Diphosphate regulation of adenosine triphosphate sensitive potassium channel in human bladder smooth muscle cells.

PURPOSE: To clarify the properties of adenosine triphosphate sensitive K+ channel in human detrusor smooth muscle we examined the effect of the representative nicotinic acid derivatives ß-nicotinamide adenine dinucleotide, cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate (Sigma-Aldrich®) on human detrusor adenosine triphosphate sensitive K+ channels. MATERIALS AND METHODS: Patch clamp procedures were done in human detrusor cells. Reverse transcriptase and real-time polymerase chain reaction were performed to clarify the subunit components of adenosine triphosphate sensitive K+ channels. RESULTS: The K+ channel opener levcromakalim induced a long lasting outward current that was inhibited by glibenclamide (Sigma-Aldrich) under the whole cell configuration. The single channel study revealed that the unitary conductance of the adenosine triphosphate sensitive K+ channel in the human detrusor was 11 pS and nucleotide diphosphates increased its open probability. Applying ß-nicotinamide adenine dinucleotide also activated the adenosine triphosphate sensitive K+ channel but applying cyclic adenosine diphosphate ribose or nicotinic acid adenine dinucleotide phosphate had little effect on channel activation. Molecular studies indicated that Kir6.1 and SUR2B were the predominant components of the adenosine triphosphate sensitive K+ channel in the human detrusor. CONCLUSIONS: To our knowledge we report the first single channel study of the adenosine triphosphate sensitive K+ channel in the human detrusor. The properties of this channel, ie unitary conductance, adenosine triphosphate sensitivity and diphosphate activation, were consistent with those of other smooth muscle organs. ß-Nicotinamide adenine dinucleotide has the potency to activate adenosine triphosphate sensitive K+ channels in the human detrusor. This channel likely has some role during ischemic conditions as well as physiological muscle motion leading to the activation of cell metabolism.

Levcromakalim and MgGDP activate small conductance ATP-sensitive K+ channels of K+ channel pore 6.1/sulfonylurea receptor 2A in pig detrusor smooth muscle cells: uncoupling of cAMP signal pathways.

Pharmacological studies have suggested the existence of ATP-sensitive K(+) (K(ATP)) channel as a therapeutic target in urinary bladders; however, electrical properties have not yet been shown. Patch-clamp techniques were applied to investigate the properties of K(ATP) channels in pig detrusor cells. In whole-cell configuration, levcromakalim, a K(ATP) channel opener, induced a long-lasting outward current in a concentration-dependent manner. The current-voltage curve of the levcromakalim-induced membrane current intersected at approximately -80 mV. This current was abolished by glibenclamide. Intracellular application of 0.1 mM GDP significantly enhanced the levcromakalim-induced membrane current, whereas cAMP did not. Furthermore, neurotransmitters related to cAMP signaling, such as calcitonin gene-related peptide, vasointestinal peptide, adenosine, and somatostatin, had little effect on the membrane current. In cell-attached configuration, levcromakalim activated K(+) channels with a unitary conductance of approximately 12 pS. When the patch configuration was changed to inside-out mode, the K(+) channel activity ran down. Subsequent application of 1 mM GDP reactivated the channels. The openings of the approximately 12 pS K(+) channels in the presence of 1 mM GDP was suppressed by ATP and glibenclamide. In reverse transcription-polymerase chain reaction, K(+) channel pore 6.1 and sulfonylurea receptor (SUR)2A were predominant in pig detrusor cells. The 12 pS K(+) channel activated by levcromakalim in pig detrusor smooth muscle cells is a K(ATP) channel. The predominant expression of SUR2A can account for the lack of effect of neurotransmitters related to cAMP.

Characterization of myelodysplastic syndrome and aplastic anemia by immunostaining of p53 and hemoglobin F and karyotype analysis: differential diagnosis between refractory anemia and aplastic anemia.

