The aim of the present study was to assess systemic circulatory and tissue activities of both the classical arm and of the alternative arm of the renin-angiotensin system (RAS) in a new transgenic rat line (TG7371) that expresses angiotensin-(1-7) (ANG 1-7)-producing fusion protein; the results were compared with the activities measured in control transgene-negative Hannover Sprague-Dawley (HanSD) rats. Plasma and tissue concentrations of angiotensin II (ANG II) and ANG 1-7, and kidney mRNA expressions of receptors responsible for biological actions of ANG II and ANG 1-7 [i.e. ANG II type 1 and type 2 (AT1 and AT2) and Mas receptors] were assessed in TG7371 transgene-positive and in HanSD rats. We found that male TG7371 transgene-positive rats exhibited significantly elevated plasma, kidney, heart and lung ANG 1-7 concentrations as compared with control male HanSD rats; by contrast, there was no significant difference in ANG II concentrations and no significant differences in mRNA expression of AT1, AT2 and Mas receptors. In addition, we found that in male TG7371 transgene-positive rats blood pressure was lower than in male HanSD rats. These data indicate that the balance between the classical arm and the alternative arm of the RAS was in male TGR7371 transgene-positive rats markedly shifted in favor of the latter. In conclusion, TG7371 transgene-positive rats represent a new powerful tool to study the long-term role of the alternative arm of the RAS in the pathophysiology and potentially in the treatment of cardio-renal diseases.

• MeSH
• angiotensin I * metabolismus   MeSH
• angiotensin II * MeSH
• kardiovaskulární nemoci metabolismus   genetika   MeSH
• krevní tlak fyziologie   MeSH
• krysa rodu rattus MeSH
• ledviny metabolismus   MeSH
• nemoci ledvin metabolismus   genetika   MeSH
• peptidové fragmenty * metabolismus   MeSH
• potkani Sprague-Dawley * MeSH
• potkani transgenní * MeSH
• protoonkogen Mas MeSH
• receptor angiotensinu typ 1 genetika   metabolismus   MeSH
• receptory spřažené s G-proteiny genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• renin-angiotensin systém * fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

WBP1L is a broadly expressed transmembrane adaptor protein involved in regulating hematopoietic stem cell function and T cell development. It interacts with NEDD4-family E3 ubiquitin ligases and regulates important chemokine receptor CXCR4. Using tandem affinity purification coupled with mass spectrometry, we identified novel WBP1L interactions with the IFNγ receptor and the Cullin-RING ubiquitin ligases CRL1β-TrCP1/2. We found that WBP1L interaction with the IFNγ receptor serves to downregulate proximal IFNγ receptor signaling in female macrophages, while the interaction with CRL1β-TrCP1/2 ubiquitin ligases regulates WBP1L protein levels. Disrupting this interaction, as well as inhibiting proteasome activity or neddylation, increased WBP1L protein levels, demonstrating that CRL1β-TrCP1/2 ubiquitin ligases regulate WBP1L protein abundance. These data provide important insights into the mechanisms controlling WBP1L function.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• HEK293 buňky MeSH
• hematopoéza * MeSH
• lidé MeSH
• makrofágy metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• myši MeSH
• proteiny s repetitivními sekvencemi beta-transducinu metabolismus   MeSH
• signální transdukce MeSH
• ubikvitinligasy * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The combination of aminophylline and salbutamol is frequently used in clinical practice in the treatment of obstructive lung diseases. While the side effects (including arrhythmias) of the individual bronchodilator drugs were well described previously, the side effects of combined treatment are almost unknown. We aimed to study the arrhythmogenic potential of combined aminophylline and salbutamol treatment in vitro. For this purpose, we used the established atomic force microscopy (AFM) model coupled with cardiac organoids derived from human pluripotent stem cells (hPSC-CMs). We focused on the chronotropic, inotropic, and arrhythmogenic effects of salbutamol alone and aminophylline and salbutamol combined treatment. We used a method based on heart rate/beat rate variability (HRV/BRV) analysis to detect arrhythmic events in the hPSC-CM based AFM recordings. Salbutamol and aminophylline had a synergistic chronotropic and inotropic effect compared to the effects of monotherapy. Our main finding was that salbutamol reduced the arrhythmogenic effect of aminophylline, most likely mediated by endothelial nitric oxide synthase activated by beta-2 adrenergic receptors. These findings were replicated and confirmed using hPSC-CM derived from two cell lines (CCTL4 and CCTL12). Data suggest that salbutamol as an add-on therapy may not only deliver a bronchodilator effect but also increase the cardiovascular safety of aminophylline, as salbutamol reduces its arrhythmogenic potential.

