12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6-8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.
- MeSH
- buněčná diferenciace účinky léků MeSH
- embryonální kmenové buňky cytologie účinky léků metabolismus MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosforylace MeSH
- kardiomyocyty cytologie metabolismus MeSH
- myši MeSH
- proteinkinasa C metabolismus MeSH
- signální transdukce účinky léků MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Papillon-Lefèvre syndrome (PLS) is characterized by nonfunctional neutrophil serine proteases (NSPs) and fulminant periodontal inflammation of unknown cause. Here we investigated neutrophil extracellular trap (NET)-associated aggregation and cytokine/chemokine-release/degradation by normal and NSP-deficient human and mouse granulocytes. Stimulated with solid or soluble NET inducers, normal neutrophils formed aggregates and both released and degraded cytokines/chemokines. With increasing cell density, proteolytic degradation outweighed release. Maximum output of cytokines/chemokines occurred mostly at densities between 2 × 107 and 4 × 107 neutrophils/cm3. Assessment of neutrophil density in vivo showed that these concentrations are surpassed during inflammation. Association with aggregated NETs conferred protection of neutrophil elastase against α1-antitrypsin. In contrast, eosinophils did not influence cytokine/chemokine concentrations. The proteolytic degradation of inflammatory mediators seen in NETs was abrogated in Papillon-Lefèvre syndrome (PLS) neutrophils. In summary, neutrophil-driven proteolysis of inflammatory mediators works as a built-in safeguard for inflammation. The absence of this negative feedback mechanism might be responsible for the nonresolving periodontitis seen in PLS.-Hahn, J., Schauer, C., Czegley, C., Kling, L., Petru, L., Schmid, B., Weidner, D., Reinwald, C., Biermann, M. H. C., Blunder, S., Ernst, J., Lesner, A., Bäuerle, T., Palmisano, R., Christiansen, S., Herrmann, M., Bozec, A., Gruber, R., Schett, G., Hoffmann, M. H. Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.
- MeSH
- chemokiny metabolismus MeSH
- cytokiny metabolismus MeSH
- dospělí MeSH
- extracelulární pasti metabolismus MeSH
- inhibitory proteas metabolismus MeSH
- ionomycin farmakologie MeSH
- kyselina močová farmakologie MeSH
- lidé MeSH
- mediátory zánětu metabolismus MeSH
- mladiství MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- NADPH-oxidasy genetika MeSH
- neutrofily účinky léků metabolismus MeSH
- parodontitida metabolismus MeSH
- proteolýza MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- zánět prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The endothelium contributes to the maintenance of vasodilator tone by releasing endothelium-derived relaxing factors, including nitric oxide (NO). In hypertension, endothelial nitric oxide synthase (eNOS) produces less NO and could be one of the contributing factors to the increased peripheral vascular resistance. Agonist-induced Ca(2+) entry is essential for the activation of eNOS. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca(2+)-permeant cation channel, is expressed in the endothelial cells and involved in the regulation of vascular tone. The present study aimed to investigate the role of TRPV4 channel in endothelium-dependent NO-mediated relaxation of the resistance artery in hypertensive rats. Using a wire myograph, relaxation response to the TRPV4 activator, 4alpha-phorbol-12,13-didecanoate (4alphaPDD) was assessed in mesenteric arteries obtained from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Compared to WKY, SHR demonstrated a significantly attenuated 4alphaPDD-induced endothelium-dependent NO-mediated relaxation. Immunohistochemical analysis revealed positive staining for TRPV4 in the endothelium of mesenteric artery sections in both WKY and SHR. Furthermore, TRPV4 mRNA and protein expressions in SHR were significantly lower than their expression levels in WKY rats. We conclude that 4alphaPDD-induced endothelium-dependent NO-mediated vasorelaxation is reduced in SHR and downregulation of TRPV4 could be one of the contributing mechanisms.
