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Biomedical subjects

A Fujimori

Publications and source records attributed to A Fujimori.

At least 55 records · Page 3Linked to original sources
PubMed1998

Murine cell line SX9 bearing a mutation in the dna-pkcs gene exhibits aberrant V(D)J recombination not only in the coding joint but also in the signal joint.

We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide "T" (9572) to "C" transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12. 2% as compared with the normal cell line. To confirm the responsibility of the dna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild type dna-pkcs cDNA was observed. SX9 is a novel dna-pkcs-deficient cell line.

Amino Acid Substitution↗
PubMed1998

Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation.

We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a lambdagt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif "IXCXE". The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2-q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication.

Amino Acid Sequence↗
PubMed1998

Chromosomal assignment of the gene for human DNA-PKcs interacting protein (KIP) on chromosome 15q25.3-q26.1 by somatic hybrid analysis and fluorescence in situ hybridization.

We report the chromosomal location of the gene for DNA-PKcs interacting protein KIP. Based on fluorescence in situ hybridization and polymerase chain reaction (PCR)-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel, this gene was mapped to q25.3-q26.1 region on chromosome 15.

Calcium-Binding Proteins↗
PubMed1998

Enhanced depressor and hyperemic responses to calcitonin gene-related peptide in spontaneously hypertensive rats.

Intravenous (i.v.) bolus injection of calcitonin gene-related peptide (CGRP) caused a depressor response, which was significantly larger in 12-week-old spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). CGRP also caused decreases in carotid and hindquarter vascular resistance, the magnitude of which was larger in the carotid than the hindquarter. In both regions, the vasodilator response to CGRP was significantly larger in SHR than WKY. Plasma CGRP level was significantly lower in SHR than WKY. These results suggest that depressor and vasodilator responses to CGRP are enhanced in SHR and that decreased plasma CGRP level in SHR may contribute to the enhanced responses.

Age Factors↗
PubMed1998

Adsorption of complement, cytokines, and proteins by different dialysis membrane materials: evaluation by confocal laser scanning fluorescence microscopy.

The membranes tested in the present study were cellulose triacetate (CTA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN). The adsorption by each membrane of albumin, IgG, C3a, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), human neutrophil elastase (HNE), and tumor necrosis factor alpha (TNFalpha) was examined and semiquantitatively graded by confocal laser scanning fluorescence microscopy (CLSFM). After clinical use the dialyzers were treated with antibodies for these proteins and cytokines. Then the samples were incubated with fluorescein isothiocyanate-labeled anti-IgG antibody and observed by CLSFM. The changes in the blood levels of C3a and cytokines were also studied. In the CTA membrane, the adsorption of these substances, except for albumin and HNE, was less than in the synthetic membranes. The PAN membrane revealed the most abundant adsorption, especially for IL-1beta, IL-6, and TNFalpha. Although a marked elevation of C3a in the blood was observed in the CTA membrane, considerable adsorption was evident in the PMMA and the PAN membranes. Because the changes in the blood levels could be affected by membrane adsorption, both the blood levels and the adsorption of the biocompatibility parameters should be evaluated when membrane biocompatibility is discussed.

Acrylic Resins↗
PubMed1998

Studies on nonpeptide angiotensin II receptor antagonists. II. Synthesis and biological evaluation of 5H-pyrazolo[1,5-b][1,2,4]triazole derivatives with a C-linked oxygen functional group at the 6-position.

2,7-Diethyl-5H-pyrazolo[1,5-b][1,2,4]triazole derivatives were synthesized and evaluated for activity as angiotensin II receptor antagonists. Replacement of the C-6 hydrogen with C-linked oxygen functional groups led to derivatives with increased in vitro activities. Among these compounds, 2,7-diethyl-5-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-5H- pyrazolo[1,5-b][1,2,4]triazole-6-carboxylic acid (2d) showed potent, insurmountable antagonism, but had poor oral potency against angiotensin II-induced pressor response in rats. In order to improve the oral activity, the carboxylic acid function of 2d was converted into a double ester. This modification afforded (+/-)-1-[(ethoxycarbonyl)oxy]ethyl 2,7-diethyl-5-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-5H- pyrazolo[1,5-b][1,2,4]-triazole-6-carboxylate (2f), which was orally active in rats, and produced a dose-dependent decrease in blood pressure when administered orally to conscious furosemide-treated dogs, with ca. 3-fold increased potency in comparison with the parent C-6 hydrogen compound.

