Presence Of Natural Inhibitors Research Articles

Molecular mechanism of selective substrate engagement and inhibitor disengagement of cysteine synthase

O-acetyl serine sulfhydrylase (OASS), referred to as cysteine synthase (CS), synthesizes cysteine from O-acetyl serine (OAS) and sulfur in bacteria and plants. The inherent challenge for CS is to overcome 4 to 6 log-folds stronger affinity for its natural inhibitor, serine acetyltransferase (SAT), as compared with its affinity for substrate, OAS. Our recent study showed that CS employs a novel competitive-allosteric mechanism to selectively recruit its substrate in the presence of natural inhibitor. In this study, we trace the molecular features that control selective substrate recruitment. To generalize our findings, we used CS from three different bacteria (Haemophilus, Salmonella, and Mycobacterium) as our model systems and analyzed structural and substrate-binding features of wild-type CS and its ∼13 mutants. Results show that CS uses a noncatalytic residue, M120, located 20 Å away from the reaction center, to discriminate in favor of substrate. M120A and background mutants display significantly reduced substrate binding, catalytic efficiency, and inhibitor binding. Results shows that M120 favors the substrate binding by selectively enhancing the affinity for the substrate and disengaging the inhibitor by 20 to 286 and 5- to 3-folds, respectively. Together, M120 confers a net discriminative force in favor of substrate by 100- to 858-folds.

Structural insights of metallo-beta-lactamase revealed an effective way of inhibition of enzyme by natural inhibitors

Metallo-beta-lactamase (MBL) is a class of enzyme that catalyzes the hydrolysis of a broad range of beta-lactam antibiotics leading to the development of drug resistance in bacteria. Inhibition of MBL is therefore pursued as a potential way to increase the susceptibility of bacteria to beta-lactam antibiotics. In this study, MBL inhibitors from natural sources such as Eupalitin, Rosmarinic acid and Luteolin are used as a potential alternative to explore their effect. The crystal structure of MBL revealed a hydrolyzed Meropenem, which was undocked from the active center pocket to get the apo-protein. The apo-protein was re-docked with substrate, three known MBL inhibitors and natural compounds to prepare the starting structure in the current work and to draw conclusions. Further, to explore the efficiency of natural inhibitors, we analyzed the dynamic behavior of the enzyme over simulation time using molecular dynamics studies. Our results suggest that MBL enzyme adopted altered conformational state in the presence of natural inhibitor. This is because, the natural inhibitors were tried to occupy a different binding pocket in the enzyme by causing positional drift from the active center pocket. Here, the different binding pocket partly comprised of active site pocket and partly by a new region explored by ligand, making it inappropriate for substrate to occupy the active site. Thus natural inhibitors may be potential entities to target MBL.AbbreviationsADMEAbsorption, Distribution, Metabolism and ExcretionBBBBlood brain barrierCHARMMChemistry at Harvard Macromolecular MechanicsCOMCenter of MassCYP2D6Cytochrome P450 2D6DSDiscovery StudioESBLExtended Spectrum Beta-lactamasesFDAFood and Drug AdministrationGLASSGlobal antimicrobial resistance surveillance systemGROMACSGROningen MAchine for Chemical SimulationsKDEKernel Density Estimation PlotsMBLMetallo-beta-lactamaseMBL-CMetallo-beta-lactamase bound to L-CaptoprilMBL-EMetallo-beta -lactamase bound to EupalitinMBL-IMetallo-beta -lactamase bound to ImipenemMBL-LMetallo-beta -lactamase bound to LuteolinMBL-RMetallo-beta -lactamase bound to Rosmarinic acidMDMolecular DynamicsMMPBSAMolecular Mechanics Poisson − Boltzmann surface areaNPTNumber of atoms in the system, Pressure of the system and Temperature of the systemnsNano secondsNVTNumber of atoms in the system, Volume of the system, and Temperature of the systemPDBProtein Data BankRgRadius of GyrationRMSDRoot Mean Square DeviationRMSFRoot Mean Square FluctuationSASASolvent Accessible Surface AreaSPC/ESimple Point ChargeWHOWorld Health OrganizationCommunicated by Ramaswamy H. Sarma

Study of Inhibiting Effect of natural products on Hard Growth Water, Valorization of lemon peel in the treatment of hard water

Hard water forms compact and adherent deposit to the walls of pipes and industrial or domestic installations. Several studies have been done in order to reduce the adverse scaling consequences and to achieve effective softening, but most of inhibitors used are chemical compounds that have damaging effects on health and the environment. There for we have used in this work an under natural alimentary product as scaling inhibitor. Our study focuses on softening hard water of Bounouara having a hardness of 58°F in the presence of lemon peel extracts as scaling inhibitor. The evaluation of scaling power of Bounouara water and inhibitory effect of lemon peel was performed by using two techniques: - scaling accelerated method to evaluate the scaling power of raw Bounouara water electrochemically in the presence of natural inhibitors (lemon peel) - Impedancemetric method to appreciate globally, using the high frequency resistance, the importance and adherence of calcium carbonate deposit. Chronoamperometric study shows that scaling time decreases by increasing of temperature. So hard water become more scale-forming in high temperatures. Application of the treatment on Bounouara water showed that total hardness inhibition requires addition of lemon peel of 0.5 g/L at 20°C and 1 g/L at 40°C. Keywords: Scaling, natural inhibitor, chronoamperometry, impedancemetry.

