Application of ultra-high voltage electron microscope tomography to 3D imaging of microtubules in neurites of cultured PC12 cells.

Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.

Short-chain fatty acids induce tissue plasminogen activator in airway epithelial cells via GPR41&43.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease generally divided based on the presence or absence of nasal polyps (NPs). One of the features of NPs is excessive fibrin deposition, which is associated with down-regulation of tissue plasminogen activator (t-PA) in NPs. As t-PA is expressed in epithelial cells, and epithelium is readily accessible to topical therapies, identifying compounds that can mediate the induction of t-PA would be a potential new strategy for the treatment of NPs. OBJECTIVE: The objective of this study was to determine whether short-chain fatty acids (SCFAs) can induce t-PA in airway epithelial cells via their known receptors GPR41 and GPR43. METHODS: We performed immunohistochemistry (IHC) to determine whether receptors for SCFAs, known as G protein-coupled receptor 41/free fatty acid receptor 3 (GPR41/FFAR3) and GPR43/FFAR2, are expressed in nasal tissue. Primary normal human bronchial epithelial (NHBE) cells were stimulated with different concentrations of SCFAs to test induction of t-PA, which was analysed by expression of mRNA and protein. Mediation of responses by SCFA receptors was evaluated by specific receptor gene silencing with siRNA. RESULTS: Immunohistochemistry study revealed that airway epithelial cells expressed GPR41 and GPR43. Acetic acid, propionic acid, butyric acid and valeric acid significantly induced t-PA expression from two- to tenfolds. The strongest inducer of t-PA from NHBE cells was propionic acid; cells stimulated with propionic acid released t-PA into the supernatant in its active form. Gene silencing of GPR41 and GPR43 revealed that induction of t-PA by SCFAs was dependent upon both GPR41 and GPR43. CONCLUSIONS AND CLINICAL RELEVANCE: Short-chain fatty acids were shown to induce airway epithelial cell expression of t-PA via GPR41 and GPR43. Topical delivery of potent compounds that activate these receptors may have value by reducing fibrin deposition and shrinking nasal polyp growth.

Novel scoring system and algorithm for classifying chronic rhinosinusitis: the JESREC Study.

BACKGROUND: Chronic rhinosinusitis (CRS) can be classified into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP displays more intense eosinophilic infiltration and the presence of Th2 cytokines. Mucosal eosinophilia is associated with more severe symptoms and often requires multiple surgeries because of recurrence; however, even in eosinophilic CRS (ECRS), clinical course is variable. In this study, we wanted to set objective clinical criteria for the diagnosis of refractory CRS. METHODS: This was a retrospective study conducted by 15 institutions participating in the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC). We evaluated patients with CRS treated with endoscopic sinus surgery (ESS), and risk of recurrence was estimated using Cox proportional hazard models. Multiple logistic regression models and receiver operating characteristics curves were constructed to create the diagnostic criterion for ECRS. RESULTS: We analyzed 1716 patients treated with ESS. To diagnose ECRS, the JESREC scoring system assessed unilateral or bilateral disease, the presence of nasal polyps, blood eosinophilia, and dominant shadow of ethmoid sinuses in computed tomography (CT) scans. The cutoff value of the score was 11 points (sensitivity: 83%, specificity: 66%). Blood eosinophilia (>5%), ethmoid sinus disease detected by CT scan, bronchial asthma, aspirin, and nonsteroidal anti-inflammatory drugs intolerance were associated significantly with recurrence. CONCLUSION: We subdivided CRSwNP in non-ECRS, mild, moderate, and severe ECRS according to our algorithm. This classification was significantly correlated with prognosis. It is notable that this algorithm may give useful information to clinicians in the refractoriness of CRS before ESS or biopsy.

[Extended thymectomy and intraoperative-intrapleural perfusion hyperthermo-chemotherapy for stage IVa invasive thymoma with myasthenia gravis].

