RESUMO
The capacity of the colon to absorb microbially produced amino acids (AAs) and the underlying mechanisms of AA transport are incompletely defined. We measured the profile of 16 fecal AAs along the rat ceco-colonic axis and compared unidirectional absorptive AA fluxes across mucosal tissues isolated from the rat jejunum, cecum, and proximal colon using an Ussing chamber approach, in conjunction with 1H-NMR and ultra-performance liquid chromatography-mass spectrometry chemical analyses. Passage of stool from cecum to midcolon was associated with segment-specific changes in fecal AA composition and a decrease in total AA content. Simultaneous measurement of up to 16 AA fluxes under native luminal conditions, with correction for endogenous AA release, demonstrated absorptive transfer of AAs across the cecum and proximal colon at rates comparable (30-80%) to those across the jejunum, with significant Na+-dependent and H+-stimulated components. Expression profiling of 30 major AA transporter genes by quantitative PCR revealed comparatively high levels of transcripts for 20 AA transporters in the cecum and/or colon, with the levels of 12 exceeding those in the small intestine. Our results suggest a more detailed model of major apical and basolateral AA transporters in rat colonocytes and provide evidence for a previously unappreciated transfer of AAs across the colonic epithelium that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues.NEW & NOTEWORTHY This study provides evidence for a previously unappreciated transfer of microbially generated amino acids across the colonic epithelium under physiological conditions that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues. The segment-specific expression of at least 20 amino acid transporter genes along the colon provides a detailed mechanistic basis for uniport, heteroexchange, Na+-cotransport, and H+-cotransport components of colonic amino acid absorption.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Colo/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Animais , Bactérias/metabolismo , Colo/microbiologia , Fezes/química , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Cinética , Ratos Sprague-Dawley , TranscriptomaRESUMO
Malylglutamate, a newly identified metabolite in earthworms, was synthesized using a traditional peptide coupling approach for assembling the amide from protected malate and glutamate precursors. The proposed structure (1) and a diastereomer were synthesized, but their NMR spectra did not match the natural sample. Further analysis of the natural sample using HMBC spectroscopy suggested an alternative attachment of the malyl moiety, and ß-malylglutamate (2) diastereomers were synthesized, L,L-2 and D,D-2. NMR spectra were an excellent match with the natural sample, and chiral-phase chromatography was employed to identify (-)-ß-l-malyl-l-glutamate (2) as the isomer native to Eisenia fetida.
Assuntos
Glutamatos/química , Glutamatos/síntese química , Oligoquetos/metabolismo , Peptídeos/síntese química , Animais , Glutamatos/metabolismo , Espectroscopia de Ressonância Magnética , Malatos/química , Peptídeos/química , EstereoisomerismoRESUMO
BACKGROUND: Pomegranate (Punica granatum L.) - a delicious fruit once used in Ayurvedic medicine - is now largely known for the antioxidant properties of its juice, which has also been considered to have health benefits against diseases such as cancer and cardiovascular diseases. These beneficial effects are associated with the fruit's high content of polyphenolic compounds. High demand and lower production levels drive pomegranate prices up, which leads to the possibility of pomegranate products being adulterated, diluted or substituted. To ensure the presence of pomegranate in various preparations labeled as containing pomegranate, a simple method was developed to screen and quantify the specific punicalagins by mass spectrometry. RESULTS: The present method was used to analyze several pure and mixed beverages from the US market, and also to quantify punicalagins in the juice of 14 pomegranate cultivars. Punicalagins were detected in all cultivars, with higher concentrations in whole fruit juices compared with aril juices. Amongst the 20 commercial beverages, punicalagins were not detected in four preparations. CONCLUSION: The liquid chromatographic-mass spectrometric method presented herein enables an easy and rapid quantification of the specific punicalagins. The latter was detected in all cultivar samples, thus supporting that punicalagin is a suitable marker of these 14 pomegranate cultivars in commercial juices. Absence of the specific marker in four commercial preparations shows the necessity of having simple and rapid methods to evaluate the presence of pomegranate in preparations. © 2019 Society of Chemical Industry.
