A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0-30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0-30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5-2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.

• MeSH
• aktivace enzymů účinky léků   MeSH
• Bacillus účinky léků   genetika   metabolismus   MeSH
• beta-mannosidasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• chlorid sodný farmakologie   MeSH
• detergenty farmakologie   MeSH
• endopeptidasy metabolismus   MeSH
• exprese genu MeSH
• genom bakteriální MeSH
• hydrolýza MeSH
• ionty MeSH
• kovy MeSH
• povrchově aktivní látky farmakologie   MeSH
• rekombinantní proteiny MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• stabilita enzymů účinky léků   MeSH
• tolerance k soli * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-mannosidasa MeSH
• chlorid sodný MeSH
• detergenty MeSH
• endopeptidasy MeSH
• ionty MeSH
• kovy MeSH
• povrchově aktivní látky MeSH
• rekombinantní proteiny MeSH

Mucin-associated microbiota are in relatively close contact with the intestinal epithelium and may thus have a more pronounced effect on host health. We have previously developed a simple mucin agar assay to simulate initial mucus colonization by intestinal microbial communities. Adherence of microbiota was estimated using flow cytometry after detachment with Triton X-100. In this study, the effect of this detergent on the cultivability of both virulent and commensal strains was investigated. Mucin attachment of selected strains was evaluated using the mucin adhesion assay. Bacteria were dislodged from the mucin surface by incubation with Triton or from the whole mucin agar layer using a stomacher. Mechanical extraction resulted in 1.24 ± 0.42, 2.69 ± 0.44, and 1.56 ± 0.85 log CFU/mL higher plate counts of Lactobacillus rhamnosus, Bacillus cereus, and Escherichia coli strains, respectively, than the chemical method. The sensitivity of bacteria to Triton varied among microbial species and strains. Among others, Triton inhibited the growth of Salmonella enterica LMG 10396 and Pseudomonas aeruginosa LMG 8029 on laboratory media, although these bacteria maintained their viability during this treatment. Only Gram-positive strains, Enterococcus hirae LMG 6399 and L. rhamnosus GG, were not affected by this detergent. Therefore, the mechanical method is recommended for the extraction of mucin-adhered bacteria that are sensitive to Triton, especially when followed by traditional cultivation techniques. However, this approach can also be recommended for strains that are not affected by this detergent, because it resulted in higher recovery of adhered L. rhamnosus GG compared to the chemical extraction.

• MeSH
• Bacteria účinky léků   růst a vývoj   MeSH
• bakteriální adheze účinky léků   MeSH
• biotest MeSH
• detergenty farmakologie   MeSH
• muciny chemie   MeSH
• oktoxynol farmakologie   MeSH
• prasata MeSH
• střevní sliznice mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• detergenty MeSH
• muciny MeSH
• oktoxynol MeSH

PRINCIPAL FINDINGS: HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. SUMMARY: Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

• MeSH
• buněčná membrána chemie   účinky léků   metabolismus   MeSH
• cetomakrogol farmakologie   MeSH
• detergenty farmakologie   MeSH
• HEK293 buňky MeSH
• hydrofobní a hydrofilní interakce MeSH
• lidé MeSH
• pertusový toxin toxicita   MeSH
• receptory opiátové delta metabolismus   MeSH
• teplota MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cetomakrogol MeSH
• detergenty MeSH
• pertusový toxin MeSH
• receptory opiátové delta MeSH

BACKGROUND: A biological scaffold from extracellular matrix can be produced by a variety of decellularization methods whose caveat consists in efficiently eliminating cells from the treated tissue. This scaffold can be used in diverse applications for tissue engineering and organ regeneration. Preservation of the extracellular matrix ultrastructure is highly desirable because of its unique architecture, contained growth factors and decreased immunological response. All of these properties provide attachment sites and adequate environment for cells colonizing this scaffold, reconstituting the decellularized organ. This review briefly describes chemical decellularization methods, evaluation of these protocols and the role of ECM in tissue engineering. CONCLUSION: Chemical decellularization is an often used method for scaffold preparation and makes possible a well-preserved three dimensional structure of extracellular matrix.

• MeSH
• detergenty farmakologie   MeSH
• extracelulární matrix účinky léků   MeSH
• lidé MeSH
• tkáňové inženýrství * MeSH
• tkáňové podpůrné struktury chemie   MeSH
• uchovávání tkání metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• detergenty MeSH

Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β-OG-selective solubility in the NKL cell line. We found that the NKG2D-DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin-bundling protein L-plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L-plastin participates in NKG2D-mediated inhibition of NK cell chemotaxis.

• Klíčová slova
• chemotaxis, membrane rafts,
• MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buňky NK cytologie   metabolismus   MeSH
• centrifugace - gradient hustoty MeSH
• chemotaxe leukocytů fyziologie   MeSH
• detergenty farmakologie   MeSH
• kultivované buňky MeSH
• lektinové receptory NK-buněk - podrodina C metabolismus   MeSH
• lektinové receptory NK-buněk - podrodina K fyziologie   MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• membránové mikrodomény účinky léků   fyziologie   MeSH
• mikrofilamenta fyziologie   MeSH
• mikrofilamentové proteiny antagonisté a inhibitory   genetika   fyziologie   MeSH
• multiproteinové komplexy MeSH
• proteom MeSH
• receptory imunologické metabolismus   MeSH
• RNA interference MeSH
• signální transdukce imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• detergenty MeSH
• HCST protein, human MeSH Prohlížeč
• KLRK1 protein, human MeSH Prohlížeč
• LCP1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina C MeSH
• lektinové receptory NK-buněk - podrodina K MeSH
• malá interferující RNA MeSH
• mikrofilamentové proteiny MeSH
• multiproteinové komplexy MeSH
• proteom MeSH
• receptory imunologické MeSH

