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1.
PLoS One ; 15(4): e0231389, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267888

RESUMO

The diagnosis of implant-associated infections is hampered due to microbial adherence and biofilm formation on the implant surface. Sonication of explanted devices was shown to improve the microbiological diagnosis by physical removal of biofilms. Recently, chemical agents have been investigated for biofilm dislodgement such as the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent dithiothreitol (DTT). We compared the activity of chemical methods for biofilm dislodgement to sonication in an established in vitro model of artificial biofilm. Biofilm-producing laboratory strains of Staphylococcus epidermidis (ATCC 35984), S. aureus (ATCC 43300), E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 53278) were used. After 3 days of biofilm formation, porous glass beads were exposed to control (0.9% NaCl), sonication or chemical agents. Quantitative and qualitative biofilm analyses were performed by colony counting, isothermal microcalorimetry and scanning electron microscopy. Recovered colony counts after treatment with EDTA and DTT were similar to those after exposure to 0.9% NaCl for biofilms of S. epidermidis (6.3 and 6.1 vs. 6.0 log10 CFU/mL, S. aureus (6.4 and 6.3 vs. 6.3 log10 CFU/mL), E. coli (5.2 and 5.1 vs. 5.1 log10 CFU/mL and P. aeruginosa (5.1 and 5.2 vs. 5.0 log10 CFU/mL, respectively). In contrast, with sonication higher CFU counts were detected with all tested microorganisms (7.5, 7.3, 6.2 and 6.5 log10 CFU/mL, respectively) (p <0.05). Concordant results were observed with isothermal microcalorimetry and scanning electron microscopy. In conclusion, sonication is superior to both tested chemical methods (EDTA and DTT) for dislodgement of S. epidermidis, S. aureus, E. coli and P. aeruginosa biofilms. Future studies may evaluate potential additive effect of chemical dislodgement to sonication.


Assuntos
Bactérias/isolamento & purificação , Biofilmes/efeitos dos fármacos , Quelantes/farmacologia , Infecções Relacionadas à Prótese/diagnóstico , Substâncias Redutoras/farmacologia , Sonicação , Bactérias/efeitos dos fármacos , Carga Bacteriana/métodos , Calorimetria , Ditiotreitol/farmacologia , Ácido Edético/farmacologia , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Humanos , Microscopia Eletrônica de Varredura , Infecções Relacionadas à Prótese/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Substâncias Redutoras/química , Cloreto de Sódio/farmacologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/fisiologia
2.
Methods Mol Biol ; 1977: 83-97, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980324

RESUMO

Reduction and alkylation are common processing steps in sample preparation for qualitative and quantitative proteomic analyses. In principle, these steps mitigate the limitations resulting from the presence of disulfide bridges. There has been recurring debate in the proteomics community around their use, with concern over negative impacts that result from overalkylation (off-target, non-thiol sites) or incomplete reduction and/or S-alkylation of cysteine. This chapter integrates findings from a number of studies on different reduction and alkylation strategies, to guide users in experimental design for their optimal use in proteomic workflows.


Assuntos
Cisteína/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteômica , Alquilantes/farmacologia , Alquilação/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Proteômica/métodos , Substâncias Redutoras/farmacologia , Fluxo de Trabalho
3.
Int J Biol Macromol ; 122: 758-769, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389529

RESUMO

The current paper reports the purification and biochemical characterization of two extracellular keratinolytic enzymes, with moderate elastolytic activity, from Bacillus amyloliquefaciens strain S13 newly isolated from the brown alga Zonaria tournefortii. The enzymes were purified to homogeneity by precipitation with (NH4)2SO4-dialysis, followed by size exclusion HPLC column, and submitted to biochemical characterization assays. The findings revealed that the pure enzymes designated KERZT-A and B were monomers with molecular masses of 28 and 47 kDa, respectively. Their identified NH2-terminal amino acid displayed high homologies with those of Bacillus keratinases. While KERZT-A was optimally active at pH 6.5 and 50 °C, KERZT-B showed optimum activity at pH 8 and 60 °C. Both enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests their belonging to the serine keratinases family. Interestingly, KERZT-A displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than KERUS from Brevibacillus brevis strain US575, NUE 12 MG (commercial enzyme), and KERZT-B unhairing keratinases. Above all, the findings indicated that KERZT-A and B enzymes seems to be an effective and an eco-friendly alternative to the conventional chemicals used for the feather keratin-biodegradation and for the unhairing of hides or skins in the leather processing industry.


