Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic F(o)F(1)-ATP synthase expressed in Escherichia coli.

F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.

Symmetrical Skin Lesions on the Gluteal Region in a Patient with Anti-Laminin-332 Mucous Membrane Pemphigoid.

Mucous membrane pemphigoid (MMP), previously called cicatricial pemphigoid, is a rare subepidermal immunobullous disorder that primarily affects the mucous membranes (1,2). MMP is divided into two major subtypes, anti-BP180-type MMP and anti-laminin-332 (previously called laminin 5 or epiligrin) MMP. Anti-laminin-332 MMP is known to be associated with malignant tumors (3), which may cause overexpression of autoantibodies and induce autoimmunity to laminin-332 (4). MMP primarily affects the mucous membranes, and widespread skin lesions are rare. In MMP, circumscribed skin lesions have been previously reported as occurring on the head, neck, and upper trunk (5). We report a case of anti-laminin-332 MMP presenting with symmetrical skin lesions characteristic of MMP on the weight-bearing areas of the gluteal region. A 66-year-old Japanese man presented with a month-long history of multiple erosions and blisters on the mucous membranes and skin, with conjunctival hyperemia, nasal obstruction, oral pain, and hoarseness of voice. Three days before the first visit, he was diagnosed with gastric cancer with liver metastasis by gastrointestinal endoscopy and abdominal ultrasound examination for tarry stool. Physical examination demonstrated erosions and tense bullae on the conjunctivae, tongue, and lips (Figure 1, a,b), as well as erosive erythematous skin lesions on the nape, right index finger, both legs, and symmetric lesions on the gluteal region (Figure 1, c). His body weight was 86 kg. Laboratory examinations showed slight liver dysfunction and elevation of C-reactive protein levels. Histopathologic examination of the skin lesions demonstrated subepidermal blisters with lymphocytic and eosinophilic infiltrates (Figure 1, d,e). Direct immunofluorescence (IF) revealed linear deposits of IgG and C3, but not IgA, along the basement membrane zone (BMZ) (Figure 1, f,g). An IgG subclass study showed IgG1 and IgG4 deposits. Indirect IF on normal human skin revealed weak positivity for IgA anti-keratinocyte cell surface antibodies and IgG anti-BMZ antibodies, which were bound to the dermal side of 1 mol/L NaCl-split skin (Figure 1, h). IgG immunoblot analyses of both normal human epidermal and dermal extracts showed negative results (including BP230, BP180, 290 kDa type VII collagen, and 200 kDa laminin-γ1). Immunoprecipitation using radio-labeled cultured keratinocyte lysate demonstrated positive reactivity with laminin-332 (Figure 1, i). We established the diagnosis of anti-laminin-332 MMP. We started treatment with oral minocycline (200 mg/day) and niacinamide (900 mg/day) with topical corticosteroids without any effect after 2 weeks of therapy. Administration of oral prednisolone (40 mg/day) with topical corticosteroids and alprostadil ointment on the skin lesions, as well as beclometasone dipropionate powder on the oral lesions resulted in significant improvement of mucocutaneous lesions within 10 days. Although the gastric cancer and liver metastasis initially responded to chemotherapy with fluorouracil and cisplatin, the patient succumbed to multiple organ failure 9 months after the initial visit. Anti-laminin-332 antibodies were originally detected by immunoprecipitation, as in our case. Immunoblotting of purified human laminin-332 have been subsequently developed, which detects the 165/145 kDa α3, 140 kDa ß3, and 105 kDa γ2 subunits of laminin-332 in various patterns (6). Today, the ELISA system uses laminin-332 preparations as adjunct diagnostic tools in MMP (7). Occasionally, a wide spectrum of autoantibodies is detected in MMP, for example, MMP with IgG antibodies to both BP180 and laminin-332, which were considered to be developed via epitope spreading. Detection of circulating IgA autoantibodies against the skin have also been reported in MMP (8). However, the pathogenic significance and mechanisms of coexistence of IgG anti-laminin-332 antibodies and IgA anti-keratinocyte cell surface antibodies found in our case are currently unknown. It is generally considered that IgG1 antibodies activate complements and are pathogenic in MMP, while IgG4 antibodies behave as blocking antibodies and are protective. In our case, direct IF revealed IgG1 and IgG4 deposits; the same was reported in a previous case report (9). The pathogenic roles of autoantibodies with different IgG subclasses need to be analyzed in further studies. Conjunctival mucosal lesions in MMP may occur by rubbing of the eyes due to irritation. Blinking subjects the conjunctivae to repeated friction. Vocal cords vibrate during breathing and speaking. The tongue moves while eating and drinking; in particular, the tip of the tongue gets into frequent contact with the inner sides of the incisor teeth. In the present case, characteristic symmetrical skin lesions were seen on the weight-bearing areas of the gluteal region on bony prominences which receive mechanical stresses in the sitting position. These skin lesions were subjected to repeated stretch and pressure stresses, but no ischemic changes were observed, such as decubitus ulcers. Therefore, the symmetrical skin lesions in the gluteal region as well as the ocular and oral mucosal lesions seen in our patient might have resulted from the same mechanism of pathogenesis. We reported a case of anti-laminin-332 MMP presenting with symmetrical gluteal skin lesions, probably induced by mechanical stress. MMP primarily affects the mucous membranes, and widespread skin lesions are rare. Our case emphasizes that clinicians need to specifically check for the presence of skin lesions on weight-bearing parts of the body during examination of patients with suspected MMP.

