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Oxygen, reactive oxygen species and tissue damage.
No full textThe diatomic molecule of oxygen contains two uncoupled electrons and can therefore undergo reduction, yielding several different oxygen metabolites, which are collectively called Reactive Oxygen Species or ROS. They are invariably produced in aerobic environments through a variety of mechanisms, which include electron "leakage" during biologic oxidations, action of flavin dehydrogenases and specific membrane associated secretion, as well as by physical activation of oxygen by irradiation, e.g. UV sun-light. Organisms have developed efficient protective mechanisms against excessive accumulation of ROS, which include superoxide anion, hydrogen peroxide and hydroxyl radical, since all these metabolites are highly reactive and affect almost every kind of organism, either directly or through conversion into other derivatives, notably NO-derived radicals or RNS. Depending on their tissue concentration they can either exert beneficial physiologic effects (control of gene expression and mitogenesis) or damage cell structures, including lipids and membranes, proteins and nucleic acids, leading to cell death. In this brief overview we summarize the present state-of-the-art, restricting the discussion to the role of ROS in physiology and pathology, not taking into account RNS. Discussion will focus on basic chemical and biochemical features of ROS, underlining how ROS can promote severe diseases, including neoplastic, cardiovascular and neurodegenerative diseases. This brief discussion should clarify the present huge interest in ROS, in the perspective to develop new and specific therapeutic approaches
C. Reactive Protein: not only a marker, but a possible physiopathologic agent in chronic vascular inflammation
No full textMassive changes in the concentration of C-reactive protein (CRP), a well recognied positie acute phase protein, are known take place rapidly in bdy fluids during the onset of inflammatory and infective diseaeses. This process results into the accumulation of CRP at the sites of the lesions and is classically closely linked to protective effects through targeted activation of complement and thereby clearance of bacteria and necrotic cell debris. Data collected during the last couple of years has documented an unexpected role of this protein as a major pathogenic element in atherosclerosis, which is now considered as an inflammation of the arterial endothelian lining. In this disease, intimal and muscolar lesions are accompained by deposition of CRP, local activation of complement, recruiment of phagocytic cells and opposite effects on the final plate stability depending on the balance between proteolytic processing and collagen deposition. Major effects are now played to reduce pharmacologically the levels of CRP in the blood stream and to counteract its local activation in order to control the development of this dangerous inflammatory disease
Effects of ligands on structure and stability of Tissue Trasglutaminase: a calorimetric and spectroscopic study
No full textTransglutaminases (Tgases) are a wide family of enzymes whose main role is to catalyze calcium dependent acyl-transfer reactions from peptidyl glutamine residues to accepting primary amines. The amine substrates can be provided by either a soluble amine (usually polyamines or alternatively histamine) and/or the aminogroups of a protein-bound lysine residue, resulting into release of proteins which are modified at glutamine residues either by simple covalent incorporation of amines or by crosslinkage through proteinase-resistant isopeptide bonds.In mammals, eight distinct Tgase isoenzymes have been identified at the genomic level. The most extensively investigated isoenzyme is the ubiquitous cytosolic isoform, denoted tissue transglutaminase. Its physiologic function is still uncertain and it has been proposed to be involved into as different cell reactions as programmed cell death death and stimulation to proliferation. Tissue transglutaminase is a bifunctional enzyme displaying also GTP hydrolysis along with the transamidating activity. These activities are switched on-off alternatively. Transamidation is usually a latent activity, thanks to a mechanism involving conformational changes triggered by calcium (an activator essential cofactor in transamidation) and GTP itself (an inhibitor). The aim of this study is to investigate the stability of the enzyme and the effects of ligands thereupon, employing heat as a simple quantificable destabilizing stimulus.In these studies we employed Tgase purified to homogeneity from human erythrocytes. In thermal inactivation experiments, Tgase was submitted to heat treatment at various temperature, measuring residual activity at timed intervals. Along with effects on activity, we investigated structural perturbations of the protein by Differential Scanning Calorimetry (DSC), with heating cycles employing thermal gradient of 0.8°C/min in a temperature range between 20°C to 90°C to study the unfolding process in the absence and in the presence of ligands. Finally, selective perturbations of domains 1 