OBJECTIVE: Transgenic mice with fluorescent protein (FP) reporters take full advantage of new in vivo imaging technologies. Therefore, we generated a TRPC5- and a TRPA1-reporter mouse based on FP C-terminal fusion, providing us with better alternatives for studying the physiology, interaction and coeffectors of these two TRP channels at the cellular and tissue level. METHODS: We generated transgenic constructs of the murine TRPC5- and TRPA1-gene with a 3*GGGGS linker and C-terminal fusion to mCherry and mTagBFP, respectively. We microinjected zygotes to generate reporter mice. Reporter mice were examined for visible fluorescence in trigeminal ganglia with two-photon microscopy, immunohistochemistry and calcium imaging. RESULTS: Both TRPC5-mCherry and TRPA1-mTagBFP knock-in mouse models were successful at the DNA and RNA level. However, at the protein level, TRPC5 resulted in no mCherry fluorescence. In contrast, sensory neurons derived from the TRPA1-reporter mice exhibited visible mTag-BFP fluorescence, although TRPA1 had apparently lost its ion channel function. CONCLUSIONS: Creating transgenic mice with a TRP channel tagged at the C-terminus with a FP requires detailed investigation of the structural and functional consequences in a given cellular context and fine-tuning the design of specific constructs for a given TRP channel subtype. Different degrees of functional impairment of TRPA1 and TRPC5 constructs suggest a specific importance of the distal C-terminus for the regulation of these two channels in trigeminal neurons.
- MeSH
- červený fluorescenční protein MeSH
- ganglion trigeminale metabolismus MeSH
- genový knockin * MeSH
- kationtové kanály TRPC * genetika metabolismus MeSH
- kationtový kanál TRPA1 * genetika metabolismus MeSH
- luminescentní proteiny * genetika metabolismus MeSH
- myši transgenní * MeSH
- myši MeSH
- rekombinantní fúzní proteiny metabolismus genetika MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.
- MeSH
- elektronová kryomikroskopie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- proteiny 14-3-3 * metabolismus chemie MeSH
- ubikvitinace MeSH
- ubikvitinligasy Nedd4 * metabolismus chemie genetika ultrastruktura MeSH
- vápník * metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondria are key to cellular energetics, metabolism, and signaling. Their dysfunction is linked to devastating diseases, including mitochondrial disorders, diabetes, neurodegenerative diseases, cardiac disorders, and cancer. Here, we present a knockout mouse model lacking the complex IV assembly factor SMIM20/MITRAC7. SMIM20-/- mice display cardiac pathology with reduced heart weight and cardiac output. Heart mitochondria present with reduced levels of complex IV associated with increased complex I activity, have altered fatty acid oxidation, and display elevated levels of ROS production. Interestingly, mutant mouse ventricular myocytes show unphysiological Ca2+ handling, which can be attributed to the increase in mitochondrial ROS production. Our study presents an example of a tissue-specific phenotype in the context of OXPHOS dysfunction. Moreover, our data suggest a link between complex IV dysfunction and Ca2+ handling at the endoplasmic reticulum through ROS signaling.
- MeSH
- endoplazmatické retikulum metabolismus MeSH
- kardiomyocyty metabolismus MeSH
- membránové proteiny * metabolismus genetika MeSH
- mitochondriální proteiny * metabolismus genetika MeSH
- myokard * metabolismus MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- oxidativní fosforylace MeSH
- proteiny dánia pruhovaného MeSH
- reaktivní formy kyslíku metabolismus MeSH
- respirační komplex IV * metabolismus MeSH
- srdeční mitochondrie metabolismus MeSH
- vápník metabolismus MeSH
- vápníková signalizace * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The sodium/calcium exchanger (NCX) type 1 has been well described in various cancers, but little is known about the other two NCX types (NCX2 and NCX3). In this study, we used the selective blocker of NCX3 - YM-244769 to investigate changes in apoptosis induction, migration, proliferation, intracellular calcium and ATP in four cancer cell lines - DLD1, HeLa, MDA-MB-231 and JIMT1. In all four cell lines we observed a concentration-dependent increase in the number of apoptotic cells, as well as reduced migration and proliferation. Induction of hypoxic conditions did not alter the response of these cells to YM-244769 in any of the above-mentioned parameters. These results indicate the role of NCX3 in cancer cell migration, proliferation and apoptosis, as inhibition of NCX1 by the specific blocker SEA0400 had no significant effect on these parameters. However, we verified the effect of NCX3 inhibition by using CRISPR/Cas9 to generate clones in which the SLC8A3 (NCX3) gene was deleted, and we obtained the same results. In addition, mitochondrial respiration was impaired in the clones with NCX3 knocked-out, suggesting that NCX3 also play a role in bioenergetics. In conclusion, we have clearly shown that NCX3 plays an important anti-apoptotic, pro-migratory and proliferative role in the cancer cells by affecting mitochondrial bioenergetics, thus supporting their survival and fate.
