1,721,196 research outputs found
Studi preliminari per l'introduzione di un sito di modulazione sodio-specifico nel tPA
No full textIl tPA è sintetizzato nelle cellule dell’endotelio vascolare e secreto nel sangue come glicoproteina a catena singola costituita da 527 residui organizzati in cinque domini strutturalifunzionali. Il substrato fisiologico accertato del tPA in vivo è un singolo legame peptidico del plasminogeno (R560-V561) che viene cosi’ attivato a plasmina. La plasmina idrolizza quindi il coagulo di fibrina formato dall’azione della trombina sul fibrinogeno. Il dominio catalitico carbossiterminale del tPA (S262-P527) contiene i residui della triade catalitica delle proteine a senna della famiglia della chimotripsina (H57(322), D192(471), S195(478)) [i residui sono indicati con la numerazione delle proteine della famiglia della chimotripsina ed in parentesi è la numerazione della sequenza del tPA]. Tra essi si annoverano le proteasi alla cascata coagulativa (trombina, proteina C, fattori VII, IX, X, XI e XII), fibrinolitica (tPA, urochinasi, plasmina) e del complemento (fattori B, D, I, C2).
E’ stato recentemente scoperto che l’attività di alcune tra queste proteasi è potenziata dal legame dello ione Na: nella trombina, ad esempio, in presenza di concentrazioni di Na fisiologiche si riscontra un innalzamento dell’attivita’ catalitica verso i substrati cromogenici e la maggiorparte dei substrati fisiologici fino ad un fattore 100 (1).
In più del 95% delle 300 sequenze di proteasi a senna presenti in banca dati in posizione 225 si riscontra o una Tyr o una Pro. La Tyr caratterizza gli enzimi modulabili da Na, mentre la Pro quelli non modulati (1). La trombina fa parte della prima classe, mentre il tPA appartiene alla seconda (2).
Il sito di legame del Na nella trombina è situato in una cavità cilindrica formata da tre segmenti 13 antiparalleli, in prossimita’ del sito di riconoscimento primario SI. Il Na è coordinato dagli ossigeni carbonilici dei residui K224 e R221a e da quattro molecole d’ acqua. L’anello fenolico del residuo Y225, distante 20 A dal sito catalitico, è ospitato in una tasca idrofobica stabilizzata da interazioni di Van der Waals con i residui V163 e V167 (3). La sostituzione dì Y225 con i rimanenti amminoacidi altera sia la specificità del Na, sia l’attività catalitica dell’enzima, tramite un meccanismo che rimane non pienamente esplicato, nonostante la risoluzione della struttura tridimensionale di 3 tra i mutanti piu’ significativi(4).
La risoluzione della struttura cristallografica del dominio catalitico del tPA ne ha confermato I’ elevata omologia strutturale (>60%) con la trombina nella regione del sito di interazione con Na (5). La presenza di una Pro in posizione 225 cambia l’orientamento del carbonile di K224 facendo mancare uno dei legami di coordinazione del Na. Tuttavia la sostituzione P225Y nel tPA non introduce un legame funzionale del Na e riduce drasticamente l’attività del tPA (6). Cio’ dimostra che il residuo Tyr in posizione 225 è necessario ma non sufficiente per determinare la specificità dell’interazione con il Na e dell’effetto allosterico. Questi dipendono da una fitta rete di interazioni cui partecipano altri residui solo in parte identificati (3, 7-11).
Per approfondire lo studio dei residui necessari alla costruzione di un sito Na specifico e di quelli richiesti per la comunicazione allosterica con il sito della catalisi sono state introdotte ulteriori mutazioni aminoacidiche puntiformi sia nella trombina che nel tPA.
L’introduzione di un sito di modulazione da Na nel dominio catalitico ha un triplice scopo:
contribuire al chiarimento del meccanismo della modulazione allosterica da Na nella trombina, enzima chiave per la regolazione della coagulazione del sangue; potenziare l’attività farmacologica del tPA, correntemente utilizzato nella terapia delI’infarto; rendere possibile i’introduzione della modulazione da Na in altre proteasi a senna del plasma, quando i meccanismi della modulazione saranno stati chiariti.