Angiotensin Receptor Antagonists↗
PubMed1998

Studies on nonpeptide angiotensin II receptor antagonists. I. Synthesis and biological evaluation of pyrazolo[1,5-b][1,2,4]triazole derivatives with alkyl substituents.

Alkyl-substituted pyrazolo[1,5-b][1,2,4]triazole derivatives were synthesized and evaluated for activity as angiotensin II receptor antagonists. Molecules with the (methylbiphenylyl)tetrazole moiety and N-5 were the preferred compounds. Ethyl substitutions at both C-2 and C-7 resulted in the optimal compound, 2,7-diethyl-5-[[2"-(1H -tetrazol-5-yl)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][1,2,4]tria zole (5n), with a pA2 value of 8.774 in rabbit aorta. In the in vivo tests, 5n inhibited the angiotensin II-induced pressor response in rats after oral administration. This compound also produced a dose-dependent decrease in blood pressure when administered orally to conscious furosemide-treated dogs, having a longer duration of action as compared to DuP753. These data suggest that 5a may be a useful agent for the treatment of angiotensin II-dependent disease, such as hypertension.

Administration, Oral↗
PubMed1998

Studies on nonpeptide angiotensin II receptor antagonists. III. Synthesis and biological evaluation of 5-alkylidene-3,5-dihydro-4H-imidazol-4-one derivatives.

5-Alkylidene-3,5-dihydro-4H-imidazol-4-one derivatives were synthesized and evaluated for activity as angiotensin II receptor antagonists. Substitutions at C-2 and C-5, respectively, with a propyl group and a 1-methylethylidene group resulted in the optimal compound, 3,5-dihydro-5-(1-methylethylidene)-2-propyl-3-[[2'-(1H-tetrazol - 5-yl)biphenyl-4-yl]methyl]-4H-imidazol-4-one (2b), with a pA2 value of 8.85 in rabbit aorta. When administered orally to rats, 2b showed a greater inhibitory effect on angiotensin II-induced pressor response than DuP 753. Compound 2b also showed a good antihypertensive effect when administered orally to conscious sodium-depleted spontaneously hypertensive rats, with a duration of action of 24 h. These data suggest that 2b may be a useful agent for the treatment of angiotensin II-dependent diseases such as hypertension.

Angiotensin II↗
PubMed1998

Studies on nonpeptide angiotensin II receptor antagonists. IV. Synthesis and biological evaluation of 4-acrylamide-1H-imidazole derivatives.

A novel series of nonpeptide angiotensin II antagonists containing the acrylamide group at the 4-position of the imidazole ring was synthesized and their antagonistic activity was examined by functional assay in rabbit aorta. The acrylamide group was selected as a large lipophilic surrogate for the chloro group of EXP3174. A structure-activity relationship study of the acrylamide moiety has shown that substitution at the 4-position with the N-methyl-3,3-dimethylacrylamide group resulted in the optimal compound, 2-butyl-4-[(3,3-dimethylacryloyl)methyl-amino]-1-[[2'-(1H-tetra zol-5- yl)biphenyl-4-yl]methyl]-1H-imidazole-5-carboxylic acid (1), which was superior to EXP3174 in vitro. Since 1 showed only poor activity against angiotensin II-induced pressor response in rats after oral administration, the carboxylic acid function of 1 was converted into prodrug esters (13). Among these, the 1-[(ethoxycarbonyl)oxy]ethyl ester (13a) showed the most potent and longest-lasting activity when given orally to rats. When administered orally to conscious furosemide-treated dogs, 13a showed an approximately 3-fold increased hypotensive activity in comparison with DuP 753. These data suggest that 13a may be an useful agent for the treatment of angiotensin II-dependent diseases, such as hypertension.

