Search

Background Here we report our surgical experiences with aberrant right subclavian artery (ARSA) aneurysm in 8 patients. Methods Eight patients underwent surgical treatment for ARSA aneurysm between March 1994 and June 2005. The age of these patients ranged from 20 to 75 years. The mean size of the ARSA aneurysm was 3.3 cm, ranging from 2 to 5 cm. The ARSA aneurysm was completely resected through a left posterolateral thoracotomy after reconstruction of the right subclavian artery through the supraclavicular approach in 4 patients (group 1). The ARSA aneurysm was excluded through a left posterolateral thoracotomy without revascularization of the right subclavian artery in 2 patients (group 2). The distal site of the ARSA aneurysm was closed followed by revascularization through a median sternotomy, and the ARSA aneurysm was left as a blind sack in 2 patients (group 3). Results None of the patients in group 1 or 3 had any postoperative complications. In group 2, 1 had a steal syndrome caused by the exclusion of the ARSA aneurysm, and the other died of sepsis 2 months after the operation. Conclusions Complete anatomical repair of the ARSA aneurysm could be performed through the combination of the supraclavicular approach and the left posterolateral thoracotomy, with excellent results. Exclusion of the ARSA aneurysm without revascularization resulted in a suboptimal outcome. Surgical results of simple closure of the ARSA followed by revascularization were uneventful, but the ARSA aneurysm was left as a blind sack.

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.

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.

The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its ‘silencing’, occurred in parallel in the nucleus.

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.

Background. This study quantified the number of intraoperative microemboli in patients undergoing aortic arch surgery using selective cerebral perfusion (SCP) in comparison with those in patients undergoing ascending aortic replacement without circulatory arrest and SCP. Methods. A transcranial Doppler monitoring of the medial cerebral artery was performed in 15 patients undergoing proximal arch replacement with SCP (SCP group) and 15 patients undergoing replacement of the ascending aorta (control group). Results. There was no significant difference in the high-intensity transient signal counts between the SCP group and the control group at any phase. In the SCP group, 4.8% of microemboli occurred during cross-clamping, and only 0.6% occurred during SCP. In the control group, 4.6% occurred during cross-clamping. Most microemboli occurred after removing the cross-clamps in both groups; 92.2% in the SCP group and 92.1% in the control group. Conclusions. The present study demonstrated that outbreak frequency of microemboli during SCP was very low, and thus implies that the risk of embolic event that may be caused by SCP is very low.

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.

A 61 year-old man was admitted to our hospital complaining of exertional dyspnea and presented with left pleural effusion. Laboratory findings showed peripheral eosinophilia and a slightly elevated inflammatory reaction. Chest X-ray film and CT revealed left pleural effusion, but there were no particular abnormal findings on the lung fields. Pleural effusion was exudative, yellowish and contained numerous eosinophils. His detailed medical history showed that he had eaten uncooked snakes, which led us to suspect parasite diseases. Multiple-dot ELISA method was performed to detect specific anti-parasite antibody. The patient was diagnosed with sparganosis after the detection of a highly positive reaction against Spirometra erinacei-europaei in both serum and pleural effusion. Parasite disease is an important consideration on differential diagnosis of eosinophilic pleural effusion, and multiple-dot ELISA method might be helpful for screening it.

The Escherichia coli MutT protein hydrolyzes 8-hydroxy-dGTP (8-OH-dGTP) in vitro, and mutT gene deficiencies cause increased spontaneous A:T-->C:G mutations. However, no direct evidence exists for enhanced mutagenicity of 8-OH-dGTP in mutT cells. In this study, 8-OH-dGTP was introduced into wild type and mutT E. coli cells, and mutations of a chromosomal gene were monitored. 8-OH-dGTP induced mutations of the rpoB gene, the degree of the mutation induction in the mutT strain being approximately 6-fold higher than that in the wild type strain. On the other hand, 2-hydroxy-dATP, which is not a substrate of the MutT protein, increased the mutation to similar degrees in the two strains. These results constitute the first evidence that the MutT protein suppresses mutation by 8-OH-dGTP in vivo.

