RESUMEN
The wildtype H-Ras protein functions as a molecular switch in a variety of cell signaling pathways, and mutations to key residues result in a constitutively active oncoprotein. However, there is some debate regarding the mechanism of the intrinsic GTPase activity of H-Ras. It has been hypothesized that ordered water molecules are coordinated at the active site by Q61, a highly transforming amino acid site, and Y32, a position that has not previously been investigated. Here, we examine the electrostatic contribution of the Y32 position to GTP hydrolysis by comparing the rate of GTP hydrolysis of Y32X mutants to the vibrational energy shift of each mutation measured by a nearby thiocyanate vibrational probe to estimate changes in the electrostatic environment caused by changes at the Y32 position. We further compared vibrational energy shifts for each mutation to the hydration potential of the respective side chain and demonstrated that Y32 is less critical for recruiting water molecules into the active site to promote hydrolysis than Q61. Our results show a clear interplay between a steric contribution from Y32 and an electrostatic contribution from Q61 that are both critical for intrinsic GTP hydrolysis.
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Guanosina Trifosfato , Electricidad Estática , Tiocianatos , Hidrólisis , Tiocianatos/química , Tiocianatos/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/química , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tirosina/química , Tirosina/metabolismo , Tirosina/genética , Mutación , Dominio Catalítico , Agua/química , Agua/metabolismo , Modelos MolecularesRESUMEN
The innate immune system employs a variety of antimicrobial oxidants to control and kill host-associated bacteria. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant that is synthesized by lactoperoxidase, myeloperoxidase, and eosinophil peroxidase at sites throughout the human body. HOSCN has potent antibacterial activity while being largely non-toxic toward human cells. The molecular mechanisms by which bacteria sense and defend themselves against HOSCN have only recently begun to be elaborated, notably by the discovery of bacterial HOSCN reductase (RclA), an HOSCN-degrading enzyme widely conserved among bacteria that live on epithelial surfaces. In this paper, I show that Ni2+ sensitizes Escherichia coli to HOSCN by inhibiting glutathione reductase and that inorganic polyphosphate protects E. coli against this effect, probably by chelating Ni2+ ions. I also found that RclA is very sensitive to inhibition by Cu2+ and Zn2+, metals that are accumulated to high levels by innate immune cells, and that, surprisingly, thioredoxin and thioredoxin reductase are not involved in HOSCN stress resistance in E. coli. These results advance our understanding of the contribution of different oxidative stress responses and redox buffering pathways to HOSCN resistance in E. coli and illustrate important interactions between metal ions and the enzymes bacteria use to defend themselves against oxidative stress. IMPORTANCE: Hypothiocyanite (HOSCN) is an antimicrobial oxidant produced by the innate immune system. The molecular mechanisms by which host-associated bacteria defend themselves against HOSCN have only recently begun to be understood. The results in this paper are significant because they show that the low molecular weight thiol glutathione and enzyme glutathione reductase are critical components of the Escherichia coli HOSCN response, working by a mechanism distinct from that of the HOSCN-specific defenses provided by the RclA, RclB, and RclC proteins and that metal ions (including nickel, copper, and zinc) may impact the ability of bacteria to resist HOSCN by inhibiting specific defensive enzymes (e.g., glutathione reductase or RclA).
