RESUMO
BACKGROUND AND OBJECTIVE: In vitro glucuronidation of 17ß-estradiol (estradiol) is often performed to assess the role of uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) in xenobiotic/drug metabolism. The objective of this study was to determine the effects of four commonly used organic solvents [i.e., dimethyl sulfoxide (DMSO), methanol, ethanol, and acetonitrile] on the glucuronidation kinetics of estradiol, which can be glucuronidated at C3 and C17 positions. METHODS: The impacts of organic solvents on estradiol glucuronidation were determined by using expressed UGT enzymes and liver microsomes from both human and animals. RESULTS: In human liver microsomes (HLM), methanol, ethanol, and acetonitrile significantly altered estradiol glucuronidation kinetics with increased Vmax (up to 2.6-fold) and CLmax (up to 2.8-fold) values. Altered estradiol glucuronidation in HLM was deduced to be attributed to the enhanced metabolic activities of UGT1A1 and UGT2B7, whose activities differ at the two glucuronidation positions. The effects of organic solvents on estradiol glucuronidation were glucuronidation position-, isozyme-, and solvent-specific. Furthermore, both ethanol and acetonitrile have a greater tendency to modify the glucuronidation activity of estradiol in animal liver microsomes. CONCLUSION: Organic solvents such as methanol, ethanol, and acetonitrile showed great potential in adjusting the glucuronidation of estradiol. DMSO is the most suitable solvent due to its minimal influence on estradiol glucuronidation. Researchers should be cautious in selecting appropriate solvents to get accurate results when assessing the metabolism of a new chemical entity.
Assuntos
Dimetil Sulfóxido , Estradiol , Etanol , Glucuronídeos , Glucuronosiltransferase , Microssomos Hepáticos , Solventes , Microssomos Hepáticos/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Estradiol/metabolismo , Estradiol/farmacologia , Glucuronosiltransferase/metabolismo , Humanos , Solventes/farmacologia , Animais , Cinética , Etanol/metabolismo , Etanol/farmacologia , Glucuronídeos/metabolismo , Dimetil Sulfóxido/farmacologia , Metanol/farmacologia , Metanol/metabolismo , Acetonitrilas/farmacologia , Acetonitrilas/metabolismoRESUMO
Hydroxynitrile lyase (HNL) from the cyanogenic millipede Oxidus gracillis (OgraHNL) is a crucial enzyme in the cyanogenesis pathway. Here, the crystal structures of OgraHNL complexed with sulfate, benzaldehyde (BA), (R)-mandelonitrile ((R)-Man), (R)-2-chloromandelonitrile ((R)-2-Cl-Man), and acetone cyanohydrin (ACN) were solved at 1.6, 1.7, 2.3, 2.1, and 2.0â Å resolutions, respectively. The structure of OgraHNL revealed that it belonged to the lipocalin superfamily. Based on this structure, positive variants were designed to further improve the catalytic activity and enantioselectivity of the enzyme for asymmetric hydrocyanation and Henry reactions.
Assuntos
Aldeído Liases , Mutagênese Sítio-Dirigida , Aldeído Liases/metabolismo , Aldeído Liases/química , Aldeído Liases/genética , Animais , Benzaldeídos/metabolismo , Benzaldeídos/química , Acetonitrilas/química , Acetonitrilas/metabolismo , Modelos Moleculares , Cristalografia por Raios X , Nitrilas/metabolismo , Nitrilas/química , EstereoisomerismoRESUMO
Many animals engage in cannibalism to supplement their diets. Among dense populations of migratory locusts, cannibalism is prevalent. We show that under crowded conditions, locusts produce an anticannibalistic pheromone called phenylacetonitrile. Both the degree of cannibalism and the production of phenylacetonitrile are density dependent and covary. We identified the olfactory receptor that detects phenylacetonitrile and used genome editing to make this receptor nonfunctional, thereby abolishing the negative behavioral response. We also inactivated the gene underlying phenylacetonitrile production and show that locusts that lack this compound lose its protection and are more frequently exposed to intraspecific predation. Thus, we reveal an anticannibalistic feature built on a specifically produced odor. The system is very likely to be of major importance in locust population ecology, and our results might therefore provide opportunities in locust management.
