The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy genetika metabolismus MeSH
- bakteriální proteiny * metabolismus MeSH
- nukleotidy metabolismus MeSH
- proteiny SecA metabolismus MeSH
- proteiny z Escherichia coli * metabolismus MeSH
- translokační kanály SEC chemie MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.
- MeSH
- HIV infekce * virologie metabolismus MeSH
- HIV-1 * MeSH
- lidé MeSH
- malá jadérková RNA * metabolismus genetika MeSH
- NAD * metabolismus MeSH
- RNA čepičky metabolismus MeSH
- RNA malá jaderná * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND & AIM: Dysfunction of skeletal muscle satellite cells might impair muscle regeneration and prolong ICU-acquired weakness, a condition associated with disability and delayed death. This study aimed to elucidate the distinct metabolic effects of critical illness and β-OH-butyrate on satellite cells isolated from these patients. METHODS: Satellite cells were extracted from vastus lateralis muscle biopsies of patients with ICU-acquired weakness (n = 10) and control group of healthy volunteers or patients undergoing elective hip replacement surgery (n = 10). The cells were exposed to standard culture media supplemented with β-OH-butyrate to assess its influence on cell proliferation by ELISA, mitochondrial functions by extracellular flux analysis, electron transport chain complexes by high resolution respirometry, and ROS production by confocal microscopy. RESULTS: Critical illness led to a decline in maximal respiratory capacity, ATP production and glycolytic capacity and increased ROS production in ICU patients' cells. Notably, the function of complex II was impaired due to critical illness but restored to normal levels upon exposure to β-OH-butyrate. While β-OH-butyrate significantly reduced ROS production in both control and ICU groups, it had no significant impact on global mitochondrial functions. CONCLUSION: Critical illness induces measurable bioenergetic dysfunction of skeletal muscle satellite cells. β-OH-butyrate displayed a potential in rectifying complex II dysfunction caused by critical illness and this warrants further exploration.
- MeSH
- adenosintrifosfát metabolismus MeSH
- dospělí MeSH
- energetický metabolismus účinky léků MeSH
- kritický stav * MeSH
- kultivované buňky MeSH
- kyselina 3-hydroxymáselná * farmakologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mitochondrie účinky léků metabolismus MeSH
- proliferace buněk účinky léků MeSH
- reaktivní formy kyslíku * metabolismus MeSH
- satelitní buňky kosterního svalu * účinky léků metabolismus MeSH
- senioři MeSH
- svalová slabost MeSH
- svalové mitochondrie účinky léků metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana, ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling.
- MeSH
- ABC transportéry * chemie genetika metabolismus MeSH
- adenosintrifosfát metabolismus MeSH
- Arabidopsis * genetika metabolismus MeSH
- brassinosteroidy * metabolismus MeSH
- konformace proteinů MeSH
- kyseliny indoloctové metabolismus MeSH
- proteiny huseníčku * chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Background: Remdesivir has recently been used more widely as an antiviral medication, possibly due to its potency against coronavirus.Aim: This study was aimed at detecting the toxicity of remdesivir on the liver and kidneys of albino rats at various doses, as well as the possibility of recovering to the normal structure of these tissues two weeks after drug discontinuation.Methods: Forty adult albino rats were divided into five groups (8 rats per group). The first group was the control group; the second group received 5 mg/kg remdesivir; the third group received 10 mg/kg for five days; and the fourth and fifth groups were withdrawal groups (treated as 2nd and 3rd groups then left for two weeks). After five days of treatment, the animals of the 1st, 2nd, and 3rd groups were sacrificed, while the animals of the withdrawal groups were killed after two weeks of drug discontinuation. Both the liver and kidneys were removed and prepared for histological examination.Results: Remdesivir-treated liver and kidneys showed histological alterations such as blood vessel congestion, mononuclear cell infiltration, and localized hepatocyte degeneration. Meanwhile, kidney sections revealed localized vacuolation of the tubular epithelium, focal glomerular tuft shrinkage with Bowman's space dilatation.Conclusion: Remdesivir is hepatotoxic and nephrotoxic mainly, at high doses. Even after drug withdrawal, structural alterations persist, particularly at high dosages, confirming that remdesivir toxicity is dose-dependent.
- Klíčová slova
- remdesivir,
- MeSH
- adenosinmonofosfát analogy a deriváty toxicita MeSH
- alanin analogy a deriváty toxicita MeSH
- antivirové látky * toxicita MeSH
- farmakoterapie COVID-19 škodlivé účinky MeSH
- histologické techniky MeSH
- játra účinky léků MeSH
- ledviny účinky léků MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.
- MeSH
- adenosintrifosfát metabolismus MeSH
- fosfofruktokinasy metabolismus MeSH
- glukoneogeneze * genetika MeSH
- glukosa metabolismus MeSH
- glycerol metabolismus MeSH
- lidé MeSH
- savci MeSH
- transaldolasa metabolismus MeSH
- Trypanosoma brucei brucei * genetika metabolismus MeSH
- uhlík metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Alpers' syndrome is an early-onset neurodegenerative disorder usually caused by biallelic pathogenic variants in the gene encoding the catalytic subunit of polymerase-gamma (POLG), which is essential for mitochondrial DNA (mtDNA) replication. The disease is progressive, incurable, and inevitably it leads to death from drug-resistant status epilepticus. The neurological features of Alpers' syndrome are intractable epilepsy and developmental regression, with no effective treatment; the underlying mechanisms are still elusive, partially due to lack of good experimental models. Here, we generated the patient derived induced pluripotent stem cells (iPSCs) from one Alpers' patient carrying the compound heterozygous mutations of A467T (c.1399G>A) and P589L (c.1766C>T), and further differentiated them into cortical organoids and neural stem cells (NSCs) for mechanistic studies of neural dysfunction in Alpers' syndrome. Patient cortical organoids exhibited a phenotype that faithfully replicated the molecular changes found in patient postmortem brain tissue, as evidenced by cortical neuronal loss and depletion of mtDNA and complex I (CI). Patient NSCs showed mitochondrial dysfunction leading to ROS overproduction and downregulation of the NADH pathway. More importantly, the NAD+ precursor nicotinamide riboside (NR) significantly ameliorated mitochondrial defects in patient brain organoids. Our findings demonstrate that the iPSC model and brain organoids are good in vitro models of Alpers' disease; this first-in-its-kind stem cell platform for Alpers' syndrome enables therapeutic exploration and has identified NR as a viable drug candidate for Alpers' disease and, potentially, other mitochondrial diseases with similar causes.
