RESUMEN
Pompe disease results in the accumulation of lysosomal glycogen in multiple tissues due to a deficiency of acid alpha-glucosidase (GAA). Enzyme replacement therapy for Pompe disease was recently approved in Europe, the U.S., Canada, and Japan using a recombinant human GAA (Myozyme, alglucosidase alfa) produced in CHO cells (CHO-GAA). During the development of alglucosidase alfa, we examined the in vitro and in vivo properties of CHO cell-derived rhGAA, an rhGAA purified from the milk of transgenic rabbits, as well as an experimental version of rhGAA containing additional mannose-6-phosphate intended to facilitate muscle targeting. Biochemical analyses identified differences in rhGAA N-termini, glycosylation types and binding properties to several carbohydrate receptors. In a mouse model of Pompe disease, glycogen was more efficiently removed from the heart than from skeletal muscle for all enzymes, and overall, the CHO cell-derived rhGAA reduced glycogen to a greater extent than that observed with the other enzymes. The results of these preclinical studies, combined with biochemical characterization data for the three molecules described within, led to the selection of the CHO-GAA for clinical development and registration as the first approved therapy for Pompe disease.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , alfa-Glucosidasas/química , alfa-Glucosidasas/farmacología , Animales , Anticuerpos/sangre , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Fibroblastos/metabolismo , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/inmunología , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Ratones , Oligosacáridos/química , Oligosacáridos/metabolismo , Unión Proteica , Conejos , Receptor IGF Tipo 2/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismoRESUMEN
Controversy exists over the potency of bone healing in the aged skeleton, and there is concern that enhancement of bone regeneration after use of bone-stimulating growth factors may not be effective in the aged. In this study, 30 skeletally mature beagles (1-2 or 10-12 years old) had titanium implants placed bilaterally in the proximal humerus for a period of 4 weeks in a model of intramembranous bone regeneration. A bony defect made at the time of surgery created a 3-mm gap between the implant surface and the host bone. Some of the implants were treated with recombinant human TGFbeta2 (rhTGFbeta2) at various does (0.32-35 microg per implant), and some served as paired controls. The dose response was similar in young and old animals. The most effective dose, 35 microg, led to a 3-fold increase in the volume fraction of new bone within the gap in both the young (p = 0.001) and old (p = 0.002) animals. At this dose, there was a 5-fold increase in osteoblast surface. While age did not significantly affect the quantity of new bone formed as assessed by backscatter scanning electron microscopy, the older animals had thinner regenerated trabeculae that tended to be spaced more closely than the younger animals. Coupled with the finding that the increase in osteoid was greater in the old animals compared with the young animals, these qualitative differences suggest that there may have been a slight delay in the rate or a defect of mineralization in the old animals.
Asunto(s)
Envejecimiento/fisiología , Regeneración Ósea/efectos de los fármacos , Regeneración Ósea/fisiología , Factor de Crecimiento Transformador beta/farmacología , Animales , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Húmero/diagnóstico por imagen , Húmero/efectos de los fármacos , Húmero/patología , Húmero/cirugía , Masculino , Prótesis e Implantes , Radiografía , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta2RESUMEN
Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, "redifferentiation" of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 10(6) cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL-6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL-6 itself were up-regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL-6 receptor, was down-regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF-beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high-density suspension culture. When articular chondrocytes were seeded in suspension at low-density (10(4) cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation.