Assuntos
Acetonitrilas , Canibalismo , Aglomeração , Gafanhotos , Feromônios , Animais , Acetonitrilas/metabolismo , Gafanhotos/genética , Gafanhotos/metabolismo , Feromônios/genética , Feromônios/metabolismoRESUMO
We evaluated the effects of a new extract (70% acetonitrile, 2E0217022196DIPFARMTDA) of Lens culinaris Medik (Terre di Altamura SRL, Altamura BA) to prevent cytotoxic damage from cisplatin, staurosporine, irinotecan, doxorubicin, and the glucocorticoid dexamethasone. The acetonitrile-water extract (range 0.1-5 mg/mL) was obtained by extracting 10 g of lentil flour with 50 milliliters of the acetonitrile-water extraction mixture in a 70:30 ratio, first for 3 h and then overnight in a shaker at room temperature. The next day, the extract was filtered and passed through a Rotavapor to obtain only the aqueous component and eliminate that with acetonitrile, and then freeze-dried to finally have the powdered extract. In vitro experiments showed that the extract prevented the cytotoxic damage induced by cisplatin, irinotecan, and doxorubicin on HEK293 and SHSY5Y cell lines after 24-96 h. In murine osteoblasts after 24-72 h of incubation time, the extract was cytoprotective against all chemicals. The extract was effective against dexamethasone, leading to synergic cell proliferation in all cell types. In bone marrow cells, the extract is cytoprotective after 72 h against doxorubicin, staurosporine, and dexamethasone. Instead, on muscle fibers, the extract has a synergic effect with chemotherapeutics, increasing cytotoxicity induced by doxorubicin and staurosporine. LC-MS attested to the existence of several phenolic structures in the extract. The most abundant families of compounds were flavonoids (25.7%) and mellitic acid (18%). Thus, the development of this extract could be implemented in the area of research related to the chemoprevention of damage to renal, neuronal, bone marrow cells, and osteoblasts by chemotherapeutics; moreover, it could be used as a reinforcer of cytotoxic action of chemotherapeutics on muscle fibers.
Assuntos
Lens (Planta) , Humanos , Animais , Camundongos , Lens (Planta)/química , Lens (Planta)/metabolismo , Água/metabolismo , Irinotecano , Cisplatino/metabolismo , Células HEK293 , Estaurosporina , Espectrometria de Massas , Acetonitrilas/metabolismo , Doxorrubicina , DexametasonaRESUMO
The evaluation of joint disease using synovial fluid is an emerging field of metabolic profiling. The analysis is challenged by multiple macromolecules which can obscure the small molecule chemistry. The use of protein precipitation and extraction has been evaluated previously, but not in synovial fluid. We systematically review the published NMR spectroscopy methods of synovial fluid analysis and investigated the efficacy of three different protein precipitation techniques: methanol, acetonitrile and trichloroacetic acid. The trichloroacetic wash removed the most protein. However, metabolite recoveries were universally very poor. Acetonitrile liquid/liquid extraction gave metabolite gains from four unknown compounds with spectral peaks at δ = 1.91 ppm, 3.64 ppm, 3.95 ppm & 4.05 ppm. The metabolite recoveries for acetonitrile were between 1.5 and 7 times higher than the methanol method, across all classes of metabolite. The methanol method was more effective in removing protein as reported by the free GAG undefined peak (44 % vs 125 %). However, qualitative evaluation showed that acetonitrile and methanol provided good restoration of the spectra to baseline. The methanol extraction has issues of a gelatinous substrate in the samples. All metabolite recoveries had a CV of > 15 %. A recommendation of acetonitrile liquid/liquid extraction was made for human synovial fluid (HSF) analysis. This is due to consistency, effective protein precipitation, recovery of metabolites and additional compounds not previously visible.
