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Familial epilepsy with auditory features (FEAF), previously known as autosomal-dominant lateral temporal lobe epilepsy (ADLTE) is a genetically heterogeneous syndrome, clinically characterized by focal seizures with prominent auditory symptoms. It is inherited with autosomal-dominant pattern with reduced penetrance (about 70%). Sporadic epilepsy with auditory features cases are more frequent and clinically indistinguishable from familial cases. One causal gene, MICAL-1, encodes MICAL-1, an intracellular multi-domain enzyme that is an important regulator of filamentous actin (F-actin) structures. Pathogenic variants in MICAL-1 account for approximately 7% of FEAF families. Here, we describe a de novo MICAL-1 pathogenic variant, p.Arg915Cys, in a sporadic case, an affected 21-year-old Italian man with no family history of epilepsy. Genetic testing was performed in the patient and his parents, using a next-generation sequencing panel. In cell-based assay, this variant significantly increased MICAL-1 oxidoreductase activity, which likely resulted in dysregulation of F-actin organization. This finding provides further support for a gain-of-function effect underlying MICAL-1-mediated epilepsy pathogenesis, as previously seen with other pathogenic variants. Furthermore, the case study provides evidence that de novo MICAL-1 pathogenic variants can occur in sporadic cases with epilepsy with auditory feature (EAF). PLAIN LANGUAGE SUMMARY: In this study, we report a new MICAL-1 pathogenic variant in a patient without family history for epilepsy, not inherited from his parents. MICAL-1 is a protein with enzymatic activity that reorganizes the structure of the cell. We proved the pathological effect of this variant by testing its enzymatic activity and found an increase of this activity. This result suggests that non-familial cases should be tested to find novel pathogenic variants in this gene.
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Epilepsia del Lóbulo Temporal , Humanos , Masculino , Adulto Joven , Epilepsia del Lóbulo Temporal/genética , Actinas/genéticaRESUMEN
YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M-1 s-1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues.
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Escherichia coli , Oxidorreductasas , Pseudomonas aeruginosa , Tiorredoxinas , Xylella , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/química , Filogenia , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/química , Xylella/enzimología , Xylella/genética , Xylella/metabolismoRESUMEN
Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.
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Proteínas de Arabidopsis , Arabidopsis , Citosol , Glutarredoxinas , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Citosol/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vías Secretoras , FilogeniaRESUMEN
Background: Xanthine oxidoreductase (XOR) inhibitor administration, known to reduce uric acid and reactive oxygen species (ROS) production, also improves vascular endothelial function (VEF). This cross-sectional study examined our hypothesis that XOR contributes to impaired VEF through ROS but not uric acid production. Methods: In 395 subjects (196 males, 199 females) without urate-lowering agent administration who underwent a health examination, plasma XOR activity was determined using our highly sensitive assay based on [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. For VEF evaluation, flow-mediated dilatation (FMD) in the brachial artery was determined by ultrasound, with physical and laboratory measurements also obtained. Results: The median values for plasma XOR activity, serum uric acid, and FMD were 26.6 pmol/h/mL, 5.4 mg/dL, and 6.2%, respectively. Simple regression analysis showed weak correlations of both log plasma XOR activity and serum uric acid level with FMD (r = -0.213, p < 0.001 and r = -0.139, p = 0.006, respectively). However, multivariable linear regression analyses revealed that log plasma XOR activity but not serum uric acid level remained associated with FMD (ß = -0.116, p = 0.037 and ß = 0.041, p = 0.549, respectively) after adjustments for various clinical parameters, with no remarkable inconsistencies for the association observed in subgroups divided based on sex or uric acid level. Finally, a series of mediation analyses showed that serum uric acid level did not meet the criteria for mediator of the association of plasma XOR activity with FMD (p = 0.538). Conclusions: These findings suggest the possibility that XOR contributes to the pathophysiology of impaired VEF through ROS but not uric acid production.
