Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• Bordetella bronchiseptica genetika   metabolismus   účinky léků   MeSH
• Bordetella pertussis genetika   patogenita   metabolismus   účinky léků   MeSH
• buněčná smrt * účinky léků   MeSH
• endoplazmatické retikulum metabolismus   účinky léků   MeSH
• homeostáza * MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• sekreční systém typu III metabolismus   genetika   MeSH
• vápník * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Východiska: Endoplazmatické retikulum (ER), organela tvořená soustavou cisteren a tubulů, je esenciální pro řadu buněčných dějů, mj. pro syntézu a transport proteinů. Pokud se chybně složené proteiny hromadí v lumen ER, dochází k rozvoji stresu ER, přičemž následnou odpovědí na narušení homeostázy je aktivace signální dráhy UPR (z angl. unfolded protein response, tj. odpověď na přítomnost nesbalených proteinů). Cílem procesu je obnovit homeostázu zvyšováním kapacity ER a jeho schopnosti skládat proteiny. K aktivaci homeostatické UPR dochází prostřednictvím některého ze tří transmembránových proteinů, kterými jsou enzym vyžadující inositol 1a (inositol-requiring enzyme 1a – IRE1a), kináza ER podobná R kináze (proteine kinase R-like ER kinase – PERK) a aktivující transkripční faktor 6 (activating transcription factor 6 – ATF6). V případě selhání pokusu o obnovu homeostázy naopak dochází prostřednictvím hyperaktivace týchž proteinů k rozvoji terminální UPR a apoptóze. Aktivace různých větví UPR byla popsána u mnoha nádorových onemocnění vč. mnohočetného myelomu (MM), který se vyznačuje maligní transformaci plazmatických buněk a zvýšenou syntézou monoklonálního imunoglobulinu, kdy je role ER zvláště podstatná. Navzdory pokrokům v léčbě MM zůstává onemocnění jen obtížně léčitelné a cílení na signální dráhy spojené s UPR by mohlo např. podpořit účinek inhibitorů proteazomu. Cíl: Tato práce si klade za cíl představit molekulární odpověď na stres ER za fyziologických okolností i v kontextu nádorových onemocnění, a to zejména s přihlédnutím k potenciálním terapeutickým cílům u MM.

Background: The endoplasmic reticulum (ER), an organelle composed of a system of cisternae and tubules, is essential for many cellular processes, including protein synthesis and transport. When misfolded proteins accumulate in the ER lumen, ER stress is induced, and the subsequent response to the disruption of homeostasis is the activation of the unfolded protein response (UPR). The purpose of this process is to restore homeostasis by increasing the capacity of the ER and its ability to fold proteins. Activation of the homeostatic UPR occurs via one of three transmembrane proteins, inositol-requiring enzyme 1a (IRE1a), protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Failure of the attempt to restore homeostasis, on the other hand, leads to the development of terminal UPR and apoptosis via hyperactivation of the same proteins. Activation of UPR has been described in many malignancies, including multiple myeloma (MM), which is characterized by malignant transformation of plasma cells and increased monoclonal immunoglobulin synthesis, where the role of the ER is of particular importance. Despite advances in the treatment of MM, the disease remains difficult to treat and targeting signaling pathways associated with the UPR could, for example, enhance the effect of proteasome inhibitors. Purpose: This review intends to present the molecular response to ER stress under physiological circumstances and in the context of cancer, particularly with regard to potential therapeutic targets in MM.