Assuntos
Metanol , Líquido Sinovial , Humanos , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Metanol/química , Espectroscopia de Ressonância Magnética/métodos , Extração Líquido-Líquido , Acetonitrilas/metabolismoRESUMO
A new method to trap catalytic intermediate species was employed with Fe-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase). ReNHase was incubated with substrates in a 23% (w/w) NaCl/H2O eutectic system that remained liquid at -20 °C, thereby permitting the observation of transient species that were present at electron paramagnetic resonance (EPR)-detectable levels in samples frozen while in the steady state. FeIII-EPR signals from the resting enzyme were unaffected by the presence of 23% NaCl, and the catalytic activity was â¼55% that in the absence of NaCl at the optimum pH of 7.5. The reaction of ReNHase in the eutectic system at -20 °C with the substrates acetonitrile or benzonitrile induced significant changes in the EPR spectra. A previously unobserved signal with highly rhombic g-values (g1 = 2.31) was observed during the steady state but did not persist beyond the exhaustion of the substrate, indicating that it arises from a catalytically competent intermediate. Distinct signals due to product complexes provide a detailed mechanism for product release, the rate-limiting step of the reaction. Assignment of the observed EPR signals was facilitated by density functional theory calculations, which provided candidate structures and g-values for various proposed ReNHase intermediates. Collectively, these results provide new insights into the catalytic mechanism of NHase and offer a new approach for isolating and characterizing EPR-active intermediates in metalloenzymes.
Assuntos
Acetonitrilas/química , Proteínas de Bactérias/química , Hidroliases/química , Ferro/química , Nitrilas/química , Rhodococcus equi/química , Acetonitrilas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Temperatura Baixa , Solventes Eutéticos Profundos/química , Teoria da Densidade Funcional , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hidroliases/genética , Hidroliases/metabolismo , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Cinética , Nitrilas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus equi/enzimologia , Cloreto de Sódio/química , Especificidade por Substrato , Água/químicaRESUMO
Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into cyanide and the corresponding aldehyde or ketone. Moreover, they catalyze the synthesis of cyanohydrin in the reverse reaction, utilized in industry for preparation of enantiomeric pure pharmaceutical ingredients and fine chemicals. We discovered a new HNL from the cyanogenic millipede, Chamberlinius hualienensis. The enzyme displays several features including a new primary structure, high stability, and the highest specific activity in (R)-mandelonitrile ((R)-MAN) synthesis (7420 U·mg-1 ) among the reported HNLs. In this study, we elucidated the crystal structure and reaction mechanism of natural ChuaHNL in ligand-free form and its complexes with acetate, cyanide ion, and inhibitors (thiocyanate or iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55 Å resolutions, respectively. The structure of ChuaHNL revealed that it belongs to the lipocalin superfamily, despite low amino acid sequence identity. The docking model of (R)-MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen bonds with R38 and K117, and the nitrile group forms hydrogen bonds with R38 and Y103. The mutational analysis showed the importance of these residues in the enzymatic reaction. From these results, we propose that K117 acts as a base to abstract a proton from the hydroxyl group of cyanohydrins and R38 acts as an acid to donate a proton to the cyanide ion during the cleavage reaction of cyanohydrins. The reverse mechanism would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida) DATABASES: Structural data are available in PDB database under the accession numbers 6JHC, 6KFA, 6KFB, 6KFC, and 6KFD.
