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Biomedical subjects

K Kohda

Publications and source records attributed to K Kohda.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of the mouse PA28 activator complex gene family: complete organizations of the three member genes and a physical map of the approximately 150-kb region containing the alpha- and beta-subunit genes.

The proteasome is a multisubunit protease responsible for the generation of peptides loaded onto MHC class I molecules. Recent evidence indicates that binding of an IFN-gamma-inducible PA28 activator complex to the 20S proteasome enhances the generation of class I binding peptides. The alpha- and beta-subunits, which constitute the PA28 activator complex in the form of an (alphabeta)3 heterohexamer, show significant amino acid sequence similarity to a protein, designated Ki or the gamma-subunit, that is capable of binding to the 20S proteasome. In this study, we describe the complete nucleotide sequences of the mouse genes, Psme1, Psme2, and Psme3, coding for the alpha-, beta-, and gamma-subunits, respectively. The overall exon-intron organizations of the three Psme genes are virtually identical, thus providing evidence that they are descended from a single ancestral gene. The promoter regions of the Psme1 and Psme2 genes contain sequence motifs that qualify as IFN-stimulated response elements, consistent with the observation that their expression is induced strongly by IFN-gamma. The Psme1 and Psme2 genes are located approximately 6 kb apart with their 3'-ends pointing toward each other on bands C2 to D1 of mouse chromosome 14, supporting the idea that they emerged by tandem duplication.

Amino Acid Sequence↗
PubMed1998

Reactions of 1-methylbenzimidazole derivatives with m-chloroperoxybenzoic acid.

Oxidation of seven 1-methylbenzimidazole (MBI) derivatives (with pKa's ranging from 1.6 to 6.0) was carried out with m-chloroperoxybenzoic acid and structures of the products formed were identified. (Condensed benzene moiety-hydroxylated)-2-(m-chlorobenzyloxy)-MBIs and 2-oxo-MBIs were obtained from MBIs with pKa's of more than 5.6 and about 3.3, respectively.

Benzimidazoles↗
PubMed1998

Inhibition of human O6-alkylguanine-DNA alkyltransferase and potentiation of the cytotoxicity of chloroethylnitrosourea by 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues.

A series of 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues of which 4(6)-benzyloxy groups were replaced with a (2-, 3-, or 4-fluorobenzyl)oxy or (2-, 3-, or 4-pyridylmethyl)oxy group, was synthesized. The abilities of these compounds to inhibit human O6-alkylguanine-DNA alkyltransferase (AGAT) in vitro and to potentiate the cytotoxicity of 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl) -3-nitrosourea (ACNU) toward HeLa S3 cells were evaluated. 2,4-Diamino-6-[(2-fluorobenzyl)oxy]-5-nitropyrimidine (3) and 2,4-diamino-5-nitro-6-(2-pyridylmethoxy)pyrimidine (6), whose ortho positions of the 6-substituent are modified, were much weaker in terms of these abilities than the corresponding meta- or para-modified compounds. These results are consistent with those of our previous study using a series of O6-benzylguanine derivatives. All 5-nitrosopyrimidine derivatives examined exerted both stronger AGAT-inhibition and ACNU-enhancement abilities than the corresponding 5-nitro derivatives. Among a variety of compounds that we have examined to date, 2,4-diamino-6-[(4-fluorobenzyl)oxy]-5-nitrosopyrimidine (10) exhibited the strongest ability to inhibit AGAT, and its magnitude was 2.5 and 50 times those of 4-(benzyloxy)-2,6-diamino-5-nitrosopyrimidine (9) and O6-benzylguanine (1), respectively. A strong positive correlation was observed between the ability to inhibit AGAT and to potentiate the cytotoxicity of ACNU. This strongly indicates that 4(6)-(benzyloxy)pyrimidine derivatives and their analogues potentiate ACNU cytotoxicity by inhibiting AGAT activity. To characterize the reactivity of test compounds, alkyl-transfer reactions were also carried out using the biomimetic alkyl-transfer system.