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Escherichia coli , Tiocianatos , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Tiocianatos/farmacología , Tiocianatos/metabolismo , Níquel/farmacología , Níquel/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Farmacorresistencia Bacteriana , Glutatión Reductasa/metabolismo , Glutatión Reductasa/genética , Antibacterianos/farmacología , Zinc/metabolismo , Zinc/farmacología , Cobre/metabolismo , Cobre/farmacologíaRESUMEN
PURPOSE: Forensic verification of cyanide (CN) poisoning by direct CN analysis in postmortem blood is challenging due to instability of CN in biological samples. CN metabolites, thiocyanate (SCN-) and 2-aminothiazoline-4-carboxylic acid (ATCA), have been proposed as more stable biomarkers, yet it is unclear if either is appropriate for this purpose. In this study, we evaluated the behavior of CN biomarkers in postmortem swine and postmortem blood to determine which serves as the best biomarker of CN exposure. METHODS: CN, SCN-, and ATCA were measured in postmortem swine (N = 8) stored at 4 °C and postmortem blood stored at 25 °C (room temperature, RT) and 37 °C (typical human body temperature, HBT). RESULTS: Following CN poisoning, the concentration of each CN biomarker increased well above the baseline. In postmortem swine, CN concentrations declined rapidly (t1/2 = 34.3 h) versus SCN- (t1/2 = 359 h, 15 days) and ATCA (t1/2 = 544 h, 23 days). CN instability in postmortem blood increased at RT (t1/2 = 10.7 h) and HBT (t1/2 = 6.6 h). SCN- and ATCA were more stable than CN at all storage conditions. In postmortem swine, the t1/2s of SCN- and ATCA were 15 and 23 days, respectively. While both the t1/2s of SCN- and ATCA were relatively lengthy, endogenous levels of SCN- were much more variable than ATCA. CONCLUSION: While there are still questions to be answered, ATCA was the most adept forensic marker of CN poisoning (i.e., ATCA produced the longest half-life, the largest increase above baseline levels, and most stable background concentrations).
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Biomarcadores , Cianuros , Animales , Cianuros/envenenamiento , Cianuros/sangre , Biomarcadores/sangre , Porcinos , Tiocianatos/envenenamiento , Tiocianatos/sangre , Tiocianatos/metabolismo , Toxicología Forense/métodos , Modelos Animales , Temperatura , Manejo de Especímenes/métodos , TiazolesRESUMEN
Hypothiocyanous acid (HOSCN) is an endogenous oxidant produced by peroxidase oxidation of thiocyanate (SCN-), an ubiquitous sulfur-containing pseudohalide synthesized from cyanide. HOSCN serves as a potent microbicidal agent against pathogenic bacteria, viruses, and fungi, functioning through thiol-targeting mechanisms, independent of currently approved antimicrobials. Additionally, SCN- reacts with hypochlorous acid (HOCl), a highly reactive oxidant produced by myeloperoxidase (MPO) at sites of inflammation, also producing HOSCN. This imparts both antioxidant and antimicrobial potential to SCN-. In this review, we discuss roles of HOSCN/SCN- in immunity and potential therapeutic implications for combating infections.
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Antiinfecciosos , Tiocianatos , Animales , Humanos , Antiinfecciosos/farmacología , Antiinfecciosos/uso terapéutico , Antiinfecciosos/química , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Ácido Hipocloroso/metabolismo , Ácido Hipocloroso/uso terapéutico , Ácido Hipocloroso/química , Oxidación-Reducción , Peroxidasa/metabolismo , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/microbiología , Tiocianatos/uso terapéutico , Tiocianatos/química , Tiocianatos/farmacología , Tiocianatos/metabolismoRESUMEN
In recent decades, there has been increasing interest in elucidating the role of sulfur-containing compounds in plant metabolism, particularly emphasizing their function as signaling molecules. Among these, thiocyanate (SCN-), a compound imbued with sulfur and nitrogen, has emerged as a significant environmental contaminant frequently detected in irrigation water. This compound is known for its potential to adversely impact plant growth and agricultural yield. Although adopting exogenous SCN- as a nitrogen source in plant cells has been the subject of thorough investigation, the fate of sulfur resulting from the assimilation of exogenous SCN- has not been fully explored. There is burgeoning curiosity in probing the fate of SCN- within plant systems, especially considering the possible generation of the gaseous signaling molecule, hydrogen sulfide (H2S) during the metabolism of SCN-. Notably, the endogenous synthesis of H2S occurs predominantly within chloroplasts, the cytosol, and mitochondria. In contrast, the production of H2S following the assimilation of exogenous SCN- is explicitly confined to chloroplasts and mitochondria. This phenomenon indicates complex interplay and communication among various subcellular organelles, influencing signal transduction and other vital physiological processes. This review, augmented by a small-scale experimental study, endeavors to provide insights into the functional characteristics of H2S signaling in plants subjected to SCN--stress. Furthermore, a comparative analysis of the occurrence and trajectory of endogenous H2S and H2S derived from SCN--assimilation within plant organisms was performed, providing a focused lens for a comprehensive examination of the multifaceted roles of H2S in rice plants. By delving into these dimensions, our objective is to enhance the understanding of the regulatory mechanisms employed by the gasotransmitter H2S in plant adaptations and responses to SCN--stress, yielding invaluable insights into strategies for plant resilience and adaptive capabilities.