Assuntos
Acetonitrilas/química , Aldeído Liases/química , Proteínas de Artrópodes/química , Artrópodes/química , Lipocalinas/química , Acetonitrilas/metabolismo , Aldeído Liases/genética , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Artrópodes/enzimologia , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ácido Iodoacético/química , Ácido Iodoacético/metabolismo , Cinética , Lipocalinas/genética , Lipocalinas/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tiocianatos/química , Tiocianatos/metabolismoRESUMO
Arylacetonitrilase from Alcaligenes faecalis ATCC8750 (NitAF) hydrolyzes various arylacetonitriles to the corresponding carboxylic acids. A systematic strategy of amino acid residue screening through sequence alignment, followed by homology modeling and biochemical confirmation was employed to elucidate the determinant of NitAF catalytic efficiency. Substituting Phe-140 in NitAF (wild-type) to Trp did not change the catalytic efficiency toward phenylacetonitrile, an arylacetonitrile. The mutants with nonpolar aliphatic amino acids (Ala, Gly, Leu, or Val) at location 140 had lower activity, and those with charged amino acids (Asp, Glu, or Arg) exhibited nearly no activity for phenylacetonitrile. Molecular modeling showed that the hydrophobic benzene ring at position 140 supports a mechanism in which the thiol group of Cys-163 carries out a nucleophilic attack on a cyanocarbon of the substrate. Characterization of the role of the Phe-140 residue demonstrated the molecular determinant for the efficient formation of arylcarboxylic acids.
Assuntos
Alcaligenes faecalis/enzimologia , Aminoidrolases/química , Aminoidrolases/metabolismo , Fenilalanina/metabolismo , Acetonitrilas/metabolismo , Alcaligenes faecalis/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoidrolases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutação , Conformação ProteicaRESUMO
Identification of MHC class I-bound peptides by immunopurification of MHC complexes and subsequent analysis by mass spectrometry is crucial for understanding T cell immunology and immunotherapy. Investigation of the steps for the MHC ligand isolation process revealed biases in widely used isolation techniques toward peptides of lower hydrophobicity. As MHC ligand hydrophobicity correlates positively with immunogenicity, identification of more hydrophobic MHC ligands could potentially lead to more effective isolation of immunogenic peptides as targets for immunotherapies. We solved this problem by use of higher concentrations of acetonitrile for the separation of MHC ligands and their respective complexes. This increased overall MHC ligand identifications by 2-fold, increased detection of cancer germline antigen-derived peptides by 50%, and resulted in profound variations in isolation efficacy between different MHC alleles correlating with the hydrophobicity of their anchor residues. Overall, these insights enabled a more complete view of the immunopeptidome and overcame a systematic underrepresentation of these critical MHC ligands of high hydrophobicity.
Assuntos
Acetonitrilas/química , Apresentação de Antígeno/imunologia , Cromatografia de Afinidade/métodos , Antígenos de Histocompatibilidade Classe I/metabolismo , Leucemia Mieloide Aguda/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Acetonitrilas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Ligantes , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Células Tumorais CultivadasRESUMO
Dichloroacetonitrile (DCAN) is one of the most toxic and common nitrogenous disinfection by-products in water treatment. It is necessary to understand how this compound can be removed. In this study, the effectiveness of vacuum ultraviolet (VUV) at 185 nm was evaluated to destroy DCAN. When water is exposed to VUV, hydroxyl radicals (HOâ¢), hydrogen atoms (Hâ¢), and hydrated electrons (eaq-) are generated. The individual contributions of these reactive species to DCAN degradation were distinguished using multiple scavengers. The results showed that eaq- was the most important species for DCAN degradation. The second-order rate constant for eaq- reacting with DCAN was calculated to be 3.16 × 1010 M-1s-1 using a quantitative structure-activity relationship (QSAR) method adopted from previous study, and determined to be 3.76 (±0.02) × 1010 M-1s-1 by competition kinetics. Although dissolved oxygen (DO) at 8 mg/L consumed 86% eaq-, the rest of eaq- still led to 93% removal of DCAN within 20 min. Chloride was the major inorganic product of DCAN degradation, while nitrate and nitrite were minor products. Quantum chemical calculation and mass balance calculation under an oxygen free condition further suggested that cleavage of C-Cl bonds was the major pathway by eaq- attack. This study demonstrated the significant role of eaq- in micropollutant destruction during VUV treatment.
