Abstract. Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex . We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains . BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain . In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synxE biosynthesis of glycosaminoglycan (GAG)' carbohydrate side chains involves an extensive series of posttranslational enzymatic reactions that occur dur ing the intracellular transport of core proteins from the ER through the Golgi complex . These reactions use specific initiation, elongation, and sulfation enzymes to modify appropriate core proteins and assemble them into mature proteoglycan molecules . For chondroitin sulfate (CS) GAGS, the carbohydrate-protein linkage region is initiated by a xylosyltransferase that transfers xylose from UDP-xylose to the hydroxyl groups of specific serine residues within a core protein. Initiation is completed by the addition oftwo galactosyl and one glucuronosyl residue through the action of galactosyltransferase I, II, and glucuronosyltransferase I, respectively. Once initiated, the repeating disaccharide backbone ofglucuronosyl and N-acetylgalactosaminyl residues is elongated by specific glucuronosyl-and N-acetylgalactosaminyltransferases . Sulfation of the repeating disaccharides by the 4-and/or 6-sulfotransferases completes the synthesis of mono-or disulfated CS (27) (see Fig . 11 thesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to ß-elimination . The ability of cells to use an exogenous acceptor, p-nitro phenyl-ß-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA . The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events . Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment .It is generally accepted that GAG synthesis is a Golgi complex-related event. Early studies using electron microscopy and radioautography showed that the Golgi complex is the major site of GAG chain synthesis (21,24,25) . Golgi-enriched preparations isolated from some cell types have also been shown to contain GAG synthesizing activity (32)...

The BMI paradigm presented here will enable researchers to investigate the interaction of plasticity after SCI and plasticity during BMI training on performance.

Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.

Fibroblast Growth Factor 2 (FGF2), an important regulator of angiogenesis, binds to endothelial cell (EC) surface fibroblast growth factor receptors (FGFR) and heparan sulfate proteoglycans (HSPG). FGF2 binding kinetics have been predominantly studied in static culture; however, the endothelium is constantly exposed to flow which may affect FGF2 binding. We therefore used experimental and computational techniques to study how EC FGF2 binding changes in flow. ECs adapted to 24 hours of flow demonstrated biphasic FGF2-HSPG binding, with FGF2-HSPG complexes increasing up to 20 dynes/cm 2 shear stress and then decreasing at higher shear stresses. To understand how adaptive EC surface remodeling in response to shear stress may affect FGF2 binding to FGFR and HSPG, we implemented a computational model to predict the relative effects of flow-induced surface receptor changes. We then fit the computational model to the experimental data using relationships between HSPG availability and FGF2-HSPG dissociation and flow that were developed from a basement membrane study, as well as including HSPG production. These studies suggest that FGF2 binding kinetics are altered in flow-adapted ECs due to changes in cell surface receptor quantity, availability, and binding kinetics, which may affect cell growth factor response.

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