RESUMO
This study introduces a simplified purification method for analyzing 82Se/78Se isotope ratios in diverse natural samples using hydride generation MC-ICP-MS. Unlike the thiol resin method, which is time-consuming and sensitive to the concentrations of reagents used at individual stages, our proposed alternative is quicker, simpler, and robust. The procedure involves coprecipitation of selenium with iron hydroxide and dissolution in hydrochloric acid. Combining hydride generation and a second cleanup stage achieves sufficient purification for Se isotope ratio measurements. The method is efficient, taking 3-4 h after sample decomposition, utilizing common reagents [HCl, Fe(NO3)3, NH4Cl] without evaporation or clean lab conditions. Results on 82Se/78Se isotope ratios in various matrices are presented, comparing them with literature data. All isotopic results have been subjected to a newly proposed state-of-the-art approach to uncertainty estimation dedicated to isotope ratio measurements. The approach is based on applying Monte Carlo simulations with consideration of different samples' results normalized by the expected value. By doing that, we obtained estimated uncertainty for any Se sample with the influence of particular measurements on the final estimation included. We employ a Monte Carlo simulation-based uncertainty estimation approach for isotope ratio measurements, providing estimated uncertainty for each selenium sample.
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As part of the development and production of pharmaceuticals, the purity of Active Pharmaceutical Ingredients stands as a fundamental parameter that significantly influences the quality, safety, and efficacy of the final drug product. Impurities in Active Pharmaceutical Ingredients are various unwanted substances that can appear during the whole manufacturing process, from raw materials to the final product. These impurities can stem from multiple sources, including starting materials, intermediates, reagents, solvents, and even degradation products resulting from exposure to environmental factors such as heat, light, or moisture. Their presence can potentially compromise the therapeutic effect of the drug, introduce unexpected side effects, or even pose safety risks to patients. This study aims to conduct the forced degradation of linagliptin and subsequently attempt to identify the resulting degradants. The degradation procedures were carried out in accordance with the guidelines of the International Committee for Harmonization. The degradation profile of linagliptin was investigated under various conditions, including acid hydrolysis, alkaline hydrolysis, oxidation, heat, and light exposure, utilizing ultra-performance liquid chromatography connected to a photo array detector. Identification and characterization of the degradation products were achieved using an ultra-performance liquid chromatography coupled with a single quadrupole detector mass spectrometer and also a liquid chromatography coupled with a high-resolution mass spectrometry. The identified degradation products demonstrate that linagliptin is particularly susceptible to degradation when exposed to acid and peroxide. Whereas, no significant degradation effects were observed under alkali, thermolytic, and photolytic conditions.
Assuntos
Linagliptina , Humanos , Espectrometria de Massas , Cromatografia Líquida/métodos , Oxirredução , Hidrólise , Cromatografia Líquida de Alta Pressão/métodos , Estabilidade de MedicamentosRESUMO
The aim of this study was to investigate how dietary modifications with pomegranate seed oil (PSO) and bitter melon aqueous extract (BME) affect mineral content in the spleen of rats both under normal physiological conditions and with coexisting mammary tumorigenesis. The diet of Sprague-Dawley female rats was supplemented either with PSO or with BME, or with a combination for 21 weeks. A chemical carcinogen (7,12-dimethylbenz[a]anthracene) was applied intragastrically to induce mammary tumors. In the spleen of rats, the selected elements were determined with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). ANOVA was used to evaluate differences in elemental composition among experimental groups. Multivariate statistical methods were used to discover whether some subtle dependencies exist between experimental factors and thus influence the element content. Experimental factors affected the splenic levels of macroelements, except for potassium. Both diet modification and the cancerogenic process resulted in significant changes in the content of Fe, Se, Co, Cr, Ni, Al, Sr, Pb, Cd, B, and Tl in rat spleen. Chemometric analysis revealed the greatest impact of the ongoing carcinogenic process on the mineral composition of the spleen. The obtained results may contribute to a better understanding of peripheral immune organ functioning, especially during the neoplastic process, and thus may help develop anticancer prevention and treatment strategies.