A 37-year-old woman diagnosed with ocular myasthenia gravis was referred to our department. Chest computed tomography (CT) showed anterior mediastinal tumor and right pleural dissemination. Extended thymectomy and right intraoperative-intrapleural perfusion hyperthermo-chemothrapy (IPHC) were performed. Pathological diagnosis was invasive thymoma type B2 and stage IVa based on Masaoka's classification. The post operative course was uneventful. The patient underwent 4 cycles of adjuvant chemotherapy with doxorubicin, cisplatin, vincristine, and cyclophosphamide (ADOC), and is free from recurrence at 12 months postoperatively.

Associations between decay-accelerating factor polymorphisms and allergic respiratory diseases.

BACKGROUND: Allergic diseases such as asthma and allergic rhinitis are major causes of morbidity in developed countries. The pathology underlying allergic respiratory diseases is considered to be IgE-mediated type I allergy characterized by mucosal inflammation that occurs in response to allergen exposure. They are common diseases involving a complex inheritance. Complement systems are known to play an important role in allergic diseases. Decay-accelerating factor (DAF) is important for the regulation of the complement system and is a good candidate for determining the susceptibility to allergic diseases. OBJECTIVE: The present study aimed to investigate whether polymorphisms in the DAF gene are associated with allergic respiratory diseases in the Japanese population. METHODS: We performed mutation screenings of DAF and conducted a tag single-nucleotide polymorphisms (SNP) association analysis for 684 unrelated adult individuals with seasonal allergic rhinitis (SAR) with Japanese ceder pollen, 188 mite-sensitive adults with asthma, and 346 unrelated non-allergic healthy controls. RESULTS: DAF is located in the tight linkage disequilibrium (LD) block spanning 62 kb. The tag SNP analysis revealed that rs10746463 was significantly associated with SAR (P=0.00033) and mite-sensitive adult asthma (P=0.044). The rs2564978 and rs3841376 haplotypes, which are located in the promoter region of DAF, were in complete LD with rs10746463 (r2=1). Luciferase reporter assays with constructs containing the 5' flanking regions of DAF showed that the plasmid with rs2564978 C/rs3841376 deletion (the risk haplotype) had a statistically significantly lower transcriptional activity than that containing the rs2564978 T/rs3841376 insertion. CONCLUSIONS: Our results suggest that DAF is one of the genes involved in conferring susceptibility to allergic respiratory diseases and show that decreased levels of DAF may be associated with the enhanced specific IgE responses occurring in allergic diseases in the Japanese population.

A G protein directly regulates mammalian cardiac calcium channels.

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.

[Emergency operation due to complications of cardiac catheterization].

From January 2001 to July 2005, 4,741 cardiac catheterizations were performed on adult patients at our institute. Five of them underwent emergency operation due to complications of cardiac catheterization. The causes of operations were acute coronary occlusion due to coronary arterial dissection in 3 patients and bleeding due to perforation of the coronary artery in 2. Intra-aortic balloon pumping was used preoperatively in 4 patients and percutaneous cardio-pulmonary support in 2. Coronary artery bypass grafting was performed on all patients. Perforation sites of the coronary arteries were closed. One patient died 4 months after surgery, due to neurological damage, but the other 4 patients recovered and have been doing well. As coronary artery disruption is one of the main complications of cardiac catheterization, it is necessary to recognize the risk of hemodynamic derangement and to give prompt and appropriate treatment.

[Two-stage repair of the transposition of great arteries with interruption/coarctation of the aorta].

We report on 10 patients who underwent two-stage repair of transposition of the great arteries (TGA) with interruption (IAA) or coarctation (CoA) of the aorta. First, an operation for aortic arch reconstruction was performed: Blalock-Park with pulmonary artery banding (PAB) for IAA (5 patients), subclavian flap with PAB for CoA (4 patients) and end-to-end anastomosis without PAB (1 patient). All survived the first operation and had no significant pressure gradient with good growth of the ascending aorta, except for the 1 case without PAB. Half of the 8 patients who underwent PAB developed migration of the PAB. The arterial switch operation (ASO) was performed 0.7-12.6 (5.6+/-4.7) months after the first surgery. One patient with an abnormal coronary artery tract was lost after ASO. Five developed pulmonary artery stenosis and 1 developed supra-aortic stenosis late after ASO. Two patients need reoperation, 1 for supra-aortic stenosis, and the other for reCoA. Two-stage repair for TGA with IAA/CoA is still a useful method with a good operative result. However, strict follow-up is necessary because of the high frequency of late morbidity.

