RESUMO
Exosomes are spherical extracellular nanovesicles with an endosomal origin and unilamellar lipid-bilayer structure with sizes ranging from 30 to 100 nm. They contain a large range of proteins, lipids, and nucleic acid species, depending on the state and origin of the extracellular vesicle (EV)-secreting cell. EVs' function is to encapsulate part of the EV-producing cell content, to transport it through biological fluids to a targeted recipient, and to deliver their cargos specifically within the aimed recipient cells. Therefore, exosomes are considered to be potential biological drug-delivery systems that can stably deliver their cargo into targeted cells. Various cell-derived exosomes are produced for medical issues, but their use for therapeutic purposes still faces several problems. Some of these difficulties can be avoided by resorting to hemisynthetic approaches. We highlight here the uses of alternative exosome-mimes involving cell-membrane coatings on artificial nanocarriers or the hybridization between exosomes and liposomes. We also detail the drug-loading strategies deployed to make them drug-carrier systems and summarize the ongoing clinical trials involving exosomes or exosome-like structures. Finally, we summarize the open questions before considering exosome-like disposals for confident therapeutic delivery.
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The impact of the polyunsaturated fatty acids (PUFAs) at physiological concentrations on the composition of eicosanoids transported within the extracellular vesicles (EVs) of rat bone marrow mesenchymal stem cells and cardiomyoblasts was reported by our group in 2020. The aim of this article was to extend this observation to cells from the cardiac microenvironment involved in the processes of inflammation, namely mouse J774 macrophages and rat heart mesenchymal stem cells cMSCs. Moreover, to enhance our capacity to understand the paracrine exchange between these orchestrators of cardiac inflammation, we investigated some machinery involved in the eicosanoid's synthesis transported by the EVs produced by these cells (including the two formerly described cells: bone marrow mesenchymal stem cells BM-MSC and cardiomyoblasts H9c2). We analyzed the oxylipin and the enzymatic content of the EVs collected from cell cultures supplemented (or not) with PUFAs. We prove that large eicosanoid profiles are exported in the EVs by the cardiac microenvironment cells, but also that these EVs carry some critical and functional biosynthetic enzymes, allowing them to synthesize inflammation bioactive compounds by sensing their environment. Moreover, we demonstrate that these are functional. This observation reinforces the hypothesis that EVs are key factors in paracrine signaling, even in the absence of the parent cell. We also reveal a macrophage-specific behavior, as we observed a radical change in the lipid mediator profile when small EVs derived from J774 cells were exposed to PUFAs. To summarize, we prove that the EVs, due to the carried functional enzymes, can alone produce bioactive compounds, in the absence of the parent cell, by sensing their environment. This makes them potential circulating monitoring entities.
Assuntos
Vesículas Extracelulares , Camundongos , Ratos , Animais , Coração , Ácidos Graxos Insaturados , Eicosanoides , InflamaçãoRESUMO
Paenibacillus mucilaginosus has widely been reported as a plant growth-promoting rhizobacteria (PGPR). However, the important genomic insights into plant growth promotion in this species remain undescribed. In this study, the genome of P. mucilaginosus G78 was sequenced using Illumina NovaSeq PE150. It contains 8,576,872 bp with a GC content of 58.5%, and was taxonomically characterized. Additionally, a total of 7337 genes with 143 tRNAs, 41 rRNAs, and 5 ncRNAs were identified. This strain can prohibit the growth of the plant pathogen, but also has the capability to form biofilm, solubilize phosphate, and produce IAA. Twenty-six gene clusters encoding secondary metabolites were identified, and the genotypic characterization indirectly proved its resistant ability to ampicillin, bacitracin, polymyxin and chloramphenicol. The putative exopolysaccharide biosynthesis and biofilm formation gene clusters were explored. According to the genetic features, the potential monosaccharides of its exopolysaccharides for P. mucilaginosus G78 may include glucose, mannose, galactose, fucose, that can probably be acetylated and pyruvated. Conservation of the pelADEFG compared with other 40 Paenibacillus species suggests that Pel may be specific biofilm matrix component in P. mucilaginosus. Several genes relevant to plant growth-promoting traits, i.e., IAA production and phosphate solubilization are well conserved compared with other 40 other Paenibacillus strains. The current study can benefit for understanding the plant growth-promoting traits of P. mucilaginosus as well as its potential application in agriculture as PGPR.
Assuntos
Paenibacillus , Paenibacillus/genética , Desenvolvimento Vegetal , Genômica , FosfatosRESUMO
We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1' whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.