P53 mutation has been reported in various solid tumors, acute leukemia and myelodysplastic syndrome (MDS), but the diagnostic significance of p53 in MDS remains to be determined. The purpose of the present paper was to examine p53 mutation and immunostaining of the same patients, because there have been few reports of simultaneous analysis of these markers. Seven p53 mutations were observed among 37 MDS and 11 cases of overt leukemia transformed from MDS (MDS-OL). Mutated p53 mainly observed in high-risk MDS had more intense p53 staining than in MDS with wild-type p53 overexpression. Aplastic anemia (AA) produced no p53 staining. The percentage of p53 staining in MDS (71%) was higher than that of mutated p53 (11%) but did not reach 100% of MDS cases studied, therefore the authors attempted to differentiate MDS, especially refractory anemia (RA) and AA, using a combination of p53 immunostaining, hemoglobin F (HbF) immunostaining and chromosome abnormality, because HbF of erythroblasts was reportedly observed in MDS RA but not in AA. Most MDS/MDS-OL (47/48) had at least one positive marker. Among 11 AA cases, only two were positive for HbF. The present results suggest that the combination of these three markers is useful to discriminate MDS from AA.

Transcriptional regulation of FKLF-2 (KLF13) gene in erythroid cells.

FKLF-2 (KLF13) was cloned from fetal globin-expressing tissues and has been shown to be abundantly expressed in erythroid cells. In this study we examined the transcriptional regulation of the KLF13 gene. A 5.5 kb 5' flanking region cloned from mouse erythroleukemia (MEL) cell genomic DNA showed that major cis regulatory activities exist in the 550 bp sequence to the unique transcription start site, and that the promoter is more active in K562 cells than in COS-7 cells. The promoter was trans-activated by co-expressed GATA-1 through the sequence containing two CCAAT motifs, suggesting that GATA-1 is involved in the abundant expression of KLF13 mRNA in the erythroid tissue. Dual action, i.e. activating effect in COS-7 and repressive effect in K562 cell, was observed on its own promoter, suggesting a feedback mechanism for the transcriptional control of the KLF13 gene in the erythroid environment. These findings provide an insight on the mechanism of inducible mRNA expression of the KLF13 gene in erythroid cells.

Loss of O6-methylguanine-DNA methyltransferase protein expression is a favorable prognostic marker in diffuse large B-cell lymphoma.

Although aberrant promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) is a favorable prognostic marker in patients with diffuse large B-cell lymphoma (DLBCL), MGMT protein expression has not been thoroughly examined. The aim of this study was to evaluate the clinical implication of MGMT protein expression and its correlation with promoter hypermethylation of the gene. We investigated MGMT protein expression by immunohistochemical analysis of 63 DLBCL patients who received cyclophosphamide as part of multidrug regimens. In addition, promoter methylation of the MGMT gene was analyzed by a methylation-specific polymerase chain reaction assay, and correlations with chemotherapeutic effect and prognosis were statistically evaluated. Immunohistochemical assay results for MGMT protein were negative in 30.2% of patients with newly diagnosed DLBCL. Immunostaining results were closely correlated with the methylation status of the promoter. Promoter DNA methylation of the gene was not detected in 34 (81.0%) of 42 tumor samples determined to be MGMT-positive DLBCL by immunostaining and was detected in 15 (88.2%) of 17 cases of MGMT-negative DLBCL. Overall survival (OS) and disease-free survival (DFS) rates were significantly higher in MGMT-negative patients than in MGMT-positive patients (5-year OS, 81.3% versus 56.6% [P = .0375]; 5-year DFS, 66.3% versus 39.9% [P = .0121]). The combined rate for complete response (CR) plus unconfirmed CR was significantly higher in MGMT-negative patients (15/19, 79.0%) than in MGMT-positive patients (25/44, 56.8%) (P = .0488). A multivariate analysis showed that absence of MGMT expression was an independent prognostic factor for OS (relative risk, 4.09; P = .0258). Lack of MGMT protein expression is associated with aberrant promoter DNA methylation and appears to be a useful marker for predicting the survival of DLBCL patients.

Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1.

The prolonged treatment with phorbol 12-myristate 13-acetate (PMA) of a human megakaryoblastic leukemia cell line, MEG-O1, induced increase of sphingosine kinase (SPHK) enzyme activity and SPHK1 protein expression as well as SPHK1 message. Protein kinase C (PKC) inhibitor prevented the PMA-induced SPHK1 gene expression. To elucidate the regulatory mechanism of this gene expression, we examined the promoter area (distal to the first exon) and its binding proteins. Luciferase analyses showed that the area of 300 bp from the first exon was sufficient for PMA-responsiveness, and that specificity protein 1 (Sp1)- and two activator protein 2 (AP-2)-binding motifs within this area were necessary for responsiveness. Inhibitors for PKC and MEK1 decreased this PMA-induced promoter activity. Electrophoresis mobility shift assay (EMSA) showed that Sp1 protein was originally bound to the Sp1 site and that two additional bands bound to the two AP-2 motifs were observed only when stimulated with PMA in MEG-O1 cells. The appearance of these bands resulted from binding to an unknown protein rather than AP-2. These results indicated that PMA up-regulates SPHK1 gene expression through PMA-responsive elements of the 5' promoter area of the gene, and suggested that PMA-mediated SPHK1 gene expression would be mediated via PKC- and ERK-dependent signal transduction pathway by binding the transcription factor to AP-2 motifs.

Ventricular afterload and ventricular work in fontan circulation: comparison with normal two-ventricle circulation and single-ventricle circulation with blalock-taussig shunts.

BACKGROUND: Recent studies have indicated that there are inherent limitations associated with Fontan physiology. However, there have been no quantitative analyses of the effects of right heart bypass on ventricular afterload, hydraulic power, and resultant overall hemodynamics. Methods and Results- During routine cardiac catheterization, aortic impedance and ventricular hydraulic power were determined, both at rest and under increased ventricular work induced by dobutamine, in 17 patients with Fontan circulation, 15 patients with a single ventricle whose pulmonary circulation was maintained only by Blalock-Taussig shunts, and 13 patients who had normal 2-ventricle circulation. Both vascular resistance (nonpulsatile load on the ventricle) and pulsatile components of ventricular afterload (represented by low-frequency impedance) were significantly higher in the Fontan group than in the other groups (P<0.01), and this was associated with decreased cardiac output in the Fontan patients. In addition, hydraulic power cost per unit forward flow was 40% lower in the 2-ventricle circulation than in the single-ventricle circulation, suggesting lower ventricular efficiency in single-ventricle circulation attributable to the lack of a pulmonary ventricle. Furthermore, in the Fontan group, beta-adrenergic reserve was markedly decreased because of a limited preload reserve. CONCLUSIONS: Fontan physiology is associated with disadvantageous ventricular power and afterload profiles and has limited ventricular reserve capacity. Thus, to improve the long-term prognosis of patients after Fontan surgery, future research should be conducted into medical interventions that can overcome these limitations inherent in Fontan circulation.

Molecular cloning and characterization of ZFF29: a protein containing a unique Cys2His2 zinc-finger motif.

We have cloned a gene, ZFF29 (zinc-finger protein of human fetal liver erythroid cells 29), from human fetal liver erythroid cells. Two types of mature mRNA were identified and designated ZFF29a and ZFF29b. In human genome the ZFF29 gene is on chromosome 9q, and the two forms are splice variants. There is a unique transcription start site, which predicts major mRNAs composed of 2485 bases for ZFF29a and 1801 bases for ZFF29b. The anticipated mRNAs were demonstrated in K562 cells, but not in any adult human tissues examined by Northern blotting. In the mouse, reverse transcription-PCR revealed that the ZFF29 mRNA is present in adult bone marrow and ovary at a higher level than in any other tissues examined. These findings suggest that ZFF29 proteins are expressed in embryonic/fetal erythroid tissues. The deduced polypeptide chains of ZFF29a and ZFF29b are composed of 306 and 350 amino acids respectively. A unique zinc-finger motif composed of two contiguous Cys(2)His(2)-type fingers is common to both forms of ZFF29. They are nuclear proteins and ZFF29b, but not ZFF29a, is an activator of erythroid gene promoters.