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Sulfuro de Hidrógeno , Plantas , Transducción de Señal , Tiocianatos , Sulfuro de Hidrógeno/metabolismo , Tiocianatos/metabolismo , Plantas/metabolismo , Gasotransmisores/metabolismo , Cloroplastos/metabolismo , Inactivación MetabólicaRESUMEN
The sulfur oxidation kinetics of an industrial strain of Acidithiobacillus caldus (At. caldus) cultured on elemental sulfur was explored in batch experiments in the absence and presence of thiocyanate (SCN-), a toxin inherent within cyanidation tailings wastewater. The Contois rate expression accurately described At. caldus sulfate generation (R2 > 0.93) and microbial growth (R2 > 0.87). For a culture maintained at 45 °C a maximum specific growth rate (µmax) of 0.105 h-1, sulfate yield from biomass (Ypx) of 4.8 × 10-9 mg SO42-.cell-1, and Contois affinity coefficient (Kx) of 1.56 × 10-8 mg S.cell-1 were established. The presence of SCN- (0 mg/L - 20 mg/L) in the bulk solution inhibited the microbial system competitively. Moreover, SCN- impeded microbial growth differentially; the rate expression was therefore partitioned with respect to SCN- concentration and inhibition constants (Ki) were determined for each region. Adaptation to discrete concentrations of SCN- (1 mg/L and 20 mg/L) improved SCN- tolerance in At. caldus; however, adapted strains exhibited reduced sulfur oxidation potential when cultured under thiocyanate-free conditions relative to the non-adapted control strain. To describe the adapted systems accurately, the Contois affinity coefficient (Kx) was revised to reflect the suspected metabolic decline. The derived Kx values increased in magnitude and affirmed an innate reduction in microbial substrate affinity or substrate adsorption capacity. Inclusion of these updated Kx constants within the rate equation suitably represented the experimental data for both adapted At. caldus strains with R2 > 0.94. Furthermore, the impact of adaptation on the inhibition kinetics and inhibition mechanism associated with SCN- exposure were reviewed. Thiocyanate inhibited sulfur oxidation non-competitively in the adapted strains, and the shift in inhibition mechanism may be attributed to the compromised metabolic state following adaptation.
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Acidithiobacillus , Aguas Residuales , Tiocianatos/metabolismo , Oxidación-Reducción , Acidithiobacillus/metabolismo , Azufre/metabolismo , Sulfatos/metabolismoRESUMEN
Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (â¼9.4 and â¼34â¯GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.