Summary: TAS-102, a new oral drug, is composed of an antitumor drug, α , α , α -trifluorothymidine (FTD), and its metabolic inhibitor, 5-chloro-6-(2-iminopyrrolidine-1-yl)methyl-2,4(1 H ,3 H )-pyrimidinedione hydrochloride (TPI). It has been reported that the oral administration of TAS-102 increases the AUC of FTD in rodents and monkeys in different manners. In this study, a pharmacokinetic model was developed, in an attempt to evaluate the bioavailability of FTD in these animals after the co-administration of TPI. Since TPI inhibits FTD metabolism competitively, a time-dependent as well as concentration-dependent model for the hepatic intrinsic clearance of FTD was developed including the time courses of both FTD and TPI. Based on this modeling, we were able to quantitatively explain the TPI dose-dependent enhancement of AUC of FTD in monkeys, while little increase was observed in rats. These results are consistent to observations that thymidine phosphorylase (TPase) is predominantly expressed in monkeys; while uridine phosphorylase (UPase) is superior to TPase in rats. Since TPase is also predominantly expressed in humans, the pharmacokinetic model developed in this study can be used to explain the bioavailability of TAS-102 in humans.

The effects of simulated microgravity on mammalian reproduction were studied using tail-suspended mice, during parabolic flight in a jet plane and in a horizontal clinostat device. In the tail-suspended mouse, which is a model of the shift of body fluid in a microgravity environment, atrophy of the testis and decrease of serum testosterone level were observed. The sperm motility decreased in the muG condition produced by the parabolic flight. There were no statistically significant differences in the efficiency of achieving normal fertilization in vitro, but there was a statistically significant decrease in the number of embryos reaching the morula and blastocyst stages after 96 hours in culture under clinostat rotation. These results suggest that the process of fertilization in vitro is not sensitive to the gravitational vector. However, the possibility exists that microgravity increases the disturbance of spermatogenesis and sperm motility or the frequency of early embryonic lethality. On the possibility of reproduction in space, there are some problems such as the cosmic radiation besides gravity. It is necessary to carry out further experiments in outer space.

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes 2-hydroxy-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. In this study, the Gly-36, Gly-37, Lys-38, Glu-43, Arg-51, Glu-52, Leu-53, Glu-55, and Glu-56 residues of Orf135, which are conserved in the three MutT-type proteins (Orf135, MutT, and MTH1), were substituted, and the enzymatic activity of these mutant proteins was examined. The mutant proteins with a substitution at the 36th, 37th, 52nd, and 56th amino acid residues completely lost their activity. On the other hand, the mutant proteins with a substitution at the 38th, 43rd, 51st, 53rd, and 55th residues could hydrolyze 5-methyl-dCTP. Some mutants with detectable activity for 5-methyl-dCTP did not hydrolyze dCTP. Activities for known substrates (5-methyl-dCTP, dCTP, 2-hydroxy-dATP, and 8-hydroxy-dGTP) were examined in detail with the four mutants, K38R, E43A, L53A, and E55Q. These results indicate the essential residues for the activity of the Orf135 protein.

A 60-year-old man was admitted to our hospital complaining of general malaise for a few months, discomfort of the right shoulder and fever in May 2003. Chest X-ray film showed an infiltrative shadow in the right lung field and chest CT scan revealed right pleural effusion. Pulmonary tuberculosis complicated with pleurisy was first suspected from the findings of high ADA level of the effusion and positive result of PPD skin test. But, microscopic examination of the specimens obtained by transbronchial lung biopsy disclosed granulomatous lesions and Mycobacterium kansasii was identified from all specimens; sputum, fluids of brushing and bronchial washing. The patient was diagnosed as pulmonary Mycobacterium kansasii infection and treated with anti-tuberculous drugs including RFP. His clinical course was good and no recurrence of pleural effusion was seen. This case was a rare case of pulmonary Mycobacterium kansasii infection complicated with pleural effusion.

Liposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracellular trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Chol-GALA), rhodamines were efficiently released and diffused into the cytosol. The addition of GALA to the Tf-L-containing medium or the encapsulation of GALA in Tf-L did not induce similar effects. These results clearly indicate that GALA must be present on the surface of liposomes to exert its function. In vitro energy transfer and dynamic light scattering experiments suggested that the endosomal escape of the encapsulates in Tf-L equipped with Chol-GALA can be attributed to pH-dependent membrane fusion. With GALA present on the surface, intracellular trafficking of liposomes after receptor-mediated endocytosis could be successfully controlled.

