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The metabolic activation of a drug to an electrophilic reactive metabolite and its covalent binding to cellular macromolecules is considered to be involved in the occurrence of idiosyncratic drug toxicity (IDT). As a cellular defense system against oxidative and electrophilic stress, phase II enzymes are known to be induced through a Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2/antioxidant response element system. We presumed that it is important for the risk assessment of drug-induced hepatotoxicity and IDTs to observe the biological responses evoked by exposure to reactive metabolites, and then investigated the mRNA induction profiles of phase II enzymes in human hepatocytes after exposure to problematic drugs associated with IDTs, such as ticlopidine, diclofenac, clozapine, and tienilic acid, as well as safe drugs such as levofloxacin and caffeine. According to the results, the problematic drugs exhibited inductive effects on heme oxygenase 1 (HO-1), which contrasted with the safe drugs; therefore, the induction of HO-1 mRNA seems to be correlated with the occurrence of drug toxicity, including IDT caused by electrophilic reactive metabolites. Moreover, glutathione-depletion and cytochrome P450 (P450)-inhibition experiments have shown that the observed HO-1 induction was triggered by the electrophilic reactive metabolites produced from the problematic drugs through P450-mediated metabolic bioactivation. Taken together with our present study, this suggests that HO-1 induction in human hepatocytes would be a good marker of the occurrence of metabolism-based drug-induced hepatotoxicity and IDT caused by the formation of electrophilic reactive metabolites.

The patient was a 54-year-old female with gastric cancer. As laparotomy showed diffuse peritoneal dissemination, only laparotomy was performed and chemotherapy was conducted with a combination of S-1 80 mg/m(2) (2 weeks administration and 1 week rest) and paclitaxel (PTX) 50 mg/m(2) (day 1, 8). After 5 courses of this regimen, endoscopy showed the tumor was reduced in size and PET-CT revealed no evidence of metastasis. She underwent total gastrectomy with D2 lymph node dissection and Roux-en Y reconstruction. Peritoneal metastasis histologically disappeared, and the final findings were T1, N0, H0, P0, CY0, M0, Stage I A, Cur A. The only adverse effect was grade 1 stomatitis during this chemotherapy. CT showed no findings of recurrence 11 months after the operation. The S-1/PTX combination chemotherapy was thought to be effective for gastric cancer with peritoneal dissemination and made curative operation possible in this case.

A 56-year-old woman was seen who had been under hemodialysis treatment. In September 2003, the patient was sent to our hospital with fever and dyspnea, and artificial respiration was initiated. Bronchoscopy detected stenosis due to compression of the bronchus. Contrast computed tomography and angiography detected a pseudoaneurysm of the right common carotid artery. We performed emergency excision of the mycotic pseudoaneurysm, which was closed with an autologous pericardial patch. We also performed median sternotomy to obtain an adequate surgical view. A perfusion tube was inserted into the internal carotid artery. The inflammatory findings and dyspnea resolved postoperatively.

We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric alpha-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity.

Genetic risk factors for ticlopidine-induced hepatotoxicity were determined in 22 Japanese patients with ticlopidine-induced hepatotoxicity and 85 Japanese patients who tolerated ticlopidine therapy without experiencing adverse reactions. There was a significant correlation between ticlopidine-induced hepatotoxicity and five human leukocyte antigen (HLA) alleles: HLA-A*3303, HLA-B*4403, HLA-Cw*1403, HLA-DRB1*1302 and HLA-DQB1*0604 (corrected probability (P)-value (Pc)<0.01). In particular HLA-A*3303 was present in 15 (68%) of the 22 patients with ticlopidine-induced hepatotoxicity and in 12 (14%) of the 85 ticlopidine-tolerant patients (odds ratio, 13.04; 95% confidence interval (CI), 4.40-38.59; the corrected P-value (Pc)=1.24 x 10(-5)). HLA-A*3303 was present in 12 (86%) of the 14 patients with ticlopidine-induced cholestatic hepatotoxicity (odds ratio, 36.50; 95% CI, 7.25-183.82, Pc=7.32 x 10(-7)). Ticlopidine-induced severe cholestatic hepatotoxicity occurred more frequently in subjects with HLA-A*3303 and its haplotype in Japanese patients. These findings may explain the high incidence of ticlopidine-induced hepatotoxicity in Japanese patients mediated via an immune-mediated mechanism.

The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.

