Biomedical subjects

Akihiko Uehara

Publications and source records attributed to Akihiko Uehara.

13 recordsLinked to original sources

PubMed2006

Chemical-reaction-based site-selective DNA cutter for PCR-free gene manipulation.

An artificial restriction DNA cutter (ARCUT), recently developed by the authors, was used to construct a fusion protein. The gene of WW-domain-containing oxidoreductase (WWOX) was cut by ARCUT just before its stop codon, and ligated to fuse the gene of enhanced green fluorescent protein (EGFP). The reading frames of two genes were adjusted to coincide each other. Throughout the manipulation, no PCR was employed. The fluorescent fusion protein was successfully expressed in mammalian cells, and showed entirely different subcellular localization from EGFP itself. Apparently, the DNA was kept completely intact during the manipulation. The man-made tool ARCUT has promising features for future biotechnology and molecular biology.

Animals↗

PubMed2006

Arrhythmogenic effects of arsenic trioxide in patients with acute promyelocytic leukemia and an electrophysiological study in isolated guinea pig papillary muscles.

BACKGROUND: Arsenic trioxide (As(2)O (3)) is a new promising regimen for patients with a relapse of acute promyelocytic leukemia (APL), but causes life-threatening arrhythmias. This study aimed to investigate the incidence and mechanism of arrythmogenesis caused by As(2)O(3). METHODS AND RESULTS: Standard 12-lead ECGs were monitored throughout As(2)O(3) therapy in 20 APL patients. As(2)O (3) (0.15 mg/kg) significantly prolonged the corrected QT interval (QTc: 445+/-7 to 517+/-17 ms, means+/-SE, p<0.01), and also increased the QTc dispersion and transmural dispersion of repolarization. Non-sustained ventricular tachycardias and torsades de pointes occurred in 4 and 1 patients, respectively. The action potentials and isometric contraction were measured in guinea pig papillary muscles during As(2)O (3) perfusion (350 micromol/L). The action potential duration was prolonged (APD(90): 150+/-11 to 195+/-12 ms at 60 min, p<0.01, n=5) and perfusion of As(2)O(3) in a low K(+) solution with a low stimulation rate augmented the prolongation of APD, and provoked early after-depolarizations and triggered activities. The prolonged exposure to As(2)O(3) induced muscle contracture, aftercontractions, triggered activities and electromechanical alternans. Tetrodotoxin or butylated hydroxytoluene partially prevented the As(2)O(3)-induced prolongation of APD. CONCLUSIONS: The prolonged QTc and spatial heterogeneity are responsible for the As(2)O(3)-induced ventricular tachyarrhythmias. In addition to prolongation of the APD, cellular Ca(2+) overload and lipid peroxidation might contribute to the electrophysiological abnormalities caused by As(2)O(3).

Action Potentials↗

PubMed2005

Genetic recombination without using either restriction enzyme or PCR.

We recently prepared artificial restriction DNA cutters (ARCUT) for site-selective scission of double-stranded DNA by combining Ce(IV)/EDTA complex with a pair of pseudo-complementary peptide nucleic acids (pcPNAs). Here we report an improved method for genetic recombination using ARCUT. The key point is to treat the scission fragments with nuclease S1 (a single-stranded DNA specific enzyme) and form blunt ends. By this procedure, these scission fragments are efficiently ligated with foreign DNA fragments having blunt ends, providing desired recombinant DNA in high yields. Neither restriction enzyme nor PCR amplification is required.

DNA Restriction Enzymes↗

PubMed2005

Construction of chimera protein by using artificial restriction DNA cutter.

We have already developed artificial restriction DNA cutter (ARCUT), which can hydrolyze double-stranded DNA site-selectively, by using Ce(IV)/EDTA in combination with two pseudo-complementary peptide nucleic acids (pcPNAs). Here, ARCUT was used to prepare a chimera protein. The gene for WW-domain containing oxidoreductase (WWOX) was clipped off by ARCUT just before its stop codon, and ligated with the gene for enhanced green fluorescent protein (EGFP). Conventional PCR for insertion of restriction enzyme site is never required.

Animals↗

PubMed2005

Gene manipulation using artificial restriction DNA cutter.

