RESUMEN
To investigate the potential of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) as a platform to support biodiversity and phylogenetic studies of psychrophilic yeasts in cold environments, the technique was employed to rapidly characterize and distinguish three psychrophilic yeasts (Rhodotorula mucilaginosa, Naganishia vishniacii, and Dioszegia cryoxerica) from three mesophilic counterparts (Saccharomyces cerevisiae Cry Havoc, S. cerevisiae California V Ale, and S. pastorianus). A detailed workflow for providing reproducible mass spectral fingerprints of low molecular weight protein/peptide features specific to the organisms studied is presented. The potential of this approach as a tool in the study of biodiversity, systematics, and phylogeny of psychrophilic microorganisms is highlighted.
Asunto(s)
Proteínas Fúngicas/química , Microbiota , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Regiones Antárticas , Rhodotorula/química , Rhodotorula/metabolismo , Saccharomyces/química , Saccharomyces/metabolismoRESUMEN
Platelets are small (1-2µm in diameter), circulating anuclear cell fragments with important roles in hemostasis and thrombosis that provide an excellent platform for studying the role of membrane components in cellular communication. Platelets use several forms of communication including exocytosis of three distinct granule populations, formation of bioactive lipid mediators, and shape change (allowing for adhesion). This work explores the role of stereochemistry and concentration of exogenous phosphatidylserine (PS) on platelet exocytosis and adhesion. PS, most commonly found in the phosphatidyl-l-serine (l-PS) form, is exposed on the outer leaflet of the cell membrane after the platelet is activated. Knowledge about the impact of exogenous phosphatidylserine on cell-to-cell communication is limited (particularly concentration and stereochemistry effects). This study found that platelets incubated in l-PS or phosphatidyl-d-serine (d-PS) are enriched to the same extent with their respective incubated PS. All levels of l-PS enrichment also showed an increase in platelet cholesterol, but only the 50µM d-PS incubation showed an increase in cholesterol. The uptake of d-PS induced the secretion of granules and manufactured platelet activating factor (PAF) in otherwise unstimulated platelets. The uptake of l-PS had a greater impact on platelet stimulation by decreasing both the amount of δ-granule secretion and the amount of PAF that was manufactured.
Asunto(s)
Plaquetas/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Fosfatidilserinas/farmacología , Factor de Activación Plaquetaria/antagonistas & inhibidores , Activación Plaquetaria/efectos de los fármacos , Animales , Plaquetas/química , Plaquetas/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Factor de Activación Plaquetaria/biosíntesis , Cultivo Primario de Células , EstereoisomerismoRESUMEN
The cellular phospholipid membrane plays an important role in cell function and cell-cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC-MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.
Asunto(s)
Plaquetas/metabolismo , Membrana Celular/metabolismo , Fosfolípidos/análisis , Adhesividad Plaquetaria/fisiología , Animales , Cromatografía Liquida/métodos , Ratones , Ratones Endogámicos C57BL , Microfluídica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1% (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 µM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.
Asunto(s)
Agmatina/análisis , Agmatina/metabolismo , Carboxiliasas/metabolismo , Pseudomonas aeruginosa/enzimología , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/métodos , Fibrosis Quística/microbiología , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Marcaje Isotópico , Límite de Detección , Mutación , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
To move beyond targeted approaches to the biochemical characterization of psychrophilic yeast and provide a more holistic understanding of the chemistry of physiological adaptation of psychrophiles at the molecular level, ultraperformance liquid chromatography combined with simultaneous acquisition of low- and high-collision energy mass spectra (UPLC/MS(e)) was employed for a preliminary comparative analysis of cell extracts of psychrophilic Antarctic yeasts Cryptococcus vishniacii CBS 10616 and Dioszegia cryoxerica CBS 10919 versus the mesophile Saccharomyces cerevisiae 'cry havoc'. A detailed workflow for providing high-confidence preliminary identifications of psychrophilic yeast-specific molecular features is presented. Preliminary identifications of psychrophile-specific features in C. vishniacii and D. cryoxerica determined with the described method include the glycerophospholipids lysophosphatidylcholine 18:2, lysophosphatidylcholine 18:3, lysophosphatidylethanolamine 18:3, and lysophosphatidylethanolamine 18:2. In addition, levels of guanosine diphosphate appear significantly elevated in cell extracts of the psychrophilic yeasts as compared to Saccharomyces cerevisiae. Finally, five psychrophilic yeast-specific peptides have been discovered. All of these are demonstrated to be glycine- and/or proline-rich, a known structural characteristic of many naturally occurring bioactive peptides. The potential of this untargeted metabolite profiling approach as a tool for knowledge discovery and hypothesis generation in the study of biodiversity and microbial adaptation is highlighted.