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Tiocianatos , Thlaspi , Tiocianatos/química , Tiocianatos/metabolismo , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Thlaspi/metabolismo , Hierro/metabolismo , Oxidación-ReducciónRESUMEN
BACKGROUND: Pressure ulcer is a severe disease in the paralyzed and aging populations. Endothelial progenitor cells (EPCs) are able to regulate ulcer healing by modulating angiogenesis, but the molecular mechanism is still obscure. Sonic hedgehog (SHH) signaling contributes to angiogenesis in various diseases and has been identified to modulate EPCs function. Here, we aimed to explore the significance of SHH signaling in EPCs function during pressure ulcers. METHODS: The EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1. Cell proliferation was detected by cell counting kit 8 (CCK-8). The DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 were analyzed by immunofluorescent analysis. The angiogenesis of EPCs was analyzed by tube formation assay. The pressure ulcers rat model was constructed, the wound injury was analyzed by H&E staining and angiogenesis was analyzed by the accumulation of CD31 based on immunofluorescent analysis. RESULTS: The expression of patched-1 and Gli-1 was enhanced by SHH activator SAG but reduced by SHH inhibitor cyclopamine in the EPCsThe PI3K, Akt, eNOS expression and the Akt phosphorylation were induced by SAG, while the treatment of cyclopamine presented a reversed result. The proliferation and migration of EPCs were enhanced by SAG but repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse the effect of SAG. CONCLUSIONS: Thus, we found that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. SHH signaling may serve as the potential target for attenuating pressure ulcers.
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Células Progenitoras Endoteliales , Úlcera por Presión , Ratas , Animales , Células Progenitoras Endoteliales/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Hedgehog/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Úlcera por Presión/metabolismo , Tiocianatos/metabolismo , Tiocianatos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Fluoresceínas/metabolismo , Fluoresceínas/farmacología , Movimiento Celular , Células CultivadasRESUMEN
Cyanogenic glycosides in forage species and the possibility of cyanide (CN) poisoning can have undesirable effects on ruminants. The literature estimates that unknown rumen bacteria with rhodanese activity are key factors in the animal detoxification of cyanogenic glycosides, as they are capable of transforming CN into the less toxic thiocyanate. Therefore, identifying these bacteria will enhance our understanding of how to improve animal health with this natural CN detoxification process. In this study, a rhodanese activity screening assay revealed 6 of 44 candidate rumen bacterial strains isolated from domestic buffalo, dairy cattle, and beef cattle, each with a different colony morphology. These strains were identified as belonging to the species Enterococcus faecium and E. gallinarum by 16S ribosomal DNA sequence analysis. A CN-thiocyanate transformation assay showed that the thiocyanate formation capacity of the strains after a 12 h incubation ranged from 4.42 to 25.49 mg hydrogen CN equivalent/L. In addition, thiocyanate degradation resulted in the production of ammonia nitrogen and acetic acid in different strains. This study showed that certain strains of enterococci substantially contribute to CN metabolism in ruminants. Our results may serve as a starting point for research aimed at improving ruminant production systems in relation to CN metabolism.
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Cianuros , Tiosulfato Azufretransferasa , Animales , Bovinos , Cianuros/metabolismo , Tiosulfato Azufretransferasa/metabolismo , Tiocianatos/metabolismo , Enterococcus/metabolismo , Rumen/microbiología , Rumiantes/metabolismoRESUMEN
Myeloperoxidase (MPO) is released by neutrophils in inflamed tissues. MPO oxidizes chloride, bromide, and thiocyanate to produce hypochlorous acid (HOCl), hypobromous acid (HOBr), and hypothiocyanous acid (HOSCN), respectively. These oxidants are toxic to pathogens, but may also react with host cells to elicit biological activity and potential toxicity. In cystic fibrosis (CF) and related diseases, increased neutrophil inflammation leads to increased airway MPO and airway epithelial cell (AEC) exposure to its oxidants. In this study, we investigated how equal dose-rate exposures of MPO-derived oxidants differentially impact the metabolome of human AECs (BEAS-2B cells). We utilized enzymatic oxidant production with rate-limiting glucose oxidase (GOX) coupled to MPO, and chloride, bromide (Br-), or thiocyanate (SCN-) as substrates. AECs exposed to GOX/MPO/SCN- (favoring HOSCN) were viable after 24 h, while exposure to GOX/MPO (favoring HOCl) or GOX/MPO/Br- (favoring HOBr) developed cytotoxicity after 6 h. Cell glutathione and peroxiredoxin-3 oxidation were insufficient to explain these differences. However, untargeted metabolomics revealed GOX/MPO and GOX/MPO/Br- diverged significantly from GOX/MPO/SCN- for dozens of metabolites. We noted methionine sulfoxide and dehydromethionine were significantly increased in GOX/MPO- or GOX/MPO/Br--treated cells, and analyzed them as potential biomarkers of lung damage in bronchoalveolar lavage fluid from 5-year-olds with CF (n = 27). Both metabolites were associated with increasing bronchiectasis, neutrophils, and MPO activity. This suggests MPO production of HOCl and/or HOBr may contribute to inflammatory lung damage in early CF. In summary, our in vitro model enabled unbiased identification of exposure-specific metabolite products which may serve as biomarkers of lung damage in vivo. Continued research with this exposure model may yield additional oxidant-specific biomarkers and reveal explicit mechanisms of oxidant byproduct formation and cellular redox signaling.