The patient was a 55-year-old female. On the diagnosis of the right acoustic tumor, a subtotal extirpation was performed. Heparinized solution was administered, and on postoperative day 7, an occlusion of the left common femoral artery was confirmed. A continuous administration of heparin was initiated after a thrombectomy. On the following day, the platelet count decreased. Following confirmation of the recurrence of thromboembolism, we again performed a thrombectomy. Considering the possibility of heparin-induced thrombocytopenia (HIT), we terminated the administration of heparin, and treatment with danaparoid and argatroban was initiated. Two days later, she redeveloped thromboembolism. After the administration of danaparoid was terminated, the platelet count improved.

Covalent binding of reactive metabolites to cytochrome P450s (P450s) often causes their mechanism-based inactivation (MBI), resulting in drug-drug interactions or toxicity. The detection and identification of the P450 sites to which reactive metabolites bind would elucidate MBI mechanisms. We describe a proteomic approach using nano-LC/linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the binding of a reactive metabolite of raloxifene, which is a known P450 3A4 inhibitor, to the P450 3A4 isozyme. LTQ-FT analyses revealed that the metabolic reaction of raloxifene in a reconstituted P450 3A4 system formed a reactive metabolite adduct to P450 3A4 apoprotein, accompanied by a mass shift of 471 Da relative to intact P450 3A4 apoprotein. The reaction mixtures were digested with trypsin, and then the tryptic digests were analyzed by nano-LC-MS/MS. This technique revealed that VWGFYDGQQPVLAITDPDMIK (position 71-91) was a tryptic peptide modified by the reactive metabolite derived from raloxifene. The site of adduction with the reactive metabolite was further postulated to be the nucleophilic OH group of Tyr-75 of P450 3A4. A proteomic approach using LTQ-FT can yield direct information on the P450 3A4 modification site without radiolabeled compounds. In addition, this information can elucidate mechanisms involved in the covalent binding of reactive metabolites and the inactivation of P450 3A4.

A 77-year-old male, who had undergone the Bentall procedure 27 years ago, was admitted to our hospital for the repair of postoperative pseudoaneurysm. This was the 3rd repair, and the pseudoaneurysm was close to the sternum. Total extracorporeal circulation was established with femorofemoral cannulation and sternotomy was performed under deep hypothermia. During sternotomy, we encountered massive hemorrhage due to injury of the aortic graft. We coped effectively with the situation utilizing temporary circulatory arrest. Aortic graft reimplantation was performed under continuous retrograde cerebral perfusion. Collapse of the suture line of the left coronary orifice was recognized and was reconstructed. The patient was discharged uneventfully on the 26th postoperative day.

We performed tricuspid valve plasty in a 72-year-old woman with pacemaker lead infection and septicemia. All the infected pacemaker system was removed under cardiopulmonary bypass. Because of advanced adhesion and infection, we needed partial resection and plasty of the tricuspid valve. Postoperative echocardiography revealed only mild tricuspid regurgitation and the recurrence of infection has been avoided. Our technique of valve plasty was useful in a patient with advanced infection of both pacemaker leads and tricuspid valve leaflets.

We present a case of disruption of the porcine aortic wall of the 27-mm Freestyle stentless bioprosthesis 5 years after the subcoronary implantation to exclude the sinus of Valsalva aneurysm of the noncoronary cusp. At the urgent reoperation, the inflow suture line was found to be intact, and therefore a new stented valve was sutured with the inflow Dacron cuff after removal of ruptured valve. The subcoronary implantation technique creates a cavity between the prosthetic and native aortic walls filled with hematoma. The outflow suture line dehiscence caused blood flow into the cavity, porcine aortic wall rupture, and leaflet destruction.

We propose a method of slice selection in solid-state MRI by combining DANTE selective excitation with magic-echo (ME) line narrowing. The DANTE RF pulses applied at the ME peaks practically do not interfere with the ME line narrowing in the combined ME DANTE sequence. This allows straightforward tailoring of the slice profile simply by introducing an appropriate modulation, such as a sinc modulation, into the flip angles of the applied DANTE RF pulses. The utility of the method has been demonstrated by preliminary experiments performed on a test sample of adamantane.

In Saccharomyces cerevisiae, acetylation of lysine 56 (Lys-56) in the globular domain of histone H3 plays an important role in response to genotoxic agents that interfere with DNA replication. However, the regulation and biological function of this modification are poorly defined in other eukaryotes. Here we show that Lys-56 acetylation in Schizosaccharomyces pombe occurs transiently during passage through S-phase and is normally removed in G(2). Genotoxic agents that cause DNA double strand breaks during replication elicit a delay in deacetylation of histone H3 Lys-56. In addition, mutant cells that cannot acetylate Lys-56 are acutely sensitive to genotoxic agents that block DNA replication. Moreover, we show that Spbc342.06cp, a previously uncharacterized open reading frame, encodes the functional homolog of S. cerevisiae Rtt109, and that this protein acetylates H3 Lys-56 both in vitro and in vivo. Altogether, our results indicate that both the regulation of histone H3 Lys-56 acetylation by its histone acetyltransferase and histone deacetylase and its role in the DNA damage response are conserved among two distantly related yeast model organisms.