• MeSH
• cílená molekulární terapie metody   MeSH
• endoplazmatické retikulum * genetika   účinky léků   MeSH
• inhibitory proteasomu farmakologie   klasifikace   terapeutické užití   MeSH
• lidé MeSH
• mnohočetný myelom * farmakoterapie   genetika   MeSH
• signální dráha UPR účinky léků   MeSH
• stres endoplazmatického retikula účinky léků   MeSH
• XBP1 analýza   účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

• MeSH
• cytoplazma účinky léků   ultrastruktura   MeSH
• dospělí MeSH
• endoplazmatické retikulum účinky léků   ultrastruktura   MeSH
• folikuly stimulující hormon farmakologie   MeSH
• indukce ovulace MeSH
• lidé MeSH
• mitochondrie účinky léků   ultrastruktura   MeSH
• mladý dospělý MeSH
• oocyty účinky léků   ultrastruktura   MeSH
• oogeneze účinky léků   fyziologie   MeSH
• segregace chromozomů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Several studies have shown that peroxynitrite (ONOO-), formed upon the reaction of •NO and O2-, is increased in many cardiovascular diseases and is detrimental to myocardial function. Proteins associated with Ca2+ homeostasis regulation in the heart may be involved in these effects. Thus, the aim of this study was to elucidate the mechanisms associated with ONOO--induced effects. We evaluated [Ca2+]i regulation, sarco/endoplasmic reticulum Ca2+- binding proteins, and phosphorylation levels of the ryanodine receptor in isolated rat myocytes. Electrical field-induced intracellular Ca2+ transients and contractions were recorded simultaneously. Myocytes superfused with 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), an ONOO- donor, decreased the amplitude of Ca2+ transients and contraction in a dose-response (1-200 μM) manner. Similarly, SIN-1 increased half-time decay in a concentration-dependent manner. Co-infusion of the ONOO- donor with FeTMPyP (1 μM), an ONOO- decomposition catalyst, inhibited the effects induced by ONOO-. Impaired sarcoplasmic reticulum Ca2+ uptake caused by ONOO- (SIN-1 200 μM) was confirmed by a reduction of caffeine-evoked Ca2+ release along with prolongation of the half-time decay. Surprisingly, ONOO- induced a spontaneous Ca2+ transient that started at the beginning of the relaxation phase and was inhibited by tetracaine. Also, reduced phosphorylation at the ryanodine receptor 2 (RyR2)-Ser-2814 site was observed. In conclusion, deficient sarco/endoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake concomitant with augmented Ca2+ release by RyR2 in myocytes may be associated with modification of myocyte Ca2+ handling by ONOO-. Thus, development of cardiac failure in diabetes, nephropathy, or hypertension may be related with elevated ONOO- in cardiac tissue.

• MeSH
• endoplazmatické retikulum účinky léků   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• intracelulární prostor metabolismus   MeSH
• kardiomyocyty účinky léků   metabolismus   MeSH
• kofein farmakologie   MeSH
• kontrakce myokardu účinky léků   MeSH
• kyselina peroxydusitá metabolismus   MeSH
• membránové transportní proteiny metabolismus   MeSH
• molsidomin analogy a deriváty   farmakologie   MeSH
• potkani Wistar MeSH
• tetrakain farmakologie   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Calcium (Ca2+) signaling and the modulation of intracellular calcium ([Ca2+]i) levels play critical roles in several key processes that regulate cellular survival, growth, differentiation, metabolism, and death in normal cells. On the other hand, aberrant Ca2+-signaling and loss of [Ca2+]i homeostasis contributes to tumor initiation proliferation, angiogenesis, and other key processes that support tumor progression in several different cancers. Currently, chemically and functionally distinct drugs are used as chemotherapeutic agents in the treatment and management of cancer among which certain anti-cancer drugs reportedly suppress pro-survival signals and activate pro-apoptotic signaling through modulation of Ca2+-signaling-dependent mechanisms. Most importantly, the modulation of [Ca2+]i levels via the endoplasmic reticulum-mitochondrial axis and corresponding action of channels and pumps within the plasma membrane play an important role in the survival and death of cancer cells. The endoplasmic reticulum-mitochondrial axis is of prime importance when considering Ca2+-signaling-dependent anti-cancer drug targets. This review discusses how calcium signaling is targeted by anti-cancer drugs and highlights the role of calcium signaling in epigenetic modification and the Warburg effect in tumorigenesis.

