National Repository of Grey Literature 67 records found  previous11 - 20nextend  jump to record: Search took 0.00 seconds. 
Adenylate cyclase toxin of bacteria Bordetella pertussis: mechanism of potassium efflux from macrophages
Pospíšilová, Eva ; Mašín, Jiří (advisor) ; Konopásek, Ivo (referee)
The adenylate cyclase toxin-hemolysin (ACT or CyaA) is a key virulence factor of Bordetella pertussis, the agent of the human respiratory disease whooping cough. CyaA penetrates phagocytes expressing the CD11b/CD18 integrin and exhibits two different activities. One toxin conformer oligomerizes in cell membrane and permeabilizes it by forming small cation-selective pores. Another toxin conformer appears to act as monomer. It forms a calcium influx path in the membrane, concomitantly with translocating into cells the adenylate cyclase enzyme domain that binds calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. We show here that CyaA causes efflux of K+ from CD11b+ cells by a mechanism that requires binding of CyaA to integrin CD11b/CD18 and permeabilization of the cellular membrane by pore-forming conformer of CyaA. Intact CyaA and the enzymatically inactive CyaA-AC- toxoid unable to generate cAMP produced the same kinetics of K+ efflux showing that elevation or signaling of cAMP had no role in this activity. The truncated CyaA variant (CyaA-AC) devoid of its invasive AC domain failed to promote K+ efflux despite a normal pore forming activity on erythrocyte and artificial membranes. However, binding of the MAb 3D1, which recognizes the distal segment of the AC domain...
Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek ; Hof, Martin (advisor) ; Fidler, Vlastimil (referee) ; Konopásek, Ivo (referee)
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
Study of the interaction between fungus Pleurotus ostreatus and bacterial cultures on the abiotic surfaces - morphological, biochemical and proteomic analysis
Kozická, Barbora ; Petráčková, Denisa (advisor) ; Konopásek, Ivo (referee)
Ligninolytic fungi are well known for their ability to degrade a wide range of xenobiotics contaminating the environment, including synthetic industrial dyes. In this work Pleurotus ostreatus was used for decolorization of a synthetic textile dye Remazol Brilliant Blue R (RBBR). To set up a model fungal "fixed-bed" bioreactor the fungus was immobilized on a polyurethane foam and artificially contaminated with a model bacterium Rhodococcus erythropolis. The development of bacterial contamination can be expected during a real application of fungal bio filters in wastewater treatment. The main aim of the work was to study interspecies interactions in the model bioreactors during the dye decolorization. Ligninolytic enzyme activities were followed in the bioreactor cultures as markers of fungal biodegradation ability. In contrast to the controls, no bacterial growth was observed in the P. ostreatus bioreactor culture liquid. The results showed that fungal laccase, pH of the culture liquid, and glucose consumption by the fungus had no effect on the bacterial growth. However, 4*105 - 1,3*106 CFU/ml of R. erythropolis was detected to be associated with the fungal solid support. The presence of these bacteria had no effect on the decolorization performance of the bioreactors. Dye decolorization efficiency...
Cytoplasmic membrane of Bacillus subtilis Regulation of the physical parameters
Beranová, Jana ; Konopásek, Ivo (advisor) ; Branny, Pavel (referee) ; Holoubek, Aleš (referee)
Bacillus subtilis, a model Gram-positive soil bacterium, employs two distinct mechanisms in its membrane adaptation to low temperature: 1) Long-term adaptation to suboptimal temperature is accomplished by increasing the ratio of anteiso- to iso-branched fatty acids in the membrane lipids. 2) After a sudden temperature decrease, the oxygen-dependent fatty acid desaturase (Des) is induced which desaturates fatty-acyl chains incorporated in membrane lipids. The transcription of the gene encoding desaturase, des, is activated by the decrease of the membrane order, via two- component system DesK-DesR. In this work, I studied the influence of cultivation conditions on the mechanisms of B. subtilis membrane adjustments for a low temperature employing fatty acid analysis, fluorescence spectroscopy, differential scanning calorimetry and methods of molecular biology. In the first part of this work, I examined the impact of the cultivation medium on the composition and biophysical features of the B. subtilis cytoplasmic membrane during growth under the optimal (40 řC) and suboptimal (20 řC) cultivation temperature. I compared the nutrient-rich complex medium containing glucose and the mineral medium supplemented with either glucose or glycerol. The results obtained showed the crucial importance of medium...
The role of RTX domain in the activity of adenylate cyclase toxin from Bordetella pertussis
Klímová, Nela ; Bumba, Ladislav (advisor) ; Konopásek, Ivo (referee)
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.
Spr0334, new protein of cell division in Streptococcus pneumoniae.
Štekerová, Nela ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...
Characterization of 32,33-didehydroroflamycoin - secondary metabolite from Streptomyces durmitorensis
Koukalová, Alena ; Černý, Jan (advisor) ; Konopásek, Ivo (referee)
Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.
Model membranes studied by advanced fluorescence techniques and molecular dynamics simulations
Melcrová, Adéla ; Hof, Martin (advisor) ; Heyda, Jan (referee) ; Konopásek, Ivo (referee)
In this thesis, we start with the description of the biophysical properties of the plasma membrane models upon signaling processess such as the increased cytoso- lic concentration of calcium ions, or posttranslational modifications of membrane proteins. Calcium signaling is characterized by a rapid increase of its cytosolic concentration. We identify calcium binding sites and characterize the binding in the plasma membrane models of increasing complexity from pure phospholipid bilayers, through cholesterol and peptide rich lipid membranes, to membranes ex- tracted from HEK293 cells. We use Time-Dependent Fluorescent Shift method, which provides direct information on hydration and mobility in defined regions of a lipid bilayer, accompanied with molecular dynamic (MD) simulations, which give molecular details of the studied interactions. The initial step of signaling mediated by PAG protein is its double palmi- toylation. We investigate changes of the biophysical properties of both the lipid membrane and the peptide itself upon the incorporation of the palmitoyls. Em- ploying all atom MD simulations, we study inter- and intramolecular interactions as well as changes in membrane hydration, thickness, or lipid ordering. The second part of the thesis, realized in a direct collaboration with a phar- macological...

National Repository of Grey Literature : 67 records found   previous11 - 20nextend  jump to record:
Interested in being notified about new results for this query?
Subscribe to the RSS feed.