Biomedical subjects

Hiroyuki Kamiya

Publications and source records attributed to Hiroyuki Kamiya.

At least 37 records · Page 2Linked to original sources

PubMed2006

[A case of pulmonary tuberculosis complicated with an orthotopic liver transplantation].

The infectious disease is one of the most important complications related to the organ transplantation. Patients using immunosuppressive agents often present atypical tuberculosis and the treatment of such case is far more difficult in some cases due to the liver damage and/or the drug interaction. We report a case of pulmonary tuberculosis in a patient of 60-year-old man using tacrolimus after an orthotopic liver transplantation. He had liver transplanted orthotopically for the long-term history of chronic hepatitis B and subsequent liver failure on January 28, 2004. An abnormal shadow was first detected on his chest X-ray film on October, 2004. He was admitted to our hospital after the smear of the gastric juice showed some acid-fast bacilli and tubercle bacilli were confirmed by polymerase chain reaction (PCR). Tuberculin skin test was positive (erythema 10 x 10) and the computed tomography (CT) scan of his chest revealed a nodular opacity with some smaller nodules scattered around in the right upper lobe. We started four anti-tuberculous drugs other than pyrazinamide (PZA) and rifampicin (RFP), which included isoniazid (INH), ethambutol (EB), streptomycin (SM), levofloxacin (LVFX). The liver enzyme was transiently elevated (AST 123 IU/I, ALT 103 IU/I) but improved after desensitization against INH. The blood concentration of tacrolimus preserved between 5 and 7 ng/ml and there was no need to change the dosage.

Humans↗

PubMed2006

[A case of eosinophilic bronchiolitis complicated with eosinophilic sinusitis].

A 42-year-old man was admitted to our hospital, complaining of dyspnea and cough for six months. Chest CT demonstrated thickening of the bronchial walls and some centrilobular nodules. From the laboratory data and the clinical course, he was first suspected to have bronchial asthma. However, it was necessary to rule out bronchiolitis due to other causes, because he had no previous asthma history and also because of the CT findings. We performed surgical lung biopsy to make a definite diagnosis. The pathological findings revealed eosinophilic bronchiolitis. While dyspnea and the eosinophilic nasal polyp improved by treatment with oral steroid therapy, the eosionophilic sinusitis and bronchiolitis relapsed after steroid tapering. Improvement and relapse of nasal and lower airway symptoms were synchronously observed. Eosinophilic bronchiolitis and eosionophilic sinusitis in this case may be considered to be a category of airway eosinophilic inflammation.

Adult↗

PubMed2005

Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment.

A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment (approximately 600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

Adenine↗

PubMed2005

Mitochondrial delivery of mastoparan with transferrin liposomes equipped with a pH-sensitive fusogenic peptide for selective cancer therapy.

Mastoparan (MP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated MP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells. The MP showed potent PT activity at concentrations above 25 microM in a homogenate of K 562 cells as well as in isolated mitochondria in the presence of phosphate. Tf-L equipped with cholesteryl GALA can release encapsulated sulforhodamine B, while Tf-L failed, as evidenced by confocal laser scanning microscopy. The MP, which was delivered with Tf-L with GALA, released cytochrome c (cyt c) from mitochondria to the cytosol, while free MP released cyt c not only to the cytosol but also extracellulary. These results demonstrate the utility of MP in Tf-L with GALA for cancer therapy.

Antineoplastic Agents↗

PubMed2005

Effects of display position of a visual in-vehicle task on simulated driving.

To determine the relative safety of onboard display positions while driving, participants performed a lane-keeping task in a driving simulator. Concurrently, they reacted to a light by pushing the brake pedal. A secondary task was projected onto a display at one of the seven different locations in the cockpit. Behavioral data, eye movements, and subjective rating scales showed that the manipulation of display information during driving disturbed drivers' performance exponentially as a function of distance between the line of sight to the outside primary task and the onboard display position. Vertical eccentricity had a greater detrimental effect than horizontal distance. Under a certain condition with a high secondary task load, reaction time of pushing the brake to the outside stimulus nearly doubled with a diagonal eccentricity of 35 degrees as compared to lower eccentricities. Subjective workload measures complement the behavioral data of clear detrimental effects with eccentricities of at least 35 degrees .

Automobile Driving↗

PubMed2005

Kinetic analysis of protein production after DNA transfection.

The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8x10(4)- and 4x10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k(el) for DNA transfection<k(el) for the luciferase protein), suggesting that a decrease in intranuclear active DNA was the main determinant of the elimination rate in this case. A preliminary pharmacokinetic model is proposed, based on the results obtained.

Animals↗

PubMed2005

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells.

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.