1. Dang, Q.D., & Di Cera, E. (1996) Proc. Nati. Acad. Sci. USA 93, 10653-1 0656.
2. WeIis, C.M., & Di Cera, E. (1992) Biochemistry 31, 11721-11730.
3. Di Cera, E., Guinto, E.R., Vindigni, A., Dang, Q.D., Ayala, Y.M., Wuyi, M., & Tulinsky, A. (1995) J. Biol. Chem. 270, 22089-22092.
4. Guinto, E.R., Caccia, S., Rose, T., Futterer, K., Waksman, G., & Di Cera, E. (1999) Proc. Nati. Acad. Sci. USA 2, 1852-1857
Structure/function correlates in variables causing hereditary angioedema
No full textHereditary angioedema (HAE) is due to mutations in C1 inhibitor (C1-INH) gene. C-INH is a serpin that controls activation of complement and contact systems. The clinical phenotype of the disease is highly variable, but the causes of such variability remain undisclosed. Identifying mutations and their molecular effect may identify structure/function as well as genotype/phenotype correlates. 492 HAE patients belonging to 196 unrelated families are regularly followed at our Department and characterized for clinical phenotype. Genotyping of these patients, now in progress, has been completed in 155. Mutations affecting residues primarily involved in the unique suicide substrate-like inhibitory mechanism of serpins have been selected for expression in Pichia pastoris. This expression system provides an average yield of 80mg per liter of culture media with wild type C1-INH. Here we present the results on the function/structure characterization of R378C variant. The patient carrying this mutation presents unusual features compared to other HAE patients: absence of typical subcutaneous angioedema, recurrent short lasting (few hours) abdominal cramps, but no major abdominal symptoms with severe pain vomiting and/or dyarrhea, variable C1-INH plasma levels with spontaneous normalization. The expressed protein has a very low yield, most likely due to an impaired secretory profile, typical of multimerization inside the cells. The inhibitory capacity of the active form is preserved, the second order inhibition constant found in progress curves was comparable to wild-type recombinant C1-INH. This results suggest that the HAE phenotype is due to a failure in general folding and structural stability. The R378C C1-INH variant could undergo different conformations (native, latent, polymer), that result in different degrees of impaired secretion and/or function, depending on specific environmental conditions
Brush border membrane vesicles from dipteran midgut: a tool for studies on nutrient absorption
Full text linkBrush border membrane vesicles (BBMV) from insects midgut can be successfully used to study several membrane phenomena, including nutrient absorption, ions permeability and insecticides mode of action. Midgut BBMV, purified from Musca domestica whole larvae, were used for the functional characterization of leucine transport. The amino acid uptake was accelerated in the presence of sodium or potassium and increased significantly when the extravesicular pH was 5.0, in agreement with the luminal pH in vivo. Radiolabelled leucine uptake was significantly reduced by an excess of cold leucine, histidine, serine and glycine, suggesting that the amino acid transporter is a broad scope carrier that does not recognize proline, glutamine and the dibasic amino acids lysine and arginine.Midgut BBMV were also obtained from homogenization of M. domestica and Bactrocera oleae adults. The final preparations showed a high enrichment in the specific activity of the BBM marker enzymes aminopeptidase N and γ-glutamyl transpeptidase, and were poorly contaminated by basolateral membranes, as indicated by the low specific activities of their marker enzyme Na+/K+ ATPase. Electron microscopy of B. oleae BBM fraction showed the presence of closed vesicles. Similar SDS-PAGE patterns, with numerous distinct bands, were detected for both B. oleae and M. domestica BBMV
Identification of variables causing different clinical expression of inherited c1-inh deficiency (hereditary angioedema)
No full textHereditary angioedema (HAE) is due to mutations in C1 inhibitor (C1-INH) gene causing its deficiency. Two phenotypic variants are known: HAE type I with low antigenic and functional plasma level of C1-INH, and type II with low C1-INH function, but normal or increased antigen due to the presence of a dysfunctional protein. Clinical expression of HAE is unpredictable despite a stable defect. We hypothesize that: i) the type of mutation in C1-INH gene and/or polymorphisms affecting the function of proteins involved in pathogenesis of symptoms could influence HAE expression; ii) the clinical response to treatment with attenuated androgens depends on the upregulation of C1-INH expression in tissues. The experimental approach is based on: i) identification of mutations responsible of HAE, definition of their structural consequences by molecular modeling, functional characterization of expressed mutant C1-INHs. ii) association of polymorphisms of coagulation Factor XII and angiotensin converting enzyme with different clinical phenotypes of HAE; iii) measurement, by real time PCR, of C1-INH mRNA from peripheral blood mononuclear cells of HAE patients on and off treatment with attenuated androgens. Mutation screening will be performed by fluorescence activated mismatch analysis.
