Effects of organic solvents on the time-dependent inhibition of CYP3A4 by diazepam.

The effects of organic solvents, acetonitrile, dimethyl sulfoxide (DMSO), and methanol, which are used to dissolve lipophilic test compounds and cytochrome P450 (P450) substrates, and carried into pre-incubation at 1% (v/v), on time-dependent inhibition of CYP3A4 by diazepam, were evaluated using human liver microsomes (HLM) and recombinant human P450 expressed microsomes (rCYPs). The inactivation kinetics of CYP3A4 by diazepam dissolved in acetonitrile and methanol were almost equal with k(inact)/K(I) values, 0.095 and 0.15 min(-1) mM(-1) for HLM and 1.1 and 1.4 min(-1) mM(-1) for rCYP3A4, respectively. In contrast, the inactivation by diazepam dissolved in 1% DMSO significantly decreased and the kinetic parameter could not be calculated. The formation rate of nordiazepam and temazepam metabolized from diazepam dissolved in DMSO were approximately half of those using substrate dissolved in acetonitrile and methanol in both HLM and rCYP3A4. Dixon plots revealed that the metabolism of diazepam in rCYP3A4 were inhibited by DMSO in a competitive or mixed-type manner with K(i) (inhibition constant) values of 6 and 24 mM for nordiazepam and temazepam, respectively. In conclusion, the time-dependent inhibition of CYP3A4 by diazepam was attenuated by DMSO, while acetonitrile and methanol had no effect. The metabolite formation profile under the conditions tested suggested that DMSO competitively inhibit the formation of the reactive metabolites of diazepam by CYP3A4. The effect of organic solvents should be taken into consideration when evaluating the in vitro time-dependent inhibition of new chemical entities.

Comparison of mechanism-based inhibition of human cytochrome P450 2C19 by ticlopidine, clopidogrel, and prasugrel.

Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4'-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite. Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, k(inact) and K(I), equal to 0.0557 min(-1) and 14.3 microM, respectively, as well as ticlopidine (0.0739 min(-1) and 3.32 microM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100 microM. The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19. Estimation of in vivo drug-drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase. In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.

[Huge right atrial myxoma].

Myxomas are account for approximately half of primary cardiac tumors, and 75% cases originate in left atrium. We report our experience of a right atrial myxoma. A 68-year-old woman was referred to us due to anorexia, general fatigue and facial edema. Echocardiogram, computed tomography (CT), magnetic resonance imaging (MRI), and catheter angiocardiogram revealed a huge tumor in right atrium. The tumor was resected completely with the attached right atrial free wall under cardiopulmonary bypass. Pathological examination showed myxomatous tissue. Postoperative course was uneventful. She discharged the hospital on the 37th day after the operation, and is now doing well without any symptoms.

[Double outlet right ventricle and pulmonary atresia with a birth weight of 1092 g].

A premature infant with double outlet right ventricle and pulmonary atresia with a birth weight of 1092 g is reported. He underwent right modified Blalock-Taussig (RMBT) shunt with an expand-polytetrafluoroethylene (ePTFE) tube of 3.0 mm in diameter between the right subclavian artery and the right pulmonary artery through right thoracotomy. Eleven days later, he had to undergo central shunt between the innominate artery and the main pulmonary trunk due to poor pulmonary blood flow. Soon after the central shunt, severe heart failure occurred due to excessive pulmonary blood flow. RMBT division was performed immediately. He finally attained definitive repair at 17 months of age. Postoperative course was uneventful and he was discharged on the 17th postoperative day.

[Severe tracheal compression resulting from atherosclerotic aortic arch aneurysm; report of a case].

A 70-year-old woman presented with severe respiratory insufficiency similar to an asthma attack. Computed tomography (CT) revealed an atherosclerotic aneurysm (maximum diameter 106 mm) on the aortic arch which resulted in a severe tracheal compression. We performed an aortic arch replacement. After the operation, we tried to manage breathing without a respirator twice without success. We then performed a tracheotomy on the 12th day after operation. The patient could breathe independently on the 19th day after the first operation. Peri-and postoperative respiratory management was difficult but the patient was discharged on the 86th day after operation without further complication.