Cell Survival↗
PubMed1998

Oxidation of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine, a 1-amino analog of MPTP, by type A and B monoamine oxidase.

A 1-amino analog of MPTP, 1(N)-amino-4-phenyl-1,2,3,6-tetrahydropyridine, was synthesized and the oxidation was examined using human synaptosomal mitochondria as sources of type A and B monoamine oxidase. An oxidation product, 1-amino-4-phenylpyridinium ion, was quantified by high-performance liquid chromatography-fluorometric detection. The amino analog was a substrate of both type A and B monoamine oxidase and the oxidation depended linearly on the enzyme amount and the reaction time with an optimal pH around 7.5. After the systemic injection of the amino analog in C57/black mice for one week, 1-amino-4-phenylpyridinium ion was detected in the brain. 1(N)-Amino-4-phenyl-1,2,3,6-tetrahydropyridine was proved to be cytotoxic to pheochromocytoma PC12 cells, and it may be a new neurotoxin bioactivated through the oxidation by type A and B monoamine oxidase.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1998

Cytotoxicity of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine and 1-amino-4-phenylpyridinium ion, 1-amino analogues of MPTP and MPP+, to clonal pheochromocytoma PC12 cells.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonism in humans after its oxidation into 1-methyl-4-phenylpyridinium ion (MPP+) by type B monoamine oxidase. The 1-amino analogues of MPTP and MPP+, 1-amino-4-phenyl-1,2,3, 6-tetrahydropyridine (APTP) and 1-amino-4-phenylpyridinium ion (APP+), were synthesized, and their cytotoxicity to clonal pheochromocytoma PC12 cells was examined using a tetrazolium formazan assay. After incubation for 48 and 72 h, both APP+ and APTP were found to be cytotoxic to PC12 cells, whereas with the N-methyl analogues, only MPP+, but not MPTP, was cytotoxic. The cytotoxicity of APTP increased with incubation time and equaled that of MPP+ after 72 h. It was found that APTP was oxidized to APP+ by type A monoamine oxidase in PC12 cells, suggesting that APP+ itself may damage the cells. In addition to APTP and APP+, N-amino analogues of N-methylisoquinolines and related derivatives were also synthesized and examined for their cytotoxicity to PC12 cells.

1-Methyl-4-phenylpyridinium↗
PubMed1998

CytA protein, a delta-endotoxin of Bacillus thuringiensis subsp. israelensis is associated with DNA.

CytA protein (27 kDa) is produced by Bacillus thuringiensis subsp. israelensis (BTI) and is contained in its inclusion bodies. We previously reported the isolation of 25 kDa portion of CytA protein (p25-CytA protein) and its strong cytotoxic activity to mammalian cells. When p25-CytA protein was applied to an anion-exchange column for further purification, three fractions (M1, M2 and M3) were separated. M1 and M2 fractions were both shown to be 25 kDa protein, while M3 was a high molecular weight complex composed of 25 kDa protein and DNA. Purification and amino acid sequence analysis showed that M1 and M2 fractions were proteins lacking 29 and 31 N-terminal amino acids from CytA protein, respectively, and M3 was M1 protein associated with DNA. DNA was detected in BTI cells co-localizing with inclusion bodies. Both M1 and M2 proteins could bind to double-stranded DNA of BTI genome in vitro; the DNA binding ability of M1 protein was higher than that of M2 protein. These results suggested that CytA protein has DNA binding ability and is associated with DNA in the mother cell.

Amino Acid Sequence↗
PubMed1998

[Diagnosis and prognosis of reactive lymphoreticular hyperplasia (RLH) or mucosa-associated lymphoid tissue (MALT) lymphoma].