Artificial restriction DNA cutter (ARCUT), which we developed recently, was used for manipulation of plasmid DNA. PCR product was inserted into pBR322 plasmid vector using ARCUT, and E. coli cells were transfected with this recombinant plasmid DNA. Successful growth of cells shows that the recombination proceeds without any unexpected mutations. Furthermore, this artificial system was applied to PCR-free construction of chimera protein.

Cerium↗

PubMed2005

[Usefulness of lung and right ventricular thallium-201 uptake during single photon emission computed tomography in exercise testing of patients with coronary artery disease].

OBJECTIVES: Increased pulmonary or right ventricular 201Tl uptake during the exercise test has been used as a marker of multivessel coronary artery disease. The most useful method for assessing the severity of coronary artery disease was evaluated among conventional evaluation of single photon emission computed tomography (SPECT), measurement of lung to heart uptake ratio (L/H), and right ventricular to left ventricular uptake ratio (RV/LV) on 201Tl images during exercise testing. METHODS: Regions-of-interest (4 X 4 pixels) were placed at the lung and the heart, and L/H was defined as mean lung uptake/mean heart uptake. Correspondingly, regions-of-interest (4 X 4 pixels) were placed at the RV and the LV, and RV/LV was defined as maximum RV uptake /maximum LV uptake. L/H and RV/LV on the initial image were analyzed in 216 patients(angiographically normal coronary arteries: 89, single-vessel disease: 82, multivessel disease: 45). The diagnostic value was evaluated using the receiver operating characteristic curve. RESULTS: All methods showed significantly higher values in patients with multivessel disease than in patients with no coronary artery disease or single-vessel disease. L/H was significantly higher in patients with prior myocardial infarction and RV/LV was significantly higher in patients without infarction. The sensitivity of only conventional SPECT evaluation for multivessel coronary artery disease was low (sensitivity 53%, specificity 94%). However, addition of evaluation of L/H and RV/LV to SPECT improved the sensitivity for multivessel coronary artery disease (sensitivity 93%, specificity 49%). CONCLUSIONS: The diagnostic sensitivity for multivessel coronary artery disease was improved by adding L/H and RV/LV to conventional evaluation of exercise 201Tl SPECT. L/H and RV/LV during exercise 201Tl imaging may provide additional information regarding the severity of coronary artery disease.

Aged↗

PubMed2004

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA.

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.

Base Sequence↗

PubMed2004

Difference in the cardioprotective mechanisms between ischemic preconditioning and pharmacological preconditioning by diazoxide in rat hearts.

BACKGROUND: Recent studies have implicated the opening of mitochondrial K(ATP) (mitoK(ATP)) channels and the production of reactive oxygen species (ROS) in the cardioprotective mechanism of ischemic preconditioning (IPC). METHODS AND RESULTS: The involvement of mitoK(ATP) channels and ROS in the cardioprotective effects of both IPC and the mitoK(ATP) channel opener diazoxide (DZ) was investigated in ischemic/reperfused rat hearts. The effects of IPC and DZ on myocardial high-energy phosphate concentrations and intracellular pH (pH(i)) were also examined using (31)P nuclear magnetic resonance spectroscopy. Although both the mitoK(ATP) channel inhibitor 5-hydroxydecanoate and the antioxidant N-acetylcysteine abolished the postischemic recovery of contractile function by DZ, neither of them inhibited that by IPC. IPC attenuated the decline in pHi during ischemia, but DZ did not (6.28+/-0.04 in IPC, p<0.05, and 6.02+/-0.05 in DZ vs 6.02 +/-0.06 in control hearts). DZ, but not IPC, reduced the decrease in ATP levels during ischemia (ATP levels at 20-min ischemia: 26.3+/-3.4% of initial value in DZ, p<0.05, and 8.1+/-3.0% in IPC vs 15.1+/-1.3% in control hearts). CONCLUSIONS: These results suggest that DZ-induced cardioprotection is related to ROS production and reduced ATP degradation during ischemia, whereas attenuated acidification during ischemia is involved in IPC-induced cardioprotection, which is not mediated through mitoK(ATP) channel opening or ROS production.

