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Biomedical subjects

K Kishi

Publications and source records attributed to K Kishi.

At least 235 records · Page 13Linked to original sources
PubMed1997

Crystal structures of substrate binding site mutants of manganese peroxidase.

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus, Phanerochaete chrysosporium, catalyzes the oxidation of MnII to MnIII. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed MnII binding site of MnP consists of a heme propionate, three acidic ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of MnII bound to the protein. Crystals grown in the absence of MnII, or in the presence of EDTA, exhibited diminished electron density at this site. Crystals grown in excess MnII exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major MnII binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined. The mutant structures lack a cation at the MnII binding site. The structure of the MnII binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.

Basidiomycota↗
PubMed1997

Developmental gene expression of gastrin receptor in rat stomach.

Gastrin, which is present in fetal plasma, may have important roles in the development of gastric mucosa, since it is not only a potent stimulator of gastric acid secretion but also a growth promoting factor. Gastrin regulates various cellular functions via its receptors on cell membrane. Therefore, in order to elucidate a role for gastrin in the development of gastrointestinal system during gestation, Northern blot analysis was performed. The results of the study suggested that gastrin receptor is mainly present on parietal cells. Furthermore, proton pump and gastrin receptor gene expressions in parietal cells were strongly stimulated by the administration of exogenous gastrin. In conclusion, gastrin may be involved in the developmental change of parietal cells through its receptors.

Animals↗
PubMed1997

Site-directed mutations at phenylalanine-190 of manganese peroxidase: effects on stability, function, and coordination.

A series of site-directed mutants, F190Y, F190L, F190I, and F190A, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium was generated by overlap extension with the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The manganese peroxidase variants (MnPs) were purified and characterized by kinetic and spectroscopic methods. At pH 4.5, the UV-vis spectra of the ferric and oxidized states of the mutant proteins were very similar to those of the wild-type enzyme. Steady-state kinetic analyses showed that the apparent Km and k(cat) values for MnII and H2O2 also were similar to the corresponding values for the wild-type MnP. The apparent Km and k(cat) values for ferrocyanide oxidation by MnP were not affected by the F190Y, F190L, or F190I mutations; however, the apparent Km value for ferrocyanide oxidation by the F190A mutant MnP was approximately 1/8 of that for the wild-type enzyme. Likewise, the apparent k(cat) value for ferrocyanide oxidation by the MnP F190A mutant was approximately 4-fold greater than the corresponding k(cat) for the wild-type MnP. The stabilities of both the native and oxidized states of MnP were significantly affected by several of the mutations at Phe190. Replacement of Phe190 by either Ile or Ala significantly destabilized the resultant proteins to thermal denaturation. Moreover, the rates of spontaneous reduction of the oxidized intermediates, MnP compounds I and II, were dramatically increased for the F190A mutant relative to the rates observed for the wild-type enzyme. The spectroscopic properties of the wild-type and F190 mutant MnPs were examined as a function of pH. At room temperature, increasing pH from 5.0 to 8.5 induced a FeIII high- to low-spin transition for all of the MnP proteins. This transition may involve direct coordination of the distal His residue to the heme iron to produce bishistidinyl coordination as suggested by magnetic circular dichroism spectroscopy. The pH at which this transition occurred was considerably lower for the F190A and F190I variants and suggests that Phe190 plays a critical role in stabilizing the heme environment of MnP.

Basidiomycota↗
PubMed1997

The fifth allele of the human deoxyribonuclease I (DNase I) polymorphism.

The fifth allele, DNASE1*5, of human deoxyribonuclease I (DNase I) has been discovered. Polymerase chain reaction fragments containing exon 5 of the DNase I gene were screened for DNA polymorphism using single-strand conformation polymorphism (SSCP) analysis. DNAs from 114 unrelated Japanese and 81 German individuals were tested and a new variant was detected. By DNA sequencing analysis, this variant was found to be caused by a heterozygous G-A transition at nucleotide position 1227 that results in a Val to Met substitution at amino acid position 92 of the mature enzyme. The nucleotide substitution was also confirmed by mismatched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. Genotyping of the variant could be carried out by three independent reactions based on PCR amplification, and phenotyping by isoelectric focusing followed by immunostaining. The results supported the presence of the fifth codominant allele, DNASE1*5, which generates a new isozyme.

