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Biomedical subjects

K Kohda

Publications and source records attributed to K Kohda.

At least 73 records · Page 4Linked to original sources
PubMed1995

Differential inactivation of O6-methylguanine-DNA methyltransferase activity by O6-arylmethylguanines.

Activity of O6-methylguanine-DNA methyltransferase (MGMT) is well related with drug resistance of tumor cells to chloroethylnitrosoureas (CENUs), because MGMT removes CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. Inactivation of MGMT is a feasible way to overcome MGMT-related resistance of tumor cells to CENUs. O6-Benzylguanine is known to be a strong depleter of MGMT. We previously reported the potentiation effect of O6-arylmethylguanine derivatives on cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), a CENU, for HeLa S3 cells. In this study, we tested the activity of these O6-arylmethylguanine derivatives as depleters of MGMT using HeLa S3 cell-free extract. The O6-arylmethylguanine derivatives tested were O6-benzylguanine (1), O6-(4-, 3-, and 2-fluorobenzyl)guanines (2-4), O6-(4, 3-, and 2-trifluoromethylbenzyl)guanines (5-7), O6-(4-, 3-, and 2-pyridylmethyl)guanines (8-10), and O6-(2- and 1-naphtylmethyl)guanines (11,12). Among these, compounds 1-3, 5, 8, 9 and 11 showed a strong MGMT depletion activity, whereas compounds 4, 6, 7, 10 and 12 were inactive. These inactive compounds, except for 6, have a substituent at the alpha position of the benzyl group (4, 7, 12) or are an alpha nitrogen analogue of 1 (10). There was a good relation (r = -0.856, p < 0.001) between the MGMT depletion activity of O6-arylmethylguanine derivatives and their potentiation activity of ACNU cytotoxicity. These results suggest that the alpha position of the benzyl group plays an important role in the interaction of O6-arylmethylguanine derivatives with MGMT to result in the inactivation of MGMT. Potent MGMT inactivators (1-3, 5, 8, 9, 11) sensitize tumor cells to CENU chemotherapy.

Antineoplastic Agents↗
PubMed1995

Wide difference between the cytotoxicity of the 11-alpha-and 11-beta-cyano analogues of tilivalline and their epimeric conversion.

Tilivalline (TV) possesses a pyrrolo[1,4]benzodiazepine nucleus and is cytotoxic toward mammalian cells. The 11-beta-cyano TV analogue (1) is about one hundred times more cytotoxic to mouse leukemia L1210 cells than TV itself. In contrast, the 11-alpha-cyano TV analogue (2), an epimer of 1, has only about one-hundredth the cytotoxicity of 1. It was found that epimerization proceeded between 1 and 2 under physiological conditions, and the cytotoxicity of 2 is thought to be caused mainly by 1 that was formed from 2 during incubation in medium.

Animals↗
PubMed1995

Mouse methyltransferase for repair of O6-methylguanine and O4-methylthymine in DNA.

cDNA for mouse O6-methylguanine-DNA methyltransferase was expressed in methyltransferase-deficient Escherichia coli mutant cells, and the overproduced mouse enzyme was purified to a homogeneous state. Using this purified product, polyclonal antibodies were prepared and used to estimate amounts of the methyltransferase protein in cells. A single cell of NIH3T3 contained 1.8 x 10(4) molecules of the methyltransferase protein. When mouse fibroblasts were immunostained, it was shown that most of the methyltransferase protein exists in the cytoplasm rather than in the nucleus. Using double-stranded oligomers containing a single O6-methylguanine or O4-methylthymine at predetermined sites, the mouse enzyme repaired O6-methylguanine and O4-methylthymine, at an almost equal efficiency. In the LacZ reversion assay, MNNG-induced A:T to G:C as well as G:C to A:T transition mutations were efficiently suppressed by the function of mouse methyltransferase, in vivo.

3T3 Cells↗
PubMed1995

CMV viraemia demonstrated in the serum of a patient with cytomegalovirus pneumonia.

We attempted to demonstrate the expression of cytomegalovirus (CMV) particles in the serum of an acute lymphocytic leukaemia patient with CMV pneumonia. The serum sample was applied to an affinity column coupled with human monoclonal antibody C23 which recognizes the envelope glycoproteins of CMV virus and neutralizes the viral activity. The DNA obtained from each fraction was amplified by double polymerase chain reaction (PCR) and analysed by gel electrophoresis. Bands were clearly observed in the eluted fraction. These results strongly suggest that CMV particles exist in the sera of patients with CMV pneumonia.