Mesenteric ischemia is a dreaded complication of acute type A aortic dissection. From January 1994 to December 2004, 134 patients with acute type A aortic dissection were operated. Eleven patients showed postoperative mesenteric ischemia. Mortality of such patients was much higher than that without mesenteric ischemia (81.8 vs. 10.6% , p < 0.0001). Preoperative mesenteric and/or lower extremity ischemia were revealed to be the risk factors of postoperative mesenteric ischemia. Our strategy to manage these patients is as follows; patients who are suffering mesenteric and/or lower extremity ischemia preoperatively, or those whose computed tomography (CT) shows stenosis, obstruction, or dissection of the superior mesenteric artery, should be recognized as high-risk patients of postoperative mesenteric ischemia. Their mesenteric circulation should be examined directly with laparotomy after the central repair. If the mesenteric circulation seems to be suboptimal, iliac artery-superior mesenteric artery bypass should be performed.

Chromatin clusters containing CENP-A, a histone H3 variant, are found in centromeres of multicellular eukaryotes. This study examines the ability of alpha-satellite (alphoid) DNA arrays in different lengths to nucleate CENP-A chromatin and form functional kinetochores de novo. Kinetochore assembly was followed by measuring human artificial chromosome formation in cultured human cells and by chromatin immunoprecipitation analysis. The results showed that both the length of alphoid DNA arrays and the density of CENP-B boxes had a strong impact on nucleation, spreading and/or maintenance of CENP-A chromatin, and formation of functional kinetochores. These effects are attributed to a change in the dynamic balance between assembly of chromatin containing trimethyl histone H3-K9 and chromatin containing CENP-A/C. The data presented here suggest that a functional minimum core stably maintained on 30-70 kb alphoid DNA arrays represents an epigenetic memory of centromeric chromatin.

Primary cardiac myxosarcoma is a rare disease; it is exceedingly rare for symptoms of systemic metastasis to precede diagnosis of the primary cardiac tumor. We describe the case of a previously healthy 60-year-old man with left atrial myxosarcoma, who had first presented with jejunal intussusception due to intestinal polyposis. Three months after resection of the jejunum, the patient experienced cerebral infarction and pulmonary edema. Further physical evaluation, which included echocardiography for the 1st time, revealed a mass in the left atrium that protruded through the mitral valve into the left ventricle. At emergency cardiac surgery, we found that the tumor involved multiple sites of the left atrium, the pulmonary veins, and the mitral anterior leaflet. Two months after surgery, the patient died of massive cerebral hemorrhage. Necropsy disclosed multiple recurrences of the cardiac myxosarcoma and widespread metastatic lesions. The intestinal polyps that had been resected originally were diagnosed, on retrospective histopathologic examination, as metastases of the myxosarcoma. In this unusual case, the metastatic lesions were the 1st clinical manifestations of a malignant cardiac tumor.

Background: Coronary artery bypass grafting (CABG) is best indicated for chronic total occlusion of the left anterior descending artery (LAD) with collaterals. We investigated angiographic changes in the collateral circulation after CABG., Methods: Preoperative and postoperative angiograms were reviewed in 42 patients who underwent grafting onto occluded LADs. We described the type, location, and size of collaterals, the Rentrop grading, and collateral frame count (CFC). Regional wall motion of the LAD area was also evaluated with the centerline method. Postoperatively, we measured the lengths of LAD proximal (Lp) and distal (Ld) to the graft anastomotic site., Results: Preoperative collaterals comprised 78 pathways (septal 42%, branch-branch 20%, atrial 19%, bridging 18%). After CABG, residual collaterals were identified, mainly through the septal pathways, in 6 patients (14%), most of whom were diabetic. The residual collaterals were a part of those which had been opacified in earlier phases of the preoperative angiograms (CFC: 17 +/- 3 vs 25 +/- 15, p = 0.01). Also, the Ld was shorter in these patients so that Lp/Ld was greater than in patients without residual collaterals (0.80 +/- 0.24 vs 0.53 +/- 0.28, p = 0.04). We found no association of residual collaterals with the improvement of LAD regional wall motion after CABG., Conclusions: Even after successful CABG, some collaterals with earlier filling of the LAD remain, mainly through the septum. Although the clinical significance remains to be clarified, complex and diffuse atherosclerosis associated with more distal graft anastomoses may contribute to maintaining collaterals after CABG to the occluded LAD, especially in diabetic patients.