Asunto(s)
Cartílago Articular/citología , Diferenciación Celular/genética , Condrocitos/citología , Condrocitos/metabolismo , Proteínas de la Matriz Extracelular , Matriz Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Adulto , Agrecanos , Northern Blotting , Cartílago Articular/metabolismo , Recuento de Células , Técnicas de Cultivo de Célula/métodos , División Celular , Células Cultivadas , Colágeno/biosíntesis , Factor de Crecimiento del Tejido Conjuntivo , Regulación de la Expresión Génica , Biblioteca de Genes , Sustancias de Crecimiento/biosíntesis , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Lectinas Tipo C , Proteoglicanos/biosíntesis , ARN Mensajero/metabolismoRESUMEN
The purposes of the present study were to determine if recombinant human transforming growth factor-beta-2 (rhTGF-beta2) enhances bone ingrowth into porous-coated implants and bone regeneration in gaps between the implant and surrounding host bone. The implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs in the presence of a 3-mm gap. In three treatment groups of animals, the test implant was treated with hydroxyapatite/tricalcium phosphate (HA/TCP) and rhTGF-beta2 in buffer at a dose per implant of 1.2 microg (n = 6), 12 microg (n = 7), or 120 microg (n = 7) and placed in the left humerus. In these same animals, an internal control implant treated only with HA/TCP and buffer was placed in the right humerus. In a non-TGF-beta treated external control group of animals (n = 7), one implant was treated with HA/TCP while the contralateral implant was not treated with the ceramic. In vitro analyses showed that approximately 15%, of the applied dose was released within 120 h with most of the release occurring in the first 24 h. The TGF-beta treated implants had significantly more bone ingrowth than the controls with the greatest effect in the 12 microg/implant group (a 2.2-fold increase over the paired internal control (P = 0.004) and a 4-fold increase over the external control (P < 0.001)). The TGF-beta treated implants had significantly more bone formation in the gap than the controls with the greatest effect in the 12 and 120 microg groups (1.8-fold increases over the paired internal controls (P = 0.003 and P = 0.012, respectively) and 2.8-fold increases over the external controls (P < 0.001 and P = 0.001, respectively)). Compared to the external controls, the internal control implants tended to have more bone ingrowth (1.9-fold increase, P = 0.066) and had significantly more bone formation in the gap (1.7-fold increase. P = 0.008). Thus, application of rhTGF-beta2 to a porous-coated implant-stimulated local bone ingrowth and gap healing in a weakly dose-dependent manner and stimulated bone regeneration in the 3-mm gap surrounding the contralateral control implant, a site remote from the local treatment with the growth factor.
Asunto(s)
Desarrollo Óseo/efectos de los fármacos , Regeneración Ósea/efectos de los fármacos , Prótesis e Implantes , Factor de Crecimiento Transformador beta/farmacología , Animales , Fenómenos Biomecánicos , Perros , Húmero/cirugía , Masculino , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/administración & dosificaciónRESUMEN
Dedifferentiated human articular chondrocytes exhibited a wide variation in their capacity to proliferate and redifferentiate in an alginate suspension culture system. The greatest extent of proliferation and redifferentiation was seen to be dependent on the formation of clonal populations of chondrocytes and correlated inversely with the initial cell seeding density. Redifferentiating chondrocytes seeded at low density (1 x 10(4) cells/ml alginate) compared with chondrocytes that were seeded at high density (1 x 10(6) cells/ml alginate) showed a nearly 3-fold higher median increase in cell number. a 19-fold greater level of type-II collagen mRNA expression, a 4-fold greater level of aggrecan mRNA expression, and a 6-fold greater level of sulfated glycosaminoglycan deposition at 4 weeks of culture. Matrix molecules from low-density cultures were assembled into chondrocyte-encapsulated, spherical extracellular matrices that were readily visualized in sections from 12-week cultures stained with antibodies against types I and II collagen and aggrecan. Ultrastructural analysis of 12-week low-density cultures confirmed the presence of thin collagen fibrils throughout the matrix.