Acrylamides↗
PubMed1997

In vivo sequencing of camptothecin-induced topoisomerase I cleavage sites in human colon carcinoma cells.

Camptothecin (CPT) is a specific topoisomerase I (top1) poison which traps top1 cleavable complexes; e.g. top1-linked DNA single-strand breaks with 5'-hydroxyl and 3'-top1 linked termini. CPT is also a potent anticancer agent and several of its derivatives have recently shown activity in the chemotherapy of solid tumors. Our aim was to apply the ligation-mediated polymerase chain reaction (LM-PCR) method to DNA extracted from CPT-treated cells in order to: (i) evaluate LM-PCR as a sensitive technique to detect in vivo CPT-induced cleavable complexes; (ii) investigate the frequency and distribution of CPT-induced DNA damage in vivo ; and (iii) compare the distribution and intensity of cleavage sites in vivo and in vitro. This report describes a protocol allowing the sequencing of top1-mediated DNA strand breaks induced by CPT in the coding strand of the 18S rRNA gene of human colon carcinoma cells. CPT or its clinical derivatives, topotecan, CPT-11, SN-38, and 9-aminocamptothecin differed in their potency and exhibited differences in their DNA cleavage pattern, which is consistent with our previous in vitro studies [Tanizawa et al . (1995) Biochemistry , 43, 7200-7206]. CPT-induced DNA cleavages induced in the presence of purified top1 were induced at the same sites in the human 18S rDNA. However, the relative intensity of the cleavages were different in vivo and in vitro. Because mammalian cells contain approximately 300 copies of the rDNA gene per genome, rDNA could be used to monitor CPT-induced DNA cleavage in different cell lines and possibly in tumor samples.

Antineoplastic Agents, Phytogenic↗
PubMed1997

The murine DNA-PKcs gene consists of 86 exons dispersed in more than 250 kb.

The DNA-PKcs gene encodes the 465-kDa catalytic subunit of DNA-dependent protein kinase (DNA-PK), which associates with heterodimeric autoantigens Ku70 and Ku80 and exhibits protein kinase activity depending on DNA double-strand breaks. The gene is also responsible for the aberration in severe combined immune deficiency (SCID) mice, which exhibit a high sensitivity to ionizing radiation and abnormal DNA rearrangement of immunoglobulin and T cell receptor genes. There is further evidence that DNA-PKcs phosphorylates various proteins involved in DNA replication, transcription, repair, and recombination. Nevertheless the structure/function relationship in this huge molecule is virtually unknown. We determined the exons and introns of the murine DNA-PKcs gene by the long-distance polymerase chain reaction method. The murine DNA-PKcs gene consists of 86 exons distributed in a region of more than 250 kb. The average size of the exons is 140 bp. All the splicing sites conform to the GT/AG rule. The SCID mutation site (Tyr4046) has been identified in exon 85. The genomic structure of the DNA-PKcs gene provides clues for the study of various functional domains in this macromolecule.

Amino Acid Sequence↗
PubMed1997

Pharmacological profile of YM358, a novel nonpeptide angiotensin AT1 receptor antagonist.

The pharmacological profile of YM358, 2,7-diethyl-5-[[2'-(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][1,2,4]tri azole potassium salt monohydrate, a novel non-peptide angiotensin AT1 receptor antagonist, was studied in vitro and in vivo. YM358 competed with [125I][Sar1, Ile8]angiotensin II for angiotensin AT1 receptors in rat liver membranes. YM358 displayed competitive kinetics and the pKi value was calculated as 8.79. In contrast, YM358 had little effect on the binding of [125I][Sar1, Ile8]angiotensin II to the angiotensin AT2 receptor in bovine cerebellum. In isolated rabbit aorta, YM358 produced a parallel rightward shift in the concentration-response curve for angiotensin II with a pA2 value of 8.82. YM358 had no effect on the contraction induced by KCl, norepinephrine, serotonin, histamine, prostaglandin F2alpha or endothelin-1 even at 10(-5) M. On the basis of pKi values in the binding assay and pA2 values in the isolated tissues, YM358 was approximately 3-10 times more potent than losartan in antagonizing angiotensin AT1 receptors. In pithed rats, intravenous administration of YM358 inhibited an increase in mean blood pressure induced by intravenous infusion of angiotensin II in a dose-dependent manner. In conscious normotensive rats, YM358 at 3-30 mg/kg p.o. inhibited the angiotensin II-induced pressor response in a dose-dependent manner. YM358 at 30 mg/kg caused maximum and complete inhibition 30 min after dosing, and inhibition lasted more than 24 h. These results demonstrate that YM358 is a potent, AT1-selective and competitive nonpeptide angiotensin receptor antagonist. Moreover, YM358 is both orally active and long-lasting. This pharmacological profile suggests that YM358 would be suitable for the treatment of cardiovascular disorders such as hypertension and chronic heart failure.

