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1.
Gene ; 893: 147927, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38374023

RESUMO

Recent semi-targeted metabolomics studies have highlighted a number of metabolites in wheat that associate with leaf rust resistance genes and/or rust infection. Here, we report the structural characterization of a novel glycosylated and partially saturated apocarotenoid, reminiscent of a reduced form of mycorradicin, (6E,8E,10E)-4,9-dimethyl-12-oxo-12-((3,4,5-trihydroxy-6-(2-hydroxyethoxy)tetrahydro-2H-pyran-2-yl)methoxy)-3-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)dodeca-6,8,10-trienoic acid, isolated from Triticum aestivum L. (Poaceae) variety 'Thatcher' (Tc) flag leaves. While its accumulation was not associated with any of Lr34, Lr67 or Lr22a resistance genes, infection of Tc with leaf rust was found to deplete it, consistent with the idea of this metabolite being a glycosylated-storage form of an apocarotenoid of possible relevance to plant defense. A comparative analysis of wheat transcriptomic changes shows modulation of terpenoid, carotenoid, UDP-glycosyltransferase and glycosylase -related gene expression profiles, consistent with anticipated biosynthesis and degradation mechanisms. However, details of the exact nature of the relevant pathways remain to be validated in the future. Together these findings highlight another example of the breadth of unique metabolites underlying plant host-fungal pathogen interactions.


Assuntos
Basidiomycota , Triticum , Triticum/genética , Triticum/microbiologia , Resistência à Doença/genética , Plantas , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Piranos
2.
Molecules ; 28(4)2023 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-36838644

RESUMO

To address the growing concern of honey adulteration in Canada and globally, a quantitative NMR method was developed to analyze 424 honey samples collected across Canada as part of two surveys in 2018 and 2019 led by the Canadian Food Inspection Agency. Based on a robust and reproducible methodology, NMR data were recorded in triplicate on a 700 MHz NMR spectrometer equipped with a cryoprobe, and the data analysis led to the identification and quantification of 33 compounds characteristic of the chemical composition of honey. The high proportion of Canadian honey in the library provided a unique opportunity to apply multivariate statistical methods including PCA, PLS-DA, and SIMCA in order to differentiate Canadian samples from the rest of the world. Through satisfactory model validation, both PLS-DA as a discriminant modeling technique and SIMCA as a class modeling method proved to be reliable at differentiating Canadian honey from a diverse set of honeys with various countries of origins and floral types. The replacement method of optimization was successfully applied for variable selection, and trigonelline, proline, and ethanol at a lower extent were identified as potential chemical markers for the discrimination of Canadian and non-Canadian honeys.


Assuntos
Mel , Mel/análise , Espectroscopia de Ressonância Magnética/métodos , Imageamento por Ressonância Magnética , Prolina , Canadá , Análise Multivariada
3.
Molecules ; 27(18)2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36144592

RESUMO

Polar lipids were extracted from residual biomass of hemp (Cannabis sativa L.) by-products with EtOH and partitioned into aqueous and chloroform fractions. The chloroform fractions were studied for their lipid composition using solid-phase extraction (SPE) followed by UHPLC/HRMS and NMR analyses. The 1H NMR and gravimetric yield of SPE indicated triacylglycerols covered ≥ 51.3% of the chloroform fraction of hemp seed hulls and hemp cake. UHPLC/HRMS analyses of remaining polar lipids led to the identification of nine diacylglycerols (DAGs), six lysophosphatidylcholines (LPCs), five lysophosphatidylethanolamines (LPEs), eight phosphatidylethanolamines (PEs), and thirteen phosphatidylcholines (PCs) for the first time from hemp seed hulls. The regiospecificity of fatty acyl substitutes in glycerol backbone of individual phospholipids were assigned by analyzing the diagnostic fragment ions and their intensities. The heat-map analysis suggested that DAG 18:2/18:2, 1-LPC 18:2, 1-LPE 18:2, PE 18:2/18:2, and PC 18:2/18:2 were the predominant molecules within their classes, supported by the fact that linoleic acid was the major fatty acid covering > 41.1% of the total fatty acids determined by GC-FID analysis. The 31P NMR analysis confirmed the identification of phospholipids and suggested PC covers ≥ 37.9% of the total phospholipid present in hemp by-products. HPLC purification led to the isolation of 1,2-dilinoleoylphosphatidylcholine and 1-palmitoyl-2-linoleoylphosphatidylcholine. These two major PCs further confirmed the UHPLC/HRMS finding.