[Advantages of starting outpatient preoperative evaluation system].

BACKGROUND: We started an outpatient preoperative evaluation system in September, 2006, and reviewed whether preoperative examination of outpatients contributed to efficiency of duties of anesthesiologists. METHODS: A questionnaire paper was delivered to each anesthesiologist before and after starting the outpatient preoperative evaluation system. Items investigated are patient background (age, sex, ASA-PS classification, planned anesthesia methods), time required, the factor that took time, the end time of preoperative examination. RESULTS: The number investigated were 245 and 240 cases before and after starting the outpatient preoperative evaluation system, individually. Most reasons why we needed time in the wards were "absence of the patient" (7.3%). We visited an average of 1.15 times to the patient in the wards. The ratio that we finished the preoperative examination after five o'clock in the evening was 29.4% before starting our system. On the other hand, we finished the preoperative examination by approximately five o'clock in the evening and time for one case was shortened from 35.5 +/- 10.9 to 29.5 +/- 8.9 minutes after starting our system. CONCLUSIONS: Outpatient preoperative evaluation raises the efficiency of duties of anesthesiologists to finish preoperative examination before hospitalization.

[Use of the GlideScope video laryngoscope in 50 pediatric patients].

BACKGROUND: The GlideScope video laryngoscope (Verathon Inc. Bothell, Washington, USA) is a relatively new device for tracheal intubation, which provides a excellent glottic visualization. We here report the clinical experience of the GlideScope (small) in 50 pediatric patients. METHODS: Tracheal intubation with GlideScope (small) was performed in 50 consecutive pediatric patients requiring orotracheal intubation for surgery. The view of glottic opening was scored according to the classification of Cormack-Lehane. The time required to intubate and the number of intubation attempts were recorded. RESULTS: In all, 50 children included 4 neonates, 8 infants under 1 year and 38 children between 1 year and 9 years. Cormack-Lehane classification 1 or 2 was obtained in 74% and 22%, respectively, and successful intubation was achieved in 48 of 50 children (96%). In remaining two babies, GlideScope failed to intubate the trachea. The mean +/- SD time for instrumentation in successful intubation at first attempt was 56.6 +/- 34.2 seconds. CONCLUSIONS: GlideScope seemed to be a novel device in pediatric patients. Further studies are required to evaluate the usefulness in neonates, small infants and children with a difficult airway.

