Colloidal-chemical characteristics of block/branched cationic and non-ionic polyamphiphiles containing poly(fluorine-alkyl methacrylate) (poly(FMA)) block and their intermolecular complexes with biopolymers were studied. The dependences of their surface activity and micelle size on the length of hydrophobic and hydrophilic blocks, as well as the length of side fluorine-alkyl branches were established. Poly(FMA)-block-poly(DMAEMA) was used for formation of interpolyelectrolyte complexes with plasmid DNA (pDNA) via their electrostatic interaction. Novel non-viral polyplexes were tested as gene delivery systems for mammalian cells. The results of DLS, TEM and MALDI-ToF studies demonstrated disaggregation of lysozyme (LYZ) aggregates in the presence of poly(FMA)-block-poly(NVP) and formation of the polyamphiphile…LYS complex possessing antibacterial action.
- MeSH
- DNA chemie metabolismus MeSH
- fluor chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- methakryláty chemie MeSH
- micely MeSH
- muramidasa chemie metabolismus MeSH
- plazmidy chemie metabolismus MeSH
- polyethylenglykoly chemie MeSH
- polymery chemie metabolismus MeSH
- technika přenosu genů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAapta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNAapta layer depended on the stripping current (Istr) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNAapta complexes occur in the Istr ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.
Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein misfolding in neurodegenerative disorders. Calorimetry has proven indispensable in this regard for recording full energetic profiles of protein unfolding and permitting data fitting based on unfolding pathway models. While both kinetic and thermodynamic protein stability are analysed by varying scan rates and reheating, the latter is rarely used in curve-fitting, leading to a significant loss of information from experiments. To extract this information, we propose fitting both first and second scans simultaneously. Four most common single-peak transition models are considered: (i) fully reversible, (ii) fully irreversible, (iii) partially reversible transitions, and (iv) general three-state models. The method is validated using calorimetry data for chicken egg lysozyme, mutated Protein A, three wild-types of haloalkane dehalogenases, and a mutant stabilized by protein engineering. We show that modelling of reheating increases the precision of determination of unfolding mechanisms, free energies, temperatures, and heat capacity differences. Moreover, this modelling indicates whether alternative refolding pathways might occur upon cooling. The Matlab-based data fitting software tool and its user guide are provided as a supplement.
Poly(3-hydroxybutyrate) (PHB) is a common carbon- and energy-storage compound simultaneously produced and degraded into its monomer 3-hydroxybutyrate (3HB) by numerous bacteria and Archae in a metabolic pathway called the PHB cycle. We investigated 3HB as a chemical chaperone capable of protecting model enzymes, namely lipase and lysozyme, from adverse effects of high temperature and oxidation. Heat-mediated denaturation of lipase in the presence or absence of 3HB was monitored by dynamic light scattering (DLS) revealing a significant protective effect of 3HB which increased as its concentration rose. Furthermore, when compared at the same molar concentration, 3HB showed a greater protective effect than the well-known chemical chaperones trehalose and hydroxyectoine. The higher protective effect of 3HB was also confirmed when employing differential scanning calorimetry (DSC) and lysozyme as a model enzyme. Furthermore, 3HB was capable of protecting lipase not only against thermal-mediated denaturation but also against oxidative damage by Cu(2+) and H2O2; its protection was higher than that of trehalose and comparable to that of hydroxyectoine. Taking into account that the PHB-producing strain Cupriavidus necator H16 reveals a 16.5-fold higher intracellular concentration than the PHB non-producing mutant C. necator PHB(-4), it might be expected that the functional PHB cycle might be responsible for maintaining a higher intracellular level of 3HB which, aside from other positive aspects of functional PHB metabolism, enhances stress resistance of bacterial strains capable of simultaneous PHB synthesis and mobilization. In addition, 3HB can be used in various applications and formulations as an efficient enzyme-stabilizing and enzyme-protecting additive.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- Cupriavidus necator chemie enzymologie metabolismus MeSH
- hydroxybutyráty metabolismus MeSH
- kyselina 3-hydroxymáselná chemie metabolismus MeSH
- muramidasa chemie metabolismus MeSH
- ochranné látky chemie metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- polyestery metabolismus MeSH
- stabilita enzymů MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Despite the high number of studies concerning seasonality of immune response in fish, information for some fish species is still scarce. Here, we assess seasonal changes in leukocyte counts and several immune parameters in three groups of farmed salmonids, i.e. brook trout (Salvelinus fontinalis), brook trout x Arctic charr hybrids (Salvelinus fontinalis x Salvelinus alpinus alpinus) and rainbow trout (Oncorhynchus mykiss) reared under the same conditions and fed with the same feed. Fish were sampled in five periods of the year (late April, early July, late August, early November and early February) and leukocyte counts, respiratory burst of blood phagocytes, lysozyme concentration in skin mucus and total complement activity were measured. Generalized linear models using fish body length as a continuous predictor and sampling period and fish species as categorical predictors, were significant for each of the parameters analysed. The highest seasonal variations in measured parameters were found in rainbow trout and lowest in hybrids. Our results confirm that measures of innate and adaptive immunity are strongly affected by season in all three groups of salmonids. The results will contribute to the improved assessment of immunocompetence in farmed fishes, essential for future sustainable development in aquaculture.
- MeSH
- fagocyty imunologie MeSH
- hlen chemie MeSH
- hybridizace genetická MeSH
- komplement metabolismus MeSH
- kůže chemie MeSH
- muramidasa metabolismus MeSH
- Oncorhynchus mykiss imunologie MeSH
- počet leukocytů veterinární MeSH
- pstruh imunologie MeSH
- respirační vzplanutí * MeSH
- roční období * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
A novel positively charged surfactant N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride enabled the reversal of the electro-osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.