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Reactive oxygen species (ROS), a type of oxygen monoelectronic reduction product, play integral roles in root growth and development. The epigenetic mechanism plays a critical role in gene transcription and expression; however, its regulation of ROS metabolism in root development is still limited. We found that the chromatin remodeling complex SWR1 regulates root length and lateral root formation in Arabidopsis. Our transcriptome results and gene ontology (GO) enrichment analysis showed that the oxidoreductase activity-related genes significantly changed in mutants for the Arabidopsis SWR1 complex components, such as arp6 and pie1, and histone variant H2A.Z triple mutant hta8 hta9 hta11. The three encoding genes in Arabidopsis are the three H2A.Z variants hta8, hta9, and hta11. Histochemical assays revealed that the SWR1 complex affects ROS accumulation in roots. Furthermore, chromatin immunoprecipitation quantitative real-time PCR (ChIP-qPCR) analysis showed that the reduced H2A.Z deposition in oxidoreductase activity-related genes caused ROS to accumulate in arp6, pie1, and hta8 hta9 hta11. H2A.Z deposition-deficient mutants decreased after the trimethylation of lysine 4 on histone H3 (H3K4me3) modifications and RNA polymerase II (Pol II) enrichment, and increased after the trimethylation of lysine 27 on histone H3 (H3K27me3) modifications, which may account for the expression change in oxidoreductase activity-related genes. In summary, our results revealed that the chromatin complex SWR1 regulates ROS accumulation in root development, highlighting the critical role of epigenetic mechanisms.
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Sequence-based screening has been widely applied in the discovery of novel microbial enzymes. However, majority of the sequences in the genomic databases were annotated using computational approaches and lacks experimental characterization. Hence, the success in obtaining the functional biocatalysts with improved characteristics requires an efficient screening method that considers a wide array of factors. Recombinant expression of microbial enzymes is often hampered by the undesirable formation of inclusion body. Here, we present a systematic in silico screening method to identify the proteins expressible in soluble form and with the desired biological properties. The screening approach was adopted in the recombinant expression of dimethyl sulfide (DMS) monooxygenase in Escherichia coli. DMS monooxygenase, a two-component enzyme consisting of DmoA and DmoB subunits, was used as a model protein. The success rate of producing soluble and active DmoA is 71% (5 out of 7 genes). Interestingly, the soluble recombinant DmoA enzymes exhibited the NADH:FMN oxidoreductase activity in the absence of DmoB (second subunit), and the cofactor FMN, suggesting that DmoA is also an oxidoreductase. DmoA originated from Janthinobacterium sp. AD80 showed the maximum NADH oxidation activity (maximum reaction rate: 6.6 µM/min; specific activity: 133 µM/min/mg). This novel finding may allow DmoA to be used as an oxidoreductase biocatalyst for various industrial applications. The in silico gene screening methodology established from this study can increase the success rate of producing soluble and functional enzymes while avoiding the laborious trial and error involved in the screening of a large pool of genes available. KEY POINTS: ⢠A systematic gene screening method was demonstrated. ⢠DmoA is also an oxidoreductase capable of oxidizing NADH and reducing FMN. ⢠DmoA oxidizes NADH in the absence of external FMN.