Assuntos
Cannabis , Cannabis/química , Clorofórmio , Cromatografia Líquida de Alta Pressão , Diglicerídeos , Ácidos Graxos , Cromatografia Gasosa-Espectrometria de Massas , Glicerol/análise , Ácidos Linoleicos , Lisofosfatidilcolinas , Espectrometria de Massas , Fosfatidilcolinas/química , Fosfatidiletanolaminas , Fosfolipídeos/análise , Triglicerídeos
4.
J Agric Food Chem ; 68(49): 14643-14651, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33252222

RESUMO

In response to the need from the food industry for new analytical solutions, a fit-for-purpose quantitative 1H NMR methodology was developed to authenticate pure coffee (100% arabica or robusta) as well as predict the percentage of robusta in blends through the study of 292 roasted coffee samples in triplicate. Methanol was chosen as the extraction solvent, which led to the quantitation of 12 coffee constituents: caffeine, trigonelline, 3- and 5-caffeoylquinic acid, lipids, cafestol, nicotinic acid, N-methylpyridinium, formic acid, acetic acid, kahweol, and 16-O-methylcafestol. To overcome the chemical complexity of the methanolic extract, quantitative analysis was performed using a combination of traditional integration and spectral deconvolution methods. As a result, the proposed methodology provides a systematic methodology and a linear regression model to support the classification of known and unknown roasted coffees and their blends.


Assuntos
Coffea/química , Espectroscopia de Ressonância Magnética/métodos , Alcaloides/análise , Cafeína/análise , Coffea/classificação , Café/química , Culinária , Análise Discriminante , Diterpenos/análise , Contaminação de Alimentos/análise , Sementes/química , Sementes/classificação
5.
Phytochemistry ; 178: 112456, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32692663

RESUMO

The gene Lr34res is one of the most long-lasting sources of quantitative fungal resistance in wheat. It is shown to be effective against leaf, stem, and stripe rusts, as well as powdery mildew and spot blotch. Recent biochemical characterizations of the encoded ABC transporter have outlined a number of allocrites, including phospholipids and abscisic acid, consistent with the established general promiscuity of ABC transporters, but ultimately leaving its mechanism of rust resistance unclear. Working with flag leaves of Triticum aestivum L. variety 'Thatcher' (Tc) and a near-isogenic line of 'Thatcher' into which the Lr34res allele was introgressed (Tc+Lr34res; RL6058), a comparative semi-targeted metabolomics analysis of flavonoid-rich extracts revealed virtually identical profiles with the exception of one metabolite accumulating in Tc+Lr34res, which was not present at comparable levels in Tc. Structural characterization of the purified metabolite revealed a phenylpropanoid diglyceride structure, 1-O-p-coumaroyl-3-O-feruloylglycerol (CFG). Additional profiling of CFG across a collection of near-isogenic lines and representative Lr34 haplotypes highlighted a broad association between the presence of Lr34res and elevated accumulations of CFG. Depletion of CFG upon infection, juxtaposed to its relatively lower anti-fungal activity, suggests CFG may serve as a storage form of the more potent anti-microbial hydroxycinnamic acids that are accessed during defense responses. Altogether these findings suggest a role for the encoded LR34res ABC transporter in modifying the accumulation of CFG, leading to increased accumulation of anti-fungal metabolites, essentially priming the wheat plant for defense.