Assuntos
Antioxidantes/química , Cromatografia Líquida de Alta Pressão/métodos , Taninos Hidrolisáveis/química , Lythraceae/química , Preparações de Plantas/química , Espectrometria de Massas em Tandem/métodos , Sucos de Frutas e Vegetais/análise , Sucos de Frutas e Vegetais/economia , Taninos Hidrolisáveis/economia , Lythraceae/classificação , Preparações de Plantas/economiaRESUMO
Earthworms ( Eisenia fetida) are vital members of the soil environment. Because of their sensitivity to many contaminants, monitoring earthworm metabolism may be a useful indicator of environmental stressors. Here, metabolic profiles of exposure to five chloroacetanilide herbicides and one enantiomer (acetochlor, alachlor, butachlor, racemic metolachlor, S-metolachlor, and propachlor) are observed in earthworm coelomic fluid using proton nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS). Multiblocked-orthogonal partial least-squares-discriminant analysis (MB-OPLS-DA) and univariate analysis were used to identify metabolic perturbations in carnitine biosynthesis, carbohydrate metabolism, lipid metabolism, nitrogen metabolism, and the tricarboxylic acid cycle. Intriguingly, stereospecific metabolic responses were observed between racemic metolachlor and S-metolachlor exposed worms. These findings support the utility of coelomic fluid in monitoring metabolic perturbations induced by chloroacetanilide herbicides in nontarget organisms and reveal specificity in the metabolic impacts of herbicide analogues in earthworms.
Assuntos
Acetamidas/metabolismo , Líquidos Corporais/química , Herbicidas/metabolismo , Oligoquetos/química , Animais , Líquidos Corporais/metabolismo , Carnitina/biossíntese , Metabolismo Energético , Monitoramento Ambiental/métodos , Cromatografia Gasosa-Espectrometria de Massas , Oligoquetos/metabolismo , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
The large intestine (cecum and colon) is a complex biochemical factory of vital importance to human health. It plays a major role in digestion and absorption by salvaging nutrients from polysaccharides via fermentation initiated by the bacteria that comprise the gut microbiome. We hypothesize that the intestinal epithelium absorbs a limited number of luminal metabolites with bioactive potential while actively excluding those with toxic effects. To explore this concept, we combined 1H NMR detection with Ussing chamber measurements of absorptive transport by rat cecum. Numerous metabolites transported across the epithelium can be measured simultaneously by 1H NMR, a universal detector of organic compounds, alleviating the need for fluorescent or radiolabeled compounds. Our results demonstrate the utility of this approach to delineate the repertoire of fecal solutes that are selectively absorbed by the cecum and to determine their transport rates.
Assuntos
Ceco/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/métodos , Animais , Feminino , Ratos , Ratos Sprague-DawleyRESUMO
The rice (Oryza sativa L.) ethylene-responsive transcription factor gene SUB1A-1 confers tolerance to prolonged, complete submergence by limiting underwater elongation growth. Upon desubmergence, SUB1A-1 genotypes rapidly recover photosynthetic function and recommence development towards flowering. The underpinnings of the transition from stress amelioration to the return to homeostasis are not well known. Here, transcriptomic and metabolomic analyses were conducted to identify mechanisms by which SUB1A improves physiological function over the 24 hr following a sublethal submergence event. Evaluation of near-isogenic genotypes after submergence and over a day of reaeration demonstrated that SUB1A transiently constrains the remodelling of cellular activities associated with growth. SUB1A influenced the abundance of ca. 1,400 transcripts and had a continued impact on metabolite content, particularly free amino acids, glucose, and sucrose, throughout the recovery period. SUB1A promoted recovery of metabolic homeostasis but had limited influence on mRNAs associated with growth processes and photosynthesis. The involvement of low energy sensing during submergence and recovery was supported by dynamics in trehalose-6-phosphate and mRNAs encoding key enzymes and signalling proteins, which were modulated by SUB1A. This study provides new evidence of convergent signalling pathways critical to the rapidly reversible management of carbon and nitrogen metabolism in submergence resilient rice.
Assuntos
Metaboloma , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Adaptação Fisiológica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Metaboloma/genética , Metabolômica , Oryza/genética , Proteínas de Plantas/genética , Brotos de Planta/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.
