Determination of free cyanide (fCN) is required for various industrial, environmental, food, and clinical samples. Enzymatic methods are not widely used in this field despite their selectivity and mild conditions. Therefore, we present here a proof of concept for new spectrophotometric enzymatic assays of fCN. These are based on the hydrolysis of fCN affording the readily detectable NADH. fCN is hydrolyzed either in one step by cyanide dihydratase (CynD) or in two steps by cyanide hydratase (CynH) and formamidase (AmiF). An advantage of the latter route is the higher activity of CynH and AmiF compared to CynD. In both cases, the resulting formate is then transformed by an NAD-dependent formate dehydrogenase (FDH). The NADH thus formed is quantified colorimetrically using a known method based on a reduction of a tetrazolium salt (WST-8) with NADH. The developed assays of fCN are selective except for formic acid interference, proceed under mild conditions, and, moreover, fCN is detoxified during the reactions. The assays proceeded in a microtiter plate format. The limit of detection (LOD) and the limit of quantification (LOQ) were lower for the three-enzyme (CynH-AmiF-FDH) method (7.00 and 21.2 µmol/L, respectively) than for the two-enzyme (CynD-FDH) method (10.7 and 32.4 µmol/L, respectively). In conclusion, the new fCN assays presented in this work are selective, high-throughput, do not require harsh conditions, and use only small amounts of chemicals and enzymes.

• Klíčová slova
• Cyanide dihydratase, Cyanide hydratase, Enzymatic assays, Formamidase, Formate dehydrogenase, Free cyanide,
• MeSH
• dehydratasy chemie   metabolismus   MeSH
• enzymatické testy * metody   MeSH
• hydrolýza MeSH
• kolorimetrie metody   MeSH
• kyanidy * analýza   metabolismus   MeSH
• limita detekce MeSH
• NAD chemie   MeSH
• spektrofotometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dehydratasy MeSH
• kyanidy * MeSH
• NAD MeSH

Dinuclear complex [Ir2(μ-L1)(η5-Cp*)2Cl2](PF6)2 (1) exhibits low micromolar cytotoxic activity in vitro in various human cancer cells (GI50 = 1.7-3.0 μM) and outperformed its mononuclear analogue [Ir(η5-Cp*)Cl(L2)]PF6 (2; GI50 > 40.0 μM); Cp* = pentamethylcyclopentadienyl, L1 = 4-chloro-2,6-bis[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl]pyridine, L2 = 5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amine. Compound 1 upregulated the Keap1/Nrf2 oxidative stress-protective pathway in the treated MV4-11 acute myeloid leukemia cells. In connection with the redox-mediated mode of action of 1, its NADH-oxidizing activity was detected in solution (1H NMR), while NAD+ remained intact (with formate as a hydride source). Surprisingly, only negligible NADH oxidation was detected in the presence of the reduced glutathione and ascorbate. Following the results of in-solution experiments, NAD(H) concentration was assessed in 1-treated MV4-11 cancer cells. Besides the intracellular NADH oxidation in the presence of 1, the induced oxidative stress also led to a decrease of NAD+, resulting in depletion of both NAD+/NADH coenzymes. The discussed findings provide new insight into the biochemical effects of catalytic anticancer compounds that induce cell death via a redox-mediated mode of action.

• Klíčová slova
• Antiproliferative activity, Complex, Dinuclear, Iridium, NADH, Reactive oxygen species,
• MeSH
• faktor 2 související s NF-E2 * metabolismus   MeSH
• iridium * chemie   farmakologie   MeSH
• komplexní sloučeniny farmakologie   chemie   chemická syntéza   MeSH
• lidé MeSH
• NAD * metabolismus   MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• protinádorové látky * farmakologie   chemie   chemická syntéza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faktor 2 související s NF-E2 * MeSH
• iridium * MeSH
• komplexní sloučeniny MeSH
• NAD * MeSH
• NFE2L2 protein, human MeSH Prohlížeč
• protinádorové látky * MeSH