Assuntos
Ascomicetos , Basidiomycota , Diglicerídeos , Resistência à Doença , Doenças das Plantas , Triticum
6.
J Nat Prod ; 80(10): 2630-2643, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-29035048

RESUMO

This report describes an approach using 1H NMR iterative full-spin analysis (HiFSA) to extract definitive structural information on unknown peptides from 1D 1H NMR data. By comparing the experimental data and HiFSA fingerprint of a known analogue, it is possible to isolate the characteristic 1H subspectrum of the different amino acids and, thus, elucidate the structure of the peptide. To illustrate this methodology, a comprehensive analysis of five new anti-Mycobacterium tuberculosis peptides (2-6), all analogues of ecumicin (1), was carried out. The method was validated by demonstrating congruence of the HiFSA-based structures with all available data, including MS and 2D NMR. The highly reproducible HiFSA fingerprints of the new ∼1600 amu peptides were generated in this process. Besides oligo-peptides, the HiFSA sequencing approach could be extended to all oligomeric compounds consisting of chains of monomers lacking H-H spin-spin coupling across the moieties. HiFSA sequencing is capable of differentiating complex oligomers that exhibit minor structural differences such as shifted hydoxyl or methyl groups. Because it employs the basic and most sensitive 1D 1H NMR experiment, HiFSA sequencing enables the exploration of peptide analogues up to at least 2000 amu, even with basic contemporary spectrometers and when only sub-milligram amounts of isolates are available.


Assuntos
Antituberculosos/isolamento & purificação , Oligopeptídeos/química , Prótons , Antituberculosos/química , Antituberculosos/farmacologia , Estrutura Molecular , Mycobacterium tuberculosis/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação
7.
Harmful Algae ; 63: 85-93, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28366404

RESUMO

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.


Assuntos
Ácido Okadáico/análise , Piranos/análise , Intoxicação por Frutos do Mar , Animais , Dinoflagelados/metabolismo , Toxinas Marinhas/análise
8.
J AOAC Int ; 99(5): 1151-62, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27524810

RESUMO

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.


Assuntos
Diarreia/complicações , Toxinas Marinhas/análise , Ácido Okadáico/análise , Piranos/análise , Padrões de Referência , Intoxicação por Frutos do Mar/complicações , Animais , Calibragem , Cromatografia Líquida/normas , Humanos , Espectroscopia de Ressonância Magnética/normas , Frutos do Mar , Espectrometria de Massas em Tandem/normas
9.
Mar Drugs ; 13(6): 3849-76, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26096274

RESUMO

Northern shrimp (Pandalus borealis) oil, which is rich in omega-3 fatty acids, was recovered from the cooking water of shrimp processing facilities. The oil contains significant amounts of omega-3 fatty acids in triglyceride form, along with substantial long-chain monounsaturated fatty acids (MUFAs). It also features natural isomeric forms of astaxanthin, a nutritional carotenoid, which gives the oil a brilliant red color. As part of our efforts in developing value added products from waste streams of the seafood processing industry, we present in this paper a comprehensive characterization of the triacylglycerols (TAGs) and astaxanthin esters that predominate in the shrimp oil by using HPLC-HRMS and MS/MS, as well as 13C-NMR. This approach, in combination with FAME analysis, offers direct characterization of fatty acid molecules in their intact forms, including the distribution of regioisomers in TAGs. The information is important for the standardization and quality control, as well as for differentiation of composition features of shrimp oil, which could be sold as an ingredient in health supplements and functional foods.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Óleos/análise , Pandalidae/química , Espectrometria de Massas em Tandem/métodos , Animais , Ácidos Graxos Ômega-3/análise , Ácidos Graxos Ômega-3/isolamento & purificação , Espectroscopia de Ressonância Magnética/métodos , Óleos/química , Óleos/isolamento & purificação , Triglicerídeos/análise , Triglicerídeos/química , Triglicerídeos/isolamento & purificação , Xantofilas/análise , Xantofilas/química , Xantofilas/isolamento & purificação
10.
J Proteome Res ; 10(11): 5102-17, 2011 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-21910437

