RESUMEN
Milk proteins serve as nutrition and affect neonate development and immunity through their bioactivity. Post-translational modifications of proteins affect their bioactivity. Glycosylation is the attachment of sugar moieties to proteins, with attachment of glycans to asparagine indicated as N-linked glycosylation. Our objective was to characterize N-linked glycosylated proteins in homogenate swine milk samples collected from sows (nâ =â 5/6) during farrowing to represent colostrum and on days 3 and 14 post-farrowing to represent transitional and mature milk, respectively. Glycopeptides were isolated with lectin-based extraction and treated with Peptide N-glycosidase F (PNGase F) to identify N-linked glycosylation sites. Purified glycopeptides were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant software was used to align spectra to Sus scrofa Uniport database to identify proteins and measure their relative abundances. Analysis of variance and Welch's t-test analysis identified glycoproteins differentially abundant between colostrum, transitional, and mature milk (false discovery rate <0.05). Shotgun proteome analysis identified 545 N-linked and glutamine, Q, -linked, glycosylation (Pâ >â 0.75 for deamidation) sites on 220 glycoproteins in sow milk. Glycoproteins were found across all three phases of swine milk production and varied by number of glycosylation sites (1-14) and in abundance and distribution between colostrum, transitional, and mature milk. Polymeric immunoglobulin receptor was the most glycosylated protein with 14 sites identified. Also highly glycosylated were casein and mucin proteins. These data are described and the relevance of glycosylated milk proteins in neonate development, such as protection against pathogens, is discussed.
Milk is essential for healthy growth and development of neonates, with proteins in milk serving as key nutrients and regulators of these processes. Protein activity is affected by modifications made to their structure including the addition of sugar groups called glycans. Here we present the characterization of sow milk proteins modification with glycan groups on asparagine and glutamine amino acids in colostrum, transitional, and mature milk of pigs. We found 220 high confidences (found in at least two sows on one day) glycoproteins, and that the abundance of glycosylated proteins varied by stage of milk production and number of glycosylated sites.
Asunto(s)
Proteoma , Espectrometría de Masas en Tándem , Embarazo , Animales , Femenino , Porcinos , Proteoma/metabolismo , Cromatografía Liquida/veterinaria , Espectrometría de Masas en Tándem/veterinaria , Lactancia , Calostro/metabolismo , Glicoproteínas/análisis , Proteínas de la Leche/metabolismo , Glicopéptidos/análisis , Glicopéptidos/metabolismoRESUMEN
Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.
Asunto(s)
Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Extractos Vegetales/química , Arabidopsis/química , Arabidopsis/enzimología , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Glicopéptidos/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Plantas/metabolismo , Polisacáridos/metabolismoRESUMEN
OBJECTIVE: Patients with craniopharyngioma (CP) frequently suffer from morbid obesity. Endocannabinoids (ECs) are involved in weight gain and rewarding behavior but have not been investigated in this context. DESIGN: Cross-sectional single-center study. METHODS: Eighteen patients with CP and 16 age- and sex-matched controls were included. Differences in endocannabinoids (2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)) and endocannabinoid-like molecules (oleoyl ethanolamide (OEA), palmitoylethanolamide (PEA), and arachidonic acid (AA) were measured at baseline and following endurance exercise. We further explored ECs-dynamics in relation to markers of HPA-axis activity (ACTH, cortisol, copeptin) and hypothalamic damage. RESULTS: Under resting conditions, independent of differences in BMI, 2-AG levels were more than twice as high in CP patients compared to controls. In contrast, 2-AG and OEA level increased in response to exercise in controls but not in CP patients, while AEA levels decreased in controls. As expected, exercise increased ACTH and copeptin levels in controls only. In a mixed model analysis across time and group, HPA measures did not provide additional information for explaining differences in 2-AG levels. However, AEA levels were negatively influenced by ACTH and copeptin levels, while OEA levels were negatively predicted by copeptin levels only. There were no significant differences in endocannabinoids depending on hypothalamic involvement. CONCLUSION: Patients with CP show signs of a dysregulated endocannabinoid system under resting conditions as well as following exercise in comparison to healthy controls. Increased 2-AG levels under resting conditions and the missing response to physical activity could contribute to the metabolic phenotype of CP patients.
Asunto(s)
Craneofaringioma , Endocannabinoides/metabolismo , Sistema Hipotálamo-Hipofisario/fisiopatología , Neoplasias Hipofisarias , Hormona Adrenocorticotrópica/metabolismo , Adulto , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/metabolismo , Estudios de Casos y Controles , Craneofaringioma/metabolismo , Craneofaringioma/fisiopatología , Estudios Transversales , Entrenamiento Aeróbico , Ejercicio Físico/fisiología , Femenino , Glicéridos/metabolismo , Glicopéptidos/metabolismo , Humanos , Hidrocortisona/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Hipotálamo/metabolismo , Hipotálamo/patología , Hipotálamo/fisiopatología , Masculino , Persona de Mediana Edad , Ácidos Oléicos/metabolismo , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/fisiopatología , Alcamidas Poliinsaturadas/metabolismo , Adulto JovenRESUMEN
Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/química , Glicopéptidos/aislamiento & purificación , Glicoproteínas/aislamiento & purificación , Lectinas/química , Neoplasias Pulmonares/química , Proteoma/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Glicoconjugados/química , Glicoconjugados/metabolismo , Glicopéptidos/clasificación , Glicopéptidos/genética , Glicopéptidos/metabolismo , Glicoproteínas/clasificación , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Humanos , Lectinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Nanopartículas de Magnetita/química , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Proteoma/clasificación , Proteoma/genética , Proteoma/metabolismo , ProteómicaRESUMEN
Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.
Asunto(s)
Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Extractos Vegetales/química , Secuencia de Aminoácidos , Arabidopsis/química , Cromatografía Liquida/métodos , Colorantes Fluorescentes/química , Glicopéptidos/química , Glicopéptidos/metabolismo , Solanum lycopersicum/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/aislamiento & purificación , Hojas de la Planta/química , Plantas Modificadas Genéticamente , Especificidad por SustratoRESUMEN
d-Glutamate (Glu) supplied by Glu racemases or d-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a d-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-l-Ala-d-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate l-Glu to UDP-MurNAc-l-Ala, providing the first example of MurD utilizing l-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal l-Glu of the product in the presence of ATP and Mg2+. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones.
Asunto(s)
Gammaproteobacteria/metabolismo , Ácido Glutámico/metabolismo , Glicopéptidos/metabolismo , Peptidoglicano/biosíntesis , Racemasas y Epimerasas/metabolismo , Gammaproteobacteria/química , Ácido Glutámico/química , Glicopéptidos/química , Peptidoglicano/químicaRESUMEN
Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have especially strong binding (apparent Kd = 0.5-1.0 nm) to the tumor-associated Tn antigen. High-resolution crystal structures were determined for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. DATABASE: Structural data are available in the PDB under the accession numbers 5T50, 5T52, 5T55, 5T54, 5T5L, 5T5J, 5T5P, and 5T5O.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Antineoplásicos Fitogénicos/química , Bauhinia/química , Lectinas de Plantas/química , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Línea Celular Tumoral , Clonación Molecular , Cristalografía por Rayos X , Dimerización , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Globósidos/química , Globósidos/metabolismo , Glicopéptidos/química , Glicopéptidos/metabolismo , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Oligosacáridos/química , Oligosacáridos/metabolismo , Extractos Vegetales/química , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping but distinct functions of EGT and MSH. Last, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.
Asunto(s)
Antioxidantes/metabolismo , Metabolismo Energético/fisiología , Ergotioneína/metabolismo , Mycobacterium tuberculosis/patogenicidad , Virulencia , Animales , Antioxidantes/análisis , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Cisteína/metabolismo , Susceptibilidad a Enfermedades , Ergotioneína/análisis , Glicopéptidos/metabolismo , Inositol/metabolismo , Pulmón/microbiología , Pulmón/patología , Macrófagos/microbiología , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Oxidación-Reducción , Análisis de Componente Principal , Espectrometría de Masas en Tándem , Factores de Transcripción/metabolismoRESUMEN
Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications.
Asunto(s)
Flores/metabolismo , Glicopéptidos/metabolismo , Hidroxiprolina/metabolismo , Prolina/metabolismo , Análisis de Secuencia de Proteína , Taraxacum/metabolismo , Secuencia de Aminoácidos , Bacterias/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Dicroismo Circular , Glicopéptidos/química , Glicopéptidos/aislamiento & purificación , Glicopéptidos/farmacología , Hidroxiprolina/química , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Peso Molecular , Prolina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We investigated whether inflammatory mediators during cecal ligation and puncture (CLP)-induced sepsis may diminish copeptin expression in magnocellular neurons, thus affecting arginine-vasopressin (AVP) synthesis. The transcript abundance of IL-1ß, IL-1R1, iNOS and HIF-1α was continuously elevated. IL-1ß, iNOS and cytochrome c protein levels progressively increased until 24h. Immunostaining for these proteins was higher at 6 and 24h, as also seen in the annexin-V assay, while copeptin was continuously decreased. This suggests that increased IL-1ß and NO levels may cause significant bioenergetics changes in magnocellular neurons, affecting copeptin expression and compromising AVP synthesis and secretion in the late phase of sepsis.
Asunto(s)
Metabolismo Energético/fisiología , Regulación de la Expresión Génica/fisiología , Glicopéptidos/metabolismo , Hipotálamo/patología , Neuronas/metabolismo , Sepsis/metabolismo , Sepsis/patología , Animales , Anexina A5/metabolismo , Arginina Vasopresina/genética , Arginina Vasopresina/metabolismo , Ciego , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Quiasma Óptico/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/metabolismo , Sepsis/etiología , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
HypSys peptides are 18-20 amino acids glycopeptide defense signal first discovered in tobacco and tomato that activate expression of defensive genes against insect-herbivores. Discovery of their orthologs in other Solanaceaous and nonsolanaceous plants demonstrated their possible ubiquitous nature and species specific functional diversity. In our continued search to establish the paradigm of defense signalling by HypSys peptides, we isolated a cDNA from potato leaves encoding putative analogs of tomato HypSys peptides flanked by conserved proteolytic cleavage sites. The gene encoding the cDNA was a member of a gene family in the tetraploid genome of potato and its expression was transcriptionally activated by wounding and methyl jasmonate. The deduced precursor protein contained a leader peptidase splice site and three putative HypSys peptides with conserved N- and C-termini along with central proline-rich motifs. In defense signalling, the three HypSys peptides elicit H2O2 generation in vivo and activate several antioxidant defensive enzymes in young potato leaves. Similar to potato systemin, the HypSys peptides activate the expression of octadecanoid pathway genes and protease inhibitors for insect defense. In addition, the HypSys peptides also activate the essential genes of the innate pathogen defense response in young potato leaves, acting as common elicitors of signalling associated with anti-herbivore and anti-pathogen defense in potato.
Asunto(s)
ADN de Plantas/genética , Glicopéptidos/genética , Precursores de Proteínas/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Plantas/metabolismo , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Glicopéptidos/metabolismo , Datos de Secuencia Molecular , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Precursores de Proteínas/metabolismo , Homología de Secuencia , Transducción de Señal , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologíaRESUMEN
In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of "robust enrichment" afforded by covalent-labeling techniques and "specificity for glycoproteins" typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status.
Asunto(s)
Compuestos de Anilina , Galactosa/metabolismo , Glicoproteínas , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismo , Compuestos de Anilina/química , Compuestos de Anilina/metabolismo , Linfocitos B/química , Linfocitos B/citología , Linfocitos B/metabolismo , Catálisis , Línea Celular , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Glicopéptidos/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Oximas/química , Oximas/metabolismo , Ácido Peryódico/metabolismoRESUMEN
Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Previous studies showed that the Gekko sulfated polysaccharide-protein complex suppressed the proliferation and migration of hepatoma cells. Gekko sulfated glycopeptide α was obtained from Gekko sulfated polysaccharide-protein complex using papain hydrolysis. Gekko sulfated glycopeptide α inhibited the proliferation and migration of SMMC-7721 cells. The secretion of IL-8 and the concentration of intracellular calcium were decreased after Gekko sulfated glycopeptide α exposure. SMMC-7721 cells in the control group showed abnormal features, with a polygonal shape, whereas this changed to a spindle shape after the treatment with Gekko sulfated glycopeptide α. Actin ï¬laments were distributed diffusely along the cell membrane in control cells, whereas those were polymerized and preferentially accumulated in the cytoplasm of treated cells. Microtubules distributed in the cytoplasm of untreated cells were located diffusely whereas those in treated cells were polymerized. Therefore, Gekko sulfated glycopeptide α inhibit the migration of hepatoma cells via reducing the secretion of IL-8 and the concentration of intracellular calcium, as well as regulating the reorganization of cytoskeleton.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Movimiento Celular/efectos de los fármacos , Glicopéptidos/farmacología , Neoplasias Hepáticas/metabolismo , Polisacáridos/farmacología , Aminoácidos/química , Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Glicopéptidos/metabolismo , Humanos , Interleucina-8/metabolismo , Espacio Intracelular/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Actinomycetes, the soil borne bacteria which exhibit filamentous growth, are known for their ability to produce a variety of secondary metabolites including antibiotics. Industrial scale production of such antibiotics is typically carried out in a multi-substrate medium where the product formation may experience catabolite repression by one or more of the substrates. Availability of reliable process models is a key bottleneck in optimization of such processes. Here we present a structured kinetic model to describe the growth, substrate uptake and product formation for the glycopeptide antibiotic producer strain Amycolatopsis balhimycina DSM5908. The model is based on the premise that the organism is an optimal strategist and that the various metabolic pathways are regulated via key rate limiting enzymes. Further, the model accounts for substrate inhibition and catabolite repression. The model is also able to predict key phenomena such as simultaneous uptake of glucose and glycerol but with different specific uptake rates, and inhibition of glycopeptide production by high intracellular phosphate levels. The model is successfully applied to both production and seed medium with varying compositions and hence has good predictive ability over a variety of operating conditions. The model parameters are estimated via a well-designed experimental plan. Adequacy of the proposed model was established via checking the model sensitivity to its parameters and confidence interval calculations. The model may have applications in optimizing seed transfer, medium composition, and feeding strategy for maximizing production.
Asunto(s)
Actinomycetales/metabolismo , Antibacterianos/metabolismo , Reactores Biológicos , Glicopéptidos/metabolismo , Modelos Estadísticos , Medios de Cultivo/metabolismo , Cinética , Fósforo/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol that is unique to actinomycetes and whose primary role is to maintain intracellular redox balance and remove toxins. MshC catalyzes the adenosine triphosphate (ATP)-dependent condensation of cysteine and glucosamine-inositol (GI) to produce cysteine-glucosamine-inositol (CGI). MshC is essential to Mycobacterium tuberculosis and therefore represents an attractive target for chemotherapeutic intervention. A screening protocol was developed to identify MshC inhibitors based on quantification of residual ATP using a coupled luminescent assay. The protocol was used to screen a library of 3100 compounds in a 384-well plate format (Z'>or=0.65). Fifteen hits (0.48%) were identified from the screen, and 2 hits were confirmed in a secondary assay that measures production of CGI. The structures of both hits contain N-substituted quinolinium moieties, and the more potent of the 2-namely, dequalinium chloride-inhibits MshC with an IC50 value of 24+/-1 microM. Further studies showed dequalinium to be an ATP-competitive inhibitor of MshC, to bind MshC with a KD of 0.22 microM, and to inhibit the growth of M. tuberculosis under aerobic and anaerobic conditions with minimum inhibitory and anaerobic bactericidal concentrations of 1.2 and 0.3 microg/mL, respectively. The screening protocol described is robust and has enabled the identification of new MshC inhibitors.
Asunto(s)
Cisteína/metabolismo , Decualinio/análisis , Decualinio/farmacología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Glicopéptidos/metabolismo , Inositol/metabolismo , Ligasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Antiinfecciosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Portadoras/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/análisis , Cinética , Mediciones Luminiscentes , Proteínas de Unión a Maltosa , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
BACKGROUND: Phenylketonuria (PKU) requires a lifelong low-phenylalanine diet that provides the majority of protein from a phenylalanine-free amino acid (AA) formula. Glycomacropeptide (GMP), an intact protein formed during cheese production, contains minimal phenylalanine. OBJECTIVE: The objective was to investigate the effects of substituting GMP food products for the AA formula on acceptability, safety, plasma AA concentrations, and measures of protein utilization in subjects with PKU. DESIGN: Eleven subjects participated in an inpatient metabolic study with two 4-d treatments: a current AA diet (AA diet) followed by a diet that replaced the AA formula with GMP (GMP diet) supplemented with limiting AAs. Plasma concentrations of AAs, blood chemistries, and insulin were measured and compared in AA (day 4) and GMP diets (day 8). RESULTS: The GMP diet was preferred to the AA diet in 10 of 11 subjects with PKU, and there were no adverse reactions to GMP. There was no significant difference in phenylalanine concentration in postprandial plasma with the GMP diet compared with the AA diet. When comparing fasting with postprandial plasma, plasma phenalyalanine concentration increased significantly with the AA but not with the GMP diet. Blood urea nitrogen was significantly lower, which suggests decreased ureagenesis, and plasma insulin was higher with the GMP diet than with the AA diet. CONCLUSIONS: GMP, when supplemented with limiting AAs, is a safe and highly acceptable alternative to synthetic AAs as the primary protein source in the nutritional management of PKU. As an intact protein source, GMP improves protein retention and phenylalanine utilization compared with AAs.
Asunto(s)
Aminoácidos/administración & dosificación , Aminoácidos/sangre , Glicopéptidos/administración & dosificación , Fenilcetonurias/sangre , Fenilcetonurias/dietoterapia , Adolescente , Adulto , Aminoácidos/metabolismo , Análisis Químico de la Sangre , Nitrógeno de la Urea Sanguínea , Niño , Seguridad de Productos para el Consumidor , Estudios Cruzados , Femenino , Glicopéptidos/efectos adversos , Glicopéptidos/metabolismo , Humanos , Insulina/sangre , Masculino , Satisfacción del Paciente , Fenilalanina/sangre , Periodo Posprandial , Resultado del Tratamiento , Adulto JovenRESUMEN
Copeptin is cleaved from the C-terminus of vasopressin (VP) prohormone. Immunohistochemical studies have revealed intense copeptin-immunoreactivity (irCOPT) in neurones of the rat hypothalamic nuclei, including paraventricular, supraoptic, suprachiasmatic, periventricular, and accessory secretory. Varicose cell processes emanated from irCOPT neurones, some of which projected caudally and traversed the internal layer of the median eminence, and terminated in the posterior pituitary. Double-labelling hypothalamic sections with copeptin antiserum and VP or oxytocin antiserum revealed an extensive overlapping of irCOPT and irVP neurones. The biological activity of human synthetic nonglycosylated copeptin or VP was evaluated in vivo and in vitro. Copeptin (1, 10, and 20 nmol/kg) injected i.v. caused no significant changes in the mean arterial pressure (MAP) and heart rate of urethane-anaesthetised rats. VP (0.1 nmol/kg) increased MAP, which was accompanied by a small decrease of the heart rate. The ratiometric fluorescence method was employed to assess changes in intracellular Ca2+ concentrations [Ca2+](i) which served as an index of the biological activity of peptides. VP (1 microM) markedly increased [Ca2+](i) of rat hypothalamic neurones or vascular smooth muscle cells, whereas copeptin (100 nm to 1 microM) caused a low amplitude, sustained increase of [Ca2+](i) in a population of hypothalamic neurones, but not in any of the vascular smooth muscle cells tested. The results obtained demonstrate that copeptin is expressed in VP neurones and that the peptide in the concentrations tested, although causing little or no detectable changes of blood pressure and heart rate in anaesthetised rats nor changes in [Ca2+](i) of cultured aortic smooth muscle cells, increases [Ca2+](i) in a small population (< 2%) of hypothalamic neurones tested, indicating that copeptin is biologically active in mammalian neurones.
Asunto(s)
Calcio/metabolismo , Glicopéptidos/metabolismo , Hipotálamo , Neuronas/metabolismo , Animales , Presión Sanguínea/fisiología , Células Cultivadas , Frecuencia Cardíaca/fisiología , Humanos , Hipotálamo/citología , Hipotálamo/metabolismo , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neuronas/citología , Oxitocina/metabolismo , Precursores de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Vasopresinas/metabolismoRESUMEN
BACKGROUND: During stress, vasopressin is a potent synergistic factor of CRH as a hypothalamic stimulator of the HPA axis. The measurements of CRH and vasopressin levels are cumbersome because of their instability and short half-life. Copeptin is a more stable peptide stoichiometrically released from the same precursor molecule. The aim of our study was to compare copeptin and cortisol levels in different stress situations. METHODS: Three groups of patients with increasing stress levels were investigated: a) healthy controls without apparent stress (n=20), b) hospitalized medical patients with moderate stress (n=25) and c) surgical patients 30 minutes after extubation, with maximal stress (n=29). In all patients we assessed cortisol and copeptin levels. Copeptin levels were measured with a new sandwich immunoassay. RESULTS: Cortisol levels in controls were (median, IQ range, 486 [397-588] nmol/L), not significantly different as compared to medical patients (438 [371-612] nmol/L, p=0.69). Cortisol levels in surgical patients after extubation were higher (744 [645-1062] nmol/L p<0.01 vs controls and medical patients). Copeptin levels in controls were 4.3 [3.2-5.5] pmol/L, which was lower as compared to medical patients (17.5 [6.4-24.1], p<0.001) and surgical patients after extubation (67.5 [37.8-110.0] pmol/L, p<0.001). The correlation between copeptin levels and cortisol was r=0.46, p<0.001. CONCLUSION: Copeptin is a novel marker of the individual stress level. It more subtly mirrors moderate stress as compared to cortisol values.
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Glicopéptidos/metabolismo , Estrés Psicológico/metabolismo , Vasopresinas/metabolismo , Hormona Adrenocorticotrópica/sangre , Adulto , Biomarcadores , Índice de Masa Corporal , Estudios de Cohortes , Femenino , Glicopéptidos/análisis , Humanos , Hidrocortisona/sangre , Hipotálamo/metabolismo , Masculino , Persona de Mediana Edad , Caracteres Sexuales , Vasopresinas/análisisRESUMEN
T cells obtained in a mouse model for rheumatoid arthritis are activated by a glycopeptide fragment from rat type II collagen (CII) bound to the class II major histocompatibility complex Aq molecule. We report a comparative model of Aq in complex with the glycopeptide CII260-267. This model was used in a structure-based design approach where the amide bond between Ala261 and Gly262 in the glycopeptide was selected for replacement with psi[COCH2], psi[CH2NH2+], and psi[(E)-CH=CH] isosteres. Ala-Gly isostere building blocks were then synthesized and introduced in CII260-267 and CII259-273 glycopeptides. The modified glycopeptides were evaluated for binding to the Aq molecule, and the results were interpreted in view of the Aq/glycopeptide model. Moreover, recognition by a panel of T-cell hybridomas revealed high sensitivity for the backbone modifications. These studies contribute to the understanding of the interactions in the ternary Aq/glycopeptide/T-cell receptor complexes that activate T cells in autoimmune arthritis and suggest possibilities for new vaccination approaches.
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Artritis Experimental/inmunología , Colágeno Tipo II/química , Glicopéptidos/química , Antígenos de Histocompatibilidad Clase II/química , Fragmentos de Péptidos/química , Receptores de Antígenos de Linfocitos T/metabolismo , Alanina/química , Animales , Células Presentadoras de Antígenos/metabolismo , Artritis Experimental/inducido químicamente , Unión Competitiva , Técnicas de Cocultivo , Glicina/química , Glicopéptidos/síntesis química , Glicopéptidos/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Hibridomas , Interleucina-2/metabolismo , Ratones , Modelos Moleculares , Estructura Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Ratas , Bazo/citología , Estereoisomerismo , Relación Estructura-Actividad , Linfocitos T/metabolismoRESUMEN
The arginine vasopressin (AVP) gene is expressed in certain cancers such as breast cancer, where it is believed to act as an autocrine growth factor. However, little is known about the regulation of the AVP protein precursor (proAVP) or AVP-mediated signaling in breast cancer and this study was undertaken to address some of the basic issues. The cultured cell lines examined (Mcf7, Skbr3, BT474, ZR75, Mcf10a) and human breast cancer tissue extract were found to express proAVP mRNA. Western analysis revealed multiple forms of proAVP protein were present in cell lysates, corresponding to those detected in human hypothalamus extracts. Monoclonal antibodies directed against different regions of proAVP bound to intact live Mcf7 and Skbr3 cells. Dexamethasone increased the amount of proAVP-associated glycopeptide (VAG) secreted by Skbr3 cells and a combination of dexamethasone, IBMX and 8br-cAMP increased cellular levels of VAG. Exogenous AVP (1, 10, and 100 nM) elevated phospho-ERK1/2 levels, and increased cell proliferation was observed in the presence of 10 nM AVP. Concurrent treatment with the V1a receptor antagonist SR49059 reduced the effects of AVP on proliferation in Mcf7 cells, and abolished it in Skbr3 cells. Results here show that proAVP components are found at the surface of Skbr3 and Mcf7 cells and are also secreted from these cells. In addition, they show that AVP promotes cancer cell growth, apparently through a V1-type receptor-mediated pathway and subsequent ERK1/2 activation. Thus, strategies for targeting proAVP should be examined for their effectiveness in diagnosing and treating breast cancer.