- MeSH
- cytochromy c chemie izolace a purifikace MeSH
- elektroforéza kapilární MeSH
- kationty chemie MeSH
- muramidasa chemie izolace a purifikace metabolismus MeSH
- myoglobin chemie izolace a purifikace MeSH
- oxid křemičitý chemie MeSH
- pankreatická ribonukleasa chemie izolace a purifikace MeSH
- povrchově aktivní látky chemie MeSH
- Publikační typ
- časopisecké články MeSH
Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression.
- MeSH
- Bacillus subtilis imunologie MeSH
- celulasy metabolismus MeSH
- Escherichia coli imunologie MeSH
- hexosaminidasy metabolismus MeSH
- messenger RNA metabolismus MeSH
- muramidasa metabolismus fyziologie MeSH
- Oligochaeta enzymologie imunologie mikrobiologie MeSH
- přirozená imunita MeSH
- proteasy metabolismus MeSH
- Saccharomyces cerevisiae imunologie MeSH
- upregulace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: An enhanced release of metals in the mouth due to galvanic cell formation is considered to be one of the causes of oral discomfort. The aim of this study was to investigate the influence of galvanic cell on salivary immune defense factors. MATERIAL AND METHODS: The levels of IgA1, IgA2, sIgA, lysozyme and antiIgA/HSP60 were evaluated in representative samples from 159 patients with galvanism, from 177 patients without galvanism and in two control groups. All the participants underwent personal history taking, clinical examination, galvanic currents measurement and saliva collection. RESULTS: Electro active dental materials were removed in 30 patients. There was a significant increase in IgA2 level, a significant decrease in antiIgA/HSP60 levels and an increase in IgA1, sIgA and lysozyme levels found after the removal of electro active restorations. Morphological symptoms disappeared in 70% of the treated patients. CONCLUSION: The study confirmed that pathologic galvanic phenomena influences the immune defense reactions in the oral cavity and thus may cause the symptoms of oral discomfort. A measurement of the galvanism and a subsequent removal of electro active restorations should become a common therapeutic procedure in the patients with oral discomfort.
- MeSH
- biologické markery metabolismus MeSH
- chaperon hsp60 imunologie metabolismus MeSH
- dospělí MeSH
- elektrogalvanismus intraorální * MeSH
- imunoglobulin A imunologie metabolismus MeSH
- kovy imunologie metabolismus MeSH
- lidé MeSH
- mitochondriální proteiny imunologie metabolismus MeSH
- muramidasa imunologie metabolismus MeSH
- odstranění implantátu MeSH
- protilátky anti-idiotypické imunologie metabolismus MeSH
- sliny imunologie metabolismus MeSH
- trvalá zubní náhrada škodlivé účinky MeSH
- ústa imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky MeSH
- práce podpořená grantem MeSH
Caenorhabditis elegans has been increasingly used to study the innate immunity and for the screening of microbe/host-specific pathogenic factors. Staphylococcus aureus-mediated infections with live C. elegans were performed on solid (full-lawn) and liquid assays. S. aureus required 90 ± 10 h for the complete killing of C. elegans, but the infection was started only after 32 h of exposure with 20% inoculum of S. aureus. The short time exposure studies revealed that, in 20% of inoculum, continuous exposure to the pathogen was required for the killing of nematode. In 100% of inoculum, only 8 h of exposure was sufficient to kill the C. elegans. To evaluate kinetically at the innate immune level, the regulation of representative candidate antimicrobial genes was investigated. Both semi-quantitative reverse transcriptase polymerase chain reaction (PCR) and real-time PCR analyses indicated the regulation of candidate immune regulatory genes of lysozyme (lys-7), cysteine protease (cpr-2), and C-type lectin (clec-60 and clec-87) family members during the course of S. aureus infections, indicating the possible contribution of the above players during the host immune response against S. aureus exposures.
- MeSH
- bakteriální léková rezistence MeSH
- Caenorhabditis elegans genetika imunologie metabolismus MeSH
- cysteinové proteasy genetika imunologie metabolismus MeSH
- exprese genu MeSH
- interakce hostitele a patogenu MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lektiny typu C genetika imunologie metabolismus MeSH
- muramidasa genetika imunologie metabolismus MeSH
- počet mikrobiálních kolonií MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- přirozená imunita MeSH
- proteiny Caenorhabditis elegans genetika imunologie metabolismus MeSH
- stafylokokové infekce genetika imunologie mikrobiologie MeSH
- Staphylococcus aureus fyziologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria.
- MeSH
- Bacillus subtilis imunologie patogenita MeSH
- bakteriální adheze MeSH
- chitinasy metabolismus MeSH
- Echinodermata genetika MeSH
- endopeptidasy metabolismus MeSH
- Escherichia coli genetika imunologie patogenita MeSH
- glukosamin analogy a deriváty imunologie metabolismus MeSH
- grampozitivní bakteriální infekce imunologie MeSH
- hydrolýza MeSH
- infekce vyvolané Escherichia coli imunologie MeSH
- interakce hostitele a patogenu MeSH
- klonování DNA MeSH
- kyseliny muramové imunologie metabolismus MeSH
- lyasy štěpící vazby C-N metabolismus MeSH
- muramidasa genetika imunologie metabolismus MeSH
- Oligochaeta enzymologie genetika imunologie MeSH
- pijavka lékařská genetika MeSH
- sekvenční homologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