[A case report of successful treatment of a child with respiratory distress using non-invasive continuous positive airway pressure via helmet].

Helmet is a new device of non-invasive continuous positive airway pressure (CPAP). Few cases have been described about usage of the helmet in children. We describe successful treatment of a child with respiratory distress using the helmet-delivered non-invasive CPAP. A 2-month-old male infant (3.1 kg) with multiple anomalies (cardiovascular, facial, and vertebral) developed respiratory distress after extubation. The helmet was well tolerated regardless of facial anomaly. Helmet CPAP started at initial settings of CPAP 8 cm H2O and FI(O2) 0.7, improved oxygenation. Pa(O2)/FI(O2) ratio increased from 106 to 316, and chest X-rays showed a marked improvement (15 hour after NPPV initiation). The helmet offers important advantage: the possibility of fitting to any children, regardless of any facial or external anomalies.

[Crisis management in pediatric anesthesia].

We describe the risk management of pediatric anesthesia. The most important risk management of pediatric anesthesia is airway and temperature management. Neonates and infants easily become hypoxic due to their insufficient functional residual capacity. Therefore airway management is most important not only during induction of anesthesia but also during maintenance of anesthesia and extubation. The management of patients' temperature, including control of room temperature should be taken into consideration. In addition, careful attention should be paid not to introduce air bubbles in any lines, especially in patients with congenital heart diseases.

Impaired spermatogenesis by testicular sarcoidosis.

Testicular involvement by sarcoidosis is a rare condition. A 23-year-old Japanese man had asymptomatic bilateral testicular lesions, which were detected by gallium scintigram, together with lesions located bilaterally in the uvea, lungs and hilar, and mediastinal lymph nodes and unilateral supraclavicular lymph nodes. Semen analysis demonstrated severely impaired spermatogenesis. Treatment with corticosteroid dramatically improved these lesions and restored spermatogenesis. This case report suggests that testicular sarcoidosis may cause male infertility.

[Use of a new video-laryngoscope (GlideScope) in patients with a difficult airway].

We describe the clinical use of a new video-laryngoscope (GlideScope, GS) in patients with a difficult airway and morbid obesity. In 4 patients with a difficult airway, showing a Cormack-Lehane grade III view with Macintosh direct laryngoscope, the glottic opening (Cormack-Lehane grade I or II) was visualized with GS. In 2 patients, showing a Cormack-Lehane grade IV view with Macintosh direct laryngoscope, Cormack-Lehane grade II view of the glottic opening was obtained. GS also provided a good view of glottic opening in a patient with morbid obesity. GS will have a profound impact on the management of the difficult airway.

Effectiveness and safety of the thrombolytic therapy for acute pulmonary thromboembolism: results of a multicenter registry in the Japanese Society of Pulmonary Embolism Research.

OBJECTIVE: To assess the effectiveness and safety of thrombolytic treatment for acute pulmonary thromboembolism (APTE), especially in the hemodynamically stable patients with right ventricular afterload stress. METHODS AND RESULTS: In a total of 221 patients with APTE, the association between thrombolytic treatment and the clinical outcomes were investigated. Thrombolysis was given to 121 patients (Thrombolytic Group), and the remaining 100 patients were treated with anticoagulation alone (Anticoagulation Group). In both patients with prolonged shock and patients who were hemodynamically stable without right ventricular afterload stress, the rate of death and recurrence of APTE in Anticoagulation Group were similar to those in Thrombolytic Group. In patients with right ventricular afterload stress, better outcomes were observed in Thrombolytic Group than in Anticoagulation Group, although the difference did not reach statistical significance. There was no significant difference in the rate of major bleeding episode between these two groups. CONCLUSION: Our results suggested that performing thrombolytic treatment in APTE patients with right ventricular afterload stress should be considered even in Japan.

Interfacial deformation between an impacting water drop and a silicone-oil surface.