Experience with the Norwood procedure without circulatory arrest.

OBJECTIVE: We evaluated a new cardiopulmonary bypass technique that allowed complete avoidance of circulatory arrest and deep hypothermia in the Norwood procedure for hypoplastic left heart syndrome. METHODS: A total of 10 patients were included in this study. The arterial line of the cardiopulmonary bypass circuit was divided in two in a Y shape; one branch was used for cerebral perfusion through the innominate artery and the other for lower body perfusion through the cannula inserted into the descending thoracic aorta. Moderate hypothermia (29 degrees C-31 degrees C rectal temperature) and high pump flow (150-180 mL. kg(-1). min(-1)) were used. A valveless conduit between the right ventricle and the pulmonary artery was used in 6 patients as an alternative pulmonary blood source to a conventional Blalock-Taussig shunt (n = 4). RESULTS: Circulatory arrest was completely avoided throughout the operation in all cases, and no complications from the new cardiopulmonary bypass technique were seen. Early deaths occurred in 3 cases. Neurologic deficits were not seen among the survivors, and the postoperative course was stable and uneventful, including satisfactory renal function. CONCLUSIONS: The Norwood procedure for hypoplastic left heart syndrome was successfully accomplished with complete avoidance of circulatory arrest by means of cerebral perfusion through the innominate artery combined with cannulation of the descending aorta. A conduit between the right ventricle and the pulmonary artery seems an excellent alternative pulmonary blood source, although right ventricular function needs to be carefully monitored.

New technique in the transfer of an anomalously originated left coronary artery to the aorta.

A 21-month-old girl with an anomalous origin of the left coronary artery underwent a transfer of the left coronary artery to the aorta using a new technique of coronary prolongation. Because the anomalous left coronary artery arising from the left anterior aspect of the pulmonary trunk was too short to reach the aorta, a simple transfer of the left coronary artery to the aorta was deemed impossible. Therefore, a transfer was performed with the help of a coronary prolongation technique using the cuff of the pulmonary trunk and an aortic flap. The new route of the left coronary artery was established anterior to the pulmonary trunk. Postoperative angiography showed a patent left coronary artery without any narrowing or kinking, as well as an improved contractility of the left ventricle. Postoperative cardiac scintigraphy showed a decreased ischemic area. This technique is thus considered applicable when the orifice of the anomalous left coronary artery is too distant from the aorta for a direct anastomosis.

Subaortic stenosis caused by anomalous papillary muscle of the mitral valve.

We report on a rare case of subaortic stenosis caused by an anomalous posteromedial papillary muscle inserting directly into the anterior mitral leaflet, which had muscular extension to the subaortic region. Resection of the muscular extension alone was ineffective. A second operation including entire resection of the anomalous papillary muscle and mitral valve replacement resulted in successful relief of the obstruction. Microscopic findings of the resected tissue were compatible with those of hypertrophic cardiomyopathy.

Norwood procedure without circulatory arrest.

In the Norwood procedure for hypoplastic left heart yndrome, the distal descending thoracic aorta was cannulated just superior to the diaphragm through median sternotomy. In combination with cerebral perfusion through the graft anastomosed to the innominate artery, which was used as a systemic-to-pulmonary shunt later, this technique enabled us to completely avoid circulatory arrest and deep hypothermia throughout the operation.

Successful staged-Fontan operation in a patient with ectopia cordis.

Ectopia cordis is a very rare and often fatal disease. We report a successful surgery case of thoracoabdominal ectopia cordis with univentricular heart. This patient underwent a three-stage Fontan procedure, a right-modified Blalock-Taussig shunt at the age of 1 month, bidirectional Glenn shunt and pulmonary arterioplasty at 2 years 8 months, and finally a total cavopulmonary connection at 4 years. This patient was discharged from the hospital in good condition and has been doing well since. Thus, ectopia cordis is not a contraindication for a Fontan operation.