MMPs are regulatory enzymes in pathways of inflammatory disorders, tissue injury, malignancies and remodelling of the lung

Members of the matrix metalloproteinase (MMP) family of zinc-dependent proteolytic enzymes regulate physiological and pathophysiological events in development, injury and repair, such as morphogenesis, vasculogenesis, remodelling of the extracellular matrix, cell migration, cleavage of cytokines, and activation of mediators and defensins. They are synthesised as secreted or transmembrane proenzymes and processed to their active forms by the removal of amino-terminal propeptides. Their activation by the cleavage of the prodomain from the latent proenzyme and the presence of natural inhibitors, such as tissue inhibitors of metalloproteinases (TIMPs), are important mechanisms that restrict the action of secreted MMPs to the micromilieu of their origin. This tight control of activation and the redundant design of the MMP system, points to their importance in the named processes. In most tissues, their constitutive expression is low and their overexpression at sites of inflammation or other pathogenic events was thought to be due the classical role of extracellular matrix degradation. However, MMPs are no longer thought of as just matrix-degrading proteins. Rather, proteolysis should be viewed as an important post-translational principle of regulating a fast spectrum of biological processes, and the functional redundancy in combination with a wide substrate overlap underscores the pivotal roles of MMPs not only as effectors but also in regulatory mechanisms. Thus, MMPs are essential in many physiological events and the increasing interest in their function originates from their contribution to inflammatory and remodelling processes, in which they regulate physical barriers, release bioactive factors, participate in membrane shedding, modulate chemokine gradients that regulate leukocyte motility, activate cells via triggering of proteinase-activated receptors (PAR), and alter the activity status of other proteases, including other MMPs. Consequently, the majority of MMP substrates are non-matrix proteins, which underscores their critical roles in regulatory processes [1, 2]. Reports implicating MMPs in destructive pathogenic …

Inibição do desenvolvimento in vitro de embriões de Coffea por cafeína exógena

A cafeína, alcalóide conhecido como 1,3,7-trimetilxantina, é encontrada em sementes quiescentes de cafeeiro, perfazendo um total de 1,1 a 1,7% em Coffea arabica L. e 2 a 3% em Coffea canephora Pierre, localizada em sua grande maioria no endosperma, na forma livre no citoplasma das células ou complexada com ácidos clorogênicos. Com função fisiológica em plantas ainda não totalmente esclarecida, a cafeína causa efeito alelopático, seja inibindo a germinação de várias espécies, seja como anti-herbívoro ou como agente pesticida natural. A lenta germinação de sementes de café ainda não foi esclarecida e várias causas são apontadas, como presença do endocarpo, baixa absorção de água e O2, presença de inibidores naturais e balanço hormonal. Embora sugeridos, estudos sobre a inibição de sementes de cafeeiro por ação da cafeína endógena e ou exógena são escassos. Assim, o presente trabalho teve como objetivo estudar o efeito da cafeína exógena sobre a germinação e o desenvolvimento de embriões de Coffea arabica L. e de Coffea canephora Pierre. O experimento foi realizado utilizando-se frutos no estádio cereja das cultivares Rubi e Apoatã IAC-2258. Após desinfestação dos frutos por 30 minutos de imersão em hipoclorito de sódio (2% i.a.) e lavagem por três vezes em água destilada e autoclavada, os embriões foram retirados e inoculados, de modo asséptico, em placas de petri com meio MS 50%, acrescido de sacarose e suplementado com diferentes concentrações de cafeína (0,00; 0,05; 0,10; 0,15; 0,20; 0,25; 0,30 e 0,40%). Os embriões foram mantidos em sala de crescimento a 27 ± 2ºC e densidade de fluxo de fótons de 13µmol.m-2.s-1, durante 23 dias, quando foram avaliados comprimento da parte aérea, comprimento de raiz e massa fresca das plântulas. Cinco dias após o cultivo, foram avaliadas a porcentagem de emissão de radículas e cotilédones, computando-se os embriões com cotilédones abertos e radículas expandidas. O delineamento experimental utilizado foi inteiramente casualizado com seis repetições por tratamento, sendo cada repetição constituída por cinco embriões. Concluiu-se que a germinação e o desenvolvimento in vitro de embriões de Coffea arabica L. e Coffea canephora Pierre são afetados por cafeína exógena. O efeito detrimental da cafeína exógena em embriões de Coffea é maior nas radículas do que nos cotilédones. Embriões de Coffea arabica L. são mais sensíveis aos efeitos negativos da cafeína exógena do que embriões de Coffea canephora Pierre. A cafeína pode contribuir para a lenta germinação de sementes e o lento desenvolvimento de plântulas de cafeeiro.

