RESUMEN
Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.
Asunto(s)
Araña Reclusa Parda/genética , Miniproteínas Nodales de Cistina/química , Insecticidas/análisis , Hidrolasas Diéster Fosfóricas/química , Venenos de Araña/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Araña Reclusa Parda/metabolismo , Secuencia Conservada , Miniproteínas Nodales de Cistina/biosíntesis , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/aislamiento & purificación , Dípteros , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Datos de Secuencia Molecular , Proteoma , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Pruebas de Toxicidad , TranscriptomaAsunto(s)
Toxicología , Toxicología , Venenos , Intoxicación , Toxinas Biológicas , Toxicología , Biología Celular , GenéticaRESUMEN
Bites from brown spiders (Loxosceles genus) have clinical manifestations including skin necrosis with gravitational spreading, and systemic involvement that may include renal failure, hemolysis, and thrombocytopenia. Mice were exposed to recombinant wild-type phospholipase-D, or to an isoform with a mutation in the catalytic domain resulting in no phospholipasic activity. Renal biopsies from mice treated with the wild-type toxin showed glomerular edema, erythrocytes and collapse of Bowman's space, edema and deposition of proteinaceous material within the tubular lumen. Ultrastructural analyses confirmed cytotoxicity by demonstrating disorders of glomerulus at foot processes and at fenestrated endothelium. Tubule alterations include deposits of amorphous material and edema, as well as an increase of epithelial cytoplasmic multivesicular bodies and electron-dense structures. There was an absence of nephrotoxicity in mice treated with the mutated toxin. Analyses of urine and blood showed that wild type toxin induced hematuria and elevation of blood urea, while treatment with mutated toxin caused no changes. Mouse lethality experiments also showed oliguria and mortality after treatment with wild-type toxin, but not following exposure to the mutated toxin. Immunofluorescence using antibodies to phospholipase-D toxin showed deposition of both toxins along the renal tubular structures as detected by confocal microscopy. Immunoblots of urine showed a 30 kDa band in samples from animals treated with wild-type toxin, but no band from mice exposed to mutated toxin. Wild-type toxin treatment caused cytoplasmic vacuolization, impaired spreading, reduction of cellular viability, and cell-cell and cell-substratum detachment in MDCK cells, while treatment with mutated isoform had no effect. Finally, there is a direct correlation between toxin activity on cell membrane phospholipids generating choline and cytotoxicity. We have defined for the first time a molecular mechanism for Loxosceles venom nephrotoxicity that is dependent on the catalytic activity of phospholipase-D toxin.
Asunto(s)
Túbulos Renales/efectos de los fármacos , Fosfolipasa D/toxicidad , Hidrolasas Diéster Fosfóricas/toxicidad , Insuficiencia Renal/inducido químicamente , Venenos de Araña/toxicidad , Animales , Dominio Catalítico/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Túbulos Renales/ultraestructura , Ratones , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Fosfolipasa D/química , Fosfolipasa D/genética , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/toxicidad , Proteinuria/inducido químicamente , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Insuficiencia Renal/patología , Venenos de Araña/química , Venenos de Araña/genéticaRESUMEN
OBJECTIVES: Cell transplantation is considered a novel approach in the treatment of myocardiopathy. The objective of this study was to evaluate the effects of autologous mononuclear stem cell therapy in doxorubicin-induced dilated myocardiopathy by conducting both functional and histopathologic analysis. METHODS: Seventy male rats were doxorubicin injected intraperitoneally for 2 weeks. At 1 month, the animals that had demonstrated left ventricular ejection fractions less than 40% were randomly divided into a mononuclear stem cell group and controls. Mononuclear stem cells were isolated. All animals underwent echocardiographic study: baseline, pre-cell therapy, and at 1 month post-cell therapy, and analyzed by the nonparametric Mann-Whitney test. Transplants were performed by subepicardial injections. Standard staining was performed. RESULTS: Twenty-three animals were randomly treated: mononuclear stem cell and control groups, with 11 rats completing the study. Cell viability was 85%. Mononuclear stem cells (n=5; 5x106 cells /300 microL medium) and control (n=6; 300 microL medium) were used. The resulting left ventricular ejection fraction in the cell therapy group was not significantly different compared with controls (p=0.54). New vessels were demonstrated in the subepicardial region. CONCLUSIONS: Autologous mononuclear stem cell therapy was not functionally effective in doxorubicin-induced dilated myocardiopathy in the animal model under study with the experimental conditions, despite occurrence of angiogenic activity.