Asunto(s)
Adaptación Fisiológica , Basidiomycota/metabolismo , Cryptococcus/metabolismo , Metaboloma , Frío , Proteínas Fúngicas/metabolismo , Lisofosfolípidos/metabolismo , Espectrometría de Masas , Saccharomyces cerevisiae/metabolismoRESUMEN
Secreted bioactive lipids play critical roles in cell-to-cell communication and have been implicated in inflammatory immune responses such as anaphylaxis, vasodilation, and bronchoconstriction. Analysis of secreted bioactive lipids can be challenging due to their relatively short lifetimes and structural diversity. Herein, a method has been developed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to quantify five cell-secreted, structurally and functionally diverse bioactive lipids (PGD2, LTC4, LTD4, LTE4, PAF) that play roles in inflammation. Sample analysis time is 5 min, and isotopically labeled internal standards are used for quantification. This method was applied to an immortal secretory cell line (RBL-2H3), a heterogeneous primary cell culture containing peritoneal mast cells, and murine platelets. In RBL cell supernatant samples, intrasample precisions ranged from 7.32-21.6%, averaging 17.0%, and spike recoveries in cell supernatant matrices ranged from 88.0-107%, averaging 97.0%. Calibration curves were linear from 10 ng mL(-1) to 250 ng mL(-1), and limits of detection ranged from 0.0348 ng mL(-1) to 0.803 ng mL(-1). This method was applied to the determination of lipid secretion from mast cells and platelets, demonstrating broad applicability for lipid measurement in primary culture biological systems.
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Metabolismo de los Lípidos , Mastocitos/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Línea Celular Transformada , Cromatografía Líquida de Alta Presión/métodos , Lípidos/química , Mastocitos/química , Ratones , Células 3T3 NIHRESUMEN
Mycobacterium tuberculosis (Mtb) lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential S-adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validated Mtb HTA as an essential enzyme and revealed a ligandable binding site. Seeking a mechanistic characterization of this enzyme, we report crucial structural details and comprehensive functional characterization of Mtb HTA. Crystallographic and mass spectral observation of the acetylated HTA intermediate and initial velocity studies were consistent with a ping-pong kinetic mechanism. Wild-type HTA and its site-directed mutants were kinetically characterized with a panel of natural and alternative substrates to understand substrate specificity and identify critical residues for catalysis. Titration experiments using fluorescence quenching showed that both substratesâacetyl-CoA and l-homoserineâengage in a strong and weak binding interaction with HTA. Additionally, substrate inhibition by acetyl-CoA and product inhibition by CoA and O-acetyl-l-homoserine were proposed to form the basis of a feedback regulation mechanism. By furnishing key mechanistic and structural information, these studies provide a foundation for structure-based design efforts around this attractive Mtb target.
Asunto(s)
Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Lisina , Acetiltransferasas/química , Metionina , Acetilcoenzima ARESUMEN
Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride/bromide ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, how NHFe-Hals preferentially bind specific anions and substrates for C-H functionalization remains unknown. Herein, using lysine halogenating BesD and HalB enzymes as model systems, we demonstrate strong positive cooperativity between anion and substrate binding to the catalytic pocket. Detailed computational investigations indicate that a negatively charged glutamate hydrogen-bonded to iron's equatorial-aqua ligand acts as an electrostatic lock preventing both lysine and anion binding in the absence of the other. Using a combination of UV-Vis spectroscopy, binding affinity studies, stopped-flow kinetics investigations, and biochemical assays, we explore the implication of such active site assembly towards chlorination, bromination, and azidation reactivities. Overall, our work highlights previously unknown features regarding how anion-substrate pair binding govern reactivity of iron halogenases that are crucial for engineering next-generation C-H functionalization biocatalysts.
