RESUMO
Isotope fractionation of metals/metalloids in biological systems is an emerging research area that demands the application of state-of-the-art analytical chemistry tools and provides data of relevance to life sciences. In this work, Se uptake and Se isotope fractionation were measured during the biofortification of baker's yeast (Saccharomyces cerevisiae)-a product widely used in dietary Se supplementation and in cancer prevention. On the other hand, metabolic labeling with 15N is a valuable tool in mass spectrometry-based comparative proteomics. For Se-yeast, such labeling would facilitate the assessment of Se impact on yeast proteome; however, the question arises whether the presence of 15N in the microorganisms affects Se uptake and its isotope fractionation. To address the above-mentioned aspects, extracellularly reduced and cell-incorporated Se fractions were analyzed by hydride generation-multi-collector inductively coupled plasma-mass spectrometry (HG MC ICP-MS). It was found that extracellularly reduced Se was enriched in light isotopes; for cell-incorporated Se, the change was even more pronounced, which provides new evidence of mass fractionation during biological selenite reduction. In the presence of 15N, a weaker preference for light isotopes was observed in both, extracellular and cell-incorporated Se. Furthermore, a significant increase in Se uptake for 15N compared to 14N biomass was found, with good agreement between hydride generation microwave plasma-atomic emission spectrometry (HG MP-AES) and quadrupole ICP-MS results. Biological effects observed for heavy nitrogen suggest 15N-driven alteration at the proteome level, which facilitated Se access to cells with decreased preference for light isotopes.
Assuntos
Saccharomyces cerevisiae , Selênio , Biofortificação , Proteoma , Transporte BiológicoRESUMO
Antimony is present in different types of plastics as a catalyzer residue and/or as a synergistic fire retardant; relatively high concentrations of this element reported in polyethylene terephthalate (PET) bottles and wrappers as well as its migration to the edible products or to different environment compartments are of concern. In this work, Sb determination is such products had been undertaken using hydride generation - microwave plasma - atomic emission spectrometry. To avoid harsh conditions typically reported for the digestion of PET, alkaline methanolysis was introduced whereas water samples were analyzed directly. Another original approach was to perform quantification by partial least squares regression (PLS1), taking spectral data from 2-nm range that comprised two emission lines (217.581 nm and less intense 217.919 nm). For PET, the calibration solutions contained Sb-free digest and covered the Sb concentration range 80-230 µg L-1. For the analysis of water, the calibration range was 0.5-10 µg L-1 and aqueous standard solutions were used. PLS1 provided reliable prediction, eliminating spectral interferences detected in the presence of PET digests and compensating for the spectral changes observed at low Sb concentrations. After standard addition to the real-world samples, the percentage recoveries were in the range 93.8-99.3% and 68-102% for PET and for bottled water, respectively. The method quantification limit for PET was 10 mg kg-1 and for water it corresponded to 0.20 µg L-1. The concentrations of Sb found in the analyzed samples were: 154-279 mg kg-1 for PET bottles and <0.5-5.30 µg L-1 for water.
Assuntos
Água Potável , Polietilenotereftalatos , Polietilenotereftalatos/química , Antimônio/química , Micro-Ondas , Análise dos Mínimos Quadrados , Água Potável/química , Análise EspectralRESUMO
In this work, a principle of flow injection analysis (FIA) was applied for sample introduction to an electrospray ionization - ion trap mass spectrometer (ESI-ITMS) with the aim to quantify chromium(III) picolinate (CrPic3) in commercial supplements by multiple reaction monitoring, and using cobalt(II) picolinate as internal standard (IS). FIA system was operated with ammonium formate 10 mmol L-1 in methanol-water (1:1, v/v) as a carrier solution at a flow rate 200 µL min-1; 100 µL injections were performed in 2-min intervals. Setting ion transitions m/z 419 â 270 and 304 â 260 for the analyte and IS, respectively, and 100 ms integration time, the method detection and quantification limits 12 ng g-1 and 40 ng g-1 of Cr (as CrPic3) in the air-dried powder. Acetonitrile extracts of the real-world samples presented varying from sample-to-sample chemical composition and IS efficiently compensated for ionization interferences. Mean results from triplicate analysis of four different supplements were obtained with relative standard deviation 0.1-4.0%, indicating acceptable precision. Trueness of the proposed FIA-ESI-ITMS/MS procedure was demonstrated by 95.8-108% percentage recoveries attained in the analysis of the CrPic3-spiked samples. For comparative purposes, total Cr was determined by ICP-MS. The quantitative results obtained indicate the necessity of analytical control of Cr(III) supplements commercially available and demonstrate that the proposed FIA-ESI-ITMS/MS procedure is well-suited for the determination of CrPic3 in such products.