OBJECTIVE: The genotoxic vesicant sulphur mustard [bis-2-(chloroethyl)sulphide] is a chemical warfare agent which is easily available due to its relatively simple synthesis. Thus, sulphur mustard is a potential agent for mass contamination. In this study, we focused on sulphur mustard toxicity and decontamination in a rat model using commercially available detergent mixtures for dermal decontamination. METHODS: Male Wistar rats were percutaneously treated with sulphur mustard and subjected to wet decontamination 2 min postexposure. Commercially produced detergents Neodekont™, Argos™, Dermogel™ and FloraFree™ were tested for their decontamination efficacy against an exposed group and their protective ratios determined. RESULTS AND CONCLUSION: The results showed that all tested detergent solutions produced an increase in the median lethal dose [LD(50) = 9.83 (5.87-13.63) mg·kg(-1)] in comparison to controls, which led to increased survival of experimental animals. In general, all tested detergents provided modest decontamination efficacy (PR = 2.0-5.7). The highest protective ratio (5.7) was consistently achieved with Argos™. Accordingly, Argos™ should be considered in further investigation of mass casualty decontamination.

• MeSH
• aplikace kožní MeSH
• chemické bojové látky toxicita   MeSH
• dekontaminace metody   MeSH
• detergenty farmakologie   MeSH
• hematurie chemicky indukované   MeSH
• kachexie chemicky indukované   MeSH
• krysa rodu Rattus MeSH
• kůže účinky léků   patologie   MeSH
• kýchání účinky léků   MeSH
• LD50 MeSH
• potkani Wistar MeSH
• průjem chemicky indukované   MeSH
• yperit toxicita   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• chemické bojové látky MeSH
• detergenty MeSH
• yperit MeSH

Misuse of various chemicals, such as chemical warfare agents, industrial chemicals or pesticides during warfare or terrorists attacks requires adequate protection. Thus, development and evaluation of novel decontamination dispositives and techniques are needed. In this study, in vitro permeation and decontamination of a potentially hazardous compound paraoxon, an active metabolite of organophosphorus pesticide parathion, was investigated. Skin permeation and decontamination experiments were carried out in modified Franz diffusion cells. Pig skin was used as a human skin model. Commercially produced detergent-based washing solutions FloraFree(™) and ArgosTM were used as decontamination means. The experiments were done under "warm", "cold", "dry" and "wet" skin conditions in order to determine an effect of various physical conditions on skin permeation of paraoxon and on a subsequent decontamination process. There was no significant difference in skin permeation of paraoxon under warm, cold and dry conditions, whereas wet conditions provided significantly higher permeation rates. In the selected conditions, decontamination treatments performed 1 h after a skin exposure did not decrease the agent volume that permeated through the skin. An exception were wet skin conditions with non-significant decontamination efficacy 18 and 28% for the FloraFree(™) and Argos(™) treatment, respectively. In contrast, the skin permeation of paraoxon under warm, cold and dry conditions increased up to 60-290% following decontamination compared to non-decontaminated controls. This has previously been described as a skin wash-in effect.

• MeSH
• časové faktory MeSH
• chemické jevy MeSH
• dekontaminace * metody   MeSH
• detergenty farmakologie   MeSH
• difuzní komory kultivační MeSH
• kožní absorpce účinky léků   fyziologie   MeSH
• kůže chemie   účinky léků   metabolismus   MeSH
• lidé MeSH
• paraoxon chemie   farmakokinetika   MeSH
• permeabilita účinky léků   MeSH
• pesticidy chemie   farmakokinetika   MeSH
• Sus scrofa MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• detergenty MeSH
• paraoxon MeSH
• pesticidy MeSH

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.

• MeSH
• aktivace enzymů MeSH
• aktivace lymfocytů MeSH
• antigeny CD45 metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• centrifugace - gradient hustoty MeSH
• detergenty farmakologie   MeSH
• membránové mikrodomény metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• signální transdukce * MeSH
• transport proteinů MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD45 MeSH
• detergenty MeSH
• PTPRC protein, human MeSH Prohlížeč
• tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.

• MeSH
• Aspergillus fumigatus účinky léků   enzymologie   MeSH
• buněčná stěna účinky léků   MeSH
• buněčné extrakty chemie   MeSH
• detergenty farmakologie   MeSH
• fungální proteiny analýza   metabolismus   MeSH
• glukosa-6-fosfátdehydrogenasa analýza   metabolismus   MeSH
• osmotický tlak MeSH
• Penicillium účinky léků   enzymologie   MeSH
• Rhodotorula účinky léků   enzymologie   MeSH
• vibrace ultrazvukové MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• buněčné extrakty MeSH
• detergenty MeSH
• fungální proteiny MeSH
• glukosa-6-fosfátdehydrogenasa MeSH

Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca(2+)-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcepsilonRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcepsilonRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.

• MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• calcimycin farmakologie   MeSH
• detergenty farmakologie   MeSH
• exocytóza MeSH
• fosfatidylseriny chemie   MeSH
• fosforylace MeSH
• inhibitory enzymů farmakologie   MeSH
• ionofory farmakologie   MeSH
• konfokální mikroskopie MeSH
• krysa rodu Rattus MeSH
• nádorové buněčné linie MeSH
• proteiny přenášející fosfolipidy metabolismus   MeSH
• tyrosin chemie   MeSH
• vanadáty farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• calcimycin MeSH
• detergenty MeSH
• fosfatidylseriny MeSH
• inhibitory enzymů MeSH
• ionofory MeSH
• pervanadate MeSH Prohlížeč
• proteiny přenášející fosfolipidy MeSH
• tyrosin MeSH
• vanadáty MeSH