[De Bakey type I aortic dissection complicating systemic lupus erythematosus; report of a case].

We report a 43-year-old patient of De Bakey type I aortic dissection [DA (I)] with a 2-year-history of systemic lupus erythematosus (SLE). He had had no treatment for SLE before the onset of dissection. Computed tomography (CT) on admission revealed DA (I), and he underwent emergency operation. Since the aortic valve and the left main coronary artery were severely damaged, aortic root replacement was performed. Coronary buttons were prepared in Carrel method and coronary reconstruction was performed in Piehler modification. After surgery, he suffered from repetitive hemolytic anemia. Corticosteroid therapy and rinsed blood transfusion were very effective for anemia. The combination with SLE and DA (I) was rare and this report of successful aortic root replacement is the second literature among English and Japanese papers.

A potent 1,4-dihydropyridine L-type calcium channel blocker, benidipine, promotes osteoblast differentiation.

During their differentiation, osteoblasts sequentially express type I collagen, alkaline phosphatase (ALP), and osteocalcin, and then undergo mineral deposition. Among dihydropyridine-type calcium channel blockers, only benidipine stimulated ALP activity of osteoblastic cells derived from neonatal mouse calvaria. To identify the molecular target of benidipine and elucidate the mechanism of action of the drug in osteoblasts, the mouse osteoblastic cell line MC3T3-E1 was used. Benidipine prompted ALP activity and ALP transcription induced by ascorbic acid, and mineral deposition by ascorbic acid and b-glycerophosphate. Benidipine, however, did not change collagen accumulation. MC3T3-E1 cells expressed the L-type Ca channel a1C subunit throughout the differentiation process, and Ca influx by potassium ions and Bay K 8644, an agonist, was strongly attenuated by benidipine. Each one of three structurally different classes of Ca channel blockers, nifedipine, verapamil, and diltiazem stimulated ALP activity, although at much higher concentrations of ca. 100 nM than benidipine, 1 pM. These results suggest that benidipine directly exerts its effect on osteoblasts and promotes osteoblast differentiation after the step of collagen accumulation by blocking the L-type Ca channel. Since benidipine blocked Ca influx more potently than the three other Ca channel blockers, the unique and potent osteoblast differentiating ability of benidipine may be due to its high affinity for Ca channel together with its high membrane retaining ability, as has been previously reported.

Successful surgical treatment of ventricular tachycardia that induced left ventricular aneurysm in 12-year-old boy.

A surgical treatment of ventricular tachycardia (VT) that induced a congenital left ventricular aneurysm (LVA) in a 12-year-old boy was performed. The VT disappeared after epicardial cryoablation and a reinforcement of LVA with a composite patch. Epicardial cryoablation, based on an intraoperative electrophysiologic study (EPS), is effective in treating VT resistant to antiarrhythmia drugs.

Predicting blood transfusion factors in coronary artery bypass surgery.

OBJECTIVE: Blood conservation has become one of the most important issues in cardiac surgery. We clarified preoperative predictors of the need for blood transfusions during coronary artery bypass graft surgery. METHODS: Subjects were 89 patients--66 men (74%) and 23 women (26%) 40 to 84 years old (mean: 66.2 +/- 8.3 years)--undergoing isolated coronary artery bypass surgery from September 1997 to December 1999. Of these, 66 patients (74%) received transfusion during hospitalization and 23 (26%) did not. Nine risk factors detected by univariate study were entered in a multivariate logistic regression model of the relationship between preoperative variables and blood transfusion. RESULTS: Independent predictors were emergency surgery (P = .0023), lower hematocrit (P = .0027), older age (P = .0043), and the presence of peripheral vascular disease (P = .0070). Optimal cutoff of hematocrit for blood transfusion was 39% and age 64 years via receiver-operating characteristics curves based on the relation between sensitivity and specificity. CONCLUSION: Patients older than 64 years with hematocrit less than 39% and/or peripheral vascular disease should be treated routinely using preoperative storage of autologous blood whenever the patient's condition permits. For patients undergoing emergency surgery, further studies are required, including lowering transfusion threshold and using determinants other than hematocrit.