Assuntos
Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Cromo , Cobalto , Suplementos Nutricionais , Ácidos Picolínicos , Reprodutibilidade dos TestesRESUMO
Sporothrixschenckii is one of the etiological agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. Its cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan, which harbors important Sporothrix-specific immunogenic epitopes. Although the peptidorhamnomannan carbohydrate moiety has been extensively studied, thus far, little is known about the protein core. Here, using LC-MS/MS, we analyzed the S.schenckii peptidorhamnomannan peptide fraction and generated mass signals of 325 proteins, most of them likely to be moonlighting proteins. Among the identified proteins, chaperonin GroEL/Hsp60 and the uncharacterized protein Pap1 were selected for further analysis. Both proteins were heterologously expressed in bacteria, and they showed adhesive properties to the extracellular matrix proteins laminin, elastin, fibrinogen, and fibronectin, although Pap1 also was bound to type-I and type-II collagen. The inoculation of concentrations higher than 40 µg of these proteins, separately, increased immune effectors in the hemolymph of Galleriamellonella larvae and protected animals from an S.schenckii lethal challenge. These observations were confirmed when yeast-like cells, pre-incubated with anti-rHsp60 or anti-rPap1 antibodies were used to inoculate larvae. The animals inoculated with pretreated cells showed increased survival rates when compared to the control groups. In conclusion, we report that Hsp60 and Pap1 are part of the cell wall peptidorhamnomannan, can bind extracellular matrix components, and contribute to the S.schenckii virulence. To our knowledge, this is the first report about moonlighting protein in the S.schenckii cell wall with an important role during the pathogen-host interaction.
RESUMO
Longstanding industrial deposits of 1-chloro-4-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene (DDT) impose environmental threat in Salamanca city, located in central Mexico. Native bacteria from this location were isolated and identified, and their potential utility for DDT biodegradation was examined. Twenty-five isolates were obtained, and cell lysates were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) with BiotyperTR; twenty-one organisms were identified at species level, and the other four were assigned to genus. The most abundant species corresponded to Bacillus (44%) and Pseudomonas genera (20%). Eight bacteria could grow in the presence of 200 mg/L of DDT. Two-week exposure of Lysinibacillus fusiformis, Bacillus mycoides, Bacillus pumilus, and Bacillus cereus to DDT 50 mg/L and 200 mg/L, caused percentage pesticide degradation in the range 41-48% and 26-31%, respectively. Other four bacteria presented lower degradation rates. Gas chromatography-mass spectrometry (GC-MS) analysis of the spent media revealed that eight isolates assisted the conversion of DDT, DDD (1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane), and DDE (1,1-dichloro-2,2-bis-(4-chlorophenyl)ethylene) to DDMU (1,1-(2-chloro-1,1-ethenediyl)-bis-(4-chlorobenzene)); however, DDNU (2,2-bis(4-chlorophenyl)ethylene), DBP (4,4'-dichlorobenzophenone(bis(4-chlorophenyl)methanone)) and DBH (bis(4-chlorophenyl)methanol) were found only for L. fusiformis, B. mycoides, B. cereus, B. marisflavi, and B. megaterium. Within the context of DDT biodegradation, the first three were the most promising isolates and further studies will be aimed at setting the experimental conditions for efficient mineralization of DDT congeners.