ATP-Binding Cassette Transporters↗

PubMed2004

Usefulness of stress myocardial perfusion imaging for evaluating asymptomatic patients after coronary stent implantation.

BACKGROUND: Stent implantation in coronary angioplasty has reduced the rate of restenosis, but many patients still undergo follow-up coronary angiography (CAG). The present study was a multi-center retrospective analysis of the usefulness of stress single photon emission computed tomography (SPECT) compared with follow-up CAG in stent-implanted patients who remained asymptomatic during the follow-up period. METHODS AND RESULTS: The study group of 103 patients underwent both SPECT and CAG at 4-9 months after stent implantation. Restenosis occurred in 20 (19%) of 106 vessel territories, and a reversible perfusion defect was found in 32 (30%) territories. Sensitivity, specificity, positive and negative predictive values, and accuracy of SPECT were 65%, 78%, 41%, 91%, and 76%, respectively. The accuracy was lower in territories with a prior myocardial infarction (71%), in the left circumflex artery (58%), and in cases with three-vessel disease (63%). The negative predictive value was high, but 7 false negative cases included 4 cases with prior myocardial infarction, and 2 cases with reversible defects in other vessel territories. CONCLUSIONS: Stress SPECT imaging is a useful tool for following up patients with coronary stent implantation, and follow-up CAG could be omitted in patients with negative SPECT imaging, no prior myocardial infarction, one- or two-vessel disease, and sufficient stress loading.

Aged↗

PubMed2004

Improvement of cardiac sympathetic innervation by renal transplantation.

UNLABELLED: Chronic renal failure (CRF) patients on dialysis frequently show reduced heart rate variability (HRV), which has been reported to be corrected by renal transplantation. Recently, (123)I-metaiodobenzylguanidine ((123)I-MIBG) scintigraphy has been used to evaluate cardiac sympathetic innervation, and uremic patients often show marked abnormalities of cardiac (123)I-MIBG uptake. We investigated whether renal transplantation can improve cardiac (123)I-MIBG uptake in patients with CRF on dialysis. METHODS: We analyzed time- and frequency-domain measures of 24-h HRV and cardiac (123)I-MIBG scintigraphy before and 1-3 mo after renal transplantation in 13 CRF patients on dialysis and in 10 control subjects. RESULTS: Both 24-h HRV and cardiac (123)I-MIBG uptake were significantly abnormal in the patients before transplantation compared with the control subjects. After transplantation, (123)I-MIBG washout rate from the myocardium significantly decreased from 46% +/- 21% to 20% +/- 22% (P = 0.006), and the heart-to-mediastinum ratio of (123)I-MIBG uptake in the late image significantly increased from 1.74 +/- 0.39 to 2.06 +/- 0.39 (P = 0.006). On the other hand, HRV measures tended to increase after transplantation but the changes did not reach statistical significance (P > 0.05). CONCLUSION: Renal transplantation provides the improvement of uremic cardiac sympathetic neuropathy assessed by (123)I-MIBG imaging, which may be a more sensitive or at least an earlier marker than HRV.

3-Iodobenzylguanidine↗

PubMed2003

Effects of cytochrome P450 inhibitors on agonist-induced Ca2+ responses and production of NO and PGI2 in vascular endothelial cells.

Production of endothelium-dependent vascular relaxing factors, such as nitric oxide (NO) and prostaglandin I2 (PGI2), and endothelium-derived hyperpolarizing factor (EDHF), is regulated in part by changes in intracellular Ca2+ concentration ([Ca2+]i) in vascular endothelial cells (ECs). Cytochrome P450 (CYP), shown to mediate endothelium-dependent hyperpolarization via epoxyeicosatrienoic acids, is one of the candidates for EDHF. In this study we tested the hypotheses that CYP might be involved in EC Ca2+ signaling and that CYP activity might be linked with production of vasodilating factors other than EDHF. To this end, structurally different CYP inhibitors including SKF 525A, econazole and miconazole were tested on primary cultured porcine aortic endothelial cells. Intracellular Ca2+ concentration was measured using the fluorescent Ca2+ indicator fura-2/AM. Bradykinin (BK, 10 nM) and thapsigargin (TG 1 microM) provoked large biphasic increases in [Ca2+], which consist of Ca2+ release from intracellular stores and transplasmalemmal Ca2+ entry. SKF 525A dose-dependently (30-100 microM) inhibited BK- and TG-stimulated Ca2+ entry, but not intracellular Ca2+ store release. Econazole (10 microM) and miconazole (10 microM) had the same effect as SKF 525A on the Ca2+ entry. SKF 525A also dose-dependently inhibited BK-stimulated production of NO and PGI2, assessed by measuring cGMP and 6-keto-PGF(1alpha) concentration. These data suggest that, in addition to its regulation of EDHF production, CYP also contributes to the regulation of other endothelium-dependent vasorelaxing factors by modifying EC Ca2+ signaling.