Assuntos
Momordica charantia , Extratos Vegetais , Óleos de Plantas , Punica granatum , Ratos Sprague-Dawley , Baço , Animais , Baço/efeitos dos fármacos , Baço/metabolismo , Feminino , Ratos , Punica granatum/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Momordica charantia/química , Óleos de Plantas/química , Óleos de Plantas/farmacologia , Suplementos Nutricionais , Sementes/química , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/metabolismoRESUMO
In this work, we synthesized and confirmed the structure of several alkaloid N-oxides using mass spectrometry and Fourier-transform infrared spectroscopy. We also investigated their reduction mechanisms using voltammetry. For the first time, we obtained alkaloid N-oxides using an oxidation reaction with potassium peroxymonosulfate as an oxidant. The structure was established based on the obtained fragmentation mass spectra recorded by LC-Q-ToF-MS. In the FT-IR spectra of the alkaloid N-oxides, characteristic signals of N-O group vibrations were recorded (bands in the range of 928 cmâ»1 to 971 cmâ»1), confirming the presence of this functional group. Electrochemical reduction studies demonstrated the reduction of alkaloid N-oxides at mercury-based electrodes back to the original form of the alkaloid. For the first time, the products of the electrochemical reduction of alkaloid N-oxides were detected by mass spectrometry. The findings provide insights into the structural characteristics and reduction behaviors of alkaloid N-oxides, offering implications for pharmacological and biochemical applications. This research contributes to a better understanding of alkaloid metabolism and degradation processes, with potential implications for drug development and environmental science.
Assuntos
Alcaloides , Técnicas Eletroquímicas , Oxirredução , Óxidos , Alcaloides/química , Óxidos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Estrutura Molecular , Espectrometria de Massas , EletrodosRESUMO
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
Alcohol dependence is characterized by the abnormal release of dopamine in the brain reward-related areas. Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that negatively regulates dopamine neurotransmission and thus is a promising target in the treatment of drug addiction. However, the role of TAAR1 in the regulation of alcohol abuse remains understudied. Here, we assessed the effect of TAAR1 activation on alcohol drinking behaviours of C57Bl/6J female mice housed in IntelliCages. The animals were administered with either vehicle or TAAR1 full selective agonist, RO5256390, and tested for alcohol consumption, alcohol preference and motivation for alcohol seeking. We found that mice with the highest preference for alcohol (high drinkers) in the RO5256390 group consumed less alcohol and had lower alcohol preference in comparison with high drinkers in the vehicle group, during 20 h of free alcohol access (FAA). We also found decreased alcohol consumption and alcohol preference comparing all animals in the RO5256390 to all animals in the vehicle group, during 20 h of FAA performed after the abstinence. These effects of RO5256390 lasted for the first 24 h after administration that roughly corresponded to the compound level in the brain, measured by mass spectrometry. Finally, we found that administration of RO5256390 may attenuate motivation for alcohol seeking. Taken together, our findings reveal that activation of TAAR1 may transiently reduce alcohol drinking; thus, TAAR1 is a promising target for the treatment of alcohol abuse and relapse.
Assuntos
Alcoolismo , Dopamina , Feminino , Camundongos , Animais , Receptores Acoplados a Proteínas G/agonistas , Consumo de Bebidas AlcoólicasRESUMO
BACKGROUND: The impact of maternal diet on mineral concentration in human milk (HM) remains unclear. The main aim of this study was to investigate the relationship between maternal dietary intake and calcium and phosphorus concentrations in HM. Furthermore, we aimed to evaluate the intake of both minerals by exclusively breastfed infants. METHODS: HM samples were obtained from 30 mothers at 6-8 weeks postpartum. Each mother was asked to express pre- and postfeeding milk four times during a 24-h period (6.00-12.00, 12.00-18.00, 18.00-24.00, 24.00-6.00). Maternal dietary assessment was based on a food frequency questionnaire and 3-day dietary records. Analysed minerals were determined using an inductively coupled plasma mass spectrometer (NexION 300D ICP mass spectrometer, Perkin Elmer SCIEX). RESULTS: The mean concentrations of calcium and phosphorus in HM samples were 278.7 ± 61.0 and 137.1 ± 21.9 mg/L, respectively, maintaining 2:1 ratio by weight. The concentration of both minerals was correlated with each other (r = 0.632, p = <0.001). The infants' mean calcium intake was 149.53 ± 36.41 mg/L, and their mean phosphorus intake was 74.62 ± 19.41 mg/L. The risk of insufficient intake of calcium was reported in 60% of infants (n = 18). Spearman's/Pearson's correlation coefficients did not reveal any correlations between HM calcium concentration and maternal diet, contrary to HM phosphorus concentration, which was positively correlated with energy (r = 0.369, p = 0.045), total protein (r = 0.464, p = 0.01), calcium (r = 385, p = 0.036), phosphorus (r = 501, p = 0.005), niacin (p < 0.001) and pyridoxine (r = 382, 0.037) intake. However, in multivariable analysis we observed that maternal dietary intake of both minerals had a positive influence on their concentration in HM. CONCLUSIONS: Maternal calcium and phosphorus intake influenced the concentration of both minerals in HM; however, the relationship was rather weak. In addition, we observed that calcium intake by most of the exclusively breastfed infants was insufficient to meet the recommended daily intake.
