Search PubMed⌕ Search

Biomedical subjects

K Kohda

Publications and source records attributed to K Kohda.

At least 55 records · Page 3Linked to original sources
PubMed1997

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.

Adenosine Triphosphate↗
PubMed1997

Enhanced cytotoxicity of alkyl viologens and N,N'-diamino analogs toward cultured murine leukemia L1210 cells under vortex-stirring with a high molecular weight polyacrylic acid.

Alkyl viologens showed cytotoxicity when incubated with cultured murine leukemia L1210 cells for 48 h, whereas they were not cytotoxic when briefly incubated for 10 min. Under the permeabilizing conditions achieved by vortex-stirring with a high molecular weight polyacrylic acid (A-119), appreciable cytotoxicity was shown even after a 10-min exposure to any of the alkyl and amino viologens examined. Acetylamino viologen showed no cytotoxicity regardless of the presence or absence of A-119. This permeabilizing procedure was demonstrated to be applicable to internalize water-soluble positively charged viologen molecules into the cell.

Acrylic Resins↗
PubMed1997

Enhanced cytotoxicity of some triterpenes toward leukemia L1210 cells cultured in low pH media: possibility of a new mode of cell killing.

Several triterpenes were tested for cytotoxicity by our contrived primary screening method using resting or dormant leukemia L1210 cells after 3 d-preculture without medium change. Some triterpenes were found to be more cytotoxic toward the 3 d-precultured resting cells than toward the growing cells in a fresh medium. These triterpenes are distinguished by highly selective cytotoxicity toward the starved resting cells unlike common anticancer agents. The highest selectivity was shown by betulinic acid, the ratio of its IC50 values toward the growing versus resting cells amounting to 175. It is suggested that this selective cytotoxicity is attributable to low pH (< or = 6.8) of the medium. It is noteworthy that betulinic acid is not cytotoxic at all in media of ordinary pH (> or = 7.0) even after a 48-h exposure. Betulinic acid might be promising as an antitumor agent toward solid tumors because the interior pH of tumor tissues is generally lower than in normal tissues.

Animals↗
PubMed1997

[Utility of daily oral administration of etoposide in 25 cases of refractory hematological malignancies].

We estimated the utility of daily oral administration of etoposide (ETOP) in 25 cases of refractory hematological malignancies who had been admitted to our hospital between February, 1988 and October, 1995. Patients were 9 cases of malignant lymphoma, 7 cases of adult T cell leukemia (ATL), 7 cases of acute leukemia, 1 case of primary macroglobulinemia, and 1 case of chronic lymphocytic leukemia (CLL). Eight cases were elderly patients over 65 years old, 7 cases were refractory to previous chemotherapies, and 13 cases were relapsed cases. ETOP was administered at 25 or 50 mg per day for at least more than 3 consecutive weeks, if the peripheral white blood cell count exceeded 1,000/microliter. Complete and partial responses were obtained in 64% of all cases, especially in 81% of malignant lymphoma and ATL. Probability of survival for 3 years of malignant lymphoma and ATL was 36.7%. As mild toxicities, 2 cases (8%) had nausea and vomiting, 2 cases (8%) had diarrhea, and 3 cases (12%) had stomatitis. Grade 3 leukocytopenia was observed in 16% of cases. Twelve out of 16 evaluable patients recovered in performance status (PS) after this therapy. Daily oral administration of ETOP might be an effective therapy for refractory hematological malignancies, especially for malignant lymphoma.

Administration, Oral↗
PubMed1997

[Diagnostic value of serum soluble interleukin-2 receptor levels in interstitial pneumonia of the patients with hematological disorders].

Serum levels of soluble interleukin-2 receptor (SIL-2R) and C-reactive protein (CRP) were measured in 12 patients with interstitial pneumonia (IP) and 10 patients with bacterial pneumonia (BP) of hematological malignancies. Mean SIL-2R levels (U/ml) were 394 +/- 140 in normal controls, 755 +/- 320 in BP, and 2328 +/- 943 in IP. A significantly higher level was found in IP than BP (p < 0.05). Moreover, the SIL-2R/CRP ratio which was over 260 in all cases of IP completely distinguished IP from BP. Levels of SIL-2R rose before the onset of IP and were closely associated with clinical course in most cases. From these results, measurement of serum SIL-2R may be useful for the diagnosis and monitoring of the clinical course of IP complicated with hematological diseases.

C-Reactive Protein↗
PubMed1997

[Study on potentiation of nitrosourea-cytotoxicity by DNA repair enzyme inhibitors in human brain tumor cells].

