Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.
- MeSH
- alfa-synuklein analýza antagonisté a inhibitory metabolismus MeSH
- benzodioxoly farmakologie MeSH
- elektroporace metody MeSH
- HeLa buňky MeSH
- intracelulární tekutina chemie účinky léků metabolismus MeSH
- lidé MeSH
- proteinové agregáty účinky léků fyziologie MeSH
- pyrazoly farmakologie MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- viabilita buněk účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The extracellular matrix (ECM) consists of proteins, glycosaminoglycans and glycoproteins, that support the dynamic interactions between cells, including intercellular communication, cell attachment, cell differentiation, cell growth and migration. As such, the ECM represents an essential and very sensitive system within the tissue microenvironment that is involved in processes such as tissue regeneration and carcinogenesis. The aim of the present review is to evaluate its diversity through Ca(2+) signaling and its role in muscle cell function. Here, we discuss some methodological approaches dissecting Ca(2+) handling mechanisms in myogenic and non-myogenic cells, e.g. the importance of Ca(2+) and calpains in muscle dystrophy. We also consider the reconstruction of skeletal muscle by colonization of decellularized ECM with muscle-derived cells isolated from skeletal muscle. Therefore, it is necessary to establish new methodological procedures based on Ca(2+) signaling in skeletal muscle cells and their effect on ECM homeostasis, allowing the monitoring of skeletal muscle reconstruction and organ repair.
Parkinson's disease (PD) is currently the second most common neurodegenerative disorder in the world. Major features of cell pathology of the disease include the presence of cytoplasmic inclusions called Lewy bodies, which are composed of aggregated proteins. The presence of Lewy's body is associated with more advanced stages of the disease when considering irreversible changes. Precise identification of the disease stage at a cellular level presents the critical tool in developing early diagnostics and/or prevention of PD. The aim of our work is to introduce sensitive microscopic analysis in living cells, focused on initial intracellular changes and thus capable to detect earlier stages of the disease.
- MeSH
- alfa-synuklein metabolismus MeSH
- biologické markery metabolismus MeSH
- buněčná inkluze metabolismus patologie MeSH
- intracelulární tekutina metabolismus MeSH
- lidé MeSH
- membránový potenciál mitochondrií fyziologie MeSH
- mitochondrie metabolismus patologie MeSH
- NAD metabolismus MeSH
- nádorové buněčné linie MeSH
- optické zobrazování metody MeSH
- Parkinsonova nemoc metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In ischemic/reperfusion (I/R) injured hearts, severe oxidative stress occurs and is associated with intracellular calcium (Ca(2+)) overload. Glucagon-Like Peptide-1 (GLP-1) analogues have been shown to exert cardioprotection in I/R heart. However, there is little information regarding the effects of GLP-1 analogue on the intracellular Ca(2+) regulation in the presence of oxidative stress. Therefore, we investigated the effects of GLP-1 analogue, (liraglutide, 10 microM) applied before or after hydrogen peroxide (H(2)O(2), 50 microM) treatment on intracellular Ca(2+) regulation in isolated cardiomyocytes. We hypothesized that liraglutide can attenuate intracellular Ca(2+) overload in cardiomyocytes under H(2)O(2)-induced cardiomyocyte injury. Cardiomyocytes were isolated from the hearts of male Wistar rats. Isolated cardiomyocytes were loaded with Fura-2/AM and fluorescence intensity was recorded. Intracellular Ca(2+) transient decay rate, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) levels were recorded before and after treatment with liraglutide. In H(2)O(2) induced severe oxidative stressed cardiomyocytes (which mimic cardiac I/R) injury, liraglutide given prior to or after H(2)O(2) administration effectively increased both intracellular Ca(2+) transient amplitude and intracellular Ca(2+) transient decay rate, without altering the intracellular diastolic Ca(2+) level. Liraglutide attenuated intracellular Ca(2+) overload in H(2)O(2)-induced cardiomyocyte injury and may be responsible for cardioprotection during cardiac I/R injury by preserving physiological levels of calcium handling during the systolic and diastolic phases of myocyte activation.
