Biomedical subjects
Publications and source records attributed to A Matsuda.
Cyclic ADP-carbocyclic-ribose (cADPcR, 2) and its several analogs were designed and synthesized as stable mimics of Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR, 1). cADPcR was stable and actually caused a significant release of Ca(2+) stronger than that of cADPR.
To investigate the mutation mechanism of purine transition in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCGAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, has been synthesized and its crystal structure determined. Two dodecamers form a B-form duplex. Electron density maps clearly show that one of the two mo(4)C residues forms a pair with a guanine residue of the opposite strand, the geometry being the canonical Watson-Crick type, and that the other mo(4)C residue forms a wobble pair with the opposite guanine residue. These two pairings are ascribed to the tautomerization of the methoxylated cytosine moieties between the amino and imino forms.
To investigate the role of divalent cations in crystal packing, four different crystals of a Dickerson-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2'-deoxy-5-formyluridine at X, were obtained under several conditions with and without divalent cations. The crystal structures are all isomorphous. The octahedrally hydrated magnesium cations found in the major groove cement the two neighbouring duplexes along the b axis. In the Mg(2+)-free crystals, a five-membered ring of water molecules occupies the same position as the magnesium site and connects the two duplexes similarly to the hydrated Mg(2+) ion. It has been concluded that water molecules can take the place of the hydrated magnesium cation in crystallization, but the magnesium cation is more effective and gives X-ray diffraction at slightly higher resolution. In all four crystals, the 5-formyluracil residues form the canonical Watson-Crick pair with adenine residues.
We examined the effects of dosage schedule on antitumor activity in vitro and in vivo to determine the optimal administration schedule for a new nucleoside antimetabolite 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106). The cytotoxicity of TAS-106 in vitro against human tumors was evaluated at three drug exposure periods. TAS-106 exhibited fairly potent cytotoxicity even with 4 h exposure, and nearly equivalent and sufficiently potent cytotoxicity with 24 and 72 h exposures. These results suggest that long-term exposure to TAS-106 will not be required to achieve maximal cytotoxicity. The antitumor activity of TAS-106 in vivo was compared in nude rat models bearing human tumors on three administration schedules, once weekly, 3 times weekly, and 5 times weekly for 2 or 4 consecutive weeks. TAS-106 showed strong antitumor activity without serious toxicity on all three schedules, but the antitumor activity showed no obvious schedule-dependency in these models. When tumor-bearing nude rats were given a single i.v. dose of [(3)H]TAS-106, tumor tissue radioactivity tended to remain high for longer periods of time as compared to the radioactivity in various normal tissues. Furthermore, when the metabolism of TAS-106 in the tumor was examined, it was found that TAS-106 nucleotides (including the active metabolite, the triphosphate of TAS-106) were retained at high concentrations for prolonged periods. These pharmacodynamic features of TAS-106 may explain the strong antitumor activity without serious toxicity, observed on intermittent administration schedules, in nude rat models with human tumors. We therefore consider TAS-106 to be a promising compound which merits further investigation in patients with solid tumors.
We studied mutational events in deoxycytidine (dCyd) kinase mRNA expression, focusing on aberrant dCyd kinase mRNA, which has been frequently observed in established cell lines resistant to antitumor dCyd nucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine (Ara-C), gemcitabine (dFdC) and 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosylcytosine (CNDAC). We describe here the expression of aberrant dCyd kinase mRNAs identified as splicing mutants. These mutants included deletions of the fifth exon in CNDAC-resistant cells (originating from HT-1080 cells), of the third exon in Ara-C-resistant cells (originating from SK-MEL-28 cells) and of the fourth exon in 2'-deoxy-2'-methylidenecytidine (DMDC)-resistant cells (originating from SK-MEL-28 cells). Various nucleoside-resistant cells originating from the same parental HT-1080 cells were established. The resulting cells expressed the same mRNA with deletion of the fifth exon, and the location of splicing was independent of the type of nucleosides used for the establishment of resistant cells. The deletion of the fifth exon in dCyd kinase seems to be a target for acquisition of resistance to antitumor cytosine nucleosides. However, distinct mutations in the dCyd kinase gene seem to be associated with acquisition of resistance to different antitumor cytosine nucleosides.