A 2-substituted purine nucleotide analog, 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), was added to a PCR mixture, to examine its mutagenic potential. The 2-OH-dATP enhanced the total mutation frequency. Interestingly, 2-OH-dATP induced both transition and transversion mutations, including A:T--G:C, A:T--C:G and G:C--T:A mutations. In contrast, other 2-substituted purine nucleotide analogs, 2-aminopurine-2'-deoxyriboside 5'-triphosphate and 2-amino-2'-deoxyadenosine 5'-triphosphate, did not affect the total mutation frequency. These results suggest that 2-OH-dATP is useful in random PCR mutagenesis for the in vitro evolution of nucleic acids and proteins, and for analyses of residues in these biomolecules.

Oligodeoxyribonucleotides containing 8-hydroxyguanine and 2-hydroxyadenine, purine lesions produced in cells by reactive oxygen species, were synthesized and inserted into vector DNAs to introduce each lesion at a predetermined site. The manipulated DNAs were transfected into living cells, and the mutants induced by each DNA lesion were collected and analyzed. In addition, the mutations induced by damaged DNA precursors with the two oxidized purine bases were studied by the use of chemically synthesized nucleoside triphosphates. In this review article, the author summarizes the mutagenic potentials of the two oxidized purine bases, by focusing on experiments examined by the author and his collaborators.

International audience; To examine the substrate recognition mechanism of the human MTH1 protein, which hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxy-dGTP, ten nucleotide analogs (8-bromo-dATP, 8-bromo-dGTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the MTH1 protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. The fact that the syn-oriented brominated nucleotides were hydrolyzed suggests that the MTH1 protein binds to deoxynucleotides adopting the syn-conformation. However, 8-hydroxy-dITP, which lacks the 2-amino group of 8-hydroxy-dGTP, was degraded with tenfold less efficiency as compared with 8-hydroxy-dGTP. In addition, deoxyisoinosine triphosphate, lacking the 6-amino group of 2-hydroxy-dATP, was hydrolyzed as efficiently as 8-hydroxy-dGTP, but less efficiently than 2-hydroxy-dATP. These results clarify the effects of the anti/syn conformation and the functional groups on the 2 and 6 positions of the purine ring on the recognition by the human MTH1 protein.

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

In this paper, we describe the synthesis of the 3′-3′-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiment. It was found that the 3′-3′-linked oligonucleotides composed of asymmetrical sequences formed the stable antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine–oligopurine sequences.

We recently found that the Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolysed 2-hydroxy-dATP (2-OH-dATP), and less efficiently, 8-hydroxy-dGTP.In this study, we examined the effects of the absence of the orf135 gene. Frequencies of spontaneous and H2O2-induced mutations were two- to three-fold higher in the orf135- strain than in the wild-type strain. These mutations include various mutations involving a G:C--T:A transversion, the same type of mutation elicited by 2-OH-dATP. Over-expression of the Orf135 protein suppressed mutations even in the wild-type strain, as well as in the orf135- strain.The mutator phenotype of bacteria lacking the Orf135 protein suggests that this protein is involved in the suppression of mutations induced by oxidized deoxynucleotides in vivo and that various MutT-type enzymes contribute to nucleotide pool sanitization.

Oxygen free radicals cause extensive chemical changes in DNA, including base and sugar modifications and strand breaks. In the present study we found that a mutagen, glyoxal, is produced by exposure of DNA to an oxygen radical forming system (5 mM FeSO4-EDTA, 37 degrees C, 60 min). It was produced with 17 times higher efficiency than 8-hydroxydeoxyguanosine. Thus it is possible that the formation of glyoxal is one of the major types of damage in DNA exposed to oxygen free radicals.