Assuntos
Bacillus amyloliquefaciens/enzimologia , Queratinas/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Estramenópilas/microbiologia , Animais , Bacillus amyloliquefaciens/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Plumas , Concentração de Íons de Hidrogênio , Hidrólise , Indústrias , Metais/farmacologia , Peso Molecular , Peptídeo Hidrolases/química , Filogenia , Substâncias Redutoras/farmacologia , Especificidade por Substrato , Temperatura
4.
Chemosphere ; 215: 815-826, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30359951

RESUMO

In order to understand the spatial distribution characteristics, loss risk and leaching behaviors of phosphorous from exposed rocks in typical small-area of Chaohu watershed, phosphorus-rich rock was selected for conducting a series of column leaching experiments to investigate the phosphorus leaching. By simulating the intermittent cycle of acid rainfall, effects of oxalic acid on the weathering of phosphate rocks were studied. Total phosphorus contents, pH and phosphate leaching capacity from phosphorus rocks were tested in the presence of oxalic acid at different dry-wet intervals. The results indicated that the cumulative release of phosphorus increased first and then decreased with the duration of dry-wet intervals increasing. Four typical kinetic equations can describe phosphorus release from phosphate rocks with the action of oxalic acid. The best fitting models were the weight function and parabolic equation, with a mean correlation coefficient R2 of 0.9727 and 0.9941, respectively, which reached significance level. Total phosphorus (TP) leaching distribution in each column showed a tendency of gradually decreasing from top to bottom except for time interval of 5 d and 7 d. Occluded-bound P (Oc-P) is the dominant form in rocks. The change point value of rocks phosphorus is 4.11  mg kg-1 after intermittent leaching, and the phosphorus loss risk is relatively large in some rocks formations.


Assuntos
Sedimentos Geológicos/análise , Ácido Oxálico/farmacologia , Fosfatos/química , Fósforo/análise , Chuva , Movimentos da Água , China , Monitoramento Ambiental , Fósforo/química , Substâncias Redutoras/farmacologia
5.
J Biol Chem ; 294(5): 1516-1528, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30514757

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of 14C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of 14C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O2 When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a "priming reduction" reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (K mR app). Theoretical analyses revealed that K mR app decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O2, H2O2, and polysaccharide concentrations.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxigenases de Função Mista/metabolismo , Polissacarídeos Bacterianos/metabolismo , Substâncias Redutoras/farmacologia , Cinética , Oxidantes/farmacologia , Oxirredução , Especificidade por Substrato
6.
Nanotechnology ; 29(47): 475604, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30191889

RESUMO

A therapeutic reduced graphene oxide (RGO) is synthesized by using fucoidan (Fu) as the reducing and surface functionalizing agent. The synthesized Fu-RGO exhibits promising characteristics for therapeutic applications such as high dispersity in aqueous media, biocompatibility, selective cytotoxicity to cancer cells, high loading capacity of the anticancer drug, and photothermal conversion effect. Therefore, Fu-GO is successfully harnessed as a combinatorial cancer treatment platform through bio-functional (Fu), chemo (doxorubicin (Dox)) and photothermal (RGO with near-infrared irradiation) modalities.


Assuntos
Antineoplásicos/farmacologia , Portadores de Fármacos/farmacologia , Grafite/farmacologia , Neoplasias/terapia , Polissacarídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/química , Terapia Combinada/métodos , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Grafite/química , Células HEK293 , Células HeLa , Humanos , Hipertermia Induzida/métodos , Raios Infravermelhos , Oxirredução , Óxidos/química , Óxidos/farmacologia , Polissacarídeos/química , Substâncias Redutoras/química , Substâncias Redutoras/farmacologia
7.
Biomed Res Int ; 2018: 5704016, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30175134