Assuntos
Bacillaceae , Bacillus , Bactérias , DDT , Microbiologia Ambiental , Espectrometria de Massas , Bacillaceae/isolamento & purificação , Bacillaceae/metabolismo , Bacillus/isolamento & purificação , Bacillus/metabolismo , Bactérias/química , Bactérias/classificação , Biodegradação Ambiental , DDT/metabolismo , Poluentes Ambientais/metabolismo , MéxicoRESUMO
Cuphea aequipetala Cav (Lythraceae) is an herb used in folk treatment for pain and inflammation. The aim of this study was to evaluate the antinociceptive and anti-inflammatory actions of an ethanol extract from the leaves and stem of Cuphea aequipetala (CAE). The antinociceptive actions of CAE (10-200 mg/kg p.o.) were assessed with the acetic acid-induced writhing, hot plate, and formalin tests. The possible mechanism of action of CAE was evaluated using inhibitors. The effects of CAE on motor coordination were assessed by the rotarod test. The in vitro anti-inflammatory actions of CAE were evaluated using LPS-stimulated primary murine macrophages, and the in vivo anti-inflammatory actions were assessed by the TPA-induced ear oedema and the carrageenan-induced paw oedema tests. The production of inflammatory mediators was estimated from both in vitro and in vivo assays. CAE showed antinociception (ED50 = 90 mg/kg) in the acetic acid test and in the second phase of the formalin test (ED50 = 158 mg/kg). Pretreatment with glibenclamide or L-NAME partially reversed the antinociception shown by the plant extract. CAE (50-200 mg/kg) did not affect motor coordination in mice. CAE increased the production of IL-10 in LPS-stimulated macrophages (EC50 = 10 pg/ml) and, in the carrageenan-induced paw oedema test (threefold increase). In conclusion, CAE induced antinociceptive effects without affecting motor coordination, probably due to the involvement of nitric oxide and ATP-sensitive K+ channels. CAE also exerts in vitro and in vivo anti-inflammatory effects by increasing the release of IL-10.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Cuphea/química , Extratos Vegetais/farmacologia , Analgésicos/administração & dosagem , Analgésicos/isolamento & purificação , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/isolamento & purificação , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Edema/tratamento farmacológico , Edema/patologia , Inflamação/tratamento farmacológico , Inflamação/patologia , Canais KATP/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Dor/tratamento farmacológico , Extratos Vegetais/administração & dosagemRESUMO
Metabolic syndrome (MetS) is a multifactor condition predisposing for diabetes, cardiovascular diseases and other degenerative disorders. Although several diagnostic criteria have been established, none of them is specific and there is a call for better pathophysiological explanation of MetS and for the discovery of molecular biomarkers. Phenotype characterization at metabolome level might be useful for both purposes. To this end, our aim was to perform comparative untargeted metabolomics of urines from MetS patients and from the control group. The study participants included 52 diagnosticated and 50 healthy individuals from Leon city in central Mexico; 23 anthropometric and clinical parameters were measured and submitted to Principal Component Analysis (PCA). The obtained PCA model allowed us for selection of 11 MetS patients and 13 control subjects, correspondingly representative for each of the two groups (clearly separated in PCA). The first morning urines from these subjects were ambulatory collected and, after methanol extraction and acidification, were submitted to capillary liquid chromatography-high resolution mass spectrometry (LC-HRMS). The obtained data were analyzed on MetaboScape® platform (Bruker Daltonics). Specifically, t-test applied to LC-HRMS data revealed several ions presenting at least 3-fold higher intensities in MetS with respect to the control samples (p < 0.05). Data analysis and complementary experiments yielded the identification of the following metabolites: indole-3-acetic acid, indole-3-acetic acid-O-glucuronide, N-(indol-3-ylacetyl) glutamine, indole-3-carbaldehyde and hydroxyhexanoycarnitine. Additionally, indole-3-carboxylic acid was annotated with 2.13-fold higher abundance in MetS patients. To assess the contribution of individual metabolites in the difference between two groups of subjects, partial least square discriminant analysis was performed for LC-HRMS data and the obtained values of variable importance in projection (VIP), confirmed the association of six above mentioned compounds with MetS. Overall, this study provides direct evidence on the disturbed catabolism of tryptophan in metabolic syndrome.
