RESUMEN
Early identification of Alzheimer's disease (AD) risk factors would aid development of interventions to delay the onset of dementia, but current biomarkers are invasive and/or costly to assess. Validated plasma biomarkers would circumvent these challenges. We previously identified the kinase DYRK1A in plasma. To validate DYRK1A as a biomarker for AD diagnosis, we assessed the levels of DYRK1A and the related markers brain-derived neurotrophic factor (BDNF) and homocysteine in two unrelated AD patient cohorts with age-matched controls. Receiver-operating characteristic curves and logistic regression analyses showed that combined assessment of DYRK1A, BDNF and homocysteine has a sensitivity of 0.952, a specificity of 0.889 and an accuracy of 0.933 in testing for AD. The blood levels of these markers provide a diagnosis assessment profile. Combined assessment of these three markers outperforms most of the previous markers and could become a useful substitute to the current panel of AD biomarkers. These results associate a decreased level of DYRK1A with AD and challenge the use of DYRK1A inhibitors in peripheral tissues as treatment. These measures will be useful for diagnosis purposes.
Asunto(s)
Enfermedad de Alzheimer/sangre , Factor Neurotrófico Derivado del Encéfalo/sangre , Homocisteína/sangre , Proteínas Serina-Treonina Quinasas/sangre , Proteínas Tirosina Quinasas/sangre , Anciano , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Biomarcadores/sangre , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Tirosina Quinasas/inmunología , Curva ROC , Quinasas DyrKRESUMEN
The use of lactic acid bacteria (LAB) as probiotics in aquaculture may improve the quality of seed production and limit the use of antibiotics in fish hatcheries. This study attempted to further characterize the candidate probiotic Lactobacillus casei X2, and the immune and physiological responses of the sea bass larvae. L. casei X2 was confirmed as a good candidate, due to its wide antibacterial spectrum against both Gram-positive and Gram-negative bacteria, and its free radical scavenging activity. In addition, if the strain did not seem able to form biofilm on abiotic surfaces, it adhered strongly to Hep-2 cells. However, these characteristics did not seem efficient in vivo. At 20 days post-hatch (dph), the expression level of CAT gene was significantly different between group fed without probiotic and the two groups treated with either Pediococcus acidilactici or L. casei. This gene was upregulated in the group treated with strain X2 and downregulated in the group with a commercial probiotic strain P. acidilactici, suggesting a better antioxidant activity with the later strain. At the same sampling date, the IL-1ß gene was upregulated in the group treated with P. acidilactici, and the HSP70 gene was overexpressed at 41 dph. As the stimulation of these two last genes, such transcriptomic indicators must be cautiously interpreted.
Asunto(s)
Lubina , Proteínas de Peces/genética , Inmunidad Innata , Lactobacillus/inmunología , Leuconostoc/inmunología , Pediococcus/inmunología , Probióticos , Alimentación Animal/análisis , Animales , Antioxidantes/metabolismo , Lubina/inmunología , Lubina/microbiología , ADN Bacteriano/genética , Dieta/veterinaria , Expresión Génica , Lactobacillus/química , Lactobacillus/genética , Leuconostoc/química , Leuconostoc/genética , Pediococcus/química , Pediococcus/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/veterinariaRESUMEN
We wish to delineate a novel, and rapidly expanding, group of inborn errors of metabolism with neurological/muscular presentations: the defects in phospholipids, sphingolipids and long chain fatty acids biosynthesis. At least 14 disorders have been described so far. Clinical presentations are diverse but can be divided into (1) diseases of the central nervous system; (2) peripheral neuropathies; and (3) muscular/cardiac presentations. (1) Leukodystrophy and/or iron deposits in basal ganglia is a common feature of phospholipase A2 deficiency, fatty acid hydroxylase deficiency, and pantothenate kinase-associated neurodegeneration. Infantile epilepsy has been reported in GM3 synthetase deficiency. Spastic quadriplegia with ichthyosis and intellectual disability are the presenting signs of the elongase 4 deficiency and the Sjogren-Larsson syndrome caused by fatty aldehyde dehydrogenase deficiency. Spastic paraplegia and muscle wasting are also seen in patients with mutations in the neuropathy target esterase gene. (2) Peripheral neuropathy is a prominent feature in PHARC syndrome due to α/ß-hydrolase 12 deficiency, and in hereditary sensory autonomic neuropathy type I due to serine palmitoyl-CoA transferase deficiency. (3) Muscular/cardiac presentations include recurrent myoglobinuria in phosphatidate phosphatase 1 (Lipin1) deficiency; cardiomyopathy and multivisceral involvement in Barth syndrome secondary to tafazzin mutations; congenital muscular dystrophy due to choline kinase deficiency, Sengers syndrome due to acylglycerol kinase deficiency and Chanarin Dorfman syndrome due to α/ß- hydrolase 5 deficiency. These synthesis defects of complex lipid molecules stand at the frontier between classical inborn errors of metabolism and other genetic diseases involving the metabolism of structural proteins.