RESUMO

Objective: To quantitatively assess the influence of reducing agents on biological macromolecules and on the possible repair of oxidative damage. Methods: Samples (antibody, enzyme, DNA, and diluted serum) were treated with reducing agents (ammonium ferrous sulfate, ascorbic acid, potassium iodide, and sodium hyposulfite) in the experimental group and with NaCl in the control group. Enzyme-linked immunosorbent assay and quantitative PCR were used to determine the activity of antibody, enzyme, and DNA. Native gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine protein structure. Reducing agents that had no inhibitory effect on biological macromolecules were selected. Antibodies were treated with oxidants to caused oxidative damage and then treated with reducing agents, and the possible repair of oxidative damage was assessed. Results: Certain concentrations of ammonium ferrous sulfate resulted in significant inhibition of antibody, enzyme, DNA, and diluted serum. Certain concentrations of ascorbic acid resulted in significant inhibition of antibody. Sodium hyposulfite and potassium iodide had no effect on antibody, enzyme, DNA, and diluted serum. The OD values in group A (in which HBsAb was treated by oxidation and then a reductant) were significantly higher than those in group B (HBsAb treated by oxidation). Conclusion: Ammonium ferrous sulfate, ascorbic acid, sodium hyposulfite, and potassium iodide had different effects on antibody, enzyme, DNA, and diluted serum. The reduction in antibody activity due to an oxidant was partially repaired by a reductant.


Assuntos
Estresse Oxidativo , Substâncias Redutoras/farmacologia , Anticorpos/efeitos dos fármacos , DNA/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Enzimas/efeitos dos fármacos , Oxidantes , Oxirredução
8.
Sci Rep ; 8(1): 12397, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120279

RESUMO

Silver nanoparticles (AgNPs) are known mainly because of their bactericidal properties. Among the different types of synthesis, there is the biogenic synthesis, which allows the synergy between the nanocomposites and substances from the organism employed for the synthesis. This study describes the synthesis of AgNPs using infusion of roots (AgNpR) and extract (AgNpE) of the plant Althaea officinalis. After the synthesis through reduction of silver nitrate with compounds of A. officinalis, physico-chemical analyzes were performed by UV-Vis spectroscopy, nanoparticles tracking analysis (NTA), dynamic light scattering (DLS) and scanning electron microscopy (SEM). Toxicity was evaluated through Allium cepa assay, comet test with cell lines, cell viability by mitochondrial activity and image cytometry and minimal inhibitory concentration on pathogenic microorganisms. Biochemical analyzes (CAT - catalase, GPx - glutathione peroxidase e GST - glutationa S-transferase) and genotoxicity evaluation in vivo on Zebrafish were also performed. AgNpE and AgNpR showed size of 157 ± 11 nm and 293 ± 12 nm, polydispersity of 0.47 ± 0.08 and 0.25 ± 0.01, and zeta potential of 20.4 ± 1.4 and 26.5 ± 1.2 mV, respectively. With regard to toxicity, the AgNpE were the most toxic when compared with AgNpR. Biochemical analyzes on fish showed increase of CAT activity in most of the organs, whereas GPx showed few changes and the activity of GST decreased. Also regarding to bactericidal activity, both nanoparticles were effective, however AgNpR showed greater activity. Althaea officinalis can be employed as reducing agent for the synthesis of silver nanoparticles, although it is necessary to consider its potential toxicity and ecotoxicity.


Assuntos
Althaea/química , Nanopartículas Metálicas , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Substâncias Redutoras/química , Substâncias Redutoras/farmacologia , Prata , Animais , Anti-Infecciosos , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Dano ao DNA/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/química , Camundongos , Testes de Sensibilidade Microbiana , Extratos Vegetais/toxicidade , Substâncias Redutoras/toxicidade , Prata/química , Toxicologia/métodos , Peixe-Zebra
9.
IET Nanobiotechnol ; 12(6): 850-856, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30104462

RESUMO

The present work is emphasised on the bio-fabrication of silver and gold nanoparticles in a single step by a microwave-assisted method using the leaf extract of Synedrella nodiflora as both reducing and stabilising agent. The synthesised nanoparticles are highly stable and show surface plasmon resonance peak at 413 and 535 nm, respectively, for silver and gold nanoparticles in UV-Vis spectrum. The functional group responsible for the reduction of metal ions were obtained from Fourier transform infrared spectroscopy. The crystalline nature of nanoparticles with face-centred cubic geometry was confirmed by the X-ray diffraction and selected area electron diffraction patterns. The morphology and sizes of the silver and gold nanoparticles were obtained from transmission electron microscopy images. The nanoparticles exhibit effective antimicrobial activities against various pathogenic strains. These antimicrobial properties were analysed by employing agar well diffusion method. The nanoparticles show significant antioxidant properties, and it was determined using 2, 2-diphenyl-1-picrylhydrazyl assay. The nanoparticles also show potent catalytic activity in the degradation of anthropogenic pollutant dyes Congo red and eosin Y by excess NaBH4. Thus, the current study demonstrates the potential use of S. nodiflora as a reducing and stabilising agent for the synthesis of silver and gold nanoparticles and their relevance in the field of biomedicine and catalysis.