Base Sequence↗
PubMed1995

Ca2+ release from Ca2+ stores, particularly from ryanodine-sensitive Ca2+ stores, is required for the induction of LTD in cultured cerebellar Purkinje cells.

1. Primary-cultured cerebellar Purkinje cells (PCs) from mouse embryos were whole cell voltage clamped, and L-glutamate (Glu) was applied iontophoretically to the dendrite. Long-term depression (LTD) of Glu-evoked currents was induced through the conjunction of repeated depolarizations and Glu applications. 2. Thapsigargin, a specific inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum, and ryanodine and ruthenium red, inhibitors of the ryanodine receptor, blocked the induction of LTD. 3. Thapsigargin and ryanodine alone did not affect influx of Ca2+ through voltage-gated Ca2+ channels and inward currents evoked by Glu applications. 4. Our results suggest that Ca2+ release from internal stores, particularly from ryanodine-sensitive stores, is necessary for the induction of LTD in cultured PCs.

Animals↗
PubMed1995

Potentiation of the cytotoxicity of chloroethylnitrosourea by O6-arylmethylguanines.

It was reported recently that monomeric O6-benzylguanine (1) acts as an alternative substrate for a DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT), and that therefore pretreatment of cells with 1 induces depletion of AGT resulting in an enhanced cytotoxic response to alkylating antitumor agents. In order to study the interaction of O6-benzylguanine derivatives with AGT and to obtain greater AGT depletion, we synthesized the following O6-arylmethylguanine derivatives and related compounds: O6-(4-, 3- and 2-fluorobenzyl)guanines (2, 3, 4), O6-(4-, 3- and 2-trifluoromethylbenzyl)guanines (5, 6, 7), O6-(4-, 3- and 2-pyridylmethyl)guanines (8, 9, 10), O6-(2- and 1-naphthylmethyl)guanines (11, 12), O6-biphenylmethylguanine (13), S and Se analogues of O6-benzylguanine (14, 15) and O6-phenylguanine (16). Ten of these are new compounds. All these compounds were tested for their potentiation of N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosou rea (ACNU) cytotoxicity using HeLa S3 and C6-1 cells. Compounds 2, 3, 5, 8, 9, 11 and 13 were active, as was 1. Compounds 7 and 12, with a substituent at the alpha position of the benzyl group, and compound 10, the alpha-nitrogen analogue of 1, were almost completely devoid of potentiating activity. These results suggest that the alpha-position of the O6-benzyl group plays an important role in the interaction of O6-benzylguanines with AGT. Of the other compounds, 4 and 6 exhibited very weak activity and 14, 15 and 16 were inactive. Possible reasons for these differences in activity are discussed in relation to the biomimetic dealkylation rates of O6-benzylguanine derivatives and the chemical characteristics of their substituents.

Alkylation↗
PubMed1995

Mutational analysis of the fic promoter recognized by RpoS (sigma 38) in Escherichia coli.

fic gene expression at the stationary phase is dependent on RpoS (sigma 38). For this paper, the fic promoter (P fic) mutants were isolated; C at the position -34 was changed into T. They did not show the stationary phase-specific expression dependent on RpoS in vivo. Furthermore, it was confirmed that they were recognized by E sigma 70 as efficiently as E sigma 38 in vitro.

Amino Acid Sequence↗
PubMed1995

Immunoblastic lymphadenopathy-like T-cell lymphoma displaying rearrangement of both IgH and TCR beta genes after 4-year follow-up of idiopathic eosinophilia.

Immunoblastic lymphadenopathy (IBL)-like T-cell lymphoma (IBL-T) occurred in a 60-year-old female after a 4-year follow-up of idiopathic eosinophilia and upper pharyngeal inflammatory tumor with infiltration of mature eosinophils. Gene analysis of tumor cells revealed rearrangement of both IgH and TCR beta genes. The patient died of lymphoma seven months after the onset of the illness, in spite of chemotherapy against lymphoma. The relationship between eosinophilia and the pathogenesis of IBL-T, as well as the significance of the rearrangement of both IgH and TCR beta genes are discussed.

Eosinophilia↗
PubMed1995

Structure-cytotoxicity relationship of tilivalline derivatives.

