST mRNA levels were consistent with the differences in p-nitrophenol and dopamine sulfations. Relative levels of hepatic ST1A3 mRNA were non-normally distributed and correlated significantly with p-nitrophenol sulfation. In addition, variant forms of ST1A3 mRNA encoding Arg213His and Met223Val were detected in human livers. With regard to Arg213His, 28 individuals who had homozygous 213Arg alleles, 15 individuals who were heterozygotes and nine homozygous 213His individuals were found by a newly established genotyping method among 52 human liver samples. Frequency of 223Val allele was apparently lower than that of 213His allele, as no homozygous 223Val individual and only three individuals who were heterozygotes (223Met/Val) were observed among 52 individuals. These results suggest that regulation of p-nitrophenol sulfation occurs at the level of gene transcription of ST1A3 which is the major transcript of the three PSULT mRNAs and that a polygenic basis for the apparent genetic polymorphism of sulfation was likely because of the existence of ST1A3 variants.

Amines↗
PubMed1998

Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa. Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased. The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T. thermophilus MutM protein was active in E.coli. The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity. Size-exclusion chromatography indicated that T. thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution. Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30%. Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C. Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.

Amino Acid Sequence↗