- MeSH
- hypoglykemika farmakologie MeSH
- intracelulární tekutina účinky léků metabolismus MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- liraglutid farmakologie MeSH
- oxidační stres účinky léků fyziologie MeSH
- peroxid vodíku toxicita MeSH
- potkani Wistar MeSH
- vápníková signalizace účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Polymeric micelles are attractive drug delivery systems for intravenously administered nonpolar drugs. Although physical parameters like size, shape and loading capacity are considered as the most important for their efficiency, here we demonstrate that the effects of serum protein interaction and characteristics of loaded compound cannot be neglected during the micelle development and design of experimental set up. Polymeric micelles prepared from amphiphilic hyaluronic acid grafted with short (hexanoic) and long fatty acids (oleic) were tested after loading with two different hydrophobic models, Nile red and curcumin. The composition of micelles affected mainly the loading capacity. Both encapsulated compounds behaved differently in the in vitro cell uptake, which was also influenced by serum concentration, where serum albumin was found to be the primary destabilizing component. This destabilization was found to be influenced by polymeric micelle concentration. Thus, the chemical structure of micelle, the properties of non-covalently loaded substance and serum albumin/polymeric micelle ratio modulate the in vitro intracellular uptake of drugs loaded in nanocarriers.
- MeSH
- buňky HT-29 MeSH
- HCT116 buňky MeSH
- intracelulární tekutina účinky léků metabolismus MeSH
- kyselina hyaluronová aplikace a dávkování metabolismus MeSH
- lidé MeSH
- micely * MeSH
- nosiče léků aplikace a dávkování metabolismus MeSH
- polymery aplikace a dávkování metabolismus MeSH
- sérový albumin aplikace a dávkování metabolismus MeSH
- systémy cílené aplikace léků metody MeSH
- vazba proteinů fyziologie MeSH
- viabilita buněk účinky léků fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Inhibitory neurotransmission plays a substantial role in encoding of auditory cues relevant for sound localization in vertebrates. While the anatomical organization of the respective afferent auditory brainstem circuits shows remarkable similarities between mammals and birds, the properties of inhibitory neurotransmission in these neural circuits are strikingly different. In mammals, inhibition is predominantly glycinergic and endowed with fast kinetics. In birds, inhibition is mediated by gamma-Aminobutiric acid (GABA) and too slow to convey temporal information. A further prominent difference lies in the mechanism of inhibition in the respective systems. In auditory brainstem neurons of mammals, [Cl(-)](i) undergoes a developmental shift causing the actions of GABA and glycine to gradually change from depolarization to the 'classic' hyperpolarizing-inhibition before hearing onset. Contrary to this, in the mature avian auditory brainstem Cl(-) homeostasis mechanisms accurately adjust the Cl(-) gradient to enable depolarizing, but still very efficient, shunting inhibition. The present review considers the mechanisms underlying development of the Cl(-) homeostasis in the auditory system of mammals and birds and discusses some open issues that require closer attention in future studies.
- MeSH
- chloridy metabolismus MeSH
- financování organizované MeSH
- GABA metabolismus účinky léků MeSH
- glycin metabolismus účinky léků MeSH
- homeostáza fyziologie MeSH
- intracelulární tekutina fyziologie metabolismus MeSH
- metaanalýza jako téma MeSH
- mozkový kmen fyziologie metabolismus MeSH
- neurotransmiterové látky antagonisté a inhibitory fyziologie metabolismus MeSH
- ptáci MeSH
- savci MeSH
- sluchová dráha fyziologie chemie MeSH
- statistika jako téma MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- akutní bolest břicha * diagnóza komplikace metabolismus MeSH
- chirurgická rána * chirurgie komplikace terapie MeSH
- extracelulární tekutina fyziologie metabolismus účinky léků MeSH
- intracelulární tekutina fyziologie metabolismus účinky léků MeSH
- komorbidita * MeSH
- kompartmenty tělních tekutin * fyziologie metabolismus účinky léků MeSH
- kongresy jako téma MeSH
- lidé středního věku MeSH
- lidé MeSH
- multiorgánové selhání diagnóza prevence a kontrola terapie MeSH
- obezita MeSH
- statistika jako téma MeSH
- syndrom systémové zánětlivé reakce diagnóza prevence a kontrola terapie MeSH
- tekutinová terapie metody využití MeSH
- tělesné tekutiny fyziologie metabolismus účinky léků MeSH
- věkové faktory MeSH
- vodní a elektrolytová rovnováha fyziologie imunologie účinky léků MeSH
- výsledky a postupy - zhodnocení (zdravotní péče) metody normy využití MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.
- MeSH
- adaptorové proteiny signální transdukční metabolismus MeSH
- antigeny CD98 - lehké řetězce fyziologie MeSH
- financování organizované MeSH
- fosforylace MeSH
- intracelulární tekutina metabolismus MeSH
- krysa rodu rattus MeSH
- mastocyty imunologie metabolismus MeSH
- molekulární sekvence - údaje MeSH
- nádorové buněčné linie MeSH
- receptory IgE fyziologie MeSH
- sekvence aminokyselin MeSH
- transportní systém aminokyselin y+ fyziologie MeSH
- tyrosin metabolismus MeSH
- vápník metabolismus MeSH
- vápníková signalizace imunologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