Assuntos
Medicago truncatula , Proteínas de Plantas/metabolismo , Proteínas Ribossômicas/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Sinorhizobium meliloti/metabolismo , Coinfecção/prevenção & controle , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Nodulação/fisiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Proteínas Ribossômicas/genética , Nódulos Radiculares de Plantas/microbiologia , Transdução de Sinais , SimbioseRESUMO
Recent works reported the relevance of cellular exosomes in the evolution of different pathologies. However, most of these studies focused on the ability of exosomes to convey mi-RNA from cell to cell. The level of knowledge concerning the transport of lipid mediators by these nanovesicles is more than fragmented. The role of lipid mediators in the inflammatory signaling is fairly well described, in particular concerning the derivatives of the arachidonic acid (AA), called eicosanoïds or lipid mediators. The aim of the present work was to study the transport of these lipids within the extracellular vesicles of rat bone marrow mesenchymal stem cells (BM-MSC) and the cardiomyoblast cell line H9c2. We were able to characterize, for the first time, complete profiles of oxilipins within these nanovesicles. We studied also the impact on these profiles, of the polyunsaturated fatty acids (PUFAs) know to be precursors of the inflammatory signaling molecules (AA, eicosapentaenoic acid EPA and Docosahexaenoic acid DHA), at physiological concentrations. By growing the progenitor cells under PUFAs supplementation, we provide a comprehensive assessment of the beneficial effect of ω-3 PUFA therapy. Actually, our results tend to support the resolving role of the inflammation that stromal cell-derived extracellular vesicles can have within the cardiac microenvironment.
Assuntos
Eicosanoides/química , Eicosanoides/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/metabolismo , Mioblastos Cardíacos/química , Mioblastos Cardíacos/metabolismo , Animais , Medula Óssea/química , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Linhagem Celular , Vesículas Extracelulares/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/química , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos , Células-Tronco Mesenquimais/efeitos dos fármacos , Mioblastos Cardíacos/efeitos dos fármacos , Oxilipinas/química , Oxilipinas/metabolismo , RatosRESUMO
Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.
Assuntos
Quitina/análogos & derivados , Quitina/metabolismo , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Transdução de Sinais/fisiologia , Ascomicetos/crescimento & desenvolvimento , Basidiomycota/crescimento & desenvolvimento , Quitosana , Ecologia , Ácidos Graxos/metabolismo , Micorrizas/fisiologia , Oligossacarídeos , Rhizobium/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Simbiose/fisiologiaRESUMO
The ß-rhizobium Cupriavidus taiwanensis is a nitrogen-fixing symbiont of Mimosa pudica. Nod factors produced by this species were previously found to be pentameric chitin-oligomers carrying common C18:1 or C16:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing terminal residue. Here, we report that, in addition, C. taiwanensis LMG19424 produces molecules where the reducing sugar is open and oxidized. We identified a novel nodulation gene located on the symbiotic plasmid pRalta, called noeM, which is involved in this atypical Nod factor structure. noeM encodes a transmembrane protein bearing a fatty acid hydroxylase domain. This gene is expressed during symbiosis with M. pudica and requires NodD and luteolin for optimal expression. The closest noeM homologs formed a separate phylogenetic clade containing rhizobial genes only, which are located on symbiosis plasmids downstream from a nod box. Corresponding proteins, referred to as NoeM, may have specialized in symbiosis via the connection to the nodulation pathway and the spread in rhizobia. noeM was mostly found in isolates of the Mimoseae tribe, and specifically detected in all tested strains able to nodulate M. pudica. A noeM deletion mutant of C. taiwanensis was affected for the nodulation of M. pudica, confirming the role of noeM in the symbiosis with this legume.
Assuntos
Cupriavidus , Mimosa , Rhizobium , Cupriavidus/classificação , Cupriavidus/genética , Genes Bacterianos/genética , Mimosa/microbiologia , Filogenia , Plasmídeos/genética , Simbiose/genéticaRESUMO
Tryptophane (Trp) labelled by 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) is very difficult to identify using CE and fluorescence detection (480 nm). Why in this article some mass spectrometry experiments show that Trp is really labelled by CBQCA as Leucine (Leu)? If the maximum of UV absorption (λmax ) is the same between Leu-CBQCA and Trp-CBQCA, the molar extinction coefficient is around 2 fold higher for Trp-CBQCA. The fluorescence of the Leu-CBQCA derivative is 50 times more important than for Trp-CBQCA. The addition of 7.5 mM of ß-cyclodextrin (ß-CD) was found to be a good mean to improve 2.1 fold the sensitivity of the Trp-CBQCA fluorescence. Using a buffer containing SDS and ß-CD in CE, a LOD of 0.7 µM of L-Trp can be reached and the ratio of the intensities between Leu, Isoleucine, Valine, Trp is 100, 21, 15, 1. Negative ESI/ MS and MS/MS of the labeled amino acids show that a loss of the carboxylate function takes place. In the presence of two enantiomers of Trp-CBQCA, we have shown that this decarboxylation is not due to the derivatization process in the solution but rather occurs in the source of the mass spectrometer.