Assuntos
Ácido Glutâmico/metabolismo , Malatos/metabolismo , Metaboloma , Metabolômica/métodos , Oligoquetos/metabolismo , Alcaloides/isolamento & purificação , Alcaloides/metabolismo , Aminobutiratos/isolamento & purificação , Aminobutiratos/metabolismo , Animais , Ecotoxicologia/métodos , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/isolamento & purificação , Espectroscopia de Ressonância Magnética , Malatos/isolamento & purificação , Ácidos Nicotínicos/isolamento & purificação , Ácidos Nicotínicos/metabolismo , Oligoquetos/química , Ornitina/análogos & derivados , Ornitina/isolamento & purificação , Ornitina/metabolismo , Espectrometria de Massas em TandemRESUMO
Global climate change has increased flooding events, which affect both natural vegetation dynamics and crop productivity. The flooded environment is lethal for most plant species because it restricts gas exchange and induces an energy and carbon crisis. Flooding survival strategies have been studied in Oryza sativa, a cultivated monocot. However, our understanding of plant adaptation to natural flood-prone environments remains scant, even though wild plants represent a valuable resource of tolerance mechanisms that could be used to generate stress-tolerant crops. Here we identify mechanisms that mediate the distinct flooding survival strategies of two related wild dicot species: Rumex palustris and Rumex acetosa. Whole transcriptome sequencing and metabolite profiling reveal flooding-induced metabolic reprogramming specific to R. acetosa. By contrast, R. palustris uses the early flooding signal ethylene to increase survival by regulating shade avoidance and photomorphogenesis genes to outgrow submergence and by priming submerged plants for future low oxygen stress. These results provide molecular resolution of flooding survival strategies of two species occupying distinct hydrological niches. Learning how these contrasting flood adaptive strategies evolved in nature will be instrumental for the development of stress-tolerant crop varieties that deliver enhanced yields in a changing climate.
Assuntos
Adaptação Fisiológica , Inundações , Regulação da Expressão Gênica de Plantas , Rumex/fisiologia , Carbono/metabolismo , Ecossistema , Etilenos/metabolismo , Perfilação da Expressão Gênica , Homeostase , Íons/metabolismo , Luz , Redes e Vias Metabólicas , Oxigênio/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Rumex/genética , Rumex/crescimento & desenvolvimento , Rumex/metabolismo , Estresse FisiológicoRESUMO
Heparin and heparan sulfate (HS) are important in mediating a variety of biological processes through binding to myriad different proteins. Specific structural elements along the polysaccharide chains are essential for high affinity protein binding, such as the 3-O-sulfated N-sulfoglucosamine (GlcNS3S) residue, a relatively rare modification essential for heparin's anticoagulant activity. The isolation of 3-O-sulfated oligosaccharides from complex mixtures is challenging because of their low abundance. Although methods such as affinity chromatography are useful in isolating oligosaccharides that bind specific proteins with high affinity, other important 3-O-sulfated oligosaccharides may easily be overlooked. Screening preparative-scale size-exclusion chromatography (SEC) fractions of heparin or HS digests using [(1)H,(15)N] HSQC NMR allows the identification of fractions containing 3-O-sulfated oligosaccharides through the unique (1)H and (15)N chemical shifts of the GlcNS3S residue. Those SEC fractions containing 3-O-sulfated oligosaccharides can then be isolated using strong anion-exchange (SAX)-HPLC. Compared with the results obtained by pooling the fractions comprising a given SEC peak, SAX-HPLC analysis of individual SEC fractions produces a less complicated chromatogram in which the 3-O-sulfated oligosaccharides are enriched relative to more abundant components. The utility of this approach is demonstrated for tetrasaccharide SEC fractions of the low molecular weight heparin drug enoxaparin facilitating the isolation and characterization of an unsaturated 3-O-sulfated tetrasaccharide containing a portion of the antithrombin-III binding sequence.