Assuntos
Anti-Infecciosos/síntese química , Antioxidantes/síntese química , Ouro/química , Química Verde/métodos , Nanopartículas Metálicas/química , Prata/química , Anti-Infecciosos/química , Antioxidantes/química , Asteraceae/química , Catálise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Substâncias Redutoras/química , Substâncias Redutoras/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
10.
Target Oncol ; 13(3): 363-370, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29644577

RESUMO

BACKGROUND: The impact of dose and simultaneous use of acid-reducing agents (ARAs) on the effectiveness of vemurafenib is unknown. OBJECTIVES: To determine the association between progression of metastatic BRAF V600 mutated melanoma and (1) dose reductions of vemurafenib and (2) simultaneous use of vemurafenib and ARAs. PATIENT AND METHODS: A retrospective cohort study of 112 first-line vemurafenib users for melanoma was conducted (March 2012-March 2016), using electronic patient records and pharmacy dispensing records of a Dutch academic hospital. Cox regression analysis was used to estimate the risk of progression with full-dose (n = 64) versus reduced-dose vemurafenib (n = 48) and with simultaneous use of vemurafenib and ARAs (n = 35) versus vemurafenib alone (n = 77). Analyses were adjusted for age and sex. RESULTS: In total, disease progression occurred in 55% of treated patients on vemurafenib, with a median progression-free survival of 6.0 (95% confidence interval [CI] 5.0-6.9) months. Compared to patients on vemurafenib alone, there was no increased risk of progression among patients requiring vemurafenib at a reduced dose or among patients receiving simultaneous therapy with vemurafenib and ARAs. In addition, there was no increased risk of progression among patients who used reduced-dose vemurafenib and ARAs versus those receiving full-dose vemurafenib as sole therapy. However, a tendency for progression was observed among patients who used full-dose vemurafenib and ARAs versus full-dose vemurafenib alone (adjusted hazard ratio [HRa] 2.37; 95% CI 0.97-5.76), which became statistically significant in a sensitivity analysis (HRa 4.56; 95% CI 1.51-13.75). CONCLUSIONS: There was no association between the use of vemurafenib in a reduced dose or the simultaneous use of vemurafenib and ARAs and the risk of progression. In addition, there was no association between the simultaneous use of vemurafenib in a reduced dose and ARAs and the risk of progression. However, patients tolerating  full-dose vemurafenib simultaneously with ARAs might have an increased risk of progression. This finding requires prospective validation.


Assuntos
Melanoma/tratamento farmacológico , Substâncias Redutoras/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Mutação , Substâncias Redutoras/farmacologia , Estudos Retrospectivos , Neoplasias Cutâneas/patologia , Vemurafenib
11.
Chemosphere ; 202: 322-329, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29574385

RESUMO

Removal/destruction of aqueous-phase octachlorodibenzo-p-dioxin (OCDD) and octachlorodibenzofuran (OCDF) via hydrodechlorination process (HDC) is experimentally evaluated over palladium/activated carbon (Pd/AC) catalyst. Pd catalyst is mainly used as active component for effectiveness in removing dioxin from wastewater. Studies on the removal of PCDD/Fs accomplished with HDC reaction in aqueous phase are limited and the influencing factors have not been clarified. In this study, high-concentration OCDD/F are selected as targets, and the effects of solvent and operating temperature on dechlorination efficiency are investigated via experimental tests. The results indicate that the highest hydrodechlorination efficiency is achieved with isopropanol as solvent. The OCDD/F removal efficiency achieved with the solution of 80% isopropanol is higher than that of 50% isopropanol, whereas the destruction efficiency of OCDD/F reveals the opposite trend. Generally, the removal and destruction efficiencies of PCDFs are higher than those of PCDDs. In addition, the activation energies of OCDD and OCDF are calculated with the Arrhenius equation as 24.8 and 23.1 kJ/mol, respectively. Stability tests are conducted with three cycles. Overall, the results indicate that a high performance (≥99%) can be achieved by combining hydrodechlorination with Pd/AC at a temperature range of 303-353 K, demonstrating that Pd/AC has good potential for removing PCDD/Fs from wastewater.


