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Akira Inoue

Publications and source records attributed to Akira Inoue.

At least 37 records · Page 2Linked to original sources
PubMed2005

Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate.

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.

Acrylic Resins↗
PubMed2005

Organic solvent tolerance of halophilic archaea, Haloarcula strains: effects of NaCl concentration on the tolerance and polar lipid composition.

Strains of halophilic archaea, Haloarcula vallismortis and two Haloarcula strains OHF-1 and OHF-2, showed high tolerance to organic solvents at high media NaCl concentrations. For example, the lowest log Pow of the solvent which allowed growth (log Pow is the common logarithm of the partition coefficient of a given solvent in a mixture of n-octanol and water) for H. vallismortis was 5.1 at 20% NaCl and 4.4 at 30% NaCl. The solvent tolerance of Haloarcula argentinensis, on the other hand, was not affected by the NaCl concentration. Cells of strains OHF-1 and OHF-2 were of triangular or irregular morphology but became spherical in cultures in NaCl media overlaid with cyclohexane (log Pow=3.4), but returned to the triangular shape when the organic solvent evaporated from the medium. When cells of strains OHF-1, OHF-2, and H. argentinensis were grown in NaCl media in the presence of n-decane, they contained less phosphatidylglycerol and more phosphatidylglycerosulfate and phosphatidylglycerophosphate methyl ester than when grown without added n-decane. When the solvent was removed from the media after cultivation, the levels of these compounds returned to their initial ones.

Cell Proliferation↗
PubMed2004

Organic solvent tolerance of halophilic alpha-amylase from a Haloarchaeon, Haloarcula sp. strain S-1.

A halophilic archaeon, Haloarcula sp. strain S-1, produced extracellular organic solvent-tolerant alpha-amylase. Molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This amylase exhibited maximal activity at 50 degrees C in buffer containing 4.3 M NaCl, pH 7.0. Moreover, the enzyme was active and stable in various organic solvents (benzene, toluene, and chloroform, etc.). Activity was not detected at low ionic strengths, but it was detected in the presence of chloroform at low salt concentrations. On the other hand, no activity was detected in the presence of ethyl alcohol and acetone.

Acetone↗
PubMed2004

A one-headed class V myosin molecule develops multiple large (approximately 32-nm) steps successively.

Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large ( approximately 36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP- and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large (approximately 32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.

Actin Cytoskeleton↗
PubMed2004

Stability of A+U-rich element binding factor 1 (AUF1)-binding messenger ribonucleic acid correlates with the subcellular relocalization of AUF1 in the rat uterus upon estrogen treatment.

The nucleocytoplasmic shuttling protein, A+U-rich element binding factor 1 (AUF1), is one of the RNA-binding proteins that specifically bind adenylate-uridylate rich elements (AREs) in mRNA 3'-untranslated regions (UTRs), and acts as a regulator of ARE-mediated mRNA degradation in the cytoplasm. We previously reported that in the female rat uterus, the levels of specific AUF1 isoform mRNAs (p40/p45) were increased by 17 beta-estradiol (E2) treatment. Therefore, we examined the role of AUF1 in the regulation of E2-mediated mRNA turnover in the rat uterus. We identified ABIN2 and Ier2/pip92 mRNAs as candidate targets of AUF1 in the rat uterus. We found that AUF1-binding elements were present in the 3'-UTR of both mRNAs and that the 3'-UTRs functioned as mRNA turnover regulatory elements. In the ovariectomized rat uterus, the nucleocytoplasmic localization of AUF1p40/p37 isoform proteins was regulated by E2. We also found that cytoplasmic AUF1-bound mRNA levels changed coincidentally with the cytoplasmic levels of AUF1p40/p37. Finally, we confirmed that the subcellular localization of AUF1p40 controlled the stability of target mRNAs in vitro, such that cytoplasmically localized AUF1p40 led to marked mRNA stabilization, whereas nuclear-localized AUF1p40 stabilized target mRNA only slightly. These results suggested that E2-inducible ARE-containing gene transcripts are regulated, at least in part, via mRNA stabilization through the nucleocytoplasmic relocalization of AUF1.

3' Untranslated Regions↗
PubMed2004

Adenovirus vector-mediated in vivo gene transfer of OX40 ligand to tumor cells enhances antitumor immunity of tumor-bearing hosts.