Preliminary results: 36 different mutations were identified in genomic DNA, 5 of them were present in more than one family. 18 of these mutations have not been previously described. In HAE type I there were 13 different stop codons, 9 different missense mutations, 3 different splicing defects, 4 large deletions and 2 deletions of 1-2 amino acids. In HAE type II all mutations were missense consisting of 4 different substitutions at the reactive site (amino acid 444) and 1 in P10 position (amino acid 436). Compared to normal controls, the amount of C1-INH mRNA was 40% in HAE type I patients (p<0.0001) and 47% in HAE type II (p<0.0001). The difference between HAE type I and type II was not statistically significant. In order to define the frequency of transcribed mutations in HAE we sequenced cDNAs from 44 patients with missense mutations, nonsense mutations and splicing defects: in 34 of them (77%) we detected a transcript originated from the mutated allele. According to the type of mutations the frequency of transcripts was: 28 of 29 in missense mutations, 6 of 11 in nonsense mutations and none in 4 splicing defects. The amount of C1-INH mRNA in transcribed mutations was 1650 (380-6500) representing 46% of normal controls while it was lower, 37% of normal (1350 range 420-3400), in mutations that were not detected in cDNA. In order to analyze patients in whom just wilde-type mRNA was expressed, we considered the 4 large deletions and found that in this type of mutations C1-INH mRNA was 23% compared to controls.
In summary: We confirmed that HAE is characterized by a high number of “private” mutations C1-INH mRNA in cytoplasm of PBMC from HAE patients was approximately 50% of that found in normal controls. Amounts of C1-INH mRNA in patients with HAE type I and type II was not significantly different. 70% of the mutations responsible for HAE are transcribed into mRNA. C1-INH mRNA is 25% of controls in mutations that cannot be transcribed. These results suggest that in patients with HAE there is a downregulation of normal C1-INH allele to approximately 50% of its functional efficiency. This fact, in addition to the heterozygous defect, is responsible for plasma levels of normal C1-INH markedly below the expected 50% and eventually exposes to the appearance of angioedema symptoms
Modulation of the association reaction between hemoglobin and carbon monoxide by proton and chloride
No full textA cryogenic technique for the isolation of the ligation intermediates in the association reaction between hemoglobin and carbon monoxide at 20 degrees C [Perrella, M., Davids, N., and Rossi-Bernardi, L. (1992) J. Biol. Chem. 267, 8744-8751] was used to study the effects of proton and chloride concentrations on the rates of the stepwise reactions. The reaction rate was observed to increase continuously in the course of the ligation process, yet the acceleration of the reaction after the binding of two ligand molecules, observed previously in 100 mM KCl, pH 7, was not observed at other pH values. At pH 6.3, such an acceleration occurred after the binding of three ligands, and at pH 8.5, a large acceleration was observed after the binding of the first ligand molecule. Greater CO binding to the beta chains was observed under all conditions, as in the previous study. The functional heterogeneity of the chains in the first ligation step increased with pH. The chloride concentration did not influence the distribution of the ligand between the alpha and beta chains at pH 6.3 and 8.5. At pH 7, less binding to the alpha chains was observed at 7 mM chloride with respect to 100 mM. The nature of the biliganded component isolated at pH 7 in 100 mM KCl and unresolved by the cryogenic technique was studied using a combination of cryogenic and noncryogenic isoelectric focusing. This component was a mixture of intermediates (alpha beta) (alpha CO beta CO), about 65%, and (alpha beta CO) (alpha CO beta), about 35%. The experimental data were compared with the distributions of intermediates calculated according to the Monod kinetic model assuming rapid and concerted transitions between two quaternary structures at each ligation step. The model provided a qualitative fit of the observed distributions of intermediates at acidic and neutral pH. A large discrepancy between the experimental observations and the predictions of the model was found at alkaline pH. The mechanism of the association reaction is discussed in the light of the available information on the tertiary/quaternary structures of the intermediates, as obtained from the studies of the deoxy/cyanomet model of ligation
The kinetics of the reaction between NO and O2 as studied by a novel approach
No full textThe kinetics of the reaction between NO and O2 was determined by measuring the time course of the decrease in the concentration of NO with a quench-flow technique. NO and O2 were mixed rapidly and reacted for periods of time varying from 10 to 50 s. A second rapid mixing with a solution containing an excess of deoxyhemoglobin and sodium hydrosulfite trapped free NO as nitrosylhemoglobin and reduced O2. The spectrum of the mixture of deoxy- and nitrosylhemoglobin was recorded within 30 s from the second mixing, before any appreciable dissociation of NO from the protein, by means of a flow-cell mounted on-line with the quench-flow apparatus. The amount of NO not consumed in the auto-oxidation reaction was calculated from the proportion of nitrosylhemoglobin in the mixture. As NO and O2 bind deoxyhemoglobin at comparable rates and NO is oxidized to nitrate by oxyhemoglobin, the ratio of hemoglobin/(NO + O2) had to be optimized to avoid the interference of this oxidation reaction. The kinetics was first and second order with respect to O2 and NO, respectively and third order overall with a rate constant k = 4 x kaq = 4 x 2.23 (+/- 0.26) x 10(6) M-2 s-1 at 20 degrees C, invariant in the pH range 7-9, in agreement with published values obtained by different methodologies
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
Remittent C1-inhibitor deficiency due to Arg378Cys mutation
No full textBackground: C1 inhibitor (C1-INH) deficiency causes hereditary angioedema. Arg378→Cys C1-INH variant was found in a patient suffering from abdominal pain and presenting markedly reduced C1-INH plasma levels episodically undergoing spontaneous normalization with concomitant disappearance of symptoms.
Purpose: To elucidate the effect of the mutation on protein function and conformation and explain the biochemical and clinical phenotype.
Methods: We expressed Arg378→Cys C1-INH variant in Pichia pastoris and performed functional and structural studies on the purified protein.
Results: Expression of C1-INH resulted in a ten fold drop in mutant protein secretion compared to wild type. Both proteins formed complexes with target proteases, but the kinetic of inhibition of the mutant was slightly diminished and this reduction increased with temperature. Gel filtration indicated that the mutant protein can form oligomers. Thermal denaturation experiments in conditions reproducing the intracellular molecular crowding demonstrated that, compared to wild-type, the mutant had higher tendency to oligomerize.
Conclusion: our findings suggest that the Arg378→Cys C1-INH variant maintains inhibitory functions against target proteases, but its structural conformation is abnormally susceptible to environmental factors that may occasionally promote protein oligomerization and interfere with its function and secretion from cells, accounting for variability in plasma levels.
Clinical Implication: We describe a new biochemical and clinical phenotype caused by a mutation in C1-INH gene
- …