Asunto(s)
Trasplante de Médula Ósea , Cardiomiopatías/terapia , Neovascularización Fisiológica , Trasplante de Células Madre , Animales , Cardiomiopatías/inducido químicamente , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Wistar , Trasplante AutólogoAsunto(s)
Toxicología , Toxicología , Biodiversidad , Venenos , Intoxicación , Toxinas Biológicas , ToxicologíaRESUMEN
Leucurolysin-a (leuc-a), a 23 kDa non-hemorrhagic metalloproteinase, is found in venom of the viper Bothrops leucurus. Here, we examine the biological consequences of leuc-a, including thrombolytic activity, direct effects on endothelial cells in culture and edematogenic activity in vivo. We demonstrate fibrinolytic activity of leuc-a, in which the protease specifically degrades alpha, beta, and gamma-gamma chains. While not causing hemorrhaging, leuc-a does cause thrombolytic activities in whole blood clots. Endothelial cells are highly resistant to leuc-a in culture. Cell viability suffered only when cells were exposed to large quantities of the protease. Nevertheless, leuc-a induces changes in cell morphology. The impact of leuc-a on cell adhesion was confirmed by an adhesion assay, in which cell adhesion to fibronectin decreased due to leuc-a. This mild cellular impact is unlike that of crude venom, where lower concentrations triggered cell death and a greater reduction in cell adhesion. Also, leuc-a increased microvessel permeability with marked edema in mice peritoneum and foot pads. These effects are similar to those of other P-I class SVPMs. These in vivo effects were weaker when crude venom was tested. In conclusion, albeit not showing significant hemorrhagic activity, leuc-a can induce a prominent edema which appears to be significant in the local effects observed after B. leucurus venom accidents.
Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/toxicidad , Fibrinólisis/efectos de los fármacos , Metaloproteasas/toxicidad , Análisis de Varianza , Animales , Adhesión Celular/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/metabolismo , Edema , Fibrina/metabolismo , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Metaloproteasas/aislamiento & purificación , Metaloproteasas/metabolismo , Microvasos/metabolismo , Conejos , Trombina/metabolismoRESUMEN
The best results of cell therapy are achieved by a greater quantity of cells, delivery to the correct place, and cell conditions of viability with proliferation and without apoptosis. The quantification of cellular growth, including proliferation and viability, has become an essential tool. The objective of this study was to analyze cell proliferation in 14-day cultures of bone marrow mesenchymal stem cells (BMMSC), skeletal muscle cells (SMC), and co-culture of both types of cells (CO). Forty-four adult Wistar male rats (250-300g) received cultured cells CO (n = 22), BMMSC (n = 10), and SMC (n = 12). All cultured cells were started with the same concentration: 5 x 10(5)/mL, under similar conditions and maintained in an incubator with 5% CO(2) at 37 degrees C, which was changed every 48 hours for 14 days. The cell count was performed in Neubauer's chamber to calculate the proliferation index (IP). Statistical analysis was performed by the nonparametric Kruskal-Wallis and Wilcoxon tests. P values <.05 were considered statistically significant. The results showed that IP was positive in all groups. In conclusion, proliferation capacity was demonstrated in all groups. SMC IP was greater than the others, although it was the most heterogeneous.
Asunto(s)
Células de la Médula Ósea/citología , Cardiopatías/terapia , Mesodermo/citología , Músculo Esquelético/citología , Miocardio/citología , Células Madre/citología , Animales , División Celular , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Wistar , Trasplante de Células MadreRESUMEN
UNLABELLED: In myocardial infarction and Chagas's disease, some physiopathological aspects are common: cardiomyocyte loss due to ischemia leads to a reduction of contractility and heart function. Different cells have been proposed for cellular cardiomioplasty. OBJECTIVE: Our goal was to evaluate the method of co-culture of skeletal muscle (SM) and mesenchymal stem cells (MSC) for cell therapy of heart failure in Chagas's disease (CD) and myocardium postinfarction (MI). METHODS: For MI, 39 rats completed the study at 1 month. Seventeen rats received cell therapy into the scar and 22 rats only medium. For CD, 15 rats completed the study at 1 month including 7 that received cell therapy and eight followed the natural evolution. All animals underwent ecocardiographic analysis at baseline and 1 month. Left ventricular, ejection fraction, end systolic, and end dyastolic volume were registered and analyzed by ANOVA. The co-culture method of SM and MSC was performed at 14 days (DMEM, with 15% FCS, 1% antibiotic, IGF-I, dexamethasone). Standard stain analysis was performed. RESULTS: For MI ejection fraction in the animals that received the co-cultured cells increased from 23.52+/-8.67 to 31.45+/-8.87 (P=.006) versus the results in the control group: 26.68+/-6.92 to 22.32+/-6.94 (P=.004). For CD, ejection fraction in animals that received the co-cultured cells increased from 31.10+/-5.78 to 53.37+/-5.84 (P<.001) versus the control group values of 36.21+/-3.70 to 38.19+/-7.03 (P=0.426). Histopathological analysis of the animals receiving co-cultured cells demonstrated the presence of myogenesis and angiogenesis. CONCLUSION: The results validated the product of SM and MSC co-cultures for treatment of diseases.