Asunto(s)
Ácidos Grasos/biosíntesis , Errores Innatos del Metabolismo Lipídico/clasificación , Fosfolípidos/biosíntesis , Esfingolípidos/biosíntesis , Animales , Humanos , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/metabolismo , Enfermedades Metabólicas/clasificación , Enfermedades Metabólicas/diagnóstico , Enfermedades Metabólicas/genética , Modelos Biológicos , Fosfolípidos/deficiencia , Esfingolípidos/deficienciaRESUMEN
The striking excess of affected males in autism spectrum disorders (ASD) suggests that genes located on chromosome X contribute to the etiology of these disorders. To identify new X-linked genes associated with ASD, we analyzed the entire chromosome X exome by next-generation sequencing in 12 unrelated families with two affected males. Thirty-six possibly deleterious variants in 33 candidate genes were found, including PHF8 and HUWE1, previously implicated in intellectual disability (ID). A nonsense mutation in TMLHE, which encodes the É-N-trimethyllysine hydroxylase catalyzing the first step of carnitine biosynthesis, was identified in two brothers with autism and ID. By screening the TMLHE coding sequence in 501 male patients with ASD, we identified two additional missense substitutions not found in controls and not reported in databases. Functional analyses confirmed that the mutations were associated with a loss-of-function and led to an increase in trimethyllysine, the precursor of carnitine biosynthesis, in the plasma of patients. This study supports the hypothesis that rare variants on the X chromosome are involved in the etiology of ASD and contribute to the sex-ratio disequilibrium.
Asunto(s)
Trastornos Generalizados del Desarrollo Infantil/genética , Cromosomas Humanos X/genética , Genes Ligados a X , Oxigenasas de Función Mixta/genética , Adulto , Estudios de Casos y Controles , Niño , Estudios de Cohortes , Exoma , Familia , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Mutación , Reacción en Cadena de la Polimerasa , Distribución por SexoRESUMEN
The diagnosis of Alzheimer's disease has long been considered a diagnosis of probability, as the definitive diagnosis can only be established by histopathological examination. However, the development of in-vivo biomarkers, considered a reflection of physiopathological processes, has changed our view of the disease. New criteria have recently been proposed that integrate such biomarkers as found in the cerebrospinal fluid (CSF) using new diagnostic tools such as magnetic resonance imaging (MRI), brain scintigraphy, FDG-positron emission tomography (PET) and PET amyloid ligand uptake studies. The value of these new criteria for the diagnosis of prodromal Alzheimer's disease and the prospect of disease-modifying drugs are also discussed.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Biomarcadores/análisis , Síntomas Prodrómicos , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/líquido cefalorraquídeo , Encéfalo/diagnóstico por imagen , Diagnóstico por Imagen/métodos , Diagnóstico Precoz , Humanos , Modelos Biológicos , Radiografía , Cintigrafía , Proteínas tau/análisis , Proteínas tau/líquido cefalorraquídeoRESUMEN
Gliomas represent 50% of primary brain tumors, and their prognosis remains poor despite the advances in diagnosis and therapeutic strategies. Low grade gliomas (LGG) are infiltrative tumors and they constantly undergo malignant transformation. Metabolic exploration of human gliomas in vivo, in animals and by using cell culture models showed important differences between tumor tissues and normal brain tissues, which can provide new markers for diagnosis, prognosis and therapeutic targets. In this study, energetic and oxidant metabolisms were explored in biopsy extracts of LGG obtained from the centre and the periphery of tumors. Metabolic pattern of these tumors was explored and the differences between the centre and the periphery pointed. Our study showed a metabolic heterogeneity between tumors, with hypermetabolic and hypometabolic profiles. Lactate to pyruvate ratio was>1, suggesting that the energy metabolism in LGG is glycolytic in nature, particularly in the centre of the tumors. Peripheral samples of tumors showed increased glucose consumption and cytochrome c oxidase activity. Lipid peroxidation and catalase activity were also increased in the periphery compared to the centre of tumors. A relationship between the main antioxidant and energy metabolism enzymes activities was observed, suggesting that periphery of tumors is more active metabolically and more resistant to free radical injury.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Metabolismo Energético , Glioma/metabolismo , Estrés Oxidativo , Adulto , Anciano , Biopsia , Encéfalo/patología , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Catalasa/metabolismo , Estudios de Cohortes , Interpretación Estadística de Datos , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Glioma/diagnóstico , Glioma/enzimología , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