• MeSH
• albuterol * farmakologie   MeSH
• aminofylin * farmakologie   MeSH
• bronchodilatancia farmakologie   MeSH
• buněčné linie MeSH
• kardiomyocyty účinky léků   metabolismus   MeSH
• lidé MeSH
• mikroskopie atomárních sil MeSH
• pluripotentní kmenové buňky účinky léků   cytologie   MeSH
• srdeční arytmie * farmakoterapie   MeSH
• srdeční frekvence účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Induction of autophagy represents an effective survival strategy for nutrient-deprived or stressed cancer cells. Autophagy contributes to the modulation of communication within the tumor microenvironment. Here, we conducted a study of the metabolic and signaling implications associated with autophagy induced by glutamine (Gln) and serum starvation and PI3K/mTOR inhibitor and autophagy inducer NVP-BEZ235 (BEZ) in the head and neck squamous cell carcinoma (HNSCC) cell line FaDu. We compared the effect of these different types of autophagy induction on ATP production, lipid peroxidation, mitophagy, RNA cargo of extracellular vesicles (EVs), and EVs-associated cytokine secretome of cancer cells. Both BEZ and starvation resulted in a decline in ATP production. Simultaneously, Gln starvation enhanced oxidative damage of cancer cells by lipid peroxidation. In starved cells, there was a discernible fragmentation of the mitochondrial network coupled with an increase in the presence of tumor susceptibility gene 101 (TSG101) on the mitochondrial membrane, indicative of the sorting of mitochondrial cargo into EVs. Consequently, the abundance of mitochondrial RNAs (mtRNAs) in EVs released by FaDu cells was enhanced. Notably, mtRNAs were also detectable in EVs isolated from the serum of both HNSCC patients and healthy controls. Starvation and BEZ reduced the production of EVs by cancer cells, yet the characteristic molecular profile of these EVs remained unchanged. We also found that alterations in the release of inflammatory cytokines constitute a principal response to autophagy induction. Importantly, the specific mechanism driving autophagy induction significantly influenced the composition of the EVs-associated cytokine secretome.

• MeSH
• adenosintrifosfát * metabolismus   MeSH
• autofagie * účinky léků   MeSH
• dlaždicobuněčné karcinomy hlavy a krku metabolismus   genetika   patologie   MeSH
• extracelulární vezikuly * metabolismus   účinky léků   MeSH
• glutamin * metabolismus   MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory hlavy a krku metabolismus   patologie   genetika   MeSH
• oxidační stres * MeSH
• RNA mitochondriální * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVE: A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. METHODS: HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. RESULTS: This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. CONCLUSIONS: This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

• MeSH
• chronická myeloidní leukemie * farmakoterapie   metabolismus   MeSH
• energetický metabolismus účinky léků   MeSH
• imatinib mesylát * farmakologie   škodlivé účinky   MeSH
• inhibitory proteinkinas farmakologie   škodlivé účinky   MeSH
• karnitin * metabolismus   farmakologie   MeSH
• lidé MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protinádorové látky škodlivé účinky   farmakologie   MeSH
• rodina nosičů rozpuštěných látek 22, člen 5 * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Hypoxia represents one of the key factors that stimulates the growth of leukemic cells in their niche. Leukemic cells in hypoxic conditions are forced to reprogram their original transcriptome, miRNome, and metabolome. How the coupling of microRNAs (miRNAs)/mRNAs helps to maintain or progress the leukemic status is still not fully described. MiRNAs regulate practically all biological processes within cells and play a crucial role in the development/progression of leukemia. In the present study, we aimed to uncover the impact of hsa-miR-155-5p (miR-155, MIR155HG) on the metabolism, proliferation, and mRNA/miRNA network of human chronic lymphocytic leukemia cells (CLL) in hypoxic conditions. As a model of CLL, we used the human MEC-1 cell line where we deleted mature miR-155 with CRISPR/Cas9. We determined that miR-155 deficiency in leukemic MEC-1 cells results in lower proliferation even in hypoxic conditions in comparison to MEC-1 control cells. Additionally, in MEC-1 miR-155 deficient cells we observed decreased number of populations of cells in S phase. The miR-155 deficiency under hypoxic conditions was accompanied by an increased apoptosis. We detected a stimulatory effect of miR-155 deficiency and hypoxia at the level of gene expression, seen in significant overexpression of EGLN1, GLUT1, GLUT3 in MEC-1 miR-155 deficient cells. MiR-155 deficiency and hypoxia resulted in increase of glucose and lactate uptake. Pyruvate, ETC and ATP were reduced. To conclude, miR-155 deficiency and hypoxia affects glucose and lactate metabolism by stimulating the expression of glucose transporters as GLUT1, GLUT3, and EGLN1 [Hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2)] genes in the MEC-1 cells.