Detailed observations have been conducted on the interfacial deformation of a silicone oil surface and a water drop falling on it. Eleven kinds of silicone oils with wide variations of kinematic viscosity, nu(T)=1-10(5) mm(2)/s, have been tested. The oil surface is disturbed by a water drop with a diameter d(L)=3.1 mm, which falls freely on it from a height of 100-1000 mm. Special attention has been directed to the maximum depth of the cavity formed on the oil surface D(M) and to the maximum diameter of the water drop spreading on the oil surface d(M). We have categorized the configurations of the oil cavity into seven patterns, and those of the water drop at the oil-water interface into five patterns. The maximum cavity depth D(M)/d(L) can be well correlated by a dimensionless group Re(TL)We(TL), where Re(TL) is Reynolds number based on d(L) and nu(T) and We(TL) is Weber number with the water density and surface tension of oil. The maximum diameter of the impacting water drop d(M)/d(L) can be correlated by the Reynolds number with a viscosity of water (Re(L)) and the Ohnesorge number (Oh). Moreover, the condition under which the impacting water drop is smashed into pieces has been also examined based on Re(L) and Oh.

Recent developments in diagnostic imaging techniques and management for acute pulmonary embolism: multicenter registry by the Japanese Society of Pulmonary Embolism Research.

OBJECTIVE: There are many reports on the diagnosis and management of acute pulmonary embolism (APE), but there have been no investigations concerning the actual conditions in which recent developments in diagnostic imaging techniques and therapies have been applied in clinical practice. The present study was designed to investigate the changes in diagnostic imaging techniques and therapies for APE. METHODS AND PATIENTS: Three hundred and nine APE patients diagnosed during January 1994-October 1997 (Group 1) were compared with 257 APE patients diagnosed during November 1997-October 2000 (Group 2) in terms of the diagnostic imaging techniques and therapies for APE. RESULTS: Compared with Group 1, pulmonary angiography and contrast-enhanced computed tomography were more frequently performed for diagnosis in Group 2 [45.3% vs 56.8% (p = 0.0069) and 13.9% vs 57.6% (p < 0.0001), respectively]. Heparin and vena cava filter were used more often in Group 2 [74.4% vs 82.1% (p = 0.033) and 18.4% vs 33.9% (p < 0.0001), respectively]. The frequency of thrombolytic therapy was unchanged between the two groups. Warfarin use in discharged patients increased from 71.9% to 83.8% (p = 0.0022). However, the examination rates for deep vein thrombosis (DVT) were low (60.8% in Group 1 and 65.4% in Group 2, p = 0.29) and unchanged using any imaging techniques. CONCLUSION: The diagnostic imaging techniques for APE increased in variety and the management has improved, while the diagnosis for DVT remains unchanged.

Rudimentary right ventricle to pulmonary artery shunt in the Norwood procedure.

We describe here successful palliative repair of tricuspid atresia, hypoplastic right ventricle, transposition of the great arteries, and hypoplastic aortic arch in a neonate. The repair consisted of the Norwood procedure with a rudimentary right ventricle to pulmonary artery shunt, which was located on the right side of a neo-aorta. This procedure could be a useful adjunct to avoid left ventriculotomy and its subsequent dysfunction.

Identification of annexin 1 as a novel autoantigen in acute exacerbation of idiopathic pulmonary fibrosis.