Assuntos
Acetonitrilas/metabolismo , Poluentes Químicos da Água/metabolismo , Cloro , Desinfecção , Elétrons , Radical Hidroxila/química , Cinética , Nitratos , Nitrogênio , Fotólise , Raios Ultravioleta , Vácuo , Poluentes Químicos da Água/análise , Purificação da Água/métodosRESUMO
Salicylic acid (SA) plays a central role in plant responses to environmental stresses. In a recent study, we suggested a third pathway for SA biosynthesis from mandelonitrile (MD) in peach plants. This pathway is an alternative to the phenylalanine ammonia-lyase pathway and links SA biosynthesis and cyanogenesis. In the present work, using biochemical approaches, we studied the effect of salt stress and Plum pox virus (PPV) infection on this proposed SA biosynthetic pathway from MD. Peach plants were submitted to salt stress and Plum pox virus (PPV) infection. We studied the levels of SA and its intermediates/precursors (phenylalanine, MD, amygdalin and benzoic acid) in in vitro shoots. Moreover, in peach seedlings, we analysed the content of H2 O2 -related enzymes, SA and the stress-related hormones abscisic acid and jasmonic acid. We showed that the contribution of this SA biosynthetic pathway from MD to the total SA pool does not seem to be important under the stress conditions assayed. Nevertheless, MD treatment not only affected the SA content, but also had a pleiotropic effect on abscisic acid and jasmonic acid levels. Furthermore, MD modulates the antioxidative metabolism via SA-dependent or -independent redox-related signalling pathways. Even though the proposed SA biosynthetic pathway seems to be functional under stress conditions, MD, and hence cyanogenic glycosides, may be operating more broadly than by influencing SA pathways and signalling. Thus, the physiological function of the proposed SA biosynthetic pathway remains to be elucidated.
Assuntos
Prunus persica , Ácido Salicílico , Estresse Fisiológico , Acetonitrilas/metabolismo , Vírus Eruptivo da Ameixa/fisiologia , Prunus persica/fisiologia , Prunus persica/virologia , Ácido Salicílico/metabolismo , Estresse Fisiológico/fisiologiaRESUMO
The arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts of the corresponding amides. The enzyme has been analysed by site-specific mutagenesis and more than 50 different variants have been generated and analysed for the conversion of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile. These comparative analyses demonstrated that single point mutations are sufficient to generate enzyme variants which hydrolyse (R,S)-mandelonitrile to (R)-mandelic acid with an enantiomeric excess (ee) of 91% or to (S)-mandelic acid with an ee-value of 47%. The conversion of (R,S)-2-phenylpropionitrile by different nitrilase variants resulted in the formation of either (S)- or (R)-2-phenylpropionic acid with ee-values up to about 80%. Furthermore, the amounts of amides that are produced from (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile could be changed by single point mutations between 2%-94% and <0.2%-73%, respectively. The present study attempted to collect and compare the results obtained during our previous work, and to obtain additional general information about the relationship of the amide forming capacity of nitrilases and the enantiomeric composition of the products.
Assuntos
Acetonitrilas/metabolismo , Aminoidrolases/metabolismo , Mutação , Pseudomonas fluorescens/enzimologia , Aminoidrolases/genética , Especificidade por SubstratoRESUMO
Acetonitrile (ACN) is a very volatile, toxic and nitrogen-rich organic compound. To enhance ACN wastewater treatment, a novel hybrid membrane-aerated bioreactor (MAB) containing aerated and non-aerated zones was established. A polypropylene hollow fiber membrane module (HF) and a silicone rubber membrane module (SR) were separately used as the bubble-free aeration diffuser and the biofilm carrier, and the non-aerated zones of these two types of reactors were packed with ceramsite. When the influent ACN loading was 1.200â¯kg/m3·d, under aeration pressures of 20â¯kPa in the HF-MAB and 40â¯kPa in the SR-MAB, ACN removal loadings of 1.116â¯kg/m3·d and 1.004â¯kg/m3·d, respectively, were achieved, and the TN (total nitrogen) removal loadings were 0.267â¯kg/m3·d and 0.246â¯kg/m3·d, respectively. In the MABs, different stratified biofilm structures of the two zones and the diffusion and counter-diffusion of oxygen synergistically promoted ACN degradation, nitrification and denitrification.