Background We developed a new instant regional myocardial blood flow (RMBF) monitor utilizing the thermal diffusion method in which the RMBF value is presented as the value inversely proportional to the thermocouple voltage output (1/V). The purposes of this study were (1) to validate the accuracy of RMBF measurement by the instant RMBF monitor in comparison with the colored microsphere method for calibration; (2) to investigate influences of it on the RMBF; and (3) to assess changes in RMBF caused by the shunt tube insertion. Methods Twenty pigs were used for this study: 4 for comparison between the instant RMBF meter and the colored microsphere method, 4 for validation of reproducibility, and 6 for measurement of RMBF during shunt tube. Results The relation between RMBF values obtained by the colored microsphere method and 1/V values by instant RMBF monitor was colored microsphere=140,992 (1/V) − 231 in epicardial layer (R 2 = 0.819) and colored microsphere=111,381 (1/V) − 165 in endocardial layer (R 2 = 0.693). The correlation coefficient and R 2 values between RMBF values measured by both methods were 0.985 and 0.839 in epicardial layer, and 0.963 and 0.679 in endocardial layer, respectively. The RMBF at each layer did not change after the attachment of the monitor. Fifteen minutes after shunt tube insertion, RMBF measured by the colored microsphere method decreased to 31.1% ( p = 0.0001) and 33.7% ( p = 0.0001) in epicardium and endocardium, respectively, and no difference was observed from the value measured by the instant RMBF monitor. Conclusions This instant RMBF monitor can provide instantaneous and continuous information of RMBF without requiring tissue examination.

Efficient nuclear entry of exogenous DNA is one of the key factors toward gene therapy success with nonviral vectors. To re-address the effects of a nuclear localization signal (NLS) peptide attached directly to DNA, we prepared three dumbbell-shaped, green fluorescent protein (GFP)-encoding DNAs containing one or two NLS peptides. The peptide was conjugated to the loop-forming oligodeoxyribonucleotides by cross-linking reactions between the peptide and a modified uracil base with a dioxaoctylamino linker, and the oligonucleotides were then ligated to the DNA molecules. The NLS-conjugated DNA dumbbells were microinjected into the cytosols and nuclei of simian COS-7 cells. In addition, unconjugated DNA dumbbells, with or without a modified uracil base, were also examined for comparison. The GFP gene was expressed with efficiencies in the order of the unmodified DNAor= the NLS-conjugated DNAthe unconjugated DNA with the base modification, with both cytosolic and intranuclear microinjections. Thus, we concluded that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus.

Background The suitability of the radial artery after transradial catheterization as a bypass conduit has been of great concern to surgeons. Methods A total of 67 patients underwent isolated coronary artery bypass grafting using the radial artery: 22 patients received preoperative transradial catheterization (group 1) and 45 patients did not receive transradial catheterization (group 2). Those patients were retrospectively reviewed. Results Patient characteristics, operative procedures, and early clinical outcome were not different between groups. The stenosis-free graft patency rates in groups 1 and 2 were 88% (16 of 18 patients) and 90% (38 of 42 patients) in the left internal thoracic artery ( p = 0.87); 77% (17 of 22 patients) and 98% (48 of 49 patients) in the radial artery ( p = 0.017); and 87% (13 of 15 patients) and 84% (21 of 25 patients) in the saphenous vein ( p = 0.42), respectively. Intimal hyperplasia of the radial artery was observed in 68% (11 of 16 patients) in group 1 and in 39% (14 of 34 patients) in group 2 ( p = 0.046). Conclusions Transradial catheterization reduced early graft patency and caused intimal hyperplasia, although it did not affect early clinical outcomes. We suggest that the use of the radial artery as a bypass conduit after transradial catheterization should be undertaken cautiously.

Background Recently skeletonization has been recognized as an alternative to pedicle harvesting of the internal thoracic artery as a technique that increases the length and caliber size of the graft compared with pedicled internal thoracic artery grafts; however, this is not yet popular for harvesting the gastroepiploic artery (GEA). We report here our experience of skeletonized GEA grafting in off-pump coronary artery bypass grafting with early clinical and angiographic results. The purpose of this study was to evaluate skeletonization of GEA grafting in off-pump coronary artery bypass grafting with a large patient volume. Methods One hundred sixty-eight patients including 131 men and 37 women (mean age, 65 years; range, 45 to 87 years) underwent the skeletonized GEA grafting in off-pump coronary artery bypass grafting. These patients represent 41% (168 of 407 patients) of those who underwent off-pump coronary artery bypass grafting operations during the same period. We used the GEA graft of choice in patients with right coronary artery lesion. Skeletonization was performed in a unique manner we developed. Results There were no in-hospital deaths among the study patients. One patient had a perioperative myocardial infarction, which was considered a result of vasospasm of the GEA graft. None of the other patients had severe morbidity. The patency rate of the skeletonized GEA graft was 98.1% (151 of 154 distal anastomoses). Conclusions This study suggests that skeletonization of the GEA graft can enlarge its caliber size and improve its flow capacity. In addition, the acceptable early clinical and angiographic outcome suggests that use of the skeletonized GEA graft in off-pump coronary artery bypass grafting surgery is safe and effective.