Several recent reports have identified lysine 56 (K56) as a novel site of acetylation in yeast histone H3. K56 acetylation is predicted to disrupt some of the histone-DNA interactions at the entry and exit points of the nucleosome core particle. This modification occurs in virtually all the newly synthesised histones that are deposited into chromatin during S-phase. Cells with mutations that block K56 acetylation show increased genome instability and hypersensitivity to genotoxic agents that interfere with replication. Removal of K56 acetylation takes place in the G2/M phase of the cell cycle and is dependent upon Hst3 and Hst4, two proteins that are related to the NAD+-dependent histone deacetylase Sir2. In response to DNA damage checkpoint activation during S-phase, expression of Hst3/Hst4 is delayed to extend the window of opportunity in which K56 acetylation can act in the DNA damage response. The high abundance of histone H3 K56 acetylation, its regulation and strategic location in the nucleosome core particle raise a number of fascinating issues that we discuss here.

Objective: Liver cirrhosis is recognized as one of the risk factors for severe complications after cardiac surgery. However, there are no established methods for risk stratification of the patients with liver cirrhosis (LC) regarding cardiac surgery. We present our experience of preoperative evaluation of liver function using asialoscintigraphy., Methods: Between April 1999 and December 2005, we evaluated preoperative liver function using asialoscintigraphy with technetium-99m galactosyl human serum albumin in four cirrhotic patients undergoing coronary artery bypass grafting (n = 2) and valve replacement (n = 2), whose etiologies of LC were alcohol abuse (n = 1) and hepatitis C virus infection (n = 3). They also underwent other tests for preoperative evaluation of liver function, including the indocyanine green (ICG) test., Results: Asialoscintigraphy revealed that the receptor index and the index of blood clearance in each patient were 0.81/0.73, 0.95/0.5, 0.82/0.62, and 0.97/0.57, respectively. These values closely correlated with the results of the ICG test. All patients were discharged alive from hospital after surgery. However, although one patient who underwent off-pump bypass had an uneventful course, three patients had major complications: pleural effusion (n = 1) and wound infection (n = 2)., Conclusion: Asialoscintigraphy is a practical, reliable method that can replace the ICG test for estimating hepatic function for risk stratification of cirrhotic patients undergoing cardiac surgery, whose mortality and morbidity are still high.

Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing alpha-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input alpha-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

We have been using magnetic resonance angiography (MRA) preoperatively to identify patients at increased risk of stroke in coronary artery bypass grafting (CABG). Based upon the intracranial MRA findings, either the off-pump or the on-pump procedure was selected. We report the results of our MRA-based strategy of CABG in 140 consecutive patients. Intracranial arterial lesions were found in 90 patients (64%). The most affected arteries were internal carotid (48%), followed by vertebral (18%) and middle cerebral (16%). Using the MRA score describing the severity of intracranial atherosclerosis, 35 patients with a score of > or = 5 underwent off-pump, while 105 patients with a score of < 5 underwent on-pump CABG. In the off-pump group, the patient age was older, the incidence of abnormal preoperative brain CT was higher, and the MRA score was higher (6.0+/-1.1 vs. 1.2+/-1.3 points) than in the on-pump groups. Even though the risk was higher in the off-pump group, the clinical outcomes were not significantly different, including the hospital mortality and the incidence of stroke and additional coronary intervention. In conclusion, the brain MRA-based selection of off-pump CABG can contribute to prevention of stroke in neurologically high-risk patients.

Background: Acetylation of histone H3 lysine 56 (K56Ac) occurs transiently in newly synthesized H3 during passage through S phase and is removed in G2. However, the physiologic roles and effectors of K56Ac turnover are unknown., Results: The sirtuins Hst3p and, to a lesser extent, Hst4p maintain low levels of K56Ac outside of S phase. In hst3 hst4 mutants, K56 hyperacetylation nears 100%. Residues corresponding to the nicotinamide binding pocket of Sir2p are essential for Hst3p function, and H3 K56 deacetylation is inhibited by nicotinamide in vivo. Rapid inactivation of Hst3/Hst4p prior to S phase elevates K56Ac to 50% in G2, suggesting that K56-acetylated nucleosomes are assembled genome-wide during replication. Inducible expression of Hst3p in G1 or G2 triggers deacetylation of mature chromatin. Cells lacking Hst3/Hst4p exhibit many phenotypes: spontaneous DNA damage, chromosome loss, thermosensitivity, and acute sensitivity to genotoxic agents. These phenotypes are suppressed by mutation of histone H3 K56 into a nonacetylatable residue or by loss of K56Ac in cells lacking the histone chaperone Asf1., Conclusions: Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.