Assuntos
Indóis , Síndrome Metabólica , Metabolômica/métodos , Triptofano , Adulto , Cromatografia Líquida/métodos , Estudos Transversais , Feminino , Humanos , Indóis/metabolismo , Indóis/urina , Masculino , Espectrometria de Massas/métodos , Síndrome Metabólica/metabolismo , Síndrome Metabólica/urina , Metaboloma/fisiologia , Análise de Componente Principal , Triptofano/metabolismo , Triptofano/urinaRESUMO
The chemoselective reaction of the C- followed by the O-centered naphthyl radicals with the more electron-deficient hypervalent bond of the diaryliodonium(III) salts is described. This discovered reactivity constitutes a new activation mode of the diaryliodonium(III) salts which enabled a one-pot doubly arylation of naphthols through the sequential C s p 2 - C s p 2 /O- C s p 2 bond formation. The naphthyl radicals were generated in the reaction by the tetramethylpiperidinyl radical (TMP·) which resulted from the homolytic fragmentation of the precursor TMP2O. Experimental and DFT calculations provided a complete panorama of the reaction mechanism.
RESUMO
The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.
Assuntos
Anti-Infecciosos/farmacologia , Brassica/química , Defensinas/farmacologia , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Bactérias/efeitos dos fármacos , Cromatografia Líquida , Defensinas/química , Defensinas/isolamento & purificação , Fungos/efeitos dos fármacos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Extratos Vegetais/química , Sementes/químicaRESUMO
The application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented for the determination of five sulfonated azo dyes in chili powders. To circumvent problems related to spectral noise and overall poor precision, acid red 88 was used as internal standard and sample cleanup was performed via ion pairing of anionic species with benzyltributylammonium bromide (BTAB) and extraction into chloroform. The key parameters influencing analytical performance were BTAB concentration, pH of the aqueous phase, amount of sample and deposition technique, concentration of 9-aminoacridine (chemical matrix), number of instant spectra per laser shot, and the raster of laser movement. The highest sample load corresponded to 100 µL of water/methanol extract taken for extraction and the method quantification limits for sunset yellow (Y6), ponceau 2R (R5), allura red (R40), and amaranth (R2) were within the range 1.50-3.10 µg g-1 (29.0 µg g-1 for tartrazine, Y5). Two-point standard addition performed in three samples yielded percentage recoveries in the range 86.4-115%; the quantification results were consistent with those obtained by HPLC-DAD. Twelve chili powders were analyzed and the results for nine of them disagreed with information provided by the manufacturers; R40 was determined in seven products at concentrations from 32.5 ± 2.1 µg g-1 to 1125 ± 73 µg g-1; Y6 and Y5 were found at lower concentrations and in fewer samples. The MALDI-TOF MS procedure can be recommended for routine control of sulfonated azo dyes in food products as a memory-free, procedurally simple, high-throughput procedure with minimal costs of instrument operation. Outline of the proposed MALDI-TOF MS procedure.