Alleles↗
PubMed1997

Isolation and partial characterization of cytoplasmic granules of LAK cells from mice showing side effects of recombinant human IL-2.

The side effects of recombinant human interleukin-2 (rhIL-2) in mice are considered to be mediated by lymphokine-activated killer (LAK) cells that have cytoplasmic granules and infiltrate into target organs. In cloned cytotoxic lymphocytes (CLs) including LAK cells in vitro, granule exocytosis is one of the mechanisms in target cell lysis, and hemolytic and BLT-serine esterase (BLT-SE) activities detected in the granules are important to the cytotoxicity of the CLs in vitro. However, no information is available on these activities of LAK granules in vivo in mice showing the side effects of rhIL-2. Therefore, the present study was designed to isolate the LAK granules from the homogenate of LAK cell-rich subcutaneous tissue in mice treated with continuous infusion of a dose of rhIL-2 that induces known toxicity. High activities in hemolysis and BLT-SE were detected in Percoll fractions from the tissue homogenate. Electron microscopic observation of these fractions revealed almost only LAK granules. These results indicate that hemolytic and BLT-SE activities are detectable in the LAK granules in vivo, suggesting that the cells have potency for cytotoxicity via their granules and are involved in the toxicity of rhIL-2 in mice. In addition, perforin, which has been identified as a hemolytic protein in CL granules in vitro, was not detected by immunoblotting in the granule fraction of the LAK cells in vivo, indicating that the hemolytic activity of the LAK granules in vivo is not due to perforin.

Animals↗
PubMed1997

Regenerating gene expression in normal gastric mucosa and indomethacin-induced mucosal lesions of the rat.

Regenerating (reg) gene expression was tested in rat gastrointestinal mucosa to investigate the role played by this gene in the healing of mucosal lesions. Expression of reg mRNA was higher in the stomach than in any other region of the gastrointestinal tract. The gastric cells that expressed reg mRNA were located in the deepest mucosal layer and were small in diameter. In an injured state following indomethacin treatment, reg gene expression was markedly augmented, accompanied by an increase of c-fos expression and healing of the mucosal lesions. These results suggest a role of the reg gene in the healing of gastrointestinal mucosal lesions.

Animals↗
PubMed1997

Brain MR imaging in patients with hepatic cirrhosis: relationship between high intensity signal in basal ganglia on T1-weighted images and elemental concentrations in brain.

In patients with hepatic cirrhosis, the globus pallidus and putamen show high intensity on T1-weighted MRI. While the causes of this high signal have been thought to include paramagnetic substances, especially manganese, no evidence for this has been presented. Autopsy in four cases of hepatic cirrhosis permitted measurement of metal concentrations in brain and histopathological examination. In three cases the globus pallidus showed high intensity on T1-weighted images. Mean manganese concentrations in globus pallidus, putamen and frontal white matter were 3.03 +/- 0.38, 2.12 +/- 0.37, and 1.38 +/- 0.24 (micrograms/g wet weight), respectively, being approximately four- to almost ten-fold the normal values. Copper concentrations in globus pallidus and putamen were also high, 50% more than normal. Calcium, iron, zinc and magnesium concentrations were all normal. The fourth case showed no abnormal intensity in the basal ganglia and brain metal concentrations were all normal. Histopathologically, cases with showing high signal remarkable atrophy, necrosis, and deciduation of nerve cells and proliferation of glial cells and microglia in globus pallidus.. These findings were similar to those in chronic manganese poisoning. On T1-weighted images, copper deposition shows no abnormal intensity. It is therefore inferred that deposition of highly concentrations of manganese may caused high signal on T1-weighted images and nerve cell death in the globus pallidus.