The MAGE-A1 gene expression is not determined solely by methylation status of the promoter region in hematological malignancies.

Tumor antigens such as MAGE-A1 are aberrantly expressed in many human tumors and could be recognized by CTL. Thus, they could be targets for cancer immunotherapy. It is presently considered that the expression of the MAGE-A1 gene is regulated by methylation of its promoter region. To estimate the possibility of activating the MAGE-A1 gene with demethylating agents with a view toward clinical use, we assessed the methylation status of its CpG-rich promoter by sodium bisulfite mapping both of samples that express the gene and those that do not. Cell lines and samples from patients with hematological malignancies were examined. Surprisingly, the methylation status of the MAGE-A1 gene did not clearly correlate with the expression of the gene. Our results indicate that the MAGE-A1 gene expression is not determined solely by the methylation status of the promoter region in hematological malignancies.

Lethal thrombus in the carotid artery during operation for acute aortic dissection with cerebral malperfusion.

A drowsy patient with acute type A aortic dissection and cerebral malperfusion required emergency operation. Because the right carotid artery was totally obstructed, cerebral perfusion was first restored by cannulating it and the left femoral artery before midline sternotomy. However, a long fresh thrombus was found flowing backward from the obstructed carotid artery. This thrombus was removed, and both arteries were connected through a Y-shaped extracorporeal circulation circuit to reperfuse the brain. During the subsequent aortic procedure, both arteries were used for arterial inflow. Such thrombi can cause grave postoperative neurologic dysfunction. Carotid artery cannulation is mandatory in such cases.

Successful treatment of ascending aortic graft infection after operation for acute aortic dissection with peripheral malperfusion.

Mediastinitis with infection of an ascending aortic graft is hard to heal and is a highly fatal complication. We had a patient in whom mediastinitis with infection of such a graft as well as an ascending aorta-femoral artery bypass graft developed after the initial operation for type A aortic dissection accompanied by peripheral malperfusion. We treated it successfully by inserting a stent into the true lumen of the thoracoabdominal aorta and using a cryopreserved homograft to replace the infected ascending aortic fabric graft.

Mid-term results of aortoplasty for dilated ascending aorta associated with aortic valve disease.

BACKGROUND: Dilatation of the ascending aorta concomitant with aortic valve disease is occasionally associated with progressive enlargement of the ascending aorta or acute aortic dissection (AAD). However, surgical procedure of choice for the aorta and its indication are controversial. PATIENTS AND METHODS: From July 1995 to August 2001, 10 patients with a moderately dilated ascending aorta (mean diameter, 52+/-4.8 mm) underwent concurrent aortic valve replacement (AVR) and aortoplasty. The aortic valve was bicuspid in eight patients. To tailor the ascending aorta 30-35 mm in diameter, the aortic wall was partially resected along the aortotomy, and the aorta was directly closed. RESULTS: Operation time and most of other perioperative variables were comparable to those of patients who underwent isolated AVR. The aortic diameter was reduced to 36.1+/-4.1 mm. Nine patients survived to hospital discharge uneventfully, but one patient developed disruption of the suture line in the aorta and died. During follow-up, no patient suffered AAD but redilatation was observed in one patient. In the two problematic patients, the ascending aorta was larger than 55 mm, and its media was histologically abnormal. CONCLUSION: In patients with dilated ascending aorta less than 55 mm in diameter, aortoplasty can be a procedure of choice. However, a prosthetic graft replacement is recommended when the diameter of the ascending aorta is larger than 55 mm.

The efficacy and safety of additional bypass graft during isolated coronary artery bypass grafting.