Assuntos
Reatores Biológicos , Águas Residuárias/química , Acetonitrilas/metabolismo , Biofilmes , Análise da Demanda Biológica de Oxigênio , Desnitrificação , Nitrificação , Nitrogênio/química , Oxigênio/metabolismo , Eliminação de Resíduos LíquidosRESUMO
Gefitinib, the first approved oral epidermal growth factor receptor (EGFR) inhibitor, has been demonstrated effective in cancers with EGFR active mutations. In this study, we established and validated a method for determining gefitinib and its main metabolites, M605211, M387783, M537194 and M523595 in patients with non-small cell lung cancer (NSCLC) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The mobile phase was water: acetonitrile (35:65, v/v) with 0.1% formic acid at a flow-rate of 0.35 mL/min, within a 3 min run time. Gefitinib and its main metabolites were separated on a X-Terra RP18 column (50 × 2.1 mm, 3.5 µm) at 40 â and subjected to mass analysis using positive electro-spray ionization (ESI). The calibration ranges of gefitinib and M523595 were 0.5-1000 ng/mL, and other compounds were 0.05-100 ng/mL with the correlation coefficients (r2) ≥ 0.99. Accuracies ranged from 92.60%-107.58 and the inter- and intra-assay precision were less than 15% for all analytes in quality control samples. There was no significant matrix effect. The ranges of extraction recoveries were 86-105% for all analytes and IS. Thirty plasmas were obtained from Sun Yat-sen university cancer center. The mean plasma concentration of (± SD) of gefitinib M537194, M523595, M387783 and M605211 were 247.18 (± 140.39) ng/mL, 7.78 (± 6.74) ng/mL, 101.09 (± 93.44) ng/mL, 1.6 (± 0.9) ng/mL and 11.63 (± 4.98) ng/mL, respectively. The validated LC/MS/MS method was effectively used in the determination of gefitinib and its four metabolites in NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/sangue , Cromatografia Líquida/métodos , Gefitinibe/sangue , Gefitinibe/metabolismo , Neoplasias Pulmonares/sangue , Plasma/metabolismo , Espectrometria de Massas em Tandem/métodos , Acetonitrilas/sangue , Acetonitrilas/metabolismo , Calibragem , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Formiatos/sangue , Formiatos/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Reprodutibilidade dos TestesRESUMO
Nitrilase-catalyzed regioselective hydrolysis of 1-cyanocyclohexaneacetonitrile (1-CHAN) is a green and efficient approach for the preparation of 1-cyanocyclohexaneacetic acid (1-CHAA), a key precursor for the synthesis of gabapentin. Here, a mesoporous biosilica particles prepared by the ethyleneamine-mediated silicification have been used as carrier for the encapsulation of nitrilase from Acidovorax facilis (NitA). The silica-encapsulated NitA (NitA@silica) with triethylenetetramine as an initiator showed the highest immobilization efficiency (98.3%) and specific activity (672.6â¯U/g). Both free and encapsulated NitA were optimally active at 40⯰C and pHâ¯7.0, however, the encapsulated enzyme exhibited wider optimum temperature range, and enhanced thermal stability compared with the free enzyme. The kinetic parameters Km and Vmax for free and encapsulated NitA were calculated to be 141â¯mM and 9.97â¯mMâ¯min-1, and 280â¯mM and 9.02â¯mMâ¯min-1, respectively. The encapsulated NitA showed good reusability and retained about 94.2% of its initial activity even after 16â¯cycles of reaction. Also, the storage experiments revealed high activity maintenance of encapsulated NitA after 17-day storage at 4⯰C. A preparative scale regioselective hydrolysis of 1-CHAN to 1-CHAA with encapsulated NitA as biocatalyst was carried out in a 2â¯L stirred bioreactor. The concentration of 1-CHAA reached 152â¯g/L after 8â¯h reaction and the conversion was 90.9%. These results showed that the encapsulation of NitA in ethyleneamine-mediated biosilica is an efficient and simple way for preparation of stable nitrilase and have a great potential for application in enzymatic production of carboxylic acids.