Basal Ganglia↗
PubMed1997

DYS19 marker in Japanese populations.

Allele frequencies of the Y chromosome-specific short tandem repeat system DYS19 (Y-27H39) were determined from blood samples obtained from 251 unrelated Japanese in Fukui and Gunma Prefectures. Five alleles that contained 10, 11, 12, 13 and 14 repeats of GATA were detected. Allele frequency distributions in Japanese populations were different from those in other Asian and Caucasian populations.

Alleles↗
PubMed1997

Helicobacter pylori induces proinflammatory cytokines and major histocompatibility complex class II antigen in mouse gastric epithelial cells.

Although Helicobacter pylori has been reported to stimulate the release of various cytokines from gastric tissue, it remains unknown whether normal and nontumorous gastric epithelial cells produce these cytokines. Therefore, in this study, we used a normal mouse gastric surface mucous cell line (GSM06) to determine whether gastric epithelial cells produce proinflammatory cytokines in response to H. pylori. The expression of MHC class II antigen was also examined, to investigate whether gastric epithelial cells participate in the immune response to H. pylori. In the study, GSM06 cells were incubated with H. pylori or its lipopolysaccharide (LPS). Proinflammatory cytokines were detected by Northern and Western blot analysis. The expression of MHC class II antigen was examined by fluorescence activated cell sorter (FACS) analysis. Genetic expression of proinflammatory cytokines such as interleukin-1alpha, tumor necrosis factor-alpha, and cytokine-induced neutrophil chemoattractant-2beta was enhanced by both intact and sonicated H. pylori, but not by H. pylori LPS. The expression of MHC class II antigen was induced by H. pylori more strongly than by interferon-gamma. We conclude that H. pylori induces the expression of proinflammatory cytokines and MHC class II antigen in gastric epithelial cells. Gastric epithelial cells may act as antigen-presenting cells and participate in the immune response to H. pylori infection.

Animals↗
PubMed1997

Distribution of prostaglandin E receptors in the rat gastrointestinal tract.

AIMS: In order to study the role of prostaglandin in the regulation of the gastrointestinal functions, gene expression of prostaglandin receptors along the rat gastrointestinal tracts were investigated. METHODS: Rats were used for the study. The combination of counterflow elutriation separation of mucosal cells and Northern blot analysis was used to detect the gene expression of prostaglandin receptors in gastrointestinal tracts. RESULTS: In small intestine and colon, prostaglandin E2 EP1 and EP3 receptor mRNAs were mainly localized in the deeper intestinal wall containing muscle layers. EP4 receptor gene expression, on the other hand, was detected in the intestinal mucosal layer. In the stomach, EP1 mRNA was detected in gastric muscle layers, whereas EP3 and EP4 receptor gene expression was mainly present in the gastric mucosal layer containing epithelial cells. In gastric epithelial cells, parietal cells were found to have both EP3 and EP4 receptors. At lower concentrations, prostaglandin E2 inhibited gastric acid secretion by parietal cells probably through EP4 receptors. At higher concentrations, however, it stimulated it. On the other hand, mucous cells possessed only EP4 receptor mRNA. CONCLUSIONS: Thus, it is suggested that prostaglandin E2 modulates gastrointestinal functions through at least three different prostaglandin receptors (EP1, EP3, and EP4), each of which has a distinct contribution in the gastrointestinal tract.

Aminopyrine↗
PubMed1997

Novel analysis of minimal residual disease in leukemia with TCR beta rearrangement--detection of monoclonality by single strand conformation polymorphism and PCR using a clonotype primer of leukemic T cell receptor beta-chain RNA.