Angiotensin II↗
PubMed1997

Antihypertensive activity of a nonpeptide angiotensin II receptor antagonist, YM358, in rats and dogs.

The antihypertensive activity of YM358, 2,7-diethyl-5-[[2'-(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][1,2,4]tri azole potassium salt monohydrate, a new nonpeptide angiotensin II receptor antagonist, was characterized in rats and dogs. In conscious rats, YM358 after a single oral administration (1-30 mg/kg) lowered blood pressure. The rank order of hypotensive potency of YM358 in conscious rats was 2-kidney, 1-clip renal hypertensive rats > spontaneously hypertensive rats > normotensive rats on the basis of maximum hypotension. YM358 also caused decreases in blood pressure in 2-kidney, 1-clip renal hypertensive dogs and furosemide-treated dogs. Repeated administration of YM358 to 2-kidney, 1-clip renal hypertensive rats for 28 days produced a stable and long-lasting antihypertensive effect without influencing circadian blood pressure and heart rate rhythms. No reflex tachycardia was observed in any animals of either species treated with YM358. Therefore, the pharmacological profile of this compound indicates that YM358 has potential as a useful antihypertensive agent.

Angiotensin II↗
PubMed1997

Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice.

The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCID-responsible gene. We isolated an ORF of the murine DNA-PKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration.

Amino Acid Sequence↗
PubMed1997

Differential cytotoxicity of clinically important camptothecin derivatives in P-glycoprotein-overexpressing cell lines.

UNLABELLED: Camptothecin and its derivatives are specific inhibitors of eukaryotic topoisomerase I (top1) and are active in cancer patients against a variety of refractory solid tumors and leukemia. PURPOSE: The present study further investigated the relationship between multidrug resistance (MDR) mediated by P-glycoproteinMDR and potential resistance to camptothecin derivatives using two experimental systems: (1) MDR KB-V1 cells selected for vinblastine resistance, and (2) NIH3T3 cells transfected with a plasmid expressing wildtype P-glycoproteinMDR multidrug transporter (NIH-MDR-G185). RESULTS: We found that both KBV-1 and NIH-MDR-G185 cells were resistant to topotecan, and that topotecan-induced cleavable complexes were reduced in KB-V1 cells, consistent with a role of P-glycoproteinMDR in cellular resistance to topotecan. By contrast, no significant resistance to camptothecin, 9-aminocamptothecin, 10, 11-methylenedioxycamptothecin, or SN-38 (the active metabolite of CPT-11) was observed in NIH-MDR-G185 cells, while KB-V1 cells were cross-resistant to these compounds but produced cleavable complexes similar to those produced by parental KB-3-1 cells. CONCLUSIONS: These results suggest that topotecan is the only camptothecin tested with significant susceptibility to MDR in cell culture, and that multidrug resistant cells such as KBV1 probably exhibit additional resistance mechanisms to camptothecins besides P-glycoproteinMDR overexpression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Local neurogenic regulation of rat hindlimb circulation: role of calcitonin gene-related peptide in vasodilatation after skeletal muscle contraction.