In 19 patients with an undesirable hemodynamic condition (n=15) or with regional asynergy and coexistent ST-T change (n=4) during isolated coronary artery bypass grafting (CABG) surgery, one (n=17) or two (n=3) additional saphenous vein grafts were placed onto left anterior descending (LAD) (n=16), right (n=4), and left circumflex (LCx) (n=2) coronary arteries. Diagnosis of the cause of the suboptimal condition was insufficient graft flow in 16 patients, and spasm of the ungrafted coronary artery in 3. Additional myocardial ischemic time was 17 9 minutes, and the graft flow was 59 25 ml/min. Additional bypass was effective in 94.5%. Eighteen patients could be weaned from cardiopulmonary bypass, and 17 (89.5%) survived and were discharged from hospital. Median duration of mechanical ventilatory support and intensive care unit stay was 15 hours and 4 days, respectively. During 63 44 months follow-up, the additional graft was occluded and the treadmill test was positive for ischemia in 2 patients, and one child patient is now considered for redo CABG. Placement of additional bypass grafts thus appeared to be an effective and relatively safe strategy, although the decision has to be made cautiously.

Surgery for active, culture negative endocarditis.

Culture negative infective endocarditis (CNE) poses very difficult problems during treatment. In this study it was found that of 132 surgically treated patients with infective endocarditis, causative organism was not identified in 46 (34.8 %). Pre- and perioperative conditions and clinical results of these patients were evaluated. CNE remained very frequent even in these years, and it did not decrease with time. Antibiotic treatment prior to microbiological examinations was commonly observed (nearly 90% orally, and 70% intravenously). In average, it took more than 2 months to establish the diagnosis of CNE after the onset, and both aortic and mitral valves were affected frequently (19.0 %). New York Heart Association functional class IV was observed significantly more commonly (61.9%) than culture positive patients. Frequencies of prosthetic valve endocarditis (12.2%), periannular abscess (36.3%), and embolism (21.4%) were similar. Infection was fairly controllable before surgery in 43.9% of CNE patients and in-hospital mortality rate was 14.3%, both of which were comparable to those of all culture positive patients. However, recurrence rate was relatively higher (10.0%). The conditions and outcomes of CNE were comparable to Staphylococcal endocarditis in some aspects, and were relatively worse than overall culture positive endocarditis.

Combined coronary artery bypass grafting and abdominal aortic aneurysm repair.

The purpose of this paper was to assess the results and feasibility of simultaneous coronary artery bypass grafting and abdominal aortic aneurysm repair. Twenty nine patients with a mean age of 65 years underwent simultaneous coronary artery bypass grafting and abdominal aortic aneurysm repair between June 1990 and March 2002. All patients had significant coronary artery disease and were considered as indicated for coronary artery bypass grafting. This was performed first in 28 patients and simultaneously with abdominal aortic aneurysm repair in one, with a mean number of grafts of 2.5, a mean aortic cross-clamp time of 40 minutes, and a mean bypass time of 115 minutes. Eight straight and 21 bifurcated grafts were employed. The total operating time averaged 400 minutes. The median postoperative hospital stay was 18 days. One patient died of stroke and mediastinitis, for a mortality rate of 3.5%. This experience suggests that combined coronary artery bypass grafting and abdominal aortic aneurysm repair is both safe and effective.

[Advantages and limitations in minimally invasive cardiac surgery].