OX40 ligand (OX40L), the ligand for OX40 on activated CD4+ T cells, has adjuvant properties for establishing effective T-cell immunity, a potent effector arm of the immune system against cancer. The hypothesis of this study is that in vivo genetic engineering of tumor cells to express OX40L will stimulate tumor-specific T cells by the OX40L-OX40 engagement, leading to an induction of systemic antitumor immunity. To investigate this hypothesis, s.c. established tumors of three different mouse cancer cells (B16 melanoma, H-2b; Lewis lung carcinoma, H-2b; and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of a recombinant adenovirus vector expressing mouse OX40L (AdOX40L). In all tumor models tested, treatment of tumor-bearing mice with AdOX40L induced a significant suppression of tumor growth along with survival advantages in the treated mice. The in vivo AdOX40L modification of tumors evoked tumor-specific cytotoxic T lymphocytes in the treated host correlated with in vivo priming of T helper 1 immune responses in a tumor-specific manner. Consistent with the finding, the antitumor effect provided by intratumoral injection of AdOX40L was completely abrogated in a CD4+ T cell-deficient or CD8+ T cell-deficient condition. In addition, ex vivo AdOX40L-transduced B16 cells also elicited B16-specific cytotoxic T lymphocyte responses, and significantly suppressed the B16 tumor growth in the immunization-challenge experiment. All of these results support the concept that genetic modification of tumor cells with a recombinant OX40L adenovirus vector may be of benefit in cancer immunotherapy protocols.

Adenoviridae↗
PubMed2004

Human deafness mutation of myosin VI (C442Y) accelerates the ADP dissociation rate.

The missense mutation of Cys(442) to Tyr of myosin VI causes progressive postlingual sensorineural deafness. Here we report the affects of the C442Y mutation on the kinetics of the actomyosin ATP hydrolysis mechanism and motor function of myosin VI. The largest changes in the kinetic mechanism of ATP hydrolysis produced by the C442Y mutation are about 10-fold increases in the rate of ADP dissociation from both myosin VI and actomyosin VI. The rates of ADP dissociation from acto-C442Y myosin VI-ADP and C442Y myosin VI-ADP are 20-40 times more rapid than the steady state rates and cannot be the rate-limiting steps of the hydrolysis mechanism in the presence or absence of actin. The 2-fold increase in the actin gliding velocity of C442Y compared with wild type (WT) may be explained at least in part by the more rapid rate of ADP dissociation. The C442Y myosin VI has a significant increase ( approximately 10-fold) in the steady state ATPase rate in the absence of actin relative to WT myosin VI. The steady state rate of actin-activated ATP hydrolysis is unchanged by the C442Y mutation at low (<10(-7) m) calcium but is calcium-sensitive with a 1.6-fold increase at high ( approximately 10(-4) m) calcium that does not occur with WT. The actin gliding velocity of the C442Y mutant decreases significantly at low surface density of myosin VI, suggesting that the mutation hampers the processive movement of myosin VI.

Actins↗
PubMed2004

Frequent activation of CArG binding factor-A expression and binding in N-methyl-N-nitrosourea-induced rat mammary carcinomas.

We previously identified a positive transcriptional element identical to human Ha-ras response element (HRE) within the promoter of the rat Ha-ras gene. We further identified CArG binding factor A (CBF-A), a member of heterogeneous nuclear ribonuclear protein (hnRNP) gene family, as a trans-acting factor that binds the HRE sequence with high affinity in rat mammary carcinoma cells. To determine if activation of CBF-A plays a role in tumor development in vivo , we investigated CBF-A expression and binding activity in rat mammary tumors induced by N-methyl-N-nitrosourea. We found that approximately 82% of tumors expressed CBF-A at levels that were 3-20 fold higher than detected in normal mammary gland. Moreover, elevated CBF-A protein levels were invariably associated with increased binding activity to the HRE. CBF-A mRNA levels in tumors were on average elevated only two fold as compared to normal mammary gland, indicating that increased CBF-A protein levels in tumors resulted from both translational and/or post-translational regulation. The level of CBF-A expression in mammary tumors was independent of Ha-ras mutational status. Together, these findings indicated that deregulation of CBF-A contributes to mammary carcinogenesis via a mechanism that is distinct from its hnRNP functions in binding and post-transcriptional regulation of RNA.