Assuntos
Benzoatos/química , Eletroforese Capilar/métodos , Corantes Fluorescentes/química , Quinolinas/química , Espectrometria de Fluorescência/métodos , Triptofano , Leucina/análise , Leucina/química , Estereoisomerismo , Espectrometria de Massas em Tandem , Triptofano/análise , Triptofano/químicaRESUMO
A complex network of pathways coordinates nodulation and epidermal root hair infection in the symbiotic interaction between rhizobia and legume plants. Whereas nodule formation was known to be autoregulated, it was so far unclear whether a similar control is exerted on the infection process. We assessed the capacity of Medicago plants nodulated by Sinorhizobium meliloti to modulate root susceptibility to secondary bacterial infection or to purified Nod factors in split-root and volatile assays using bacterial and plant mutant combinations. Ethylene implication in this process emerged from gas production measurements, use of a chemical inhibitor of ethylene biosynthesis and of a Medicago mutant affected in ethylene signal transduction. We identified a feedback mechanism that we named AOI (for Autoregulation Of Infection) by which endosymbiotic bacteria control secondary infection thread formation by their rhizospheric peers. AOI involves activation of a cyclic adenosine 3',5'-monophosphate (cAMP) cascade in endosymbiotic bacteria, which decreases both root infectiveness and root susceptibility to bacterial Nod factors. These latter two effects are mediated by ethylene. AOI is a novel component of the complex regulatory network controlling the interaction between Sinorhizobium meliloti and its host plants that emphasizes the implication of endosymbiotic bacteria in fine-tuning the interaction.
Assuntos
Etilenos/metabolismo , Medicago truncatula/microbiologia , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Sinorhizobium meliloti/fisiologia , Simbiose , Proteínas de Bactérias/metabolismo , Modelos Biológicos , Epiderme Vegetal/microbiologia , Nodulação , Compostos Orgânicos Voláteis/metabolismoRESUMO
Macrophage-mediated cytotoxicity is controlled by surface receptor expression and activation. Despite the numerous studies documenting the role of macrophage C-type lectin receptors (CLR) in pathogen elimination, little is known about their contribution to antitumor responses. Here, we report that IL13 inhibits T-cell lymphoma and ovarian adenocarcinoma development in tumor-bearing mice through the conversion of tumor-supporting macrophages to cytotoxic effectors, characterized by a CLR signature composed of dectin-1 and mannose receptor (MR). We show that dectin-1 and MR are critical for the recognition of tumor cells through sialic acid-specific glycan structure on their surface and for the subsequent activation of macrophage tumoricidal response. Finally, we validated that IL13 antitumor effect mediated by dectin-1 and MR overexpression on macrophages can extend to various types of human tumors. Therefore, these results identify these CLRs as potential targets to promote macrophage antitumor response and represent an attractive approach to elicit tumor-associated macrophage tumoricidal properties.
Assuntos
Interleucina-13/genética , Lectinas Tipo C/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/genética , Neoplasias/etiologia , Neoplasias/metabolismo , Receptores de Superfície Celular/genética , Animais , Arginase/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Interleucina-13/metabolismo , Lectinas Tipo C/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Knockout , Ácido N-Acetilneuramínico/metabolismo , Necrose/genética , Necrose/imunologia , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Lipids are naturally occurring organic compounds that can be classified into a number of types based on their solubility in nonpolar organic solvents, and are generally insoluble in water. The great structural variety of these various types of lipids has led them to be components of many different biological substances such as oils, waxes, cellular membranes, tissues and biological fluids. The use of capillary electrophoresis (CE) for the study of lipids during the past 30 years has been relatively rare when compared to its use for other classes of biomolecules, primarily due to their insolubility in water. However, a number of interesting studies have been conducted, and as part of this review, we will present the different approaches that were used, which mainly consist of micellar kinetic chromatography and non-aqueous CE. The main advantages of the use of these techniques compared to GC is the very simple sample preparation that is required and, compared to LC, the very robust and quick separations that can be obtained. In this review, we present the various methods that have been reported in the literature that have been used for the study of fatty acids, phospholipids, glycerides, eicosanoids and sterols, with the inclusion of various tables presenting descriptions of the CE methods used as well as the methods of detection, including UV absorbance, fluorescence, mass spectrometry, and conductivity. This review aims to demonstrate that CE can be easily used for the analysis of lipids.