RESUMO

One of the greatest strengths of "-omics" technologies is their ability to capture a molecular snapshot of multiple cellular processes simultaneously. Transcriptomics, proteomics, and metabolomics have, individually, been used in wide-ranging studies involving cell lines, tissues, model organisms, and human subjects. Nonetheless, despite the fact that their power lies in the global acquisition of parallel data streams, these methods continue to be employed separately. We highlight work done to merge transcriptomics and metabolomics technologies to study zebrafish (Danio rerio) embryogenesis. We combine information from three bioanalytical platforms, that is, DNA microarrays, (1)H nuclear magnetic resonance ((1)H NMR), and mass spectrometry (MS)-based metabolomics, to identify and provide insights into the organism's developmental regulators. We apply a customized approach to the analysis of such time-ordered measurements to provide temporal profiles that depict the modulation of metabolites and gene transcription. Initially, the three data sets were analyzed individually but later they were fused to highlight the advantages gained through such an integrated approach. Unique challenges posed by fusion of such data are discussed given differences in the measurement error structures, the wide dynamic range for the molecular species, and the analytical platforms used to measure them (i.e., fluorescence ratios, NMR, and MS intensities). Our data analysis reveals that changes in transcript levels at specific developmental stages correlate with previously published data with over 90% accuracy. In addition, transcript profiles exhibited trends that were similar to the accumulation of metabolites over time. Profiles for metabolites such as choline-like compounds (Trimethylamine-N-oxide, phosphocholine, betaine), creatinine/creatine, and other metabolites involved in energy metabolism exhibited a steady increase from 15 hours post fertilization (hpf) to 48 hpf. Other metabolite and transcript profiles were transiently rising and then falling back to baseline. The "house keeping" metabolites such as branched chain amino acids exhibited a steady presence throughout embryogenesis. Although the transcript profiling corresponds to only 16 384 genes, a subset of the total number of genes in the zebrafish genome, we identified examples where gene transcript and metabolite profiles correlate with one another, reflective of a relationship between gene and metabolite regulation over the course of embryogenesis.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos , Peixe-Zebra/embriologia , Algoritmos , Aminoácidos/metabolismo , Animais , Blástula/metabolismo , Proteínas de Peixes/genética , Gástrula/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Metabolômica , Análise Multivariada , Análise de Componente Principal , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
11.
Mol Biosyst ; 7(7): 2181-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21547298

RESUMO

Urinary tract obstruction (UTO) results in renal compensatory mechanisms and may progress to irrecoverable functional loss and histologic alterations. The pathophysiology of this progression is poorly understood. We identified urinary metabolite alterations in a rodent model of partial and complete UTO using (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy. Principal component analysis (PCA) was used for classification and discovery of differentiating metabolites. UTO was associated with elevated urinary levels of alanine, succinate, dimethylglycine (DMG), creatinine, taurine, choline-like compounds, hippurate, and lactate. Decreased urinary levels of 2-oxoglutarate and citrate were noted. The patterns of alteration in partial and complete UTO were similar except that an absence of elevated urinary osmolytes (DMG and hippurate) was noted in complete UTO. This pattern of metabolite alteration indicates impaired oxidative metabolism of the mitochondria in renal proximal tubules and production of renal protective osmolytes by the medulla. Decreased production of osmolytes in complete obstruction better elucidates the pathophysiology of progression from renal compensatory mechanisms to irrecoverable changes. Further confirmation of these potential biomarkers in children with UTO is necessary.


Assuntos
Metaboloma , Obstrução Ureteral/metabolismo , Obstrução Ureteral/urina , Animais , Creatinina/sangue , Modelos Animais de Doenças , Feminino , Espectroscopia de Ressonância Magnética , Masculino , Concentração Osmolar , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Obstrução Ureteral/sangue , Obstrução Ureteral/patologia
12.
Anal Bioanal Chem ; 398(5): 2243-52, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20827466