This review reports recent observations concerning specificities of the cellular energy metabolism in cerebral tissues that highlight on characteristics of that of glial tumours, such as the association of metabolic alterations aggressiveness of these tumours. Compared to normal cerebral tissue, glial tissue exhibits both a relative independence towards oxygen and substrate furnitures and thus vascularization, as well as the metabolic co-operation of neurons and glial cells within the tumour. Occurrence of a Warburg effect could explain such metabolic autonomy that might be associated to genetic changes observed in gliomas. Characteristics of the glycolytic metabolism within glioma tissue therefore may be novel land therapeutic approaches for the treatment of these tumours.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Metabolismo Energético , Glioma/metabolismo , Adulto , Factores de Edad , Animales , Astrocitos/metabolismo , Astrocitos/fisiología , Encéfalo/patología , Neoplasias Encefálicas/epidemiología , Neoplasias Encefálicas/patología , Niño , Modelos Animales de Enfermedad , Glioma/epidemiología , Glioma/genética , Glioma/patología , Glucosa/metabolismo , Glucólisis , Humanos , Lactatos/metabolismo , Metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Estadificación de NeoplasiasRESUMEN
Arabino-Galactan Proteins (AGPs) were isolated from Chios mastic gum (CMG) by using a buffer containing 0.1 M NaCl, 20 mM Tris-HCl, pH 7.5. Protein analytical methods, combined with specific procedures for carbohydrate characterization, indicated the presence of highly glycosylated protein backbone. In particular, staining by Yariv reagent of the electrophoretically separated molecules revealed the existence of arabinose and galactose and such a modification is characteristic for AGPs. After experiments involving extensive dialysis of the isolated extracts against water and atomic absorption, there was evidence of the existence of zinc ions that are probably covalently bound to the AGPs. By using anion-exchange chromatography, capillary electrophoresis, colorimetric methods and GC-MS, it was found that the extracts were separated into three major populations (A, B, and C), which were consistent with their respective negative charge content namely, uronic acid. The characterization of neutral sugars that was investigated with GC-MS showed the existence of arabinose and galactose in different amounts for each group. Experiments concerning the inhibition of growth of Helicobacter pylori in the presence of AGPs, as is shown for other CMG constituents, showed that the extracts of at least 1.4 g CMG affected the viability of the bacterium. There is no evidence as to whether the AGPs provoke abnormal morphologies of H. pylori, as is reported for the total CMG, or for O-glycans that possess terminal alpha1, 4-linked N-acetylglucosamine and are expressed in the human gastric mucosa; this has to be further investigated.
Asunto(s)
Galactanos/química , Pistacia/química , Pistacia/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Resinas de Plantas/química , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/metabolismo , Antibacterianos/farmacología , Cromatografía por Intercambio Iónico , Electroforesis Capilar , Cromatografía de Gases y Espectrometría de Masas , Glicosilación , Helicobacter pylori/efectos de los fármacos , Resina Mástique , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Solubilidad , Agua/químicaRESUMEN
Adsorption properties of gram-scale samples of different kind of arc discharge nanotubes were studied, namely: (A) raw collaret collected on the cathode, (B) raw soots collected on the lateral reactor wall, (C) thermally treated soot, and (D) thermally then chemically treated soot. The morphology, structure, and composition of these materials were characterized by SEM, TEM, TGA, and BET. In addition, hydrogen adsorption isotherms were recorded experimentally for A, B, and D samples over the pressure range of 0 to 55 bar at ambient temperature. Our experiments indicated a maximum-yet weak-hydrogen storage at room temperature of approximately 0.13 H2 wt% for the purified product (D).