- MeSH
- arteriae mesentericae účinky léků metabolismus patofyziologie MeSH
- cévní endotel účinky léků metabolismus MeSH
- down regulace účinky léků fyziologie MeSH
- forboly farmakologie MeSH
- hypertenze metabolismus patofyziologie MeSH
- kationtové kanály TRPV agonisté metabolismus MeSH
- krysa rodu rattus MeSH
- orgánové kultury - kultivační techniky MeSH
- potkani inbrední SHR MeSH
- potkani inbrední WKY MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- vazodilatace účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- arginin farmakologie MeSH
- bilirubin farmakologie MeSH
- hem * farmakologie MeSH
- HIV-1 * fyziologie účinky léků MeSH
- latence viru fyziologie MeSH
- lidé MeSH
- oxid uhelnatý MeSH
- RNA virová genetika metabolismus MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Ultra-weak photon emission (UPE) is light emitted spontaneously by biological systems without the use of specific luminescent complexes. UPE is emitted in the near-UV/UV-Vis/near-IR spectra during oxidative metabolic reactions; however, the specific pathways involved in UPE remain poorly understood. Here, we used HL-60 cells, a human promyelocytic cell line that is often used to study respiratory burst, as a model system to measure UPE kinetics together with metabolic changes. HL-60 cells were differentiated into neutrophil-like cells by culturing in all-trans-retinoic acid for 7days. We then used a targeted metabolomics approach with capillary electrophoresis-mass spectrometry to profile intracellular metabolites in HL-60 cells and to investigate the biochemical changes based on the measured UPE profile. Our analysis revealed that the levels of specific metabolites, including putrescine, creatine, β-alanine, methionine, hydroxyproline, serine, and S-adenosylmethionine, were significantly altered in HL-60 cells after inducing respiratory burst. A comparison with recorded UPE data revealed that the changes in putrescine, glutathione, sarcosine, creatine, β-alanine, methionine, and hydroxyproline levels were inversely correlated with the change in UPE intensity. These results suggest that these metabolic pathways, particular the methionine pathway, may play a role in the observed changes in UPE in HL-60 cells and therefore demonstrate the potential for using UPE to monitor metabolic changes.
Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) β, a GPI-anchored protein belonging to the folate receptor family. As FRβ shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRβ, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRβ in the plasma membrane of human FRβ(+) macrophages and FRβ-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRβ: that is, we report functional interactions of FRβ with receptors mediating cellular adhesion, in particular the CD11b/CD18 β2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRβ(+) human primary macrophages and THP-1 cells to collagen in comparison with their FRβ(-) counterparts. We further show that FRβ is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRβ as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen.
- MeSH
- antigeny CD11b fyziologie MeSH
- antigeny CD18 fyziologie MeSH
- buněčná adheze MeSH
- folátový receptor 2 fyziologie MeSH
- kolagen farmakologie MeSH
- kultivované buňky MeSH
- kyselina listová metabolismus MeSH
- lidé MeSH
- makrofágy fyziologie MeSH
- pohyb buněk MeSH
- proliferace buněk MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Altered gap junctional intercellular communication (GJIC) has been associated with chemical carcinogenesis, where both chemical tumor promoters and chemopreventive agents (CPAs) are known to conversely modulate GJIC. The aim of this study was to investigate whether attenuation of chemically inhibited GJIC represents a common outcome induced by different CPAs, which could be effectively evaluated using in vitro methods. Rat liver epithelial cells WB-F344 were pretreated with a CPA for either 30 min or 24 h, and then exposed to GJIC-inhibiting concentration of a selected tumor promoter or environmental toxicant [12-O-tetradecanoylphorbol-13-acetate (TPA), lindane, fluoranthene, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), perfluorooctanoic acid (PFOA), or pentachlorophenol]. Out of nine CPAs tested, quercetin and silibinin elicited the most pronounced effects, preventing the dysregulation of GJIC by all the GJIC inhibitors, but DDT. Metformin and curcumin attenuated the effects of three GJIC inhibitors, whereas the other CPAs prevented the effects of two (diallyl sulfide, emodin) or one (indole-3-carbinol, thymoquinone) GJIC inhibitor. Significant attenuation of chemically induced inhibition of GJIC was observed in 27 (50%) out of 54 possible combinations of nine CPAs and six GJIC inhibitors. Our data demonstrate that in vitro evaluation of GJIC can be used as an effective screening tool for identification of chemicals with potential chemopreventive activity.