Assuntos
Hidrocarbonetos Clorados/química , Incineração/métodos , Dibenzodioxinas Policloradas/química , Substâncias Redutoras/farmacologia , Catálise , Halogenação , Dibenzodioxinas Policloradas/isolamento & purificação
12.
J Proteome Res ; 17(4): 1636-1646, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29498529

RESUMO

The expansion of biomedical and therapeutic applications of silver nanoparticles (AgNPs) raises the need to further understand their biological effects on human cells. In this work, NMR metabolomics has been applied to reveal the metabolic effects of AgNPs toward human hepatoma (HepG2) cells, which are relevant with respect to nanoparticle accumulation and detoxification. Cellular responses to widely disseminated citrate-coated AgNPs (Cit30) and to emergent biogenic AgNPs prepared using an aqueous plant extract as reducing and stabilizing agent (GS30) have been compared with a view to assess the influence of nanoparticle coating on the metabolic effects produced. Subtoxic concentrations (IC5 and IC20) of both nanoparticle types caused profound changes in the cellular metabolome, suggesting adaptations in energy production processes (glucose metabolism and the phosphocreatine system), antioxidant defenses, protein degradation and lipid metabolism. These signatures were proposed to reflect mainly metabolism-mediated protective mechanisms and were found to be largely common to Cit30 and GS30 AgNPs, although differences in the magnitude of response, not captured by conventional cytotoxicity assessment, were detected. Overall, this study highlights the value of NMR metabolomics for revealing subtoxic biological effects and helping to understand cell-nanomaterial interactions.


Assuntos
Fígado/metabolismo , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Ácido Cítrico/farmacologia , Excipientes/farmacologia , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Substâncias Redutoras/farmacologia
13.
Int J Biol Macromol ; 113: 565-574, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29501840

RESUMO

Extracellular alkaline protease producing salt tolerant alkaliphilic actinobacteria, Nocardiopsis xinjiangensis strain OM-6 was isolated from Okha Madi (OM) site of coastal Gujarat, India. The purified protease was stable even at 70°C in 100mM Ca2+ with Kd=20×10-3 and t1/2=34min. The activation energies (E), enthalpy (∆H*) and entropy (∆S*) for protease deactivation were 29.35kJ/mol, 26.68kJ/mol and -186.22J/mol, respectively in 200mM Ca2+. The ∆G* for protease deactivation was 97.63kJ/mol at 50°C in 100mM Ca2+. OM-6 protease exhibited enhanced residual activities up to 103%, 70%, 144% and 119% with SDS, CTAB, Tween 80 and Triton X-100, respectively after 2h of incubation at 40°C. Interestingly, residual activity of OM-6 protease increased by 450% and 559% in 50mM H2O2 and 10mM ß-mercaptoethanol respectively even after 2h of incubation. Moreover, protease retained 100% of its original activity with H2O2 and ß-mercaptoethanol at highest concentration after 24h. The protease retained more than 60% of original activity with 1% w/v of each commercial detergent even after 2h at 40°C. These unique properties of protease make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Detergentes/farmacologia , Endopeptidases/química , Endopeptidases/metabolismo , Actinobacteria/efeitos dos fármacos , Actinobacteria/crescimento & desenvolvimento , Estabilidade Enzimática/efeitos dos fármacos , Espaço Extracelular/enzimologia , Gelatina/farmacologia , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Oxirredução , Substâncias Redutoras/farmacologia , Sais/farmacologia , Tensoativos/farmacologia , Termodinâmica
14.
J Biol Chem ; 293(10): 3593-3606, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29352103

RESUMO

Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent lipid rafts when platelets are activated. Using metabolic labeling and hydroxylamine (HA)/HCl treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on cysteine 275, 279, 280, 282, 283, and 285, and SNAP-23 was modified on cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [3H]palmitate into the t-SNAREs increased although the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [3H]palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release, as well as P-selectin exposure in platelets treated with the acyltransferase inhibitor cerulenin or the thioesterase inhibitor palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose- and time-dependent manner whereas palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin, suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.