Several means of analyzing minimal residual disease (MRD) in leukemia involving the rearranged T cell receptor (TCR) gene have been described. We investigated MRD in leukemia with TCR beta rearrangement by examining TCR beta-chain RNA. A complementary DNA (cDNA) corresponding to the variable region of the TCR beta-chains originating from the peripheral blood or bone marrow from four patients was amplified. Single strand conformation polymorphism (SSCP) analysis of amplified cDNA showed that all four patients had monoclonal leukemia with TCR beta rearrangement; two patients had Vbeta2+ leukemia, another patient had Vbeta14+ leukemia and the other had Vbeta9+ leukemia. Flow cytometry supported this finding. Sequencing of the Vbeta2-complementarity determining region 3 (CDR3), Vbeta9-CDR3 and Vbeta14-CDR3 revealed monoclonality. To investigate MRD using TCR beta-chain RNA, cDNA from each patient was diluted with the cDNA of a healthy person and amplified using a specific CDR3 clonotype primer. A band in the ethidium bromide-stained agarose gel was detected from samples diluted 10,000-fold. SSCP analysis determined which V region gene was utilized in monoclonal leukemic cells. The leukemic cell specific TCR, determined in such a manner, may be a target for immunotherapy. Because the MRD of T cell malignancy can be easily examined once the CDR3 clonotype primer is made, this novel analysis is considered to be a useful method.

Adolescent↗
PubMed1997

Characterization and sensitivity to interleukin 2 and interferon alpha of leukemic cells from a patient with large granular lymphocytic leukemia associated with chronic active Epstein-Barr virus infection.

A patient presented with chronic large granular lymphocyte leukemia associated with chronic active Epstein-Barr virus infection (CAEBV). Cell cycle analysis revealed a minimal growth compatible with chronic lymphocytic leukemia After 5 months of treatment, the patient died from acute transformation of the leukemia. Cell harvested during chronic phase were analyzed for sensitivity to interleukin 2 (IL-2) and interferon alpha (IFN alpha) in vitro by means of surface phenotyping and cell cycle assay. IL-2 induced remarkable growth of the cells, whereas IFN alpha did not confer a growth advantage. Since IFN alpha was expected to have no growth induction effect on the leukemia cells, it was administered to the patient to treat the CAEBV.

Antineoplastic Agents↗
PubMed1997

Regional excitatory and inhibitory amino acid concentrations in pentylenetetrazol kindling and kindled rat brain.

We determined regional concentrations of excitatory and inhibitory amino acids in pentylenetetrazol (PTZ) kindling and kindled rat brains in order to investigate the mechanisms responsible for the PTZ kindling process and kindled state. Compared with control rats, PTZ kindling rats had significantly higher concentrations of aspartate in the striatum and the temporal cortex 24 h after the 14th injection of PTZ. Glutamate and GABA concentrations were also significantly higher in the brainstem of PTZ kindling rats 24 h after the 14th injection of PTZ. These findings suggest that the striatum and temporal cortex may participate in a pathway for propagation of the PTZ kindling process, and that the brainstem may be a primary site of PTZ kindling epileptogenesis or a part of the pathways for propagation. On the other hand, the concentrations of amino acids in PTZ kindled rats and controls did not differ 4 weeks after the 14th injection. This suggests that the alterations we detected in amino acid metabolism are not related to retention of the PTZ kindled state, and that some other mechanism for it must exist.

Alanine↗
PubMed1997

Scoop biopsy of intracaval tumor thrombi: a preliminary report of a minimally invasive technique to obtain large samples.

PURPOSE: The purpose of this study is to safely improve the yield of intracaval biopsy. MATERIALS AND METHOD: A co-axial system was composed of an inner catheter with a smoothly tapered tip with a shark jaw, and an outer sheath. The biopsy procedure consisted of four steps: (1) the tip was stuck into the target thrombi; (2) the inner sheath was advanced deeply into the target and the jaw was opened within the mass; (3) the outer sheath was advanced to envelope the inner cather; (4) then the system was withdrawn. After simulation experiments seven patients underwent this scoop biopsy. RESULTS: A simulation experiment proved that this technique brought much larger samples with minimal damage of the target surface. Seven patients who had been suspected of intracaval tumor thrombi underwent this procedure and confidential pathological examination without any complications. CONCLUSION: This scoop biopsy procedure was thought to be helpful in obtaining large samples safely.