Identification of shiga toxin-producing Escherichia coli possessing insertionally inactivated Shiga toxin gene.

We have investigated the Shiga toxin genes of Shiga toxin-producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes. As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS). This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper. On the other hand, both Shiga toxin 2 genes were different (98.3% identity). These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution. Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, i.e., the insertion can be reflected in the size of amplicon.

Effects of various antihypertensive drugs on the function of osteoblast.

Several studies have suggested that high blood pressure is associated with the risk of bone loss. Since various antihypertensive drugs are in wide use for the treatment of hypertension, it is important to investigate the influences of these drugs on bone metabolism. Osteoblasts play a pivotal role in the regulation of bone formation. During differentiation, they sequentially express type I collagen, alkaline phosphatase (ALP), other bone matrix proteins, and finally undergo mineral deposition. In this study, we examined the effects of various antihypertensive drugs on the function of osteoblast using clonal MC3T3-E1 cells. Drugs examined include dihydropyridine-type calcium channel blockers (benidipine, amlodipine, and nifedipine), angiotensin-converting enzyme (ACE) inhibitors (captopril, lisinopril, and enalapril), and angiotensin II receptor type1 (AT1) antagonists (TCV-116 and KW-3433). None of the ACE inhibitors or AT1 antagonists affected ALP activity or cellular DNA content significantly. In contrast, benidipine, amlodipine, and nifedipine increased ALP activity when used in amounts 1 pM, 100 nM, and 100 nM, respectively. Benidipine blocked calcium influx through the L-type voltage dependent calcium channel of MC3T3-E1 more potently than amlodipine or nifedipine. These calcium channel blockers did not change collagen accumulation. Benidipine significantly increased in vitro mineralization at a concentration of 1 nM and higher, while amlodipine did so at 1 microM and nifedipine did not. Comparison of the effective concentration of each calcium channel blocker in our study with the reported maximum serum concentration of each drug suggests that benidipine, but not amlodipine or nifedipine, promotes mineral deposition in human.

[Experiences of minimally invasive great saphenous vein graft harvest using with endopath].

We have experienced 20 cases of minimally invasive great saphenous vein graft harvest using with endoscopy, Endopath, from March 1999. As we experienced cases, we can harvest great saphenous vein graft, about 30-40 cm in size, from only two 4-cm incisions for about 50 minutes. There are no wound infection, pain, and edema. Great saphenous vein graft harvesting with Endopath is less invasive, painless after surgery and makes patients satisfied about cosmetic problem.

Spontaneous reactivation of Shiga toxins in Escherichia coli O157:H7 cells caused by transposon excision.

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes (stx2) of Escherichia coli O157:H7. Using PCR amplification, we detected the wild-type stx2 genes in colonies of E. coli O157:H7 which possessed stx2 genes inactivated by insertion of IS1203v. This suggests that IS1203v is excised from the inactivated stx2 genes in E. coli O157:H7. We isolated the cells possessing the wild-type stx2 genes, and confirmed Stx2 productivities by reversed passive latex agglutination. We also analyzed the frequency of the appearance of the Stx2-producing cells using a quantitative PCR method. As a result, the frequency was 3.00 x 10(-6) with culturing for 24 h at 37 degrees C, and this increased to 8.83 x 10(-5) when E. coli O157:H7 possessing the inactivated stx2 genes was transformed by an expression plasmid harboring the IS1203v transposase. These results showed that some Stx2-nonproducing E. coli O157:H7 strains could be spontaneously changed into Stx2-producing cells.

A mutant sarcosine oxidase in which activity depends on flavin adenine dinucleotide.