RESUMO

The production and certification of a series of azaspiracid (AZA) calibration solution reference materials is described. Azaspiracids were isolated from contaminated mussels, purified by preparative liquid chromatography and dried under vacuum to the anhydrous form. The purity was assessed by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The final concentration of each AZA in a CD(3)OH stock solution was determined by quantitative NMR spectroscopy. This solution was then diluted very accurately in degassed, high purity methanol to a concentration of 1.47 ± 0.08 µmol/L for CRM-AZA1, 1.52 ± 0.05 µmol/L for CRM-AZA2, and 1.37 ± 0.13 µmol/L for CRM-AZA3. Aliquots were dispensed into argon-filled glass ampoules, which were immediately flame-sealed. The calibration solutions are suitable for method development, method validation, calibration of liquid chromatography or mass spectrometry instrumentation and quality control of shellfish monitoring programs.


Assuntos
Furanos/análise , Toxinas Marinhas/análise , Piranos/análise , Frutos do Mar , Compostos de Espiro/análise , Animais , Bivalves/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Soluções/química
13.
Magn Reson Chem ; 47 Suppl 1: S96-104, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19731396

RESUMO

The global analysis of metabolites can be used to define the phenotypes of cells, tissues or organisms. Classifying groups of samples based on their metabolic profile is one of the main topics of metabolomics research. Crisp clustering methods assign each feature to one cluster, thereby omitting information about the multiplicity of sample subtypes. Here, we present the application of fuzzy K-means clustering method for the classification of samples based on metabolomics 1D (1)H NMR fingerprints. The sample classification was performed on NMR spectra of cancer cell line extracts and of urine samples of type 2 diabetes patients and animal models. The cell line dataset included NMR spectra of lipophilic cell extracts for two normal and three cancer cell lines with cancer cell lines including two invasive and one non-invasive cancers. The second dataset included previously published NMR spectra of urine samples of human type 2 diabetics and healthy controls, mouse wild type and diabetes model and rat obese and lean phenotypes. The fuzzy K-means clustering method allowed more accurate sample classification in both datasets relative to the other tested methods including principal component analysis (PCA), hierarchical clustering (HCL) and K-means clustering. In the cell line samples, fuzzy clustering provided a clear separation of individual cell lines, groups of cancer and normal cell lines as well as non-invasive and invasive tumour cell lines. In the diabetes dataset, clear separation of healthy controls and diabetics in all three models was possible only by using the fuzzy clustering method.


Assuntos
Algoritmos , Metabolômica , Urina/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Lógica Fuzzy , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Análise de Componente Principal , Ratos
14.
Environ Sci Technol ; 43(1): 219-25, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19209610

RESUMO

Several fundamental requirements must be met so that NMR-based metabolomics and the related technique of metabonomics can be formally adopted into environmental monitoring and chemical risk assessment. Here we report an intercomparison exercise which has evaluated the effectiveness of 1H NMR metabolomics to generate comparable data sets from environmentally derived samples. It focuses on laboratory practice that follows sample collection and metabolite extraction, specifically the final stages of sample preparation, NMR data collection (500, 600, and 800 MHz), data processing, and multivariate analysis. Seven laboratories have participated from the U.S.A., Canada, U.K., and Australia, generating a total of ten data sets. Phase 1 comprised the analysis of synthetic metabolite mixtures, while Phase 2 investigated European flounder (Platichthys flesus) liver extracts from clean and contaminated sites. Overall, the comparability of data sets from the participating laboratories was good. Principal components analyses (PCA) of the individual data sets yielded ten highly similar scores plots for the synthetic mixtures, with a comparable result for the liver extracts. Furthermore, the same metabolic biomarkers that discriminated fish from clean and contaminated sites were discovered by all the laboratories. PCA of the combined data sets showed excellent clustering of the multiple analyses. These results demonstrate that NMR-based metabolomics can generate data that are sufficiently comparable between laboratories to support its continued evaluation for regulatory environmental studies.