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Escherichia coli , Oxigenasas de Función Mixta , Escherichia coli/genética , Escherichia coli/metabolismo , Mononucleótido de Flavina/metabolismo , Oxigenasas de Función Mixta/metabolismo , NAD/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , SulfurosRESUMEN
Flavodoxin-like proteins (Fld-LPs) are an important constituent of the oxidative stress defense system in several organisms and highly conserved from bacteria to humans. These proteins possess NAD(P)H:quinone oxidoreductase activity and convert quinones to hydroquinones through two-electron reduction, using NAD(P)H and quinone as electron donor and acceptor, respectively. Purified yeast and bacterial Fld-LPs exhibit NAD(P)H:quinone oxidoreductase activity in vitro. Here, we describe a protocol to measure oxidoreductase activity of Fld-LPs that are present in extracts of whole cells. We have recently shown that the assembly and activity of a Fld-LP, CgPst2, is regulated by an aspartyl protease-mediated cleavage of its C-terminus in the pathogenic yeast Candida glabrata. Mutant yeast where the CgPST2 gene was deleted lacked cellular NAD(P)H:quinone oxidoreductase activity and displayed elevated susceptibility to menadione stress. The protocol described herein is based on the measurement of NADH oxidation (conversion of NADH to NAD+) by endogenous Fld-LPs in the presence of quinone menadione. This assay can be performed with whole cell lysates prepared by the mechanical lysis of C. glabrata cells and does not require expression and purification of Fld-LPs from a heterogeneous system, thereby allowing researchers to study the effect of different posttranslational modifications and varied structural states of Fld-LPs on their enzymatic activities. Since many FLP-LPs are known to exist in dimeric and tetrameric states possessing differential activities, our efficient and easy-to-use assay can reliably detect and validate their quinone reductase activities. Although we have used menadione with CgPst2 enzyme in our study, the protocol can easily be modified to examine the presence of Fld-LPs with specificity for other quinones. As this assay does not require many expensive chemicals, it can readily be scaled up and adapted for other medically important fungi and potentially be a useful tool to characterize fungal oxidative stress response systems and screen inhibitors specific for fungal Fld-LPs, thereby contributing to our understanding of fungal pathogenesis mechanisms.
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The inhibition of platelet aggregation, and the activity of oxidoreductases and microhemocirculation in a burn wound on the treatment of burns with wound dressings based on bacterial nanocellulose (BC)-zinc oxide nanoparticles (ZnO NPs)-betulin diphosphate (BDP) were studied. The control of the treatment by BC-ZnO NPs-BDP on burned rats by the noninvasive DLF method showed an increase in perfusion and the respiratory component in wavelet spectra, characterizing an improvement in oxygen saturation in the wound. The study on the volunteers' blood found the inhibition of ADP-induced platelet aggregation by 30-90%. Disaggregation depends on the dose under the action of the ionized form of BDP and ZnO NPs-BDP in a phosphate buffer; it was reversible and had two waves. It was shown on rats that the specific activity of LDHreverse and LDHdirect (control-intact animals) on day 21 of treatment increased by 11-38% and 23%, respectively. The LDHreverse/LDHdirect ratio increased at BC-ZnO NPs-BDP treatment, which characterizes efficient NAD+ regeneration. AlDH activity increased significantly in the first 10 days by 70-170%, reflecting the effectiveness of the enzyme and NAD+ in utilizing toxic aldehydes at this stage of burn disease. The activities of GR and G6PDH using NADP(H) were increased with BC-ZnO NPs-BDP treatment.
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Vendajes , Quemaduras/terapia , Agregación Plaquetaria/efectos de los fármacos , Triterpenos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Acetobacteraceae/química , Animales , Quemaduras/fisiopatología , Celulosa/química , Difosfatos/química , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Reductasa/metabolismo , Humanos , Flujometría por Láser-Doppler , Masculino , Nanopartículas del Metal/química , NADP/metabolismo , Agregación Plaquetaria/fisiología , Ratas Wistar , Espectroscopía Infrarroja por Transformada de Fourier , Triterpenos/químicaRESUMEN
The effects of natural compounds on biofilm formation have been extensively studied, with the goal of identifying biofilm formation antagonists at sub-lethal concentrations. Salicylic and cinnamic acids are some examples of these compounds that interact with the quinone oxidoreductase WrbA, a potential biofilm modulator and an antibiofilm compound biomarker. However, WrbA's role in biofilm development is still poorly understood. To investigate the key roles of WrbA in biofilm maturation and oxidative stress, Escherichia coli wild-type and ∆wrbA mutant strains were used. Furthermore, we reported the functional validation of WrbA as a molecular target of salicylic and cinnamic acids. The lack of WrbA did not impair planktonic growth, but rather affected the biofilm formation through a mechanism that depends on reactive oxygen species (ROS). The loss of WrbA function resulted in an ROS-sensitive phenotype that showed reductions in biofilm-dwelling cells, biofilm thickness, matrix polysaccharide content, and H2O2 tolerance. Endogenous oxidative events in the mutant strain generated a stressful condition to which the bacterium responded by increasing the catalase activity to compensate for the lack of WrbA. Cinnamic and salicylic acids inhibited the quinone oxidoreductase activity of purified recombinant WrbA. The effects of these antibiofilm molecules on WrbA function was proven for the first time.