The mechanism of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) action was investigated in human lymphoblastoid CEM cells and myeloblastic leukemia ML-1 cells. CNDAC was metabolized to its 5'-triphosphate and incorporated into DNA, which was associated with inhibition of DNA synthesis. After incubation of cells with [(3)H]CNDAC, metabolites were detected in 3'-->5' phosphodiester linkage and at the 3' terminus of cellular DNA. Specific enzymatic hydrolysis of DNA demonstrated that the parent nucleoside and its 2'-epimer 2'-C-cyano-2'-deoxy-2-ribo-pentofuranosylcytosine accounted for approximately 65% of the total analogs incorporated into DNA and essentially all of the drug in the 3'-->5' phosphodiester linkage. In contrast, all detectable radioactivity at 3' termini was associated with 2'-C-cyano-2',3'-didehydro-2',3'-dideoxycytidine. This de facto DNA chain-terminating nucleotide arises from an electronic characteristic and cleavage of the 3'-phosphodiester bond subsequent to the addition of a nucleotide to the incorporated CNDAC moiety by beta-elimination, a process that generates a single strand break in DNA. Investigation of the biological consequences of these actions indicated that, after incubation with cytostatic concentrations of CNDAC, cell cycle progression was delayed during S phase, but that cells arrested predominantly in the G(2) phase. This differed from the S phase-arresting actions of ara-C and gemcitabine, other deoxycytidine analogs that inhibit DNA replication but do not cause strand breaks. Thus, once incorporated into DNA, the CNDAC molecule appears to act by a dual mechanism that 1) delays the progress of further DNA replication, but 2) upon addition of a deoxynucleotide results in the conversion of the incorporated analog to a de facto DNA chain terminator at the 3' terminus of a single strand break. It is likely that DNA strand breaks trigger cell cycle arrest in G(2).
We have found that milnacipran, a clinically useful antidepressant due to its inhibition of the re-uptake of serotonin (5-HT) and noradrenaline, is also a non-competitive NMDA-receptor antagonist. Based on the cyclopropane structure of milnacipran, conformationally restricted analogs were designed and synthesized. Of these analogs, (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) is 30-fold stronger than milnacipran as an NMDA-receptor antagonist with virtually no inhibitory effect on the neurotransmitter re-uptake. PPDC was identified as a new class of NMDA-receptor antagonist because it has a mode of action different from that of the previous antagonists; it selectivly binds the GluRepsilon3/GluRzeta1 and GluRepsilon4/GluRzeta1 subtype receptors in an agonist-independent allosteric manner. Functional assays of PPDC with the Xenopus oocytes system and cultured mouse neurons under voltage-clamp conditions confirmed that it acts as a potent NMDA-receptor antagonist. PPDC effectively protected against NMDA-induced neurotoxicity in both cultured mouse cerebral cortex and delayed neuronal death in a gerbil ischemic model. It was also active in a reserpine-treated mouse Parkinsons disease model. Thus, PPDC may be a candidate for a clinically useful NMDA-receptor antagonist, since the development of previous NMDA-receptor antagonists as drugs has been hindered by various undesirable side effects.
We reviewed 80 patients admitted to our hospital who were diagnosed with acute epiglottitis between January 1995 and March 1999, and their clinical features, evolution and treatments were analysed. No sexual predominance was found, and there was no patient younger than 16 years of age. The patient fatality rate was 1.3 per cent, and the hospitalization period was markedly longer than those of other reports.
In the sexual reproduction of Paramecium tetraurelia, the somatic nucleus (macronucleus) undergoes massive genomic rearrangement, including gene amplification and excision of internal eliminated sequences (IESs), in its normal developmental process. Strain d4-662, one of the pawn mutants, is a behavioral mutant of P. tetraurelia that carries a recessive allele of pwB662. ThepwB gene in the macronucleus of the strain has an insertion of the IES because a base substitution within the IES prevents its excision during gene rearrangement. Cultures of this strain frequently contain cells reverting to the wild type in the behavioral phenotype. The mutant and revertant cells maintained stable clonal phenotypes under the various environmental conditions examined unless they underwent sexual reproduction. After sexual reproduction, both mutant and revertant produced 2.7-7.1% reverted progeny. A molecular analysis performed on the macronuclear DNA of the mutant and revertant of d4-662 showed that much less than 1% of the mutant IES was precisely excised at every sexual reproduction of the strain. Therefore, the alternative phenotype of strain d4-662 seems to be caused by an alternative excision of the mutant IES.