The mutagenicity of oxidized dATP, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP mainly elicited a G x C to A x T transition, and a G x C to T x A transversion to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.

To elucidate the behavior of DNA polymerase eta against the oxidized purine nucleotides, we determined the utilization efficiency of 2-hydroxy-dATP and 8-hydroxy-dGTP by the recombinant yeast DNA polymerase eta using the primer extension assay with the synthetic oligonucleotide template-primers, and compared those by DNA polymerase alpha. Results indicate that DNA polymerase eta incorporates 2-hydroxy-dATP opposite template G in addition to template T and 8-hydroxy-dGTP opposite A in addition to C, respectively. Kinetic analysis revealed that the rate of mutation caused by 2-OH-dATP and 8-OH-dGTP with DNA polymerase eta should be much higher than those with DNA polymerase alpha.

5-Formyluracil, a major oxidized form of thymine, was incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the DNA replication efficiency and the mutation frequency of 5-formyluracil in simian COS-7 cells were investigated. 5-Formyluracil did not block DNA replication and was weakly mutagenic in simian cells. 5-Formyluracil primarily elicited base substitutions at the modified positions.

We developed a coronary active perfusion system (CAPS) to avoid myocardial ischemia during off-pump coronary artery bypass (OPCAB). The purpose of this study was to determine the optimal timing of CAPS perfusion, and to investigate any adverse effects when it is driven within the systolic phase. Twenty-four pigs were divided into four groups: (A) systolic, (B) early-diastolic, (C) middle-diastolic, and (D) late-diastolic perfusion. Myocardial blood flow (MBF), hemodynamic and mechanical data were measured during 30 minutes of CAPS perfusion. MBF in group A was lower than in the other groups (p0.001), but no significant differences were observed among groups B to D. End-systolic pressure-volume relation (Mw) and preload recruitable stroke work relation (Ees) in group A was lower than in groups B to D (p0.01 and p0.05, respectively), but no significant differences were observed among groups B to D. CAPS could maintain regional MBF and left ventricular function if it was driven only within the diastolic phase and required no strict adjustment, but CAPS has an adverse effect when it is driven in the systolic phase.

The optimization of drug disposition in the body leads to an increase in its therapeutic effect and to a decrease in adverse effects. Liposomes can serve as a potential drug carrier for achieving this. However, the behavior of a drug carrier system under in vivo conditions is complex. Therefore, a more complete understanding of the pharmacokinetics of liposomes themselves, as well as that of the encapsulated drug, is required. The optimization of the pharmacokinetics of liposomes can be performed by linking a pharmacodynamic model of the free drugs that are encapsulated into liposomes. Sensitivity analysis was applied to optimize the delivery system to maximize the antitumor effect of liposomal doxorubicin (DOX). Advanced technology for ligand-mediated selective targeting and intracellular targeting is also introduced for antitumor agents and for gene delivery systems.

Cerebral Thrombemboli with left ventricular origin are occasionally seen in patients with post-infarction left ventricular aneurysm or dilatativer cardiomyopathy of non-ischemic cause. Freedom of medical history of cardiac disease and corresponding symptoms may delay the proper diagnosis, particularly in patients with distinct neurological symptoms and normal findings in electrocardiogram or cerebral MRI. We report on a rare case of long standing neurological symptoms and late diagnosis of a left ventricular thrombus without clinical symptoms or medical history of cardiac disease. The patient underwent a thrombus extraction via left ventricular apical approach. He was discharged from hospital after an uneventful course in our clinic with remaining mild neurological symptoms that were partially recurrent under physiotherapy and logopedic therapy in the course of the following two years. An echocardiographical evaluation of cardiac function and exclusion of a cardiac source of emboli as a first line diagnostic tool may have led to an early diagnosis. Therefore, it should be implemented in the routine examination, independent of cardiac history or present cardiac symptoms.