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Fibrosis Quística , Tiocianatos , Humanos , Preescolar , Tiocianatos/metabolismo , Peroxidasa/metabolismo , Bromuros , Cloruros , Oxidantes/metabolismo , Antioxidantes , Ácido Hipocloroso/metabolismo , Células Epiteliales/metabolismo , MetabolómicaRESUMEN
It is a big challenge to bioremediate thiocyanate pollution in the gold extraction heap leaching tailings and surrounding soils with high contents of arsenic and alkali. Here, a novel thiocyanate-degrading bacterium Pseudomonas putida TDB-1 was successfully applied to completely degrade 1000 mg/L thiocyanate under a high arsenic (400 mg/L) and alkaline condition (pH = 10). It also leached the contents of thiocyanate from 1302.16 to 269.72 mg/kg in the gold extraction heap leaching tailings after 50 h. The maximum transformation rates of S and N in thiocyanate to the two finial products of SO42- and NO3- were 88.98 % and 92.71 %, respectively. Moreover, the genome sequencing confirmed that the biomarker gene of thiocyanate-degrading bacterium, CynS was identified in the strain TDB-1. The bacterial transcriptome revealed that critical genes, such as CynS, CcoNOQP, SoxY, tst, gltBD, arsRBCH and NhaC, etc. in the thiocyanate degradation, S and N metabolisms, and As and alkali resistance were significantly up-regulated in the groups with 300 mg/L SCN- (T300) and with 300 mg/L SCN- and 200 mg/L As (TA300). In addition, the protein-protein interaction network showed that the glutamate synthase encoding by gltB and gltD served as central node to integrate the S and N metabolism pathways with thiocyanate as substrate. The results of our study provide a novel molecular level insight for the dynamic gene expression regulation of thiocyanate degradation by the strain TDB-1 with a severe arsenic and alkaline stress.
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Arsénico , Pseudomonas putida , Pseudomonas putida/metabolismo , Tiocianatos/metabolismo , OroRESUMEN
Thiocyanate-containing wastewater harms ecosystems and can cause serious damage to animals and plants, so it is urgent to treat it effectively. In this study, a new efficient thiocyanate-degrading consortium was developed and its degradation characteristics were studied. It was found that up to 154.64 mM thiocyanate could be completely degraded by this consortium over 6 days of incubation, with a maximum degradation rate of 1.53 mM h-1. High-throughput sequencing analysis showed that Thiobacillus (77.78%) was the predominant thiocyanate-degrading bacterial genus. Plant toxicology tests showed that the germination index of mung bean and rice seeds cultured with media obtained after thiocyanate degradation by the consortium increased by 94% and 84.83%, respectively, compared with the control group without thiocyanate degradation. Cytotoxicity tests showed that thiocyanate without degradation significantly decreased the Neuro-2a cell activity and mitochondrial membrane potential; induced reactive oxygen species generation and apoptosis; increased the cellular Ca2+ concentration; and damaged the cell nucleus and DNA. Furthermore, the thiocyanate degradation products produced the consortium were almost totally non-toxic, revealing the same characteristics as those of the control using distilled water. This study shows that the consortium has a high degradation efficiency and detoxification characteristics, as well as great application potential in bioremediation of industrial thiocyanate-containing wastewater.