Asunto(s)
Cristalización/métodos , Electroquímica/métodos , Transferencia de Energía , Hidrógeno/química , Hidrógeno/aislamiento & purificación , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestructura , Absorción , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Nanotecnología/métodos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The adsorption of equimolar binary mixtures of hydrogen-carbon dioxide, hydrogen-methane, and methane-carbon dioxide in porous material models is determined by grand canonical Monte Carlo simulations. The material models have an adsorbent surface similar to that of nanofibers with a herringbone structure. Our main result, which is relevant for hydrogen purification and carbon dioxide capture, is that the adsorption selectivities calculated for the mixtures can differ significantly from those deduced from simulations of the adsorption of pure gases, in particular, when one of the adsorbed gases presents a capillary condensation induced by confinement within the pore network. A comparison of our data is also made with theoretical models used in the literature for predicting the properties of the mixture adsorption.
RESUMEN
Staphylococcus epidermidis is a major nosocomial pathogen, even though it is a member of the normal bacterial flora of skin and the mucous membranes. A major complication is the development of biofilms on implanted medical devices. Diagnosis of coagulase-negative staphylococcal infections relies on the presence of clinical manifestation of infections and on microbiologic evidence, usually obtained after the removal of the biomaterial. Solid-phase immunoassays have not yet been used for routine diagnosis of coagulase-negative staphylococcal infections and distinction between pathogenic and normal cocci. The enzyme immunoassays developed in the last decade are presented in this review article. Serodiagnosis has been attempted by determining antibodies against bacterial cells, mixtures of S. epidermidis slime antigens and discrete slime antigens. Detection or typing of staphylococcal cells has been performed by specific antibodies and lectins. There is still a long way until the application of such assays in the routine clinical laboratory and large clinical studies are necessary.
Asunto(s)
Coagulasa/química , Inmunoensayo/métodos , Infecciones Estafilocócicas/diagnóstico , Anticuerpos Antibacterianos , Biopelículas , Ensayo de Inmunoadsorción Enzimática/métodos , Predicción , Grecia , Humanos , Infecciones Relacionadas con Prótesis/diagnóstico , Infecciones Relacionadas con Prótesis/microbiología , Infecciones Estafilocócicas/epidemiología , Staphylococcus epidermidisRESUMEN
Interaction of basic fibroblast growth factor (bFGF) with heparin/heparan sulfate proteoglycans protects the growth factor against proteolytic degradation and is essential for its cellular activity. Although the structural requirements of heparin and heparan sulfate for the high-affinity binding to bFGF have been extensively examined, studies on intact heparin proteoglycans are limited. In this report, the purity and the binding ability of a heparin proteoglycan-like molecule-the heparin-bovine serum albumin (heparin-BSA) conjugate-was examined using capillary zone electrophoresis (CZE). Furthermore, the affinity of bFGF binding to the heparin-BSA conjugate was studied using an enzyme solid-phase assay. Chondroitin sulfate, dermatan sulfate, hyaluronan, heparan sulfate and variously sulfated disaccharides derived from heparin and heparan sulfate were also studied for their ability to compete with the binding of bFGF to heparin. Heparin-BSA conjugate was synthesized by reductive amination and, following precipitation with 1.5 vols of ethanol-sodium acetate, it was obtained free of contaminating heparin. Heparin-BSA-bFGF conjugate was obtained following incubation of heparin-BSA with bFGF for 2 h at 37 degrees C. Intact heparin, heparin-BSA and heparin-BSA-bFGF conjugates were completely resolved by CZE using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Competitive solid phase assay showed that, among the glycosaminoglycans tested, heparin exhibits the highest affinity binding to bFGF (IC(50) = 6.4 nM). Heparan sulfate showed a lower affinity as compared with that of heparin, whereas all other glycosaminoglycans and heparin/heparan sulfate-derived disaccharides tested showed minute effects. The developed CZE method is rapid and accurate and can be easily used to identify bFGF-interacting heparin preparations of biopharmaceutical importance.