Nitrosoureas are antitumor alkylating agents widely used in the chemotherapy of malignant brain tumors. However, the effectiveness of adjuvant nitrosourea chemotherapy has proved inadequate, failing to provide any significant prolongation of survival time. One of the reasons for the poor results is a drug resistance system in the form of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). O6-alkylguanine derivatives are well known to be inhibitors of MGMT, and inactivation of MGMT by these derivatives leads to increased tumor cell sensitivity to nitrosoureas. In this study, the authors tested the ability of O6-benzylguanine, O6-(4-, 3- and 2-fluorobenzyl) guanines, O6-(4-, 3- and 2-trifluoromethylbenzyl) guanines, O6-(4-, 3- and 2-pyridylmethyl) guanines and O6-(2- and 1-naphthylmethyl) guanines to reduce MGMT activity in SF-188 cell-free extract by using [3H] methylated substrate DNA and analyzed their enhancing effect on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl) -3-nitrosourea hydrochloride (ACNU) by using a calorimetric cytotoxicity assay. The MGMT activity in the SF-188 cell-free extract was 944 +/- 43 fmol/mg protein (Mean +/- SD, n = 5). O6-(4- and 3-fluorobenzyl) guanines were found to be more effective in inactivating MGMT than O6-benzylguanine. O6-(4-trifluoromethylbenzyl) guanine considerably reduced MGMT activity as did O6-benzylguanine. O6-(3-trifluoromethylbenzyl) guanine, O6-(4- and 3-pyridylmethyl) guanines, and O6-(2-naphthylmethyl) guanine were intermediately effective, but O6-(2-fluorobenzyl) guanine, O6-(2-trifluoromethylbenzyl) guanine and O6-(1-naphthylmethyl) guanine were less effective. ACNU cytotoxicity in SF-188 cells was strongly enhanced by pretreatment with O6-(4- and 3-fluorobenzyl) guanines and O6-(4-trifluoromethylbenzyl) guanine and moderately enhanced by O6-(3- trifluoromethylbenzyl) guanine and O6-(4- and 3-pyridylmethyl) guanines, but not enhanced by O6-(2-fluorobenzyl) guanine, O6-(2-trifluoromethylbenzyl) guanine and O6-(1-naphthylmethyl) guanine. The test compounds were not cytotoxic at concentrations between 0.5 and 5.0 microM. The enhancing effects on ACNU cytotoxicity were consistent with the inhibition of MGMT activity after two-hour pretreatment with O6-arylmethylguanine derivatives. These results indicate that the 2-position of the O6-benzyl group plays an important role in the inactivation of the MGMT activity and the potentiation of ACNU cytotoxicity.

Antineoplastic Agents↗
PubMed1996

A novel enzyme enantio-selectively synthesizes (R)salsolinol, a precursor of a dopaminergic neurotoxin, N-methyl(R)salsolinol.

In the human brain, only (R)enantiomer of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline ((R)salsolinol) and N-methyl-salsolinol, a dopaminergic neurotoxin, were detected, suggesting their enzymatic biosynthesis. This paper reports the isolation and characterization of a novel enzyme, which enantio-selectively synthesizes (R)salsolinol from dopamine and acetaldehyde. Dopamine, acetaldehyde, formaldehyde and pyruvic acid were the substrates of this synthase, whereas N-methyldopamine, adrenaline, noradrenaline and L-DOPA were not. The possible function of this enzyme under physiological and pathological conditions in the brain is discussed.

Brain↗
PubMed1996

Comparison of chemically induced DNA breakage in cellular and subcellular systems using the comet assay.

The alkaline comet assay, employing a single cell gel electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual mammalian cells. In this report, we describe a modified version of this assay which we used to assess DNA damage as a result of treating lysed cells with genotoxic and antimetabolic agents. By means of this modified assay, DNA is no longer held under the regulation of any metabolic pathway or membrane barrier. Using 3 direct-acting agents, hydrogen peroxide, N-methyl-N-nitrosourea, and bleomycin, we were able to induce increased DNA migration by both the standard and modified comet assays. In contrast, with 4-nitroquinoline 1-oxide, 5-fluorouracil, and methotrexate, which require cellular enzymatic activity to induce DNA damage, we succeeded in inducing increased DNA migration using the standard comet assay conditions only. In some cases, the modified comet assay might be helpful in analyzing chemical and biological characteristics of genotoxic agents when performed in combination with the standard comet assay.

4-Nitroquinoline-1-oxide↗
PubMed1996

Enhancement effect of O6-fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells.