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INTRODUCTION: Xanthine oxidoreductase (XOR) activity plays an important role as a pivotal source of reactive oxygen species, which is associated with cardiovascular disease (CVD) events. Patients with CKD have increased risk of CVD events. In the present study, factors associated with plasma XOR activity in pre-dialysis CKD patients were investigated. METHODS: In this cross-sectional study, plasma XOR activity in 118 pre-dialysis CKD patients (age 68 [57-75] years; 64 males, 26 with diabetes mellitus [DM]) was determined using a newly established highly sensitive assay based on (13C2,15N2) xanthine and liquid chromatography/triple quadrupole mass spectrometry. RESULTS: Plasma glucose, hemoglobin A1c, and estimated glomerular filtration (eGFR) were significantly and positively correlated with plasma logarithmically transformed XOR (ln-XOR) activity. In multiple regression analyses, eGFR and hemoglobin A1c or plasma glucose were significantly, independently, and positively associated with plasma ln-XOR activity after adjusting for several confounders. Plasma XOR activity was significantly higher in CKD patients with (n = 26) than in those without (n = 92) DM (62.7 [32.3-122] vs. 25.7 [13.4-45.8] pmol/h/mL, p < 0.001). A total of 38 patients were taking uric acid-lowering drugs. Multiple regression analysis of CKD patients not administered uric acid-lowering drugs (n = 80) showed no significant association between eGFR and plasma ln-XOR activity. In contrast, association between glycemic control and plasma ln-XOR activity was significant even in CKD patients without uric acid-lowering drug treatment. CONCLUSIONS: These results indicate the importance of glycemic control in CKD patients in regard to decreased XOR, possibly leading to a decrease in CVD events.
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Glucemia/análisis , Insuficiencia Renal Crónica/sangre , Xantina Deshidrogenasa/sangre , Anciano , Estudios Transversales , Diálisis , Femenino , Hemoglobina Glucada/análisis , Control Glucémico , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The common reed (Phragmites australis) is a dominant species in the coastal wetlands of the Chinese Yellow River Delta, where it tolerates a wide range of salinity. Recent environmental changes have led to the increase of soil salinity in this region, which has degraded much of the local vegetation. Clones of common reeds from the tidal marsh may have adapted to local high salinity habitat through selection on genes and metabolic pathways conferring salt tolerance. This study aims to reveal molecular mechanisms underlying salt tolerance in the tidal reed by comparing them to the salt-sensitive freshwater reed under salt stress. We employed comparative transcriptomics to reveal the differentially expressed genes (DEGs) between these two types of common reeds under different salinity conditions. The results showed that only three co-expressed genes were up-regulated and one co-expressed gene was down-regulated between the two reed types. On the other hand, 1,371 DEGs were exclusively up-regulated and 285 DEGs were exclusively down-regulated in the tidal reed compared to the control, while 115 DEGs were exclusively up-regulated and 118 DEGs were exclusively down-regulated in the freshwater reed compared to the control. From the pattern of enrichment of transcripts involved in salinity response, the tidal reed was more active and efficient in scavenging reactive oxygen species (ROS) than the freshwater reed, with the tidal reed showing significantly higher gene expression in oxidoreductase activity. Furthermore, when the reeds were exposed to salt stress, transcripts encoding glutathione metabolism were up-regulated in the tidal reed but not in the freshwater reed. DEGs related to encoding glutathione reductase (GR), glucose-6-phosphate 1-dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PD), glutathione S-transferase (GST) and L-ascorbate peroxidase (LAP) were revealed as especially highly differentially regulated and therefore represented candidate genes that could be cloned into plants to improve salt tolerance. Overall, more genes were up-regulated in the tidal reed than in the freshwater reed from the Yellow River Delta when under salt stress. The tidal reed efficiently resisted salt stress by up-regulating genes encoding for oxidoreductase activity and glutathione metabolism. We suggest that this type of common reed could be extremely useful in the ecological restoration of degraded, high salinity coastal wetlands in priority.