Consistent with the hypothesis that pulmonary epithelial apoptosis is the key to the acute exacerbation of idiopathic pulmonary fibrosis (IPF), we conducted serological identification of Ags by recombinant expression cloning (SEREX) analysis using type II alveolar cell carcinoma (A549) cell lines to identify disease-related Abs. In a survey of Abs to the recombinant autoantigens identified by SEREX analysis, five Abs were identified as novel candidates for the acute exacerbation of IPF. Abs to annexin 1 were detected in 47 and 53% of the sera and bronchoalveolar lavage materials from patients with acute exacerbation of IPF. Some identical TCR Vbeta genes were identified in sequential materials obtained at 1-3 mo in all 10 acute exacerbation IPF cases, suggesting that some infiltrating CD4-positive T cells sharing limited epitopes expand by Ag-driven stimulation during disease extension. The CDR3 region of these identical TCR Vbeta genes showed high homology with the N-terminal portion of annexin 1, including in the HLA-DR ligand epitopes predicted by TEPITOPE analysis. By Western blotting analysis and observation of the CD4-positive T cell responses in bronchoalveolar lavage samples, the N-terminal portion of annexin 1 was cleaved and found to induce marked proliferative responses of CD4-positive T cells in three patients. Our study demonstrates that annexin 1 is an autoantigen that raises both Ab production and T cell response in patients with acute exacerbation of IPF, and that the N-terminal portion of annexin 1 plays some role in the pathogenesis of acute exacerbation in IPF patients.

Ascending to descending aorta bypass for middle aortic syndrome.

Though stenoses of the descending aorta and its branches are seen with congenital anomalies or systemic inflammation, occlusion of the descending aorta is extremely rare. A patient with an occluded hypoplastic descending thoracic aorta required re-operation because of graft failure between the descending thoracic aorta and the infrarenal abdominal aorta. The etiology of the aortic occlusion in this case is unknown, but inflammation, such as Takayasu disease, is speculated.

Clinical path modeling in XML for a web-based benchmark test system for medication.

Many hospitals have introduced the Clinical Path (Path) to improve medical procedures. A Path is a way to manage care and check lists for a certain disease, providing a useful tool for hospital management. Paths can help hospitals reduce the duration of hospitalization and variations in care of patients while increasing hospital revenue. Nowadays, Paths are made by each hospital and there is no standard format. Benchmark testing between Paths used by different hospitals is important for evaluating medical practices, in order to develop and improve more effective practices. However, as the formats used in Paths are not standardized, benchmark testing of Paths is no easy task. To start benchmark testing of Paths, we compare medication in Paths and introduce description rules of medication in XML. Based on these, we developed a prototype system that enables us to compare the difference of medications in Paths prescribed between multiple of hospitals.

Electronic clinical path system based on semistructured data model using personal digital assistant for onsite access.

Clinical Paths (Paths) have been introduced by different hospitals for patient care management. An Electronic Clinical Path (ECP) with onsite access provision seems to improve the efficiency of medical staffs because they can share vast medical information about patients at a time and also can reuse accumulated data easily, which is impossible with paper-based Path. Data model is the basis for implementing ECP. However, there is no established model for ECP. The purpose of this study is to introduce a model for ECP and implement an ECP with onsite access system. We introduced a Semistructured Data Model (SSDM) for ECP, and implemented a Web application system based on this model using Personal Digital Assistant (PDA) as inputting device. Our system functioned as expected with wireless LAN, and users handled the data on bedside using PDA. By introducing SSDM, we showed the correspondence between schema of Paths and implementation of ECP.

Incidence and clinical characteristics of chronic pulmonary thromboembolism in Japan compared with acute pulmonary thromboembolism: results of a multicenter registry of the Japanese Society of Pulmonary Embolism Research.

The incidence of acute pulmonary thromboembolism (APTE) in Japan is quoted as being extremely low compared with the United States, and the incidence and clinical characteristics of chronic pulmonary thromboembolism (CPTE) in Japan is unknown, so this study investigated these aspects of CPTE in 309 patients with APTE and 68 patients with CPTE. The ratio of the incidence of CPTE to APTE was 0.22 and there was no significant difference in age or sex between the APTE and CPTE patients. All of the predisposing factors for pulmonary thromboembolism, except for thrombophilia, were more frequently seen in the patients with APTE. There are some differences in the incidence and clinical characteristics of CPTE compared with APTE between Japanese and American patients in Japan, suggesting that the pathogenesis of CPTE in Japan may differ from that in the USA.