From 1979 to June 2005, 90 patients aged 65 or older underwent aortic valve replacement with 19-mm prosthetic valve. They were 84 women and 6 men, with a mean age of 74. The mean body surface area was 1.35 m2. Bioprosthetic valves were implanted in 77 patients (85.6%). In-hospital mortality was 2.2% (2 of 90). There were 13 late deaths. New York Heart Association (NYHA) functional class improved to class I in most of survivors. Survival rates for 5 and 10 years were 84.9 and 71.2%, respectively. The outcome of aortic valve replacement with 19-mm prosthetic valve in elderly patients was excellent.

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.

Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells. To investigate the relationship among centromeric, transcriptionally permissive and non-permissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by beta geo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1alpha and trimethyl histone H3-K9 were enriched at the non-transcribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.

Significant venous hypercarbia has been reported in septic shock and circulatory failure. Cardiopulmonary bypass also impairs systemic and pulmonary blood perfusion. The objective of this study was to determine the clinical significance of the increased venous-arterial CO2 tension gradient resulting from venous hypercarbia after cardiopulmonary bypass. On arrival in the intensive care unit, venous and arterial CO2 tensions were measured in the radial and pulmonary arteries in 140 consecutive patients who had undergone coronary (n = 79), valve (n = 34), aortic (n = 20), and other (n = 7) surgery under cardiopulmonary bypass. The mean venous-arterial CO2 tension gradient was 5.0 +/- 3.3 mm Hg (range, 7.7 to 15.7 mm Hg). By linear regression analysis, the factors that significantly correlated with venous-arterial CO2 tension gradient were bypass duration, aortic crossclamp time, initial arterial lactate level, transpulmonary arteriovenous lactate difference, arterial bicarbonate level, base excess, cardiac index, mixed venous O2 saturation, O2 delivery, O2 consumption, and the peak value of creatine kinase. The venous-arterial CO2 tension gradient may reflect impaired perfusion and anaerobic metabolism induced by cardiopulmonary bypass and could be a simple and useful indicator for patient management after surgery under cardiopulmonary bypass.

The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations in the ASPM gene which is involved in the regulation of neurogenesis. The predicted gene product contains two putative N-terminal calponin-homology (CH) domains and a block of putative calmodulin-binding IQ domains common in actin binding cytoskeletal and signaling proteins. Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analyses reveal that ASPM is widely expressed in fetal and adult tissues and upregulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat (HOR) structure. Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N- and C-termini of ASPM. In a western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the spindle poles during mitosis. This finding suggests that MCPH is the consequence of an impairment in mitotic spindle regulation in cortical progenitors due to mutations in ASPM.

DNA breaks are extremely harmful lesions that need to be repaired efficiently throughout the genome. However, the packaging of DNA into nucleosomes is a significant barrier to DNA repair, and the mechanisms of repair in the context of chromatin are poorly understood. Here we show that lysine 56 (K56) acetylation is an abundant modification of newly synthesized histone H3 molecules that are incorporated into chromosomes during S phase. Defects in the acetylation of K56 in histone H3 result in sensitivity to genotoxic agents that cause DNA strand breaks during replication. In the absence of DNA damage, the acetylation of histone H3 K56 largely disappears in G2. In contrast, cells with DNA breaks maintain high levels of acetylation, and the persistence of the modification is dependent on DNA damage checkpoint proteins. We suggest that the acetylation of histone H3 K56 creates a favourable chromatin environment for DNA repair and that a key component of the DNA damage response is to preserve this acetylation.