We studied the diagnostic significance of immunohistolochemical staining and immunoglobuline gene rearrangement of jumbo-biopsy specimen from 14 patients with stomach lesions which were difficult to distingwish between reactive lymphoreticular hyperplasia (RLH) and mucosa-associated lymphoid tissue lymphoma (MALT lymphoma). We also investigated Helicobacter pylori (HP) infection in the patients and followed their clinical courses from a mean observation period of 3 years and 4 months following initial diagnosis. As a result, 7 of the 14 cases were diagnosed as having MALT lymphoma. All of them were resected, and then the diagnosis was confirmed. Metastasis was found in 2 cases. The other 7 cases were diagnosed as RLH. A favorable prognosis during follow up without any treatment supported the belief that they were non-malignant lesions. HP infections were observed on 83% of the RLH cases and 57% of MALT lymphoma cases. In one of the case of RLH, the lesion disappeared after eradication of HP.

Aged↗
PubMed1997

Preparation of (R)-N-methylsalsolinol via reversed-phase high-performance liquid chromatography using beta-cyclodextrin as a mobile-phase additive.

A new preparation method was devised using beta-cyclodextrin as a mobile-phase additive in a reversed-phase high-performance liquid chromatography system. Preparative separation of the biologically active (R)-enantiomer was achieved from racemic N-methylsalsolinol. Beta-cyclodextrin was removed completely in good yield by acid extraction and solid-phase extraction. By a slight modification, this method will be applicable to the isolation of various types of biologically important enantiomers.

Chromatography, High Pressure Liquid↗
PubMed1997

Phosphorylation-dependent regulation of N-methyl-D-aspartate receptors by calmodulin.

The N-methyl-D-aspartate (NMDA) receptor plays important roles in synaptic plasticity and brain development. The NMDA receptor subunits have large intracellular domains in the COOH-terminal region that may interact with signal-transducing proteins. By using the yeast two-hybrid system, we found that calmodulin interacts with the COOH terminus of the NR1 subunit and inactivates the channels in a Ca2+-dependent manner. Here we show that protein kinase C (PKC)-mediated phosphorylation on serine residues of NR1 decreases its affinity for calmodulin. This suggests that PKC-mediated phosphorylation of NR1 prevents calmodulin from binding to the NR1 subunit and thereby inhibits the inactivation of NMDA receptors by calmodulin. In addition, we show that stimulation of metabotropic glutamate receptor 1alpha, which potentiates NMDA channels through PKC, decreases the ability of NR1 to bind to calmodulin. Thus, our data provide clues to understanding the basis of cross-talk between two types of receptors, metabotropic glutamate receptors and the NR1 subunit, in NMDA channel potentiation.

Amino Acid Sequence↗
PubMed1997

Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(III) complex.

A complex of an independent L chain from anti-mesotetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90 degrees C). Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment. The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone. Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115-146) and hydrophilic region (147-189) in CL are important for thermostability and activity, respectively. Tertiary structure of L-zyme was predicted by AbM. Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity. Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively. Distance between proximal His and distal His in L-zyme is 9.09 A, whereas that of ARP is 7.8 A.

Animals↗
PubMed1997

PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes.

The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-gamma (IFN-gamma)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 alpha- and beta-subunits. The deduced amino acid sequences of the alpha- and beta-subunits were 49% identical. We also determined the primary structure of the mouse PA28 gamma-subunit (Ki antigen), a protein of unknown function structurally related to the alpha- and beta-subunits. The amino acid sequence identity of the gamma-subunit to the alpha- and beta-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the alpha- and beta-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-gamma-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the gamma-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a gamma-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-gamma-inducible alpha- and beta-subunits emerged by gene duplication from a gamma-subunit-like precursor.

Amino Acid Sequence↗
PubMed1997

Structural analysis and chromosomal localization of the mouse Psmb5 gene coding for the constitutively expressed beta-type proteasome subunit.

The proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex class I molecules. Accumulated evidence indicates that, upon stimulation with interferon-gamma (IFN-gamma), three beta-type subunits, designated LMP2, LMP7, and PSMB10, are incorporated into the 20S proteasome by displacing the housekeeping beta-type subunits designated PSMB6, PSMB5, and PSMB7, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. In the present study, we determined the organization of the mouse gene Psmb5, coding for the PSMB5 subunit. Psmb5 is made up of three exons, spanning approximately 5 kilobases. Its exon-intron organization differs radically from those of the other IFN-gamma-regulated, beta-type subunit genes including Lmp7 with which Psmb5 is believed to share an immediate common ancestor. The structure of the mouse Psmb5 gene is identical to that of its recently characterized human counterpart. Thus, the unique organization of the gene coding for the PSMB5 subunit appears to have been established before mammalian radiation. As well as the Psmb5 gene, the mouse genome contains a processed pseudogene designated Psmb5-ps. Interspecific backcross mapping showed that Psmb5 maps close to the Gtrgal2 locus on chromosome 14 and that Psmb5-ps is located in the vicinity of the Psme3 locus on chromosome 11. These results were confirmed by fluorescent in situ hybridization analysis that localized Psmb5 to band C2 to proximal D1 of chromosome 14 and Psmb5-ps to band D of chromosome 11.

Amino Acid Sequence↗
PubMed1997

Long-term survival and differentiation of human peripheral blood CD34+ cells in SCID mice.

Human peripheral blood progenitor cells (PBPC) are currently used as a source of hematopoietic reconstitution by autologous transplantation after myeloabrative chemotherapy for malignancies. PBPC would also be useful for allogeneic transplantation since the collection of PBPC is much safer than that of bone marrow stem cells (BM). For allogeneic transplantation, it is imperative to confirm that PBPC contains self-renewable stem cells that can sustain a long lasting hematopoiesis. In the present study, we examined the reconstitution of human hematopoiesis in severe combined immunodeficiency (SCID) mice by transplanting peripheral blood CD34+ cells in which the neo gene was transduced as a marker. In 2 of 4 mice receiving PB-CD34+ cell transplantation, the neo gene appeared as early as 4 weeks and lasted as long as 24 weeks in all DNA preparations of bone marrow, peripheral blood and spleen cells from the SCID mice, while in 2 of 4 mice receiving BM-CD34+ cell transplantation, although the neo gene also lasted as late as 24 weeks, it did not appear as early as in the mice receiving PB-CD34+ cell transplantation. A similar observation was noted in clinical trials, i.e. the white blood cell and platelet recovered earlier by transplantation of PBPC than of BM. In mice who had the neo gene, we were also able to demonstrate by FACS the presence of human lineage specific antigen in the cells as late as 24 weeks after transplantation with PB-CD34+ cells, and the presence of human IgG in the sera 10 weeks after transplantation. These findings indicate that PB-CD34+ cells contain long-term repopulating stem cells which undergo differentiation in SCID mice.

Animals↗
PubMed1997

Substrate specificity of human O6-methylguanine-DNA methyltransferase for O6-benzylguanine derivatives in oligodeoxynucleotides.

To investigate the substrate specificity of human O6-methylguanine-DNA methyltransferase (MGMT) for O6-benzylguanine (6BG) derivatives incorporated in oligodeoxynucleotides, we prepared 25-mer lengths of sequences containing various 6BG derivatives and their related compounds and then measured the ability of these derivatives to inactivate MGMT in vitro. Oligodeoxynucleotides containing a 6BG, O6-(2-fluorobenzyl)guanine (2F-6BG), O6-(3-fluorobenzyl)guanine (3F-6BG), O6-(4-fluorobenzyl)guanine (4F-6BG), O6-benzylhypoxanthine (6BH), or O6-methylguanine (6MG) were all good substrates for MGMT, and no obvious differences were observed among them. Oligodeoxynucleotides containing N2-isobutyrylated 6BG and 6MG showed only a slightly reduced capacity for inactivating MGMT compared to N2-nonmodified forms of these derivatives. No obvious differences were observed in the corresponding double-stranded and single-stranded oligodeoxynucleotides. MGMT substrate specificity for the 6BG derivatives in the oligodeoxynucleotide was found to be quite different from that seen in our previous study [Mineura, K., et al. (1994) Int. J. Cancer 58, 706-712; (1995) Int. J. Cancer 63, 148-151. Kohda, K., et al. (1995) Biol. Pharm. Bull. 18, 424-430] and others [Moschel, R. C., et al. (1992) J. Med. Chem. 35, 4486-4491. Chae, M.Y., et al. (1994) J. Med. Chem. 37, 342-347] using the corresponding free bases. In brief, (i) 6BG, 3F-6BG, and 4F-6BG greatly inhibited human MGMT, whereas 2F-6BG, 6BH, and 6MG displayed much weaker activity; (ii) any modifications at the 2-amino group of the 6BG resulted in severe reductions in the ability to inactivate MGMT. These results obtained by the experiments using oligodeoxynucleotides and free bases suggest that human MGMT has low substrate specificity for 6BGs in oligodeoxynucleotides. Conformational changes in human MGMT which favor binding to oligodeoxynucleotides containing 6BG derivatives and the subsequent transfer of their benzyl groups may account for the difference in substrate specificity between the incorporated 6BG derivatives and their free base form.