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Uridine/cytidine kinase which converts uridine and cytidine to their corresponding monophosphates is a rate-limiting enzyme involved in the salvage pathway of pyrimidine synthesis. We isolated cDNA encoding the enzyme from human fibrosarcoma cells, then determined its nucleotide sequence by the 5'-RACE method followed by confirmation employing the human genome DNA library. The isolated uridine/cytidine kinase cDNA (UCK cDNA) consisted of 786 nucleotides encoding 261 amino acids and was found to have approximately 70% homology with mouse UCK cDNA. Northern blot analysis of human leukemia RNAs with labeled UCK gene showed a single band at 1.6 kb to be UCK mRNA, and southern blot analysis of the UCK cDNA after digestion with BamHI, SacI and XbaI enzymes showed four band signals, suggesting the UCK gene to have at least 4 exons. A truncated form of UCK cDNA was expressed as the His-tag conjugated protein in Escherichia coli. The expressed and purified protein specifically converted uridine and cytidine to their corresponding monophosphates and also phosphorylated antitumor nucleosides such as 5-fluorouridine, cyclopentenyl-cytosine and 3'-C-ethynylcytidine. The present results suggest that our cloned human UCK cDNA encodes the correct amino acid sequence for UCK protein, showing high intracellular phosphorylation activity forward natural and synthetic pyrimidine nucleosides.
A 41-year-old man visited his doctor in May 2000 because of a sore throat and high fever. His symptoms did not improve, despite administration of antibiotics and nonsteroidal anti-inflammatory drugs. Since a chest X-ray examination revealed an anterior mediastinal bulky tumor, he was referred and admitted to our hospital on June 21, 2000. The peripheral white blood cell count was 44,540/microliter with 74% myeloblasts. Bone marrow aspiration revealed a hypercellular marrow with 82% myeloblasts, which were negative for peroxidase and alpha-naphthyl butylate esterase staining. Blast cells were positive for CD7, CD13, CD33, CD34, and HLA-DR, and negative for CD56. A needle biopsy specimen of the mediastinal tumor consisted of myeloblasts. We diagnosed the patient as having CD7 (+) acute myeloid leukemia (AML) (M0) with a bulky mediastinal mass based on the surface marker analysis, although the clinical features resembled myeloid/NK precursor acute leukemia. The patient achieved a complete remission after two courses of induction therapy. We are planning an allogeneic stem cell transplantation during his first remission because of the high risk of relapse.
The results of the clinical studies of insulin lispro carried out in Japan to confirm the pharmacodynamics, pharmacokinetics, efficacy and safety, which were recently released were summarized in this article. When compared to human regular insulin, single subcutaneous injection to healthy persons showed that insulin lispro was adsorbed and disappeared more rapidly in blood stream and blood lowering action revealed more early. The potency of hypoglycemic action was stronger. Long-term multicenter, randomized, comparative, parallel, open-label study to diabetic patients of both type 1 and type 2 by multiple injection method showed that insulin lispro resulted in the significant improvement of 2 hour-postprandial blood glucose level 12 and 24 weeks after the initiation of treatment. HbA1c level did not change between two groups during the study. Insulin lispro did not give any adverse effects during 48 week-study. These studies support the conclusion that insulin lispro is useful and effective in the treatment of diabetes as a short-acting insulin.
Excluding chronic myelomonocytic leukemia, a total of 92 consecutive patients with myelodysplastic syndrome showing less than 20% blasts in the bone marrow were analyzed. We evaluated the clinical significance of the WHO and MDS 2000 classifications by reviewing each MDS patient according to the classification. The WHO criteria classified the MDS patients into 36 with RA, 22 with RCMD and 33 with RAEB, whereas according to the MDS 2000 criteria there were 19 RAEB-I patients and 15 RAEB-II patients. Based on the WHO classification, the RCMD patients had higher platelet counts and percentages of blasts among BM cells than the RA patients (P = 0.0018, P = 0.0001). Twenty percent of the RA patients, 44.8% of the RCMD patients, and 70.8% of the RAEB patients had cytogenetic abnormalities. Among them, the poor karyotype was present in 6.7% of the RA patients, 21.0% of the RCMD patients and 41.6% of the RAEB patients. The rate of acute leukemia death was 14.3% in the RA patients, 67.7% in the RAEB patients and 50.0% in the RCMD patients. Analysis of survival times revealed significant differences between RA and RCMD patients (P = 0.0482). The clinical features of RCMD patients were intermediate between those of RAEB and RA patients. There was no difference between the clinical features of the RAEB-I and RAEB-II patients in the MDS 2000 classification.