- MeSH
- apoptóza účinky léků MeSH
- lidé MeSH
- mitochondrie metabolismus účinky léků MeSH
- nádorové buněčné linie MeSH
- nádory * metabolismus patologie genetika MeSH
- pohyb buněk účinky léků MeSH
- proliferace buněk účinky léků MeSH
- pumpa pro výměnu sodíku a vápníku * metabolismus genetika antagonisté a inhibitory MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Alterations in the excitability of dorsal root ganglion (DRG) neurons are critical in the pathogenesis of acute and chronic pain. Neurotransmitter release from the terminals of DRG neurons is regulated by cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1), both activated by anandamide (AEA). In our experiments, the AEA precursor N-arachidonoylphosphatidylethanolamine (20:4-NAPE) was used to study the modulation of nociceptive DRG neurons excitability using K+-evoked Ca2+ transients. Intrathecal administration was used to evaluate in vivo effects. Application of 20:4-NAPE at lower concentrations (10 nM - 1 μM) decreased the excitability of DRG neurons, whereas the higher (10 μM) increased it. Both effects of 20:4-NAPE were blocked by the N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor LEI-401. Similarly, lower concentrations of externally applied AEA (1 nM - 10 nM) inhibited DRG neurons, whereas higher concentration (100 nM) did not change it. High AEA concentration (10 μM) evoked Ca2+ transients dependent on TRPV1 activation in separate experiments. Inhibition of the CB1 receptor by PF514273 (400 nM) prevented the 20:4-NAPE- and AEA-induced inhibition, whereas TRPV1 inhibition by SB366791 (1 μM) prevented the increased DRG neuron excitability. In behavioral tests, lower 20:4-NAPE concentration caused hyposensitivity, while higher evoked mechanical allodynia. Intrathecal LEI-401 prevented both in vivo effects of 20:4-NAPE. These results highlight anti- and pro-nociceptive effects of 20:4-NAPE mediated by CB1 and TRPV1 in concentration-dependent manner. Our study underscores the complexity of endocannabinoid signaling in pain transmission modulation and highlights 20:4-NAPE as a potential therapeutic target, offering new insights for developing analgesic strategies.
- MeSH
- endokanabinoidy farmakologie metabolismus MeSH
- fosfatidylethanolaminy * farmakologie MeSH
- fosfolipasa D * metabolismus antagonisté a inhibitory MeSH
- kationtové kanály TRPV metabolismus MeSH
- krysa rodu rattus MeSH
- kyseliny arachidonové * farmakologie MeSH
- neurony * účinky léků metabolismus MeSH
- polynenasycené alkamidy farmakologie MeSH
- potkani Sprague-Dawley MeSH
- receptor kanabinoidní CB1 metabolismus MeSH
- spinální ganglia * účinky léků metabolismus cytologie MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Elevated plasma homocysteine (Hcy) levels lead to hyperhomocysteinemia, a condition associated with various neurological disorders affecting multiple brain regions, including the hippocampus. In this study, we investigated the effects of exposing cultured rat hippocampal neurons to Hcy concentrations corresponding to mild, moderate, and severe hyperhomocysteinemia. A short 24-hour exposure had minimal effects, whereas prolonged exposure up to 14 days moderately enhanced hippocampal excitability without altering the gene expression of voltage-dependent calcium, sodium, or potassium channels or intracellular calcium levels. These findings suggest that Hcy-induced changes in neuronal excitability may contribute to neuropathologies associated with hyperhomocysteinemia.