The covalent flavin attachment site in the Arthrobacter sarcosine oxidase (cysteine at position 318) was replaced with serine, and the mutational effect of C318S was analyzed. Wild type and C318S with a C-terminal 6-histidine tag were constructed and homogeneously purified by the single step. The covalently binding to flavin was not essential to the enzyme activity because the C318S mutant exhibited extremely weak activity. Moreover, the activity of the mutant was recovered in the presence of flavin adenine dinucleotide (FAD), and significantly increased as the concentration of FAD increased. This dependence of the mutant on FAD indicates that the noncovalent binding of free FAD to the mutant enzyme is reversible.

Regression of left main coronary ostium stenosis after surgical revascularization and steroid therapy.

We describe a patient in whom stenosis of the left main coronary ostium completely regressed after steroid treatment following surgical revascularization. A 46-year-old woman with unstable angina underwent double coronary artery bypass grafting. Although she did not fulfill diagnostic criteria for Takayasu's disease, we began postoperative steroid therapy on postoperative day 14 based on clinical manifestations and histological findings. Coronary angiography 33 days after surgery showed the ostial stenosis of the left main coronary artery had disappeared. Steroid therapy for suspected Takayasu's disease should be considered even after surgical revascularization.

[A case of acute pulmonary embolism fourteen days after hysterectomy].

We describe a 54-year-old female with acute pulmonary embolism. She showed a syncope attack and hypotension fourteen days after hysterectomy for uterine cancer. Preoperative echocardiogram revealed that thrombus in the right atrium was coming and going through the tricuspid valve. Emergent pulmonary embolectomy through the sternotomy under cardiopulmonary bypass was performed 4.5 hours after the diagnosis without homologous blood transfusion. Postoperative perfusion scintigram 20 days after the operation showed normal filling of the both lungs except for the localized defect at the distal portion of the right middle lobe. The patient was discharged on the 22nd postoperative day and she has been followed up with anticoagulation therapy. It is essential that we have the opportunity to salvage an otherwise helpless situation by a high index of suspicion and a prompt surgical intervention.

Reactivation of insertionally inactivated Shiga toxin 2 genes of Escherichia coli O157:H7 caused by nonreplicative transposition of the insertion sequence.

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes of Escherichia coli O157:H7. We analyzed the transpositional mechanism of IS1203v in order to investigate whether the Shiga toxin 2 genes inactivated by IS1203v could revert to the wild type. When the transposase activity of IS1203v was enhanced by artificial frameshifting, IS1203v was obviously excised from the Shiga toxin 2 gene in a circular form. The IS1203v circle consisted of the entire IS1203v, but an extra 3-bp sequence (ATC) intervened between the 5' and 3' ends of IS1203v. The extra 3-bp sequence was identical to a direct repeat which was probably generated upon insertion. Moreover, we detected the Shiga toxin 2 gene with a precise excision of IS1203v. In the wild-type situation, the transposition products of IS1203v could be observed by PCR amplification. These results show that IS1203v can transpose in a nonreplicative manner and that the Shiga toxin gene inactivated by this insertion sequence can revert to the wild type.

Giant extracardiac unruptured aneurysm of the sinus of Valsalva: a case report.

Extracardiac unruptured aneurysm of the sinus of Valsalva (ASV) is rare and difficult to diagnose accurately by echocardiography or cardiac catheterization preoperatively. A 63-year-old woman, with dyspnea and palpitations, diagnosed with aortic regurgitation (AR) with congestive heart failure and extracardiac unruptured ASV, was referred to our hospital for surgical repair. The unruptured ASV was well visualized by magnetic resonance imaging (MRI), and diagnosed as an extracardiac type. Surgical repair was performed by aortic valve replacement and aneurysmectomy. It was concluded that early surgical repair of extracardiac ASV should be considered to prevent sudden death, and MRI is an accurate and useful method for preoperative diagnosis.

Left atrial myxoma associated with acute myocardial infarction.

We describe a patient with left atrial myxoma associated with acute myocardial infarction. Since hemodynamics were impaired even with the support of an intra-aortic balloon pump, the patient underwent removal of the tumor concomitant with coronary artery bypass grafting to the right coronary artery on the fifth day from infarction onset. In circumstances of life-threatening of myxoma associated with acute myocardial infarction, removal of myxoma with coronary artery bypass should be performed in an acute phase of myocardial infarction.