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1.
J Vasc Interv Radiol ; 27(8): 1242-1250.e3, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27363296

RESUMEN

PURPOSE: To elucidate the mechanism of action of intradiscal oxygen-ozone therapy for herniated intervertebral disc therapy. METHODS: Ozone's mechanism of action was investigated using 3 approaches: mathematical models of intervertebral disc space to explore the relationship between disc pressure and volume; ozonolysis experiments using glycosaminoglycans (GAGs) from a Chinese hamster ovary cell line that were similar in composition to GAGs found in human nucleus pulposus; and experiments in which live Yucatan miniature pigs received various concentrations of percutaneous, image-guided intradiscal oxygen-ozone treatment and were examined (after sacrifice) with histology and semiquantitative analysis of disc cytokine concentrations. RESULTS: Engineering calculations support observations that a small (6%) disc volume reduction can result in considerable (9.84%) intradiscal pressure reduction. Porcine disc histology and Chinese hamster ovary GAG ozonolysis results showed that administered ozone reacted with and fragmented disc proteoglycans, reducing disc volume through disc dehydration. Cytokine analysis of porcine discs found that each of 4 cytokines measured (interleukin [IL]-1ß, IL-6, IL-8, and tumor necrosis factor α) increased in concentration after 2 wt% ozone treatment. CONCLUSIONS: Oxygen-ozone therapy breaks down proteoglycan GAGs that maintain disc osmotic pressure, dehydrating the nucleus pulposus and reducing intervertebral disc volume. This is likely a primary mechanism by which ozone relieves nerve root compression and alleviates herniated disc-related pain. Additionally, 2 wt% ozone appears to interact with intradiscal cytokines, generating an antiinflammatory response that may contribute to symptom improvement.


Asunto(s)
Antiinflamatorios/administración & dosificación , Desplazamiento del Disco Intervertebral/tratamiento farmacológico , Disco Intervertebral/efectos de los fármacos , Vértebras Lumbares/efectos de los fármacos , Ozono/administración & dosificación , Animales , Células CHO , Simulación por Computador , Cricetulus , Citocinas/metabolismo , Modelos Animales de Enfermedad , Módulo de Elasticidad , Glicosaminoglicanos/metabolismo , Inyecciones Espinales , Disco Intervertebral/metabolismo , Disco Intervertebral/patología , Disco Intervertebral/fisiopatología , Desplazamiento del Disco Intervertebral/metabolismo , Desplazamiento del Disco Intervertebral/patología , Desplazamiento del Disco Intervertebral/fisiopatología , Vértebras Lumbares/metabolismo , Vértebras Lumbares/patología , Vértebras Lumbares/fisiopatología , Modelos Biológicos , Núcleo Pulposo/efectos de los fármacos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patología , Presión Osmótica , Porcinos , Porcinos Enanos
2.
Methods Mol Biol ; 1229: 43-8, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25325943

RESUMEN

Heparan sulfate (HS) plays numerous important roles in biological systems through their interactions with a wide array of proteins. Structural biology studies of heparan sulfate are often challenging due to the heterogeneity and complexity of the HS molecules. Radioisotope metabolic labeling of HS in cellular systems has enabled the elucidation of HS structures as well as the interactions between HS and proteins. However, radiolabeled structures are not amenable for advanced structural glycobiology studies using sophisticated instruments such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The utilization of stable isotope-enriched HS precursors is an appealing approach to overcome these challenges. The application of stable isotope-enriched HS precursors has facilitated the HS structural analysis by NMR spectroscopy and mass spectrometry. Herein we describe a simple method to prepare isotopically enriched HS precursors.


Asunto(s)
Heparitina Sulfato/química , Heparitina Sulfato/síntesis química , Marcaje Isotópico/métodos , Conformación de Carbohidratos , Isótopos de Carbono , Células Cultivadas , Isótopos de Nitrógeno , Radioisótopos de Azufre/metabolismo , Tritio/metabolismo
3.
Methods Mol Biol ; 1229: 209-19, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25325956

RESUMEN

Heparin and heparan sulfate (HS) glycosaminoglycans have important roles in anticoagulation, human development, and human diseases. HS C5-epimerase, which catalyzes the epimerization of GlcA to IdoA, is a crucial enzyme involved in the biosynthesis of heparin-related biomolecules. Here, we describe a detailed method for measuring the total activity of HS C5-epimerase that involves the following steps: H/D exchange upon epimerization of the substrate with HS C5-epimerase, low-pH nitrous acid treatment of the substrate, the separation of low-pH nitrous acid-cleaved disaccharides using HPLC, and mass spectrometry analysis. This nonradioactive method is rapid and sensitive and, importantly, allows us to study the reversible nature of HS C5-epimerase.