Assuntos
Plaquetas/metabolismo , Cisteína/metabolismo , Exocitose , Processamento de Proteína Pós-Traducional , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Acilação/efeitos dos fármacos , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Cisteína/química , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Humanos , Hidroxilamina/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Oxirredução , Selectina-P/metabolismo , Ácido Palmítico/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Qa-SNARE/química , Proteínas Qb-SNARE/química , Proteínas Qc-SNARE/química , Substâncias Redutoras/farmacologia , Propriedades de Superfície/efeitos dos fármacos , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismo , Trítio
15.
Int J Biol Macromol ; 108: 1176-1184, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28919530

RESUMO

Bacillus nealsonii PN-11 produces thermo-alkalistable mannanase and protease active in wide temperature and pH range. Optimization of coproduction of protease and mannanase from this strain and application of cocktail of these enzymes as detergent additives were studied. On optimization mannanase yield of 834Ug-1 (11.12 fold increase) and protease yield of 70Ug-1 (4.7 fold increase) could be obtained in a single fermentation. Purification and characterization of mannanase have been done earlier and protease was done during this study and has a molecular mass of 48kDa. pH and temperature optima for protease were 10.0 and 65°C respectively. It was completely stable at 60°C for 3h and retained >80% of activity at pH 11.0 for 1h. Both the enzymes were compatible with detergents individually and in a combination. The wash performance of the detergent on different type of stains improved when protease or mannanase were used individually. However destaining was more efficient when a combination of mannanase and protease was used.


Assuntos
Bacillus/metabolismo , Biotecnologia/métodos , Detergentes/química , Fermentação , Peptídeo Hidrolases/biossíntese , beta-Manosidase/biossíntese , Quelantes/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Oxidantes/farmacologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Substâncias Redutoras/farmacologia , Tensoativos/farmacologia , beta-Manosidase/química , beta-Manosidase/metabolismo
16.
Sci Rep ; 7(1): 12775, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28986540

RESUMO

The irritant receptor TRPA1 was suggested to mediate analgesic, antipyretic but also pro-inflammatory effects of the non-opioid analgesic acetaminophen, presumably due to channel activation by the reactive metabolites parabenzoquinone (pBQ) and N-acetyl-parabenzoquinonimine (NAPQI). Here we explored the effects of these metabolites on the capsaicin receptor TRPV1, another redox-sensitive ion channel expressed in sensory neurons. Both pBQ and NAPQI, but not acetaminophen irreversibly activated and sensitized recombinant human and rodent TRPV1 channels expressed in HEK 293 cells. The reducing agents dithiothreitol and N-acetylcysteine abolished these effects when co-applied with the metabolites, and both pBQ and NAPQI failed to gate TRPV1 following substitution of the intracellular cysteines 158, 391 and 767. NAPQI evoked a TRPV1-dependent increase in intracellular calcium and a potentiation of heat-evoked currents in mouse spinal sensory neurons. Although TRPV1 is expressed in mouse hepatocytes, inhibition of TRPV1 did not alleviate acetaminophen-induced hepatotoxicity. Finally, intracutaneously applied NAPQI evoked burning pain and neurogenic inflammation in human volunteers. Our data demonstrate that pBQ and NAQPI activate and sensitize TRPV1 by interacting with intracellular cysteines. While TRPV1 does not seem to mediate acetaminophen-induced hepatotoxicity, our data identify TRPV1 as a target of acetaminophen with a potential relevance for acetaminophen-induced analgesia, antipyresia and inflammation.


Assuntos
Acetaminofen/metabolismo , Capsaicina/farmacologia , Metaboloma , Canais de Cátion TRPV/metabolismo , Animais , Benzoquinonas/farmacologia , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Cisteína/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Iminas/farmacologia , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Dor/fisiopatologia , Fosforilação/efeitos dos fármacos , Substâncias Redutoras/farmacologia , Reflexo/efeitos dos fármacos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Pele/patologia , Canais de Cátion TRPV/agonistas
17.
FEBS J ; 284(24): 4314-4327, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29076625

RESUMO

Heme d1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd1 nitrite reductase of many denitrifying bacteria. In the course of heme d1 biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18-didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d1 molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S-adenosyl-l-methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron-sulfur clusters. While the N-terminal [4Fe-4S] cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18-didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.