Asunto(s)
Electroforesis Capilar/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/aislamiento & purificación , Proteoglicanos/aislamiento & purificación , Heparina/metabolismo , Proteoglicanos/metabolismoRESUMEN
Staphylococcus epidermidis is an important cause of bacterial keratitis. Certain S. epidermidis strains produce an extracellular slime layer rich in an acidic polysaccharide with a molecular size of 20 kDa (20-kDa PS). We have demonstrated that the level of 20-kDa PS-specific antibodies significantly rises after establishment of slime-producing S. epidermidis bacteraemia and, furthermore, that rabbit polyclonal antibodies to 20-kDa PS opsonize cells of slime-producing S. epidermidis to a great degree and promote their clearance by polymorphonuclear cells (Arch. Biochem. Biophys. 342 (1997) 389; J. Pharm. Biomed. Anal. 22 (2000) 1029). The purpose of this study was to examine the protective and therapeutic effects both of active immunization, using 20-kDa PS as antigen, and of passive administration of specific antibodies towards the 20-kDa PS in a rabbit keratitis model. For active immunization, 20 rabbits were subcutaneously immunized with 20-kDa PS, whereas for passive immunization specific polyclonal IgG antibodies against 20-kDa PS were administered to 20 rabbits 1 day before induction of infection. Clinical observations were made weekly for 1 month and levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by an enzyme immunoassay. The levels of specific anti-20-kDa PS IgG in serum and aqueous humor following either active or passive immunization were significantly higher as compared with control groups (P<0.001). Although, actively immunized rabbits showed significantly less corneal damage than control animals, passively immunized ones were significantly better protected as compared with both control and those actively immunized. Obtained results suggest that 20-kDa PS plays crucial role in the pathogenesis of S. epidermidis keratitis and that both types of immunization significantly protect against corneal S. epidermidis pathology and damage.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Queratitis/inmunología , Proteínas de Unión al GTP Monoméricas/uso terapéutico , Fosfoproteínas/uso terapéutico , Infecciones Estafilocócicas/inmunología , Staphylococcus epidermidis/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Humor Acuoso/inmunología , Femenino , Técnicas para Inmunoenzimas , Queratitis/tratamiento farmacológico , Proteínas de Unión al GTP Monoméricas/inmunología , Fosfoproteínas/inmunología , Conejos , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
Immunoglobulins are present in most tissues and plasma and play crucial role in immune system. Alteration of the levels of the immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3 and IgG4) is an indication of a disturbed immunological response. The aim of the present study was the development of a capillary electrophoresis (CE) method for the analysis of IgG subclasses in respect to their variable kappa and lambda chains. Various analytical conditions and CE modes, including capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and micellar electrokinetic capillary chromatography (MECC) have been thoroughly studied. CZE was found to be the most convenient way to separate IgG subclasses. Three of the human IgG subclasses were resolved using uncoated fused-silica and 50 mM phosphate, pH = 9.3, as operating buffer at 20 kV and detection at 214 nm. IgG1kappa was completely separated from IgG2kappa and IgG3kappa, whereas IgG2kappa co-migrated with IgG4kappa, which is the minor IgG subclass. Under the same conditions IgG4lambda was completely separated from IgG1lambda, IgG2lambda and IgG3lambda, enabling the identification of the various lambda chains. The developed CE method is rapid and can be applied to the identification of the major immunoglobulin G subclasses in respect to their variable kappa and lambda chains.
Asunto(s)
Electroforesis Capilar/métodos , Inmunoglobulina G/análisis , Cadenas kappa de Inmunoglobulina/análisis , Cadenas lambda de Inmunoglobulina/análisis , Humanos , Inmunoglobulina G/clasificaciónRESUMEN
Glycosaminoglycans are biologically significant carbohydrates which either as free chains (hyaluronan) or constituents of proteoglycans (chondroitin/dermatan sulfates, heparin, heparan sulfate and keratan sulfate) participate and regulate several cellular events and (patho)physiological processes. Capillary electrophoresis, due to its high resolving power and sensitivity, has been successfully used for the analysis of glycosaminoglycans. Determination of compositional characteristics, such as disaccharide sulfation pattern, is a useful prerequisite for elucidating the interactions of glycosaminoglycans with matrix effective molecules and, therefore, essential in understanding the biological functions of proteoglycans. The interest in the field of characterization of such biologically important carbohydrates is soaring and advances in this field will signal a new revolution in the area of glycomics equivalent to that of genomics and proteomics. This review focuses on the capillary electrophoresis methods used to determine the disaccharide pattern of glycosaminoglycans in various biologic samples as well as advances in the sequence analysis of glycosaminoglycans using both chromatographic and electrophoretic techniques.