Asunto(s)
Alginatos/farmacología , Cartílago Articular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Medios de Cultivo/farmacología , Proteínas de la Matriz Extracelular , Adulto , Agrecanos , Cartílago Articular/crecimiento & desarrollo , Cartílago Articular/ultraestructura , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , División Celular/fisiología , Condrocitos/metabolismo , Condrocitos/ultraestructura , Colágeno/genética , Colágeno/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Glicosaminoglicanos/metabolismo , Humanos , Lectinas Tipo C , Masculino , Persona de Mediana Edad , Proteoglicanos/genética , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Sulfatos/metabolismoRESUMEN
Commercially available human plasma-derived preparations of the serine protease inhibitor antithrombin (AT) were shown to contain low levels of oxidation, and we sought to determine whether oxidation might be a means of regulating the protein's inhibitory activity. A recombinant form of AT, with similarly low levels of oxidation as purified, was treated with hydrogen peroxide in order to study the effect of oxidation, specifically methionine oxidation, on the biochemical properties of this protein. AT contains two adjacent methionine residues near the reactive site loop cleaved by thrombin (Met314 and Met315) and two exposed methionines that border on the heparin binding region of AT (Met17 and Met20). In forced oxidations with hydrogen peroxide, the methionines at 314 and 315 were found to be the most susceptible to oxidation, but their oxidation did not affect either thrombin-inhibitory activity or heparin binding. Methionines at positions 17 and 20 were significantly oxidized only at higher concentrations of peroxide, at which point heparin affinity was decreased. However at saturating heparin concentrations, activity was only marginally decreased for these highly oxidized samples of AT. Structural studies indicate that highly oxidized AT is less able to undergo the complete conformational change induced by heparin, most probably due to oxidation of Met17. Since this does not occur in less oxidized, and presumably more physiologically relevant, forms of AT such as those found in plasma preparations, oxidation does not appear to be a means of controlling AT activity.
Asunto(s)
Antitrombina III/metabolismo , Heparina/metabolismo , Metionina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dicroismo Circular , Humanos , Peróxido de Hidrógeno/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The objective of these studies was to characterize the macrophage mannose receptor binding and pharmacological properties of carbohydrate remodeled human placental-derived and recombinant beta-glucocerebrosidase (pGCR and rGCR, respectively). These are similar but not identical molecules that were developed as enzyme replacement therapies for Gaucher disease. Both undergo oligosaccharide remodeling during purification to expose terminal mannose sugar residues. Competitive binding data indicated carbohydrate remodeling improved targeting to mannose receptors over native enzyme by two orders of magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At 37 degrees C, 95% of the total macrophage binding was mannose receptor specific. In vivo, pGCR and rGCR were cleared from circulation by a saturable pathway. The serum half-life (t1/2) was 3 minutes when less than saturable amounts were injected intravenously (IV) into mice. Twenty minutes postdose, beta-glucocerebrosidase activity increased over endogenous levels in all tissues examined. Fifty percent of the injected activity was recovered. Ninety-five percent of recovered activity was in the liver. Parenchymal cells (PC), Kupffer cells (KC), and liver endothelium cells (LEC) were responsible for 75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR and for 76%, 11%, and 12%, respectively, of the hepatocellular uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2 = 2 days) and KC (t1/2 = 3 days).
Asunto(s)
Enfermedad de Gaucher/terapia , Glucosilceramidasa/química , Glucosilceramidasa/metabolismo , Lectinas Tipo C , Lectinas de Unión a Manosa , Animales , Endotelio Vascular/metabolismo , Femenino , Glucosilceramidasa/aislamiento & purificación , Glucosilceramidasa/farmacocinética , Semivida , Humanos , Inyecciones Intravenosas , Cinética , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Macrófagos Peritoneales/metabolismo , Receptor de Manosa , Ratones , Ratones Endogámicos BALB C , Oligosacáridos/química , Oligosacáridos/metabolismo , Placenta/enzimología , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Especificidad por Sustrato , Distribución TisularRESUMEN
Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma-derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.
Asunto(s)
Antitrombina III/análisis , Antitrombina III/genética , Antitrombina III/aislamiento & purificación , Animales , Animales Modificados Genéticamente , Femenino , Cabras , Humanos , Proteínas de la Leche/análisis , Proteínas de la Leche/genética , Proteínas de la Leche/aislamiento & purificación , Ingeniería de Proteínas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.