Assuntos
Cromatografia em Gel/métodos , Enoxaparina/análise , Espectroscopia de Ressonância Magnética/métodos , Oligossacarídeos/isolamento & purificação , Antitrombina III/metabolismo , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Enoxaparina/química , Heparitina Sulfato/análise , Heparitina Sulfato/química , Hidrogênio , Dados de Sequência Molecular , Isótopos de Nitrogênio , Oligossacarídeos/química , Oligossacarídeos/metabolismoRESUMO
Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state (17)Oâ quadrupole central-transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143â kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π-bonding network, particularly at the adjacent α-carbon site.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Triptofano Sintase/química , Domínio Catalítico , Cristalografia por Raios XRESUMO
Glucosamine is an important constituent of the heterogeneous glycosaminoglycans heparin and heparan sulfate occurring in N-acetylated and N-sulfated forms, and as the unmodified amine. Though the (1)H and (15)N NMR chemical shifts of N-acetyl- and N-sulfoglucosamine residues have been extensively characterized, this study provides the first direct NMR characterization of the amine groups of glucosamine and 3-O-sulfoglucosamine in aqueous solution. The solvent exchange properties of the amine protons are examined, and the possibility of a salt bridge between the sulfate and amine groups of 3-O-sulfoglucosamine is explored through (1)H NMR pKa measurements but is not supported by the experimental results.
Assuntos
Aminas/química , Glucosamina/química , Heparitina Sulfato/química , Espectroscopia de Ressonância Magnética/métodos , Monossacarídeos/química , Água/químicaRESUMO
Final effluent samples from 10 southern California (United States) wastewater treatment facilities, employing four distinct treatment schemes, were surveyed for selected pharmaceuticals, personal care products (PPCPs), alkylphenols, and 21 of their halogenated disinfection byproducts. Chlorinated and brominated standards and isotopically labeled internal standards were synthesized and purified to confirm and more accurately quantify selected disinfection byproducts of salicylic acid, bisphenol A, gemfibrozil, naproxen, diclofenac, technical 4-nonylphenol, and 4-tert-octylphenol using high-performance liquid chromatography and tandem mass spectrometry. Concentrations of parent compounds ranged from <10 to 3830 ng/L (gemfibrozil), and those of chloro/bromo byproducts ranged from <4 to 370 ng/L (dibromo nonylphenol). The highest concentrations of parent compounds were measured in effluent that was not subjected to tertiary treatment. The chlorinated and brominated byproduct concentration may be affected by the influent concentration of parent compounds, hydraulic retention times, and chlorine contact times. Salicylic acid was readily halogenated, which is evident from the ratio of halogenated to nonhalogenated species. There were no measured chlorinated byproducts of bisphenol A despite occasionally high concentrations of the parent compound. Not surprisingly, higher concentrations of most brominated species were measured in the treatment plant with the highest bromide concentrations. These results demonstrate the occurrence of novel halogenated byproducts of PPCPs that have limited toxicological data and significant uncertainty with regard to their risk to ecological systems.
Assuntos
Cosméticos/análise , Monitoramento Ambiental , Preparações Farmacêuticas/análise , Águas Residuárias/química , Poluentes da Água/análise , Purificação da Água/métodos , Compostos Benzidrílicos/análise , California , Cromatografia Líquida de Alta Pressão , Halogenação , Fenóis/análise , Espectrometria de Massas em TandemRESUMO
Oxygen deficiency, caused by flooding of all or a portion of a plant, leads to significant gene regulatory and metabolic responses associated with survival. When oxygen-deprived in light, aerial organs and root systems respond in distinct manners because of their respective autotrophy and heterotrophy, as well as intrinsic differences in cell biology and organ function. To better understand organ-specific responses to oxygen deficiency, we monitored changes in the metabolome of roots and shoots of Arabidopsis thaliana seedlings using gas chromatography-mass spectrometry and (1) H-nuclear magnetic resonance spectroscopy. Only roots accumulated high amounts of γ-aminobutyrate (GABA) and lactate, whereas both organs accumulated alanine (Ala) upon hypoxia. Meta-analysis of gene regulation data revealed higher induction of mRNAs coding for fermentative enzymes in roots as compared with shoots. However, the elevation in GABA level was not correlated with changes in transcript abundance, supporting the proposal that post-translational mechanisms are important in metabolic acclimation to hypoxia. The biosynthesis, degradation and function of GABA and Ala during oxygen deprivation and re-aeration is discussed. Finally, a systematic survey of low-oxygen mediated regulation of genes associated with primary metabolism across organs and cell types reveals exciting new avenues for future studies.