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Tiocianatos , Aguas Residuales , Aguas Residuales/toxicidad , Tiocianatos/toxicidad , Tiocianatos/metabolismo , Ecosistema , Bacterias/metabolismo , Biodegradación AmbientalRESUMEN
Thiocyanate is a common pollutant in gold mine, textile, printing, dyeing, coking and other industries. Therefore, thiocyanate in industrial wastewater is an urgent problem to be solved. This paper reviews the chemical properties, applications, sources and toxicity of thiocyanate, as well as the various treatment methods for thiocyanate in wastewater and their advantages and disadvantages. It is emphasized that biological systems, ranging from laboratory to full-scale, are able to successfully remove thiocyanate from factories. Thiocyanate-degrading microorganisms degrade thiocyanate in autotrophic manner for energy, while other biodegrading microorganisms use thiocyanate as a carbon or nitrogen source, and the biochemical pathways and enzymes involved in thiocyanate metabolism by different bacteria are discussed in detail. In the future, degradation mechanisms should be investigated at the molecular level, with further research aiming to improve the biochemical understanding of thiocyanate metabolism and scaling up thiocyanate degradation technologies from the laboratory to a full-scale.
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Tiocianatos , Aguas Residuales , Aguas Residuales/química , Tiocianatos/metabolismo , Bacterias/metabolismo , Nitrógeno/metabolismo , Residuos Industriales/análisis , Eliminación de Residuos Líquidos/métodosRESUMEN
Thyroid disruptors are found in food, atmosphere, soil, and water. These contaminants interfere with the thyroid function through the impairment of thyroid hormone synthesis, plasma transport, peripheral metabolism, transport into the target cells, and thyroid hormone action. It is well known that iodide uptake mediated by the sodium-iodide symporter (NIS) is the first limiting step involved in thyroid hormones production. Therefore, it has been described that several thyroid disruptors interfere with the thyroid function through the regulation of NIS expression and/or activity. Perchlorate, nitrate, and thiocyanate competitively inhibit the NIS-mediated iodide uptake. These contaminants are mainly found in food, water and in the smoke of cigarettes. Although the impact of the human exposure to these anions is highly controversial, some studies indicated their deleterious effects in the thyroid function, especially in individuals living in iodine deficient areas. Considering the critical role of thyroid function and the production of thyroid hormones for growth, metabolism, and development, this review summarizes the impact of the exposure to these NIS-inhibitors on thyroid function and their consequences for human health.
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Contaminantes Ambientales , Percloratos , Humanos , Percloratos/toxicidad , Percloratos/metabolismo , Tiocianatos/metabolismo , Tiocianatos/farmacología , Nitratos/metabolismo , Nitratos/farmacología , Glándula Tiroides/metabolismo , Contaminantes Ambientales/metabolismo , Yoduros/metabolismo , Yoduros/farmacología , Hormonas Tiroideas , Agua/metabolismoRESUMEN
Thiocyanate (SCN-) alters the potency of certain agonists for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, and dysfunctions in AMPA receptor signaling are considered to underlie a number of neurological diseases. While humans may be exposed to SCN- from the environment, including food sources, a carrier-mediated system transports SCN- from the brain into the blood and is an important regulator of SCN- distribution in the central nervous system. The assessment of this SCN- efflux system in the brain would thus be useful for understanding the mechanisms underlying the neurotoxicity of SCN- and for elucidating the relationship between the efflux system and brain diseases. However, the currently available technique for studying SCN- efflux is severely limited by its invasiveness. Here, we describe the development of a SCN- protracer, 9-pentyl-6-[11C]thiocyanatopurine ([11C]1), to overcome this limitation. [11C]1 was synthesized by the reaction of the iodo-precursor and [11C]SCN- or the reaction of the disulfide precursor with [11C]NH4CN. The protracer [11C]1 entered the brain after intravenous injection into mice and was rapidly metabolized to [11C]SCN-, which was then eliminated from the brain. The efflux of [11C]SCN- was dose-dependently inhibited by perchlorate, a monovalent anion, and the highest dose caused an 82% reduction in the efflux rate. Our findings demonstrate that [11C]1 can be used for the noninvasive and quantitative assessment of the SCN- efflux system in the brain.