Alkylating Agents↗
PubMed2004

N-terminal modification and its effect on the biochemical characteristics of Akazara scallop tropomyosins expressed in Escherichia coli.

Akazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala- (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, nf-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar alpha-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca(2+)-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca(2+)-regulation together with troponin.

Actins↗
PubMed2004

Failure to proliferate and mitotic arrest of CDK11(p110/p58)-null mutant mice at the blastocyst stage of embryonic cell development.

The CDK11(p110) protein kinases are part of large-molecular-weight complexes that also contain RNA polymerase II, transcriptional elongation factors, and general pre-mRNA splicing factors. CDK11(p110) isoforms may therefore couple transcription and pre-mRNA splicing by their effect(s) on certain proteins required for these processes. The CDK11(p58) kinase isoform is generated from the CDK11(p110) mRNA through the use of an internal ribosome entry site in a mitosis-specific manner, suggesting that this kinase may regulate the cell cycle during mitosis. The in vivo role and necessity of CDK11(p110/p58) kinase function during mammalian development were examined by generating CDK11(p110/p58)-null mice through targeted disruption of the corresponding gene using homologous recombination. While heterozygous mice develop normally, disruption of both CDK11(p110/p58) alleles results in early embryonic lethality due to apoptosis of the blastocyst cells between 3.5 and 4 days postcoitus. Cells within these embryos exhibit both proliferative defect(s) and a mitotic arrest. These results are consistent with the proposed cellular functions of the CDK11(p110/p58) kinases and confirm that the CDK11(p110/p58) kinases are essential for cellular viability as well as normal early embryonic development.

Animals↗
PubMed2003

Randomized study of dexamethasone treatment for delayed emesis, anorexia and fatigue induced by irinotecan.

The prevention of post-chemotherapy symptoms such as delayed emesis, anorexia, and fatigue induced by irinotecan has not been studied. We compared the effects of dexamethasone (Dex) with those of a placebo on these symptoms in a randomized study. Seventy patients scheduled to receive irinotecan chemotherapy were enrolled in the study and randomly divided into a treatment or a placebo group. In the treatment group, 8 mg of Dex were administered on days 2-4 after the start of chemotherapy. All patients in both groups received Dex and granisetron for prophylaxis against acute emesis on day 1. We evaluated 68 patients (35 receiving Dex, 33 receiving the placebo). Although delayed emesis was completely prevented in most of patients in both groups (Dex, 82.9%; placebo, 78.8%), anorexia and fatigue were more completely prevented in those in the Dex group (Dex, 62.9% and 77.1%, placebo, 39.4% and 57.6%, respectively). The effect of Dex on improving simultaneous prophylaxis against all three symptoms was almost significant (Dex, 60.0%; placebo, 36.4%; P=0.058). The safety profiles of the two groups were not discernibly different. These results suggest that treatment with Dex may be beneficial to reduce post-chemotherapy symptoms induced by irinotecan, specifically anorexia and fatigue, with acceptable toxicities.

Adult↗
PubMed2003

Identification of S1 proteins B2, C1 and D1 as AUF1 isoforms and their major role as heterogeneous nuclear ribonucleoprotein proteins.

AUF1 (A+U-rich RNA binding factor) participates in the rapid decay of mRNAs in the cytoplasm. It is sometimes called heterogeneous nuclear ribonucleoprotein (hnRNP) D0; however, evidence for its characterization as an hnRNP protein has been scarce. S1 proteins A-D are those selectively extracted at pH 4.9 from isolated nuclei pretreated with either RNase A or DNase I. In the present study we identified S1 ('first supernatant') proteins B2, C1 and D1 with p45, p40 and p37 AUF1s respectively, by microsequencing and product analysis of transfected cDNAs. We found, further, that more than 96% of the S1 proteins occurred in the nucleus, and localized largely in RNase-sensitive structures. B2 was confined in the nucleus and C1 directly bound to heterogeneous nuclear RNAs (hnRNAs). These B2 and C1 proteins formed hnRNP structures responsible for the 33 S, and, to lesser extent, the 40 S particles, which were liberated upon mild nucleolytic cleavage. On the other hand, D1 and the remainder of C1 were associated with nuclease-hypersensitive sites of hnRNAs, and comprised the major cytoplasmic AUF1s that may be involved in mRNA decay. Two-dimensional immunoblotting resolved each S1 isoform into up to six spots or more, and suggested that the previous uncertain relationship of hnRNP D0 and hnRNP D is resolved in terms of charge differences and differential splicing arising from one gene. The present results thus indicate that S1 proteins B2, C1 and D1 are identical with AUF1 proteins, but largely occur as hnRNP proteins in the nucleus. That hnRNP D0 is indeed an hnRNP protein was verified.