Assuntos
Acetonitrilas/química , Aminoidrolases/química , Aminoidrolases/metabolismo , Biocatálise , Cicloexanos/química , Nitrilas/química , Trientina/química , Acetonitrilas/metabolismo , Cápsulas , Cicloexanos/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Nitrilas/metabolismo , Dióxido de Silício/química , Estereoisomerismo , Especificidade por Substrato , TemperaturaRESUMO
We recently identified unique caterpillar-induced plant volatile compounds emitted from apple leaves infested with the larvae of various leafroller species. In subsequent field tests, binary blends of phenylacetonitrile+acetic acid and 2-phenylethanol+acetic acid were found to be attractive to a range of tortricid leafroller species (Tortricidae: Tortricinae) in both the Southern and Northern Hemispheres. In this work, the caterpillar-induced plant volatiles from the apple-leafroller system were tested in two vineyards in Spain and Hungary for their attractiveness to the grape frugivore Lobesia botrana (Tortricidae: Olethreutinae). As seen for Tortricinae species, a binary blend of phenylacetonitrile+acetic acid attracted significantly more male and female L. botrana to traps than acetic acid or blank lures. Traps baited with other caterpillar-induced plant volatile compounds (benzyl alcohol, 2-phenylethanol, indole, and (E)-nerolidol, each as a binary blend with acetic acid) did not catch significantly more moths than traps containing acetic acid alone. The catches of male and female moths support an optimistic future for new products in female tortricid surveillance and control that are based on combinations of kairomone compounds released from larval-damaged foliage.
Assuntos
Ácido Acético/farmacologia , Acetonitrilas/farmacologia , Interações Hospedeiro-Parasita , Lepidópteros/efeitos dos fármacos , Vitis/parasitologia , Compostos Orgânicos Voláteis/farmacologia , Ácido Acético/metabolismo , Acetonitrilas/metabolismo , Animais , Lepidópteros/patogenicidade , Malus/metabolismo , Malus/parasitologia , Compostos Orgânicos Voláteis/metabolismoRESUMO
Many aggregating animals use aposematic signals to advertise their toxicity to predators. However, the coordination between aposematic signals and toxins is poorly understood. Here, we reveal that phenylacetonitrile (PAN) acts as an olfactory aposematic signal and precursor of hypertoxic hydrogen cyanide (HCN) to protect gregarious locusts from predation. We found that PAN biosynthesis from phenylalanine is catalyzed by CYP305M2, a novel gene encoding a cytochrome P450 enzyme in gregarious locusts. The RNA interference (RNAi) knockdown of CYP305M2 increases the vulnerability of gregarious locusts to bird predation. By contrast, the elevation of PAN levels through supplementation with synthetic PAN increases the resistance of solitary locusts to predation. When locusts are attacked by birds, PAN is converted to HCN, which causes food poisoning in birds. Our results indicate that locusts develop a defense mechanism wherein an aposematic compound is converted to hypertoxic cyanide in resistance to predation by natural enemies.
Assuntos
Acetonitrilas/metabolismo , Aves , Doenças Transmitidas por Alimentos , Gafanhotos/metabolismo , Cianeto de Hidrogênio/intoxicação , Comportamento Predatório , Olfato/fisiologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Técnicas de Silenciamento de Genes , Interferência de RNARESUMO
A novel bioflocculant, A-GS408, produced by Klebsiella oxytoca GS-4-08 cultured in acetonitrile (ACN) as sole nitrogen source was investigated in this study. A complete degradation of 1 g l-1 of ACN was achieved in 350 h, and 4.6 g of crude A-GS408 can be obtained in one litter of synthetic medium. The as-prepared bioflocculant exhibits good flocculation efficiency (over 90%) toward Kaolin solution with the aid of Fe3+. Chemical analysis showed that the bioflocculant was mainly composed of polysaccharides (46.3%) and proteins (20.6%). FTIR and XPS results indicated the abundant carboxyl, amine and hydroxyl groups in A-GS408 s, which play an important role on Pd2+ and Cu2+ adsorption. The adsorption of heavy metals can be well stimulated by Freundlich isotherm equation, and the Kf was up to 439.2 mg1-1/n l1/n g-1 and 112.2 mg1-1/n l1/n g-1 for Pb2+ and Cu2+, respectively. The kinetic fitting results proved that the adsorption of heavy metals by A-GS408 was chemisorption. This study may provide a new method for preparation of bioflocculant, which can not only degrade toxic compound i.e., acetonitrile, but also can reuse considerable nitrogen source from nitrile-containing wastewater.
