RESUMEN
Basic fibroblast growth factor (bFGF) modulates gingival growth, and its release from heparan sulfate (HS) in the extracellular matrix (ECM) governs local tissue bioavailability. We identified a heparin/HS interacting protein (HIP/L29) that recognizes specific HS sequences. We hypothesize that HIP/L29, by modulating the interactions of bFGF with HS chains on proteoglycans, could regulate bFGF bioavailability. To investigate interactions between bFGF and HIP/L29, we isolated and cultured fibroblasts from normal gingiva and overgrown gingiva from patients on cyclosporine (CSA). bFGF significantly stimulated gingival fibroblast proliferation with or without heparin. Recombinant human HIP/L29 dramatically decreased bFGF-induced proliferation, but did not alter responses to insulin-like growth factor-1 (IGF-1). Analysis of mitogen-activated protein kinase (MAPK) phosphorylation patterns showed that bFGF stimulation of p44 (Erk-1), but not p42 (Erk-2), also was inhibited by HIP/L29 in a dose-dependent manner. Together, these results support our hypothesis that HIP/L29 modulates the bioavailability and action of bFGF.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/fisiología , Encía/crecimiento & desarrollo , Proteínas Ribosómicas/fisiología , Análisis de Varianza , Animales , Disponibilidad Biológica , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Fibroblastos/citología , Encía/citología , Encía/efectos de los fármacos , Hiperplasia Gingival/inducido químicamente , Hiperplasia Gingival/fisiopatología , Sustancias de Crecimiento/farmacología , Sustancias de Crecimiento/fisiología , Humanos , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Recombinantes/farmacología , Proteínas Ribosómicas/farmacología , Estadísticas no ParamétricasRESUMEN
For several years, interferon alpha-1, also known as interferon alpha-D, has been studied for treatment of various viral diseases, such as hepatic fibrosis caused by hepatitis B, herpes simplex virus keratitis, and bovine respiratory diseases in calves. Currently, recombinant human interferon alpha-D (rHuIFNalphaD) is expressed intracellularly in Escherichia coli or secreted by Bacillus subtilis and Saccharomyces cerevisiae. In this report, we describe the process of obtaining a relatively high-yield secretion of biologically active recombinant rHuIFNalphaD using the Pichia pastoris system. The process produced as high as 0.7 mg of purified protein per 20 ml of shake culture of rHuIFNalphaD with better bioactivity than the commercially available rHuIFNalphaD molecule produced in E. coli.
Asunto(s)
Interferón-alfa , Pichia/genética , Secuencia de Aminoácidos , Antivirales/aislamiento & purificación , Secuencia de Bases , Bioensayo , Clonación Molecular , Humanos , Metanol/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesisRESUMEN
Proteins with affinity to heparin under physiological conditions were isolated from bovine cerebral cortex. First, the extract of cerebral cortex was applied to a chondroitin polysulfate column under physiological conditions. Then, the pass-through fraction was applied to a heparin column. Among the bands on SDS polyacrylamide gel electrophoresis of the fraction bound to the heparin column, the major one was identified as fructose 1,6-bisphosphate aldolase (FPA), a cytosolic enzyme involved in the glycolytic pathway. The results indicated that FPA is a heparin-binding protein which exhibits no affinity to chondroitin polysulfate. The results of affinity chromatographies revealed that FPA binds to intact heparin and modified heparins desulfated at C2 OH of the iduronic acid residue or at C6 OH or C2 NH2 of the glucosamine residue. When 6-O-desulfated heparin was employed as the affinity ligand, a single peak having FPA activity was isolated from the extract of bovine cerebral cortex. By further Mono Q chromatography and Superdex gel-filtration, five isoenzymes were purified with more than 50% recovery. These isoenzymes were identified as FPA A4, A3C1, A2C2, A1C3, and C4 by native electrophoresis with and without 4 M urea and subsequent amino acid sequence analysis. The use of 6-O-desulfated heparin affinity chromatography thus facilitated the purification of FPA.
Asunto(s)
Fructosa-Bifosfato Aldolasa/química , Heparina/química , Animales , Bovinos , Cromatografía de Afinidad , Fructosa-Bifosfato Aldolasa/metabolismo , Heparina/metabolismo , Ligandos , Unión ProteicaRESUMEN
Heparin affinity chromatography of synthetic peptide fragments mimicking tissue factor pathway inhibitor (TFPI) indicated that the minimal heparin binding sequence consists of 12 amino acid residues located at the C-terminal tail. Within this minimal sequence, Arg-257 and Arg-259 appeared to contribute most significantly to interaction with heparin. Affinity chromatography of TFPI using immobilized heparin derivatives regiospecifically desulfated at O-6 of the glucosamine residue, N-2 of the glucosamine residue, and/or O-2 of the iduronic acid residue indicated that all the sulfate groups in heparin appeared to be required for TFPI-heparin interaction. Among them, however, the 6-O-sulfate groups appeared to make the largest contribution to the interaction, while the 2-O-sulfate groups contributed the least. In vitro experiments on the inhibition of factor Xa by TFPI enhanced with native and chemically modified heparins afforded similar results.