Animals↗

PubMed2005

New NTP analogs: the synthesis of 4'-thioUTP and 4'-thioCTP and their utility for SELEX.

The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631-635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).

Base Sequence↗

PubMed2005

Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein.

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H(2)O(2). These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.

Amino Acid Substitution↗

PubMed2005

Cytokine induction by a bacterial DNA-specific modified base.

Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N(6)-methyladenine (N(6)-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N(6)-MeA induced cytokines when injected into mice. Co-injection of N(6)-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N(6)-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N(6)-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine.

Adenine↗

PubMed2005

Dual hydrolysis of diphosphate and triphosphate derivatives of oxidized deoxyadenosine by Orf17 (NtpA), a MutT-type enzyme.

To determine whether the Orf17 (NtpA) protein of Escherichia coli, a MutT-type enzyme, functions as a hydrolyzing enzyme for a damaged deoxyribonucleotide, we purified the recombinant Orf17 protein and incubated it with oxidized deoxyribonucleotides. Of the deoxyribonucleoside 5'-triphosphates tested, 8-hydroxy-2'-deoxyadenosine 5'-triphosphate was hydrolyzed by this protein. Unexpectedly, the Orf17 protein degraded 8-hydroxy-2'-deoxyadenosine 5'-diphosphate 2.3-fold more efficiently than the corresponding triphosphate. Thus, this protein is the first MutT-type enzyme that hydrolyzes both the triphosphate and diphosphate derivatives of a deoxyribonucleoside, with similar efficiencies. These results suggest that the Orf17 protein may be involved in the hydrolysis of oxidized dATP and dADP.

Cloning, Molecular↗

PubMed2005

Increased SFHR gene correction efficiency with sense single-stranded DNA.

BACKGROUND: The correction of a mutated gene by the small fragment homologous replacement (SFHR) method is a highly attractive approach for gene therapy. However, the current SFHR method with a heat-denatured double-stranded PCR fragment yielded a low correction efficiency. METHODS: Single-stranded (ss) DNA fragments were prepared from ss phagemid DNA and tested in a gene correction assay with an inactivated Hyg-EGFP fusion gene, as a model target. RESULTS: A 606-nt sense, ss DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. CONCLUSIONS: These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes.

Animals↗

PubMed2005

Off-pump CABG with synchronized arterial flow ensuring system.

PURPOSE: We developed a synchronized, arterial-flow, ensuring system to perform coronary anastomoses safely without any ischemia-related event. DESCRIPTION: Arterial blood is removed from the femoral artery. The resulting blood passes a switching valve and is pumped out to a syringe pump. This pump controller provides pulsatile arterial blood flow synchronized with the diastolic phase on an electrocardiogram. The arterial blood is perfused to the coronary artery through a fine flexible cannula during anastomosis. EVALUATION: From February 1999, 524 consecutive patients were operated on using the synchronized arterial flow ensuring system. Mean duration for each anastomosis was 7.6 +/- 3.3 minutes (range, 4 to 20 min). There were no intraoperative fatal arrhythmias, ventricular arrhythmias, or short-run or hemodynamic deterioration during anastomoses. No hospital death was observed, and postoperative myocardial infarction occurred in 2 patients (0.4%). Postoperative angiography showed a 98.1% patency rate. CONCLUSIONS: The early clinical and angiographical results for off-pump CABG with the synchronized arterial flow ensuring system were excellent without mortality. We believe that off-pump CABG can be more safely performed using the synchronized arterial flow ensuring system based on our favorable results.

Aged↗

PubMed2005

Mutagenesis induced by oxidized DNA precursors: roles of Y family DNA polymerases in Escherichia coli.

To reveal the roles of Y family DNA polymerases in the mutagenesis induced by oxidatively damaged DNA precursors, 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP (8-OH-dGTP) were introduced into Escherichia coli strains deficient in the Y family polymerases, DNA polymerase IV (pol IV, encoded by the dinB gene) and DNA polymerase V (pol V, encoded by the umuDC locus). The mutation induced by 2-OH-dATP, but not that induced by 8-OH-dGTP, occurred less frequently in the dinB- strain than in the wild-type (wt) strain, suggesting the involvement of pol IV in the mutagenesis by 2-OH-dATP. Expression of pol IV from plasmid enhanced the mutagenesis by 2-OH-dATP in the dinB- strain. This enhancement depends on the polymerase activity since the expression of a mutant pol IV lacking the polymerase activity did not increase the mutations induced by 2-OH-dATP. In contrast, both 2-OH-dATP and 8-OH-dGTP caused mutations more efficiently in the umuDC- strain than in the wt strain, suggesting that the umuDC gene products suppressed the mutagenesis by these oxidized DNA precursors. The DNA polymerase activity was not required for the suppressive effects because expression of the umuDC gene products lacking the polymerase activity also suppressed the mutagenesis. These results suggest that the E. coli pol IV was involved in mutagenesis by 2-OH-dATP and that the umuDC gene products play suppressive role(s) in the mutagenesis by damaged nucleotides.