Assuntos
Acetonitrilas/metabolismo , Klebsiella oxytoca/metabolismo , Metais Pesados/isolamento & purificação , Purificação da Água/métodosRESUMO
Phosphodiesterase 4 (PDE4) inhibitors with potential activities for CNS disorders provide a new therapeutic strategy for depression. To discover PDE4 inhibitors with anti-neuroinflammation activities, reliable three-dimensional quantitative structure-activity relationship (3D-QSAR) models on our previous reported catecholic PDE4 inhibitors was built with a statistically significant cross-validated coefficient (q2 ), conventional coefficient (r2 ), and good predictive capabilities based on the molecular docking results, using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods. Based on the analysis of CoMFA and CoMSIA contour maps, a series of 2-(3,4-dialkoxyphenyl)-2-(substituted pyridazin-3-yl) acetonitriles 16a-i was designed and synthesized. Among these compounds, compound 16a exhibited good inhibitory activities toward PDE4B1 and PDE4D7 with mid-nanomolar IC50 values and potential anti-neuroinflammation activity in BV-2 cells. Docking simulation of compound 16a in the PDE4 catalytic domain activity pocket revealed that compound 16a maybe assumed a "V-shaped" conformation, extending the side chain to S-pocket.
Assuntos
Acetonitrilas/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Desenho de Fármacos , Inibidores da Fosfodiesterase 4/química , Relação Quantitativa Estrutura-Atividade , Acetonitrilas/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Simulação de Acoplamento Molecular , Inibidores da Fosfodiesterase 4/metabolismoRESUMO
Salinity is considered as one of the most important abiotic challenges that affect crop productivity. Plant hormones, including salicylic acid (SA), are key factors in the defence signalling output triggered during plant responses against environmental stresses. We have previously reported in peach a new SA biosynthetic pathway from mandelonitrile (MD), the molecule at the hub of the cyanogenic glucoside turnover in Prunus sp. In this work, we have studied whether this new SA biosynthetic pathway is also present in plum and the possible role this pathway plays in plant plasticity under salinity, focusing on the transgenic plum line J8-1, which displays stress tolerance via an enhanced antioxidant capacity. The SA biosynthesis from MD in non-transgenic and J8-1 micropropagated plum shoots was studied by metabolomics. Then the response of J8-1 to salt stress in presence of MD or Phe (MD precursor) was assayed by measuring: chlorophyll content and fluorescence parameters, stress related hormones, levels of non-enzymatic antioxidants, the expression of two genes coding redox-related proteins, and the content of soluble nutrients. The results from in vitro assays suggest that the SA synthesis from the MD pathway demonstrated in peach is not clearly present in plum, at least under the tested conditions. Nevertheless, in J8-1 NaCl-stressed seedlings, an increase in SA was recorded as a result of the MD treatment, suggesting that MD could be involved in the SA biosynthesis under NaCl stress conditions in plum plants. We have also shown that the plum line J8-1 was tolerant to NaCl under greenhouse conditions, and this response was quite similar in MD-treated plants. Nevertheless, the MD treatment produced an increase in SA, jasmonic acid (JA) and reduced ascorbate (ASC) contents, as well as in the coefficient of non-photochemical quenching (qN) and the gene expression of Non-Expressor of Pathogenesis-Related 1 (NPR1) and thioredoxin H (TrxH) under salinity conditions. This response suggested a crosstalk between different signalling pathways (NPR1/Trx and SA/JA) leading to salinity tolerance in the transgenic plum line J8-1.