• MeSH
• antitumorózní látky farmakologie   terapeutické užití   MeSH
• apoptóza účinky léků   MeSH
• cílená molekulární terapie metody   MeSH
• endoplazmatické retikulum účinky léků   metabolismus   MeSH
• lidé MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• nádory farmakoterapie   metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• vápník metabolismus   MeSH
• vápníková signalizace účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Although patients with lower urinary tract symptoms constitute a large and still growing population, understanding of bladder detrusor muscle physiology remains limited. Understanding the interactions between the detrusor smooth muscle cells and other bladder cell types (e.g. interstitial cells, IC) that may significantly contribute to coordinating and modulating detrusor contractions represents a considerable challenge. Computer modeling could help to elucidate some properties that are difficult to address experimentally; therefore, we developed in silico models of detrusor smooth muscle cell and interstitial cells, coupled through gap junctions. The models include all of the major ion conductances and transporters described in smooth muscle cell and interstitial cells in the literature. The model of normal detrusor muscle (smooth muscle cell and interstitial cells coupled through gap junctions) completely reproduced the experimental results obtained with detrusor strips in the presence of several pharmacological interventions (ryanodine, caffeine, nimodipine), whereas the model of smooth muscle cell alone (without interstitial cells) failed to reproduce the experimental results. Next, a model of overactive bladder, a highly prevalent clinical condition in both men and women with increasing incidence at older ages, was produced by modifying several processes as reported previously: a reduction of Ca(2+)-release through ryanodine receptors and a reduction of Ca(2+)-dependent K(+)-conductance with augmented gap junctional coupling. This model was also able to reproduce the pharmacological modulation of overactive bladder. In conclusion, a model of bladder detrusor muscle was developed that reproduced experimental results obtained in both normal and overactive bladder preparations. The results indicate that the non-smooth muscle cells of the detrusor (interstitial cells) contribute significantly to the contractile behavior of bladder detrusor muscle and should not be neglected. The model suggests that reduced Ca(2+)-release through ryanodine receptors and Ca(2+)-dependent K(+)-conductance together with augmented gap junctional coupling might play a major role in overactive bladder pathogenesis.

• MeSH
• Ca2+-ATPasy metabolismus   MeSH
• endoplazmatické retikulum účinky léků   metabolismus   MeSH
• hyperaktivní močový měchýř patofyziologie   MeSH
• lidé MeSH
• membránové potenciály účinky léků   fyziologie   MeSH
• močový měchýř cytologie   fyziologie   MeSH
• myocyty hladké svaloviny účinky léků   fyziologie   MeSH
• počítačová simulace * MeSH
• ryanodin farmakologie   MeSH
• sarkoplazmatické retikulum účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Resistant cancer phenotype is a key obstacle in the successful therapy of prostate cancer. The primary aim of our study was to explore resistance mechanisms in the advanced type of prostate cancer cells (PC-3) and to clarify the role of autophagy in these processes. We performed time-lapse experiment (48 hours) with ROS generating plumbagin by using multimodal holographic microscope. Furthermore, we also performed the flow-cytometric analysis and the qRT-PCR gene expression analysis at 12 selected time points. TEM and confocal microscopy were used to verify the results. We found out that autophagy (namely mitophagy) is an important resistance mechanism. The major ROS producing mitochondria were coated by an autophagic membrane derived from endoplasmic reticulum and degraded. According to our results, increasing ROS resistance may be also accompanied by increased average cell size and polyploidization, which seems to be key resistance mechanism when connected with an escape from senescence. Many different types of cell-cell interactions were recorded including entosis, vesicular transfer, eating of dead or dying cells, and engulfment and cannibalism of living cells. Entosis was disclosed as a possible mechanism of polyploidization and enabled the long-term survival of cancer cells. Significantly reduced cell motility was found after the plumbagin treatment. We also found an extensive induction of pluripotency genes expression (NANOG, SOX2, and POU5F1) at the time-point of 20 hours. We suppose, that overexpression of pluripotency genes in the portion of prostate tumour cell population exposed to ROS leads to higher developmental plasticity and capability to faster respond to changes in the extracellular environment that could ultimately lead to an alteration of cell fate.