and 2 of the enzyme were monitored by tryptophan fluorescence emission between 310 and 370 nm, with excitation at 295 nm, at constant temperature increments. The calculated ratio of fluorescence emission at two wavelengths (at 350 and 330 nm, marked F350/F330) was a suitable measure of the loss of protein tertiary structure and was employed to measure ligand-induced conformational changes and their relationships to thermal stability. The regulation of tissue transglutaminase is achieved through conformational changes triggered by the modulatory ligands calcium (an essential activator) and GTP (an inhibitor), through induction of conformational changes. In this report we address the thermodynamics of these effects by means of a combination of thermal treatment, spectroscopic measurements and differential scanning calorimetry. Our data confirm that binding of ligands induce large conformational changes through movement of domains, identified by distinct unfolding transitions in DSC thermograms. In particular they demonstrate that the protein structure is characterized by tight interaction between the N-terminal and the C-terminal domains in the absence of ligands, a reinforcement of the interaction in the presence of the inhibitor GTP and a losening in the presence of calcium ions, with appreciable changes in thermal stability, hindering or disclosing the active site to the substrate. Also addition of heparin did not by itself significantly modify the thermogram profile but it prevented completely the aggregation of the protein which is usually observed late in the fusion profile after the unfolding process is completed
Structural-functional relationships in muscle Glycogen Synthase
No full textGlycogen Synthase (GS) and glycogenin catalyze the biosynthesis of mammalian glycogen. GS itself plays the pivotal role and is highly regulated. The great relevance of GS in overall glycogen metabolism, homeostasis of glycaemia, the pathogenesis of diabetic complications emphasizes the necessity for a better understanding of the regulatory mechanisms. This goal is presently prevented by the lack of structural information, which are essentially limited to the primary structure, the oligomeric state and the location of the phosphorylation regulatory sequences. Our present efforts are directed to define the domain structure of the active and the inactive state, taking into account the effects of the ligands.
Studies were perfume on purified GS by means of i) thermal inactivation kinetics; ii) spectrofluorimetric analysis in the tryptophan emission spectrum and iii) Differential Scanning Calorimetry (DSC). Experiments were carried out on phosphorylated and dephosphorylated GS in the absence and in the presence of ligands. In this preparation, glycogenin is regularly present at tight-binding complex with GS.
Experiments of thermal inactivation on both GS a and b, revealed marked differences in their inactivation kinetics. Spectra of tryptophan fluorescence emission were characterized by a biphasic profile with a maximum at 332 and 356 nm. UDO-glucose and glucose 6-Pbring about a moderate quenching of tryptophan fluorescence, with parallel contribution by buried and exposed tryptophan, suggesting that ligand induced conformational changes do not grossly modify the protein ternary structure. DSC Thermograms of unfolding purified GS a demonstrate a complex pattern with main transition at 62 C°, consisting with a structure with multiple domains with slightly different melting temperatures. AT variance, unfolding of the phosphorilated b form takes place at lower temp. (Tm 56 C°), indicative of consistent conformational changes following phosphorilation. We are now investigating effects of ligands on the DSC unfolding profile. Finally, limited proteolysis of the GS-glycogenin complex indicated extensive degradation of glycogenin, with only limited nicking of GS, as if glycogenin is located in a more superficial position in the native protein complex.
The present experiments confirm that GS is a multidomain protein and that its complex regulation of catalytic activity is linked to conformational changes triggered by allosteric or by covalent modification
Isotope effect as a probe of the catalytic rilevance of coenzyme isomerization in the enzyme 6-phosphogluconate dehydrogenase from sheep liver
No full textInterection with heparin protects tissue transglutaminase against inactivation by heating and by proteolysis
No full textThe considerable affinity of tissue transglutaminase for heparin was the basis for use of heparin-based affinity matrices for enzyme purification. Interaction of transglutaminase with heparin might mimic the physiological binding to membrane heparan sulfates, accounting for the limited but significant fraction of enzyme exposed at cell surface to crosslink ECM proteins. Exploring effects of heparin on transglutaminase activity and stability, we have noted that heparin only slightly affects activity in vitro, but the protein against heat treatment and proteolysis
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
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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