Non-competitive cyclic peptides for targeting enzyme-substrate complexes.

Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.

Betti reaction enables efficient synthesis of 8-hydroxyquinoline inhibitors of 2-oxoglutarate oxygenases.

There is interest in developing potent, selective, and cell-permeable inhibitors of human ferrous iron and 2-oxoglutarate (2OG) oxygenases for use in functional and target validation studies. The 3-component Betti reaction enables efficient one-step C-7 functionalisation of modified 8-hydroxyquinolines (8HQs) to produce cell-active inhibitors of KDM4 histone demethylases and other 2OG oxygenases; the work exemplifies how a template-based metallo-enzyme inhibitor approach can be used to give biologically active compounds.

Short communication: opposing effects of lactoferrin on the proliferation of fibroblasts and epithelial cells from bovine mammary gland.

Lactoferrin is present in several physiologic fluids, including milk and colostrum. Recently, evidence has accumulated that lactoferrin acts as a regulator of cell proliferation. Lactoferrin mRNA and protein levels in bovine mammary glands are known to markedly increase after cessation of milking. To clarify the role of bovine lactoferrin (bLF) in mammary involution and remodeling during dry periods, we investigated whether bLF affects the proliferation of cultured cells derived from bovine mammary gland and examined the mechanism underlying the proliferative response to bLF. Addition of bLF to the culture medium increased the proliferation of bovine mammary stromal fibroblasts (bMSF), but decreased that of bovine mammary epithelial cells (bMEC). Proliferation was significantly increased in the bMSF treated with bLF (100µg/mL or greater) as compared with unstimulated cells. The maximal proliferative effect of bLF on bMSF occurred at 1,000µg/mL, such that the proliferation of the bLF-stimulated bMSF was approximately 2.5 times that of unstimulated cells. The bLF increased the production of proliferating cell nuclear antigen and rapid phosphorylation of the p44/p42 mitogen-activated protein kinase in bMSF, but not in bMEC. The bLF-induced proliferation and production of proliferating cell nuclear antigen in bMSF was suppressed by U0126, a specific inhibitor of mitogen-activated protein kinase. Furthermore, treatment with bLF for 24h decreased the mRNA levels of the 3 isoforms of transforming growth factor ß in bMSF (16-66%) but upregulated those in bMEC (122-157%). These opposite effects of bLF on the proliferation of epithelial and fibroblast cells and their expression of transforming growth factor ß may play a crucial role in bovine mammary involution and remodeling.

Disc displacement and changes in condylar position.

OBJECTIVES: To determine whether disc displacement (DD) can be a factor causing changes in condylar position in the glenoid fossa, using limited cone beam CT (LCBCT) images taken in the same time period as MRI that verified DD. METHODS: The study included 60 joints in 57 male and female subjects aged 12-20 years (mean age 14.8 years). Subjects fulfilling the inclusion criteria were enrolled from a pool of post-orthodontic patients, and divided into four groups according to their disc status confirmed by MRI: partial DD (PDD), total DD with reduction (TDDWR), lateral DD (LDD) and medial DD (MDD). Changes in joint space from previously reported norms were measured on LCBCT images. RESULTS: In PDD, the condyles were displaced posteriorly in the fossae with a mean anterior space of 2.7 ± 0.5 mm (normal 1.3 ± 0.2 mm) and a posterior space of 1.8 ± 0.4 mm (normal 2.1 ± 0.3 mm). In TDDWR, the condyles were displaced not only posteriorly as observed in PDD, but also vertically with a reduced superior space of 1.9 ± 0.4 mm (normal 2.5 ± 0.5 mm). In LDD, the lateral space was significantly increased to 2.5 ± 0.3 mm (normal 1.8 ± 0.4 mm), while central and medial spaces were significantly decreased to 2.2 ± 0.5 mm (normal 2.7 ± 0.5 mm) and 1.7 ± 0.4 mm (normal 2.4 ± 0.5 mm), respectively. In MDD, the medial space was increased and the lateral and central spaces were significantly decreased. CONCLUSIONS: These results indicate that DD in adolescents and young adults can cause the condyle to change its position in the fossa with alterations in joint space which depend on the direction and extent of DD.

Botulinum toxin-A use in paediatric hypertonia: Canadian practice patterns.