Enzymatic proteolysis has been implicated in diverse neuropathological conditions, including acute/subacute ischemic brain injuries and chronic neurodegeneration such as Alzheimer disease and Parkinson disease. Calcium-dependent proteases, calpains, have been intensively analyzed in relation to these pathological conditions, but in vivo experiments have been hampered by the lack of appropriate experimental systems for a selective regulation of the calpain activity in animals. Here we have generated transgenic (Tg) mice that overexpress human calpastatin, a specific and the only natural inhibitor of calpains. In order to clarify the distinct roles of these cell death-associated cysteine proteases, we dissected neurodegenerative changes in these mice together with Tg mice overexpressing a viral inhibitor of caspases after intrahippocampal injection of kainic acid (KA), an inducer of neuronal excitotoxicity. Immunohistochemical analyses using endo-specific antibodies against calpain- and caspase-cleaved cytoskeletal components revealed that preclusion of KA-induced calpain activation can rescue the hippocampal neurons from disruption of the neuritic cytoskeletons, whereas caspase suppression has no overt effect on the neuritic pathologies. In addition, progressive neuronal loss between the acute and subacute phases of KA-induced injury was largely halted only in human calpastatin Tg mice. The animal models and experimental paradigm employed here unequivocally demonstrate their usefulness for clarifying the distinct contribution of calpain and caspase systems to molecular mechanisms governing neurodegeneration in adult brains, and our results indicate the potentials of specific calpain inhibitors in ameliorating excitotoxic neuronal damages.

Objective: Surgical treatment of thoracic aortic surgery in patients with coronary artery disease was investigated., Methods: Between 1990 and April 2003, 330 patients underwent elective thoracic aortic surgery. Fifty-six patients who underwent aortic root reconstruction were excluded and 274 patients were examined. Fifty-four (20%) patients showed concomitant coronary artery disease. Ten had undergone coronary revascularization previously; and 3 underwent coronary revascularization [2 coronary artery bypass grafting (CABG), 1 percutaneous transluminal coronary angioplasty (PTCA)] before aortic surgery. Twenty-three patients underwent elective CABG simultaneously and 2 patients had additional coronary artery bypass because of cardiac ischemia during operation. The number of patients who underwent thoracic aortic surgery including Asc Ao+AVR was 2, hemi arch 1, total arch 15, distal arch 5, distal arch+LV aneurysmectomy 1, and thoracoabdominal Ao 1. Two patients underwent coronary revascularization with arterial grafts and the others with SVG grafts., Results: There was one hospital death (4%). In patients without coronary bypass, 2 patients suffered cardiac ischemic events., Conclusion: Our thoracic aortic operations with concomitant CABG using SVG were overall successful. Our current strategies for thoracic aortic surgery in patients with concomitant coronary artery disease include conducting a dipyridamole myocardial perfusion-imaging test first in patients not at risk of coronary artery disease, and if the test is positive, coronary angiography is performed and aggressive coronary revascularization is conducted where possible.

Aims: In this clinical study, we compared two groups of age-matched patients, AAI and DDD, to evaluate the clinical benefits of AAI pacing in patients with sick sinus syndrome (SSS) and normal atrioventricular (AV) conduction., Methods and Results: Ninety-five patients with SSS implanted with AAI pacemakers were compared with 101 SSS patients implanted with DDD pacemakers. Mortality, chronic atrial fibrillation, lead survival rates, and reoperation rates were compared by Kaplan-Meier analysis. Eight AAI devices were switched to DDD due to high-degree (grade 2-3) AV block. The incidence of high-degree AV block was 1.104%/year, with a freedom rate of 88.6% at 10 years. There were no significant differences between the two groups in survival rates (87.8% in AAI vs. 93.4% in DDD at 10 years), freedom from atrial fibrillation (93.6% vs. 90.6%), or freedom from reoperation (71.3% vs. 76.3%). On the other hand, lead failure was twice as frequent in the DDD group than in the AAI group (relative risk=2.045, P=0.0382)., Conclusion: AAI pacing, a simple system using a single lead and single-chamber pacemaker, can achieve a clinical outcome similar to that of the DDD mode in patients with SSS and normal AV conduction.

Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.

An 11-year-old boy (weight 20 kg, height 124 cm), who was survived from ventricular fibrillation due to hypertrophic cardiomyopathy, admitted to our institution for implantable cardioveter defibrillator (ICD) implantation. We implanted a transvenous single coil lead and a device (Medtronic model 6943, GEM II VR 7229 Cx) in the subpectoral pocket. We selected this system because of less restriction on normal cardiac function, low operative morbidity, and expectation of long-term defibrillation threshold stability. Subpectoral implantation is cosmetically acceptable comparing with abdominal area. Lead insertion by cut-down technique is feasible and recommended to avoid lead-related complications. ICDs are infrequently used in pediatric patients and prospective study with long-term follow-up will be required to ascertain the prognosis for young survivors from sudden cardiac death.

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.