Asunto(s)
Carbohidrato Epimerasas/metabolismo , Medición de Intercambio de Deuterio/métodos , Pruebas de Enzimas/métodos , Heparitina Sulfato/metabolismo , Espectrometría de Masas/métodos , Animales , Biocatálisis , Carbohidrato Epimerasas/aislamiento & purificación , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Disacáridos/metabolismo , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Humanos , Ácido Idurónico/química , Ácido Idurónico/metabolismo , Células Sf9
4.
Anal Bioanal Chem ; 401(1): 237-44, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21573838

RESUMEN

Heparan sulfate (HS) proteoglycans regulate a number of biological functions in many systems. Most of the functions of HS are attributed to its unique structure, consisting of sulfated and non-sulfated domains, arising from the differential presence of iduronyl and glucuronyl residues along the polysaccharide chain. A single glucuronyl C5-epimerase enzyme acts on HS precursors, converts glucuronyl residues into iduronyl residues, and modulates subsequent biosynthetic steps in vivo. Previously, the ratios of non-sulfated epimers within the polysaccharide chain have been calculated by resolving radiolabeled GlcA-(A)Man(R) and IdoA-(A)Man(R) disaccharides using a tedious paper chromatography technique. This radioactive assay, based on measuring either the release or incorporation of (3)H at C5 carbon of uronyl residues of (3)H-labeled HS precursor substrate, has been in use over three decades to characterize the action of HS C5-epimerase. We have developed a non-radioactive assay to estimate the epimerase activity through resolving GlcA-(A)Man(R) and IdoA-(A)Man(R) disaccharides on high-performance liquid chromatography in conjunction with hydrogen/deuterium exchange upon epimerization protocol-liquid chromatography mass spectrometry (DEEP-LC-MS). Utilizing this new, non-radioactive-based assay, DEEP-LC-MS, we were able to determine the extent of both forward and reverse reactions on the same substrate catalyzed by C5-epimerase. The results from this study also provide insights into the action of C5-epimerase and provide an opportunity to delineate snapshots of biosynthetic events that occur during the HSPG assembly in the Golgi.


Asunto(s)
Carbohidrato Epimerasas/metabolismo , Heparitina Sulfato/metabolismo , Espectrometría de Masas/métodos , Animales , Línea Celular , Cromatografía Liquida/métodos , Deuterio , Medición de Intercambio de Deuterio/métodos , Disacáridos/aislamiento & purificación , Heparina/metabolismo , Hidrógeno , Insectos/enzimología , Proteoglicanos/metabolismo
5.
Carbohydr Res ; 345(15): 2228-32, 2010 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-20832774

RESUMEN

The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin and heparan sulfate oligosaccharide or disaccharide fragments using simple 1D (1)H NMR spectroscopy is often complicated by the extensive signal overlap. (13)C NMR signals, on the other hand, overlap less and therefore, (13)C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific (13)C-labeled heparin and heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly (13)C- and (15)N-labeled precursors were also produced and characterized by (13)C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of (13)C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% (13)C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome-proteome interactions.


Asunto(s)
Heparina/química , Heparitina Sulfato/química , Espectroscopía de Resonancia Magnética/métodos , Polisacáridos/química , Espectrometría de Masa por Ionización de Electrospray/métodos
6.
Glycoconj J ; 27(6): 625-33, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20717719

RESUMEN

Glycosaminoglycans (GAG) play decisive roles in various cardio-vascular & cancer-associated processes. Changes in the expression of GAG fine structures, attributed to deregulation of their biosynthetic and catabolic enzymes, are hallmarks of vascular dysfunction and tumor progression. The wide spread role of GAG chains in blood clotting, wound healing and tumor biology has led to the development of modified GAG chains, GAG binding peptides and GAG based enzyme inhibitors as therapeutic agents. Xylosides, carrying hydrophobic aglycone, are known to induce GAG biosynthesis in various systems. Given the important roles of GAG chains in vascular and tumor biology, we envision that RGD-conjugated xylosides could be targeted to activated endothelial and cancer cells, which are known to express α(v)ß(3) integrin, and thereby modulate the pathological processes. To accomplish this vision, xylose residue was conjugated to linear and cyclic RGD containing peptides using click chemistry. Our results demonstrate that RGD-conjugated xylosides are able to prime GAG chains in various cell types, and future studies are aimed toward evaluating potential utility of such xylosides in treating myocardial infarction as well as cancer-associated thrombotic complications.