Mixed type of total anomalous pulmonary venous connection.

BACKGROUND: The mixed type of total anomalous pulmonary venous connection is a rare condition in which some diagnostic and surgical problems still remain to be solved. METHODS: In 9 patients a single pulmonary vein was connected to the systemic vein at a site different from the drainage site of the confluence of three other pulmonary veins. In 2 other patients, four pulmonary veins made a confluence which had two drainage sites. Correct diagnosis was made in all 7 patients who received cardiac catheterization but only in 5 of the 9 patients by color Doppler echocardiography. Total correction was performed in 3 patients and the single anomalous pulmonary vein was left uncorrected in 8 other patients. RESULTS: There were two in-hospital deaths. Seven patients with a single residual anomalous pulmonary vein have been in good condition without clinical symptoms of congestive heart failure or pulmonary hypertension. CONCLUSIONS: Diagnosis of mixed type of total anomalous pulmonary venous correction by echocardiography is sometimes difficult. When a mixed type is suspected, cardiac catheterization is recommended if the condition of the patient permits it. A single anomalous pulmonary vein may be left uncorrected without serious complications, but close observation is needed to prevent congestive heart failure and pulmonary vascular obstructive disease.

Selective cerebral perfusion technique during aortic arch repair in neonates.

We describe selective cerebral perfusion techniques for repair of the aortic arch in neonates. These techniques may help protect the brain from ischemic injury caused by a cessation of cerebral perfusion for aortic arch reconstruction in patients with hypoplastic left heart syndrome or interrupted aortic arch.

Postoperative cardiac rhythms with superior-septal approach and lateral approach to the mitral valve.

BACKGROUND: The superior-septal approach provides an excellent view of the mitral valve and therefore has received considerable interest. However, the safety of this approach is controversial because it requires division of the sinus node artery in most cases. METHODS: Postoperative cardiac rhythms were analyzed in 152 consecutive patients who underwent mitral valve procedures between January 1992 and February 1995 with a conventional right lateral left atriotomy (group 1, n = 69) or the superior-septal approach (group 2, n = 83). Follow-up ranged from 2 to 38 months, and the mean follow-up was 16.1 months in group 1 and 13.8 months in group 2. RESULTS: The mortality rate was similar in the two groups (1.4% in group 1 and 1.2% in group 2), and the causes of death were not related to the left atriotomy. At discharge, 96% of the patients in group 1 who were in sinus rhythm preoperatively and 78% of those in group 2 remained in sinus rhythm. At the last follow-up, 88% of these patients in group 1 and 83% in group 2 remained in sinus rhythm. Among the patients in atrial fibrillation or junctional rhythm before operation, 12% in group 1 and 11% in group 2 had regained sinus rhythm at the last follow-up. There were no significant differences in these values. CONCLUSIONS: Although the incidence of dysrhythmias was higher with the superior-septal approach in the early postoperative period, this approach provides an excellent operative view of the mitral valve and similar results in terms of late postoperative cardiac rhythms as the right lateral left atriotomy.

The effects of caffeine on the electrical properties of isolated, single rat ventricular cells.

The effects of caffeine on ionic currents in isolated, single cells from rat ventricles were compared with the effects of adrenaline. In current-clamp experiments, caffeine (1.0 to 10.0 mM) increased both the height of the plateau and the duration of the action potential. Both caffeine and adrenaline increased the amplitude and duration of the slow responses. Voltage-clamp experiments reveal that the repolarization phase of the action potential in isolated cells is determined mainly by the slow inward current (Is), which has voltage-dependent kinetics similar to the Is in intact cardiac tissue: No evidence of a time-dependent outward current (Ix), or a hyperpolarization-induced inward current (Ih) or (If) was found, although a time-independent background current (Ik1) was observed. Caffeine increased the amplitude of Is, but did not change its time course, its voltage dependency (of steady-state inactivation and activation), or its apparent reversal potential. Ik1 is not affected by the caffeine. The results indicated that the effects of caffeine were nearly identical to those of adrenaline. The effects of caffeine on the electrical properties of single cells from rat ventricles derive chiefly from its action on Is channels.