Aged↗
PubMed1997

Oxidant-induced activation of nuclear factor-kappa B in cultured guinea pig gastric epithelial cells.

The aim of this study was to reveal oxidant-sensitive components in gastric epithelial cells, which may regulate inflammatory processes in gastric mucosa. Gel mobility shift assay showed that treatment of cultured guinea pig gastric epithelial cells with hydrogen peroxide or diamide produced a KB oligonucleotide-protein complex within 5 min. The binding proteins consisted of a p50/p65 heterodimer, which was identified by immunosupershift, UV cross-linking, and immunoprecipitation analyses. Immunocytochemical study demonstrated that surface epithelial cells and parietal cells expressed p500 and p65 mainly in the cytosol, and the oxidants rapidly initiated the nuclear translocation of the components. The oxidants caused the up-regulation of p105 (a p50 precursor) synthesis and the expression of inducible nitric oxide synthase mRNA. These results suggest that the oxidant-sensitive p50/p65 heterodimer in gastric epithelial cells may play an important role in transcriptional activation of genes involved in inflammatory responses of the stomach.

Animals↗
PubMed1997

Second allogeneic bone marrow transplantation for post-transplant leukemia relapse: results of a survey of 66 cases in 24 Japanese institutes.

To assess the consequence of second BMT (BMT2) for leukemia relapse after allogeneic BMT, we analyzed the clinical course of 66 recipients who were treated by BMT2 in Japan. Diagnoses included 29 ANLL, 27 ALL, six CML and four MDS. Durations between the first BMT (BMT1) to relapse and BMT1 to BMT2 were 13.5 +/- 13.7 months and 17.4 +/- 13.9 months, respectively. Donors for BMT2 were replaced in 11 cases. Thirty-one patients were in CR (or CP) at BMT2. Earlier deaths were observed in those who received BMT2 within 12 months after BMT1, mostly caused by regimen-related toxicity and infections. Overall leukemia-free survival rate was 28% at 2 years and 16% at 4 years. Factors influencing the poor prognosis after BMT2 were early (<6 months) relapse, early (<12 months) BMT2, not in remission at BMT2, and ALL. Intensified conditioning did not affect either remission duration or LFS. Among the 39 cases observed for more than 100 days, 18 developed chronic GVHD (cGVHD) and showed longer remission duration than those without cGVHD. Our analysis indicates that BMT2 as treatment for leukemia relapse is effective in selected cases, and exploration of pre-BMT treatment and post-BMT immunotherapy is warranted.

Adolescent↗
PubMed1997

Primary familial polycythaemia associated with a novel point mutation in the erythropoietin receptor.

Primary familial and congenital polycythaemia (PFCP) is a rare disease characterized by congenital erythrocytosis inherited in an autosomal dominant fashion. Recently, mutations in the erythropoietin receptor (EpoR) have been identified in PFCP families. We describe a Japanese family with an autosomal dominant inheritance of PFCP. An in vitro colony assay demonstrated hypersensitivity of erythroid progenitors to erythropoietin (Epo) in affected family members. Sequence analysis of RT-PCR products amplified from the C-terminal region of EpoR transcripts in affected family members revealed that they were all heterozygous for C and T bases at position 5986, which suggested a genetic mutation (C to T) on one allele of EpoR. This mutation gave rise to a translation termination codon TAG at amino acid 435. Thus, the resulting EpoR is a truncated protein product lacking all 74 amino acids downstream of the mutation. To date, all genetic mutations affecting a family with PFCP, including this one, have been located in the cytoplasmic negative regulatory region of the EpoR. All mutations gave rise to truncated Epo receptors between Tyrosine 427 and Tyrosine 455. The phosphotyrosines in this region of EpoR have been demonstrated to be binding sites for SHP-1 phosphatase. Therefore PFCP is presumably brought about as a result of genetic mutations which cause the loss of the SHP-1 binding site in the cytoplasmic region of EpoR.

Base Sequence↗