Alzheimer's disease (AD) and sporadic Creutzfeldt-Jakob disease (sCJD) are both characterized by extracellular pathologically conformed aggregates of amyloid proteins-amyloid β-protein (Aβ) and prion protein (PrPSc), respectively. To investigate the potential morphological colocalization of Aβ and PrPSc aggregates, we examined the hippocampal regions (archicortex and neocortex) of 20 subjects with confirmed comorbid AD and sCJD using neurohistopathological analyses, immunohistochemical methods, and confocal fluorescent microscopy. Our data showed that extracellular Aβ and PrPSc aggregates tended to be, in most cases, located separately, and "compound" plaques were relatively rare. We observed PrPSc plaque-like structures in the periphery of the non-compact parts of Aβ plaques, as well as in tau protein-positive dystrophic structures. The AD ABC score according to the NIA-Alzheimer's association guidelines, and prion protein subtype with codon 129 methionine-valine (M/V) polymorphisms in sCJD, while representing key characteristics of these diseases, did not correlate with the morphology of the Aβ/PrPSc co-aggregates. However, our data showed that PrPSc aggregation could dominate during co-aggregation with non-compact Aβ in the periphery of Aβ plaques.
- MeSH
- Alzheimerova nemoc patologie MeSH
- amyloidní beta-protein genetika metabolismus MeSH
- amyloidní plaky patologie MeSH
- Creutzfeldtova-Jakobova nemoc patologie MeSH
- extracelulární prostor chemie MeSH
- jednonukleotidový polymorfismus genetika MeSH
- kodon genetika MeSH
- komorbidita MeSH
- lidé středního věku MeSH
- lidé MeSH
- mozek patologie MeSH
- neurony patologie MeSH
- pilotní projekty MeSH
- proteinové agregáty * MeSH
- proteiny tau metabolismus MeSH
- PrPSc proteiny genetika metabolismus MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Many microalgal species produce a wide range of highly-value products which are interesting for biotechnological applications. Cultivation of microalgal species Dictyosphaerium pulchellum and Dictyosphaerium tetrachotomum, strains Růžicka and Fott resulted yields of 0.2, 0.7 and 1.8 g/L of extracellular biopolymers (EPSs), respectively. All biopolymers were shown to be anionic proteoglycans. The sugar composition analyses of all EPSs showed high contents of hexoses and the presence of partially methylated monosaccharide residues, i.e. hexoses, and deoxy hexoses. The dominant sugar component of all EPSs was found to be galactose. Extracellular microalgal biopolymers were subjected to immunobiological and immunotoxicological evaluation using murine melanoma cancer cells B16, murine fibroblast cell line NIH-3T3, murine macrophages cell line RAW 264.7 and skin construct EpiDerm™ (EPI-200). The EPSs exerted the antiproliferative effectivity; treatment of EPS induced proinflammatory cytokines TNF-α, IL-6, IL-12, IL-1β and IL-17, also engaged in anti-cancer immunity. Immunotoxicological studies revealed their non-toxic character and safe application on EpiDerm™.
- MeSH
- biopolymery chemie farmakologie toxicita MeSH
- buňky NIH 3T3 MeSH
- Chlorophyta chemie MeSH
- extracelulární prostor chemie MeSH
- imunologické faktory chemie farmakologie toxicita MeSH
- kůže účinky léků MeSH
- mikrořasy chemie MeSH
- myši MeSH
- RAW 264.7 buňky MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Microalgae organisms are of interest for many biotechnology applications due to the production of a wide range of biologically active compounds. Incubation of Wollea saccata in a large scale afforded a mucilaginous, high molecular weight biopolymer composed of carbohydrate, protein and phenolic compounds. Sugar moiety was rich in hexoses (60%) and 6-deoxyhexoses (31%), while only 9% of pentoses was identified. Methylation analysis revealed about 40 types of methylated sugar derivatives, suggesting a very complex structure of Wollea biopolymer. Pharmacological studies revealed new pharmacodynamic properties of cyanobacteria biopolymer, i.e. antitussive and bronchodilatory. Biopolymer was able to suppress the cough reflex induced by chemical tussigen, but its effect was lower than that of codeine, the strongest antitussive agent. The bronchodilatory effect was similar or higher than the effect of salbutamol, a bronchodilatory drug used in a clinical practice. In pharmacological studies, there were no signs of toxicity or side effects in the animals following administration of Wollea biopolymer.
Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- buněčná membrána metabolismus MeSH
- extracelulární prostor chemie MeSH
- glykosylace MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- Jurkat buňky MeSH
- lidé MeSH
- lipoylace * MeSH
- membránové proteiny chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- transport proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U ml(-1)), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U ml(-1)). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5-5.0, and K (M) and V (max) values were 62 μM and 15.8 μmol min(-1) l(-1), respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K (i) of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0-6.0, respectively. The combination of relatively low molecular mass and low K (M) value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.