BACKGROUND: This study aims to assess current practices of Canadian physicians providing botulinum toxin-A (BoNT-A) treatments for children with hypertonia and to contrast these with international "best practice" recommendations, in order to identify practice variability and opportunities for knowledge translation. METHODS: Thirteen Canadian physicians assembled to develop and analyze results of a cross-sectional electronic survey, sent to 50 physicians across Canada. RESULTS: Seventy-eight percent (39/50) of physicians completed the survey. The most frequently identified assessment tools were Gross Motor Function Classification System, Modified Tardieu Scale and neurological examination. Goal-setting tools were infrequently utilized. Common indications for BoNT-A injections and the muscles injected were identified. Significant variability was identified in using BoNT-A for hip displacement associated with hypertonia. The most frequent adverse event reported was localized weakness; 54% reporting this "occasionally" and 15% "frequently". Generalized weakness, fatigue, ptosis, diplopia, dysphagia, aspiration, respiratory distress, dysphonia and urinary incontinence were reported rarely or never. For dosage, 52% identified 16 Units/kg body weight of Botox® as maximum. A majority (64%) reported a maximum 400 Units for injection at one time. For localization, electrical stimulation and ultrasound were used infrequently (38% and 19% respectively). Distraction was the most frequently used pain-management technique (64%). CONCLUSIONS: Canadian physicians generally adhere to international best practices when using BoNT-A to treat paediatric hypertonia. Two knowledge-translation opportunities were identified: use of individualized goal setting prior to BoNT-A and enhancing localization techniques. Physicians reported a good safety profile of BoNT-A in children.

Spatiotemporal classification of environmental monitoring data in the Yeongsan River basin, Korea, using self-organizing maps.

Environmental monitoring data for planning, implementing and evaluating the Total Maximum Daily Loads (TMDL) management system have been measured at about 8-day intervals in a number of rivers in Korea since 2004. In the present study, water quality parameters such as Suspended Solids (SS), Biochemical Oxygen Demand (BOD), Dissolved Oxygen (DO), Total Nitrogen (TN), and Total Phosphorus (TP) and the corresponding runoff were collected from six stations in the Yeongsan River basin for six years and transformed into monthly mean values. With the primary objective to understand spatiotemporal characteristics of the data, a methodologically systematic application of a Self-Organizing Map (SOM) was made. The SOM application classified the environmental monitoring data into nine clusters showing exclusively distinguishable patterns. Data frequency at each station on a monthly basis identified the spatiotemporal distribution for the first time in the study area. Consequently, the SOM application provided useful information that the sub-basin containing a metropolitan city is associated with deteriorating water quality and should be monitored and managed carefully during spring and summer for water quality improvement in the river basin.

[Ascending aortic aneurysm and aortic valve regurgitation long after operative repair of coarctation of the aorta: report of a case].

We report a case of surgical treatment for ascending aortic aneurysm and aortic valve regurgitation (AR) 24 years after operative repair of coarctation of the aorta (CoA). The patient was a 32-year-old man who had undergone operative repair of CoA and patent ductus arteriosus (PDA) ligation when 8 years old, and was followed since then. However, since 14 years after the operation, dilation of his ascending aorta and AR was observed. Then the AR deteriorated and the ascending aorta dilated, and at 24 years after operation he had symptoms of heart failure. So we performed ascending aorta replacement and aortic root replacement (reimplantation). Despite primary success of the operative repair of CoA, however. 9% of patients develop aortic aneurysms long after the operation. Therefore, long-term follow-up is needed after repair of coarctation of the aorta.

[Ventricular septal defect with aortic valvular regurgitation complicated with infective endocarditis; report of a case].

We report a case of ventricular septal defect (VSD) associated with infective endocarditis complication of aortic and pulmonary valvular regurgitation. He received treatment for pyelonephritis and after 2 months he was suffering from congestive heart failure. Echocardiography showed vegetation on the aortic valve and the anterior semilunar cusp of the pulmonary valve. He was referred to our department for surgical repair. Aortic valvular replacement, pulmonary valvular repair and direct closure of VSD were performed. The postoperative course was uneventful and the patient discharged at 28 postoperative day.

[Noonan syndrome with atrial septal defect and hypertrophic cardiomyopathy].

Noonan syndrome is one of the most common nonchromosomal syndromes seen in children with congenital heart disease. The major cause of death is congestive heart failure. We report a case of Noonan syndrome with hypertrophic cardiomyopathy and arterial septal defect (ASD). Since 10 years old he had been suffering from congestive heart failure. ASD patch closure with a fenestrated flap valve patch was performed at 12 years of age. After operation, left-to-right shunt flow through the patch gradually decreased and he was discharged at 19 postoperative day in good condition.

[A low birth weight infant (904 g) associated with ventricular septal defect].

Published data has suggested that low body weight is risk factor for surgical intervention in congenital heart disease. We report a case of a low birth weight infant with ventricular septal defect (VSD) and pulmonary hypertension (PH). A female infant was born at 31 weeks' gestation. The birth weight was 904 g. At birth she was suffering from congestive heart failure. We diagnosed VSD and PH. We performed pulmonary artery banding at 23 days of age. She accepted the band at a circumference of 15.5 mm. And we performed VSD patch closure and de-banding at 175 days of age. After operation she was on good course and discharged at 19 postoperative day.