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Leite Humano , Fósforo na Dieta , Lactente , Feminino , Humanos , Leite Humano/metabolismo , Aleitamento Materno , Cálcio/análise , Cálcio/metabolismo , Fósforo/análise , Fósforo/metabolismo , Cálcio da Dieta , Minerais/análise , Minerais/metabolismo , Dieta , LactaçãoRESUMO
Significant advances in the technological development of mass spectrometry in the field of proteomics and the generation of extremely large amounts of data require a very critical approach to assure the validity of results. Commonly used procedures involved liquid chromatography followed by high-resolution mass spectrometry measurements. Proteomics analysis is used in many fields including the investigation of the metabolism of biologically active substances in organisms. Thus, there is a need to care about the validity of the obtained results. In this work, we proposed a standardized protocol for proteomic analysis using liquid chromatography-high-resolution mass spectrometry, which covers all of these analytical steps to ensure the validity of the results. For this purpose, we explored the requirements of the ISO/IEC 17025:2017 standard as a reference document for quality control in biochemistry research-based mass spectrometry.
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Proteômica , Proteômica/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida/métodos , Controle de QualidadeRESUMO
Boron has gained significant attention in medical research due to its B-10 isotope's high cross section for the reaction with thermal neutrons, generating ionizing particles that can eliminate cancer cells, propelling the development of boron neutron capture therapy (BNCT) for cancer treatment. The compound 4-borono-L-phenylalanine (BPA) has exhibited potential in BNCT clinical trials. Enhancing BPA uptake in cells involves proposing L-amino acid preloading. This study introduces a novel analytical strategy utilizing ICP-MS and single cell ICP-MS (SC-ICP-MS) to assess the effectiveness of L-tyrosine and L-phenylalanine preloading on human non-small cell lung carcinoma (A549) and normal Chinese hamster lung fibroblast (V79-4) models, an unexplored context. ICP-MS outcomes indicated that L-tyrosine and L-phenylalanine pre-treatment increased BPA uptake in V79-4 cells by 2.04 ± 0.74-fold (p = 0.000066) and 1.46 ± 0.06-fold (p = 0.000016), respectively. Conversely, A549 cells manifested heightened BPA uptake solely with L-tyrosine preloading, with a factor of 1.24 ± 0.47 (p = 0.028). BPA uptake remained higher in A549 compared to V79-4 regardless of preloading. SC-ICP-MS measurements showcased noteworthy boron content heterogeneity within A549 cells, signifying diverse responses to BPA exposure, including a subset with notably high BPA uptake. This study underscores SC-ICP-MS's utility in precise cellular boron quantification, validating cellular BPA uptake's heterogeneity.