Good long-term results with the bioprosthetic valves for patients 70 years old and older have been reported. However, because the average lifespan is increasing, we aimed to clarify whether patients older than 70 may still be candidates for valve re-replacement. Seventy-one patients 70 years old and older, who received a total of 81 bioprosthetic valve replacements during 73 procedures between 1988 and 2000, were reviewed. There were 8 hospital and 7 late deaths. Ten-year actual survival after valve replacement was 73.5%, and 82.8% when hospital deaths were excluded. During the follow-up period, 2 patients received mitral valve re-replacement. Ten years of freedom from reoperation were found in 66.7% for all valves and in 50.0% for mitral valves. The average lifespan in Japan is currently 77.64 years for men and 84.62 years for women; therefore, valve degeneration in patients who receive bioprosthesis replacement in their early 70s should be anticipated.

To avoid myocardial ischemia during off-pump coronary artery bypass, we developed a coronary active perfusion system (CAPS) that perfuses arterial blood to the coronary artery at the diastolic phase of the cardiac cycle by a syringe pump system. We report herein the details and compare CAPS with a passive shunt circuit from the femoral artery (FA shunt), regarding the physiology of the coronary artery. Six pigs were used for this study. After CAPS or FA shunt perfusion was established, coronary flow and coronary pressure were measured. The coronary flows in the native coronary artery, FA shunt perfusion, and CAPS perfusion with syringe pump stroke volume settings ranging from 0.1 to 0.4 ml were 27.2 ′ 3.0, 4.1 ′ 1.5, 12.7 ′ 1.2, 24.8 ′ 1.9, 33.3 ′ 1.6, and 42.2 ′ 1.9 ml/min, respectively. Mean coronary pressures under FA shunt perfusion and CAPS perfusion with stroke settings from 0.1 to 0.4 ml were 23.7 ′ 4.6, 48.8 ′ 5.0, 61.3 ′ 7.5, 70.8 ′ 5.4, and 82.3 ′ 5.2 mm Hg, respectively. CAPS had an advantage over the FA shunt regarding coronary flow with safe coronary pressure. Using CAPS, an off-pump coronary artery bypass can be performed safely without myocardial ischemia.

To visualize the intracellular trafficking of exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be "naked" DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus.

To examine the mutagenicity of 5-formylcytosine (5-fC), an oxidation product of 5-methylcytosine (5-mC), 5-fC was incorporated into predetermined sites of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5'-AFGCGT-3' and 5'-ACGFGT-3' (F represents 5-fC), the recognition site for the restriction enzyme MluI (5'-ACGCGT-3'). 5-fC was incorporated into the template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and analyzed after a second transfection into Escherichia coli. 5-fC weakly blocked DNA replication in mammalian cells. The 5-fC residues were mutagenic, with mutation frequencies in double-stranded vectors of 0.03-0.28%. The mutation spectrum of 5-fC was broad, and included targeted (5-fC-->G, 5-fC-->A, and 5-fC-->T) and untargeted mutations. These results suggest that the oxidation of 5-mC results in mutations at and around the modified sites.

Objectives: To avoid myocardial ischemia in off-pump coronary artery bypass, we developed a coronary active perfusion system (CAPS). The purposes of this study were to investigate the changes in myocardial blood flow and tissue oxygenation during CAPS perfusion, and to validate the ventricular arrthymias and hemodynamic deterioration preventing the effect. Methods: Sixteen pigs were divided into a CAPS perfusion group (group C) and a simple coronary occlusion group (group O). The left anterior descending coronary artery was snared in both groups and 30 min of CAPS perfusion was performed in group C. Results: Ventricular arrhythmias were not observed in group C, but occurred in seven out of eight pigs in group O (P ¼ 0:003). None of the hemodynamic variables changed in group C, but they deteriorated in group O. Myocardial blood flow, saturation, and hemoglobin plus myoglobin concentration were maintained with a baseline level in group C, but decreased significantly in group O (P , 0:001). Conclusion: CAPS is a reliable method to avoid myocardial ischemia during coronary occlusion and it may be useful for off-pump coronary artery bypass q 2002 Elsevier Science B.V. All rights reserved.