Alternative Splicing↗
PubMed2003

Determination of human myosin III as a motor protein having a protein kinase activity.

The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actin-based motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with Kactin of 112 microm and Vmax of 0.34 s-1, indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 +/- 0.05 microm/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.

Actins↗
PubMed2003

Severe acute interstitial pneumonia and gefitinib.

Gefitinib is an oral selective inhibitor of the epidermal growth factor receptor tyrosine kinase that is an effective treatment for patients with advanced non-small cell lung cancer who do not respond to platinum-based chemotherapy. We assessed four patients who had non-small cell lung cancer causing severe acute interstitial pneumonia in association with gefitinib. Although two patients recovered after treatment with steroids, the other two died from progressive respiratory dysfunction. On the basis of autopsies and bilateral distribution of diffuse ground-glass opacities in chest CTs, we diagnosed diffuse alveolar damage, which was consistent with acute interstitial pneumonia. Patients with interstitial pneumonia also had other pulmonary disorders such as previous thoracic irradiation and poor performance status. Physicians should be aware of the alveolar damage induced by gefitinib, especially for patients with these characteristic features.

Adult↗
PubMed2003

Development of a polymer bileaflet valve to realize a low-cost pulsatile blood pump.

The final goal of this study is to realize a low-cost pulsatile blood pump especially for patients with acute heart failure or postoperative low cardiac output syndrome. In support of the pump, two types of polymer bileaflet valves with different configuration of the valve seats were developed. Influence of the leaflet thickness on the hydrodynamics of the prototype was preliminarily investigated among 70 microm, 100 microm, and 150 microm. As to the valves with the thinner leaflets, buckling of the leaflets was observed, which induced a large amount of regurgitation at valve closure. However, by thickening the leaflet to 150 microm, the mean flow of the prototype and the second model could be successfully comparable to the Medtronic-Hall valve. Moreover, accelerated fatigue tests showed that reinforcement of the valve seat with the additional spokes in the second model extended the durability by four times as compared with the prototype, equivalent to an in vivo duration of over one month.

Cardiac Output, Low↗
PubMed2003

Study of paclitaxel and dose escalation of cisplatin in patients with advanced non-small cell lung cancer.

BACKGROUND: We conducted a dose-finding and feasibility study in which we administered a fixed dose 3-h infusion of paclitaxel and an escalating dose of cisplatin in Japanese patients with advanced non-small cell lung cancer. METHODS: Chemotherapy consisted of fixed dose (210 mg/m(2)) paclitaxel given over 3 h on day 1 and an escalating dose of cisplatin on day 2, every 3-4 weeks. The dose of cisplatin was 40 mg/m(2) at level 1, 60 mg/m(2) at level 2 and 80 mg/m(2) at level 3. RESULTS: Between October 1999 and February 2001, 24 patients were enrolled and 58 cycles were administered. The major hematological toxicities were leukopenia and neutropenia. Grade 4 neutropenia developed in 83.3%, 66.7% and 83.3% of patients at the dose levels of 1, 2 and 3, respectively. The major non-hematological toxicities consisted of alanine aminotransferase (ALT) elevation and peripheral neuropathy. Grade 3 ALT elevation was observed in two of the 12 patients at level 3, but both recovered within 3 days. The peripheral neuropathy was sensory-dominant and frequent, and it was almost tolerable. The maximum tolerated dose was not identified even at the highest dose of paclitaxel (210 mg/m(2)) and cisplatin (80 mg/m(2)) administered in the study. The recommended dose was determined to be paclitaxel 210 mg/m(2) on day 1 and cisplatin 80 mg/m(2) on day 2, every 3-4 weeks. Seven partial responses were observed in the 24 patients. CONCLUSIONS: The combination of paclitaxel 210 mg/m(2) and cisplatin 80 mg/m(2) was found to be a well-tolerated active regimen in Japanese patients with advanced non-small cell lung cancer.

Adult↗