afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptmyces coelicolor A3(2)

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3′ to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the ΔafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation ofafsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues

An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus

In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high end-to-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX–AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST–AfsR-g); the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX–AfsK-g in the presence of [γ-32P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX–AfsK-g phosphorylated serine and threonine residues of GST–AfsR-g in the presence of [γ-32P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1%, but did form spores on mannitol- and glycerol-containing media; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism

Protein serine/threonine kinases in signal transduction for secondary metabolism and morphogenesis in Streptomyces

A number of proteins in the Gram-positive bacterial genus Streptomyces are phosphorylated on their serine/threonine and tyrosine residues in response to developmental phases. AfsR is one of these proteins and acts as a transcriptional factor in both the regulation of secondary metabolism in Streptomyces coelicolor A3(2) and morphological differentiation in Streptomyces griseus. In S. coelicolor A3(2), AfsR is phosphorylated on its serine and threonine residues by more than three protein kinases whose kinase activity is enhanced by means of autophosphorylation on their serine and threonine residues. The degree of autophosphorylation of AfsK is regulated by KbpA which, by binding directly to the kinase domain of AfsK, inhibits its autophosphorylation. Phosphorylation of AfsR enhances its DNA-binding activity and causes it to bind the promoter elements, including –35, of afsS, thus resulting in activation of afsS transcription. ATPase activity of AfsR is essential for this transcriptional activation, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. afsS, encoding a 63-aminoacid protein, then activates transcription of actII-ORF4, a pathway-specific transcriptional activator in the actinorhodin biosynthetic gene cluster, in an as yet unknown way. Distribution of the afsK-afsR systems in a wide variety of Streptomyces species and the presence of many phosphorylated proteins in a given Streptomyces strain suggest that the signal transduction via not only two-component regulatory systems but also serine/ threonine kinases generally regulates secondary metabolism and morphogenesis in this genus

Dissection of lung parenchyma using electrocautery is a safe and acceptable method for anatomical sublobar resection

BACKGROUND: Anatomic sublobar resection is being assessed as a substitute to lobectomy for primary lung cancers. However, persistent air leak after anatomic sublobar resection is prevalent and increasing surgical morbidity and costs. The use of electrocautery is being popularized recently in anatomic sublobar resection. We have retrospectively evaluated the safety and efficacy of intersegmental plane dissection using electrocautery. METHODS: Between April 2009 to September 2010, 47 patients were treated with segmentectomy for clinical T1N0M0 non-small cell lung cancers. The intersegmental plane was dissected using electrocautery alone or in combination with staplers. We evaluated the methods of dividing intersegmental plane (electrocautery alone or combination with electrocautery and staplers), intraoperative blood loss, duration of chest tube placement, duration of surgery, preoperative FEV(1.0) %, incidence of prolonged air leak, length of postoperative hospital stay, postoperative pulmonary function at 6 months after surgery and the cost for sealing intersegmental plane. RESULTS: Among the 47 patients, 22 patients underwent intersegmental plane dissection with electrocautery alone and 25 patients did in combination with electrocautery and staplers. The mean number of stapler cartridges used was only 1.3 in electrocautery and staplers group. Mean age, gender, number of patients whose FEV(1)% < 70 % were similar between two groups. There was no statistical difference between electrocautery alone and combination with electrocautery and staplers group in duration of surgery (282 vs. 290 minutes), intraoperative blood loss (203 vs.151 ml), duration of chest tube placement (3.2 vs. 3.1 days), postoperative hospital stay (11.0 vs.10.0 days), postoperative loss of FEV(1.0) (13 vs.8 %), loss of FVC (11 vs. 6 %) or incidence of minor postoperative complications [9 % (2/22) vs. 16 % (4/25), p = 0.30)]. However, incidence of prolonged air leak was higher in electrocautery alone group than in combination with electrocautery and staplers group [14 % (3/22) vs. 4 % (1/25), p = 0.025)]. The cost of materials for sealing air leaks amounted to €964 per patient in the electrocautery alone group and €1594 per patient in combination with electrocautery and staplers group. CONCLUSIONS: The number of patients with prolonged air leak was higher in the electrocautery alone group. The use of staplers in addition to electrocautery may lead to reduced prolonged air leak. However, the use of electrocautery for intersegmental plane dissection appeared to be safe with acceptable postoperative complications and effective in reducing costs

Phenotypic convergence in bacterial adaptive evolution to ethanol stress

Stability of ethanol tolerance. Strain F at the end point (2,500 h) and at 576 h was cultivated for 200 generations absent ethanol stress. After the cultivation, ethanol tolerance was evaluated by measuring specific growth rates in 5 % ethanol stress (red bars). The growth rates under ethanol stress were similar to those before the non-stress cultivation (blue bars) and were significantly higher than that of the parent strain. (PDF 976 kb

Proximal (entry) tear of dissecting aortic aneurysm visualized by three-dimensional echocardiography

AbstractJ Thorac Cardiovasc Surg 2002;124:1245-

Activity Based Costing / Activity Based Managementを用いた院外持ち込み画像データの効果的な取り込み方法

Background: With filmless operation becoming widespread in Japanese hospitals, medical images provided for referral purposes are frequently stored using portable media such as compact discs. Few facilities import brought-in data into their own Picture Archiving and Communication Systems (PACS) with a clearly defined responsible creator at the time of data storage. Currently, no literature analyzing this series of tasks exists. Objective: This study aimed to determine the best workflow for importing brought-in images into PACS in order to optimize management and costs. Methods: We created three different importation operational workflows (post-medical examination importation, pre-medical examination importation, and pre-medical examination importation using Temporary PACS for storage), and clarified the decision maker, who decides when to lock and prevent further image modifications, and the responsible creator of the image. Task analysis of each operation was carried out using Activity-Based Costing and Activity-Based Management, methodologies used in the field of management accounting. Results: Task analyses revealed that post-examination importation increased the burden on physicians, in addition to increasing operational costs. It was also shown that an explicit responsible creator did not exist with pre-examination importation. When pre-examination importation was conducted using Temporary PACS, physicians were both decision makers and responsible creators. Moreover, the burden on physicians was low and the costs could also be kept low. Conclusion: We analyzed importation tasks involved in bringing external images into PACS. Our results indicate that, from the perspective of management and costs, it is most efficient to import images using Temporary PACS servers