The centromere is an essential functional domain responsible for the correct inheritance of eukaryotic chromosomes during cell division. Eukaryotic centromeres include the highly conserved centromere-specific histone H3 variant, CENP-A, which has provided a powerful tool for investigating the recruitment of centromere components. However, the trigger that targets CENP-A to a specific genomic locus during centromere assembly remains unknown. Although, on rare occasions, CENP-A chromatin may assemble at non-centromeric DNA, all normal human centromeres are assembled and maintained on alpha-satellite (alphoid) DNA. The importance of alphoid DNA and CENP-B binding sites (CENP-B boxes), typical of normal human centromere DNA configurations, has been demonstrated through their requirement in de novo centromere assembly and Human Artificial Chromosome (HAC) assays. Mechanisms to link the centromere tightly to specific genomic sequences exist in humans and the two yeast species.

To investigate the mechanism of chromatin assembly at human centromeres, we isolated cultured human cell lines in which a transfected alpha-satellite (alphoid) YAC was integrated ectopically into the terminal region of host chromosome 16, where it was stably maintained. Centromere activity of the alphoid YAC was suppressed at ectopic locations on the host chromosome, as indicated by the absent or reduced assembly of CENP-A and -C. However, long-term culture in selective medium, or short-term treatment with the histone deacetylase inhibitor Trichostatin A (TSA), promoted the re-assembly of CENPA, -B and -C at the YAC site and the release of minichromosomes containing the YAC integration site. Chromatin immunoprecipitation analyses of the re-formed minichromosome and the alphoid YAC-based stable human artificial chromosome both indicated that CENP-A and CENP-B assembled only on the inserted alphoid array but not on the YAC arms. On the YAC arms at the alphoid YAC integration sites, TSA treatment increased both the acetylation level of histone H3 and the transcriptional level of a marker gene. An increase in the level of transcription was also observed after long-term culture in selective medium. These activities, which are associated with changes in chromatin structure, might reverse the suppressed chromatin state of the YAC at ectopic loci, and thus might be involved in the epigenetic change of silent centromeres on ectopic alphoid loci.

Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation. Three subtypes of HP1, namely HP1alpha, beta, and gamma, have been identified in humans. In this study, using yellow fluorescent protein (YFP) fusion constructs, we examined the intracellular localization of human HP1 subtypes during the cell cycle. During interphase, all three HP1 subtypes were localized to centromeric heterochromatin and to promyelocytic leukemia (PML) nuclear bodies. Different preferences, however, were observed among the subtypes: during interphase HP1beta localized most preferentially to centromeric heterochromatin, whereas HP1alpha and gamma were more preferentially localized to PML nuclear bodies. During metaphase, only HP1alpha, was localized to the centromere. We thus determined which molecular domains of HP1 were necessary for their intracellular localization. Our results showed that the C-terminal fragment (amino acid residues 101-180) of HP1alpha was necessary for localization to the metaphase centromere and the N-terminal fragment (amino acid residues 1-76) of HP1beta was necessary for localization to the interphase centromere. Interestingly, simultaneous observations of residues 101-180 of HP1alpha and residues 1-76 of HP1beta in living HeLa cells revealed that during late prophase, the HP1beta fragment dissociated from centromeric regions and the HP1alpha fragment accumulated in centromeric regions. These results indicate that different specific regions of human HP1alpha and HP1beta mediate localization to metaphase and interphase centromeric regions resulting in association of different subtypes of HP1 with the centromere at different times during the cell cycle.

The Polycomb group (PcG) gene products form complexes that regulate chromatin configuration to mediate cellular memory to postmitotic somatic cells and postmeiotic oocytes in Drosophila melanogaster. Structural and functional similarities of PcG proteins between invertebrates and vertebrates suggest mammalian PcG proteins may be involved to imprint transcriptional status at various loci into postmitotic and postmeiotic daughter cells. To address molecular mechanisms underlying PcG-mediated cellular memory, it might be a prerequisite to understand subcellular localization of PcG proteins during mitosis and meiosis. In this study, we analyzed subcellular localization of Rae28/Ph1 and Ring1B by using newly generated monoclonal antibodies in mitotic somatic cells and meiotic mouse oocytes. Results suggest that Rae28/Ph1 and Ring1B dissociate from the chromatin upon its condensation in mitotic prophase in the U2-OS human osteosarcoma cell line. During maturation of oocytes, significant alterations of Rae28/Ph1 and Ring1B localization are concordant with configuration changes of the chromatin at the germinal vesicle stage of meiotic prophase. Importantly, dissociation of Rae28/Ph1 and Ring1B from the chromatin temporally correlates with transcriptional arrest both in mitosis and meiosis. Present and previous observations suggest molecular mechanisms required for mitotic regulation of RNA polymerase II could be involved in dissociation of PcG proteins.