Objectives: There has been no method which can measure regional myocardial blood flow in real-time and continuously. The purpose of this study was to validate myocardial blood flow measurement using a thermal diffusion method. For this purpose, myocardial blood flow measurement was performed using the thermal diffusion method and the electrolytic hydrogen clearance method. Methods: Seven pigs were used for this study, six were for comparison between the thermal diffusion and electrolytic hydrogen clearance methods, and one was for demonstration of myocardial blood flow measurement using the thermal diffusion method on a beating heart coronary artery bypass model with ischemic preconditioning. Results: A good correlation was found between myocardial blood flow values obtained by the electrolytic hydrogen clearance method and 1/V values obtained by a thermal diffusion probe, the correlation coefficient was 0.841 (P < 0.001). During the beating heart coronary artery bypass, the regional myocardial blood flow was recorded in real-time and continuously. Conclusions: This study demonstrated measurement of regional myocardial blood flow using the thermal diffusion method for the first time and simultaneous measurement using the electrolytic hydrogen clearance method for calibration. It provided a real-time and continuous myocardial blood flow measurement and has a potential to contribute to progress in beating-heart surgery.

We examined the effects of troglitazone, one of thiazolidinedione derivatives on human basophilic leukemia cell line KU812. Troglitazone caused the suppression of cell growth, which was suggested to result from the decrease in cyclin E and the hyperphosphorylated form of retinoblastoma tumor suppressor gene product (pRb). In addition, troglitazone caused a decrease in histamine secretion due to the reduced expression of histidine decarboxylase mRNA. Peroxisome proliferator-activated receptor (PPAR)-gamma mRNA was undetectable by reverse transcription-polymerase chain reaction (RT-PCR) in KU812 cells. These findings suggested that troglitazone suppressed cell growth and histamine synthesis independently of PPARgamma.

A nucleotide pool sanitizing enzyme, the human MTH1 protein, hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxyd-GTP. To examine the substrate recognition mechanism of the MTH1 protein, ten nucleotide analogs (8-bromo-dATP, 8-bromod-GTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. This results suggests the importance of the anti/syn-conformation and the functional groups on the 2 and 6-positions of the purine ring.

We prepared a series of linearized DNA duplexes of various lengths in order to examine the effects of topology and the size of exogenous, plasmid-derived DNAs on transgene expression. These linearized DNA duplexes were capped at each end with a highly stable loop (5'-GCGAAGC-3') to produce a dumbbell-shaped construct that is refractory to exonuclease digestion in comparison to the analogous uncapped DNA duplexes. Intranuclear microinjection of the DNA dumbbells into simian COS-7 cells allowed the expression of the green fluorescent protein (GFP) gene on the linearized molecules, which was expressed five- to 10-fold more than that on the circular DNA of the same size. In addition, the expression by the dumbbell DNA was higher than that by the circular plasmid for at least 14 days. Interestingly, the size of the dumbbell DNA affected the transgene expression upon their microinjection into cell nuclei. The GFP expression efficiency increased with decreasing DNA size below a DNA size of 5.7 kb. The effects of topology and size on the expression of DNAs transfected with cationic lipids are similar to those of DNAs microinjected into cell nuclei. In contrast, microinjection into the cytosol showed the inverse size dependency over a range of 2.3 to 9.4 kb. Thus, transcription of a transgene in the nucleus, but not endocytosis or nuclear entry, was influenced by the exogenous DNA structure, and this was the primary determinant of transgene expression upon transfection under our conditions. These results indicate that small, linearized DNA duplexes that are end-capped with a highly stable loop (dumbbell-shaped DNA) would be very useful for nonviral gene therapy.

To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5′-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides. Of the nucleotides tested, 2-hydroxydeoxyadenosine 5′-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5′-triphosphate, were hydrolyzed by this protein. These damaged deoxynucleotides elicit transversion mutations in E. coli (Inoue, M., Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, H. (1998) J. Biol. Chem. 273, 11069–11074). These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides.

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T-->C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T-->C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.