RESUMO
The impact of Cr(VI) in sunflower roots has been studied, focusing on the oxidation of polyunsaturated fatty acids. Plants were grown hydroponically in the presence of 0, 1.0, 5.0 and 25â¯mgCr L-1. Methanolic root extracts were analyzed by capillary liquid chromatography coupled through negative electrospray ionization to a quadrupole-time of flight mass spectrometry (capHPLC-ESI-QTOF-MS). Using partial least squares algorithm, eighteen features strongly affected by Cr(VI) were detected and annotated as linoleic acid (LA), alpha-linolenic acid (ALA) and sixteen oxidation products containing hydroperoxy-, epoxy-, keto-, epoxyketo- or hydroxy-functionalities, all of them classified as oxylipins. Inspection of the MS/MS spectra acquired for features eluting at different retention times but assigned as a sole compound, confirmed isomers formation: three hydroperoxy-octadecadienoic acids (HpODE), two oxo-octadecadienoic acids (OxoODE) and four epoxyketo-octadecenoic acids (EKODE). Around 70% of metabolites in sunflower LA metabolic pathway were affected by Cr(VI) stress and additionally, four EKODE isomers not included in this pathway were found in the exposed roots. Among ALA-derived oxylipins, 13-epi-12-oxo-phytodienoic acid (OPDA) is of relevance, because of its participation in the activation of secondary metabolism. The abundances of all oxylipins were directly dependent on the Cr(VI) concentration in medium; furthermore, autooxidation of LA to HpODE isomers was observed after incubation with Cr(VI). These results point to the direct involvement of Cr(VI) in non-enzymatic oxidation of fatty acids; since oxylipins are signaling molecules important in plant defensive response, their synthesis under Cr(VI) exposure sustains the ability of sunflower to grow in Cr(VI)-contaminated environments.
Assuntos
Carcinógenos Ambientais/farmacologia , Cromo/farmacologia , Ácidos Graxos Insaturados/metabolismo , Helianthus/metabolismo , Metabolômica , Raízes de Plantas/metabolismo , Espectrometria de Massas em Tandem/métodos , Helianthus/efeitos dos fármacos , Helianthus/crescimento & desenvolvimento , Oxirredução , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimentoRESUMO
RATIONALE: Quantification of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is challenging yet attractive, due to micro-scale procedural simplicity, high throughput and lack of memory effects. Since these features are important while analyzing trace elements in quality control schemes, MALDI-TOFMS was used for the determination of copper (Cu) and lead (Pb) in tequila with quantification carried out by partial least squares regression (PLS2) and by univariate calibration (UC). METHODS: In the proposed procedure, Bi(III) was added as internal standard (IS), diethyldithiocarbamate complexes were formed (pH 7.4) and extracted into chloroform; after solvent evaporation and re-constitution in acetonitrile, the sample was co-crystallized with α-cyano-4-hydroxycinnamic acid on a steel target. From the acquired mass spectra, UC was performed using IS-normalized signals of the monoisotopic ions of analytes, and the m/z range 350-513 was used for PLS2. Accuracy was tested by recovery experiments and by inductively coupled plasma (ICP)-MS analysis. RESULTS: When compared with direct analyte signal measurements, application of IS yielded enhanced analytical performance using either UC or PLS2; the method quantification limits were: 11.1 µg L-1 , 23.4 µg L-1 for Cu and 89.8 µg L-1 , 97.1 µg L-1 for Pb, respectively. In tequila, MALDI-TOFMS and ICP-MS provided consistent results for Cu (165-2599 µg L-1 ); Pb was not detected in any sample by MALDI-TOFMS, yet recoveries obtained after standard addition were indicative of acceptable accuracy (400 µg L-1 Pb added; recoveries: 91.2-108% for UC and 98.8-120% for PLS2). CONCLUSIONS: New experimental evidence has been provided supporting the inclusion of trace metals quantification within a range of MALDI-TOFMS applications. Slightly better results were obtained for UC as compared with PLS2 yet both methods can be recommended for testing the compliance of Cu and Pb levels with Official Mexican Norm. Of note, while using PLS2, there is no need for signal integration nor for IS normalization.