The introduction of endoscopic technology to cardiovascular surgery was significantly delayed compared to abdominal and lung surgery, although it has been gradually introduced in this field during the past decade in closure of patent ductus arteriosus, repair of the vascular ring, implantation of pacemaker leads or AICD, and pericardectomy. Endoscopic technology also started to be used in harvesting saphenous vein grafts (SVG) and the left internal thoracic artery for coronary artery bypass grafting(CABG) from the mid-1990s. Although complete endoscopic surgery has not yet been established in the major field of standard cardiovascular surgery, many cardiac surgeons attempt to minimize the size of chest wounds with 6- to 8-cm skin incisions, which is called minimally invasive cardiac surgery (MICS) or minimally invasive direct coronary artery bypass (MIDCAB). Complete endoscopic cardiac surgeries were performed utilizing the Zeus system and Da Vinci system at the end of the 20th century. Another method to minimize the invasiveness of CABG is to perform it without cardiopulmonary bypass, so-called off-pump coronary artery bypass (OPCAB). Currently, less-invasive procedures are mainly applied for relatively simple cardiac surgeries, although these procedures are also potentially effective to avoid postoperative cerebral or respiratory complications in high-risk patients. MICS is effective in reducing the size of surgical wounds and in decreasing intraoperative blood loss. On the other hand, the duration of anesthesia and surgery can be prolonged due to technical difficulty, and the risk of unsatisfactory anastomosis or incomplete revascularization can also be increased. The cardiopulmonary bypass circuit utilized for MICS requires a more complicated system including negative pressure venous drainage. The detection of accidental trouble during surgery, which is related to the extracorporeal circulation or the MICS procedure itself, can be delayed due to the limited surgical view. MICS procedures carry additional risks related to the more complicated cardiopulmonary bypass system and small surgical wound. We must be deliberate in determining the indications for MICS and obtain complete informed consent from patients when we perform MICS, including informing them of the additional risks related to the MICS procedure itself and the possibility of conversion to standard open-heart surgery.

[Left ventricular assist system].

Two types of artificial heart, the total artificial heart (TAH) and ventricular assist system (VAS), have been utilized in patients with end-stage heart failure waiting for heart transplantation (bridge to transplantation). The TAH is a system to replace the native heart, whereas the VAS is a system to support the left ventricle (LVAS), right ventricle (RVAS), or both ventricles (BVAS) while maintaining native cardiac function. There are two types of VAS, paracorporeal VAS and implantable VAS. Implantable VAS devices such as Novacor LVAS and HeartMate LVAS are only available for LVAS, although paracorporeal VAS, such as Toyobo VAS, Zeon VAS, and Thoratec VAS, are available for RVAS, LVAS, or BVAS. Due to recent advances in the VAS, the "patient discharge program" from hospital has been promoted for improvement of the quality of life (QOL) and reduction of medical costs. VAS also have been utilized as "bridges to recovery" for native hearts or for "semipermanent use" in patients without indications for heart transplantation, and superior clinical results of VAS therapy compared with conservative therapy have been reported in terms of one-year survival rate and improvement of QOL. Recently, several inexpensive VAS systems with an axial or centrifugal pump have been developed and the initial clinical trials of these systems have recently started in Western countries. Another remarkable technological advance in VAS is the Lion Heart (Arrow) which is a totally implantable VAS system the includes an energy transmission system. We expect that recent technological progress in VAS will improve the survival and QOL in patients with end-stage heart failure.

Endogenous cardiac troponin T modulates Ca(2+)-mediated smooth muscle contraction.

Mechanisms linked to actin filaments have long been thought to cooperate in smooth muscle contraction, although key molecules were unclear. We show evidence that cardiac troponin T (cTnT) substantially contributes to Ca(2+)-mediated contraction in a physiological range of cytosolic Ca(2+) concentration ([Ca(2+)](i)). cTnT was detected in various smooth muscles of the aorta, trachea, gut and urinary bladder, including in humans. Also, cTnT was distributed along with tropomyosin in smooth muscle cells, suggesting that these proteins are ready to cause smooth muscle contraction. In chemically permeabilised smooth muscle of cTnT(+/-) mice in which cTnT reduced to ~50%, the Ca(2+)-force relationship was shifted toward greater [Ca(2+)](i), indicating a sizeable contribution of cTnT to smooth muscle contraction at [Ca(2+)](i) < 1 µM. Furthermore, addition of supplemental TnI and TnC reconstructed a troponin system to enhance contraction. The results indicated that a Tn/Tn-like system on actin-filaments cooperates together with the thick-filament pathway.