1. The mechanism of neurogenic regulation of skeletal muscle circulation was studied in the hindlimb of anaesthetized rats in vivo. Regional blood flow (RBF) of the hindlimb was recorded with a pulsed Doppler flow probe positioned in the iliac artery. 2. A short period (1 min) of sciatic nerve stimulation at 10 Hz caused a sustained increase in RBF (from 2.0 +/- 0.2 to 3.7 +/- 0.2 kHz at the peak), but no appreciable change in either MBP or HR, suggesting that the nerve stimulation produced local vasodilatation of the peripheral vasculature. The hyperaemic response reached a peak within 15 s and characteristically remained above the basal level for more than 5 min after the cessation of nerve stimulation. The response was regarded as a secondary response brought about by the contraction of skeletal muscles since (+)-tubocurarine (0.73 micromol kg(-1), i.a.) almost abolished it. 3. Lignocaine (43 micromol kg(-1), i.a.) and capsaicin (0.33 micromol kg(-1), i.a.) significantly suppressed the hyperaemic response to skeletal muscle contraction, suggesting that capsaicin-sensitive sensory nerves contribute to the hyperaemia. In contrast, an inhibitor of NO synthase, N(omega)-nitro-L-arginine methyl ester (1 micromol kg(-1) min(-1), i.v.), did not affect the hyperaemic response. 4. Serum levels of calcitonin gene-related peptide (CGRP) in iliac venous effluent significantly increased from 51 +/- 4 to 77 +/- 5 fmol ml(-1) during the hyperaemic response to skeletal muscle contraction. A bolus injection of CGRP (300 pmol kg(-1), i.a.) induced a long-lasting increase in RBF of the hindlimb. Moreover, CGRP(8-37) (100 nmol kg(-1) min(-1), i.v.), a specific CGRP1 receptor antagonist, significantly suppressed the hyperaemic response, especially the sustained phase of the response which was almost abolished by this antagonist. 5. These results suggest that CGRP, which is released from peripheral endings of capsaicin-sensitive sensory nerves, partly mediates the hyperaemia evoked by skeletal muscle contraction of the rat hindlimb.

Animals↗
PubMed1997

Local neurogenic regulation of rat hindlimb circulation: CO2-induced release of calcitonin gene-related peptide from sensory nerves.

1. The mechanism of release of calcitonin gene-related peptide (CGRP) from sensory nerves in response to skeletal muscle contraction was investigated in the rat hindlimb in vivo and in vitro. 2. In the anaesthetized rat, sciatic nerve stimulation at 10 Hz for 1 min caused a hyperaemic response in the hindlimb. During the response, partial pressure of CO2 in the venous blood effluent from the hindlimb significantly increased from 43 +/- 3 to 73 +/- 8 mmHg, whereas a small decrease in pH and no appreciable change in partial pressure of O2 were observed. 3. An intra-arterial bolus injection of NaHCO3 (titrated to pH 7.2 with HCl), which elevated PCO2 of the venous blood, caused a sustained increase in regional blood flow of the iliac artery. Capsaicin (0.33 micromol kg(-1), i.a.) and a specific calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8-37), (100 nmol kg(-1) min(-1), i.v.) significantly suppressed the hyperaemic response to NaHCO3. Neither ND(omega)-nitro-L-arginine methyl ester (1 micromol kg(-1) min(-1), i.v.) nor indomethacin (5 mg kg(-1), i.v.) affected the response. 4. The serum level of CGRP-like immunoreactivity in the venous blood was significantly increased by a bolus injection of NaHCO3 (pH = 7.2) from 50 +/- 4 to 196 +/- 16 fmol ml(-1). 5. In the isolated hindlimb perfused with Krebs-Ringer solution, a bolus injection of NaHCO3 (pH = 7.2) caused a decrease in perfusion pressure which was composed of two responses, i.e., an initial transient response and a slowly-developing long-lasting one. CGRP(8-37) significantly inhibited the latter response by 73%. 6. These results suggest that CO2 liberated from exercising skeletal muscle activates capsaicin-sensitive perivascular sensory nerves locally, which results in the release of CGRP from their peripheral endings, and then the released peptide causes local vasodilatation.

Animals↗