102 research outputs found
Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen
No full textModulation of the proteolytic activity of matrix metalloproteinase-2
(gelatinase A) on fibrinogen
Susanna MONACO*, Magda GIOIA*†, Janet RODRIGUEZ‡, Giovanni Francesco FASCIGLIONE*, Donato DI PIERRO*,
Giulio LUPIDI§, Ludwig KRIPPAHL_, Stefano MARINI* and Massimo COLETTA*†1
*Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy, †Interuniversity Consortium on the Metal
Chemistry in Biological Systems (CIRCMSB), P.za Umberto I 1, I-70100 Bari, Italy, ‡Laboratorio de Farmacologia, Centro de Quimica Farmaceutica, 200 y 21, Atabey, 21, Atabey,
Playa, P.O. Box 16042, La Habana, Cuba, §Department of Molecular, Cellular and Animal Biology, University of Camerino, Via F. Camerini 2, I-62032 Camerino, Italy, and
_Departamento de Quimica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal
The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37◦C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the β-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation intermediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour,which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism. Key words: fibrinogen, fragmentation, gelatinase A, kinetics, molecular modelling, pH-dependence
Valutazione degli effetti dell'alimentazione biologica sui parametri metabolici
No full textValutazione degli effetti dell'alimentazione biologica sui parametri metabolici.
Contesto: Sono pochi gli studi disponibili sull’uomo relativi all’intervento nutrizionale sul
confronto dei prodotti alimentari biologici e convenzionali. Molti ritengono che l’alimentazione
basata sull’utilizzo di prodotti di origine biologica risulti essere più sana rispetto all’utilizzo di
prodotti convenzionali.
Obiettivo: L'obiettivo di questo studio è stato quello di verificare gli effetti di una dieta
mediterranea controllata, basata sull’utilizzo di prodotti organici rispetto alla stessa con prodotti
alimentari convenzionali, su valori di Capacità Antiossidante Totale plasmatica, Malondialdeide,
sul metabolismo cellulare e stato redox, e sulla composizione corporea, al fine di prevenire su base
nutrizionale le malattie degenerative croniche.
Razionale: Sedici uomini italiani, nove di essi soggetti sani e sette uomini uremici, di età compresa
tra 30-65 anni, sono stati arruolati. Parametri Antropometrici e BIA di tutti i partecipanti sono stati
misurati. La capacità antiossidante totale (TAC) plasmatica è stata valutata mediante il metodo
ORAC; la concentrazione della MDA, l'energia metabolica e lo stato redox cellulare sono stati
misurati mediante metodo HPLC. La dieta abituale di assunzione è stata stimata durante i 12 mesi
precedenti da un questionario semiquantitativo di frequenza alimentare. La capacità antiossidante
alimentare è stata valutata con metodo ORAC.
Risultati: L’analisi Orac di cibi biologici e convenzionali, in generale, ha dimostrato un aumento
significativo della capacità antiossidante alimentare del gruppo dei prodotti biologici. Non sono
state osservate notevoli differenze per quanto riguarda la composizione del corpo, ma sono stati
osservati un evidente cambiamento per quanto riguarda la distribuzione dell’acqua corporea. La
capacità antiossidante del plasma ha mostrato un aumento significativo, dopo il consumo di
alimentazione biologica. Inoltre non vi era alcuna differenza significativa tra i valori di plasma con
l'eccezione di una significativa riduzione dei valori di fosforo nel sangue. Nei soggetti uremici
inoltre, sono aumentate la fosforilazione dell’AMP e dell’ADP e una significativa più elevata
concentrazione di ATP (27%, p <0.005), una notevole diminuzione della MDA valori (36%, p
<0,005) sono stati osservati dopo la dieta biologica.
Conclusioni: Il nostro studio dimostra chiaramente che una dieta mediterranea biologica è stato un
notevole sostegno per la prevenzione e il controllo delle malattie degenerative croniche a base
nutrizionale, in grado di ridurre i radicali liberi indotti dalla perossidazione lipidica, apportare
l'aumento della TAC e migliorare il metabolismo energetico cellulare.The effects of Organic Diet on metabolic parameters.
ABSTRACT
Background: Few nutritional intervention studies in humans are available on the comparison of
organic and conventional food products. Many considers the organic foods healthier than the
conventional foods.
Objective: The aim of this study was to verify the effects of a controlled Mediterranean diet,
consisting of organic versus conventional foods, on plasma values of Total Antioxidant Capacity,
Malondialdehyde, on metabolic front and redox cell state, and body composition, in order to
prevent of chronic degenerative diseases based nutritional.
Design: Sixteen Caucasian Italian, of them nine were healthy subjects and seven uremic men, aged
30-65 years, were recruited. Anthropometric and BIA parameters of all the participants were
measured. Baseline plasma total antioxidant capacity was assessed by ORAC method; MDA
concentration, metabolic energy front and redox cell state by HPLC method have been measured at
baseline and after organic diet consumption. Usual dietary intakes during the previous 12 months
were estimated by a semiquantitative food-frequency questionnaire. Food antioxidant capacity was
assessed by ORAC method.