Assuntos
Terapia por Captura de Nêutron de Boro , Fenilalanina , Cricetinae , Animais , Humanos , Fenilalanina/química , Tirosina , Boro/farmacologia , Análise Espectral , Compostos de Boro/químicaRESUMO
BACKGROUND Cinacalcet is a calcium-sensing receptor agonist that is clinically approved for the treatment of secondary hyperparathyroidism in chronic kidney disease and hypercalcemia in patients with parathyroid carcinoma. This study aimed to use quantitative mass spectrometry-based label-free proteomics to evaluate the effects of cinacalcet on protein expression in rat brains and livers. MATERIAL AND METHODS We randomly assigned 18 Wistar rats to 2 groups: an untreated control group (n=6) and a group treated with cinacalcet at a dose corresponding to the maximum dose used in humans (2 mg/kg/body weight, 5 days/week) divided into 7-day (n=6) and 21-day (n=6) treatment subgroups. A mass-spectrometry-based label-free quantitative proteomics approach using peptides peak area calculation was used to evaluate the changes in protein expression in examined tissues. Bioinformatics analysis of quantitative proteomics data was done using MaxQuant and Perseus environment. RESULTS No changes in protein expression were revealed in the 7-day treatment subgroup. We detected 10 upregulated and 3 downregulated proteins in the liver and 1 upregulated protein in the brain in the 21-day treatment subgroup compared to the control group. Based on Gene Ontology classification, all identified differentially expressed proteins were indicated as molecular functions involved in the enzyme regulator activity (36%), binding (31%), and catalytic activity (19%). CONCLUSIONS These findings indicate that long-term cinacalcet therapy can impair phase II of enzymatic detoxication and can cause disturbances in blood hemostasis, lipid metabolism, and inflammatory mediators or contribute to the acceleration of cognitive dysfunction; therefore, appropriate patient monitoring should be considered.
Assuntos
Proteômica , Receptores de Detecção de Cálcio , Animais , Encéfalo/metabolismo , Cálcio , Cinacalcete/farmacologia , Cinacalcete/uso terapêutico , Humanos , Fígado/metabolismo , Espectrometria de Massas , Naftalenos , Hormônio Paratireóideo , Ratos , Ratos Wistar , Receptores de Detecção de Cálcio/metabolismoRESUMO
In many pharmaceuticals, a hydrogen atom or hydroxyl group is replaced by a fluorine to increase bioavailability and biostability. The fate of fluorine released from fluorine-containing drugs is not well investigated. The aim of this study was to examine possible fluorination of proteins in rat liver and brain after administration of the fluorinated drug cinacalcet. We assigned 18 Wistar rats to a control group (n = 6) and a group treated with cinacalcet (2 mg kg-1/body weight, 5 days/week), divided into 7 day (n = 6) and 21 day (n = 6) treatment subgroups. Fluorinated proteins were identified using a free proteomics approach; chromatographic separation and analysis by high-resolution mass spectrometry; peptide/protein identification using the Mascot search algorithm; manual verification of an experimentally generated MS/MS spectrum with the theoretical MS/MS spectrum of identified fluorinated peptides. Three fluorinated proteins (spectrin beta chain; carbamoyl-phosphate synthase [ammonia], mitochondrial; 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 1) were identified in the liver and four (spectrin beta chain, dihydropyrimidinase-related protein 4, prominin-2, dihydropyrimidinase-related protein 4) in the brain tissue after 21 days of cinacalcet treatment, but not in the control group. Introduction of fluorine into an organism by administration of fluorinated drugs results in tissue-specific fluorination of proteins.
Assuntos
Flúor , Halogenação , Animais , Encéfalo , Cinacalcete , Fluoretos , Flúor/química , Fígado , Preparações Farmacêuticas , Ratos , Ratos Wistar , Espectrina , Espectrometria de Massas em TandemRESUMO
In this work, a method for the accurate and precise determination of the Ge isotope ratio in synthetic water and natural samples of geological origin using multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) with hydride generation was developed. The method was based on the liquid-liquid extraction of Ge to eliminate all elements affecting the generation of germanium hydrides. The standard-sample bracketing method was used to correct instrumental bias. Registration of analytical signal in time-resolved mode gave way to choose signals with best parameters and improved the precision of the results. Controlling the pH by using acetic buffer boosted the sensitivity by nearly five times in comparison to hydride generation methods suggested by other authors. The newly developed method is much simpler and quicker, does not need laborious Ge separation with ion-exchange resins, and thanks to its superior sensitivity, allows measurements of the Ge isotopic ratio in materials with relatively low Ge content. Delta values of the 74Ge/70Ge isotope ratio were measured in standard reference materials for which reference values were available in the GeoREM database. We demonstrated that the accuracy and precession of this method are equally good or better than methods proposed by other authors.