[Aortoesophageal fistula due to thoracic aortic aneurysm; report of a case].

We report a 74-year-old man with aortoesophageal fistula due to aotic aneurysm. He underwent 2 stage operations. At the 1st operation the graft replacement of thoracic aorta and esophagectomy were performed. Inflammatory reactions improved with systemic administration of antibiotics and continuous irrigation of the thoracic cavity. On the 21st postoperative day, the esophagus was reconstructed by gastrointestinal interposition technique via ante-thoracic route. On the 58th post operative day he was discharged.

[New method for preparing proximal anastomotic system].

Heartstring is a useful device. However, the device failure at the time of loading the seal into the delivery device is a troublesome issue. To avoid this problem, we invent a new method using 2 tourniquets made of 5 mm-wide woven Teflon tapes and plastic tubes. Using our method, the loading procedure became easier and more reliable.

[Successful surgical management of papillary muscle rupture following acute myocardial infarction in an elderly patient].

Papillary muscle rupture is rare but catastrophic complication of acute myocardial infarction. We report a 91-year-old woman who underwent successful management of papillary muscle rupture following acute myocardial infarction. She was transferred to our hospital because of severe pulmonary edema and cardiogenic shock. Echocardiography revealed severe mitral valve regurgitation due to total rupture of anterolateral papillary muscle. After intubation, intraaortic balloon pumping (IABP) and percutaneous cardiopulmonary support (PCPS) insertion, mitral valve replacement was successfully performed. She was discharged 134 days after operation. In papillary muscle rupture deteriorating hemodynamics, early diagnosis and immediate cardiopulmonary support are required before surgical treatment. She was, to the best of our knowledge, the oldest among the reported cases of successful surgical treatment of papillary muscle rupture in Japan.

Structure and mechanism of arylamine N-acetyltransferases.

Arylamine N-acetyltransferases (NATs) are a family of phase II drug-metabolising enzymes which are important in the biotransformation of various aromatic and heterocyclic amines and hydroxylamines, arylhydrazines and arylhydrazides. NATs are present in a wide range of eukaryotes and prokaryotes. Humans have two functional NAT isoforms, both of which are highly polymorphic. The pharmacogenetics of NATs is an area which has been extensively studied. The determination of the X-ray crystal structure of NAT from Salmonella typhimurium led to the identification of the catalytically essential triad of residues: Cys-His-Asp, which is present in all functional NAT enzymes. Recent co-crystallisation data and in silico docking studies of NAT from Mycobacterium smegmatis with substrates and inhibitors have aided the identification of important contact residues within the active site. The X-ray crystal structures of four prokaryotic NAT proteins have now been determined, and these have been used to generate structural models of eukaryotic NATs, providing valuable insight into their active-site architecture. In addition to aiding crystallographic experiments, recent progress in the production of recombinant prokaryotic and eukaryotic NATs has allowed comparative studies of the kinetics and activity profiles of these enzymes. In this review we present an overview of recent structural and activity studies on NAT enzymes, and we outline how in silico methods may be used to predict NAT protein-ligand interactions based on the current knowledge.

First Report of Pythium aphanidermatum on Basella rubra in Japan.