Assuntos
Meio Ambiente , Peixes/metabolismo , Cooperação Internacional , Metabolômica , Animais , Espectroscopia de Ressonância Magnética , Análise Multivariada , Análise de Componente Principal , Extratos de Tecidos/metabolismo , Poluentes Químicos da Água/análise
15.
J Nat Prod ; 71(9): 1518-23, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18698820

RESUMO

Chemical analyses of plankton and highly toxic mussel samples collected in eastern Canada during an intense bloom of the dinoflagellate Alexandrium tamarense established the presence of a complex mixture of paralytic shellfish poisoning (PSP) toxins. Application of a newly developed technique, hydrophilic interaction liquid chromatography-mass spectrometry, confirmed the identities of the known toxins and revealed the presence in the mussels of five saxitoxin analogues (M1-M5) that were not present in the plankton. Four of these compounds were isolated and their structures established by tandem mass spectrometry, 1D- and 2D-NMR spectroscopy, and chemical interconversion experiments. One of these was found to be 11beta-hydroxysaxitoxin (M2), while the other three were found to be new saxitoxin analogues, namely, 11beta-hydroxy-N-sulfocarbamoylsaxitoxin (M1), 11,11-dihydroxy-N-sulfocarbamoylsaxitoxin (M3), and 11,11-dihydroxysaxitoxin (M4). Compound M5 remains unidentified because of insufficient material for characterization.


Assuntos
Bivalves/química , Dinoflagelados/química , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Saxitoxina/química , Saxitoxina/isolamento & purificação , Animais , Canadá , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Saxitoxina/análogos & derivados
16.
J Org Chem ; 71(23): 8724-31, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-17080999

RESUMO

Biosynthetic origins of the cyclic imine toxin 13-desmethyl spirolide C were determined by supplementing cultures of the toxigenic dinoflagellate Alexandrium ostenfeldii with stable isotope-labeled precursors [1,2-13C2]acetate, [1-13C]acetate, [2-13CD3]acetate, and [1,2-13C2,15N]glycine and measuring the incorporation patterns by 13C NMR spectroscopy. Despite partial scrambling of the acetate labels, the results show that most carbons of the macrocycle are polyketide-derived and that glycine is incorporated as an intact unit into the cyclic imine moiety. This work represents the first conclusive evidence that such cyclic imine toxins are polyketides and provides support for biosynthetic pathways previously defined for other polyether dinoflagellate toxins.


Assuntos
Dinoflagelados/metabolismo , Compostos Macrocíclicos/metabolismo , Toxinas Marinhas/metabolismo , Compostos de Espiro/metabolismo , Animais , Isótopos de Carbono , Dinoflagelados/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/isolamento & purificação , Espectroscopia de Ressonância Magnética/métodos , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Estrutura Molecular , Sensibilidade e Especificidade , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Estereoisomerismo
17.
J Nat Prod ; 69(7): 983-7, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16872129

RESUMO

Using LC/MS methodology, spirolides were detected in two clonal isolates of Alexandrium ostenfeldii isolated from Limfjorden, Denmark. Examination of the LC/MS profiles of extracts from these Danish cultures revealed the presence of two dominant peaks representing two previously unidentified spirolide components and one minor peak identified as the previously reported desmethyl spirolide C (1). Culturing of these clonal strains, LF 37 and LF 38, of A. ostenfeldii resulted in the accumulation of sufficient cell biomass to allow for the isolation and structure elucidation of two new spirolides, 13,19-didesmethylspirolide C (2) and spirolide G (3). While 2 was found to differ from 1 only in that it contained one less methyl group, 3 was the first spirolide to be isolated that contained a 5:6:6-trispiroketal ring system. The effect of this new feature on the toxicity of 3 relative to other spirolides is presently being pursued.