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Isótopos , Extração Líquido-Líquido , Isótopos/análise , Espectrometria de Massas , Análise EspectralRESUMO
The aim of the study was to verify in a cardio-oncological model experiment if conjugated linoleic acids (CLA) fed to rats with mammary tumors affect the content of selected macro- and microelements in their myocardium. The diet of Sprague-Dawley females was supplemented either with CLA isomers or with safflower oil. In hearts of rats suffering from breast cancer, selected elements were analyzed with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). In order to better understand the data trends, cluster analysis, principal component analysis and linear discriminant analysis were applied. Mammary tumors influenced macro- and microelements content in the myocardium to a greater extent than applied diet supplementation. Significant influences of diet (p = 0.0192), mammary tumors (p = 0.0200) and interactions of both factors (p = 0.0151) were documented in terms of Fe content. CLA significantly decreased the contents of Cu and Mn (p = 0.0158 and p = 0.0265, respectively). The level of Ni was significantly higher (p = 0.0073), which was more pronounced in groups supplemented with CLA. The obtained results confirmed antioxidant properties of CLA and the relationship with Se deposition. Chemometric techniques distinctly showed that the coexisting pathological process induced differences to the greater extent than diet supplementation in the elemental content in the myocardium, which may impinge on cardiac tissue's susceptibility to injuries.
Assuntos
Antioxidantes/farmacologia , Ácidos Linoleicos Conjugados/farmacologia , Neoplasias Mamárias Animais/dietoterapia , Miocárdio/química , Animais , Quimiometria/métodos , Cobre/química , Cobre/isolamento & purificação , Feminino , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Mamárias Animais/química , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Manganês/química , Manganês/isolamento & purificação , Espectrometria de Massas , Miocárdio/metabolismo , Níquel/química , Níquel/isolamento & purificação , Ratos , Selênio/química , Selênio/isolamento & purificaçãoRESUMO
In this work, a method for the accurate and precise determination of 82Se/78Se isotope ratio in natural samples of environmental and biological origin, using multicollector inductively coupled plasma mass spectrometry in a wet plasma mode without using neither hydride generation nor separation of Se, was developed. It was based on the optimized regression model with standard-sample bracketing (ORM-SSB) to efficiently correct instrumental isotopic fractionation/mass bias and matrix effects. In addition, three mass bias correction models of SSB alone, SSB combined with internal standard (IS-SSB), and ORM-SSB were compared for the Se isotope ratio measurements. NIST SRM 987 Sr was used as an internal standard, and the reproducibility of the results obtained with the proposed method was verified by measuring NIST SRM 3149 standard over different days (nine independent measurement sessions). Delta values of the 82Se/78Se isotope ratio were measured in selenium-enriched yeast-certified reference material SELM-1, natural selenomethionine samples, and model solutions of artificial seawater. Solutions obtained after thiol resin treatment were measured to demonstrate the applicability of the proposed method in eliminating matrix effects due to residual of thiol resin in the sample solutions. Among three mass bias correction models, ORM-SSB correction model proved to be the best to eliminate the matrix effects and instrumental drift. IS-SSB model offered also a good precision but was slightly less accurate. Both models showed good robustness against effects of different sample matrices. Finally, the SSB alone could not be recommended for Se isotope analysis as it produces inaccurate and imprecise results.