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In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The Km values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414 mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.
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Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/aislamiento & purificación , Methylobacterium/enzimología , Oxidorreductasas de Alcohol/metabolismo , Biodegradación Ambiental , Coenzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Formaldehído/metabolismo , Concentración de Iones de Hidrógeno , Metanol/metabolismo , NAD/metabolismo , Estructura Cuaternaria de Proteína , Temperatura , Zinc/químicaRESUMEN
Coppicing consists of periodically cutting back tree stems to ground level to stimulate the growth of multiple stems from the stool. In Central Europe, many coppiced forests were abandoned at the beginning of the last century owing to a decline in the demand for charcoal and wood. This was assumed to enable the forests to recover and the properties to become similar to those of unmanaged forest (high forest). Most studies on abandoned coppiced forest have focused on forest recovery, while soil recovery has generally been overlooked. With the aim of filling this gap, this study investigated the effect of coppicing abandonment on soil recovery by analysing the changes in soil enzyme activities (dehydrogenase, ß-glucosidase, invertase, urease, acid phosphatase and arylsulphatase). Two differently managed sessile oak (Quercus petraea) forests were selected for study: a former coppice forest, abandoned >90â¯years ago, and an undisturbed forest. The analytical data were compared to assess the degree of recovery of the soil in the abandoned coppice forest. The soil organic matter content was two times lower in the abandoned coppice than in the high forest, suggesting that organic matter depletion due the past coppicing is a long-term effect. All of the absolute enzyme activities were also two times lower in the abandoned coppice forest soil than in the high forest soil. However, the specific enzyme activities were similar in both types of soil. This indicates that metabolic activity is similar in both soil types, suggesting that it either recovers faster than organic matter and soil enzyme activity or that, despite the depletion in organic matter and enzyme activities, metabolic activity was sustained in coppiced forest soil. However, in the latter case this would imply that organic matter and soil enzymes were lost in exactly the same proportion, which is highly improbable.
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Conservación de los Recursos Naturales , Monitoreo del Ambiente/métodos , Bosques , Microbiología del Suelo , Quercus , Suelo , ÁrbolesRESUMEN
OBJECTIVE: To explore the disturbed molecular functions and pathways in clear cell renal cell carcinoma (ccRCC) using Gibbs sampling. METHODS: Gene expression data of ccRCC samples and adjacent non-tumor renal tissues were recruited from public available database. Then, molecular functions of expression changed genes in ccRCC were classed to Gene Ontology (GO) project, and these molecular functions were converted into Markov chains. Markov chain Monte Carlo (MCMC) algorithm was implemented to perform posterior inference and identify probability distributions of molecular functions in Gibbs sampling. Differentially expressed molecular functions were selected under posterior value more than 0.95, and genes with the appeared times in differentially expressed molecular functions ≥5 were defined as pivotal genes. Functional analysis was employed to explore the pathways of pivotal genes and their strongly co-regulated genes. RESULTS: In this work, we obtained 396 molecular functions, and 13 of them were differentially expressed. Oxidoreductase activity showed the highest posterior value. Gene composition analysis identified 79 pivotal genes, and survival analysis indicated that these pivotal genes could be used as a strong independent predictor of poor prognosis in patients with ccRCC. Pathway analysis identified one pivotal pathway - oxidative phosphorylation. CONCLUSIONS: We identified the differentially expressed molecular functions and pivotal pathway in ccRCC using Gibbs sampling. The results could be considered as potential signatures for early detection and therapy of ccRCC.