Asunto(s)
Glicoconjugados/metabolismo , Glicosaminoglicanos/metabolismo , Glicósidos/metabolismo , Oligopéptidos/metabolismo , Animales , Células CHO , Bovinos , Línea Celular Tumoral , Cromatografía por Intercambio Iónico , Cricetinae , Cricetulus , Células Endoteliales/metabolismo , Glicoconjugados/química , Glicosaminoglicanos/química , Glicósidos/química , Humanos , Oligopéptidos/química
7.
Carbohydr Res ; 345(2): 250-6, 2010 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-19945695

RESUMEN

We report the preparation of size-defined [(15)N]N-acetylheparosan oligosaccharides from Escherichia coli-derived (15)N-enriched N-acetylheparosan. Optimized growth conditions of E. coli in minimal media containing (15)NH(4)Cl yielded [(15)N]N-acetylheparosan on a preparative scale. Depolymerization of [(15)N]N-acetylheparosan by heparitinase I yielded resolvable, even-numbered oligosaccharides ranging from disaccharide to icosaccharide. Anion-exchange chromatography-assisted fractionation afforded size-defined [(15)N]N-acetylheparosan oligosaccharides identifiable by ESI-TOFMS. These isotopically labeled oligosaccharides will prove to be valuable research tools for the chemoenzymatic synthesis of heparin and heparan sulfate oligosaccharides and for the study of their structural biology.


Asunto(s)
Heparitina Sulfato/química , Oligosacáridos/química , Heparina/química , Isótopos de Nitrógeno/química , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad
8.
J Biol Chem ; 284(38): 25842-53, 2009 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-19628873

RESUMEN

Glycosaminoglycan (GAG) biosynthesis requires numerous biosynthetic enzymes and activated sulfate and sugar donors. Although the sequence of biosynthetic events is resolved using reconstituted systems, little is known about the emergence of cell-specific GAG chains (heparan sulfate, chondroitin sulfate, and dermatan sulfate) with distinct sulfation patterns. We have utilized a library of click-xylosides that have various aglycones to decipher the mechanism of GAG biosynthesis in a cellular system. Earlier studies have shown that both the concentration of the primers and the structure of the aglycone moieties can affect the composition of the newly synthesized GAG chains. However, it is largely unknown whether structural features of aglycone affect the extent of sulfation, sulfation pattern, disaccharide composition, and chain length of GAG chains. In this study, we show that aglycones can switch not only the type of GAG chains, but also their fine structures. Our findings provide suggestive evidence for the presence of GAGOSOMES that have different combinations of enzymes and their isoforms regulating the synthesis of cell-specific combinatorial structures. We surmise that click-xylosides are differentially recognized by the GAGOSOMES to generate distinct GAG structures as observed in this study. These novel click-xylosides offer new avenues to profile the cell-specific GAG chains, elucidate the mechanism of GAG biosynthesis, and to decipher the biological actions of GAG chains in model organisms.


Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Glicosaminoglicanos/biosíntesis , Glicósidos/inmunología , Animales , Células CHO , Secuencia de Carbohidratos , Cricetinae , Cricetulus , Glicosaminoglicanos/análisis , Glicosaminoglicanos/química , Glicósidos/análisis , Glicósidos/química
9.
J Biol Chem ; 283(43): 28881-7, 2008 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-18708345

RESUMEN

Proteoglycans (PGs) are composed of a protein moiety and a complex glycosaminoglycan (GAG) polysaccharide moiety. GAG chains are responsible for various biological activities. GAG chains are covalently attached to serine residues of the core protein. The first step in PG biosynthesis is xylosylation of certain serine residues of the core protein. A specific linker tetrasaccharide is then assembled and serves as an acceptor for elongation of GAG chains. If the production of endogenous GAG chains is selectively inhibited, one could determine the role of these endogenous molecules in physiological and developmental functions in a spatiotemporal manner. Biosynthesis of PGs is often blocked with the aid of nonspecific agents such as chlorate, a bleaching agent, and brefeldin A, a fungal metabolite, to elucidate the biological roles of GAG chains. Unfortunately, these agents are highly lethal to model organisms. Xylosides are known to prime GAG chains. Therefore, we hypothesized that modified xylose analogs may able to inhibit the biosynthesis of PGs. To test this, we synthesized a library of novel 4-deoxy-4-fluoroxylosides with various aglycones using click chemistry and examined each for its ability to inhibit heparan sulfate and chondroitin sulfate using Chinese hamster ovary cells as a model cellular system.


Asunto(s)
Sulfatos de Condroitina/fisiología , Heparitina Sulfato/fisiología , Animales , Brefeldino A/farmacología , Células CHO , Secuencia de Carbohidratos , Supervivencia Celular , Sulfatos de Condroitina/antagonistas & inhibidores , Cricetinae , Cricetulus , Glicósidos/química , Heparitina Sulfato/antagonistas & inhibidores , Modelos Biológicos , Modelos Químicos , Datos de Secuencia Molecular , Polisacáridos/química , Proteoglicanos/química
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