- MeSH
- antikarcinogenní látky farmakologie MeSH
- DDT toxicita MeSH
- epitelové buňky účinky léků MeSH
- fluoreny toxicita MeSH
- fluorokarbony toxicita MeSH
- hexachlorcyklohexan toxicita MeSH
- játra cytologie účinky léků MeSH
- kapryláty toxicita MeSH
- karcinogeny toxicita MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- kurkumin farmakologie MeSH
- metformin farmakologie MeSH
- mezerový spoj účinky léků metabolismus MeSH
- mezibuněčná komunikace účinky léků MeSH
- potkani inbrední F344 MeSH
- tetradekanoylforbolacetát toxicita MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mechanisms controlling the metabotropic γ-aminobutyric acid receptor (GABAB) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly down-regulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABAB(1b)subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABABin a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- endocytóza účinky léků MeSH
- HEK293 buňky MeSH
- hydrazony farmakologie MeSH
- lidé MeSH
- luciferasy genetika metabolismus MeSH
- luminescentní proteiny genetika metabolismus MeSH
- lyzozomy metabolismus MeSH
- molekulární sekvence - údaje MeSH
- posttranslační úpravy proteinů * MeSH
- proteinkinasa C genetika metabolismus MeSH
- proteolýza MeSH
- receptory GABA-B genetika metabolismus MeSH
- reportérové geny MeSH
- sekvence aminokyselin MeSH
- signální transdukce MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- thiolesterasa ubikvitinu genetika metabolismus MeSH
- ubikvitin genetika metabolismus MeSH
- ubikvitinace MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The present study provides a comprehensive data on the antioxidant, antimicrobial and neutrophil-modulating activities of extracts from six medicinal plants--blackberry (Rubus fruticosus) leaves, chokeberry (Aronia melanocarpa) leaves, hawthorn (Crataegus monogyna) leaves, lady's mantle (Alchemilla glabra) aerial parts, meadowsweet (Filipendula ulmaria) aerial parts and raspberry (Rubus idaeus) leaves. In order to analyze the antioxidant activity of the herbs, several methods (ORAC, TRAP, HORAC and inhibition of lipid peroxidation) were used. Blackberry leaves and meadowsweet extracts revealed the highest antioxidant activities via all methods. All extracts studied blocked almost completely the opsonized zymosan particle-activated ROS production by neutrophils from human whole blood. On the other hand, the effect of extracts on phorbol myristate acetate-activated ROS production was much milder and even nonsignificant in the case of chokeberry leaves. This latter result suggests that extracts (apart from their antioxidative activity) interfere with the signaling cascade of phagocyte activation upstream of the protein kinase C activation. The antimicrobial activity of the investigated extracts against 11 human pathogens was investigated using three different methods. Meadowsweet and blackberry leaves extracts had the highest antimicrobial effect and the lowest minimal inhibiting concentrations (MICs) against the microorganisms tested.
- MeSH
- antiinfekční látky izolace a purifikace farmakologie MeSH
- antioxidancia izolace a purifikace farmakologie MeSH
- gramnegativní bakterie účinky léků růst a vývoj MeSH
- grampozitivní bakterie účinky léků růst a vývoj MeSH
- léčivé rostliny chemie MeSH
- lidé MeSH
- listy rostlin chemie MeSH
- makrofágy cytologie účinky léků metabolismus MeSH
- mikrobiální testy citlivosti MeSH
- nadzemní části rostlin chemie MeSH
- neutrofily cytologie účinky léků metabolismus MeSH
- opsoniny farmakologie MeSH
- peroxidace lipidů účinky léků MeSH
- polyfenoly izolace a purifikace farmakologie MeSH
- primární buněčná kultura MeSH
- reaktivní formy kyslíku antagonisté a inhibitory metabolismus MeSH
- rostlinné extrakty izolace a purifikace farmakologie MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Resveratrol-3,5,4'-trihydroxystilbene-possesses antioxidant activities in vitro. It dose-dependently inhibited the generation of peroxyl, hydroxyl, peroxides, and lipid peroxidation products in cell free systems. Oxidative burst of whole human blood stimulated with PMA, fMLP, OpZ, and A23187 was inhibited in a concentration-dependent way, indicating suppression of both receptor and nonreceptor activated chemiluminescence by resveratrol. Results from isolated human neutrophils revealed that resveratrol was active extracellularly as well as intracellularly in inhibiting the generation of reactive oxygen species. Liberation of ATP and analysis of apoptosis showed that in the concentration of 100 μM, resveratrol did not change the viability and integrity of isolated neutrophils. Western blot analysis documented that resveratrol in concentrations of 10 and 100 μM significantly decreased PMA-induced phosphorylation of PKC α/β II. Dose-dependent inhibition of nitrite production and iNOS protein expression in RAW 264.7 cells indicated possible interference of resveratrol with reactive nitrogen radical generation in professional phagocytes. The results suggest that resveratrol represents an effective naturally occurring substance with potent pharmacological effect on oxidative burst of human neutrophils and nitric oxide production by macrophages. It should be further investigated for its pharmacological activity against oxidative stress in ischaemia reperfusion, inflammation, and other pathological conditions, particularly neoplasia.
- MeSH
- buněčné linie MeSH
- dusitany metabolismus MeSH
- fagocyty účinky léků enzymologie metabolismus MeSH
- látky reagující s kyselinou thiobarbiturovou metabolismus MeSH
- lidé MeSH
- luminiscenční měření MeSH
- luminol metabolismus MeSH
- myši MeSH
- neutrofily účinky léků metabolismus MeSH
- peroxidace lipidů účinky léků MeSH
- proteinkinasy metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- respirační vzplanutí účinky léků MeSH
- scavengery volných radikálů metabolismus MeSH
- separace buněk MeSH
- stilbeny farmakologie MeSH
- synthasa oxidu dusnatého, typ II metabolismus MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