Asunto(s)
Cartílago Articular/citología , Cartílago Articular/patología , Condrocitos/citología , Adolescente , Adulto , Diferenciación Celular/fisiología , Niño , Preescolar , Condrocitos/química , Condrocitos/enzimología , Proteoglicanos Tipo Condroitín Sulfato/genética , Colágeno/análisis , Colágeno/genética , Matriz Extracelular/química , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Humanos , Hipertrofia , Lectinas Tipo C , Persona de Mediana Edad , Morfogénesis/fisiología , Sondas de Oligonucleótidos , Fenotipo , Proteoglicanos/genética , ARN Mensajero/análisis , VersicanosRESUMEN
Prolactin (PRL) is an immunomodulator that has been demonstrated to enhance immune responses both in vitro and in vivo. Prolactin enhances the proliferative response of lymphoid cells to both nonspecific mitogens and specific antigens and increases their production of IL-2 and interferon-gamma. Studies were performed to examine whether recombinant human prolactin (r-hPRL) also acts as a growth factor for B cell hybridomas. Prolactin was able to stimulate proliferation of murine B cell hybridomas in a dose-dependent manner and enhanced their proliferation in response to IL-4, IL-5, and IL-6. This increase in proliferation resulted in an overall increase in antibody production. Studies were also undertaken to examine the effect of PRL with transforming growth factor beta (TGF-beta), an immunosuppressive cytokine. Hybridoma cell lines incubated with TGF-beta demonstrated a dose-dependent decrease in proliferation. Variability in the degree of inhibition was observed among the various hybridomas in their responsiveness to TGF-beta. The addition of r-hPRL to the cultures reversed the antiproliferative effects of TGF-beta. The mechanism by which PRL can overcome the anti-proliferative effect of TGF-beta is under investigation. These findings provide an additional rationale for using r-hPRL clinically in immunosuppressed patients in certain disease settings such as AIDS and cancer, where overexpression of TGF-beta has been implicated in disease development and progression.
Asunto(s)
Linfocitos B/efectos de los fármacos , Prolactina/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Adyuvantes Inmunológicos/farmacología , Animales , Formación de Anticuerpos/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/inmunología , División Celular/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Hibridomas , Interleucina-4/farmacología , Interleucina-5/farmacología , Interleucina-6/farmacología , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
The Epicel ASAProgram service generates autologous keratinocyte grafts used for the closure of full-thickness wounds in moderately and severely burned patients. The manufacturing process used to generate Epicel service autografts (ESA) is based upon the keratinocyte co-culture technique described by Rheinwald and Green which employs murine Swiss 3T3/J2 fibroblasts as feeder cells. Recently, a technique has been described that employs a polyurethane wound dressing, HydroDerm (HD, Innovative Technologies, Ltd), as a delivery vehicle for cultured keratinocytes intended for autologous grafting. We have examined the practical feasibility of this technique and report on testing the ability of HD to support keratinocyte growth and epithelium formation in vitro, at the air-liquid interface (ALI), and in vivo, after grafting to full-thickness wounds created on the backs of athymic (Swiss Nu/Nu) mice. The results demonstrate that keratinocytes grow on the HD dressing in Gibco SFM at a rate that is approximately 15 per cent of that observed when cells are cultivated on tissue culture (TC) plastic using standard techniques, yet the cells retain their proliferative capacity and form an epithelium in vitro when cultivated at the ALI on a dermal substrate. Keratinocyte-seeded HD membranes were also transferred to full-thickness wounds in athymic mice. Animals grafted with cells seeded to HD developed human epithelium, as revealed by species-specific detection of involucrin and evolved a normal attachment to the wound substratum, as demonstrated through the expression of dermally opposed laminin and alpha 6 beta 4 integrin. The ability of keratinocytes to maintain proliferative potential after seeding onto HD and their ability to form a properly oriented epithelium in vitro and in vivo suggests that this wound dressing may be useful as a vehicle for autologous keratinocyte grafting and help to provide earlier epithelial coverage to the burned patient. However, because of the slow proliferation rate of keratinocytes on HydroDerm, timely graft delivery would be best achieved by combining cell expansion via the Rheinwald and Green culture system, followed by the seeding of cells onto HydroDerm in a reduced calcium medium for subsequent autologous grafting.