Asunto(s)
Trasplante de Células/fisiología , Enfermedad de Chagas/terapia , Cardiopatías/terapia , Músculo Esquelético/citología , Mioblastos/citología , Células Madre/citología , Animales , Enfermedad de Chagas/fisiopatología , Técnicas de Cocultivo , Diástole , Modelos Animales de Enfermedad , Cardiopatías/fisiopatología , Ratas , Ratas Wistar , Regeneración , Reproducibilidad de los Resultados , Sístole , Función Ventricular IzquierdaRESUMEN
The venom of the brown spider is remarkable because it causes dermonecrotic injury, hemorrhagic problems, hemolysis, platelet aggregation and renal failure. The mechanism by which the venom causes hemorrhagic disorders is poorly understood. Rabbits intradermally exposed to the venom showed a local hemorrhage starting 1 h after inoculation and reaching maximum activity between 2 and 3 days. Biopsies examined by light and transmission electron microscopy showed subendothelial blebs, vacuoles and endothelial cell membrane degeneration in blood vessels, plasma exudation into connective tissue, and fibrin and thrombus formation within blood vessels. Loxosceles intermedia venom incubated with fibrinogen partially degrades Aalpha and Bbeta chains of intact fibrinogen, and significantly cleaves all Aalpha, Bbeta and gamma chains when they were separated or when fibrinogen is denatured by boiling. Proteolytic kinetic studies showed that the Aalpha chain is more susceptible to venom hydrolysis than the Bbeta chain. The fibrinogenolysis is blocked by ethylenediamine tetraacetic acid and 1,10-phenanthroline, but not by other protease inhibitors. Human plasma incubated with the venom had coagulation parameters such as prothrombin time, activated partial thromboplastin time and thrombin time increased. Through molecular sieve chromatography, we isolated a venom toxin of 30 kDa with fibrinogenolytic activity. We propose that the local and systemic hemorrhagic disorders evoked in loxoscelism are consequences of direct venom fibrinogenolysis together with cytotoxicity to subendothelial structures and endothelial cells in blood vessels.
Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Fibrinógeno/efectos de los fármacos , Venenos de Araña/toxicidad , Animales , Coagulación Sanguínea/efectos de los fármacos , Vasos Sanguíneos/patología , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Endotelio Vascular/ultraestructura , Fibrinógeno/metabolismo , Hemorragia/inducido químicamente , Humanos , Cinética , Microscopía Electrónica , Inhibidores de Proteasas/farmacología , Conejos , Venenos de Araña/análisis , Venenos de Araña/farmacología , Toxinas Biológicas/química , Toxinas Biológicas/aislamiento & purificaciónRESUMEN
Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues.
Asunto(s)
Membrana Basal/efectos de los fármacos , Matriz Extracelular/enzimología , Hemostasis/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/envenenamiento , Serina Endopeptidasas/efectos de los fármacos , Venenos de Araña/envenenamiento , Arañas , Animales , Humanos , Serina Endopeptidasas/análisis , Serina Endopeptidasas/metabolismoRESUMEN
Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues
Asunto(s)
Humanos , Animales , Membrana Basal/efectos de los fármacos , Endopeptidasas/metabolismo , Matriz Extracelular/efectos de los fármacos , Hemostasis/efectos de los fármacos , Venenos de Araña/enzimología , Arañas , Endopeptidasas/análisis , Venenos de Araña/química , Venenos de Araña/toxicidadRESUMEN
The effect of brown spider (Loxosceles intermedia) venom on endothelial cells was investigated in vivo and in vitro. Morphological and ultrastructural observations by light microscopy and transmission electron microscopy showed that the venom acts in vivo upon vessel endothelial cells of rabbits that were intradermally injected, evoking vessel instability, cytoplasmic endothelial cell vacuolization, and blebs. Likewise, treatment of rabbit endothelial cells in culture with the venom led to loss of adhesion of the cells to the substrate. Endothelial cells in culture were metabolically radiolabeled with sodium [35S]-sulfate and the sulfated compounds (proteoglycans and sulfated proteins) from medium, cell surface, and extracellular matrix (ECM) were analyzed. Agarose gel electrophoresis and SDS-PAGE showed that the venom is active on the ECM and on cell surface proteoglycans, shedding these molecules into the culture medium. In addition, when purified heparan sulfate proteoglycan (HSPG) and purified laminin-entactin (LN/ET) complex were incubated with the venom we observed a partial degradation of the protein core of HSPG as well as the hydrolysis of entactin. The above results suggest that the L. intermedia venom has a deleterious effect on the endothelium of vessels both in vivo and in culture, removing important constituents such as HSPG and entactin that are involved in the adhesion of endothelial cells and of subendothelial ECM organization.