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Algoritmos , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Cadenas de Markov , Método de Montecarlo , TranscriptomaRESUMEN
In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2) = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 µL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.
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Radioisótopos de Carbono/química , Pruebas de Enzimas/métodos , Radioisótopos de Nitrógeno/química , Ácido Úrico/química , Ácido Úrico/metabolismo , Xantina Deshidrogenasa/metabolismo , Animales , Cromatografía Liquida , Humanos , Espectrometría de Masas , Ratones , Ácido Úrico/farmacología , Xantina Deshidrogenasa/sangreRESUMEN
Macrophage Migration Inhibitory Factor (MIF) is the first human cytokine reported and was thought to have a central role in the regulation of inflammatory responses. Homologs of this molecule have been reported in bacteria, invertebrates and plants. Apart from cytokine activity, it also has two catalytic activities viz., tautomerase and di-sulfide oxidoreductase, which appear to be involved in immunological functions. The CXXC catalytic site is responsible for di-sulfide oxidoreductase activity of MIF. We have recently reported thiol-disulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti (Wba-MIF-2), although it lacks the CXXC motif. We hypothesized that three conserved cysteine residues might be involved in the formation of di-sulfide oxidoreductase catalytic site. Homology modeling of Wba-MIF-2 showed that among the three cysteine residues, Cys58 and Cys95 residues came in close proximity (3.23Å) in the tertiary structure with pKa value 9, indicating that these residues might play a role in the di-sulfide oxidoreductase catalytic activity. We carried out site directed mutagenesis of these residues (Cys58Ser & Cys95Ser) and expressed mutant proteins in Escherichia coli. The mutant proteins did not show any oxidoreductase activity in the insulin reduction assay, thus indicating that these two cysteine residues are vital for the catalytic activity of Wba-MIF-2.
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Cisteína/metabolismo , Factores Inhibidores de la Migración de Macrófagos/inmunología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Oxidorreductasas/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Wuchereria bancrofti/enzimología , Wuchereria bancrofti/inmunología , Animales , Dominio Catalítico , Humanos , Oxidación-ReducciónRESUMEN
Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/min/mg of protein, respectively (mean±SD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Xantina Deshidrogenasa/metabolismo , Animales , Marcaje Isotópico , Ratones Endogámicos ICRRESUMEN
Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a ß-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.
RESUMEN
Abrogating an unwanted immune response toward a specific antigen without compromising the entire immune system is a hoped-for goal in immunotherapy. Instead of manipulating dendritic cells and suppressive regulatory T cells, depleting effector T cells or blocking their co-stimulatory pathways, we describe a method to specifically inhibit the presentation of an antigen eliciting an unwanted immune reaction. Inclusion of an oxidoreductase motif within the flanking residues of MHC class II epitopes polarizes CD4(+) T cells to cytolytic cells capable of inducing apoptosis in antigen presenting cells (APCs) displaying cognate peptides through MHC class II molecules. This novel function results from an increased synapse formation between both cells. Moreover, these cells eliminate by apoptosis bystander CD4(+) T cells activated at the surface of the APC. We hypothesize that they would thereby block the recruitment of cells of alternative specificity for the same autoantigen or cells specific for another antigen associated with the pathology, providing a system by which response against multiple antigens linked with the same disease can be suppressed. These findings open the way toward a novel form of antigen-specific immunosuppression.
RESUMEN
Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11ß-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group.