Asunto(s)
Quemaduras/cirugía , Queratinocitos/trasplante , Membranas Artificiales , Poliuretanos , Células 3T3/citología , Animales , Antígenos de Superficie/análisis , Vendajes , Calcio/administración & dosificación , Adhesión Celular , Recuento de Células , División Celular , Células Cultivadas , Medios de Cultivo , Técnicas de Cultivo , Epitelio/fisiología , Epítopos/análisis , Estudios de Factibilidad , Humanos , Integrina alfa6beta4 , Integrinas/análisis , Queratinocitos/citología , Queratinocitos/fisiología , Laminina/análisis , Ratones , Ratones Desnudos , Vehículos Farmacéuticos , Precursores de Proteínas/análisis , Piel/citología , Piel/lesiones , Piel/patología , Especificidad de la Especie , Trasplante AutólogoRESUMEN
During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growth factor-beta (TGF-beta), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal fibroblasts grow to confluence and then cease proliferation. In this study, culture conditions with TGF-beta 1 have been delineated that promote human fibroblasts to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF-beta was added thrice weekly to normal human dermal fibroblasts, the cells proliferated and stratified up to 16 cell layers thick within the culture dish, producing a tissue-like fibroplasia. TGF-beta stimulated both DNA synthesis as measured by 3H-thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti-fibronectin antibodies which inhibited both basal and TGF-beta-stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with beta-amino-proprionitrile (BAPN) did not inhibit basal or TGF-beta stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum factors, TGF-beta stimulates postconfluent, fibronectin matrix-dependent, fibroblast growth creating a fibroplasia-like tissue in vitro.
Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de Heridas , Aminopropionitrilo/farmacología , Anticuerpos Bloqueadores/farmacología , Proteínas Sanguíneas/farmacología , División Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Medios de Cultivo/farmacología , ADN/biosíntesis , Fibroblastos/citología , Fibroblastos/metabolismo , Fibronectinas/inmunología , Humanos , Masculino , Piel/citologíaRESUMEN
Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor beta (TGF-beta) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-beta 1 or TGF-beta 2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-beta and IGF-I.
Asunto(s)
Cartílago Articular/fisiología , Colágeno/genética , Proteínas de la Matriz Extracelular , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteoglicanos/genética , Factor de Crecimiento Transformador beta/administración & dosificación , Adulto , Agrecanos , Biomarcadores , Células Cultivadas , Medios de Cultivo , Humanos , Insulina/administración & dosificación , Lectinas Tipo C , Persona de Mediana Edad , Factores de TiempoRESUMEN
The Transforming Growth Factor-betas (TGF-beta) are a group of multifunctional proteins whose cellular sites of production and action are widely distributed throughout the body, including the central nervous system (CNS). Within the CNS, various isoforms of TGF-beta are produced by both glial and neural cells. When evaluated in either cell culture or in vivo models, the various isoforms of TGF-beta have been shown to have potent effects on the proliferation, function, or survival of both neurons and all three glial cell types, astrocytes, microglia and oligodendrocytes. TGF-beta has also been shown to play a role in several forms of acute CNS pathology including ischemia, excitotoxicity and several forms of neurodegenerative diseases including multiple sclerosis, Parkinson's disease, AIDS dementia and Alzheimer's disease.
Asunto(s)
Sistema Nervioso Central/irrigación sanguínea , Sistema Nervioso Central/fisiología , Isquemia/etiología , Enfermedades Neurodegenerativas/etiología , Factor de Crecimiento Transformador beta/fisiología , Complejo SIDA Demencia/etiología , Enfermedad de Alzheimer/etiología , Animales , Astrocitos/fisiología , Sistema Nervioso Central/lesiones , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/etiología , Expresión Génica , Humanos , Isquemia/genética , Isquemia/fisiopatología , Microglía/fisiología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/etiología , Esclerosis Múltiple/fisiopatología , Enfermedades Neurodegenerativas/fisiopatología , Oligodendroglía/fisiología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/etiología , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta/genéticaRESUMEN