Asunto(s)
Citotoxinas/farmacología , Endotelio Vascular/citología , Hidrolasas Diéster Fosfóricas/farmacología , Venenos de Araña/farmacología , Animales , Membrana Basal/química , Línea Celular , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Proteoglicanos de Heparán Sulfato/metabolismo , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Microscopía Electrónica , ConejosRESUMEN
Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1, 10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.
Asunto(s)
Membrana Basal/efectos de los fármacos , Membrana Basal/ultraestructura , Hidrolasas Diéster Fosfóricas/toxicidad , Serina Endopeptidasas/toxicidad , Venenos de Araña/toxicidad , Animales , Electroforesis en Gel de Poliacrilamida , Heparitina Sulfato/química , Humanos , Inmunohistoquímica , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/efectos de los fármacos , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Necrosis , Trasplante de Neoplasias , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Proteoglicanos/química , Conejos , Sarcoma Experimental/patología , Piel/patologíaRESUMEN
High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia venom to linear gradient 3-20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0. with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, librinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia venom probably involved in the harmful effects of the venom.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , Serina Endopeptidasas/química , Venenos de Araña/química , Venenos de Araña/enzimología , Animales , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Gelatina/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Hidrolasas Diéster Fosfóricas/toxicidad , Conejos , Serina Endopeptidasas/toxicidad , Inhibidores de Serina Proteinasa/farmacología , Venenos de Araña/toxicidad , Especificidad por Sustrato , Tripsina/farmacologíaRESUMEN
The brown spider, genus Loxosceles, is becoming of great medical importance, with envenomation (Loxoscelism) occurring throughout the world. The biological activities of the brown spider venom usually include dermonecrotic lesions at the bite site accompanied by hemolytic and haemorrhagic effects and also by renal failure. The objective of the present study was to describe the histology of the venom gland of L. intermedia using glands from adult spiders which were investigated by light microscopy, using immunohistochemical and staining methods, by transmission electron microscopy, and by scanning electron microscopy. The organization of the venom gland of Loxosceles intermedia follows the general architecture of spiders' venom glands. Using light microscopy and transmission electron microscopy we observed that the venom glands of L. intermedia present two layers of striated muscle fibers, an external layer and an internal layer in touch with an extracellular matrix which is a basement membrane structure and a fibrillar collagen matrix separating the muscular region from epithelial cells of the venom gland. Muscle cells are multinucleated, with nuclei peripherally placed and their cytoplasm rich in sarcoplasmic reticulum, myofibrills and continuous Z lines. By using scanning electron microscopy we can detect muscular cells from external layer as branching cells. Epithelial cells have their cytosol extremely rich in rough endoplasmic reticulum, mitochondria collection, Golgi apparatus, interdigitating membranes and secretory vesicles that ultimately accumulate the venom, a complex protein mixture.