- MeSH
- hipokampus * cytologie účinky léků MeSH
- homocystein * farmakologie MeSH
- iontové kanály * genetika metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- neurony * účinky léků metabolismus cytologie MeSH
- potkani Sprague-Dawley MeSH
- regulace genové exprese * účinky léků MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Accumulating evidence suggests that manganese oxide nanoparticles (NPs) show multiple enzyme-mimicking antioxidant activities, which supports their potential in redox-targeting therapeutic strategies for diseases with impaired redox signaling. However, the systemic administration of any NP requires thorough hemocompatibility testing. In this study, we assessed the hemocompatibility of synthesized Mn3O4 NPs, identifying their ability to induce spontaneous hemolysis and eryptosis or impair osmotic fragility. Concentrations of up to 20 mg/L were found to be safe for erythrocytes. Eryptosis assays were shown to be more sensitive than hemolysis and osmotic fragility as markers of hemocompatibility for Mn3O4 NP testing. Flow cytometry- and confocal microscopy-based studies revealed that eryptosis induced by Mn3O4 NPs was accompanied by Ca2+ overload, altered redox homeostasis verified by enhanced intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), and a decrease in the lipid order of cell membranes. Furthermore, Mn3O4 NP-induced eryptosis was calpain- and caspase-dependent.
- MeSH
- buněčná membrána * metabolismus účinky léků MeSH
- eryptóza * účinky léků MeSH
- erytrocyty účinky léků metabolismus MeSH
- hemolýza účinky léků MeSH
- kalpain * metabolismus MeSH
- kaspasy * metabolismus MeSH
- lidé MeSH
- nanočástice * chemie MeSH
- oxidy * farmakologie chemie MeSH
- reaktivní formy dusíku * metabolismus MeSH
- reaktivní formy kyslíku * metabolismus MeSH
- sloučeniny manganu * farmakologie chemie MeSH
- vápník * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The neuropeptide B/W signalling system (NPB/W) has been identified in multiple body regions and is integral to several physiological processes, including the regulation of food intake and energy homeostasis. Recently, it has also been detected in human skin; however, its specific functions in this context remain to be thoroughly investigated. This study aims to identify the expression of neuropeptides B/W receptor 1 (NPBWR1) and neuropeptides B/W receptor 2 (NPBWR2) in human dermal fibroblasts of mesenchymal origin using genomic and proteomic techniques. We will also investigate the role of these receptors in cell proliferation and calcium signalling. METHODS: The mRNAs for NPBWR1 and NPBWR2 were detected using quantitative PCR (qPCR) analysis and further validated by western blot and immunofluorescence analyses. Additionally, we synthesised ligands for these receptors, specifically hNPB (25-53) and hNPW (33-62), to investigate their effects on cell proliferation and intracellular calcium levels in human fibroblasts. RESULTS: Our results demonstrated that hNPW (33-62) has anti-proliferative effect on human dermal fibroblasts and concentration of 0.1-μmol/L can significantly decrease intracellular calcium levels (p < 0.05). CONCLUSION: This finding suggests a potential role for the NPB/W signalling system in pathologies associated with impaired calcium handling, such as fibrosis. Furthermore, we observed that the proliferation of human fibroblasts was not affected by hNPB (25-53). Our findings could lead to the development of new therapeutic strategies for various skin conditions and improved wound healing.