Assuntos
Proteínas de Bactérias/metabolismo , Heme/análogos & derivados , Proteínas com Ferro-Enxofre/metabolismo , Nitrato Redutase/metabolismo , Propionatos/metabolismo , Rhodobacteraceae/enzimologia , S-Adenosilmetionina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Catálise , Cromatografia Líquida de Alta Pressão , Ditionita/farmacologia , Heme/biossíntese , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/isolamento & purificação , Modelos Químicos , Estrutura Molecular , Mutagênese Sítio-Dirigida , Nitrato Redutase/genética , Nitrato Redutase/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Substâncias Redutoras/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato , Tetrapirróis/metabolismo
18.
J Biol Chem ; 292(45): 18469-18485, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-28939771

RESUMO

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of the expansion segment 7 (ES7), a putative regulatory region located on the surface of the 60S ribosomal subunit. Our data also show that ES7c is cleaved at early stages of the gene expression program that enables cells to successfully counteract oxidative stress and is not a prerequisite or consequence of apoptosis. Moreover, the 60S subunits containing ES7c-cleaved rRNA cofractionate with intact subunits in sucrose gradients and repopulate polysomes after a short starvation-induced translational block, indicating their active role in translation. These results demonstrate that ES7c cleavage in rRNA is an early and sensitive marker of increased ROS levels in yeast cells and suggest that changes in ribosomes may be involved in the adaptive response to oxidative stress.


Assuntos
Regulação Fúngica da Expressão Gênica , Estresse Oxidativo , Polirribossomos/enzimologia , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/enzimologia , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hormese , Cinética , Conformação de Ácido Nucleico , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidases/genética , Peroxidases/metabolismo , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Clivagem do RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Fúngico/química , RNA Ribossômico/química , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Substâncias Redutoras/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esferoplastos/efeitos dos fármacos , Esferoplastos/enzimologia , Esferoplastos/crescimento & desenvolvimento , Esferoplastos/fisiologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
19.
J Virol Methods ; 248: 1-6, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28532602

RESUMO

Research involving biosafety level 3 pathogens such as West Nile virus (WNV) is often limited by the limited space and technical constraints of these environments. To conduct complex analytical studies outside of high containment, robust and reliable inactivation methods are needed that maintain compatibility with downstream assays. Here we report the inactivation of WNV in spiked serum samples using a commercially available SDS-PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification proteins differentially expressed in the serum of mice experimentally infected with WNV.


Assuntos
Proteínas Sanguíneas/metabolismo , Detergentes/farmacologia , Temperatura Alta , Proteômica/métodos , Substâncias Redutoras/farmacologia , Soro/virologia , Inativação de Vírus , Vírus do Nilo Ocidental/fisiologia , Animais , Tampões (Química) , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Camundongos , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Ensaio de Placa Viral , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/efeitos dos fármacos
20.
Biochem Biophys Res Commun ; 482(3): 419-425, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28212725

RESUMO

Disruption of redox homeostasis is a key phenotype of many pathological conditions. Though multiple oxidizing compounds such as hydrogen peroxide are widely recognized as mediators and inducers of oxidative stress, increasingly, attention is focused on the role of lipid hydroperoxides as critical mediators of death and disease. As the main component of cellular membranes, lipids have an indispensible role in maintaining the structural integrity of cells. Excessive oxidation of lipids alters the physical properties of cellular membranes and can cause covalent modification of proteins and nucleic acids. This review discusses the synthesis, toxicity, degradation, and detection of lipid peroxides in biological systems. Additionally, the role of lipid peroxidation is highlighted in cell death and disease, and strategies to control the accumulation of lipid peroxides are discussed.


Assuntos
Morte Celular/fisiologia , Peroxidação de Lipídeos/fisiologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Peróxidos Lipídicos/metabolismo , Peróxidos Lipídicos/toxicidade , Inibidores de Lipoxigenase/farmacologia , Redes e Vias Metabólicas , Oxirredução , Substâncias Redutoras/farmacologia
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