Flow cytometric analysis of gelatinase B (MMP-9) activity using immobilized fluorescent substrate on microspheres.

Gelatinase B is a member of the metalloproteinase family of enzymes that degrade the extracellular matrix under normal and pathological conditions, including autoimmune diseases and tumor cell dissemination. In the present work, we describe a simple and reliable method that allows qualitative and quantitative measurements of a specific enzymatic reaction (mediated here by gelatinase B) by flow cytometry using fluorochrome-labeled substrate coated on polystyrene microspheres. Using this approach, proteolytic degradation can be monitored by the decrease of the fluorescence emitted by the microspheres following incubation with the enzyme. In most of the experiments, the signal-to-noise ratio between autofluorescent microspheres and those coated with the fluorescein isothiocyanate (FITC)-labeled substrate was near 500. This ratio allows one to measure accurately the enzymatic activity in the presence of chemical or biological inhibitors. FITC labeling and passive adsorption of substrates on the solid support did not cause significant conformational changes or steric hindrance that would interfere with the proteolytic activity of gelatinase B. This method constitutes a powerful tool for the measurement, on a routine basis, of the net activity resulting from the balance between the gelatinase B activity and the presence of natural inhibitors and for the identification of new metalloproteinase inhibitors that could suppress the excessive proteolytic activity that characterizes many disease processes.

Flow cytometric analysis of gelatinase B (MMP‐9) activity using immobilized fluorescent substrate on microspheres

Gelatinase B is a member of the metalloproteinase family of enzymes that degrade the extracellular matrix under normal and pathological conditions, including autoimmune diseases and tumor cell dissemination. In the present work, we describe a simple and reliable method that allows qualitative and quantitative measurements of a specific enzymatic reaction (mediated here by gelatinase B) by flow cytometry using fluorochrome-labeled substrate coated on polystyrene microspheres. Using this approach, proteolytic degradation can be monitored by the decrease of the fluorescence emitted by the microspheres following incubation with the enzyme. In most of the experiments, the signal-to-noise ratio between autofluorescent microspheres and those coated with the fluorescein isothiocyanate (FITC)-labeled substrate was near 500. This ratio allows one to measure accurately the enzymatic activity in the presence of chemical or biological inhibitors. FITC labeling and passive adsorption of substrates on the solid support did not cause significant conformational changes or steric hindrance that would interfere with the proteolytic activity of gelatinase B. This method constitutes a powerful tool for the measurement, on a routine basis, of the net activity resulting from the balance between the gelatinase B activity and the presence of natural inhibitors and for the identification of new metalloproteinase inhibitors that could suppress the excessive proteolytic activity that characterizes many disease processes. © 1996 Wiley-Liss, Inc.

IL-1β, a strong mediator for glucose uptake by rheumatoid and non-rheumatoid cultured human synoviocytes

Higher basal 2-deoxy- d-glucose uptake in rheumatoid synovial cells than in non-rheumatoid synovial cells, was found to be associated with an increased interleukin-1β (IL-1β) secretion (respectively 850 ± 238 vs. 8.3 ± 2.4 pg/24 h/10 5 cells, mean ± S.E.M.). When exogenous human recombinant IL-1β was added to cultures, a marked stimulation of 2-deoxy- d-glucose uptake was performed by both human synovial cultured cells, in a time-dependent and dose-dependent manner (IL-1β0–100 ng/ml). In non-rheumatoid synoviocytes, stimulation occurred 1–3 h following the addition of 1 ng/ml interleukin-1β and increased up to 24 hours (respectively +150% and +261.4% after 6 and 24 hours association time). Rheumatoid synovial cells were less sensitive to 1 ng/ml IL-1β (respectively +80% and +146.4%). IL-1β increased significantly the V max for 2-deoxy- d-glycose uptake by synovial cells, with no change in the K m. This effect was protein synthesis-dependent, and not secondary to prostaglandin E2 synthesis or cell growth. IL-1β possesses an important effect on glucose homeostasis in synovial cells, which could be indirect and/or regulated by the presence of natural inhibitors.

Proteolytic capacity in human plasma. I. Measurement of proteolytic activity in the presence of natural inhibitors and study of the inter-individual variations.

The maximal proteolytic activity in plasma that can be obtained by activation with urokinase and in the presence of natural inhibitors is designated proteolytic capacity. A modified caseinolytic method measuring this parameter has been described. In a normal population large variations between individuals were found, and this is probably due to differences in the plasminogen-plasmin level. The caseinolytic method correlated well with in vitro thrombolysis and with fibrinogenolysis in plasma. Serum had a higher proteolytic capacity than plasma. Pregnancy and the oral contraceptives caused a pronounced rise in proteolytic capacity, whereas thyrotoxicosis caused a decrease in the capacity. Two healthy donors, one with zero proteolytic capacity and a plasminogen level of 35–45%, and one with very high proteolytic capacity and a plasminogen level of about 130% were studied in particular. No increased tendency for thrombosis was found in persons with very low or zero proteolytic capacity.