Antineoplastic Agents↗
PubMed1997

Synthesis and miscoding specificity of oligodeoxynucleotide containing 8-phenyl-2'-deoxyguanosine.

Aryl radicals and arenediazonium ions are suspected to react with cellular DNA, resulting in C8-arylguanine adducts. 8-Phenyl-2'-deoxyguanosine (8-PhdG) was synthesized as a model adduct by reacting dG with benzenediazonium chloride and incorporated into oligodeoxynucleotides using phosphoramidite techniques. A site-specifically modified oligodeoxynucleotide containing a single 8-PhdG was then used as a template for primer extension reactions catalyzed by the intact (exo+) or 3'-->5' exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha (pol alpha). Although primer extensions catalyzed by the Klenow fragments were retarded at the position of 8-PhdG, most of the primer extension passed the lesion to form the fully extended products. In contrast, primer extensions catalyzed by pol alpha were strongly blocked opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-PhdG. The exo- Klenow fragment incorporated primarily dCMP, the correct base, opposite 8-PhdG, along with small amounts of incorporation of dAMP. Two-base deletions were also observed. In contrast, the exo+ Klenow fragment incorporated dCMP opposite the lesion. When pol alpha was used, 8-PhdG promoted small amounts of misincorporation of dAMP and dGMP as well as one- and two-base deletions. The duplex containing 8-PhdG.dG was thermally and thermodynamically more stable than dG.dG. The duplex containing 8-PhdG.dA was thermodynamically more stable than dG.dA. We conclude that 8-PhdG is a weak miscoding lesion, capable of generating G-->T and G-->C transversions and deletions in cells.

Animals↗
PubMed1997

An endogenous dopaminergic neurotoxin, N-methyl-(R)-salsolinol, induces DNA damage in human dopaminergic neuroblastoma SH-SY5Y cells.

Recently, an endogenous neurotoxin, 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl-(R)-salsolinol], was found to elicit parkinsonism in rats with selective depletion of dopamine neurons in the substantia nigra without necrotic tissue reaction. The mechanism of the cell death was examined by detection of DNA damage using a single-cell gel electrophoresis (comet) assay in human dopaminergic neuroblastoma SH-SY5Y cells. Only N-methylsalsolinol was found to induce DNA damage, whereas other catechol isoquinolines, such as (R)-salsolinol, (S)-salsolinol, and 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion, did not. The (R)-enantiomer of N-methylsalsolinol damaged DNA much more profoundly than the (S)-enantiomer. Cycloheximide protected the cells from DNA damage, suggesting that an apoptotic process may account for the DNA damage. Morphological changes indicating apoptotic cell death were also confirmed. Antioxidants and deprenyl reduced DNA damage, indicating that the damage was initiated by oxidative stress and that neuroprotection by deprenyl may be partially ascribed to its prevention of DNA damage. Apoptosis induced by neurotoxins may be a mechanism underlying the cell death of dopamine neurons in the substantia nigra of Parkinson's disease.

Antioxidants↗