O6-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O6-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O6-fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O6-benzylguanine and O6-phenylguanine. The O6-(4- and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O6-benzylguanine. In contrast, O6-(2-fluorobenzyl)guanine and O6-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O6-(4- and 3-fluorobenzyl) guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O6-(2-fluorobenzyl)guanine and O6-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O6-fluorobenzylguanine derivatives. O6-Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O6-benzylguanine and were powerful MGMT inactivators. The chemical features of the O6-benzyl group are a biologically important determinant in the reaction evolution with MGMT.

Antineoplastic Agents↗
PubMed1996

Pulmonary veno-occlusive disease accompanied by microangiopathic hemolytic anemia 1 year after a second bone marrow transplantation for acute lymphoblastic leukemia.

Although hepatic veno-occlusive disease (HVOD) is a common complication of allogenic bone marrow transplantation (BMT), pulmonary veno-occlusive disease (PVOD) is very rare. Only three cases have been described in the literature. We report the case of a 19-year-old woman who developed PVOD accompanied by microangiopathic hemolytic anemia (MAHA) and hemolytic uremic syndrome (HUS) 1 year after a second BMT for relapsed acute lymphoblastic leukemia (ALL). Autopsy examination revealed obstruction of the small pulmonary veins with edematous thickening of the intima. These findings are compatible with PVOD. Pulmonary GVHD and pulmonary aspergillosis were also observed. Various etiologic factors have been implicated in PVOD after BMT. We postulate that pulmonary GVHD and pulmonary infection including aspergillosis played an important role in the occurrence of both PVOD and HUS in our patient. Microangiopathic cytokines released in response to the GVHD and infection may damage the intima of microvessels that were previously injured by the two BMT. Despite appropriate therapy, the microangiopathic process was irreversible and the patient died. Thus, measures must be taken to prevent and treat PVOD after BMT.

Adolescent↗
PubMed1996

Synthesis, miscoding specificity, and thermodynamic stability of oligodeoxynucleotide containing 8-methyl-2'-deoxyguanosine.

8-Methyl-2'-deoxyguanosine (8-MedG) was synthesized by reacting dG under the methyl radical generating system and incorporated into oligodeoxynucleotides using phosphoramidite techniques. The site-specifically modified oligodeoxynucleotide containing a single 8-MedG was then used as a template for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo-) Klenow fragment of Escherichia Coli DNA polymerase I and mammalian DNA polymerase alpha. Primer extension catalyzed by the exo- Klenow fragment readily passed the 8-MedG lesion in the template while that catalyzed by pol alpha was retarded opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-MedG. Both DNA polymerases incorporated primarily dCMP, the correct base opposite the lesion, along with small amounts of incorporation of dGMP and dAMP. In addition, two-base deletion was observed only when the exo- Klenow fragment was used. The thermodynamic stability of 8-MedG in the duplex was also studied. The duplex containing 8-MedG:dG was more thermally and thermodynamically stable than that of dG:dG. The duplex containing 8-MedG:dA was more thermodynamically stable than that of dG:dA. We conclude that 8-MedG is a miscoding lesion and capable of generating G --> C and G --> T transversions and deletion in cells.

Chemical Phenomena↗
PubMed1996

Synergetic cytotoxicity of bleomycin and polyhydric alcohols: DNA strand breakage evaluated by "comet assay".

DNA strand breakage induced in cultured cells by bleomycin (BLM) was remarkably enhanced in the presence of glycerol or polyvinyl alcohol which produces an enhancement effect on BLM cytotoxicity, but not in the presence of alcohols such as methanol, ethanol, or polyethylene glycol which do not produce enhanced cytotoxicity. The comet assay, a useful analytical method for evaluating DNA strand breakage, clearly demonstrated that combinations of BLM and polyhydric alcohols induced serious DNA damage in the whole cell populations compared with those induced by treatment with BLM alone. The comet assay also successfully showed distributions of cell populations with various degrees of DNA damage. These results suggested that an increase in DNA damage induced in the presence of polyhydric alcohols might be responsible for the enhancement of BLM cytotoxicity.

Alcohols↗
PubMed1996

Cytotoxicity of fluoroethylating agents is potentiated by O6-benzylguanine.