Adenosine Triphosphate↗

PubMed2005

Conversion of nucleotide sequence with single-stranded DNA fragment prepared from phagemid DNA.

The correction of a mutated gene is a highly attractive approach for gene therapy. This in vivo mutagenesis method will also be an effective tool in biotechnology. However, the current small fragment homologous replacement (SFHR) method with a heat-denatured double-stranded PCR fragment yielded the low correction efficiency. Single-stranded DNA fragments were prepared from single-stranded phagemid DNAs and tested in a gene correction assay with a Hyg-EGFP fusion gene inactivated by a substitution mutation, as a model target. A 606-nt sense, single-stranded DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. On the other hand, correction of frameshift mutations with the sense single-stranded DNA fragment were 2-3-fold as efficient as that with the PCR fragment. These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes.

Bacteriophages↗

PubMed2005

Correction of frameshift mutations with single-stranded and double-stranded DNA fragments prepared from phagemid/plasmid DNAs.

We recently found that a heat-denatured, double-stranded DNA fragment, prepared from plasmid DNA (dsHES), and a sense single-stranded DNA fragment, prepared from single-stranded phagemid DNA (fSense), corrected an inactivated hygromycin-resistance and enhanced green fluorescence protein fusion (Hyg-EGFP) gene containing a base substitution (G:C to C:G) mutation 2-fold and more than 10-fold, respectively, more efficiently than the conventional PCR fragment (pcrHES), in the small fragment homologous replacement method. In this study, we tested the abilities of these new DNA fragments to correct Hyg-EGFP genes inactivated by one base insertion (+G) and deletion (-C) mutations. In contrast to its activity with the substitution mutation, the fSense fragment showed similar efficiencies to those of the dsHES fragment in the correction of frameshift mutations. For the correction of the insertion mutation, the efficiencies were in the order of dsHES (0.21%)>or=fSense (0.18%)>pcrHES (0.08%). In the case of the correction of the deletion mutation, the efficiencies were in the order of fSense (0.27%)>or=dsHES (0.19%)>pcrHES (0.12%). These results suggest that sense single- and double-stranded DNA fragments prepared from phagemid and plasmid DNAs, respectively, have the potential to correct frameshift mutations.

Animals↗

PubMed2005

Effect of methylated adenine in plasmid DNA on transgene expression in mice.

Cationic lipid-mediated transfer of DNA is promising in gene therapy. However, one disadvantage with this approach is the induction of an inflammatory response, which may decrease transgene expression. Recently, we found that plasmid DNA containing N6-methyladenine (N6-MeA), a bacterium-specific modified base, induced cytokine twice as efficiently as plasmid DNA without N6-MeA, when complexed with cationic lipids. Thus, plasmid DNA without N6-MeA might express a transgene more efficiently than that containing N6-MeA in vivo. To evaluate the effects of adenine methylation on transgene expression in vivo, we injected luciferase-encoding plasmid DNA, complexed with cationic lipids or a cationic polymer, intravenously into mice. When the plasmid DNA-cationic lipid complexes were injected, the luciferase expression from the methylated and unmethylated plasmids was similar, although cytokine was more efficiently elicited by the methylated DNA than the unmethylated DNA. Hydrodynamics-based injections of plasmid DNA-cationic polymer complexes did not induce cytokine, and the luciferase expression from the unmethylated plasmid was slightly lower than that from the methylated plasmid DNA. These results suggest that the presence of N6-MeA did not reduce transgene expression in vivo.

Adenine↗

PubMed2005

In vivo mutagenicities of damaged nucleotides produced by nitric oxide and ionizing radiation.

To evaluate the in vivo mutagenicities of damaged DNA precursors (deoxyribonucleoside 5'-triphosphates) produced by exposure to nitric oxide (NO) and ionizing radiation, five damaged deoxyribonucleotides (deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, dUTP, and 8-hydroxy-dATP) were introduced into competent Escherichia coli cells. Their mutagenic potentials were assayed using the chromosomal rpoB gene as a mutagenesis target. In contrast to 8-hydroxy-dGTP and 2-hydroxy-dATP, which were examined in an earlier study, none of these damaged deoxyribonucleotides significantly increased the rpoB mutant frequency. These results suggest that these five damaged deoxyribonucleotides are weakly mutagenic in vivo if at all. Thus their contributions to mutations induced by NO and ionizing radiation may be small.

Deoxyribonucleotides↗