Asunto(s)
Disacáridos/análisis , Electroforesis Capilar/métodos , Glicosaminoglicanos/química , Secuencia de CarbohidratosRESUMEN
A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per-O-benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS<1.98%) and a linearity range from 5 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.
Asunto(s)
Electroforesis Capilar/métodos , Glicoproteínas/química , Ácido N-Acetilneuramínico/análisis , Femenino , Humanos , Ácido N-Acetilneuramínico/sangre , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Proteoglycans are a major family of glycoconjugates which participate in and regulate several cellular events and biological functions. Their glycan chains determine their physicochemical and biological properties. Capillary electrophoresis, because of its high resolving power and sensitivity, has been successfully used for the analysis of carbohydrates. The monosaccharide constituents, the disaccharide sulfation pattern, and the uronic acid distribution within glycan chains of proteoglycans determine their interactions with matrix effectors and are responsible for numerous effects. Determination of the chemical composition and identification of key structural components and domains of glycans are, therefore, essential in understanding the biological functions of proteoglycans. In this report an overview of the capillary electrophoresis methods used to analyze and characterize the structure of the glycan chains of proteoglycans is presented.
Asunto(s)
Carbohidratos/análisis , Microquímica/instrumentación , Proteoglicanos/química , Secuencia de Carbohidratos , Disacáridos/análisis , Electroforesis Capilar , Microquímica/métodos , Monosacáridos/análisis , Oligosacáridos/análisis , Análisis de Secuencia/métodosRESUMEN
Microemulsion electrokinetic capillary chromatography (MEEKC) is a capillary electrophoresis technique in which neutral and ionized species can be resolved according to their partitioning into moving oil droplets present in the operating buffer. In this report, we present for the first time the application of MEEKC in the analysis of glycosaminoglycans. An efficient method for the separation of the variously sulfated delta-disaccharides obtained following digestion of chondroitin and dermatan sulfates with chondro/ dermato lyases and derivatization with 2-aminoacridone is described. Nonsulfated, mono-, di-, and trisulfated delta-disaccharides were completely separated using the microemulsion octane/butan-1-ol/Sodium dodecyl sulfate (SDS) in 10 mM borate buffer, pH 9.3, at 25 kV. Agreement of the obtained disaccharide composition with literature values showed that MEEKC can be used for the analysis of glycosaminoglycans.
Asunto(s)
Disacáridos/análisis , Glicosaminoglicanos/análisis , Sulfatos/análisis , Animales , Cartílago/química , Cromatografía Capilar Electrocinética Micelar/métodos , BallenasRESUMEN
Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.
Asunto(s)
Dermatán Sulfato/química , Electroforesis Capilar/métodos , Aminoacridinas , Proteínas Bacterianas/metabolismo , Condroitina ABC Liasa/metabolismo , Condroitín Liasas/metabolismo , Condroitinasas y Condroitín Liasas/metabolismo , Disacáridos/análisis , Femenino , Ácido Glucurónico/análisis , Humanos , Ácido Idurónico/análisis , Leiomioma/química , Miometrio/química , Oligosacáridos/análisis , Polisacáridos/química , Estructura Terciaria de Proteína , Especificidad por Sustrato , Neoplasias Uterinas/químicaRESUMEN
PURPOSE: To quantify the concentrations of basic fibroblast growth factor (bFGF) and hyaluronan (HA) in the aqueous humor of patients with the exfoliation syndrome (XFS) or exfoliative glaucoma (XFG). METHODS: Aqueous humor bFGF and HA levels were measured in 13 patients with XFS and in 7 patients with XFG. The results were compared with those obtained from 17 healthy controls. RESULTS: Mean bFGF levels were significantly higher in the XFG patients than those in the XFS patients, which in turn were higher than the bFGF levels in the healthy individuals. Aqueous humor HA levels in both patients with the XFS and the XFG were significantly higher compared to the controls. CONCLUSION: We suggest that bFGF plays an important role in the pathogenesis of XFS and XFG, as well as in the synthesis of secreted HA, which may result in connective tissue degradation that affects the ocular anterior segment.