O6-Benzylguanine (BG) is a potent depleter of a repair enzyme O6-alkylguanine-DNA alkyltransferase. Pretreatment of cells with BG potentiates the cytotoxicity of chloroethylating anti-cancer agents. In this study we used HeLa S3 cells to examine the cytotoxic potentiation of 39 compounds after BG pretreatment. Compounds tested included anti-cancer agents and carcinogens, and among them only the cytotoxicity of methylating, chloroethylating and fluoroethylating agents was potentiated. This is the first description of the cytotoxic potentiation of fluoroethylating agents. Potentiation ratios were found to vary even among compounds possessing the same alkylating group. By pretreatment with 10 microM of BG, the cytotoxicity of methylating agents such as N-methyl-N-nitrosourea and streptozotocine was potentiated 3.7 and 9.4 fold, respectively. For chloroethylating agents, the potentiation ratios were 3.5 for N-chloroethyl-N-nitrosourea, 8.6 for N-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N'-(2-chloroethyl)-N'-nitroso urea (ACNU), 2.2 for N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), 3.0 for N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU) and 5.2 for chloroethyl methanesulfonate. With respect to fluoroethylating agents, the potentiation ratios were 7.2 for N-fluoroethyl-N-nitrosourea, 2.0 for N-cyclohexyl-N'-fluoroethyl-N'-nitrosourea and 5.5 for fluoroethyl methanesulfonate. No effect was observed with the bromoethylating agent, N-bromoethyl-N-nitrosourea. There was no potentiation of the cytotoxicity of anti-cancer agents such as mitomycin C (MMC), cisplatin (CDDP), 5-fluorouracil (5FU), bleomycin (BLM), prednisolone, camptothecin, etoposide, methotrexate or vinblastine. A possible mechanism for the cytotoxic potentiation of the test compounds by BG pretreatment is discussed.

Antineoplastic Agents↗
PubMed1996

Syndrome of the sea-blue histiocyte.

A 39-year-old male was admitted with fever, systemic lymph node swelling, liver dysfunction and mild splenomegaly. Liver biopsy specimen showed histiocytic aggregation in portal areas. These histiocytes were closely packed with granules, dyed sea-blue with May-Giemsa staining. Further microscopical examination of lymph nodes, gastro-intestinal tract and bone marrow also revealed the accumulation of sea-blue histiocytes. Activities of lipid metabolic enzymes were normal and hematopoietic diseases which are sometimes accompanied by secondary sea-blue histiocytosis were ruled out. We diagnosed this case as syndrome of the sea-blue histiocyte.

Adult↗
PubMed1996

The effect of Helicobacter pylori on gastric acid secretion by isolated parietal cells from a guinea pig. Association with production of vacuolating toxin by H. pylori.

BACKGROUND: One of the features of Helicobacter pylori infection in the human stomach seems to be disordered gastric acid secretion. The effect of vacuolating toxin (VT) produced by H. pylori on gastric acid secretion was examined. METHODS: VT(+)(toxigenic) and VT(-)(nontoxigenic) strains of H. pylori were cultured in brucella broth. The culture supernatant was added to isolated parietal cells, and acid secretion and intracellular adenosine 3'5'-cyclic phosphate (cAMP) and Ca2+ levels were measured with the 14C-aminopyrine (14C-AP) method, with 125I radioimmunoassay (RIA), and with the fura-2 fluorescence method, respectively. RESULTS: In the VT(+) strain a considerable inhibitory effect on 14C-AP accumulation was observed. However, the VT(-) strain had no significant effect on intracellular c-AMP and Ca2+. CONCLUSIONS: The VT(+) strain of H. pylori has an inhibitory effect on gastric acid secretion, whereas the VT(-) strain does not. This inhibitory effect was not associated with the response of second messengers. It is speculated that VT produced by H. pylori has a direct action on H(+)-K+ adenosine triphosphatase in parietal cells.

Animals↗
PubMed1996

[Evaluation of endoscopic pirarubicin-Lipiodol emulsion injection therapy for gastric cancer].

To investigate the effect on gastric cancer and metastatic lymph node, an emulsion made of pirarubicin and lipiodol mixture was injected around the lesion of the gastric cancer using gastrointestinal endoscopy. At the site of emulsion injection and lymph node, the concentration of the THP Lipiodol emulsion was enough despite injection more than 7 days before. This targeting therapy for metastatic lymph nodes was considered effective.

Adult↗
PubMed1995

Thermostable peroxidase activity with a recombinant antibody L chain-porphyrin Fe(III) complex.

In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso-tetrakis(4-carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03-1, 13-1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03-1 and Mab13-1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13-1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13-1L chain and TCPP Fe(III) was 90 degrees C, while that the TCPP Fe(III) alone was 60 degrees C.

Amino Acid Sequence↗