Asunto(s)
Arañas/anatomía & histología , Animales , Epitelio/ultraestructura , Inmunohistoquímica , Microscopía Electrónica , Músculos/citología , Músculos/ultraestructuraRESUMEN
Working with Mel-85 (a human melanoma cell line), we have been able to detect a laminin-binding molecule with an apparent molecular mass of 100/110 kDa (Mel-85-LBM). Reduction with beta-mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interaction seems to be very specific since Mel-85-LBM binds laminin, but not fibronectin, vitronectin or type I collagen in affinity chromatography experiments. The molecule has a negative net charge at physiological pH and binds laminin in a divalent cation dependent way. Mel-85-LBM was metabolically radiolabeled with sodium [35S]-sulfate and chemical beta-elimination of purified Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sensitive to chondroitinase ABC digestion. These findings show that this molecule is a chondroitin sulfate proteoglycan. The location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiserum raised against purified Mel-85-LBM, that specifically reacts with just one molecule by western blotting among Mel-85 total cell extract as well as produces a positive signal by flow cytometry and a fluorescence profile of Mel-85 cells adhered on laminin.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Laminina/metabolismo , Melanoma/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Movimiento Celular , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Humanos , Melanoma/patología , Unión Proteica , Células Tumorales CultivadasRESUMEN
Loxosceles spp. (brown spider) envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site (a hallmark of accidents) and, to a lesser extent, thrombocytopenia, hemolysis and disseminated intravascular coagulation in some cases. Using lectin-immunolabeling, lectin-affinity chromatography, glycosidase and proteinase K treatments we were able to identify several venom N-glycosylated proteins with high-mannose oligosaccharide structures, complex-type glycoconjugates such as fucosylated glycans, but no galactose or sialic acid residues as complex sugars or glycosaminoglycan residues. Working with enzymatically or chemically deglycosylated venom we found that platelet aggregation (thrombocytopenic activity) as well as the fibronectinolytic and fibrinogenolytic (haemorrhagic) effects of the venom were sugar-independent when compared to glycosylated venom. Nevertheless, zymograph analysis in co-polymerized gelatin gels showed that enzymatic N-deglycosylation of loxolysin-B, a high-mannose 32-35 kDa glycoprotein of the venom with gelatinolytic metalloproteinase activity, caused a reduction of approximately 2 kDa in its molecular weight and a reduction of the gelatinolytic effect to a residual activity of 28% when compared to the glycosylated molecule, indicating a post-translational glycosylation-dependent gelatinolytic effect. Analysis of the dermonecrotic effect of the chemically or enzymatically N-deglycosylated venom detected only residual activity when compared with the glycosylated control. Thus, the present report suggests that oligosaccharide moieties play a role in the destructive effects of brown spider venom and opens the possibility for a carbohydrate-based therapy.
Asunto(s)
Oligosacáridos/química , Enfermedades de la Piel/inducido químicamente , Venenos de Araña/química , Animales , Carbohidratos/farmacología , Cromatografía de Afinidad , Electroforesis , Fibrinógeno/efectos de los fármacos , Fibronectinas/efectos de los fármacos , Glicosilación , Humanos , Immunoblotting , Necrosis , Agregación Plaquetaria , Conejos , Enfermedades de la Piel/etiología , Venenos de Araña/metabolismo , ArañasRESUMEN
By studying Loxosceles intermedia (Brown spider) venom we were able to detect a proteolytic action on fibronectin and fibrinogen but an inability to degrade full length laminin, type I and type IV collagens. By studying enzyme inhibitors we observed that divalent metal chelators as EDTA and 1,10-phenanthroline completely blocked this cleaving action whereas serine-protease inhibitors, thiol-protease inhibitor and acid-protease inhibitor showed little or no effect on the proteolytic activity of the venom indicating involvement of a metalloproteinase. Zymogram analysis of venom detected a 35 kDa molecule with gelatinolytic activity. The metalloproteinase nature was further supported by its sensitivity to 4-aminophenyl mercuric acetate (APMA) treatment which decreased its molecular weight to 32 kDa, inhibition of its gelatinolytic effect by 1,10-phenanthroline and its elution from gelatin-sepharose affinity beads. In addition, zymogram experiments using fibronectin and fibrinogen as substrates detected a fibronectinolytic and fibrinogenolytic band at 28 kDa which changed its electrophoretic mobility to 20 kDa band after organomercurial treatment. The inhibitory effect of 1,10 phenanthroline and APMA sensitivity on this proteolytic effect confirmed the presence of a second metalloproteinase in the venom. The data presented herein describe two invertebrate metalloproteinases in L. intermedia venom with different specificities one gelatinolytic and another, fibronectinolytic and fibrinogenolytic, probably involved in the harmful effects of the venom.
Asunto(s)
Metaloendopeptidasas/aislamiento & purificación , Venenos de Araña/análisis , Animales , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Gelatina/metabolismo , Metaloendopeptidasas/metabolismo , Hidrolasas Diéster Fosfóricas/análisis , Venenos de Araña/enzimologíaRESUMEN
Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.
Asunto(s)
Glicosaminoglicanos/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Fibronectina/metabolismo , Adhesión Celular , Electroforesis en Gel Bidimensional , Heparitina Sulfato/metabolismo , Humanos , Conformación Proteica , Sulfatos/metabolismo , Células Tumorales CultivadasRESUMEN
Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha 5 beta 1 integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation and a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.