RESUMEN
Carbon dots (CDs) are emerging as the material of choice in a range of applications due to their excellent photoluminescence properties, ease of preparation from inexpensive precursors, and low toxicity. However, the precise nature of the mechanism for the fluorescence is still under debate, and several molecular fluorophores have been reported. In this work, a new blue fluorophore, 5-oxopyrrolidine-3-carboxylic acid, was discovered in carbon dots synthesized from the most commonly used precursors: citric acid and urea. The molecular product alone has demonstrated interesting aggregation-enhanced emission (AEE), making it unique compared to other fluorophores known to be generated in CDs. We propose that this molecular fluorophore is associated with a polymer backbone within the CDs, and its fluorescence behavior is largely dependent on intermolecular interactions with the polymers or other fluorophores. Thus, a new class of non-traditional fluorophores is now relevant to the consideration of the CD fluorescence mechanism, providing both an additional challenge to the community in resolving the mechanism and an opportunity for a greater range of CD design schemes and applications.
RESUMEN
Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD−HPCA and H200N−HPCA, respectively) with O(2). A blue intermediate forms within 20 ms of mixing of O(2) with H200N−HPCA (H200N(Int1)(HPCA)). Parallel mode electron paramagnetic resonance and Mössbauer spectroscopies show that this intermediate contains high-spin Fe(III) (S = 5/2) antiferromagnetically coupled to a radical (S(R) = 1/2) to yield an S = 2 state. Together, optical and Mössbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200N(Int1)(HPCA) decays over the next 2 s, possibly through an Fe(II) intermediate (H200N(Int2)(HPCA)), to yield the product and the resting Fe(II) enzyme. Reaction of FeHPCD−HPCA with O(2) results in rapid formation of a colorless Fe(II) intermediate (FeHPCD(Int1)(HPCA)). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCD(Int1)(HPCA) suggests it is an intermediate occurring after O(2) activation and attack. The similar Mössbauer parameters for FeHPCD(Int1)(HPCA) and H200N(Int2)(HPCA) suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O(2) via the iron does occur, leading to aromatic ring cleavage.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Brevibacterium flavum/enzimología , Dioxigenasas/química , Dioxigenasas/metabolismo , Compuestos Ferrosos/metabolismo , Oxígeno/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Brevibacterium flavum/química , Brevibacterium flavum/genética , Dioxigenasas/genética , Transporte de Electrón , Compuestos Ferrosos/química , Cinética , Modelos Moleculares , Oxígeno/química , Unión ProteicaRESUMEN
A method has been developed for rapid quantification of nine glycolytic intermediates using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS/MS) to monitor the metabolism of glucose during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is significantly less than traditional ion exchange liquid chromatography assays or enzymatic assays. Complete glycolytic intermediate analysis by LC/MS/MS can be achieved in less than 7 min per sample. Quantification is accomplished using isotopically labeled glucose, glucose-6-phosphate, and pyruvate as internal standards. In addition, a method to deconvolute peak areas of coeluting structural isomers based on unique product ion ratios has been developed to allow accurate quantification of the individual isomers 2-phosphoglycerate and 3-phosphoglycerate, as well as glucose-6-phosphate and fructose-6-phosphate. Intrasample precisions for glycolytic intermediate measurements in cell-free extracts using this method vary between 0.9% and 11.8%, averaging 3.5% (RSD). Calibration curves are linear over the range 0.1-100 microg/mL, and detection limits are estimated at 2-49 ng/mL. Spike recoveries in cell extracts vary from 53% to 127% averaging 91%. This method has the potential to demonstrate correlation of glycolytic intermediate flux to microbial production profiles toward acceleration of the bioprocess development cycle.
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Cromatografía Líquida de Alta Presión/métodos , Glucosa/metabolismo , Glucólisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Fermentación , Fructosafosfatos/análisis , Glucosa/análisis , Glucosa-6-Fosfato/análisis , Ácidos Glicéricos/análisis , Isomerismo , Marcaje IsotópicoRESUMEN
We report the use of ultra high performance liquid chromatography (UPLC) coupled with acquisition of low- and high-collision energy mass spectra (MSe) to explore small molecule compositions that are unique to either enriched-autophagosomes or secretions of chemically activated murine mast cells. Starting with thousands of features, each defined by a chromatographic retention time, m/z values and ion intensities, manual examination of the extracted ion chromatograms (XIC) of chemometrically selected features was essential to eliminate false positives, occurring at rates of 33, 14 and 37% in samples of three biological systems. Forty-six percent of features that passed the XIC-based checkpoint, had IDs in compound databases used here. From these, 19% of IDs had experimental high-collision energy MSe spectra that were in agreement with in-silico fragmentation. The importance of this second checkpoint was highligthed through validation with selected commercially available standards. This work illustrates that checkpoints in data processing are essential to ascertain reliability of unbiased metabolomic studies, thereby reducing the risk of generating 'false identifications' which are is a major concern as 'omics' data continue to proliferate and be used as platforms to lauch novel biological hypotheses.