Tilivalline (TV) possesses a pyrrolo[1,4]benzodiazepine nucleus and an indole substituent in its structure, and is cytotoxic toward mouse leukemia L1210 cells. In order to obtain more effective compounds, we have previously synthesized a variety of TV analogues. In this study, the cytotoxicity of these compounds has been evaluated. Among the compounds tested, the 11-beta-cyano compound, a TV analogue bearing the cyano group instead of the indole group, is approximately 100 times more cytotoxic than TV itself. The alpha-epimer of this cyano compound has about one-hundredth the cytotoxicity of the 11-beta-cyano compound. Structure-cytotoxicity relationships are discussed, including the effects of stereoisomers (alpha and beta) at the 11 position and substitution at the benzene moiety of TV.

Animals↗
PubMed1995

[Two cases of adult T-cell leukemia . lymphoma (ATL) with long-term remission by chronic daily administration of low-dose etoposide].

We tried long-term oral administration of low-dose etoposide (LDE) for seven patients with adult T-cell leukemia . lymphoma (ATL) at Asahikawa Red Cross Hospital in 1988. Two long-term survivors are presented. Case 1: A 53-year-old man was diagnosed as acute ATL in 1987. Because VEPA therapy was not effective, LDE (50 mg/day) therapy was started. Five months after entering CR, a lymphoma recurrence in the skin was observed. Retreatment with LDE and radiation therapy was effective, but he died of reprogression of ATL three years and seven months following initial diagnosis. Case 2: A 43-year-old woman was diagnosed as acute ATL in 1989. With LDE (50 mg/day) therapy for 40 days, she entered CR. The lymphoma recurred in the central nervous system (CNS) and skin in 1992. After radiation of CNS and LDE therapy, she showed CR again. She is alive now over five years and eight months after diagnosis.

Administration, Oral↗
PubMed1994

[Change in lobar uptake of 99mTc-galactosyl serum albumin after transarterial embolization therapy].

Liver function was studied by hepatic scintigraphy with 99mTc-galactosyl serum albumin (GSA) before and after transarterial embolization (TAE) therapy in 16 patients with hepatocellular carcinoma. The percent uptake of GSA in liver was measured separately in the treated and non-treated areas. GSA uptake increased in both areas. The change was more marked in the non-embolized lobe than the embolized lobe. We conclude that the increase in GSA uptake reflects the regeneration of liver cells after TAE.

Carcinoma, Hepatocellular↗
PubMed1994

Enhancing effect of O6-alkylguanine derivatives on chloroethylnitrosourea cytotoxicity toward tumor cells.

O6-Alkylguanine derivatives are well known as chemical modulators of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Depletion of the enzyme by these derivatives leads to increase sensitivity of tumor cells to chloroethylnitrosoureas. We tested the effect of O6-methylguanine, O6-benzylguanine, O6-(p-methylbenzyl)guanine, O6-(p-chlorobenzyl)guanine, O6-(p-methoxybenzyl)guanine, O6-methylhypoxanthine and O6-benzylhypoxanthine on the sensitivity of tumor cell lines to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) using a colorimetric cytotoxicity assay. The sensitivity of MGMT-proficient tumor cells including HeLA S3, C6-1, C6-2/ACNU, U-138 MG and U-373 MG cells was greatly enhanced by 2 hr pretreatment of 10-100 microM O6-benzylguanine, O6-(p-methylbenzyl)guanine and O6-(p-chlorobenzyl)guanine, but not by O6-methylguanine or O6-methylhypoxanthine. O6-(p-methylbenzyl)guanine moderately sensitized the 2 cell lines, HeLa S3 and C6-1, tested in our study to ACNU cytotoxicity. O6-Benzylhypoxanthine at the high concentration (100 microM) sensitized, to some extent, 3 MGMT-proficient cell lines. Lesser degrees of enhancement by the O6-benzylguanine derivatives were noted in MGMT-deficient tumor cells. Biological effects of O6-alkylguanine derivatives on enhancing ACNU cytotoxicity of tumor cells suggest that the exocyclic 2-amino and O6-benzyl groups in O6-benzylguanine skeleton are both essential for the inhibition of MGMT activity.

Animals↗
PubMed1994

Potential of O6-methylguanine or O6-benzylguanine in the enhancement of chloroethylnitrosourea cytotoxicity on brain tumours.