• Publikační typ
• časopisecké články MeSH

A series of new indole-pyrazole hybrids 8a-m were synthesized through the palladium-catalyzed ligandless Heck coupling reaction from easily accessible unsubstituted, methoxy- or fluoro-substituted 4-ethenyl-1H-pyrazoles and 5-bromo-3H-indoles. These compounds exerted cytotoxicity to melanoma G361 cells when irradiated with blue light (414 nm) and no cytotoxicity in the dark at concentrations up to 10 μM, prompting us to explore their photodynamic effects. The photodynamic properties of the example compound 8d were further investigated in breast cancer MCF-7 cells. Evaluation revealed comparable anticancer activities of 8d in both breast and melanoma cancer cell lines within the submicromolar range. The treatment induced a massive generation of reactive oxygen species, leading to different types of cell death depending on the compound concentration and the irradiation intensity.

• MeSH
• fotochemoterapie * MeSH
• fotosenzibilizující látky * farmakologie   chemická syntéza   chemie   MeSH
• indoly * farmakologie   chemie   chemická syntéza   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• nádory prsu farmakoterapie   patologie   MeSH
• palladium chemie   farmakologie   MeSH
• protinádorové látky * farmakologie   chemická syntéza   chemie   MeSH
• pyrazoly * farmakologie   chemická syntéza   chemie   MeSH
• reaktivní formy kyslíku * metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Adhesion G protein-coupled receptors (aGPCRs) play an important role in neurodevelopment, immune defence and cancer; however, their role throughout viral infections is mostly unexplored. We have been searching for specific aGPCRs involved in SARS-CoV-2 infection of mammalian cells. In the present study, we infected human epithelial cell lines derived from lung adenocarcinoma (Calu-3) and colorectal carcinoma (Caco-2) with SARS-CoV-2 in order to analyse changes in the level of mRNA encoding individual aGPCRs at 6 and 12 h post infection. Based on significantly altered mRNA levels, we identified four aGPCR candidates-ADGRB3/BAI3, ADGRD1/GPR133, ADGRG7/GPR128 and ADGRV1/GPR98. Of these receptors, ADGRD1/GPR133 and ADGRG7/GPR128 showed the largest increase in mRNA levels in SARS-CoV-2-infected Calu-3 cells, whereas no increase was observed with heat-inactivated SARS-CoV-2 and virus-cleared conditioned media. Next, using specific siRNA, we downregulated the aGPCR candidates and analysed SARS-CoV-2 entry, replication and infectivity in both cell lines. We observed a significant decrease in the amount of SARS-CoV-2 newly released into the culture media by cells with downregulated ADGRD1/GPR133 and ADGRG7/GPR128. In addition, using a plaque assay, we observed a reduction in SARS-CoV-2 infectivity in Calu-3 cells. In summary, our data suggest that selected aGPCRs might play a role during SARS-CoV-2 infection of mammalian cells.