- MeSH
- fibroblasty * metabolismus MeSH
- kultivované buňky MeSH
- kůže * metabolismus cytologie MeSH
- lidé MeSH
- neuropeptidy metabolismus genetika MeSH
- proliferace buněk * MeSH
- receptory neuropeptidů metabolismus genetika MeSH
- signální transdukce MeSH
- vápník * metabolismus MeSH
- vápníková signalizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Osteoporóza je chronické metabolické onemocnění kostí, které vzniká důsledkem nerovnováhy mezi kostní novotvorbou a resorpcí. Kostní remodelace je dynamický proces ovlivněný genetickými faktory, výživou a pohybovou aktivitou. Vápník, fosfor a vitamin D jsou ústředními nutričními faktory v prevenci a léčbě osteoporózy, ale vzhledem ke složitosti kostní tkáně je nezbytné zohlednit i další nutrienty. Článek shrnuje význam jednotlivých živin, diskutuje rizika spojená s nevhodnými stravovacími návyky, rostoucí spotřebou průmyslově zpracovaných potravin a zdůrazňuje význam celkového životního stylu v prevenci osteoporózy. Přehled poskytuje ucelený obraz na význam jednotlivých živin v primární prevenci osteoporózy s ohledem na aktuální trendy a nabízí praktická doporučení pro podporu zdraví kostí.
Osteoporosis is a chronic metabolic bone disease that results from an imbalance between bone formation and resorption. Bone remodeling is a dynamic process influenced by genetic factors, nutrition and physical activity. Calcium, phosphorus, and vitamin D are key nutritional factors in the prevention and treatment of osteoporosis, but due to the complexity of bone tissue, other nutrients must also be considered. This article summarizes the importance of each nutrient, discusses the risks associated with inappropriate dietary habits, increasing consumption of ultra-processed foods and emphasizes the importance of overall lifestyle in the prevention of osteoporosis. This review offers a comprehensive view of the role of individual nutrients in the primary prevention of osteoporosis in the context of current trends and provides practical recommendations for maintaining bone health.
- MeSH
- fosfor metabolismus MeSH
- hořčík metabolismus MeSH
- lidé MeSH
- nenasycené mastné kyseliny metabolismus MeSH
- osteoporóza * prevence a kontrola MeSH
- proteiny metabolismus MeSH
- průmyslově zpracované potraviny MeSH
- vápník metabolismus MeSH
- vitaminy metabolismus terapeutické užití MeSH
- živiny MeSH
- Check Tag
- lidé MeSH
Hemolysis and eryptosis contribute to anemia encountered in patients undergoing chemotherapy. Eicosapentaenoic acid (EPA) is an omega-3 dietary fatty acid that has anticancer potential by inducing apoptosis in cancer cells, but its effect on the physiology and lifespan of red blood cells (RBCs) is understudied. Human RBCs were exposed to anticancer concentrations of EPA (10-100 ?M) for 24 h at 37 °C. Acetylcholinesterase (AChE) activity and hemolysis were measured by colorimetric assays whereas annexin-V-FITC and forward scatter (FSC) were employed to identify eryptotic cells. Oxidative stress was assessed by H2DCFDA and intracellular Ca2+ was measured by Fluo4/AM. EPA significantly increased hemolysis and K+ leakage, and LDH and AST activities in the supernatants in a concentration-dependent manner. EPA also significantly increased annexin-V-FITC-positive cells and Fluo4 fluorescence and decreased FSC and AChE activity. A significant reduction in the hemolytic activity of EPA was noted in the presence extracellular isosmotic urea, 125 mM KCl, and polyethylene glycol 8000 (PEG 8000), but not sucrose. In conclusion, EPA stimulates hemolysis and eryptosis through Ca2+ buildup and AChE inhibition. Urea, blocking KCl efflux, and PEG 8000 alleviate the hemolytic activity of EPA. The anticancer potential of EPA may be optimized using Ca2+ channel blockers and chelators to minimize its toxicity to off-target tissue. Keywords: EPA, Eryptosis, Hemolysis, Calcium, Anticancer.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory * farmakologie MeSH
- eryptóza účinky léků MeSH
- erytrocytární membrána * účinky léků metabolismus MeSH
- erytrocyty účinky léků metabolismus MeSH
- fosfatidylseriny * metabolismus MeSH
- hemolýza * účinky léků MeSH
- kyselina eikosapentaenová * farmakologie MeSH
- lidé MeSH
- vápník metabolismus MeSH
- vápníková signalizace * účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