The purine analogues O6-methylguanine and O6-benzylguanine are well-known as a chemical modulator of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Inactivation of the enzyme by O6-methylguanine or O6-benzylguanine is expected to enhance sensitivity of tumours to chloroethylnitrosoureas. We studied the effect of O6-methylguanine or O6-benzylguanine pretreatment on cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in brain tumour cells and transplanted brain tumours. Two-hour exposure of O6-methylguanine at higher concentrations (500 microM, 1,000 microM) increased ACNU cytotoxicity by only 2 times in ACNU-resistant C6-1 brain tumour cells. O6-Benzylguanine at concentrations between 10 and 100 microM markedly enhanced the cytotoxic effect. The ACNU sensitivity of the tumour cels pretreated with O6-benzylguanine was 5-40 times that of the cells without O6-benzylguanine. Neither O6-methylguanine nor O6-benzylguanine appreciably enhanced ACNU cytotoxicity of 9 L cells, which were originally sensitive to ACNU. Intracarotid ACNU with O6-methylguanine or O6-benzylguanine decreased proliferating activity of transplanted C6-1 brain tumours significantly during 48 hours. O6-Benzylguanine pretreatment resulted in a greater degree of suppression for a long time. The C6-1 tumours treated only with intracarotid ACNU showed a transient inhibition and a rapid regrowth during 24 hours after the treatment. These results indicate that O6-methylguanine or O6-benzylguanine increases ACNU cytotoxicity and may be feasible for effective combination therapy with chloroethylnitrosourea in the chemotherapy of malignant brain tumours.

Animals↗
PubMed1994

Intracellular channels.

Intracellular channels are located on the membranes of intracellular organelles and are involved in ion transfer, within the cytosolic compartments, in response to internal stimuli. Recently, various types of inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca(2+)-release channels, mitochondrial voltage-dependent anion channels, and a vesicular Cl- channel have been molecularly cloned and characterized, and their functional roles in the central nervous system are beginning to be clarified.

Animals↗
PubMed1994

Inhibitory effect of fatty acids on 8-hydroxydeoxyguanosine formation in calf thymus DNA treated with bleomycin-Fe(II).

The effect of fatty acids on 8-hydroxydeoxyguanosine (8-OH-dG) formation in calf thymus DNA treated with bleomycin (BLM)-Fe(II) in vitro was studied. The formation of 8-OH-dG was greatly reduced in the presence of stearic acid (18:0) or oleic acid (18:1), however, it increased to the control level with an increase in the number of unsaturated bonds of fatty acids (18:2, 18:3). This increase in 8-OH-dG formation may have resulted from the indirect oxidation of DNA by the peroxidized unsaturated fatty acids formed by BLM-Fe(II). The inhibitory effect of saturated fatty acids on 8-OH-dG formation was observed with those possessing more than 12 methylene chains. Stearic acid also inhibited peroxidation of unsaturated fatty acids (18:2, 18:3) treated with BLM-Fe(II), however, it had no effect on ribose degradation of DNA treated with BLM-Fe(II). These results suggest that different active oxygen species are probably involved in DNA degradation and in formation of 8-OH-dG in DNA and of peroxidized products in fatty acids.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Demethylation of O6-methylguanine in DNA by O6-methylguanine-DNA methyltransferase: synthesis and characterization of O6,9-dimethylguanine coordinated with cis-platin at the N7-position.

In order to investigate the chemical characteristics of O6-methylguanines, the substrate of O6-methylguanine-DNA methyltransferase, cis[PtCl(NH3)2(O6,9-dimethylguanine-7)]NO3, a 1:1 complex of cis-platin with O6,9-dimethylguanine, was synthesized and its demethylation rate, pKa, X-ray structure and cytotoxicity were determined.

Animals↗
PubMed1994

[Evaluation of polymerase chain reaction for diagnosis of Helicobacter pylori infection].

To evaluate diagnostic tests of Helicobacter pylori (H. pylori), we compared polymerase chain reaction (PCR) for the specific detection of H. pylori, culture and positivity of serum antibody against the bacteria in 59 patients with gastroduodenal disease. PCR assay was designed to amplify 203bp of DNA fragments. PCR detected 10pg of H. pylori DNA, or 40 CFU of the bacteria. PCR was positive in 82.1% of histological gastritis with neutrophil infiltration. Culture and serum antibody to H. pylori were positive in 65.0%, and 94.1% of those patients. Detection rate by PCR was significantly higher than that by the culture methods in patients with chronic gastritis. These results demonstrate that PCR is a sensitive diagnostic test for H. pylori infection.

Adult↗