Cyanophycin (multi-L-arginyl-poly-L-aspartate), a water-insoluble reserve polymer of cyanobacteria, is a product of nonribosomal peptide synthesis. The purification of cyanophycin synthetase of the cyanobacterium Anabaena variabilis is described. In sodium dodecylsulfate/polyacrylamide gel electrophoresis, the enzyme preparation shows one band with an apparent molecular mass of 100 kDa. The native enzyme has an apparent molecular mass of approximately 230 kDa, as determined by size-exclusion chromatography, suggesting that the active form is a homodimer. During catalysis, ATP is converted to ADP. The gene coding for cyanophycin synthetase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The C-terminal 60% of the deduced amino acid sequence of cyanophycin synthetase show sequence similarity to enzymes of the superfamily of ligases involved in the biosynthesis of murein and of folyl-poly(γ-glutamate). Cells of Escherichia coli harbouring the gene on a plasmid express active synthetase and accumulate cyanophycin-like material. The results prove that a single enzyme catalyzes the de novo synthesis of cyanophycin.Keywords : non-ribosomal peptide synthesis; cyanobacteria ; cyanophycin synthetase; purification; heterologous expression.Most cyanobacteria contain the nitrogen-rich reserve mateFrom the cyanobacterium Anabaena cylindrica, Simon [9] has enriched an enzyme that synthesizes cyanophycin in vitro rial multi-L-arginyl-poly (L-aspartic acid) (trivial name, cyanoand has studied the basic properties of the biosynthetic reaction. phycin), which is deposited in the cytoplasm in the form of gran-The substrates of the reaction are the constituent amino acids Lules [1Ϫ4]. In this polymer, nearly all β-carboxy groups of a arginine and L-aspartic acid, Mg-ATP, and cyanophycin as poly(aspartate) backbone are linked to A-amino groups of argiprimer [9]. In a strict sense, the assay measures an elongation nine residues by iso-peptide bonds, resulting in an approximate reaction. A thiol reagent such as 2-mercaptoethanol and K ϩ were 1:1 stoichiometry of aspartate/arginine [1,5,6]. The polymer is found to be required for full activity [9]. The latter properties the product of nonribosomal peptide synthesis [7]. Its molecular are, in addition to the formation of iso-peptide bonds, reminismass, as estimated by SDS/PAGE, in a given cyanobacterial specent of the requirements of the biosynthesis of glutathione [10] cies is not uniform, but ranges over about 25Ϫ100 kDa [1]. Cyaand of poly(γ-D-glutamate) [11]. nophycin is also present in filamentous cyanobacteria such asIn this communication, we describe the purification of cyaAnabaena which differentiate, in a semiregular pattern, so-called nophycin synthetase from the cyanobacterium Anabaena varheterocysts, cells specialized for fixation of N 2 under aerobic iabilis American Type Culture Collection (ATCC) 29413. It is conditions. In these species, cyanophycin may not only serve as demonstrated that a homodimer of a 100-kDa protein incorpoa reserve material,...
Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix®, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell–extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix®, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.
BackgroundWith an average prescription rate of 50%, in German primary care antibiotics are still too frequently prescribed for respiratory tract infections. The over-prescription of antibiotics is often explained by perceived patient pressure and fears of a complicated disease progression. The CHANGE-2 trial will test the effectiveness of two interventions to reduce the rate of inappropriate antibiotic prescriptions for adults and children suffering from respiratory tract infections in German primary care.Methods/DesignThe study is a three-arm cluster-randomized controlled trial that measures antibiotic prescription rates over three successive winter periods and reverts to administrative data of the German statutory health insurance company AOK. More than 30,000 patients in two regions of Germany, who visit their general practitioner or pediatrician for respiratory tract infections will be included. Interventions are: A) communication training for general practitioners and pediatricians and B) intervention A plus point-of-care testing. Both interventions are tested against usual care. Outcome measure is the physicians’ antibiotic prescription rate for respiratory tract infections derived from data of the health insurance company AOK. Secondary outcomes include reconsultation rate, complications, and hospital admissions.DiscussionMajor aim of the study is to improve the process of decision-making and to ensure that patients who are likely to benefit from antibiotics are treated accordingly. Our approach is simple to implement and might be used rapidly among general practitioners and pediatricians. We expect the results of this trial to have major impact on antibiotic prescription strategies and practices in Germany, both among general practitioners and pediatricians.Trial registrationThe study is registered at the Current Controlled Trials Ltd (ISRCTN01559032)
BackgroundCalculation of individual risk is the cornerstone of effective cardiovascular prevention. arriba is a software to estimate the individual risk to suffer a cardiovascular event in 10 years. Prognosis and the absolute effects of pharmacological and lifestyle interventions help the patient make a well-informed decision. The risk calculation algorithm currently used in arriba is based on the Framingham risk algorithm calibrated to the German setting. The objective of this study is to evaluate and adapt the algorithm for the target population in primary care in Germany.Methods/designarriba-pro will be conducted within the primary care scheme provided by a large health care insurer in Baden-Württemberg, Germany. Patients who are counseled with arriba by their general practitioners (GPs) will be included in the arriba-pro cohort. Exposure data from the consultation with arriba such as demographic data and risk factors will be recorded automatically by the practice software and transferred to the study centre. Information on relevant prescription drugs (effect modifiers) and cardiovascular events (outcomes) will be derived from administrative sources.DiscussionThe study is unique in simulating a therapy naïve cohort, matching exactly research and application setting, using a robust administrative data base, and, finally, including patients with known cardiovascular disease who have been excluded from previous studies.Trial registrationThe study is registered with Deutsches Register Klinischer Studien (DRKS00004633).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.