Prokop, Lukáš*
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1. vyd. 134 s. : il. ; 21 cm
Publikace je učební oporou stejnojmenného vzdělávácího kurzu,který připravuje zdravotníky k tomu aby byli schopni poskytovat první psychickou pomoc. Práce ve zdravotnictví znamená kontakt s nemocemi,ztrátami, někdy i lidským neštěstím či smrtí. Tyto situace doprovází smutek, truchlení, bezradnost, stavy akutní stresové reakce a jiné průvodní jevy. Bývají přítomny nejen u samotných pacientů, například při sdělení závažné diagnózy, ale také u jejích blízkých.
- MeSH
- horké linky MeSH
- krizová intervence MeSH
- profesionální etika MeSH
- psychický stres MeSH
- sebevražda MeSH
- smrt MeSH
- studium ošetřovatelství MeSH
- zákonodárství ošetřovatelské MeSH
- zdravotnická komunikace MeSH
- zdravotničtí pracovníci MeSH
- Publikační typ
- učebnice MeSH
- Konspekt
- Veřejné zdraví a hygiena
- NLK Obory
- veřejné zdravotnictví
- psychologie, klinická psychologie
Substrate specificity is a key feature of enzymes determining their applicability in biomaterials and biotechnologies. Experimental testing of activities with novel substrates is a time-consuming and inefficient process, typically resulting in many failures. Here, we present an experimentally validated in silico method for the discovery of novel substrates of enzymes with a known reaction mechanism. The method was developed for a model system of biotechnologically relevant enzymes, haloalkane dehalogenases. On the basis of the parametrization of six different haloalkane dehalogenases with 30 halogenated substrates, mechanism-based geometric criteria for reactivity approximation were defined. These criteria were subsequently applied to the previously experimentally uncharacterized haloalkane dehalogenase DmmA. The enzyme was computationally screened against 41,366 compounds, yielding 548 structurally unique compounds as potential substrates. Eight out of 16 experimentally tested top-ranking compounds were active with DmmA, indicating a 50% success rate for the prediction of substrates. The remaining eight compounds were able to bind to the active site and inhibit enzymatic activity. These results confirmed good applicability of the method for prioritizing active compounds-true substrates and binders-for experimental testing. All validated substrates were large compounds often containing polyaromatic moieties, which have never before been considered as potential substrates for this enzyme family. Whereas four of these novel substrates were specific to DmmA, two substrates showed activity with three other tested haloalkane dehalogenases, i.e., DhaA, DbjA, and LinB. Additional validation of the developed screening strategy with the data set of over 200 known substrates of Candida antarctica lipase B confirmed its applicability for the identification of novel substrates of other biotechnologically relevant enzymes with an available tertiary structure and known reaction mechanism.
- MeSH
- chemické databáze MeSH
- fungální proteiny chemie metabolismus MeSH
- hydrolasy chemie genetika metabolismus MeSH
- lipasa chemie metabolismus MeSH
- počítačová simulace MeSH
- preklinické hodnocení léčiv metody MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 μM and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization.
- MeSH
- hydrolasy antagonisté a inhibitory chemie MeSH
- inhibitory enzymů chemie izolace a purifikace metabolismus MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- Mycobacterium tuberculosis enzymologie MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many enzymes contain tunnels and gates that are essential to their function. Gates reversibly switch between open and closed conformations and thereby control the traffic of small molecules-substrates, products, ions, and solvent molecules-into and out of the enzyme's structure via molecular tunnels. Many transient tunnels and gates undoubtedly remain to be identified, and their functional roles and utility as potential drug targets have received comparatively little attention. Here, we describe a set of general concepts relating to the structural properties, function, and classification of these interesting structural features. In addition, we highlight the potential of enzyme tunnels and gates as targets for the binding of small molecules. The different types of binding that are possible and the potential pharmacological benefits of such targeting are discussed. Twelve examples of ligands bound to the tunnels and/or gates of clinically relevant enzymes are used to illustrate the different binding modes and to explain some new strategies for drug design. Such strategies could potentially help to overcome some of the problems facing medicinal chemists and lead to the discovery of more effective drugs.
- MeSH
- cílená molekulární terapie * MeSH
- enzymy metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- racionální návrh léčiv MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
As in other areas of experimental science, operation of electrophysiological laboratory, design and performance of electrophysiological experiments, collection, storage and sharing of experimental data and metadata, analysis and interpretation of these data, and publication of results are time consuming activities. If these activities are well organized and supported by a suitable infrastructure, work efficiency of researchers increases significantly. This article deals with the main concepts, design, and development of software and hardware infrastructure for research in electrophysiology. The described infrastructure has been primarily developed for the needs of neuroinformatics laboratory at the University of West Bohemia, the Czech Republic. However, from the beginning it has been also designed and developed to be open and applicable in laboratories that do similar research. After introducing the laboratory and the whole architectural concept the individual parts of the infrastructure are described. The central element of the software infrastructure is a web-based portal that enables community researchers to store, share, download and search data and metadata from electrophysiological experiments. The data model, domain ontology and usage of semantic web languages and technologies are described. Current data publication policy used in the portal is briefly introduced. The registration of the portal within Neuroscience Information Framework is described. Then the methods used for processing of electrophysiological signals are presented. The specific modifications of these methods introduced by laboratory researches are summarized; the methods are organized into a laboratory workflow. Other parts of the software infrastructure include mobile and offline solutions for data/metadata storing and a hardware stimulator communicating with an EEG amplifier and recording software.
- Publikační typ
- časopisecké články MeSH
An ageing population is a typical feature of many developed countries across the world. Analyzed from a biomedical and philosophical point of view, this phenomenon is also a potential risk factor for social sustainability of communities. The association between ageing and cancer seems to be more than apparent. Therefore, the further increase of epidemic-like incidence of malignant tumors in a population can be expected in the near future. Elderly people usually suffer from age-dependent diseases, and such polymorbidity can seriously affect the treatment of malignant tumors. Such an impending situation may be associated with multiple medical, social and economic issues. This article summarizes data about the possible molecular mechanism influencing rapid spreading of tumors in the elderly population. Reduction of the activity of DNA repair machinery is a likely genetic cause. Besides this, even epigenetic mechanisms can influence this process. In this context, the role of cancer stroma in controlling multiple biological properties of tumors is a prospective target for translational research with potential therapeutic outcomes.
BACKGROUND: Heterologous expression systems based on promoters inducible with isopropyl-β-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacI(Q)/P(lacUV5)-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, side-reactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked. RESULTS: The physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer's chemical properties. CONCLUSIONS: IPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect. Importantly, we show that induction with lactose, the natural inducer of P lac , dramatically lightens the burden without reducing the efficiency of the synthetic TCP degradation pathway. This suggests that lactose may be a better inducer than IPTG for the expression of heterologous pathways in E. coli BL21(DE3).
The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.
- MeSH
- bodová mutace MeSH
- design s pomocí počítače * MeSH
- embryonální kmenové buňky cytologie metabolismus MeSH
- fibroblastový růstový faktor 2 chemie genetika metabolismus MeSH
- lidé MeSH
- počítačová simulace MeSH
- proteinové inženýrství * MeSH
- řízená evoluce molekul MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
