1,720,975 research outputs found
Reaction of phenylglyoxal with arginine groups in D-amino acid oxidase from Rhodotorula gracilis
No full textD-Amino-acid oxidase from Rhodotorula gracilis was irreversibly inactivated by phenylglyoxal in a biphasic process. The fast phase was completed in less than 1 min. Its extent was linearly dependent on phenylglyoxal concentration and was not influenced by the presence of FAD or benzoate, a pseudo-substrate. The second phase of inactivation was due to a simple second-order reaction. The presence of FAD exerted only partial protection; the second-order rate constants of inactivation were 8.3 M-1 min-1 for holoprotein and 18.0 M-1 min-1 for apoprotein. The addition of benzoate completely protected against this second phase of inactivation. Efforts to isolate the enzyme modified at a single arginine residue at the end of the fast phase were unsuccessful, but analysis of the enzyme isolated at the end of the slow phase identified an arginine residue, protected by benzoate, that is highly conserved in all D-amino-acid oxidases and corresponds to Arg283 in the pig kidney enzyme. Modification of this residue is directly involved in the inactivation process during the slow phase. This arginine may represent the basic residue ion pairing with the carboxylate group of the substrate or the residue interacting with the flavin N1-C2 = O locus
Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis
No full textStudies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site
Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH
No full textDespite a number of studies, the formation of the Michaelis complexes between ferredoxin-NADP (+) reductases and NADP(H) eluded detailed investigations by rapid kinetic techniques because of their high formation rates. Moreover, the reversible nature of the reaction of hydride ion transfer between these enzymes and NADPH prevented the obtainment of reliable estimates of the rate constant of the hydride transfer step. Here we show that by working at a high salt concentration, the mechanism of the reaction with NADPH of FprA, a Mycobacterium tuberculosis homologue of adrenodoxin reductase, is greatly simplified, making it amenable to investigation by rapid reaction techniques. The approach presented herein allowed for the first time the observation of the formation of the Michaelis complex between an adrenodoxin reductase-like enzyme and NADPH, and the determination of the related rate constants for association and dissociation. Furthermore, the rate constant for the reaction of hydride ion transfer between NADPH and FAD could be unambiguously assessed. It is proposed that the approach described should be applicable to other ferredoxin reductase enzymes, providing a valuable experimental tool for the study of their kinetic properties
Identification of Lys116 as the target of N-ethylmaleimide inactivation of ferredoxin:NADP+ oxidoreductase
No full textOxidized ferredoxin:NADP+ oxidoreductase (FNR) was slowly and irreversibly inactivated by N-ethylmaleimide. Complete protection against inactivation was afforded by saturating concentrations of NADP+. In the presence of NADPH, a rapid inhibition of the enzyme ensued; however, this inhibition was found to be reversible. In the tryptic map of the flavoprotein, modified with N-ethyl[2,3-14C]maleimide in oxidizing conditions, a unique radioactive peptide was found. Its sequence comprised residues 110-117 of the enzyme: Lys116 was shown to be the residue alkylated by N-ethylmaleimide. It is noteworthy that the same residue of FNR was found to be modified by 5-dimethylaminoaphthalene-1-sulfonyl(dansyl) chloride at the putative NADP(H)-binding site [Cidaria, D., Biondi, P. A., Zanetti, G. & Ronchi, S. (1985) Eur. J. Biochem. 146, 295-299]. Furthermore, the data reported here demonstrate that the sulfhydryl groups of FNR are not involved in enzyme inactivation by N-ethylmaleimide
Creazione di un sistema di valutazione della qualità del processo di triage
No full textn letteratura, la maggior parte degli studi in materia di valutazione della qualità del triage si rifà a valutazioni parziali come la soddisfazione dei pazienti e il rispetto dei tempi1,2. Poiché la complessità del triage si estende oltre questi indicatori, emerge la necessità di creare uno strumento che valuti tale processo nella sua globalità. Nel 2007 abbiamo costruito a tale scopo una griglia, basata sulla letteratura internazionale, sui criteri di qualità Joint Commission e sulla normativa italiana in materia di triage. Sono stati successivamente pubblicati i risultati ottenuti tramite l’applicazione dello strumento3. Alla luce dei recenti aggiornamenti internazionali in materia di triage, si ritiene utile proporre una revisione dello strumento
Building a quality evaluation system for the nursing triage process
No full textBuilding a Quality Evaluation System for the Nursing Triage Process
Gadda G., Bollini G., Lolli A., Destrebecq A., Terzoni S.
In literature there are no studies regarding the quality evaluation of the whole triage process; there is a strong need for an appropriate tool, since most systems consider patients’ satisfaction and triage time only. We created a new measurement grid, with several indicators, aimed at evaluating many other aspects of the triage process. Every indicator is associated with a score, and the total score defines the quality level of the nursing triage process. This new system has been tested successfully in a major Milan hospital in 2009, resulting in a peer-reviewed publication.Conclusions: we think this could be an important tool in the Italian ERs. Right now, it seems the only one available to assess the whole triage process
Creazione di un sistema di valutazione della qualità del processo di triage infermieristico
No full textIn literature there are no studies regarding the quality evaluation of the whole triage process; however, since it is necessary to evaluate what really happens everyday in the Emergency Rooms, as well as verifying the daily level of throughput, there is a strong need for an appropriate tool. Measuring the quality of triage means improving the caring process. This article presents a new measurement grid, aimed at realizing a Quality Evaluation of Nursing Care. Currently, the most widespread systems used for the QENC are the Australian Triage Scale, the Canadian triage and Acuity Scale and the Italian Group of Triage Scale. The global/biphasic triage system was used in our study because it seems the most accurate, according to literature. There are several indicators that evaluate many aspects of the triage process; every single indicator has a score. The sum of the scores defines the quality level of the nursing triage process. Our paper discusses the application of this score in a major Milan hospital, based upon a preliminary study
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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