Assuntos
Adaptação Fisiológica , Arabidopsis/fisiologia , Carbono/metabolismo , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Oxigênio/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Redes e Vias Metabólicas , Metaboloma , Especificidade de Órgãos , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Proteoma , Estresse Fisiológico , Transcriptoma , Água/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
The anticoagulant properties of heparin stem in part from high-affinity binding to antithrombin-III (AT-III) inducing a 300-fold increase in its inhibitory activity against the coagulation protease factor Xa. The minimal structural requirements for AT-III binding are contained in the rare heparin pentasaccharide sequence containing a 3,6-O-sulfated N-sulfoglucosamine residue. ACE is used in this work to measure the relative AT-III binding affinities of the low molecular weight heparins (LWMHs) dalteparin, enoxaparin, and tinzaparin and the synthetic pentasaccharide drug fondaparinux (Arixtra). Determination of the AT-III binding affinities of the LWMHs is complicated by their inherent structural heterogeneity and polydispersity. The fractional composition of 3-O-sulfo-N-sulfoglucosamine residues was determined for each drug substance using 2D NMR and used in the interpretation of the ACE results.
Assuntos
Antitrombina III/metabolismo , Eletroforese Capilar/métodos , Heparina de Baixo Peso Molecular/metabolismo , Espectroscopia de Ressonância Magnética/métodosRESUMO
An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface.
Assuntos
Éteres Cíclicos/química , Bicamadas Lipídicas/química , Proteínas/química , Resorcinóis/química , Sequência de Aminoácidos , Ânions/química , Dados de Sequência Molecular , Ligação Proteica , Proteínas/metabolismo , Eletricidade EstáticaRESUMO
Natural disasters such as drought, extreme temperatures, and flooding can severely impact crop production. Understanding the metabolic response of crops threatened with these disasters provides insights into biological response mechanisms that can influence survival. In this study, a comparative analysis of GC-MS and (1)H NMR results was conducted for wild-type and tolerant rice varieties stressed by up to 3 days of submergence and allowed 1 day of postsubmergence recovery. Most metabolomics studies are conducted using a single analytical platform. Each platform, however, has inherent advantages and disadvantages that can influence the analytical coverage of the metabolome. In this work, a more thorough analysis of the plant stress response was possible through the use of both (1)H NMR and GC-MS. Several metabolites, such as S-methyl methionine and the dipeptide alanylglycine, were only detected and quantified by (1)H NMR. The high dynamic range of NMR, as compared with that of the GC-TOF-MS used in this study, provided broad coverage of the metabolome in a single experiment. The sensitivity of GC-MS facilitated the quantitation of sugars, organic acids, and amino acids, some of which were not detected by NMR, and provided additional insights into the regulation of the TCA cycle. The combined metabolic information provided by (1)H NMR and GC-MS was essential for understanding the complex biochemical and molecular response of rice plants to submergence.
Assuntos
Metabolômica , Oryza/metabolismo , Proteínas de Plantas/análise , Estresse Fisiológico , Metabolismo dos Carboidratos , Carboidratos/análise , Cromatografia Líquida/métodos , Ciclo do Ácido Cítrico , Dipeptídeos/análise , Dipeptídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Análise de Componente Principal , Vitamina U/análise , Vitamina U/metabolismoRESUMO
Heparin and heparan sulfate (HS) are members of a biologically important group of highly anionic linear polysaccharides called glycosaminoglycans (GAGs). Because of their structural complexity, the molecular-level characterization of heparin and HS continues to be a challenge. The work presented herein describes an emerging approach for the analysis of unfractionated and low molecular weight heparins, as well as porcine and human-derived HS. This approach utilizes the untapped potential of (15)N NMR to characterize these preparations through detection of the NH resonances of N-sulfo-glucosamine residues. The sulfamate group (1)H and (15)N chemical shifts of six GAG microenvironments were assigned based on the critical comparison of selectively modified heparin derivatives, NMR measurements for a library of heparin-derived oligosaccharide standards, and an in-depth NMR analysis of the low molecular weight heparin enoxaparin through systematic investigation of the chemical exchange properties of NH resonances and residue-specific assignments using the [(1)H,(15)N] HSQC-TOCSY experiment. The sulfamate microenvironments characterized in this study include GlcNS(6S)-UA(2S), ΔUA(2S)-GlcNS(6S), GlcNS(3S)(6S)-UA(2S), GlcNS-UA, GlcNS(6S)-red(α), and 1,6-anhydro GlcNS demonstrating the utility of [(1)H,(15)N] HSQC NMR spectra to provide a spectroscopic fingerprint reflecting the composition of intact GAGs and low molecular weight heparin preparations.
Assuntos
Glucosamina/análogos & derivados , Heparina/química , Heparitina Sulfato/química , Configuração de Carboidratos , Células Cultivadas , Glucosamina/química , Células HEK293 , Heparitina Sulfato/isolamento & purificação , Humanos , Espectroscopia de Ressonância Magnética/normas , Isótopos de Nitrogênio , Prótons , Padrões de ReferênciaRESUMO
We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.
Assuntos
Peptídeos Cíclicos/farmacologia , Drusas Retinianas/imunologia , Epitélio Pigmentado da Retina/metabolismo , Células Cultivadas , Ativação do Complemento , Humanos , Drusas Retinianas/tratamento farmacológico , Drusas Retinianas/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/embriologiaRESUMO
In exploring the capability of nuclear magnetic resonance (NMR) spectroscopy for pomegranate juice analysis, the eight aromatic singlet resonances of α- and ß-punicalagin were clearly identified in the (1)H NMR spectra of juice samples. The four downfield resonances were found to be sensitive to small pH changes around pH 3.50 where the NMR spectra of the juice samples were recorded. To understand this unusual behavior, the (1)H and (13)C resonance assignments of the punicalagin anomers were determined in aqueous solution and pH titrations with UV and (1)H NMR detection carried out to characterize the acid-base properties of punicalagin over the pH range 2-8. Simultaneous fitting of all of the pH-sensitive (1)H NMR signals produced similar but significantly different pKa values for the first two deprotonation equilibria of the gallagic acid moiety of the punicalagin α- (pKa1 = 4.57 ± 0.02, pKa2 = 5.63 ± 0.03) and ß- (pKa1 = 4.36 ± 0.01, pKa2 = 5.47 ± 0.02) anomers. Equivalent pKa values, (α : 6.64 ± 0.01, ß : 6.63± 0.01) were measured for the third deprotonation step involving the ellagic acid group, in good agreement with a prior literature report. The punicalagin anomer equilibrium readjusts in parallel with the proton dissociation steps as the pH is raised such that ß-punicalagin becomes the most abundant anomer at neutral pH. The unusual upfield shifts observed for the glucose H3 and H5 resonances with increasing pH along with the shift in the α/ß anomer equilibrium are likely the consequence of a conformational rearrangement.
Assuntos
Bebidas/análise , Taninos Hidrolisáveis/química , Lythraceae/química , Espectroscopia de Ressonância Magnética/métodos , Concentração de Íons de Hidrogênio , IsomerismoRESUMO
A chemometric technique, visual interpretation of z-score ratios (VIZR), written in the open source code R, has been developed to identify metabolic differences between individual biosamples and a control group. To demonstrate the capabilities of VIZR, 49 urine samples were collected from healthy volunteers: 41 samples were collected randomly following a normal dietary routine and 7 test samples were collected after dietary supplementation with either ibuprofen or alcoholic beverages. An eighth test sample was prepared by 50% dilution of a control sample. Sample analysis was conducted by (1)H nuclear magnetic resonance (NMR) spectroscopy and the collected data were subjected to VIZR analysis, which successfully discriminated each of the 8 test samples from the 41 control samples. In addition, VIZR analysis revealed the NMR spectral regions responsible for the disparity between the individual test samples and the control group. The self-normalizing nature of the VIZR calculation provides a robust analysis independent of dilution effects, which is especially important in urine analyses. Potential applications of VIZR include high-throughput data analysis for toxicological profiling, disease diagnosis, and biomarker identification in any type of biosample for which a control dataset can be established. Although demonstrated herein for the statistical analysis of (1)H NMR data, the VIZR program is platform independent and could be applied to digitized metabolic datasets acquired using other techniques including hyphenated mass spectrometry measurements.