Results: ORAC analysis of conventional and organic foods in general was showed a significant
increase of the antioxidant capacity of the organic group of foods. No significant differences were
observed with regard body composition, but were observed an evident change with regard the body
water distribution. Plasma antioxidant capacity was significant increased, after the consumption of
organic diet. Moreover there was no significant difference between plasma values with the
exception of a significant reduction of blood phosphorus values. In uremic subjects, AMP and ADP
phosphorylation were increased and a significant higher concentration of ATP (+27%, p<0.005),
finally a significant decrease of MDA values ( 36%, p<0.005)were observed after organic diet.
Conclusions: Our study clearly demonstrates that a Mediterranean organic diet was a considerable
support for the prevention and control of chronic degenerative diseases based nutritional, able to
reduce free radical-induced lipid peroxidation, increasing the TAC and improving the metabolic
and energetic cellular appearance
Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin
No full textSomatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ)1–40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ1–40) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10–25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an ‘open–closed’ equilibrium
Application of Electronic Noses for Disease Diagnosis and Food Spoilage Detection
No full textOver the last twenty years, newly developed chemical sensor systems (socalled“electronic noses") have odour analyses made possible. This paper describes theapplications of these systems for microbial detection in different fields such as medicineand the food industry, where fast detection methods are essential for appropriatemanagement of health care. Several groups have employed different electronic noses forclassification and quantification of bacteria and fungi to obtain accurate medicaldiagnosis and food quality control. So far, detection and identification of bacterial andfungal volatiles have been achieved by use of e-noses offering different correctclassification percentages. The present review includes examples of bacterial and fungalspecies producing volatile compounds and correlated to infectious diseases or fooddeterioration. The results suggest the possibility of using this new technology both inmedical diagnostics and in food control management
Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts.
No full textAIMS:Use of an electronic nose (zNose(TM)) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media. METHODS AND RESULTS: Cultures of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and yeasts (two Candida albicans strains) were grown on agar and in broth media and incubated for 24 h at 37 degrees C. Headspace samples from microbial cultures were analysed by the zNose(TM), a fast gas chromatography-surface acoustic wave detector. Olfactory images of volatile production patterns were observed to be different for the various species tested after 24 h. Moreover, some strains (two K. pneumoniae, two C. albicans) did not show changes in volatile production patterns within our species.
CONCLUSIONS: Our experiments demonstrate that the electronic nose system can recognize volatile production patterns of pathogens at species level. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results, although preliminary, promise exciting challenges for microbial diagnostics
Determination of the in vitro activity of fluconazole against yeast strains using HPLC.
No full textThe aim of this study was to evaluate the in vitro activity of fluconazole against two yeast strains by determining the fluconazole
content via high-performance liquid chromatography (HPLC). Candida krusei ATCC 6258 and Saccharomyces cerevisiae were
tested for fluconazole susceptibility by dilution tests. Fluconazole measurements by HPLC were performed in cultures of the strains
at 6, 12, 24 and 48 h. In C. krusei ATCC 6258 cultures, 88.94% of the fluconazole was recovered at 48 h with respect to the level at
time 0 h. The drug exhibited major activity at 12 and 24 h. An increase in cell number and elevated concentration of the drug were
detected at 48 h. In S. cerevisiae cultures, the measurements revealed a fluconazole content of approximately 51.25% at 12, 24 and
48 h with respect to the level at time 0 h. However, the percentage of fluconazole detected and the minimal inhibitory concentration
(MIC) value determined by agar dilution tests suggest a potential for fluconazole resistance in this susceptible strain. The decreases
in fluconazole in C. krusei ATCC 6258 and S. cerevisiae cultures at each time point suggest a mechanism of resistance that is not correlated
to efflux pumps
Role of proteolytic enzymes in the COVID-19 infection and promising therapeutic approaches
No full textIn the Fall of 2019 a sudden and dramatic outbreak of a pulmonary disease (Coronavirus Disease COVID-19), due to a new Coronavirus strain (i.e., SARS-CoV-2), emerged in the continental Chinese area of Wuhan and quickly diffused throughout the world, causing up to now several hundreds of thousand deaths. As for common viral infections, the crucial event for the viral life cycle is the entry of genetic material inside the host cell, realized by the spike protein of the virus through its binding to host receptors and its activation by host proteases; this is followed by translation of the viral RNA into a polyprotein, exploiting the host cell machinery. The production of individual mature viral proteins is pivotal for replication and release of new virions. Several proteolytic enzymes either of the host and of the virus act in a concerted fashion to regulate and coordinate specific steps of the viral replication and assembly, such as (i) the entry of the virus, (ii) the maturation of the polyprotein and (iii) the assembly of the secreted virions for further diffusion. Therefore, proteases involved in these three steps are important targets, envisaging that molecules which interfere with their activity are promising therapeutic compounds. In this review, we will survey what is known up to now on the role of specific proteolytic enzymes in these three steps and of most promising compounds designed to impair this vicious cycle
Multiple functions of insulin-degrading enzyme: a metabolic crosslight?
No full textInsulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as Î2-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the âaggregopathiesâ and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitinâproteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulin in vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the âquality controlâ machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes
Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains
No full textProteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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