RNA 5'-modification with NAD+/NADH (oxidized/reduced nicotinamide adenine dinucleotide) has been found in bacteria, eukaryotes and viruses. 5'-NAD is incorporated into RNA by RNA polymerases (RNAPs) during the initiation of synthesis. It is unknown (i) which factors and physiological conditions permit substantial NAD incorporation into RNA in vivo and (ii) how 5'-NAD impacts gene expression and the fate of RNA in bacteria. Here we show in Escherichia coli that RNA NADylation is stimulated by low cellular concentration of the competing substrate ATP, and by weakening ATP contacts with RNAP active site. Additionally, RNA NADylation may be influenced by DNA supercoiling. RNA NADylation does not interfere with posttranscriptional RNA processing by major ribonuclease RNase E. It does not impact the base-pairing between RNAI, the repressor of plasmid replication, and its antisense target, RNAII. Leaderless NADylated model mRNA cI-lacZ is recognized by the 70S ribosome and is translated with the same efficiency as triphosphorylated cI-lacZ mRNA. Translation exposes the 5'-NAD of this mRNA to de-capping by NudC enzyme. We suggest that NADylated mRNAs are rapidly degraded, consistent with their low abundance in published datasets. Furthermore, we observed that ppGpp inhibits NudC de-capping activity, contributing to the growth phase-dependency of NADylated RNA levels.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• bakteriální RNA metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy metabolismus   genetika   MeSH
• endoribonukleasy metabolismus   genetika   MeSH
• Escherichia coli * genetika   metabolismus   MeSH
• messenger RNA metabolismus   genetika   MeSH
• NAD * metabolismus   MeSH
• posttranskripční úpravy RNA MeSH
• proteiny z Escherichia coli metabolismus   genetika   MeSH
• proteosyntéza MeSH
• RNA čepičky * metabolismus   MeSH
• stochastické procesy MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• bakteriální RNA MeSH
• DNA řízené RNA-polymerasy MeSH
• endoribonukleasy MeSH
• messenger RNA MeSH
• NAD * MeSH
• proteiny z Escherichia coli MeSH
• ribonuclease E MeSH Prohlížeč
• RNA čepičky * MeSH

Maintenance of NAD pools is critical for neuronal survival. The capacity to maintain NAD pools declines in neurodegenerative disease. We identify that low NMNAT2, the critical neuronal NAD producing enzyme, drives retinal susceptibility to neurodegenerative insults. As proof of concept, gene therapy over-expressing full length human NMNAT2 is neuroprotective. To pharmacologically target NMNAT2, we identify that epigallocatechin gallate (EGCG) can drive NAD production in neurons through an NMNAT2 and NMN dependent mechanism. We confirm this by pharmacological and genetic inhibition of the NAD-salvage pathway. EGCG is neuroprotective in rodent (mixed sex) and human models of retinal neurodegeneration. As EGCG has poor drug-like qualities, we use it as a tool compound to generate novel small molecules which drive neuronal NAD production and provide neuroprotection. This class of NMNAT2 targeted small molecules could have an important therapeutic impact for neurodegenerative disease following further drug development.

• MeSH
• genetická terapie metody   MeSH
• katechin * analogy a deriváty   farmakologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• NAD * metabolismus   MeSH
• neurodegenerativní nemoci farmakoterapie   metabolismus   genetika   MeSH
• neurony * metabolismus   účinky léků   MeSH
• neuroprotektivní látky * farmakologie   MeSH
• nikotinamidnukleotidadenylyltransferasa * metabolismus   genetika   MeSH
• retina metabolismus   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• epigallocatechin gallate MeSH Prohlížeč
• katechin * MeSH
• NAD * MeSH
• neuroprotektivní látky * MeSH
• nikotinamidnukleotidadenylyltransferasa * MeSH
• NMNAT2 protein, human MeSH Prohlížeč

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
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá jadérková RNA * MeSH
• NAD * MeSH
• RNA čepičky MeSH
• RNA malá jaderná * MeSH

Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana, which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments.

• Klíčová slova
• Krebs cycle, NAD+, NADP+, TCA cycle, cofactor preference, isocitrate dehydrogenase,
• MeSH
• isocitrátdehydrogenasa * genetika   metabolismus   MeSH
• isocitráty metabolismus   MeSH
• NAD * metabolismus   MeSH
• NADP metabolismus   MeSH
• protein - isoformy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• isocitrátdehydrogenasa * MeSH
• isocitráty MeSH
• isocitric acid MeSH Prohlížeč
• NAD * MeSH
• NADP MeSH
• protein - isoformy 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.

• Klíčová slova
• Alpers' disease, NAD+, NR, cortical organoids, induced pluripotent stem cells, mitochondrial function,
• 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
• Názvy látek
• DNA polymeráza gama MeSH
• mitochondriální DNA MeSH
• NAD MeSH
• niacinamid MeSH
• nicotinamide-beta-riboside MeSH Prohlížeč
• pyridinové sloučeniny * MeSH