Basella rubra L. (Indian spinach, Japanese name Tsurumurasaki) is cultivated worldwide as an ornamental and its aerial parts are consumed as a vegetable and health food. A severe rot of leaves, stems, and roots was found on B. rubra cv. Midori grown in a commercial field at Naruto-City (34°11'N, 134°36(E) in Tokushima Prefecture, Japan from May to September 2004. More than 50% of approximately 2,100 plants in the field were destroyed by the disease. Soft, black lesions appeared initially at the base of the stems in 2- to 5-month-old plants and enlarged gradually upward and downward within 2 days after plant injury caused by heavy rainfall. A fluffy, white mass of mycelium appeared on the surface of lesions under moist conditions. A Pythium species was routinely isolated from rotted stems and roots and identified as Pythium aphanidermatum (Edson) Fitzpatrick on the basis of its morphology on a grass leaf water culture (2). Characteristics of isolate OPU743 (NBRC No. 101556, MAFF No. 239847) included hyphae as much as 10 µm wide, terminal oogonia 17.8 to 28.8 µm in diameter, and monoclinous or diclinous antheridia 8.8 to 10.9 µm wide, either terminal or intercalary, with one or rarely two per oogonium. Oospores were aplerotic, 13.5 to 22.6 µm in diameter. Sporangia were terminal or occasionally intercalary, and either inflated filamentous hyphae or complexes of swollen hyphal branches were present in cultures. Cardinal temperatures for growth on potato carrot agar were 10°C minimum, 37°C optimum, and 40°C maximum with a daily radial growth rate of 32.9 mm at 25°C. Pathogenicity tests were conducted on potted 3-month-old B. rubra (cv. Midori). A wound (1 mm deep and 5 mm long made by a razor) on the surface of the stem of the plant was inoculated with an 8-mm-diameter agar disk of isolate OPU743 (grown at 25°C for 48 h on potato dextrose agar) attached to a stem of the plant using a paraffin film. The inoculated plants were placed in transparent plastic bags and kept in a growth chamber at 24 to 26°C with continuous light (82 to 126 µmol·m-2·s-1). The experiment was done four times with three plants in each experiment. The same number of plants was used for the noninoculated control. Dark brown rot of stems and leaves developed on 66.7% of inoculated plants within 2 days after inoculation. P. aphanidermatum reisolated from diseased tissues was morphologically identical to the original isolate OPU743. Noninoculated control plants showed no symptoms. P. aphanidermatum has been described on B. rubra in Brazil (1), but has not been reported from other regions of the world. To our knowledge, this is the first report of P. aphanidermatum on B. rubra in Japan. References: (1) C. F. Robbs. A Lavoura 74:43, 1972. (2) A. J. Van Der Plaats-Niterink. Stud. Mycol. 21:1, 1981.

A case of bullous pemphigoid associated with autoantibodies targeting antigenic sites other than the NC16a domain of BP180.

Bullous pemphigoid (BP) is associated with autoantibodies to the 180-kDa BP antigen (BP180), and the antigenic site exists on noncollagenous 16a (NC16a) domain of BP180. We now report a male BP patient whose IgG autoantibodies did not react against the NC16a domain of BP180 by either immunoblotting or ELISA, whereas they did react with BP180 extracted from normal human keratinocytes. Anti-BP180 cicatricial pemphigoid was ruled out due to the lack of conjunctival mucosal involvement and the absence of scarring in the oral cavity. Our findings indicate that there is an antigenic reactive region other than NC16a on the extracellular domain of BP180. There have been few reports describing detailed clinical features of BP caused by autoantibodies targeting antigenic sites other than the NC16a domain. We conclude that it is difficult to differentiate their clinical features from those associated with autoantibodies targeting the NC16a domain of BP180.

Effects of famotidine, mosapride and tandospirone for treatment of functional dyspepsia.

BACKGROUND: An effective therapeutic strategy for functional dyspepsia (FD) has not been well-established. AIM: We investigated and compared the therapeutic effects of famotidine, mosapride and tandospirone for the control of dyspeptic symptoms. METHODS: Fully examined FD patients of outpatient clinics at seven different medical centres were enrolled in the study. They were randomly assigned to three groups based on the type of drug administered: famotidine, mosapride and tandospirone. The effects of treatment over 4 weeks were assessed by visual analogue scales. RESULTS: All of the drugs showed beneficial effects, although famotidine was the most effective for symptom relief, which was significantly greater than tandospirone, while the effect of mosapride was similar to that of famotidine. No subtype of FD showed a better response to a particular type of drug. CONCLUSIONS: For the treatment of FD, famotidine demonstrated the best therapeutic effect, followed by mosapride, while that of tandospirone was significantly lower.

The origins of polypoidal choroidal vasculopathy.

BACKGROUND/AIM: There are two theories on the pathogenesis of polypoidal choroidal vasculopathy (PCV): variants in choroidal neovascularisation (CNV) and inner choroidal vessel abnormalities. On indocyanine green angiography (IGA) with a video camera system, PCV has a characteristic appearance, but inadequate image quality has made detailed interpretation difficult. This study aims to improve imaging, using confocal scanning laser ophthalmoscopy (SLO), to elucidate the pathogenesis of PCV. METHODS: High speed IGA with confocal SLO of 45 eyes (44 patients) showed typical PCV findings of a branching vascular network and polypoidal lesions. RESULTS: Vessels comprising branching networks began to fill simultaneously with the surrounding choroidal arteries in 38 eyes. Small numbers of vessels filling within a branching network, in the arterial and arteriovenous phases of IGA, showed focal dilatation, constriction, and tortuousity. Vessel abnormalities, corresponding to polypoidal lesions, existed within a network in eight eyes and included loops similar in calibre to network vessels, and numerous microaneurysmal dilatations of small vessels. Vessel pulsation was seen in 24 eyes. CONCLUSION: PCV is caused by inner choroidal vessel abnormalities, not CNV.