- MeSH
- DNA polymeráza gama MeSH
- indukované pluripotentní kmenové buňky * MeSH
- lidé MeSH
- mitochondriální DNA genetika MeSH
- mitochondriální nemoci * MeSH
- mutace MeSH
- NAD genetika MeSH
- niacinamid analogy a deriváty MeSH
- pyridinové sloučeniny * MeSH
- Schilderova difuzní cerebroskleróza * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Oxalate degradation is one of lactic acid bacteria's desirable activities. It is achieved by two enzymes, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). The current study aimed to screen 15 locally isolated lactic acid bacteria to select those with the highest oxalate degradation ability. It also aimed to amplify the genes involved in degradation. MRS broth supplemented with 20 mM sodium oxalate was used to culture the tested isolates for 72 h. This was followed by an enzymatic assay to detect remaining oxalate. All isolates showed oxalate degradation activity to variable degrees. Five isolates demonstrated high oxalate degradation, 78 to 88%. To investigate the oxalate-degradation potential of the selected isolates, they have been further tested for the presence of genes that encode for enzymes involved in oxalate catabolism, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). Three strains showed bands with the specific OXC and FRC forward and reverse primers designated as (SA-5, 9 and 37). Species-level identification revealed Loigolactobacillus bifermentans, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum. Preliminary results revealed that the tested probiotic strains harbored both oxc and frc whose products are putatively involved in oxalate catabolism. The probiotic potential of the selected strains was evaluated, and they showed high survival rates to both simulated gastric and intestinal fluids and variable degrees of antagonism against the tested Gram-positive and negative pathogens and were sensitive to clarithromycin but resistant to both metronidazole and ceftazidime. Finally, these strains could be exploited as an innovative approach to establish oxalate homeostasis in humans and prevent kidney stone formation.
- MeSH
- acylkoenzym A * MeSH
- CoA-transferasy genetika metabolismus MeSH
- karboxylyasy * genetika MeSH
- lidé MeSH
- oxaláty metabolismus MeSH
- probiotika * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The adenylyl cyclase (AC) signaling pathway is suggested to be a key regulator of immune system functions. However, specific effects of cyclic adenosine monophosphate (cAMP) on T helper (Th) cell differentiation and functions are unclear. The involvement of cAMP in the Th cell differentiation program, in particular the development of Th1, Th2, and Th17 subsets, was evaluated employing forskolin (FSK), a labdane diterpene well known as an AC activator. FSK mediated an elevation in Th1-specific markers reinforcing the Th1 cell phenotype. The Th2 differentiation was supported by FSK, though cell metabolism was negatively affected. In contrast, the Th17 immunophenotype was severely suppressed leading to the highly specific upregulation of CXCL13. The causality between FSK-elicited cAMP production and the observed reinforcement of Th2 differentiation was established by using AC inhibitor 2',5'-dideoxyadenosine, which reverted the FSK effects. Overall, an FSK-mediated cAMP increase affects Th1, Th2 and Th17 differentiation and can contribute to the identification of novel therapeutic targets for the treatment of Th cell-related pathological processes.
- MeSH
- aktivace lymfocytů * MeSH
- AMP cyklický * MeSH
- buněčná diferenciace MeSH
- buňky Th17 MeSH
- kolforsin farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
Kangrelor v dnešní době představuje jediný intravenózní inhibitor destičkového receptoru P2Y12 s klinicky prokázanou účinností pro snížení ischemických komplikací perkutánní koronární intervence (PCI). Dle souhrnu údajů o léčivém přípravku (SPC) je kangrelor doporučen u pacientů podstupující PCI, kteří nebyli léčení perorálním P2Y12 inhibitorem, nebo u nichž tato terapie není možná nebo vhodná. V následující kazuistice bude představen případ vysoce rizikového pacienta s komplexním intervenčním výkonem za použití tohoto přípravku (1).
Kangrelor represents the only intravenous inhibitor of the platelet receptor P2Y12 with clinically proven effectiveness in reducing ischemic complications of percutaneous coronary intervention (PCI). According to the Summary of Product Characteristics (SPC), Kangrelor is recommended for patients undergoing PCI who have not been treated with an oral P2Y12 inhibitor or for whom this therapy is not possible or suitable. In the following case study, a high-risk patient undergoing a complex interventional procedure using this preparation will be presented (1).
- Klíčová slova
- Kangrelor,
- MeSH
- adenosinmonofosfát farmakologie terapeutické užití MeSH
- akutní koronární syndrom farmakoterapie MeSH
- infarkt myokardu * diagnóza terapie MeSH
- inhibitory agregace trombocytů farmakologie terapeutické užití MeSH
- lidé MeSH
- purinergní receptory P2Y - antagonisté * farmakologie terapeutické užití MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- Publikační typ
- kazuistiky MeSH