RESUMO
Human lead (Pb) exposure induces many adverse health effects, including some related to lead accumulation in organs. Although lead bio-distribution in the body has been described, the molecular mechanism underlying distribution and excretion is not well understood. The transport of essential and toxic metals is principally mediated by proteins. How lead affects the expression of metal transporter proteins in the principal metal excretory organs, i.e., the liver and kidney, is unknown. Considering that co-administration of melatonin and lead reduces the toxic effects of lead and lead levels in the blood in vivo, we examined how lead and co-administration of lead and melatonin affect the gene and protein expression of metal transporter proteins (ZIP8, ZIP14, CTR1 and DMT1) in these organs. Rats were exposed intraperitoneally to lead or lead-melatonin. Our results show that Pb exposure induces changes in the protein and gene expression of ZIP8, ZIP14 and CTR1. Alterations in the copper/zinc ratio found in the blood, liver and kidney were likely related to these changes. With DMT1 expression (gene and protein), a positive correlation was found with lead levels in the kidney. Co-administration of melatonin and lead reduced lead-induced DMT1 expression through an unknown mechanism. This effect of melatonin relates to reduced lead levels in the blood and kidney. The metal transport protein function and our results suggest that DMT1 likely contributes to lead accumulation in organs. These data further elucidate the effects of lead on Cu and Zn and the molecular mechanism underlying lead bio-distribution in animals.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Cobre/análise , Regulação da Expressão Gênica/efeitos dos fármacos , Chumbo/farmacologia , Melatonina/farmacologia , Zinco/análise , Animais , Proteínas de Transporte/metabolismo , Chumbo/análise , Masculino , Espectrometria de Massas , Melatonina/análise , Ratos , Ratos WistarRESUMO
OBJECTIVE: The goal of this work was to set up a high throughput procedure for the determination of fatty acid methyl esters (FAMEs) in cosmetic castor oils using flow injection - electrospray ionization - high resolution mass spectrometry, and to demonstrate the need of such analysis for the quality control purposes. METHODS: The sample aliquot was mixed with isooctane:chloroform (1:1) and submitted to transesterification; the obtained FAMEs were appropriately diluted using water:isopropanol:acetonitrile (20:50:30) with addition of sodium formate which served as an internal standard, lock mass calibrant and promoted the formation of sodium adducts during electrospray ionization (ESI). The principle of flow injection analysis (FIA) was applied for sample introduction to an ESI - quadrupole- time of flight mass spectrometer (ESI-QTOFMS). The carrier solution was composed of water:isopropanol:acetonitrile (20:50:30). From the acquired MS data, flowgrams of the extracted [M+Na]+ ions were obtained using the following m/z values for individual FAMEs: 293.2451 (C16:0); 315.2295 (C18:3); 317.2451 (C18:2); 319.2608 (C18:1); 321.2764 (C18:0); 335.2557 (C18:1,OH); 349.3077 (C20:0); 377.3390 (C22:0) and m/z 226.9515 for IS. Baseline-subtracted and filtered signals were integrated and the list of peaks intensities was exported to Excel, where calibration functions were obtained and quantification carried out. Gas chromatography with a flame ionization detector (GC-FID) was used as an alternative analytical tool. RESULTS: The calibration detection limits for FAMEs of unsaturated fatty acids were in the range 3.61 - 8.62 µg L-1 and for saturated compounds in the range 8.51 - 82.4 µg L-1 . The results obtained for commercial were in good agreement with GC-FID data; among nine cosmetic oils analyzed, three contained low concentrations of ricinoleic acid (C18:1, OH), indicating adulteration of castor bean oil with other vegetable oils. CONCLUSION: Application of FIA for the sample introduction to ESI-QTOFMS enabled for reliable determination of FAMEs in cosmetic oils with sampling frequency of thirty per hour as compared to two samples per hour achievable using GC-FID. The proposed procedure is especially well suited for FAMEs of unsaturated fatty acids that are primary components of castor triacylglycerides, and contribute to desirable properties of any cosmetic oil. This article is protected by copyright. All rights reserved.
RESUMO
The use of soluble and highly oxidizing Ag(III) in the form of the tetrahydroxoargentate ion Ag(OH)4- is reported for the oxidation of surrogate organic recalcitrant dyes (i.e., rhodamine 6G (Rh6G) and fluorescein (Fl)). The possible use of Ag(OH)4- for the treatment of these and other refractory compounds is assessed. Such dyes were selected due to their common occurrence, stability, refractory nature, the relatively high toxicity of Rh6G, and their structural similarity to Fl. Several reaction intermediates/products were identified. The results showed that the highly oxidizing tetrahydroxoargentate anion was capable of degrading these recalcitrant dyes. Furthermore, the final degradation products do not represent a higher environmental risk than the original surrogates themselves. In addition, the partial mineralization of the dyes was proven.
Assuntos
Corantes/química , Poluentes Ambientais/química , Prata/química , Íons/química , Oxirredução , Rodaminas/químicaRESUMO
The non-appropriate conditions faced by nutritionally stressed bacteria propitiate error-prone repair events underlying stationary-phase- or stress-associated mutagenesis (SPM). The genetic and molecular mechanisms involved in SPM have been deeply studied but the biochemical aspects of this process have so far been less explored. Previous evidence showed that under conditions of nutritional stress, non-dividing cells of strain B. subtilis YB955 overexpressing ribonucleotide reductase (RNR) exhibited a strong propensity to generate true reversions in the hisC952 (amber), metB5 (ochre) and leuC425 (missense) mutant alleles. To further advance our knowledge on the metabolic conditions underlying this hypermutagenic phenotype, a high-throughput LC-MS/MS proteomic analysis was performed in non-dividing cells of an amino acid-starved strain, deficient for NrdR, the RNR repressor. Compared with the parental strain, the level of 57 proteins was found to increase and of 80 decreases in the NrdR-deficient strain. The proteomic analysis revealed an altered content in proteins associated with the stringent response, nucleotide metabolism, DNA repair, and cell signaling in amino acid-starved cells of the ∆nrdR strain. Overall, our results revealed that amino acid-starved cells of strain B. subtilis ∆nrdR that escape from growth-limiting conditions exhibit a complex proteomic pattern reminiscent of a disturbed metabolism. Future experiments aimed to understand the consequences of disrupting the cell signaling pathways unveiled in this study, will advance our knowledge on the genetic adaptations deployed by bacteria to escape from growth-limiting environments.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteoma , Proteômica , Ribonucleotídeo Redutases/genética , Aminoácidos/metabolismo , Cromatografia Líquida , Mutagênese , Nucleotídeos/metabolismo , Proteômica/métodos , Estabilidade de RNA , Estresse Fisiológico , Espectrometria de Massas em TandemRESUMO
2,4-Diacetylphloroglucinol (DAPG) (1) is a phenolic polyketide produced by some plant-associated Pseudomonas species, with many biological activities and ecological functions. Here, we aimed at reconstructing the natural history of DAPG using phylogenomics focused at its biosynthetic gene cluster or phl genes. In addition to around 1500 publically available genomes, we obtained and analyzed the sequences of nine novel Pseudomonas endophytes isolated from the antidiabetic medicinal plant Piper auritum. We found that 29 organisms belonging to six Pseudomonas species contain the phl genes at different frequencies depending on the species. The evolution of the phl genes was then reconstructed, leading to at least two clades postulated to correlate with the known chemical diversity surrounding DAPG biosynthesis. Moreover, two of the newly obtained Pseudomonas endophytes with high antiglycation activity were shown to exert their inhibitory activity against the formation of advanced glycation end-products via DAPG and related congeners. Its isomer, 5-hydroxyferulic acid (2), detected during bioactivity-guided fractionation, together with other DAPG congeners, were found to enhance the detected inhibitory activity. This report provides evidence of a link between the evolution and chemical diversity of DAPG and congeners.