RESUMO
Matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix, make a major contribution to the progression of a vast number of diseases, such cancer or epilepsy. Although several MMP inhibitors (MMPi) have been developed to date for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and, most importantly, the presence of severe side effects. The latter can be explained by their lack of selectivity of these inhibitors. In this regard, MMPs' family members have a high structural homology, which challenge the development of selective inhibitors for a specific MMP. Here, we have used in silico calculations and in vitro data to design MMPi that selectively target gelatinases (MMP-2 and MMP-9) and have the capacity to cross the blood-brain barrier. Following this approach, we obtained compound 40 that shows high proteolytic stability and low cytotoxicity. This compound may be of particular interest for the treatment of central nervous diseases such epilepsy or Alzheimer's disease, where gelatinase activity is increased. Our data show the specificity of compound 40 for recombinant MMP-9 and MMP-2 and endogenous MMP-9 from rat hippocampal cell cultures, and reveals its permeability across the blood-brain barrier in vivo.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Desenho de Fármacos , Gelatinases/antagonistas & inibidores , Ácidos Hidroxâmicos/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Gelatinases/metabolismo , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/química , Estrutura Molecular , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A fit for purpose analytical protocol was designed towards searching for low molecular weight seleno-compounds in sprouts. Complementary analytical techniques were used to collect information enabling the characterization of selenium speciation. Conceiving the overall characterization of the behavior of selenium, inductively plasma optical mass spectrometry (ICP-MS) was used to determine the total selenium content in entire sprouts as well as in selected extracts or chromatographic fractions. Then, high-performance liquid chromatography combined with ICP-MS (HPLC-ICP-MS) was used to evaluate the presence of inorganic and organic seleno-compounds, with the advantages of being very sensitive towards selenium, but limited by available selenium standard compounds. Finally, ultra-high performance liquid chromatography electrospray ionization triple quadrupole mass spectrometry (UHPLC-ESI-QqQ-MS/MS) and UHPLC-ESI-Orbitrap-MS/MS were used for the confirmation of the identity of selected compounds and identification of several unknown compounds of selenium in vegetable sprouts (sunflower, onion, radish), respectively. Cultivation of plants was designed to supplement sprouts with selenium by using solutions of selenium (IV) at the concentration of 10, 20, 40, and 60 mg/L. The applied methodology allowed to justify that vegetable sprouts metabolize inorganic selenium to a number of organic derivatives, such as seleno-methylselenocysteine (SeMetSeCys), selenomethionine (SeMet), 5'-seleno-adenosine, 2,3-DHP-selenolanthionine, Se-S conjugate of cysteine-selenoglutathione, 2,3-DHP-selenocysteine-cysteine, 2,3-DHP-selenocysteine-cysteinealanine, glutathione-2,3-DHP-selenocysteine, gamma-Glu-MetSeCys or glutamyl-glycinyl-N-2,3-DHP-selenocysteine.
Assuntos
Extratos Vegetais/química , Plântula/química , Compostos de Selênio/química , Selênio/química , Cisteína/química , Peso Molecular , Espectrometria de Massas em TandemRESUMO
Relatively few studies have been focused so far on magnesium-isotope fractionation during plant growth, element uptake from soil, root-to-leaves transport and during chlorophylls biosynthesis. In this work, maize and garden cress were hydroponically grown in identical conditions in order to examine if the carbon fixation pathway (C4, C3, respectively) might have impact on Mg-isotope fractionation in chlorophyll-a. The pigment was purified from plants extracts by preparative reversed phase chromatography, and its identity was confirmed by high-resolution mass spectrometry. The green parts of plants and chlorophyll-a fractions were acid-digested and submitted to ion chromatography coupled through desolvation system to multiple collector inductively coupled plasma-mass spectrometry. Clear preference for heavy Mg-isotopes was found in maize green parts (∆26Mgplant-nutrient 0.65, 0.74 for two biological replicates, respectively) and in chlorophyll-a (∆26Mgchlorophyll-plant 1.51, 2.19). In garden cress, heavy isotopes were depleted in green parts (∆26Mgplant-nutrient (-0.87)-(-0.92)) and the preference for heavy isotopes in chlorophyll-a was less marked relative to maize (∆26Mgchlorophyll-plant 0.55-0.52). The observed effect might be ascribed to overall higher production of energy in form of adenosine triphosphate (ATP), required for carbon fixation in C4 compared to C3, which could reduce kinetic barrier and make equilibrium fractionation prevailing during magnesium incorporation to protoporphyrin ring.