Aging is a natural process of gradual decrease in physical and mental capacity. Biological age (accumulation of changes and damage) and chronological age (years lived) may differ. Biological age reflects the risk of various types of disease and death from any cause. We selected potential biomarkers of aging - telomerase, AGEs, GDF11 and 15 (growth differentiation factor 11/15), sirtuin 1, NAD+ (nicotinamide adenine dinucleotide), inflammasome NLRP3, DNA/RNA damage, and klotho to investigate changes in their levels depending on age and sex. We included 169 healthy volunteers and divided them into groups according to age (under 35; 35-50; over 50) and sex (male, female; male and female under 35; 35-50, over 50). Markers were analyzed using commercial ELISA kits. We found differences in values depending on age and gender. GDF15 increased with age (under 30 and 35-50 p < 0.002; 35-50 and over 50; p < 0.001; under 35 and over 50; p < 0.001) as well as GDF11 (35-50 and over 50; p < 0.03; under 35 and over 50; p < 0.02), AGEs (under 30 and 35-50; p < 0.005), NLRP3 (under 35 over 50; p < 0.03), sirtuin 1 (35-50 and over 50; p < 0.0001; under 35 and over 50; p < 0.004). AGEs and GDF11 differed between males and females. Correlations were identified between individual markers, markers and age, and markers and sex. Markers that reflect the progression of biological aging vary with age (GDF15, GDF11, AGEs, NLRP3, sirtuin) and sex (AGEs, GDF11). Their levels could be used in clinical practice, determining biological age, risk of age-related diseases and death of all-causes, and initiating or contraindicating a therapy in the elderly based on the patient's health status.

• Klíčová slova
• AGEs, Aging, DNA/RNA damage, GDF15, NLRP3, Sirtuin 1,
• MeSH
• biologické markery MeSH
• DNA MeSH
• kostní morfogenetické proteiny MeSH
• lidé MeSH
• NAD * MeSH
• produkty pokročilé glykace MeSH
• protein NLRP3 MeSH
• růstové diferenciační faktory metabolismus   MeSH
• senioři MeSH
• sirtuin 1 MeSH
• stárnutí genetika   MeSH
• telomerasa * MeSH
• zdravotní stav MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• DNA MeSH
• GDF11 protein, human MeSH Prohlížeč
• kostní morfogenetické proteiny MeSH
• NAD * MeSH
• produkty pokročilé glykace MeSH
• protein NLRP3 MeSH
• růstové diferenciační faktory MeSH
• sirtuin 1 MeSH
• telomerasa * MeSH

Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

• Klíčová slova
• SIRT3, honokiol, mitochondria, nicotinamide adenine dinucleotide, pulmonary hypertension,
• MeSH
• fibroblasty metabolismus   MeSH
• lidé MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• NAD metabolismus   MeSH
• plicní hypertenze * patologie   MeSH
• sirtuin 3 * genetika   metabolismus   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• honokiol MeSH Prohlížeč
• mitochondriální proteiny MeSH
• NAD MeSH
• SIRT3 protein, human MeSH Prohlížeč
• sirtuin 3 * MeSH

Animal and human feces typically include intestinal sulfate-reducing bacteria (SRB). Hydrogen sulfide and acetate are the end products of their dissimilatory sulfate reduction and may create a synergistic effect. Here, we report NADH and NADPH peroxidase activities from intestinal SRB Desulfomicrobium orale and Desulfovibrio piger. We sought to compare enzymatic activities under the influence of various temperature and pH regimes, as well as to carry out kinetic analyses of enzymatic reaction rates, maximum amounts of the reaction product, reaction times, maximum rates of the enzyme reactions, and Michaelis constants in cell-free extracts of intestinal SRB, D. piger Vib-7, and D. orale Rod-9, collected from exponential and stationary growth phases. The optimal temperature (35 °C) and pH (7.0) for both enzyme's activity were determined. The difference in trends of Michaelis constants (Km) during exponential and stationary phases are noticeable between D. piger Vib-7 and D. orale Rod-9; D. orale Rod-9 showed much higher Km (the exception is NADH peroxidase of D. piger Vib-7: 1.42 ± 0.11 mM) during the both monitored phases. Studies of the NADH and NADPH peroxidases-as putative antioxidant defense systems of intestinal SRB and detailed data on the kinetic properties of this enzyme, as expressed by the decomposition of hydrogen peroxide-could be important for clarifying evolutionary mechanisms of antioxidant defense systems, their etiological role in the process of dissimilatory sulfate reduction, and their possible role in the development of bowel diseases.

• MeSH
• antioxidancia * MeSH
• buněčné extrakty MeSH
• Desulfovibrio * MeSH
• lidé MeSH
• NAD MeSH
• NADP MeSH
• obranné mechanismy MeSH
• peroxidasy MeSH
• sírany MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia * MeSH
• buněčné extrakty MeSH
• NAD MeSH
• NADP MeSH
• NADPH peroxidase MeSH Prohlížeč
• peroxidasy MeSH
• sírany MeSH