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Percloratos , Receptores AMPA , Animales , Aniones , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Disulfuros/metabolismo , Humanos , Ratones , Percloratos/metabolismo , Receptores AMPA/metabolismo , Tiocianatos/metabolismo , Tiocianatos/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.
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Antiinfecciosos , Tiocianatos , Animales , Disulfuros , Hemo , Humanos , Peróxido de Hidrógeno/farmacología , Lactoperoxidasa , Mamíferos/metabolismo , NAD , Oxidantes/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Tiocianatos/metabolismo , Tiocianatos/farmacología , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
Hypothiocyanite and hypothiocyanous acid (OSCN-/HOSCN) are pseudohypohalous acids released by the innate immune system which are capable of rapidly oxidizing sulfur-containing amino acids, causing significant protein aggregation and damage to invading bacteria. HOSCN is abundant in saliva and airway secretions and has long been considered a highly specific antimicrobial that is nearly harmless to mammalian cells. However, certain bacteria, commensal and pathogenic, are able to escape damage by HOSCN and other harmful antimicrobials during inflammation, which allows them to continue to grow and, in some cases, cause severe disease. The exact genes or mechanisms by which bacteria respond to HOSCN have not yet been elucidated. We have found, in Escherichia coli, that the flavoprotein RclA, previously implicated in reactive chlorine resistance, reduces HOSCN to thiocyanate with near-perfect catalytic efficiency and strongly protects E. coli against HOSCN toxicity. This is notable in E. coli because this species thrives in the chronically inflamed environment found in patients with inflammatory bowel disease and is able to compete with and outgrow other important commensal organisms, suggesting that HOSCN may be a relevant antimicrobial in the gut, which has not previously been explored. RclA is conserved in a variety of epithelium-colonizing bacteria, implicating its HOSCN reductase activity in a variety of host-microbe interactions. We show that an rclA mutant of the probiotic Limosilactobacillus reuteri is sensitive to HOSCN and that RclA homologs from Staphylococcus aureus, Streptococcus pneumoniae, and Bacteroides thetaiotaomicron all have potent protective activity against HOSCN when expressed in E. coli.
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Proteínas de Escherichia coli , Escherichia coli , Oxidorreductasas , Tiocianatos , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Tiocianatos/química , Tiocianatos/metabolismoRESUMEN
Isothiocyanates, such as sulforaphane and iberin, derived from glucosinolates (GLS) in cruciferous vegetables, are known to prevent and suppress cancer development. GLS can also be converted by bacteria to biologically inert nitriles, such as sulforaphane-nitrile (SFN-NIT) and iberin-nitrile (IBN-NIT), but the role of the gut microbiome in this process is relatively undescribed and SFN-NIT excretion in humans is unknown. An ex vivo fecal incubation model with in vitro digested broccoli sprouts and 16S sequencing was utilized to explore the role of the gut microbiome in SFN- and IBN-NIT production. SFN-NIT excretion was measured among human subjects following broccoli sprout consumption. The fecal culture model showed high inter-individual variability in nitrile production and identified two sub-populations of microbial communities among the fecal cultures, which coincided with a differing abundance of nitriles. The Clostridiaceae family was associated with high levels, while individuals with a low abundance of nitriles were more enriched with taxa from the Enterobacteriaceae family. High levels of inter-individual variation in urine SFN-NIT levels were also observed, with peak excretion of SFN-NIT at 24 h post broccoli sprout consumption. These results suggest that nitrile production from broccoli, as opposed to isothiocyanates, could be influenced by gut microbiome composition, potentially lowering efficacy of cruciferous vegetable interventions.