Assuntos
Endófitos/química , Floroglucinol/análogos & derivados , Piper/microbiologia , Plantas Medicinais/microbiologia , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , Pseudomonas/química , Ácidos Cumáricos/química , Ácidos Cumáricos/isolamento & purificação , México , Estrutura Molecular , Família Multigênica , Floroglucinol/química , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Piper/genética , Componentes Aéreos da Planta/química , Plantas Medicinais/genética , Policetídeos/química , EstereoisomerismoRESUMO
Invertebrate immune priming is a growing field in immunology. This phenomenon refers to the ability of invertebrates to generate a more vigorous immune response to a second encounter with a specific pathogen and can occur within and across generations. Although the precise mechanism has not been elucidated, it has been suggested that methylation of DNA is a cornerstone for this phenomenon. Here, using a novel method of analytical chemistry (a reversed-phase liquid chromatography procedure) and the beetle Tenebrio molitor as a model system, we did not find evidence to support this hypothesis taking into account the percentage of methylated cytosine entities in DNA (5mdC) within or across generations. However, we found a lower percentage of methylated cytosine entities in RNA (5mC) within but not across generations in immune priming experiments with adults against the bacteria Micrococcus lysodeikticus and larvae against the fungus Metarhizium anisopliae. To our knowledge, this is the first report suggesting a role of differential methylation on RNA during immune priming within generations.
RESUMO
It has been reported that glycation of human serum albumin (HSA) changes its capability for copper binding whereas the increase of free copper might have an impact on protein glycation - a key process in diabetes progression. In this work, proteomic analysis of non-glycated HSA and HSA glycated with methylglyoxal (MGo) in the absence or in the presence of Cu(ii) (0.1; 1.0; 5.0 mg Cu L-1) has been undertaken. Trypsin hydrolysates were subjected to capillary HPLC-ESI-QTOF-MS and MS/MS. Raw data were analyzed using two proteomic platforms: MaxQuant () and ProteinScape (Bruker). Considering seven MGo-derived modifications, the sequence coverage was 98% for non-modified HSA and ≥93% for HSA incubated with MGo or MGo + Cu(ii). Peptide mapping yielded 76 identical peptides in all samples though important differences were found between non-modified HSA and protein glycated with or without Cu(ii). Overall, 46 peptides with residues from 1 to 3 modified were detected/sequenced; the MGo-derived modifications found were: hydroimidazolone, argpyrimidine, Nε-carboxyethyl-lysine and S-carboxyethyl-cysteine; 39 modified sites were identified (22 on arginine, 12 on lysine, and 5 on cysteine) and among them, 27 were common for ProteinScape and MaxQuant. The count of the modified peptides and the comparative analysis of their abundance in different samples indicated that Cu(ii) at physiological and sub-physiological concentrations inhibited HSA glycation as compared to the glycation of the Cu-devoid protein; at higher concentrations (5 mg Cu L-1), this inhibitory effect tends to be inverted. The results obtained suggest that increased protein glycation might be associated with Cu-deficiency and with excessive Cu(ii) concentrations, calling for more detailed studies performed on real-world samples with a strict control of copper concentration.
Assuntos
Cromatografia Líquida/métodos , Cobre/farmacologia , Espectrometria de Massas/métodos , Aldeído Pirúvico/química , Albumina Sérica/química , Albumina Sérica/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Técnicas In VitroRESUMO
The application of capHPLC-ESI-QTOF-MS and MS/MS to study the impact of Cr(VI) on metabolites profile in Helianthus annuus is reported. Germinated seeds were grown hydroponically in the presence of Cr(VI) (25 mgCr/L) and root extracts of the exposed and control plants were analyzed by untargeted metabolomic approach. The main goal was to detect which metabolite groups were mostly affected by Cr(VI) stress; two data analysis tools (ProfileAnalysis, Bruker, and online XCMS) were used under criteria of intensity threshold 5 · 104 cps, fold change ≥ 5, and p ≤ 0.01, yielding precursor ions. Molecular formulas were assigned based on data processing with two computational tools (SIRIUS and MS-Finder); annotation of candidate structures was performed by database search using CSI:FingerID and MS-Finder. Even though ultimate identification has not been achieved, it was demonstrated that secondary metabolism became activated under Cr(VI) stress. Among 42 candidate compounds returned from database search for seven molecular formulas, ten structures corresponded to isocoumarin derivatives and eleven were sesquiterpenes or sesquiterpene lactones; three benzofurans and four glycoside or pyrane derivatives of phenolic compounds were also suggested. To gain further insight on the effect of Cr(VI) in sunflower, isocoumarins and sesquiterpenes were selected as the target compounds for future study.