1,721,012 research outputs found
BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF HUMAN CARBONIC ANHYDRASES
No full textCarbonic anhydrases (CAs. EC 4.2.1.1) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and proton. In humans, 15 isozymes have been described with different subcellular localization. Indeed, CA I-III, VII, and XIII are cytosolic, CA IV, IX, XII and XIV are membrane-bound, CA VA and CA VB are mitochondrials, and CA VI is secreted. Human CA isozymes are extensively distributed in several tissues and organs where, as modulators of pH and ion transport, they take part in a variety of physiological and pathological processes. As a result, in the last years many of these enzymes have become important therapeutic targets for pharmaceutical research. However, given the high degree of sequence and structure similarity among the different isoforms, to date most of the CA-directed drugs developed lack of selectivity and consequently present many side-effects. X-Ray crystallography is one of the most useful instruments in the structure-based drug design of selective molecules able to interact with a target enzyme. Indeed, there are several examples in the literature where knowledge of the crystallographic structure of an enzyme allowed the design of molecules able to interact with specific residues, thus regulating enzyme biological activity. This is why in recent years there has been an extensive research effort focusing on the crystal structure resolution of all catalytically active alfa-CA isoforms, with the result that most of the human CA isoforms have been so far structurally characterized. As a part of a general research project based on the structure-based drug design of isoform-selective CA inhibitors (CAIs), my Ph.D thesis has been focused on the study of the biochemical and structural features of two cytosolic CA isoforms, namely hCA VII and hCA I, which among the 15 human isoforms have been poorly investigated. hCA VII was the sole cytosolic isozyme, that at the beginning of this Ph.D thesis was not yet structurally characterized. This enzyme, similarly to hCA II, is a very efficient catalyst for hydration of carbon dioxide, being 10-50 times more active compared to other two cytosolic isoforms, hCA I and hCA XIII. However, in contrast to hCA II which is widely spread in human tissues, CA VII has a more limited distribution, being localized mainly in some brain tissues of humans and rats, in stomach, duodenum, colon, liver and skeletal muscle of mice. In the first part of my Ph.D thesis a complete biochemical characterization of this enzyme was carried out. Interestingly, these studies highlighted the capability of two reactive cysteines to be S-glutathionylated during the purification procedures. Since S-glutathionylation was reported in vivo also for another cytosolic CA isozyme, namely CA III, and was associated to a protective response to oxidative stress, this phenomenon was investigated in details also for CA VII. Such studies showed that Cys183 and Cys217 were involved in adduct formation. These reactive cysteines were mutagenized and the corresponding double mutant (C183S/C217S) was expressed. The native enzyme, its double mutant and the S-glutathionylated adduct were fully characterized for their CO2 hydration, esterase and phosphatase activity. These kinetic studies indicated that the modification of Cys183 and/or Cys217 by glutathione does not have a relevant impact on the active site of the enzyme, causing rather small differences in its specific activity. Moreover, an important observation was that hCA VII was highly effective as esterase and phosphatase, compared to other cytosolic CA isoforms, such as CA I, II, III and XIII. These findings seem to indicate that the observed S-glutathionylation, if present in vivo, is not involved in the regulation of the enzyme catalytic activity but rather, as observed for hCA III, can help hCA VII to function as an oxygen radical scavenger to protect cells from oxidative damage. Further in vivo studies are currently underway to investigate this issue. The X-ray crystallographic structure of a hCA VII mutated form in complex with a classical sulfonamide inhibitor, namely acetazolamide, was also solved. A detailed comparison of the obtained structure with those already reported for other CA isozymes provided novel insights into the catalytic properties of this protein family and offered the basis for a mutagenesis approach aimed at determining the contribution of the active site single residues to the enzyme catalytic efficiency. Moreover, on the basis of the structural differences detected within the active site of the various CA isoforms, further prospects for the design of isozyme-specific CA inhibitors have been obtained. hCA I was one of the first members of the CA family to be identified. Even though the 3D structure of this enzyme was first characterized already in 1975, only few structural studies on complexes formed with different inhibitors have been reported so far. Since these studies are fundamental for the drug design of isoform-selective inhibitors, part of this thesis has been dedicated to the structural characterization of a complex that hCA I forms with topiramate (TPM), which is a molecule of pharmacological interest for the treatment of epilepsy. The analysis of the structure of the complex showed that, upon binding of the inhibitor, the active site of hCA I undergoes a profound reorganization, which has never been observed in any other CA/inhibitor complex and might therefore be useful in designing CA inhibitors. Moreover, the comparison with hCA II/TPM and hCA VA/TPM complex structures, previously investigated, showed that a different H-bond network together with the movement of active site residues to accommodate the inhibitor may account for the difference of inhibition constants of TPM towards different CA isozymes. These data may be helpful in the design of CAIs selective for various isozymes
Characterization of Phenolic Compounds in Virgin Olive Oil and their Effect on the Formation of Carcinogenic/Mutagenic Heterocyclic Amines in a Model System
No full textRole of Antioxidants in the Protection from Aging-Related Diseases
Get PDFEditorial on a special issue focused on antioxidants in the Protection from Aging-Related Disease
Identification of non-specific Lipid Transfer Protein gene family members in Solanum lycopersicum and insights into the features of Sola l 3 protein
Get PDFNon-specific lipid transfer proteins (nsLTPs) are characterized by an eight-cysteine motif backbone that is stabilized by four disulphide bonds. The strong interest towards this protein family is mainly due to the fact that nsLTPs are involved in many biological processes and have been identified as major human allergens. Since tomato (Solanum lycopersicum L.) is one of the most consumed and allergenic vegetables, a full characterization of this family is needed. In this study, hidden Markov model profiles were used to identify nsLTPs within the tomato protein complement. Following manual curation, 64 nsLTP genes were classified into six sub-families. Furthermore, nsLTP gene structure, distribution and arrangement along tomato chromosomes were investigated. Available RNA-seq expression profile data and Real-Time PCR analyses were used to derive expression patterns of tomato nsLTPs in different tissues/organs. Non-specific LTP genes with high level of expression in tomato fruits were filtered out since they could play a key role in tomato allergenicity. Among these genes was Solyc10g075090 that encodes the allergen Sola l 3. Finally, cloning, heterologous expression, purification and biochemical characterization of the recombinant protein Sola l 3 was performed
Characterization of Phenolic Compounds in Virgin Olive Oil and their Effect on the Formation of Carcinogenic/Mutagenic Heterocyclic Amines in a Model System
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Peptidi biostimolanti e bioprotettivi e loro impiego in agricoltura
No full textLa presente invenzione si riferisce a nuovi peptidi isolati aventi attività biostimolante e bioprotettiva contro lo stress abiotico e biotico nelle piante, nonché a composizioni comprendenti detti peptidi. Preferibilmente, i peptidi secondo l'invenzione provengono da piante di Solanum lycopersicum (pomodoro) e sono prodotti in modo ricombinante o sintetico. Viene anche descritto l'uso di detti peptidi e/o di detta composizione per aumentare la resistenza a stress biotici e/o abiotici in una pianta
Biostimulant and bioprotective peptides and their use in agriculture
No full textThe present invention relates to novel isolated peptides having biostimulant and bioprotective activity against abiotic and biotic stress in plants, as well as to compositions comprising said peptides. Preferably, the peptides according to the invention are from Solanum lycopersicum (tornato) plants and are produced in a recombinant or synthetic manner. The use of said peptides and/or said composition to increase resistance to biotic and/or abiotic stress in a plant is also described
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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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