- MeSH
- Coriolaceae chemie enzymologie genetika MeSH
- extracelulární prostor chemie enzymologie genetika MeSH
- fungální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- kinetika MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- transport proteinů MeSH
- xylosidasy chemie genetika izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-D: -glucosaminide, N,N´-diacetyl-β-D: -chitobiose, or N,N´,N˝-triacetyl-β-D: -chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.
- MeSH
- 2D gelová elektroforéza MeSH
- acetylglukosaminidasa chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- chitinasy chemie genetika metabolismus MeSH
- Clostridium chemie enzymologie genetika izolace a purifikace MeSH
- extracelulární prostor chemie enzymologie genetika MeSH
- feces mikrobiologie MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Focal cortical dysplasias (FCDs) of the brain are recognized as a frequent cause of intractable epilepsy. To contribute to the current understanding of the mechanisms of epileptogenesis in FCD, our study provides evidence that not only cellular alterations and synaptic transmission, but also changed diffusion properties of the extracellular space (ECS), induced by modified extracellular matrix (ECM) composition and astrogliosis, might be involved in the generation or spread of seizures in FCD. The composition of the ECM in FCD and non-malformed cortex (in 163 samples from 62 patients) was analyzed immunohistochemically and correlated with the corresponding ECS diffusion parameter values determined with the real-time iontophoretic method in freshly resected cortex (i.e. the ECS volume fraction and the geometrical factor tortuosity, describing the hindrances to diffusion in the ECS). The ECS in FCD was shown to differ from that in non-malformed cortex, mainly by the increased accumulation of certain ECM molecules (tenascin R, tenascin C, and versican) or by their reduced expression (brevican), and by the presence of an increased number of astrocytic processes. The consequent increase of ECS diffusion barriers observed in both FCD type I and II (and, at the same time, the enlargement of the ECS volume in FCD type II) may alter the diffusion of neuroactive substances through the ECS, which mediates one of the important modes of intercellular communication in the brain - extrasynaptic volume transmission. Thus, the changed ECM composition and altered ECS diffusion properties might represent additional factors contributing to epileptogenicity in FCD.
- MeSH
- astrocyty metabolismus MeSH
- brevican analýza MeSH
- difuze MeSH
- dítě MeSH
- dospělí MeSH
- extracelulární matrix chemie metabolismus MeSH
- extracelulární prostor chemie metabolismus MeSH
- iontoforéza metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- malformace mozkové kůry metabolismus patologie MeSH
- mladiství MeSH
- mladý dospělý MeSH
- nemoci mozku metabolismus patologie MeSH
- neokortex patologie MeSH
- předškolní dítě MeSH
- tenascin analýza MeSH
- versikany analýza MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The structure of the H107R variant of the extracellular domain of the mouse natural killer cell receptor NKR-P1A has been determined by X-ray diffraction at 2.3 Å resolution from a merohedrally twinned crystal. Unlike the structure of the wild-type receptor in space group I4(1)22 with a single chain per asymmetric unit, the crystals of the variant belonged to space group I4(1) with a dimer in the asymmetric unit. Different degrees of merohedral twinning were detected in five data sets collected from different crystals. The mutation does not have a significant impact on the overall structure, but led to the binding of an additional phosphate ion at the interface of the molecules.
- MeSH
- extracelulární prostor chemie MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- lektinové receptory NK-buněk - podrodina B chemie genetika MeSH
- molekulární modely MeSH
- mutace MeSH
- myši MeSH
- terciární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The main goal of this research was to investigate how different factors influence membrane fouling. The impact of the different concentrations of activated sludge and the amount of extracellular polymer substances (EPS) were monitored. Two pilot plants with submerged membrane modules (hollow fiber and flat sheet) were operated and the raw wastewater was used. Humic substances were identified as the major components of EPS in the activated sludge (more than 34%) in both pilot plants. As the basic constituent in permeate, humic substances were identified as the most dominant components in the effluent (61%) in both pilot plants. Conversely, proteins were mostly analyzed in permeate and supernatant below the detection limit. The total amount of EPS [mgg(-1) (VSS)] was similar for concentrations of activated sludge 6, 10 and 14 g L(-1). Carbohydrates were identified as the component of EPS which tends most to clog membranes.
- MeSH
- biopolymery biosyntéza MeSH
- bioznečištění MeSH
- extracelulární prostor chemie MeSH
- kinetika MeSH
- kvartérní amoniové sloučeniny analýza MeSH
- membrány umělé MeSH
- měření biologické spotřeby kyslíku MeSH
- odpad tekutý - odstraňování metody MeSH
- pilotní projekty MeSH
- rozpustnost MeSH
- tlak MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 degrees C, respectively. The enzyme was stable at pH 3.5-10.5 and was still active at 95 degrees C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.