Recently, we developed a regio- and stereoselective method for introducing a vinyl group at the position beta to a hydroxyl group in halohydrins or alpha-phenylselenoalkanols via a radical atom-transfer cyclization reaction with a vinylsilyl group as a temporary connecting radical-acceptor tether. The synthesis of 2'-deoxy-2'-C-vinyl- and 2'-deoxy-2'-C-hydroxymethyluridines (7 and 8, respectively) and the corresponding 2'-deoxycytidine congeners (10 and 11, respectively), which were designed as potential antitumor and/or antiviral agents, was achieved using this radical atom-transfer cyclization as the key step. When the 2'-deoxy-2'-iodo-5'-O-monomethoxytrityl (MMTr) uridine derivative 19a, bearing a vinylsilyl group at the 3'-hydroxyl group, was heated with (Me(3)Sn)(2) and AIBN in benzene, the corresponding radical atom-transfer product was generated, which in turn was successively treated with tetrabutylammonium fluoride and TBSCl/imidazole to give the desired 2'-deoxy-5'-O-MMTr-3'-O-TBS-2'-C-vinyluridine (25). Compound 25 was successfully converted into the target 2'-deoxy-2'-branched pyrimidine ribonucleosides 7, 8, 10, and 11.
3,7-Anhydro-D-glycero-D-ido-octitol 1,5,6-trisphosphate (5) was designed as a novel IP(3)-receptor ligand having a C-glycosidic structure and was synthesized via a radical cyclization reaction with a temporary connecting vinylsilyl tether as the key step. The phenyl 2-O-dimethylvinylsilyl-3,4, 6-tri-O-benzyl-1-seleno-beta-D-glucopyranoside (7), in the usual (4)C(1)-conformation, was successively treated with Bu(3)SnH/AIBN and under Tamao oxidation conditions to give a mixture of five C-glycosidic products. On the other hand, similar successive treatment of the corresponding 3,4-di-O-TBS-protected substrates 13 and 24, which were in an unusual (1)C(4)-conformaion due to the steric repulsion between the bulky silyl protecting groups, gave the desired 1alpha-C-glycosides 18 and 25, respectively, as the major products. Thus, the course of the radical cyclization was effectively controlled by a change in the conformation of the pyranose ring into a (1)C(4)-form due to steric repulsion between the adjacent bulky TBS-protecting groups at the 3- and 4-hydroxyl groups. From 25, the target 5 was synthesized via phosphorylation of the hydroxyls by the phosphoramidite method. The C-glycoside trisphosphate 5 has significant binding affinity for IP(3) receptor of calf cerebella.
An efficient synthesis of cyclic IDP-carbocyclic-ribose (3) and its 8-bromo derivative 6, as stable mimics of cyclic ADP-ribose, was achieved, and a condensation reaction with phenylthiophosphate-type substrate 15 or 16 to form an intramolecular pyrophosphate linkage was a key step. N-1-Carbocyclic-ribosylinosine derivative 28 and the corresponding 8-bromo congener 24 were prepared via condensation between N-1-(2,4-dinitrophenyl)inosine derivative 17 and a known optically active carbocyclic amine 18. Compounds 24 and 28 were then converted to the corresponding 5"-phosphoryl-5'-phenylthiophosphate derivatives 15 and 16, respectively, which were substrates for the condensation reaction to form an intramolecular pyrophosphate linkage. Treatment of 8-bromo substrate 15 with I2 or AgNO3 in the presence of molecular sieves 3A (MS 3A) in pyridine at room temperature gave the desired cyclic product 12 quantitatively, while the yield was quite low without MS. The similar reaction of 8-unsubstituted substrate 16 gave the corresponding cyclized product 32 in 81% yield. Acidic treatment of these cyclic pyrophosphates 12 and 32 readily gave the targets 6 and 3, respectively. This result suggests that the construction of N-1-substituted hypoxanthine nucleoside structures from N-1-(2,4-dinitrophenyl)inosine derivatives and the intramolecular condensation by activation of the phenylthiophosphate group with I2 or AgNO3/MS 3A combine to provide a very efficient route for the synthesis of analogues of cyclic ADP-ribose such as 3 and 6. Thus, this may be an entry to a general method for synthesizing biologically important cyclic nucleotides of this type.
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