• MeSH
• analýza hlavních komponent MeSH
• autofagie účinky léků   MeSH
• buněčná sebeobnova * účinky léků   MeSH
• časosběrné zobrazování MeSH
• endoplazmatické retikulum účinky léků   metabolismus   MeSH
• entóza účinky léků   MeSH
• inhibiční koncentrace 50 MeSH
• lidé MeSH
• metastázy nádorů MeSH
• mezibuněčná komunikace účinky léků   MeSH
• mitofagie účinky léků   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty genetika   patologie   MeSH
• naftochinony farmakologie   MeSH
• oxidační stres * účinky léků   MeSH
• průtoková cytometrie MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• velikost buňky účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.

• MeSH
• autofagie účinky léků   genetika   MeSH
• benzylaminy farmakologie   MeSH
• chinazoliny farmakologie   MeSH
• endoplazmatické retikulum účinky léků   ultrastruktura   virologie   MeSH
• lidé MeSH
• mikrotubuly účinky léků   ultrastruktura   virologie   MeSH
• nádorové buněčné linie MeSH
• neurony účinky léků   ultrastruktura   virologie   MeSH
• nokodazol farmakologie   MeSH
• primární buněčná kultura MeSH
• replikace viru účinky léků   MeSH
• sirolimus farmakologie   MeSH
• tomografie elektronová MeSH
• virion účinky léků   růst a vývoj   ultrastruktura   MeSH
• viry klíšťové encefalitidy účinky léků   růst a vývoj   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Adipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. METHODS: Preadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. RESULTS: Exposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1-24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. CONCLUSIONS: Acute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes.

• MeSH
• buněčná diferenciace * MeSH
• endoplazmatické retikulum účinky léků   metabolismus   MeSH
• fosforylace MeSH
• fyziologický stres * MeSH
• lidé MeSH
• lipidy biosyntéza   MeSH
• signální dráha UPR MeSH
• thapsigargin farmakologie   MeSH
• tukové buňky cytologie   MeSH
• tunikamycin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Aflatoxins are potent hepatocarcinogen in animal models and suspected carcinogen in humans. The most important aflatoxin in terms of toxic potency and occurrence is aflatoxin B1 (AFB1). In this review, we mainly summarized the key metabolizing enzymes of AFB1 in animals and humans. Moreover, the interindividual and the interspecies differences in AFB1 metabolism are highly concerned. In human liver, CYP3A4 plays an important role in biotransforming AFB1 to the toxic product AFB1-8,9-epoxide. In human lung, CYP2A13 has a significant activity in metabolizing AFB1 to AFB1-8,9-epoxide and AFM1-8,9-epoxide. The epoxide of AFB1-8,9-epoxide could conjugate with glutathione to reduce the toxicity by glutathione-S-transferase (GST). In poultry species, CYP2A6, CYP3A37, CYP1A5, and CYP1A1 are responsible for bioactivation of AFB1. There are interindividual variations in the rate of activation of aflatoxins in various species, and there are also differences between children and adults. The age and living regions are important factors affecting resistance of species to AFB1. The rate of AFB1-8,9-epoxide formation and its conjugation with glutathione are key parameters in interspecies and interindividual differences in sensitivity to the toxic effect of AFB1. This review provides an important information for key metabolizing enzymes and the global metabolism of aflatoxins in different species.

• MeSH
• aflatoxin B1 metabolismus   toxicita   MeSH
• druhová specificita MeSH
• endoplazmatické retikulum účinky léků   enzymologie   MeSH
• játra účinky léků   enzymologie   metabolismus   MeSH
• karcinogeny životního prostředí metabolismus   toxicita   MeSH
• lidé MeSH
• metabolická inaktivace MeSH
• plíce účinky léků   enzymologie   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• respirační sliznice účinky léků   enzymologie   metabolismus   MeSH
• střevní sliznice účinky léků   enzymologie   metabolismus   MeSH
• systém (enzymů) cytochromů P-450 genetika   metabolismus   MeSH
• toxikokinetika MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH