RESUMEN
BACKGROUND: PGs are involved in cellular communication and cancer biology. The role of CS in melanoma and fibrosarcoma cell lines was explored by using chondroitin AC lyase (PsPL8A). METHODS: The proliferation of mouse fibroblast L929, human melanoma (SK-Mel 28) and fibrosarcoma (HT-1080) cell lines after treatment with chondroitin AC lyase (PsPL8A) was studied by MTT assay. The mode of cell death was studied by Annexin-V FITC using flow cytometry and fluorescence microscopy. The alteration in mitochondrial cell potential was studied by JC-1 dye using fluorescence microscopy and flow cytometry. RESULTS: Treatment of L929 cells with PsPL8A imparts no cytotoxicity and showed no alteration in proliferation with nearly 95-98% cell viability. An overall 58% and 59% inhibition of SK-Mel 28 and HT-1080 cell proliferation was observed with 1.3 µM of PsPL8A after 24 h of incubation. The PsPL8A (1.3 µM) treated SK-Mel 28 and HT-1080 cells showed significant green fluorescence with annexin-V FITC under fluorescence microscopy and 56.6% and 35.5% apoptosis, respectively by flow cytometry analysis. The results of fluorescence microscopy and flow cytometry of SK-Mel 28 and HT-1080 upon treatment with PsPL8A (1.3 µM) for 24 h, gave green fluorescence due to dissipation of mitochondrial potential with JC-1 dye. CONCLUSIONS: Chondroitin AC lyase (PsPL8A) displayed anti-tumor potential against human melanoma SK-Mel 28 and fibrosarcoma HT-1080 cell lines, while the mouse fibroblast L929 cells were unaffected.
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Antineoplásicos/farmacología , Proteínas Bacterianas/farmacología , Condroitín Liasas/farmacología , Fibrosarcoma/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Pedobacter/enzimología , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos/aislamiento & purificación , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Condroitín Liasas/aislamiento & purificación , Condroitín Liasas/toxicidad , Fibrosarcoma/patología , Humanos , Melanoma/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Neoplasias Cutáneas/patologíaRESUMEN
We examined the effects of chondroitinases on the release of dermatan sulfate (DS)-induced arginine amidase (AA) from rabbit ear artery. DS-induced AA release was significantly decreased by treatment with chondroitinase ABC (ABCase) in the rabbit ear artery. On the other hand, Chondroitinase ACII (ACIIase) enhanced spontaneous and DS-induced AA release. Heat-inactivated ABCase and ACIIase did not affect spontaneous and DS-induced AA release. Furthermore, ABCase, but not ACIIase and heat-inactivated chondroitinases, degraded DS. These results indicate that the facilitatory effect of DS-induced AA release from the rabbit ear artery is affected by the molecular size of DS.
Asunto(s)
Anticoagulantes/farmacología , Arterias/efectos de los fármacos , Condroitín Liasas/farmacología , Dermatán Sulfato/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Arterias/metabolismo , Condroitina ABC Liasa/farmacología , Dermatán Sulfato/química , Oído , Conejos , Relación Estructura-ActividadRESUMEN
Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.
Asunto(s)
Membrana Celular/metabolismo , Glicosaminoglicanos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Condroitín Liasas/farmacología , Sulfatos de Condroitina/farmacología , Citometría de Flujo , Glicósidos/farmacología , Proteínas Fluorescentes Verdes/genética , Heparina/farmacología , Liasa de Heparina/farmacología , Humanos , Microscopía Confocal , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.
Asunto(s)
Movimiento Celular/fisiología , Glicosaminoglicanos/fisiología , Neutrófilos/enzimología , Fosfolipasas A/fisiología , Azepinas/farmacología , Benzoquinonas/farmacología , Movimiento Celular/efectos de los fármacos , Quimiotaxis/fisiología , Condroitín Liasas/farmacología , Flavobacterium/enzimología , Heparina/farmacología , Liasa de Heparina/farmacología , Humanos , Técnicas In Vitro , Leucotrieno B4/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Fosfolipasas A2 , Inhibidores de Agregación Plaquetaria/farmacología , Polisacárido Liasas/farmacología , Triazoles/farmacologíaRESUMEN
Proteoglycans have been implicated in the invagination and formation of various embryonal cavitied primordia. In this paper the expression of chondroitin sulphate proteoglycan (CSPG) is analysed in the lens primordium during lens vesicle formation, and demonstrate that this proteoglycan has a specific distribution pattern with regard to invagination and fusion processes in the transformation of placode into lens vesicle. More specifically, CSPG was detected in: (1) the apical surface of lens epithelial cells, where early CSPG expression was observed in the whole of the lens placode whilst in the vesicle phase it was restricted to the posterior epithelium; (2) intense CSPG expression in the basal lamina, which remained constant for the entire period under study; (3) CSPG expression in the intercellular spaces of the lens primordium epithelium, which increased during the invagination of the primordium and which at the vesicle stage was more evident in the posterior epithelium; and (4) CSPG expression on the edges of the lens placode both prior to and during fusion. Treatment with beta- D -xyloside causes significant CSPG depletion in the lens primordium together with severe alterations in the invagination and fusion of the lens vesicle; this leads to the formation of lens primordia which in some cases remain practically flat or show partial invagination defects or fusion disruption. Similar results were obtained by enzyme digestion with chondroitinase AC but not with type II heparinase, which indicates that alterations induced by beta- D -xyloside were due to interference in CSPG synthesis. The findings demonstrate that CSPG is a common component of the lens primordium at the earliest developmental stages during which it undergoes specific modifications. It also includes experimental evidence to show that 'in vivo' CSPG plays an important role in the invagination and fusion processes of the lens primordium.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Cristalino/embriología , Animales , Membrana Basal/embriología , Membrana Basal/metabolismo , Embrión de Pollo , Condroitín Liasas/farmacología , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Técnica del Anticuerpo Fluorescente , Glicósidos/farmacología , Cápsula del Cristalino/embriología , Cápsula del Cristalino/metabolismo , Cristalino/efectos de los fármacos , Cristalino/metabolismo , Microscopía Confocal , Morfogénesis/fisiología , Polisacárido Liasas/farmacologíaRESUMEN
In the current study, a glycosaminoglycan lyase, chondroitinase B, was used to study the role of dermatan sulfate proteoglycans on human dermal fibroblast proliferation. Pretreatment with chondroitinase B significantly decreased fibroblast proliferative responses to serum (20% to 55%). In contrast, heparinase III and chondroitinase AC were less effective in inhibiting fibroblast proliferation to serum. Analysis of glycosaminoglycans on chondroitinase B-treated fibroblasts confirmed that dermatan sulfate was removed from fibroblasts by this enzyme. Chondroitinase B treatment also decreased proliferation to basic fibroblast growth factor (bFGF) by 20% and reduced receptor binding by 25%. Heparinase III inhibited bFGF binding by 73%, but decreased proliferation to bFGF by only 21%. Chondroitinase AC had no effect on bFGF proliferation or binding. These data suggest that dermatan sulfate proteoglycans play a significant role in the control of human dermal fibroblast proliferation.
Asunto(s)
Dermatán Sulfato/fisiología , Fibroblastos/citología , Piel/citología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Condroitín Liasas/farmacología , Proteoglicanos Tipo Condroitín Sulfato/deficiencia , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Dermatán Sulfato/deficiencia , Dermatán Sulfato/metabolismo , Interacciones Farmacológicas , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Polisacárido Liasas/metabolismo , Polisacárido Liasas/farmacología , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
The urinary glycoprotein that inhibits calcium oxalate (CaOx) crystallization in vitro shows a structural similarity to urinary trypsin inhibitor (UTI; recently termed bikunin), the light chain of inter-alpha-trypsin inhibitor (I alpha I). The functional domains of I alpha I involved in its inhibitory activity of CaOx crystallization have been investigated using isolated intact domains of I alpha I produced from controlled proteolytic digests of the protein. The fragments investigated include the heavy chains of I alpha I, UTI, chondroitinase AC-treated UTI, and the carboxyl-terminal domain of UTI (termed HI-8). The effects of I alpha I and its fragments on the inhibitory activity of CaOx crystallization were evaluated in vitro using CaOx crystal aggregation and growth assays, and seeded crystal generation assay as well as using crystal matrix protein generation assay. UTI, but not the heavy chains of I alpha I, had a discernible effect on CaOx crystallization inhibitory activity. Less requirement of the carbohydrate moiety of UTI is implicated by the observation that chondroitinase AC-treated UTI fragment was also found to inhibit CaOx crystallization with almost the same activity as UTI. HI-8 also efficiently inhibited CaOx crystallization, while I alpha I showed a weak inhibitory activity. The results are almost consistent with a seed crystal generation assay and a crystal adsorption inhibition assay, in which I alpha I or its derivatives inhibits prothrombin fragment 1 (F1) adsorption to CaOx crystals. In conclusion, these results suggest that the part of the I alpha I protein responsible for inhibition of CaOx crystallization is the carboxyl-terminal domain of UTI.
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alfa-Globulinas/química , alfa-Globulinas/farmacología , Oxalato de Calcio/química , Glicoproteínas de Membrana , Inhibidor de la Tripsina de Soja de Kunitz , Oxalato de Calcio/antagonistas & inhibidores , Oxalato de Calcio/orina , Condroitín Liasas/farmacología , Cristalización , Glicoproteínas/efectos de los fármacos , Glicoproteínas/farmacología , Humanos , Concentración Osmolar , Proteínas/análisis , Protrombina/metabolismoRESUMEN
Vessel wall subendothelial extracellular matrix, a dense mesh formed of collagens, fibronectin, laminin, and proteoglycans, has important roles in lipid and lipoprotein retention and cell adhesion. In atherosclerosis, vessel wall heparan sulfate proteoglycans (HSPG) are decreased and we therefore tested whether selective loss of HSPG affects lipoprotein retention. A matrix synthesized by aortic endothelial cells and a commercially available matrix (Matrigel; , Rutherford, NJ) were used. Treatment of matrix with heparinase/heparitinase (1 U/ml each) increased LDL binding by approximately 1.5-fold. Binding of lipoprotein (a) [Lp(a)] to both subendothelial matrix and Matrigel(R) increased 2-10-fold when the HSPG were removed by heparinase treatment. Incubation of endothelial cells with oxidized LDL (OxLDL) or lysolecithin resulted in decreased matrix proteoglycans and increased Lp(a) retention by matrix. The effect of OxLDL or lysolecithin on endothelial PG was abolished in the presence of HDL. The decrease in matrix HSPG was associated with production of a heparanase-like activity by OxLDL-stimulated endothelial cells. To test whether removal of HSPG exposes fibronectin, a candidate Lp(a) binding protein in the matrix, antifibronectin antibodies were used. The increased Lp(a) binding after HSPG removal was inhibited 60% by antifibronectin antibodies. Similarly, the increased Lp(a) binding to matrix from OxLDL-treated endothelial cells was inhibited by antifibronectin antibodies. We hypothesize that atherogenic lipoproteins stimulate endothelial cell production of heparanase. This enzyme reduces HSPG which in turn promotes Lp(a) retention.
Asunto(s)
Endotelio/metabolismo , Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismo , Lipoproteína(a)/metabolismo , Lipoproteínas LDL/metabolismo , Proteoglicanos/metabolismo , Animales , Bovinos , Células Cultivadas , Condroitín Liasas/farmacología , Colágeno/metabolismo , Combinación de Medicamentos , Endotelio/efectos de los fármacos , Fibronectinas/metabolismo , Proteoglicanos de Heparán Sulfato , Liasa de Heparina , Laminina/metabolismo , Lipoproteínas HDL/farmacología , Lipoproteínas LDL/farmacología , Lisofosfatidilcolinas/farmacología , Oxidación-Reducción , Polisacárido Liasas/farmacologíaRESUMEN
STUDY DESIGN: The biomechanical effects of chondroitinase ABC and chymopapain related to spinal segmental instability were investigated using a canine model, as well by as radiologic and histologic analyses. OBJECTIVES: To evaluate the biomechanical, radiologic, and histologic affects on the lumber intervertebral disc of chondroitinase ABC compared with chymopapain. SUMMARY OF BACKGROUND DATA: No study on the biomechanical effects of chondroitinase ABC has been reported. METHODS: Forty-eight lumbar intervertebral discs in eight beagles were randomly assigned to three groups and received one of three materials: chondroitinase ABC, chymopapain, or buffered saline, using a lateral percutaneous procedure. One week after injection, the animals were killed and the lumbar spinal motion segments were removed. Spinal segmental instability after chemonucleolysis was evaluated in spinal motion segments without posterior elements. Radiologic and histologic changes were also investigated. RESULTS: Spinal segmental instability and disc space narrowing were more greater in the chymopapain group than in the chondroitinase ABC group. Destruction of nucleus and anulus proteoglycans, indicated by loss of safranin-O staining, was less intense in chondroitinase ABC-injected discs. CONCLUSIONS: Chondroitinase ABC results in less spinal segmental instability, disc space narrowing, and destruction of proteoglycans in intervertebral disc matrix than chymopapain.
Asunto(s)
Condroitín Liasas/farmacología , Quimopapaína/farmacología , Quimiólisis del Disco Intervertebral , Disco Intervertebral/fisiopatología , Vértebras Lumbares/fisiopatología , Columna Vertebral/fisiopatología , Animales , Fenómenos Biomecánicos , Perros , Diseño de Equipo , Disco Intervertebral/diagnóstico por imagen , Disco Intervertebral/efectos de los fármacos , Disco Intervertebral/patología , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/efectos de los fármacos , Vértebras Lumbares/patología , Masculino , Docilidad , RadiografíaRESUMEN
Different types of proteoglycans (PGs) from adult worms of Schistosoma haematobium, were sequentially extracted using chaotropic agents under associative conditions (0.5 M GnCl), dissociative conditions (4 M GnCl) and detergents (Triton X-100 and SDS). The extracts were designated F1, F2, F3 and F4, respectively. The highest amount of uronic acid and carbohydrate was detected in the associative extract (F1) while the highest amount of protein was detected in the SDS extract (F4). Agarose polyacrylamide gel electrophoresis (A-PAGE) indicated the presence of a different PG in each extract with different electrophoretic mobilities. Agarose gel electrophoresis of glycosaminoglycan (GAG) separated from GnCl, associative and dissociative extracts, and the residue suggested the presence of dermatan sulphate in the two extracts and the residue, in addition to a GAG-like material found in the associative extract only. This glycosaminoglycan showed resistance to digestion with all mucopolysaccharidases and nitrous acid treatment. Gel filtration chromatography of associative extract on Sepharose CL-6B indicated the presence of three main uronic acid peaks (P1, P2 and P3). Chondroitin sulphate was the main GAG that could be detected in peak one (P1). Peak two (P2) contains carbohydrate and uronic acid but has no protein or absorbance at 280 nm. P2 has two types of GAGs: dermatan sulphate and a GAG-like material. The role of this PG in helping the adult schistosomes in evading immobilization by the host blood clotting cascade is discussed. Antibodies to peak one and peak two were detected in hamster sera infected with S. haematobium and S. mansoni using the ELISA test. The specificity of peak two was found to be evident in its low cross-reactivity (18.9%) when confronted with S. mansoni infected sera.
Asunto(s)
Glicosaminoglicanos/química , Proteoglicanos/química , Schistosoma haematobium/química , Animales , Antígenos Helmínticos/análisis , Sangre/parasitología , Condroitina ABC Liasa/farmacología , Condroitín Liasas/farmacología , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glicosaminoglicanos/inmunología , Glicosaminoglicanos/aislamiento & purificación , Ácido Nitroso/farmacología , Proteoglicanos/aislamiento & purificación , Schistosoma haematobium/inmunología , Esquistosomiasis Urinaria/sangre , Esquistosomiasis Urinaria/inmunología , Esquistosomiasis mansoni/inmunologíaRESUMEN
PURPOSE: Synchrotron x-ray diffraction experiments indicate that corneas with macular corneal dystrophy (MCD) contain unusual 4.6-A periodic repeats thought to reside in proteoglycans or glycosaminoglycans. Recently the 4.6-A x-ray reflection was found to be significantly diminished after incubation of MCD specimens in buffer containing chondroitinase ABC or N-glycanase. We examined the sulfated proteoglycans in these glycosidase-digested MCD corneas. METHODS: Transmission electron microscopy was used in conjunction with cuprolinic blue-staining for sulfated proteoglycans. RESULTS: Incubation of an MCD specimen in enzyme buffer left both small and large proteoglycan filaments in the stromal matrix, whereas incubation in the presence of chondroitinase ABC removed these molecules from the tissue. Incubation in buffer containing N-glycanase, on the other hand, removed the large proteoglycan filaments from the MCD stroma but left unaffected the small collagen-associated proteoglycans. CONCLUSION: These results are consistent with the interpretation that 4.6-A periodic repeats in MCD corneas reside in large sulfated proteoglycan filaments (or aggregates thereof) that may contain chondroitin/dermatan sulfate and keratan sulfate or keratan components.
Asunto(s)
Distrofias Hereditarias de la Córnea/patología , Sustancia Propia/ultraestructura , Proteoglicanos/ultraestructura , Condroitín/análisis , Condroitín/ultraestructura , Condroitín Liasas/farmacología , Colorantes , Distrofias Hereditarias de la Córnea/metabolismo , Sustancia Propia/química , Medios de Cultivo , Dermatán Sulfato/análisis , Dermatán Sulfato/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Histocitoquímica , Humanos , Indoles , Sulfato de Queratano/análisis , Sulfato de Queratano/ultraestructura , Microscopía Electrónica , Compuestos Organometálicos , Proteoglicanos/análisis , Difracción de Rayos XRESUMEN
In chick skin, nerve arcades develop around the base of feathers. In order to understand the mechanisms of their formation, we have tried to dissociate arcade formation from feather morphogenesis in various ways. Nerve patterns were analysed (1) in hydrocortisone-treated embryos that are partially devoid of feathers, (2) after retinoic acid treatment that produces ectopic feathers, (3) in dorsal root ganglia-skin co-cultures. Whenever tested, immunochemistry revealed that nerve arcades form around chondroitin sulphate proteoglycan-rich areas. Hydrocortisone treatment modifies the distribution of two out of three chondroitin sulphate proteoglycan epitopes tested, as well as the shapes of the feathers and nerve arcades, but not fibronectin, tenascin or laminin localizations. Chondroitinase digestion in co-cultures eliminated the nerve arcade formation and produced abnormally thin feathers, but nevertheless with a normal spatial distribution. Thus, chondroitin sulphate proteoglycans are probably not involved in the overall arrangement of feathers, but appear to play a fundamental role in both the formation of nerve arcades and the morphogenesis of the feather.
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Sulfatos de Condroitina/análisis , Plumas/embriología , Piel/inervación , Animales , Western Blotting , Embrión de Pollo , Condroitín Liasas/farmacología , Técnicas de Cocultivo , Colágeno/análisis , Plumas/efectos de los fármacos , Plumas/inervación , Fibronectinas/análisis , Inmunohistoquímica , Laminina/análisis , Piel/química , Piel/efectos de los fármacos , Piel/embriología , Tenascina/análisis , Tretinoina/farmacologíaRESUMEN
When embryonic thalamic neurons are plated onto living slices of mouse forebrain, cell attachment and neurite outgrowth on different layers of the developing cerebral cortex vary dramatically, in ways that correlate with the timing and pattern of thalamocortical innervation. These layer-specific differences can be eliminated from embryonic day 16 slices by enzymatic removal of chondroitin sulfate (CS). The cortical plate (a zone avoided by thalamic axons in vivo) possesses inhibitory activity (anti-adhesive, neurite repelling) and the intermediate zone and subplate (in which thalamic axons normally grow) possess stimulatory activity (adhesive, neurite promoting), both of which are chondroitinase sensitive. These opposing activities appear not to reflect the presence of different CS proteoglycans (CSPGs) in different zones, but rather the presence of differentially localized CS-binding molecules, which can be competed away by soluble CS. This model reconciles conflicting reports on the actions of CSPGs in neural development, and suggests a role for CSPGs in the organization of matrix-bound cues in the brain.
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Corteza Cerebral/embriología , Sulfatos de Condroitina/fisiología , Neuronas/fisiología , Tálamo/embriología , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Corteza Cerebral/citología , Condroitín Liasas/farmacología , Embrión de Mamíferos/fisiología , Matriz Extracelular/metabolismo , Técnicas In Vitro , Ratones/embriología , Neuritas/fisiología , Prosencéfalo/efectos de los fármacos , Tálamo/citologíaRESUMEN
CD44 is an integral membrane glycoprotein that is a principal receptor for hyaluronan and plays a role in cell-extracellular matrix interactions. Recent studies of melanomas in mouse models have suggested that increased CD44 expression by these tumors may relate to metastatic potential. Immunohistochemical expression of CD44 (standard [s] and variant [v6]) in benign and malignant nevomelanocytic lesions was assessed in formalin-fixed, paraffin-embedded tissue and was correlated with histological parameters and prognostic factors. Cases included benign nevi (three junctional, four compound, five intradermal, five blue, six Spitz, one deep penetrating), architecturally disordered (dysplastic) nevi (three, and primary (22) and metastatic melanomas (eight). All of the benign lesions showed diffuse and essentially uniform membrane staining of CD44s in nevomelanocytic cells, regardless of lesion size, depth, or extent of dermal involvement. In contrast, semiquantitative analysis (0 to 3+) of the primary melanomas showed heterogeneous and decreased staining of CD44s, which inversely correlated with lesion size (-0.569) and depth of invasion (-0.622 and -0.617 for Breslow's depth and Clark's level, respectively). These results were significant at P < .05. CD44s expression in metastases paralleled that of their respective primaries. None of the benign nevomelanocytic lesions showed CD44v6 staining. In contrast, all of the malignant nevomelanocytic lesions showed cytoplasmic staining of the tumor cells. Pretreatment with chondroitinase did not alter CD44s staining. CD44s expression by immunohistochemical determination is uniform in benign nevomelanocytic lesions. Malignant melanomas show decreased, heterogeneous staining that inversely correlates with increasing size, depth, and level of invasion. CD44 expression may be a prognostic indicator in malignant melanomas. Tumor staining with anti-chondroitin sulfate monoclonal antibodies suggests that CD44s may be expressed as a chondroitin sulfate proteoglycan in primary melanomas.
Asunto(s)
Receptores de Hialuranos/análisis , Nevo Pigmentado/inmunología , Adulto , Anciano , Condroitín Liasas/farmacología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Piel/inmunologíaRESUMEN
The glycosaminoglycans of sciatic nerves recovering from crush-injury were studied in adult guinea pigs and compared with those of non-injured mature neural tissues. The glycosaminoglycans were recovered from the 1,900 g supernatant and pellet of the tissue homogenates and assayed for hexuronate contents and susceptibilities to hyaluronidase, chondroitinase ABC, and nitrous acid. In the normal brain and central nerve tracts, the glycosaminoglycans were distributed both in the supernatant and pellet fractions; the brain showed a predominance of chondroitin sulphates but the tracts showed a predominance of heparan sulphates. Twice as much glycosaminoglycans were found in normal sciatic nerves, only in the pellet fraction and with heparan sulphate predominant. In the 2 weeks post-crush, progressive increase in hexuronate was observed, due mainly to additional chondroitin sulphate forms in the supernatant; the pellet fraction in the same period was however similar to the untreated controls in relative abundance of glycosaminoglycan classes and hexuronate content. At 4 weeks post-crush, although the total hexuronate returned to the control level, a significant proportion of glycosaminoglycans remained in the supernatant fraction. Evidence is thus provided for the need to modulate the glycosaminoglycan expression pattern in adult neural tissue to allow post-traumatic tissue remodelling and axonal regrowth.
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Glicosaminoglicanos/aislamiento & purificación , Regeneración Nerviosa , Nervio Ciático/fisiología , Animales , Química Encefálica , Adhesión Celular , Condroitín Liasas/farmacología , Sulfatos de Condroitina/aislamiento & purificación , Dermatán Sulfato/aislamiento & purificación , Cobayas , Heparitina Sulfato/aislamiento & purificación , Ácido Hialurónico/aislamiento & purificación , Hialuronoglucosaminidasa/farmacología , Compresión Nerviosa , Ácido Nitroso/farmacología , Nervio Olfatorio/química , Nervio Óptico/química , Especificidad de Órganos , Nervio Ciático/química , Hidróxido de Sodio/farmacologíaRESUMEN
PURPOSE: To examine the distribution of proteoglycans in the exfoliation materials in order to investigate the nature of the materials. METHODS: The anterior parts of two eyes with exfoliation syndrome were examined by electron microscopy after staining with cupromeronic blue (cmb). Some specimens were treated with enzymes and/or nitrous acid prior to staining. The effects of the enzymes were evaluated statistically by counting the density of the cmb-positive filaments in the exfoliation materials, using a computer. One eye with exfoliation syndrome stained with alcian blue was observed with light microscopy. RESULTS: Exfoliation materials were observed along the epithelial cells of the iris and ciliary body, and in the trabecular meshwork and zonules. In tissue specimens treated with cmb, electron-dense filaments were seen associated with the exfoliation materials. Microfibrils in the trabecular meshwork and iris, and zonular fibrils themselves were free of any filament staining, while the exfoliation materials located closely to the fibrils contained the electron-dense filaments. In the tissue specimens treated with chondroitinase AC, chondroitinase B, chondroitinase ABC or nitrous acid before cmb staining, the amount of the filament associated with exfoliation materials decreased in comparison to the controls. Digestion with keratinase did not demonstrate any significant changes in staining. A combination treatment with chondroitinase ABC and nitrous acid eliminated almost all filaments associated with the exfoliation materials. In the eye stained with alcian blue, the zonules that did not stain for the dye demonstrated an accumulation of exfoliation materials that stained strongly for alcian blue. CONCLUSIONS: Exfoliation materials contain chondroitin sulfate, dermatan sulfate, heparan sulfate proteoglycans. Depositions of proteoglycans on the microfibrils may be closely associated with the formation of exfoliation materials.
Asunto(s)
Cuerpo Ciliar/ultraestructura , Síndrome de Exfoliación/metabolismo , Glicósido Hidrolasas , Iris/ultraestructura , Proteoglicanos/metabolismo , Malla Trabecular/ultraestructura , Anciano , Anciano de 80 o más Años , Azul Alcián , Condroitín Liasas/farmacología , Cuerpo Ciliar/efectos de los fármacos , Cuerpo Ciliar/metabolismo , Colorantes , Síndrome de Exfoliación/patología , Femenino , Humanos , Indoles , Iris/efectos de los fármacos , Iris/metabolismo , Masculino , Ácido Nitroso/farmacología , Compuestos Organometálicos , Proteoglicanos/efectos de los fármacos , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
STUDY DESIGN: This study demonstrated the chemonucleolytic effects of chondroitinase ABC and its histologic and biochemical background. OBJECTIVES: To determine the course of chondroitinase ABC action on normal rabbit discs, and to find its minimum effective dosage. SUMMARY OF BACKGROUND DATA: No previous study has assessed the chemonucleolytic action of chondroitinase ABC in a time- and dose-dependent manner. This study also investigated the biochemical causes of radiologic and histologic changes in the discs. METHODS: Rabbits were injected with 4 U of pharmaceutical-grade chondroitinase ABC intradiscally. They were radiologically and histologically observed, and biochemical analyses of the discs were conducted on days 1, 3, 5, 7, and 10 postinjection in the time course study. Different doses of chondroitinase ABC were injected, and radiologic observations and water content of the discs were measured in the dose-finding study. RESULTS: The time course study revealed that the chondroitin sulfate content of discs significantly decreased from day 1 postinjection until the end of the experimental period. The weight and water content of the nucleus pulposus decreased on day 3, and disc space narrowing was observed from the day after injection. The dose-finding study showed that a dose of 0.0002 U/disc still induced disc space narrowing and a decrease in water content. CONCLUSIONS: Chondroitinase ABC is estimated to have a chemonucleolytic effect at least by day 3 postinjection at a dose level of 0.0002 U/disc or higher in rabbits.
Asunto(s)
Condroitín Liasas/farmacología , Quimiólisis del Disco Intervertebral , Disco Intervertebral/efectos de los fármacos , Animales , Condroitín Liasas/metabolismo , Sulfatos de Condroitina/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Ácido Hialurónico/metabolismo , Disco Intervertebral/química , Disco Intervertebral/diagnóstico por imagen , Sulfato de Queratano/metabolismo , Tamaño de los Órganos , Conejos , Radiografía , Factores de Tiempo , Agua/metabolismoRESUMEN
The mitogenic action of insulin-like growth factors (IGFs) on target cells is determined by interaction with signaling IGF-I receptors and modulated by interactions with IGF-binding proteins (IGFBPs). IGFBP-3, an abundant IGFBP that binds IGF-I and IGF-II with high affinity, can form soluble inhibitory complexes with the IGFs that prevent them from binding to IGF-I receptors. Alternatively, IGFBP-3 can bind to the cell surface and possibly potentiate IGF action or act independently of the IGFs. Previous studies showed that heparin inhibited IGFBP-3 binding to the cell surface and increased its accumulation in the medium, suggesting that it might act as a competitive inhibitor of IGFBP-3 binding to structurally similar heparan sulfate proteoglycans on the cell surface. We evaluated this hypothesis by binding 125I-labeled recombinant glycosylated human IGFBP-3 to human fetal skin fibroblasts (GM-10) and to C6 rat glioma cells at 12 C. Heparin inhibited [125I]IGFBP-3 binding more effectively than chondroitin sulfate and dextran sulfate. Complete digestion of cell surface heparan sulfate and chondroitin sulfate glycosaminoglycans using heparitinase and chondroitinase ABC, however, did not significantly decrease IGFBP-3 binding. Quantitative removal was demonstrated by analysis of parallel cultures of cells whose glycosaminoglycans had been biosynthetically labeled using Na2 35SO4. These results suggested that IGFBP-3 did not bind to heparan sulfate glycosaminoglycans on the cell surface, and that the inhibition of IGFBP-3 binding by heparin most likely resulted from its direct interaction with the heparin-binding domains of IGFBP-3. When [125I]IGFBP-3 was incubated with GM-10 fibroblasts or C6 glioma cells at 37 C for 4 h, only 10% of the bound ligand remained associated with the cell surface; approximately 90% of the cell-associated radio-activity was internalized and could be recovered in lysates of acid-washed cells. Incubation with IGF-I or heparin decreased the total cell-associated radioactivity, but did not affect internalization. These results suggest that direct interaction of heparin or IGF-I with IGFBP-3 inhibits its ability to bind to the surface of GM-10 fibroblasts and C6 glioma cells.
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Glioma/metabolismo , Heparina/farmacología , Heparitina Sulfato/fisiología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/antagonistas & inhibidores , Proteoglicanos/fisiología , Piel/metabolismo , Animales , Condroitín Liasas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glioma/patología , Proteoglicanos de Heparán Sulfato , Antagonistas de Heparina/farmacología , Liasa de Heparina , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Polisacárido Liasas/farmacología , Ratas , Piel/citología , Piel/embriología , Células Tumorales Cultivadas/efectos de los fármacosAsunto(s)
Cartílago Articular/inmunología , Epítopos , Osteoartritis/inmunología , Anciano , Anticuerpos Monoclonales/inmunología , Cartílago Articular/efectos de los fármacos , Condroitín Liasas/farmacología , Sulfatos de Condroitina/inmunología , Femenino , Humanos , Técnicas Inmunológicas , Coloración y EtiquetadoRESUMEN
Women are particularly susceptible to malaria during first and second pregnancies, even though they may have developed immunity over years of residence in endemic areas. Plasmodium falciparum-infected red blood cells (IRBCs) were obtained from human placentas. These IRBCs bound to purified chondroitin sulfate A (CSA) but not to other extracellular matrix proteins or to other known IRBC receptors. IRBCs from nonpregnant donors did not bind to CSA. Placental IRBCs adhered to sections of fresh-frozen human placenta with an anatomic distribution similar to that of naturally infected placentas, and this adhesion was competitively inhibited by purified CSA. Thus, adhesion to CSA appears to select for a subpopulation of parasites that causes maternal malaria.