RESUMEN
Novel approaches to protein measurement based on mass spectrometry are being developed that challenge more traditional methods. This review summarizes the emergence of mass spectrometry as a tool for clinical protein, peptide, and amino acid determination. Specific applications of mass spectrometry to the measurement of transferrin, transthyretin, glycated hemoglobin, and homocysteine will be discussed, as will the limitations of the technology, and future directions for clinical protein measurement.
Asunto(s)
Aminoácidos/análisis , Espectrometría de Masas/métodos , Péptidos/análisis , Proteínas/análisis , Hemoglobinas/análisis , Hemoglobinas/genética , Homocisteína/sangre , Homocisteína/genética , Humanos , Recién Nacido , Tamizaje Masivo , Espectrometría de Masas/economía , Espectrometría de Masas/normas , Errores Innatos del Metabolismo/prevención & control , Prealbúmina/análisis , Prealbúmina/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transferrina/análisis , Transferrina/genéticaRESUMEN
Monitoring amino acid metabolism during fermentation has significant potential from the standpoint of strain selection, optimizing growth and production in host strains, and profiling microbial metabolism and growth state. A method has been developed based on rapid quantification of underivatized amino acids using liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) to monitor the metabolism of 20 amino acids during microbial fermentation. The use of a teicoplanin-based chiral stationary phase coupled with electrospray tandem mass spectrometry allows complete amino acid analyses in less than 4 min. Quantification is accomplished using five isotopically labeled amino acids as internal standards. Because comprehensive chromatographic separation and derivatization are not required, analysis time is significantly less than traditional reversed- or normal-phase LC-based amino acid assays. Intra-sample precisions for amino acid measurements in fermentation supernatants using this method average 4.9% (R.S.D.). Inter-day (inter-fermentation) precisions for individual amino acid measurements range from 4.2 to 129% (R.S.D.). Calibration curves are linear over the range 0-300 microg/ml, and detection limits are estimated at 50-450 ng/ml. Data visualization techniques for constructing semi-quantitative fermentation profiles of nitrogen source utilization have also been developed and implemented, and demonstrate that amino acid profiles generally correlate with observed growth profiles. Further, cellular growth events, such as lag-time and cell lysis can be detected using this methodology. Correlation coefficients for the time profiles of each amino acid measured illustrate that while several amino acids are differentially metabolized in similar fermentations, a select group of amino acids display strong correlations in these samples, indicating a sub-population of analytes that may be most useful for fermentation profiling.
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Aminoácidos/metabolismo , Cromatografía Liquida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Calibración , Fermentación , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
A method has been developed for simultaneous identification of soyasaponins and soy isoflavones in soy products, based on liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Soy-based nutraceutical products were analyzed by LC/ESI-MS with detection of protonated and sodiated molecular ions, as well as characteristic fragment ions for these compounds. Soy isoflavones were characterized by a strong protonated molecular ion in addition to corresponding [aglycone + H](+) ions. Monitoring the soyasaponin-specific protonated aglycone and dehydrated aglycone ions throughout the chromatogram provided a unique fingerprint for soyasaponin content in the samples. This mass spectrometric fingerprint also allowed immediate classification of the soyasaponin analytes as group A or B soyasaponins, based on the unique masses of aglycone ions observed for each class. Quantification of soyasaponin B(b) in soy-derived materials, based on the use of a purified soyasaponin B(b) standard and a glycyrrhizin internal standard, has been accomplished.
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Cromatografía Líquida de Alta Presión/métodos , Glycine max/química , Isoflavonas/análisis , Ácido Oleanólico/análogos & derivados , Saponinas/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Isoflavonas/química , Ácido Oleanólico/análisis , Ácido Oleanólico/química , Saponinas/químicaRESUMEN
The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.