Assuntos
Eletroforese Capilar/métodos , Lipídeos/análise , Condutividade Elétrica , Lipídeos/química , Análise EspectralRESUMO
The structural variation of symbiotic signals released by rhizobia determines the specificity of their interaction with legume plants. Previous studies showed that Sinorhizobium strains from different phylogenetic lineages had different symbiotic performance on certain cultivated soybeans. Whether they released similar or different symbiotic signals remained unclear. In this study, we compared their nod and exo gene clusters and made a detailed structural analysis of Nod factors and EPS by ESI-MS/MS and two dimensions NMR. Even if there are some differences among nod or exo gene clusters; they produced much conserved Nod factor and EPS compositions. The Nod factors consist of a cocktail of ß-(1, 4)-linked tri-, tetra-, and pentamers of N-acetyl-D-glucosamine (GlcNAc). The C2 position on the non-reducing terminal end is modified by a lipid chain that contains 16 or 18 atoms of carbon-with or without unsaturations-, and the C6 position on the reducing residue is decorated by a fucose or a 2-O-methylfucose. Their EPS are composed of glucose, galactose, glucuronic acid, pyruvic acid in the ratios 5:1:2:1 or 6:1:2:1. These findings indicate that soybean cultivar compatibility of Sinorhizobium strains does not result from Nod factor or EPS structure variations. The structure comparison of the soybean microbionts with other Sinorhizobium strains showed that Nod factor structures of soybean microbionts are much conserved, although there are no specific genes shared by the soybean microsymbionts. EPS produced by Sinorhizobium strains are different from those of Bradyrhizobium. All above is consistent with the previous deduction that Nod factor structures are related to host range, while those of EPS are connected with phylogeny.
RESUMO
One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced. Treatment of the data by the counting of double-stranded DNA T29 sequences containing a maximum of two mismatches reveals a good correlation with the enrichment factor (fE). This factor is the ratio of the number of aptamer sequences found in the collected complex sample divided by the total number of sequencing reads (aptamer and non-aptamer) plus the quantity of T29 molecules (spiked into a DNA library) injected into CE.
Assuntos
Aptâmeros de Nucleotídeos/química , Eletroforese Capilar/métodos , Quadruplex G , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Biblioteca Gênica , Trombina/análiseRESUMO
In the tenth edition of this article focused on recent advances in amino acid analysis using capillary electrophoresis, we describe the most important research articles published on this topic during the period from June 2015 to May 2017. This article follows the format of the previous articles published in Electrophoresis. The new developments in amino acid analysis with CE mainly describe improvements in CE associated with mass spectrometry. Focusing on applications, we mostly describe clinical works, although metabolomics studies are also very important. Finally, works focusing on amino acids in food and agricultural applications are also described.
Assuntos
Aminoácidos/análise , Animais , Testes de Química Clínica/métodos , Técnicas Eletroquímicas/métodos , Eletroforese Capilar/métodos , Análise de Alimentos/métodos , Humanos , Metabolômica/métodos , Técnicas Analíticas Microfluídicas/métodos , Sensibilidade e Especificidade , EstereoisomerismoRESUMO
We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.
Assuntos
Rhizobiaceae/fisiologia , Sophora/fisiologia , Simbiose/fisiologia , Mutação , Nodulação/genética , Nodulação/fisiologia , Sophora/genética , Sophora/microbiologiaRESUMO
Tris-Acetate buffer is currently used in the selection and the characterization of ssDNA by capillary electrophoresis (CE). By applying high voltage, the migration of ionic species into the capillary generates a current that induces water electrolysis. This phenomenon is followed by the modification of the pH and the production of Tris derivatives. By injecting ten times by capillary electrophoresis ssDNA (50 nM), the whole oligonucleotide was degraded. In this paper, we will show that the Tris buffer in the running vials is modified along the electrophoretic process by electrochemical reactions. We also observed that the composition of the metal ions changes in the running buffer vials. This phenomenon, never described in CE, is important for fluorescent ssDNA analysis using Tris buffer. The oligonucleotides are degraded by electrochemically synthesized species (present in the running Tris vials) until it disappears, even if the separation buffer in the capillary is clean. To address these issues, we propose to use a sodium phosphate buffer that we demonstrate to be electrochemically inactive.