• MeSH
• adenokarcinom plic * genetika   virologie   patologie   metabolismus   MeSH
• Caco-2 buňky MeSH
• COVID-19 * genetika   virologie   metabolismus   MeSH
• lidé MeSH
• messenger RNA * genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory plic genetika   virologie   patologie   metabolismus   MeSH
• receptory spřažené s G-proteiny * metabolismus   genetika   MeSH
• SARS-CoV-2 * genetika   fyziologie   metabolismus   MeSH
• upregulace * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cévní mozková příhoda, infarkt myokardu a plicní embolie představují významnou zdravotní zátěž pro současnou společnost, přičemž jejich hlavní příčinou jsou krevní sraženiny a poškození vnitřní výstelky cév (endotelu). Přesná a rychlá diagnostika těchto sraženin, zejména určení jejich stáří, je klíčová pro optimální volbu léčebného postupu, jako je trombolýza (farmakologické rozpuštění sraženiny) nebo trombektomie (mechanické odstranění sraženiny). Stávající diagnostické metody však nedosahují požadované úrovně přesnosti a efektivity. Tento článek zkoumá potenciál jodovaných nanočástic (IoNP) na bázi polyjodovaných biodegradabilních polymerů, které mohou cíleně vyhledávat specifické složky, jako je fibrin, a umožnit tak vizualizaci krevních sraženin pomocí rentgenových zobrazovacích metod, jako je počítačová tomografie (CT) nebo skiaskopie. Nanočástice mají schopnost nejen zobrazit sraženiny, ale také odhadnout jejich stáří, což by mohlo výrazně podpořit moderní teranostický přístup, který kombinuje diagnostiku a terapii. Článek také demonstruje možnosti testování farmakokinetiky těchto teranostik pomocí modelového organismu (potkana), využívající metodu laserové ablace s hmotnostní spektrometrií s indukčně vázaným plazmatem. Tento přístup přispívá k lepšímu porozumění biodegradability potenciálních léčiv a představuje klíčový krok v preklinickém hodnocení.

Stroke, myocardial infarction, and pulmonary embolism represent a significant health burden for modern society, with their primary causes being blood clots and damage to the endothelial lining of blood vessels. Accurate and rapid diagnosis of these clots, particularly determining their age, is crucial for optimal treatment selection, such as thrombolysis (pharmacological dissolution of the clot) or thrombectomy (mechanical removal of the clot). However, existing diagnostic methods do not achieve the required levels of accuracy and efficiency. This article explores the potential of iodinated nanoparticles (IoNPs) based on polyiodinated biodegradable polymers, which can selectively target specific components such as fibrin, thereby enabling visualization of blood clots using X-ray imaging techniques like computed tomography (CT) or fluoroscopy. The nanoparticles have the capability not only to visualize clots but also to estimate their age, which could significantly support a modern theranostic approach that combines diagnosis and therapy. The article also demonstrates the possibilities of testing the pharmacokinetics of these theranostics using a model organism (rat), employing the method of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This approach contributes to a better understanding of the biodegradability of potential therapeutics and represents a key step in preclinical evaluation.

• MeSH
• cévní mozková příhoda diagnostické zobrazování   terapie   MeSH
• diagnostické zobrazování MeSH
• kontrastní látky MeSH
• lidé MeSH
• nanočástice MeSH
• teranostická nanomedicína * metody   MeSH
• trombektomie MeSH
• trombolytická terapie MeSH
• trombóza diagnostické zobrazování   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

GABAB receptors (GBRs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GBRs regulate fast synaptic transmission by gating Ca2+ and K+ channels via the Gβγ subunits of the activated G protein. It has been demonstrated that auxiliary GBR subunits, the KCTD proteins, shorten onset and rise time and increase desensitization of receptor-induced K+ currents. KCTD proteins increase desensitization of K+ currents by scavenging Gβγ from the channel, yet the mechanism responsible for the rapid activation of K+ currents has remained elusive. In this study, we demonstrate that KCTD proteins preassemble Gβγ at GBRs. The preassembly obviates the need for diffusion-limited G protein recruitment to the receptor, thereby accelerating G protein activation and, as a result, K+ channel activation. Preassembly of Gβγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gβ subunits. The binding site is shared between Gβ1 and Gβ2, limiting the interaction of KCTD proteins to these particular Gβ isoforms. Substituting residues in the KCTD binding site of Gβ1 with those from Gβ3 hinders the preassembly of Gβγ with GBRs, delays onset and prolongs rise time of receptor-activated K+ currents. The KCTD-Gβ interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs.

• MeSH
• draslíkové kanály metabolismus   genetika   MeSH
• gating iontového kanálu * fyziologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• proteiny vázající GTP - beta-podjednotky * metabolismus   genetika   MeSH
• proteiny vázající GTP - gama-podjednotky * metabolismus   genetika   MeSH
• receptory GABA-B * metabolismus   genetika   MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH