RESUMEN
Recent studies have reported very low capacity during sterile filtration of glycoconjugate vaccines due to rapid fouling of the sterile filter. The objective of this study was to explore the potential for significantly increasing the capacity of the sterile filter through the use of an appropriate prefilter. Data were obtained using prefilters with different pore size and chemistry, with the sterile filtration performed at constant filtrate flux using 0.22 µm nominal pore size Durapore® polyvinylidene difluoride membranes. Prefiltration through 5 µm pore size Durapore® or Nylon prefilters nearly eliminated the fouling of the sterile filter, leading to more than a 100-fold reduction in the rate of pressure increase for the sterile filter. This dramatic improvement in sterile filter performance was due to the removal of large components (greater than 1 µm in size) as confirmed by dynamic light scattering. These results demonstrate the potential of using large pore size prefilters to significantly enhance the performance of the sterile filtration process for the production of important glycoconjugate vaccines.
Asunto(s)
Filtración , Glicoconjugados , Vacunas Conjugadas , Contaminación de Medicamentos/prevención & control , Filtración/métodos , Filtración/normas , Glicoconjugados/análisis , Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Membranas Artificiales , Porosidad , Vacunas Conjugadas/análisis , Vacunas Conjugadas/química , Vacunas Conjugadas/aislamiento & purificaciónRESUMEN
The exploitation of recombinant enzymes for the synthesis of complex carbohydrates is getting increasing attention. Unfortunately, the analysis of the resulting products often requires advanced methods like nuclear magnetic resonance spectroscopy and mass spectrometry. Here, we use the capsule polymerases Cps4B and Cps11D from Actinobacillus pleuropneumoniae serotypes 4 and 11, respectively, as examples for the in vitro synthesis of capsule polymers similar to those used in glycoconjugate vaccine formulations. We demonstrate how substrate turnover in an enzymatic reaction can be analyzed by HPLC-based anion exchange chromatography and provide the protocol for separation and detection of UV-active polymer. Moreover, we describe how UV-inactive polymer can be separated and visualized using polyacrylamide gel electrophoresis followed by combined alcian blue-silver staining.
Asunto(s)
Antígenos Bacterianos/química , Cápsulas Bacterianas/enzimología , Glicoconjugados/síntesis química , Polisacáridos/síntesis química , Vacunas Conjugadas/química , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glicoconjugados/inmunología , Glicoconjugados/aislamiento & purificación , Polímeros/síntesis química , Polisacáridos/inmunología , Polisacáridos/aislamiento & purificación , Vacunas Conjugadas/inmunología , Vacunas Conjugadas/aislamiento & purificaciónRESUMEN
Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).
Asunto(s)
Galectina 3/aislamiento & purificación , Glicoconjugados/aislamiento & purificación , Pruebas de Inhibición de Hemaglutinación/normas , Pruebas de Hemaglutinación/normas , Proteoglicanos/aislamiento & purificación , Animales , Proteínas Sanguíneas , Bovinos , Electroforesis en Gel de Poliacrilamida/métodos , Eritrocitos/química , Eritrocitos/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Filtración/métodos , Harina/análisis , Galectina 3/química , Galectina 3/genética , Galectina 3/metabolismo , Galectinas , Glicoconjugados/química , Glicosilación , Humanos , Unión Proteica , Proteoglicanos/química , Conejos , Glycine max/química , Tiroglobulina/farmacología , Xanthosoma/químicaRESUMEN
Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.
Asunto(s)
Flavonoides/farmacocinética , Glicoconjugados/aislamiento & purificación , Microsomas Hepáticos/metabolismo , Orthosiphon/química , Acetilación , Animales , Bilis/química , Biotransformación , Heces/química , Flavonoides/sangre , Flavonoides/orina , Microbioma Gastrointestinal/fisiología , Glicoconjugados/metabolismo , Hidrogenación , Masculino , Metilación , Oxidación-Reducción , Extractos Vegetales/química , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Flavonoids represent an important class of natural products with a central role in plant physiology and human health. Their accurate annotation using untargeted mass spectrometry analysis still relies on differentiating similar chemical scaffolds through spectral matching to reference library spectra. In this work, we combined molecular network analysis with rules for fragment reactions and chemotaxonomy to enhance the annotation of similar flavonoid glyconjugates. Molecular network topology progressively propagated the flavonoid chemical functionalization according to collision-induced dissociation (CID) reactions, as the following chemical attributes: aglycone nature, saccharide type and number, and presence of methoxy substituents. This structure-based distribution across the spectral networks revealed the chemical composition of flavonoids across intra- and interspecies and guided the putatively assignment of 64 isomers and isobars in the Chrysobalanaceae plant species, most of which are not accurately annotated by automated untargeted MS2 matching. These proof of concept results demonstrate how molecular networking progressively grouped structurally related molecules according to their product ion scans, abundances, and ratios. The approach can be extrapolated to other classes of metabolites sharing similar structures and diagnostic fragments from tandem mass spectrometry.
Asunto(s)
Chrysobalanaceae/química , Flavonoides/aislamiento & purificación , Glicoconjugados/aislamiento & purificación , Glicósidos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Chrysobalanaceae/metabolismo , Flavonoides/química , Flavonoides/clasificación , Glicoconjugados/química , Glicoconjugados/clasificación , Glicósidos/química , Glicósidos/clasificación , Glicosilación , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The purpose of this study was to compare the application of several extraction methods, including cold extraction (CAE), hot extraction (HAE), ultrasonic-assisted extraction (UAE), and microwave-assisted extraction (MAE), all carried out in 0.1â¯M NaOH, under their respective best parameters, for obtaining products from leaf of Fragaria vesca L. The extracts with the highest anticoagulant activity were purified by multi-step extraction procedure and separated by gel permeation chromatography, giving rise to the macromolecular complexes. They were subsequently structurally characterized using colorimetric methods, FT-IR, GC-MS, and NMR (1H and 1H/13C HSQC), as polyphenolic-polysaccharide conjugates with the anticoagulant activity. The polysaccharide parts of the conjugates obtained by different extraction procedures were found to vary significantly. The most selective in their activity were the glycoconjugates extracted in UAE and MAE processes, i.e. arabinogalactan and pectin-like conjugates, respectively. In terms of their anticoagulant activity all of them were non-direct factor Xa inhibitors mediated by antithrombin.
Asunto(s)
Fragaria/química , Extractos Vegetales/química , Hojas de la Planta/química , Polifenoles/química , Polifenoles/aislamiento & purificación , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Glicoconjugados/farmacología , Extracción Líquido-Líquido/métodos , Microondas , Polifenoles/farmacología , Polisacáridos/farmacología , Análisis Espectral , Ondas UltrasónicasRESUMEN
The parotoid gland of bufonids is characterized as a specialized integument region, formed by different gland types. The secretion elaborated by the largest glandular alveoli has been related to animal chemical defense and is constituted by granular protein content, associated with a basophilic and alcianophilic material with features of glycoconjugates. This study aimed to identify and characterize the glycoconjugates in the secretion of the largest granular gland of the parotoid gland of Rinella icterica by histochemical and immunohistochemical techniques at light microscopy, biochemical methods, and nuclear magnetic resonance spectroscopy. Our results showed that the glycoconjugate content contains a mixture of chondroitin6sulfate (C6S) and chondroitin-non-sulfate (C0S). Thus, chondroitin sulfate probably plays an important role in gland physiology, probably protecting the protein content while inside the secretory portion.
Asunto(s)
Acetilgalactosamina/química , Bufonidae/metabolismo , Sulfatos de Condroitina/química , Ácido Glucurónico/química , Glicoconjugados/química , Glándula Parótida/química , Acetilgalactosamina/aislamiento & purificación , Animales , Brasil , Bufonidae/anatomía & histología , Secuencia de Carbohidratos , Sulfatos de Condroitina/aislamiento & purificación , Ácido Glucurónico/aislamiento & purificación , Glicoconjugados/aislamiento & purificación , Masculino , Glándula Parótida/anatomía & histología , Glándula Parótida/fisiologíaRESUMEN
Individual monosaccharides can be linked in a variety of different combinations to form complex glycoconjugates. In contrast to DNA and proteins, glycoconjugate synthesis does not follow any template but is the consequence of the concerted action of various enzymes such as transferases and glycosidases . Thus, tools for glycoconjugate sequencing need to differentiate individual monosaccharide identity, linkage and anomericity to investigate and understand glycoconjugate function. In this chapter we provide a concise overview on the most commonly used and robust tools to separate and characterise glycoconjugate isomers.
Asunto(s)
Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Polisacáridos/químicaRESUMEN
INTRODUCTION: 18F-fluoroglycosylation via oxime formation is a chemoselective and mild radiolabeling method for sensitive molecules. Glycosylation can also improve the bioavailability, in vivo kinetics, and stability of the compound in blood, as well as accelerate clearance of biomolecules. A typical synthesis procedure for 18F-fluoroglycosylation with [18F]FDG (2-deoxy-2-[18F]fluoro-d-glucose) and [18F]FDR (5-deoxy-5-[18F]fluoro-d-ribose) involves two HPLC (high performance liquid chromatography) purifications: one after 18F-fluorination of the carbohydrate to remove its labeling precursor, and a second one after the oxime formation step to remove the aminooxy precursor. The two HPLC purifications can be time consuming and complicate the adaptation of the synthetic strategy in nuclear medicine applications and automated synthesis. We have developed a procedure in which SPE (solid phase extraction) and resin purification methods replace both of the needed HPLC purification steps. METHODS: We used [18F]FDR and [18F]FDG as prosthetic groups to radiolabel two aminooxy-modified model molecules, a tetrazine and a PSMA (prostate specific membrane antigen) inhibitor. After fluorination, the excess carbohydrate precursor was removed by derivatizing it with 4,4'-dimethoxytrityl chloride (DMT-Cl). The DMT moiety increases the hydrophobicity of the unreacted precursor making the separation from the fluorinated precursor possible with simple C18 Sep-Pak cartridge. For removal of the aminooxy precursor, we used a commercially available aldehyde resin (AminoLink, Thermo Fisher Scientific). C18 Sep-Pak SPE cartridge was used to separate [18F]FDR and [18F]FDG from the 18F-fluoroglycoconjugate end product. RESULTS: [18F]FDR and [18F]FDG were efficiently purified from their precursors, free fluorine-18, and other impurities. The aldehyde resin quantitatively removed the unreacted aminooxy precursors after the oxime formation. The fluorine-18 labeled oxime end products were obtained with high radiochemical purity (>99%) and molar activity (>600â¯GBq⯵mol-1). CONCLUSIONS: We have developed an efficient cartridge purification method for producing high molar activity 18F-glycoconjugates synthesized via oxime formation.
Asunto(s)
Fraccionamiento Químico/instrumentación , Radioisótopos de Flúor/química , Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Oximas/química , Compuestos de Anilina/química , Glicosilación , Cinética , Tomografía de Emisión de Positrones , Piridinas/química , RadioquímicaRESUMEN
Glycoconjugates extracted from Genipa americana leaves (PE-Ga) were separated into two fractions, denominated as PFI and PFII (total carbohydrate: 23-36%/uronic acid: 9-30%; protein:4-5%; polyphenols:0.776-0.812 mg/g), mainly composed by arabinose, galactose and uronic acid and presenting high (PFI) and low (PFII) molecular weight (based on polyacrylamide electrophoresis gel and gel permeation chromatography). Uronic acid was also detected by FT-IR (wavenumbers: 1410 and 1333 cm-1) and NMR (α-GalpA). Deproteinization of glycoconjugates showed reduced protein and polyphenol levels with loss of its biological effects. PE-Ga and PFII prolonged clotting time-aPTT (3.6 and 1.8x), while PE-Ga and PFI inhibited by 48% (100 µg/µL) the ADP-induced platelet aggregation. In vivo, these glycoconjugates at 1 mg/kg inhibited (37-53%) venous thrombus formation (4.7 ± 0.1 mg) and increased bleeding time (PE-Ga and PFI:3.0x; PFII:1.7x vs. PBS:906 ± 16.7 s). In conclusion, the arabinogalactan-rich glycoconjugate of G. americana leaves, containing uronic acid, present antiplatelet, anticoagulant (intrinsic/common pathway) and antithrombotic effects, with low hemorrhagic risk.
Asunto(s)
Anticoagulantes/farmacología , Fibrinolíticos/farmacología , Galactanos/farmacología , Glicoconjugados/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Rubiaceae/química , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Coagulación Sanguínea/efectos de los fármacos , Fibrinolíticos/química , Fibrinolíticos/aislamiento & purificación , Galactanos/química , Galactanos/aislamiento & purificación , Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Voluntarios Sanos , Humanos , Hojas de la Planta/química , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Ratas , Ratas Wistar , Trombosis de la Vena/tratamiento farmacológicoRESUMEN
Glycans present in biological glycoconjugates have several structural and functional roles. Elucidation of glycan structure and biological function is critical to understand their role in physiological and pathogenic process, enabling the development of diagnostic methods and disease treatment. Immobilized glycosidases are powerful tools for glycan analysis, as they are able to remove specific carbohydrates without altering the protein structure. Here we describe the individual immobilization of Aspergillus oryzae ß-galactosidase and Canavalia ensiformis α-mannosidase onto agarose and silica magnetic nanoparticles activated with cyanate ester groups. High immobilization yields (70-90%) were achieved, keeping above 60% of its original activity. Immobilized glycosidases were effective in the selective deglycosylation of model glycoproteins and a Fasciola hepatica lysate, evidenced by a decrease in specific lectin recognition of 40-50% after enzymatic deglycosylation. Immobilized glycosidases were reused for several deglycosylation cycles without loss of effectiveness. Their use was extended to the elucidation of the glycan role of native glycoconjugates. A decrease in the recognition of lactoferrin treated with α-mannosidase by a C-type lectin receptor, DC-SIGN was found. Also the specific deglycosylation of a F. hepatica lysate demonstrated the relevance of mannosylated glycans in the induction of Th2/Treg immune responses during the infection. Our results show successful immobilization of specific glycosidases in nano-supports and validate their utility to identify glycans biological functions.
Asunto(s)
Enzimas Inmovilizadas/química , Glicoconjugados/análisis , Glicómica , Nanopartículas de Magnetita , alfa-Manosidasa/química , beta-Galactosidasa/química , Animales , Aspergillus oryzae/enzimología , Médula Ósea/metabolismo , Canavalia/enzimología , Bovinos , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/metabolismo , Enzimas Inmovilizadas/metabolismo , Fasciola hepatica/metabolismo , Glicoconjugados/aislamiento & purificación , Glicoproteínas/metabolismo , Glicosilación , Lactoferrina/metabolismo , Lectinas/metabolismo , Lectinas Tipo C/metabolismo , Ratones Endogámicos BALB C , Polisacáridos/metabolismo , Receptores de Superficie Celular/metabolismo , alfa-Manosidasa/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Polysaccharide conjugates were alkali-extracted from green tea (TPC-A). Although it contained 11.80% covalently binding proteins, TPC-A could not bind to the Coomassie Brilliant Blue dyes G250 and R250. TPC-A had no expected characteristic absorption peak of protein in the UV-vis spectrum scanning in the range of 200-700 nm. The UV-vis wavelength of 280 nm was not suitable to detect the presence of the protein portion of TPC-A. The zeta potential of TPC-A merely presented the negative charge properties of polysaccharides instead of the acid-base property of its protein section across the entire pH range. Furthermore, TPC-A was more stable when the pH of solution exceeded 4.0. In addition, no precipitation or haze was generated in the TPC-A/(-)-epigallocatechin gallate (EGCG) mixtures during 12 h storage. TPC-A has emulsifying activity, which indicated that its protein moiety formed hydrophobic groups. Thus, it was proposed that some physical properties of TPC-A protein were shielded by its olysaccharide, since the protein moiety was wrapped by its polysaccharide chains.
Asunto(s)
Glicoconjugados/química , Proteínas de Plantas/química , Polisacáridos/química , Té/química , Catequina/análogos & derivados , Catequina/química , Glicoconjugados/aislamiento & purificación , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Extracción Líquido-Líquido/métodos , Polisacáridos/aislamiento & purificación , Colorantes de Rosanilina , Hidróxido de Sodio , Espectrofotometría UltravioletaRESUMEN
Radioprotective potential of the polyphenolic glycoconjugates, isolated from flowers of Sanguisorba officinalis L. (So) and Erigeron canadensis L. (Ec), and from leaves of Fragaria vesca L. (Fv) and Rubus plicatus Whe. Et N. E. (Rp) as well as their aglycones (SoA, EcA, FvA and RpA, respectively), against γ-radiation-induced lipid peroxidation in human plasma and DNA damage in lymphocytes, were investigated in vitro. These properties were assessed by measuring the concentration of thiobarbituric acid reactive substances (TBARS) and using the alkaline comet assay, and were compared to the protective effects of rutin (R) and quercetin (Q). Cytotoxicity of the glycoconjugates/aglycones towards L929 mouse fibroblasts and human lymphocytes were also measured. Plant products from S. officinalis, similar to Q, were able to reduce the most radiation-induced lipid peroxidation as well as DNA damage and extent of oxidative damage to the DNA basis. Contrary to the pure flavonoids, where Q was shown to be significantly more effective than its glycoside R, the results did not show more benefit with application of SoA/EcA over So/Ec in terms of lipid peroxidation inhibition. Moreover, glycoconjugates Ec and So showed much higher capacity in protecting lymphocytes against radiation-induced genotoxicity which may suggest that between the polyphenolic and polysaccharide parts exist some synergistic effects. There were no significant differences between Fv versus FvA or Rp versus RpA in terms of the provided radioprotection. Summarizing, plant glycoconjugates isolated by the multi-step method offered sufficient radioprotection. In addition, they possess many advantages, compared to the synthetic polyphenolic compounds or the plant extracts, such as water-solubility and minor toxicity.
Asunto(s)
Glicoconjugados/química , Polifenoles/química , Protectores contra Radiación/farmacología , Rosaceae/química , Animales , Antioxidantes/química , Asteraceae/química , Asteraceae/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Ensayo Cometa , ADN/química , ADN/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Rayos gamma , Glicoconjugados/aislamiento & purificación , Glicoconjugados/farmacología , Humanos , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/efectos de la radiación , Ratones , Plantas Medicinales/química , Plantas Medicinales/metabolismo , Quercetina/farmacología , Protectores contra Radiación/química , Protectores contra Radiación/aislamiento & purificación , Rosaceae/metabolismo , Rutina/farmacologíaRESUMEN
Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) expressed on a variety of DCs, is a C-type lectin receptor that recognizes glycans on a diverse range of pathogens, including parasites. The interaction of DC-SIGN with pathogens triggers specific signaling events that modulate DC-maturation and activity and regulate T-cell activation by DCs. In this work we evaluate whether F. hepatica glycans can immune modulate DCs via DC-SIGN. We demonstrate that DC-SIGN interacts with F. hepatica glycoconjugates through mannose and fucose residues. We also show that mannose is present in high-mannose structures, hybrid and trimannosyl N-glycans with terminal GlcNAc. Furthermore, we demonstrate that F. hepatica glycans induce DC-SIGN triggering leading to a strong production of TLR-induced IL-10 and IL-27p28. In addition, parasite glycans induced regulatory DCs via DC-SIGN that decrease allogeneic T cell proliferation, via the induction of anergic/regulatory T cells, highlighting the role of DC-SIGN in the regulation of innate and adaptive immune responses by F. hepatica. Our data confirm the immunomodulatory properties of DC-SIGN triggered by pathogen-derived glycans and contribute to the identification of immunomodulatory glyans of helminths that might eventually be useful for the design of vaccines against fasciolosis.
Asunto(s)
Células Dendríticas/inmunología , Fasciola hepatica/química , Fasciola hepatica/inmunología , Glicoconjugados/metabolismo , Interacciones Huésped-Patógeno , Factores Inmunológicos/metabolismo , Linfocitos T/inmunología , Animales , Moléculas de Adhesión Celular , Anergia Clonal , Células Dendríticas/efectos de los fármacos , Glicoconjugados/aislamiento & purificación , Humanos , Factores Inmunológicos/aislamiento & purificación , Lectinas Tipo C , Receptores de Superficie CelularRESUMEN
Radioprotective effects of the water-soluble polyphenolic glycoconjugates, isolated from flowers of Sanguisorba officinalis L.(SO) and Erigeron canadensis L.(EC), and from leaves of Fragaria vesca L. (FV) and Rubus plicatus Whe. Et N. E. (RP), against γ-radiation-induced toxicity in human peripheral blood lymphocytes were investigated. Cell treatment with glycoconjugates (1, 5 and 25µg/mL) prior exposure to 10/15Gy radiation resulted in concentration-dependent reduction of DNA damage including oxidative DNA lesions (comet assay), substantial inhibition of lipid peroxidation (TBARS) and restoration of superoxide dismutase and S-glutathione transferase activities. Glycoconjugates isolated from SO and EC ensured better protection versus these from RP and FV, with the SO product potential comparable to that of the reference quercetin. Strong antioxidant/radioprotective activity of the SO and EC glycoconjugates could be attributed to high abundance of syringol-type and ferulic acid units in their matrices, respectively. Moreover, polyphenolic glycoconjugates (25µg/mL), including RP and FV products, significantly decreased DNA damage when applied post-radiation suggesting their modulating effects on DNA repair pathways. Preliminary data on the glycoconjugate phenolic structural units, based on GLC/MS of the products of pyrolysis and in situ methylation, in relation to application of plant products as potential radioprotectors is promising and deserves further investigation.
Asunto(s)
Antioxidantes/farmacología , Asteraceae/química , Glicoconjugados/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Protectores contra Radiación/farmacología , Rosaceae/química , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Ensayo Cometa , Ácidos Cumáricos/análisis , Ácidos Cumáricos/química , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Rayos gamma , Glutatión Transferasa/metabolismo , Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de la radiación , Extractos Vegetales/química , Cultivo Primario de Células , Pirogalol/análogos & derivados , Pirogalol/análisis , Pirogalol/química , Quercetina/farmacología , Protectores contra Radiación/química , Protectores contra Radiación/aislamiento & purificación , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Although like proteins, polysaccharides are synthesized by enzymes, unlike proteins there is no template. This means that they are polydisperse, do not generally have compact folded structures, and are often very large with greater nonideality behavior in solution. This chapter considers the relevant analytical ultracentrifuge methodology available for characterizing these and related carbohydrate-based systems and information this methodology supplies, in terms of sizes, shapes, and interactions using a comprehensive range of examples, including glycoconjugates and lignins. The relevance and potential of recent software developments such as SEDFIT-MSTAR, the Extended Fujita algorithm, and HYDFIT are considered.
Asunto(s)
Glicoconjugados/aislamiento & purificación , Lignina/aislamiento & purificación , Polisacáridos/aislamiento & purificación , Conformación de Carbohidratos , ADN/química , ADN/aislamiento & purificación , Glicoconjugados/química , Lignina/química , Peso Molecular , Polisacáridos/química , Unión Proteica , Proteínas/química , Proteínas/aislamiento & purificación , Programas Informáticos , Ultracentrifugación/métodosRESUMEN
Thermomonas hydrothermalis is a Gram-negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell-wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure-dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system-an antagonistic effect, as shown in studies of LPS structure-activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo-oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.
Asunto(s)
Gammaproteobacteria/química , Glicoconjugados/química , Lípidos/química , Lipopolisacáridos/antagonistas & inhibidores , Oligosacáridos/química , Secuencia de Carbohidratos , Glicoconjugados/aislamiento & purificación , Glicoconjugados/farmacología , Células HEK293 , Humanos , Lípido A/química , Lípidos/aislamiento & purificación , Lípidos/farmacología , Datos de Secuencia Molecular , Oligosacáridos/aislamiento & purificación , Oligosacáridos/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Glicoconjugados/aislamiento & purificación , Humanos , Leche Humana/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría Ultravioleta , Espectrometría de Masas en TándemRESUMEN
From the air-dried Wild strawberry (Fragaria vesca L., family Rosaceae) leaves five water-soluble glycoconjugates Fv I-V by different extraction conditions have been isolated. Effects of extraction steps/agents on chemical composition and anticoagulant activity of Fv I-V were examined. Dark brown F. vesca conjugates Fv I-V were recovered in 4.5-8.4% yields, based on dry herb. Isolates were composed of carbohydrate, phenolic and protein components. Fv I-V displayed on HPLC broad molecule-mass distribution patterns with dominance of low molecule-masses 9-14 kDa. Their carbohydrate parts revealed high hexuronic acids content (35-60%) while the dominant neutral sugars - galactose, arabinose and rhamnose were found in lower amounts and indicated the presence of rhamnogalacturonans associated with arabinogalactans in all F. vesca preparations. In all Fv I-V isolates high polyphenolic contents were determined, whereas proteins were found in low amounts only. In in vitro experiments on human pooled plasma Fv I-V showed at higher concentrations complete inhibition of plasma clot formation and the most active conjugates in aPTT, PT and TT tests were shown to be Fv I and Fv III, containing the highest amounts of phenolics.
Asunto(s)
Anticoagulantes , Coagulación Sanguínea/efectos de los fármacos , Fragaria/química , Glicoconjugados , Anticoagulantes/administración & dosificación , Anticoagulantes/química , Galactanos/química , Galactanos/aislamiento & purificación , Glicoconjugados/administración & dosificación , Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Ácidos Hexurónicos/química , Ácidos Hexurónicos/aislamiento & purificación , Humanos , Espectroscopía de Resonancia Magnética , Hojas de la Planta/químicaRESUMEN
Mycobacterium avium complex (MAC) is a group of non-tuberculous mycobacteria that cause tuberculosis-like diseases in humans. Unlike Mycobacterium tuberculosis, MAC expresses high levels of glycopeptidolipids (GPLs) containing a well-defined tetrapeptide-amino alcohol core, composed of D-phenylalanine, D-allo-threonine, D-alanine, and L-alaninol, that is modified with a fatty acid and sugar residues. Surprisingly, however, a careful scrutiny of the mass spectrum of MAC GPLs revealed the presence of ions that could not readily accountable for the known GPL structure. The magnitude of the ions was increased prominently when GPLs were isolated from the valine-supplemented culture, and the ions representing the authentic GPL species were diminished, suggesting the possibility that the basic structure of the peptide backbone might be altered in response to the exogenously added valine. Indeed, further mass spectrometry (MS)/MS and gas chromatography-MS analysis indicated a substitution of D-valine for the N-terminal D-phenylalanine of the tetrapeptide core, and the presence of D-valine and the absence of D-phenylalanine was confirmed by high-performance liquid chromatography, using the derivatized amino acid residues that were released from the tetrapeptide. Finally, specific antibodies to the purified valine-containing GPL species were detected in the serum of a MAC-infected guinea pig. Therefore, these results identify a new molecular species of MAC GPLs with immunogenic potential.