6-Ketoprostaglandin F1 alpha↗

PubMed2003

Protective effects of hydrogen peroxide against ischemia/reperfusion injury in perfused rat hearts.

Among the several mechanisms proposed for ischemic preconditioning (IPC), generation of reactive oxygen species (ROS) is reported to be involved in the cardioprotective effects of IPC. The present study was designed to investigate whether repetitive exposure to hydrogen peroxide (H(2)O(2)) can protect the myocardium against subsequent ischemia/reperfusion injury, and whether the H(2)O(2)-induced cardioprotection is related to the preservation of energy metabolism. Langendorff-perfused rat hearts were exposed to two, 5 min episodes of IPC or to various concentrations of H(2)O(2) twice and then to 35 min global ischemia and 40 min reperfusion. Using (31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy, cardiac phosphocreatine (PCr) and ATP and intracellular pH (pH(i)) were monitored. IPC and the treatment with 2 micromol/L H(2)O(2) significantly improved the post-ischemic recovery of left ventricular developed pressure (LVDP) and the PCr and ATP compared with those of the control ischemia/reperfusion (LVDP: 36.9 +/-7.4% of baseline in control hearts, 84.0+/-3.5% in IPC, 65.4+/-3.8% in H(2)O(2); PCr: 51.1+/-5.3% in control hearts, 81.4+/-5.5% in IPC, 81.7+/-5.2% in H(2)O(2); ATP: 12.3+/-1.6% in control hearts; 30.0+/-2.8% in IPC, 28.6+/-2.3% in H(2)O(2), mean +/- SE, p<0.05). However, lower (0.5 micromol/L) or higher (10 micromol/L) concentration of H(2)O (2) had no effect. There were significant linear correlations between mean LVDP and high-energy metabolites after 40 min reperfusion in H(2)O(2)-treated hearts. In IPC-treated hearts, the mean LVDP was greater than that in the 2 micromol/L H(2)O(2)-treated hearts under similar levels of high-energy metabolites. IPC also ameliorated intracellular acidification (6.38+/-0.03 in control hearts, 6.65+/-0.04 in IPC, p<0.05), but treatment with H(2)O(2) did not affect pH(i) during ischemia (6.40+/-0.05 in H(2)O(2)). In conclusion, H(2)O(2) had protective effects against ischemia/reperfusion injury and the effects were related to the preservation of energy metabolism. IPC could have additional protective mechanisms that are associated with the amelioration of intracellular acidosis during ischemia.

Adenosine Triphosphate↗

PubMedDate not supplied

Coronary ectasia with slow flow related to apical hypertrophic cardiomyopathy--a case report.

Coronary ectasia (CE) has been reported to be associated with a high risk of coronary events and caused by several etiologies. The authors present a patient with CE who was noted to have an ECG abnormality in routine health check. Coronary angiography revealed diffuse ectasia in all 3 coronary arteries. The flow of contrast medium was slow and focal squeezing signs were observed in the left coronary artery. Echocardiography indicated mild apical hypertrophy and significant reduction of coronary flow reserve. Exercise thallium scintigraphy exhibited a transient perfusion defect in the inferior region. This is the first case report that a mild apical hypertrophy induced severe CE with slow coronary flow. The authors recommend a long-term prophylaxis of thrombotic complications with antiplatelet agents in patients with CE.

Blood Flow Velocity↗