Asunto(s)
Brassica/química , Microbioma Gastrointestinal , Glucosinolatos/metabolismo , Isotiocianatos/metabolismo , Nitrilos/metabolismo , Sulfóxidos/metabolismo , Clostridiaceae , Enterobacteriaceae , Femenino , Humanos , Masculino , Brotes de la Planta/química , Tiocianatos/metabolismoRESUMEN
The simultaneous detection of cyanide (CN), thiocyanate (SCN), and selenocyanate (SeCN) by a HPLC-fluorescence detector (FLD) with the post-column König reaction was recently reported. SCN and SeCN are also detectable by HPLC-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) because sulfur and selenium can be detected, respectively, without any pre- or post-treatment. ICP-MS has high sensitivity for selenium and sulfur detection and is robust to sample matrices. In this study, we compared HPLC-FLD with the post-column König reaction and HPLC-ICP-MS in terms of SCN and SeCN detection sensitivity and linearity. The limit of detection (LOD) for SCN indicated that HPLC-FLD with the post-column König reaction was 354 times more sensitive than HPLC-ICP-MS. Likewise, the LOD for SeCN indicated that HPLC-FLD was 51 times more sensitive than HPLC-ICP-MS. These results demonstrated that HPLC-FLD was a more suitable technique for SeCN and SCN detection than HPLC-ICP-MS. We previously reported that SeCN was generated in selenite-exposed mammalian cells to detoxify excess selenite. HPLC-FLD with the post-column König reaction enabled good separation and detection for quantifying SCN and SeCN in mammalian cell lines exposed to selenite. The intracellular SCN and SeCN concentrations determined by this technique suggested differences in the metabolic capacity for selenite to form SeCN among the cell lines. In addition, since the amount of intracellular SCN and SeCN were significantly decreased by pretreatment of myeloperoxidase (MPO) inhibitors, SCN and SeCN were resulted from the interaction of sulfur and selenium with endogenous CN, respectively, generated with MPO.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cianatos/análisis , Espectrometría de Masas/métodos , Compuestos de Selenio/análisis , Espectrometría de Fluorescencia/métodos , Tiocianatos/análisis , Cianatos/metabolismo , Células Hep G2 , Humanos , Límite de Detección , Modelos Lineales , Compuestos de Selenio/metabolismo , Tiocianatos/metabolismoRESUMEN
Despite improvements in revascularization after a myocardial infarction, coronary disease remains a major contributor to global mortality. Neutrophil infiltration and activation contributes to tissue damage, via the release of myeloperoxidase (MPO) and formation of the damaging oxidant hypochlorous acid. We hypothesized that elevation of thiocyanate ions (SCN-), a competitive MPO substrate, would modulate tissue damage. Oral dosing of rats with SCN-, before acute ischemia-reperfusion injury (30 min occlusion, 24 h or 4 week recovery), significantly reduced the infarct size as a percentage of the total reperfused area (54% versus 74%), and increased the salvageable area (46% versus 26%) as determined by MRI imaging. No difference was observed in fractional shortening, but supplementation resulted in both left-ventricle end diastolic and left-ventricle end systolic areas returning to control levels, as determined by echocardiography. Supplementation also decreased antibody recognition of HOCl-damaged myocardial proteins. SCN- supplementation did not modulate serum markers of damage/inflammation (ANP, BNP, galectin-3, CRP), but returned metabolomic abnormalities (reductions in histidine, creatine and leucine by 0.83-, 0.84- and 0.89-fold, respectively), determined by NMR, to control levels. These data indicate that elevated levels of the MPO substrate SCN-, which can be readily modulated by dietary means, can protect against acute ischemia-reperfusion injury.