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Carboxiliasas/metabolismo , Interacciones Huésped-Patógeno , Inflamación/metabolismo , Pulmón/enzimología , Pseudomonas aeruginosa/enzimología , Agmatina/metabolismo , Animales , Biopelículas , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/microbiología , Estudios Longitudinales , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Mutagénesis , Mutación , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Fenotipo , Neumonía Bacteriana/metabolismo , Estudios Prospectivos , Esputo/metabolismo , Esputo/microbiologíaRESUMEN
Mast cells play a significant role in both the innate and adaptive immune response; however, the tissue-bound nature of mast cells presents an experimental roadblock to performing physiologically relevant mast cell experiments. In this work, a heterogeneous cell culture containing primary culture murine peritoneal mast cells (MPMCs) was studied to characterize the time-dependence of mast cell response to allergen stimulation and the time- and concentration-dependence of the ability of the heterogeneous MPMC culture to uptake and degranulate exogenous serotonin using high performance liquid chromatography (HPLC) coupled to an electrochemical detector. Additionally, because mast cells play a central role in asthma, MPMCs were exposed to CXCL10 and CCL5, two important asthma-related inflammatory cytokines that have recently been shown to induce mast cell degranulation. MPMC response to both allergen exposure and cytokine exposure was evaluated for 5-HT secretion and bioactive lipid formation using ultraperformance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer (UPLC-MS/MS). In this work, MPMC response was shown to be highly regulated and responsive to subtle alterations in a complex environment through time- and concentration-dependent degranulation and bioactive lipid formation. These results highlight the importance of selecting an appropriate mast cell model when studying mast cell involvement in allergic response and inflammation.
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Citocinas/inmunología , Mastocitos/inmunología , Serotonina/inmunología , Animales , Degranulación de la Célula , Células Cultivadas , Quimiocina CCL5/inmunología , Quimiocina CXCL10/inmunología , Inmunoglobulina E/inmunología , Inflamación/inmunología , Mastocitos/citología , Ratones , Ratones Endogámicos C57BL , Espectrometría de Masas en TándemRESUMEN
There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.(1) Here, we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Ciclooctanos/química , Difosfatos/química , Diterpenos/química , Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Ciclooctanos/metabolismo , Difosfatos/metabolismo , Diterpenos/metabolismo , Péptidos/metabolismo , Prenilación de Proteína , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
A method has been developed for rapid quantification of organic acids using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS-MS) to monitor the metabolism of 10 organic acids during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is less than traditional ion chromatography assays, with complete organic acid analyses by UPLC/ESI-MS-MS being achieved in less than 3 min. Quantification is accomplished using nine isotopically labeled organic acids as internal standards. Intrasample precisions for organic acid measurements in fermentation supernatants using this method average 8.9% (RSD). Calibration curves are linear over the range of 0.06-100 microg/mL, and detection limits are estimated at 0.06-1 microg/mL. This method has the potential to demonstrate correlation of organic acid consumption and production by microorganisms with observed growth profiles, novel media formulations, and cellular growth events. Data visualization software has been used to profile organic acid levels during fermentation and correlate these profiles to nutrient supplementation protocols employed during microbial production. The potential use of this capability in computational modeling and simulation of microbial metabolism to accelerate the bioprocess development cycle is recognized.
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Ácidos/análisis , Cromatografía Líquida de Alta Presión/métodos , Compuestos Orgánicos/análisis , Espectrometría de Masas en Tándem/métodos , Ácidos/metabolismo , Fenómenos Fisiológicos Bacterianos , Fermentación , Marcaje Isotópico , Compuestos Orgánicos/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Stable isotope labeling of an intracellular chemical precursor or metabolite allows direct detection of downstream metabolites of that precursor, arising from novel enzymatic activity of interest, using metabolite profiling liquid chromatography-mass spectrometry. This approach allows the discrimination of downstream metabolites produced from novel enzymatic activity from the unlabeled form of the metabolite arising from native metabolic processes within the cell. Even for the case in which a given product of novel enzymatic activity is a transient, the novel enzymatic activity that produced it can be demonstrated to exist indirectly by identification of product-specific downstream metabolites. Therefore, direct or indirect discovery of novel enzymatic machinery engineered within a cell can be accomplished without a requirement for direct product purification or identification. The application of this approach to confirm the presence of a novel metabolic activity, alanine 2,3-aminomutase, obtained by mutagenesis and selection are discussed. The advantages of metabolite profiling approaches to metabolic engineering in terms of accelerating enzyme discovery and development of intellectual property will also be highlighted.