An enzymatic debriding preparation was formulated with purified enzyme derived from a crude pineapple stem extract. The primary component of this preparation was the sulfhydryl protease ananain which represented >/=85% of the proteolytic activity. The remaining proteolytic activity in the preparation was contributed by a co-purifying homologous cysteine protease comosain. Taken together these two proteases provided a protein purity of greater than 95% as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This ananain-based enzyme preparation exhibited both gelatinolytic and fibrinolytic activity in vitro. Ananain-based enzyme preparation was formulated in a hydrophilic cream vehicle at concentrations ranging from 115 to 260 U/gm vehicle. Ananain-based enzyme preparation formulated in this fashion is referred to as Vianain debriding agent. Vianain was applied to partial-thickness cutaneous burn wounds produced in the skin of domestic pigs. A maximum of two 4-hour applications of Vianain provided complete debridement of eschar from the partial-thickness burn wounds as judged by light and electron microscopic analyses of biopsy specimens harvested before and after debridement. Wounds debrided with Vianain exhibited more rapid reepithelialization as compared with wounds that were not debrided. Wounds on pigs that were hyperimmunized to ananain-based enzyme preparation before burning and debridement with Vianain exhibited a similar enhancement in reepithelialization as compared with wounds treated with vehicle alone. The capacity of Vianain to debride necrotic tissue was also evaluated in a guinea pig ischemic ulcer model. Full-thickness ischemic lesions were created on the back of guinea pigs. Vianain was applied to the hardened necrotic tissue for 6 hours per day for up to a maximum of 5 days. Complete debridement of these wounds was accomplished within 4 to 5 days. Treatment of ischemic cutaneous ulcerations in this animal model with two commercially available enzyme-debriding agents provided little or no debridement of the necrotic tissue. In vitro, Vianain treatment of surgically debrided human tissue samples, obtained from patients with burn injury or cutaneous ulcers, showed that the protease preparation was effective in rapidly digesting these necrotic tissues.
RESUMEN
An inducible gene amplification system was utilized to study the effects of overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) in vitro. BTS, a monkey kidney cell line expressing a temperature-sensitive simian virus 40 (SV-40) large T antigen was stably transfected at the nonpermissive temperature with a plasmid containing an SV-40 origin of replication and the cDNA for either the wild-type CFTR or the mutant G551D-CFTR. Shift of the isolated cell lines to the permissive temperature resulted in induction and accumulation to high levels of the CFTR plasmid, mRNA, and protein. However, high-level expression of CFTR was transient in both BTS-CFTR and BTS-G551D cells due to a decrease in their respective levels of CFTR mRNA. Because G551D-CFTR only exhibits residual Cl channel activity, this suggests that the observed downregulation with BTS-G551D cells may have been induced by either the physical presence of high amounts of CFTR or some low threshold level of Cl- channel activity. Examination of cell growth properties revealed a correlation between high-level expression of wild-type CFTR and growth arrest of the cells at the G2/M phase. However, similar induction of the G551D-CFTR mutant showed only a slight growth inhibition and little enrichment of cells at the G2/M phase. Cytofluorographic analysis further revealed that BTS-CFTR cells were significantly larger than parental BTS or BTS-G551D cells at all stages of the cell cycle. These results indicate that CFTR overexpression is capable of inducing its own downregulation and that high levels of Cl- channel activity can result in increased cell volume and subsequent cell growth abnormalities.
Asunto(s)
Células/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Animales , División Celular , Línea Celular , Fenómenos Fisiológicos Celulares , Células/citología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fase G2 , Haplorrinos , Riñón/citología , Riñón/metabolismo , Mitosis , Mutación , ARN Mensajero/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, alpha-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al. (Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R. (1992) Cell 68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[alpha-32P]ATP with half-maximal binding at 25 and 50 microM, respectively. These values compare well with those reported for 8-N3-[alpha-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins.
Asunto(s)
Canales de Cloruro/fisiología , Fibrosis Quística/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Membrana Dobles de Lípidos , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Proteínas Recombinantes/aislamiento & purificación , SpodopteraRESUMEN
Sequence homology between the alpha-subunits of G-proteins and other GTP-binding proteins and certain regions within the nucleotide binding domains (NBDs) of cystic fibrosis transmembrane conductance regulator (CFTR) indicates that these protein structures may be similar. A sequence alignment of the NBDs of CFTR and NBDs from other membrane transporters, forms the basis of a structural model. This model predicts that one of the conserved sequences GGQR, within which a number of CF mutations occur, forms part of the nucleotide binding pocket and serves as an ON/OFF conformational switch as observed in GTP binding proteins. Furthermore, based on subtle sequence differences between the first and second NBDs of CFTR and from structure-activity data, we suggest that the nucleotide binding site environments of the two NBDs are different.