Objectives: We sought to test the effectiveness of ultra-short-acting cardioselective beta-blockade, landiolol hydrochloride, for warm heart surgery. Methods: The isolated perfused rat heart preparation was used. After preischemic measurement of cardiac function, 3 min of coronary infusion of crystalloid cardioplegic solution (378C) with landiolol hydrochloride of various concentrations (1, 2.5, 5, and 10 mmol/ l) or without it (control group) was performed, followed by 30 min of warm ischemic arrest. Finally, postischemic function was measured. Results: The percentage recoveries of heart rate in hearts receiving 0, 1, 2.5, 5, and 10 mmol/l landiolol hydrochloride were 89.4 ^ 3.4%, 90.9 ^ 1.7%, 89.6 ^ 1.8%, 83.4 ^ 3.3%, and 74.3 ^ 1.9% (P , 0:05 vs. 0, 1, and 2.5 mmol/l groups), respectively. The percentage recoveries of aortic flow were 55.6 ^ 3.1%, 62.8 ^ 3.3%, 75.0 ^ 4.2% (P , 0:05 vs. 0 and 10 mmol/l groups), 65.3 ^ 5.3%, and 51.6 ^ 4.0%, respectively. Similar recovery profiles were observed with the first derivative of the rise in aortic pressure, stroke volume and stroke work. The total amount of coronary effluent in the hearts receiving 5 or 10 mmol/l was lower than in the other groups. Conclusions: Landiolol hydrochloride has the potential to enhance postischemic cardiac function after the warm cardioplegic arrest. The optimal concentration for maximum postischemic functional recovery was 2.5 mmol/l, and recoveries of aortic flow and heart rate decreased in hearts receiving 5 mmol/l or more. q 2001 Elsevier Science B.V. All rights reserved.

To test the possibility that the Escherichia coli MutY or MutM protein acts as a 2-hydroxyadenine (2-OH-Ade) glycosylase, we treated double-stranded oligodeoxyribonucleotides containing 2-OH-Ade with the E. coli MutY or MutM protein in vitro. We found that a strand with 2-OH-Ade was a very poor substrate of MutY, irrespective of the base in the complementary strand. Moreover, a strand containing adenine or guanine opposite 2-OH-Ade was also rarely cleaved by MutY. The cleavage of oligonucleotides with 2-OH-Ade by MutM was not observed. These results indicate that neither MutY nor MutM plays an important role in the removal of 2-OH-Ade from DNA.

Oligodeoxyribonucleotides containing a DNA lesion were synthesized and inserted into vector DNAs to introduce the lesion at a predetermined site. The manipulated DNAs were transfected into living mammalian cells and the mutants induced by the DNA lesion were collected and analyzed. This approach was applied to various DNA lesions produced in cells by reactive oxygen species, chemicals, and ultraviolet light. In addition, the mutations induced by damaged DNA precursors were studied by the use of chemically synthesized nucleoside triphosphates. In this review article, the author summarizes the results obtained by these types of experiments, focusing on two oxidatively damaged compounds, 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) and 2-hydroxyadenine (1,2-dihydro-2-oxoadenine).

Oligodeoxyribonucleotides containing a DNA lesion were synthesized and inserted into vector DNAs to introduce the lesion at a predetermined site. The manipulated DNAs were transfected into living mammalian cells and the mutants induced by the DNA lesion were collected and analyzed. This approach was applied to various DNA lesions produced in cells by reactive oxygen species, chemicals, and ultraviolet light. In addition, the mutations induced by damaged DNA precursors were studied by the use of chemically synthesized nucleoside triphosphates. In this review article, the author summarizes the results obtained by these types of experiments, focusing on two oxidatively damaged compounds, 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) and 2-hydroxyadenine (1,2-dihydro-2-oxoadenine).

The insertion specificities of an oxidized dATP analogue, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I. In contrast to our previous observation that mammalian DNA polymerase alpha incorporated the oxidized nucleotide opposite T and C, these two E. coli DNA polymerases incorporated 2-OH-dATP opposite T and G on the DNA template. Steady-state kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times more frequently opposite T than opposite G. On the other hand, the incorporation of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2 orders of magnitude more efficient than that opposite G. These results indicate that the misinsertion specificity of 2-OH-dATP differs between replicative and repair-type DNA polymerases, and provide a biochemical basis for the mutations induced by 2-OH-dATP in E. coli.