Assuntos
Clorofila A/análise , Lepidium sativum/crescimento & desenvolvimento , Magnésio/química , Zea mays/crescimento & desenvolvimento , Ciclo do Carbono , Fracionamento Químico , Clorofila A/química , Cromatografia de Fase Reversa , Hidroponia , Isótopos/química , Lepidium sativum/química , Extratos Vegetais/análise , Extratos Vegetais/química , Zea mays/químicaRESUMO
TAR DNA-binding protein 43 (TDP-43) is a hallmark of some neurodegenerative disorders, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43-related pathology is characterized by its abnormally phosphorylated and ubiquitinated aggregates. It is involved in many aspects of RNA processing, including mRNA splicing, transport, and translation. However, its exact physiological function and role in mechanisms that lead to neuronal degeneration remain elusive. Transgenic rats that were characterized by TDP-43 depletion in neurons exhibited enhancement of the acquisition of fear memory. At the cellular level, TDP-43-depleted neurons exhibited a decrease in the short-term plasticity of intrinsic neuronal excitability. The induction of long-term potentiation in the CA3-CA1 areas of the hippocampus resulted in more stable synaptic enhancement. At the molecular level, the protein levels of an unedited (R) FLOP variant of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR1 and GluR2/3 subunits decreased in the hippocampus. Alterations of FLOP/FLIP subunit composition affected AMPAR kinetics, reflected by cyclothiazide-dependent slowing of the decay time of AMPAR-mediated miniature excitatory postsynaptic currents. These findings suggest that TDP-43 may regulate activity-dependent neuronal plasticity, possibly by regulating the splicing of genes that are responsible for fast synaptic transmission and membrane potential.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Hipocampo/metabolismo , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Espinhas Dendríticas/metabolismo , Ratos , Ratos Transgênicos , Receptores de AMPA/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Highly oxygenated molecules (HOMs) are a class of compounds associated with secondary organic aerosols exhibiting high oxygen to carbon (O:C) ratios and often originating from the oxidation of biogenic compounds. Here, the photooxidation and ozonolysis of isoprene were examined under a range of conditions to identify HOM tracers for aged isoprene aerosol. The HOM tracers were identified as silylated derivatives by gas chromatography-mass spectrometry and by detecting their parent compounds by liquid chromatography-high resolution mass spectrometry. In addition to the previously observed methyltetrols and 2-methylglyceric acid, seven tracer compounds were identified, including 2-methyltartronic acid (MTtA), 2-methylerythronic acid (2MeTrA), 3-methylerythronic acid (3MeTrA), 2-methylthreonic acid (2MTrA), 3-methylthreonic acid (3MTrA), erythro-methyltartaric acid (e-MTA), and threo-methyltartaric acid (t-MTA). The molecular structures were confirmed with authentic standards synthesized in the laboratory. The presence of some of these HOMs in the gas and particle phases simultaneously provides evidence of their gas/particle partitioning. To determine the contributions of aged isoprene products to ambient aerosols, we analyzed ambient PM2.5 samples collected in the southeastern United States in summer 2003 and at two European monitoring stations located in Zielonka and Godów (Poland). Our findings show that methyltartaric acids (MTA) and 2- and 3-methylthreonic acids (and their stereoisomers) are representative of aged isoprene aerosol because they occur both in the laboratory chamber aerosol obtained and in ambient PM2.5. On the basis of gas chromatography-mass spectrometry (GC-MS) analysis, their concentrations were found to range from 0.04 ng for 3-methylthreonic acid to 6.3 ng m-3 for methyltartaric acid at the southeast site in Duke Forest, NC, USA.
Assuntos
Poluentes Atmosféricos , Hemiterpenos , Aerossóis , Butadienos , Hidroxiácidos , Pentanos , Sudeste dos Estados UnidosRESUMO
Aldehyde dehydrogenase 3B2 (ALDH3B2) gene contains a premature termination codon, which can be skipped or suppressed resulting in full-length protein expression. Alternatively, the longest putative open reading frame starting with the second in-frame start codon would encode short isoform. No unequivocal evidence of ALDH3B2 expression in healthy human tissues is available. The aim of this study was to confirm its expression in human placenta characterized by the highest ALDH3B2 mRNA abundance. ALDH3B2 DNA and mRNA were sequenced. The expression was investigated using western blot. The identity of the protein was confirmed using mass spectrometry (MS). The predicted tertiary and quaternary structures, subcellular localization, and phosphorylation sites were assessed using bioinformatic analyses. All DNA and mRNA isolates contained the premature stop codon. In western blot analyses, bands corresponding to the mass of full-length protein were detected. MS analysis led to the identification of two unique peptides, one of which is encoded by the nucleotide sequence located upstream the second start codon. Bioinformatic analyses suggest cytoplasmic localization and several phosphorylation sites. Despite premature stop codon in DNA and mRNA sequences, full-length ALDH3B2 was found. It can be formed as a result of premature stop codon readthrough, complex phenomenon enabling stop codon circumvention.