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.

Kearns-Sayre syndrome is regarded as a type of mitochondrial encephalomyopathy accompanied with mitochondrial DNA abnormality of the muscle. Diagnosis of this disease is based upon the progressive external ophthalmoplegia, atypical retinal pigmentation and cardiac conduction block. We report two clinical cases of this disorder treated with permanent pacemaker implantation at a 20 year old man (patient 1) and a 27 years old woman (patient 2). Patient 1 with bifascicular block at 19 years old progressed into complete heart block at 20 years old. Patient 2 with complete heart block was occurred "torsade de pointes." Several problems of this disease in permanent pacing should be considered the patients' small size, pacing mode selection and coexistence of congenital heart disease. Routine electrocardiography is recommended for these patients and bifascicular block in this disease constitutes a definite indication for prophylactic pacemaker implantation.

There is not yet agreement about the optimal size of the prostheses in aortic and mitral valve replacement with Manouguian's technique. In this technique, the aortic prosthetic valve can be pushed upon the mitral prosthesis which may cause dysfunction of the aortic prosthetic valve. The aim of this study was to clarify the size of the prostheses needed to avoid dysfunction of the aortic prosthetic valve. Three patients underwent aortic and mitral valve replacement through this procedure. Two of them had active aortic and mitral valve endocarditis. Aortomitral continuity involved with abscesses could be approached and completely excised using this technique. All patients survived the operation, but 1 of them suffered aortic mechanical valve dysfunction for the reason stated. Anatomical analysis of the geometrical relation of the 2 prosthetic valves suggests that the mitral annulus should be enlarged less than 25 mm to avoid dysfunction of the aortic prosthetic valve.

The products of the Polycomb group of genes form complexes that maintain the state of transcriptional repression of several genes with relevance to development and in cell proliferation. We have identified Ring1B, the product of the Ring1B gene (Rnf2 - Mouse Genome Informatics), by means of its interaction with the Polycomb group protein Mel18. We describe biochemical and genetic studies directed to understand the biological role of Ring1B. Immunoprecipitation studies indicate that Ring1B form part of protein complexes containing the products of other Polycomb group genes, such as Rae28/Mph1 and M33, and that this complexes associate to chromosomal DNA. We have generated a mouse line bearing a hypomorphic Ring1B allele, which shows posterior homeotic transformations of the axial skeleton and a mild derepression of some Hox genes (Hoxb4, Hoxb6 and Hoxb8) in cells anterior to their normal boundaries of expression in the mesodermal compartment. By contrast, the overexpression of Ring1B in chick embryos results in the repression of Hoxb9 expression in the neural tube. These results, together with the genetic interactions observed in compound Ring1B/Mel18 mutant mice, are consistent with a role for Ring1B in the regulation of Hox gene expression by Polycomb group complexes.

Cyclin-dependent protein kinases (Cdks) in eukaryotic cells work as a key enzyme at various points in the cell cycle. At the onset of S phase, active S-phase Cdks (S-Cdks) are essential for chromosomal DNA replication. Although several replication proteins are phosphorylated in a Cdk-dependent manner, the biological effects of phosphorylation of these proteins on the activation of DNA replication have not been elucidated. Here we show that Sld2 (ref. 4) (also known as Drc1; ref. 5), one of the replication proteins of budding yeast (Saccharomyces cerevisiae), is phosphorylated in S phase in an S-Cdk-dependent manner, and mutant Sld2 lacking all the preferred Cdk phosphorylation sites (All-A) is defective in chromosomal DNA replication. Moreover, the complex that contains, at least, Sld2 and Dpb11 (ref. 6) (the Sld2-Dpb11 complex) is formed predominantly in S phase; the All-A protein is defective in this complex formation. Because this complex is suggested to be essential for chromosomal DNA replication, it seems likely that S-Cdk positively regulates formation of the Sld2-Dpb11 complex and, consequently, chromosomal DNA replication.

Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain. We previously demonstrated that neprilysin is the major Abeta-degrading enzyme in vivo. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating Abeta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on Abeta metabolism. The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wild-type neprilysin, but not the mutant, led to significant decreases in both the Abeta40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expression also resulted in reducing cell-associated Abeta, which could be more neurotoxic than extracellular Abeta. These results indicate that the manipulation of neprilysin activity in neurons, the major source of Abeta in the brain, would be a relevant strategy for controlling the Abeta levels and thus the Abeta-associated pathology in brain tissues.