Assuntos
DNA de Cadeia Simples/análise , Eletroforese Capilar/métodos , Oligonucleotídeos/análise , Soluções Tampão , Cromatografia Líquida de Alta Pressão , DNA de Cadeia Simples/química , Técnicas Eletroquímicas , Eletroforese em Gel de Poliacrilamida , Fluoresceínas/química , Fluorescência , Espectrometria de MassasRESUMO
Native laser-induced fluorescence using UV lasers associated to CE offers now a large related literature, for now 30 years. The main works have been performed using very expensive Ar-ion lasers emitting at 257 and 275 nm. They are not affordable for routine analyses, but have numerous applications such as protein, catecholamine, and indolamine analysis. Some other lasers such as HeCd 325 nm have been used but only for few applications. Diode lasers, emitting at 266 nm, cheaper, are extensively used for the same topics, even if the obtained sensitivity is lower than the one observed using the costly UV-Ar-ion lasers. This review presents various CE or microchips applications and different UV lasers used for the excitation of native fluorescence. We showed that CE/Native UV laser induced fluorescence detection is very sensitive for detection as well as small aromatic biomolecules than proteins containing Trp and Tyr amino acids. Moreover, it is a simple way to analyze biomolecules without derivatization.
Assuntos
Aminoácidos/análise , Eletroforese Capilar/métodos , Neurotransmissores/análise , Catecolaminas/análise , Fluorescência , Lasers , Proteínas/análise , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
Soil-dwelling, nitrogen-fixing rhizobia signal their presence to legume hosts by secreting lipo-chitooligomers (LCOs) that are decorated with a variety of chemical substituents. It has long been assumed, but never empirically shown, that the LCO backbone is synthesized first by NodC, NodB, and NodA, followed by addition of one or more substituents by other Nod proteins. By analyzing a collection of in-frame deletion mutants of key nod genes in the bacterium Rhizobium sp. IRBG74 by mass spectrometry, we were able to shed light on the possible substitution order of LCO decorations, and we discovered that the prevailing view is probably erroneous. We found that most substituents could be transferred to a short chitin backbone prior to acylation by NodA, which is probably one of the last steps in LCO biosynthesis. The existence of substituted, short chitin oligomers offers new insights into symbiotic plant-microbe signaling.
Assuntos
Quitina/análogos & derivados , Lipopolissacarídeos/genética , Rhizobium/genética , Acilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitina/análise , Quitina/química , Quitina/isolamento & purificação , Quitosana , Lipopolissacarídeos/metabolismo , Espectrometria de Massas , Estrutura Molecular , Mutação , Oligossacarídeos , Rhizobium/químicaRESUMO
Pulsed lasers are widely used in capillary electrophoresis (CE) studies to provide laser induced fluorescence (LIF) detection. Unfortunately pulsed lasers do not give linear calibration curves over a wide range of concentrations. While this does not prevent their use in CE/LIF studies, the non-linear behavior must be understood. Using 7-hydroxycoumarin (7-HC) (10-5000 nM), Tamra (10-5000 nM) and tryptophan (1-200 µM) as dyes, we observe that continuous lasers and LEDs result in linear calibration curves, while pulsed lasers give polynomial ones. The effect is seen with both visible light (530 nm) and with UV light (355 nm, 266 nm). In this work we point out the formation of byproducts induced by pulsed laser upon irradiation of 7-HC. Their separation by CE using two Zeta LIF detectors clearly shows that this process is related to the first laser detection. All of these photodegradation products can be identified by an ESI-/MS investigation and correspond to at least two 7HC dimers. By using the photodegradation model proposed by Heywood and Farnsworth (2010) and by taking into account the 7-HC results and the fact that in our system we do not have a constant concentration of fluorophore, it is possible to propose a new photochemical model of fluorescence in LIF detection. The model, like the experiment, shows that it is difficult to obtain linear quantitation curves with pulsed lasers while UV-LEDs used in continuous mode have this advantage. They are a good alternative to UV pulsed lasers. An application involving the separation and linear quantification of oligosaccharides labeled with 2-aminobezoic acid is presented using HILIC and LED (365 nm) induced fluorescence.