Assuntos
Dinoflagelados/química , Iminas , Toxinas Marinhas , Compostos de Espiro , Animais , Dinamarca , Iminas/química , Iminas/isolamento & purificação , Iminas/farmacologia , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/farmacologia , Estrutura Molecular , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/farmacologia
18.
J Exp Zool A Comp Exp Biol ; 305(6): 480-8, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16506225

RESUMO

At seawater temperatures below 1 degrees C, rainbow smelt (Osmerus mordax) accumulate plasma levels of glycerol up to 400 mM. Aspects of the synthesis of glycerol in liver and its regulation were previously investigated, but the pathways leading to glycerol synthesis remained unconfirmed. Here, we report nuclear magnetic resonance (NMR) studies which elucidate, in more detail, the fuel sources for rapid glycerol synthesis in rainbow smelt. Initial NMR analysis of liver homogenates from fish held at cold (-1 degrees C) temperatures and from fish transferred from 8 degrees C to -1 degrees C showed elevated glycerol, whereas those from fish held at 8 degrees C had far lower glycerol levels. These results confirm a temperature-responsive glycerol synthesis and show that NMR is a suitable approach to investigate the phenomenon. Further studies with fish held at low temperature and injected with labelled L-[2,3-(13)C(2)] alanine or D-[U-(13)C(6)]glucose revealed conversion of both alanine and glucose to glycerol. (13)C spectra showed satellites ((1)J(CC)=41.1 Hz) about the glycerol resonances indicating intact incorporation of a (13)C-(13)C unit in liver glycerol of fish injected with L-[2,3-(13)C(2)]alanine and a (13)C-(13)C-(13)C unit in liver glycerol of fish injected with D[U-(13)C(6)]glucose. Thus, glycerol can be efficiently produced directly from amino acid precursors by glyceroneogenesis, which is an abbreviated gluconeogenesis process leading to glycerol through dihydroxyacetone phosphate (DHAP). Glucose can also be metabolised to glycerol via an abbreviated form of glycolysis that similarly leads to glycerol through DHAP.


Assuntos
Alanina/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Fígado/metabolismo , Osmeriformes/metabolismo , Animais , Isótopos de Carbono , Deutério , Glicerol/química , Espectroscopia de Ressonância Magnética , Temperatura
19.
Dis Aquat Organ ; 65(2): 107-14, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16060263

RESUMO

1H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon.


Assuntos
Aeromonas salmonicida , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Furunculose/veterinária , Salmo salar , Animais , Análise Química do Sangue/métodos , Colina/sangue , Doenças dos Peixes/sangue , Furunculose/sangue , Furunculose/metabolismo , Lipoproteínas/sangue , Espectroscopia de Ressonância Magnética , Análise de Componente Principal
20.
Anal Chem ; 77(10): 3123-31, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15889900

RESUMO

We examine the use of external standards for quantitative measurement by 1H NMR of solution concentrations of natural products and other low molecular weight, hydrogen-containing compounds and show that precision and accuracy ca. 1% is obtainable with a commercial 11.7 T spectrometer when standards and analytes are contained in separate but identical sealed precision glass NMR tubes. Numerous factors contributing to the intensity of the NMR signals are evaluated. Precise measurements of 360 degrees pulse lengths for each sample provide direct corrections for variations in probe Q-factor that enable samples in different solvents to be compared, provided single-coil excitation and detection is used throughout. Samples need not be prepared in deuterated solvents if the 1H spectra of the solvents are simple enough for peak suppression by presaturation. The approach is particularly suitable for hazardous materials kept in sealed tubes and for the preparation of certified calibration solution reference materials for use with LC-MS and other techniques where deuterated solvents should be avoided.


Assuntos
Produtos Biológicos/análise , Eucariotos/química , Espectroscopia de Ressonância Magnética/métodos , Toxinas Marinhas/análise , Soluções/química , Calibragem , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Deutério/química , Compostos Heterocíclicos com 3 Anéis/análise , Compostos Heterocíclicos com 3 Anéis/química , Hidrocarbonetos Cíclicos/análise , Hidrocarbonetos Cíclicos/química , Iminas/análise , Iminas/química , Ácido Caínico/análogos & derivados , Ácido Caínico/análise , Ácido Caínico/química , Espectroscopia de Ressonância Magnética/normas , Toxinas Marinhas/química , Ácido Okadáico/análise , Ácido Okadáico/química , Reprodutibilidade dos Testes , Saxitoxina/análise , Saxitoxina/química , Sensibilidade e Especificidade , Solventes/química
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