RESUMEN
Surface coat proteins (SCPs) of entomopathogenic nematodes are implicated in the suppression/evasion of host immune responses, which is required for successful host colonization. Steinernema glaseri NC strain SCPs suppressed immune responses in oriental beetle larvae (Exomala orientalis), a susceptible host for S. glaseri, in a dosage-dependent manner, thus protecting Heterorhabditis bacteriophora from being killed in the same host. Melanization of H. bacteriophora decreased from 92+/-5% in the untreated check to 1+/-3% when protected by injection of 230ng of S. glaseri SCPs. As the SCPs dosage increased, freely moving H. bacteriophora increased from 3+/-4% in the untreated group to 57+/-15% with an SCPs dose of 940ng. At 2h and in the absence of SCPs, 8% and 11% of hemocytes of E. orientalis were stained by propidium iodide and Hoechst, respectively. When exposed to 300ng/microl SCPs, 70% and 96% were stained, respectively. At 6h, propidium iodide stained 37% and 92% of the hemocytes without and with SCPs, respectively. In contrast, more than 90% of the cells were stained by Hoechst with or without SCPs. As native proteins, two isolated S. glaseri SCPs had an immunosuppressive effect; they were each composed of 38kDa (PI=4.6) and 56kDa (PI=3.6) subunits. SCP peptides were sequenced using LC-MS/MS and the mass fingerprints obtained with MALDI-TOF-MS; there were no significant matches found in peptide databases, which suggests that the SCPs studied are novel proteins. Twelve cDNA sequences were derived based on short peptides and 7 of them had no significant match against the Caenorhabditis elegans protein database. One of the cDNA matched an unknown C. elegans protein and the remaining 4 cDNAs matched proteins of C. elegans and Brugia malayi.
Asunto(s)
Escarabajos/parasitología , Proteínas de la Membrana/aislamiento & purificación , Nematodos/fisiología , Animales , Cromatografía Liquida , Hemocitos/metabolismo , Larva/parasitología , Proteínas de la Membrana/química , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
Reproduction of entomopathogenic nematodes requires that they escape recognition by a host's immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS-PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.
Asunto(s)
Insectos/parasitología , Nematodos/patogenicidad , Infecciones por Nematodos/inmunología , Animales , Susceptibilidad a Enfermedades , Proteínas del Helminto/análisis , Hemocitos/parasitología , Interacciones Huésped-Parásitos , Insectos/inmunología , Nematodos/clasificación , Nematodos/crecimiento & desarrollo , Nematodos/inmunología , Infecciones por Nematodos/parasitología , Especificidad de la Especie , VirulenciaRESUMEN
Since osteoblast proliferation is critical for bone development, the effect of bone extracellular matrix (ECM) proteins on osteoblast signaling and proliferation in serum-free medium was investigated. Proliferation was highest in primary rat calvarial osteoblasts cells grown on fibronectin but less on type I collagen; osteonectin and poly-L-lysine did not support early proliferation. Fibronectin and type I collagen binding requires integrins, whereas cell adhesion to osteonectin or poly-L-lysine does not involve integrins. Therefore, the role of integrins in osteoblast signaling, leading to the induction of AP-1 transcription factors (c-fos and c-jun) which are important in cell proliferation, was studied. c-fos and c-jun message levels were increased at 60 min in osteoblasts plated onto fibronectin or collagen, but not in cells on osteonectin or poly-L-lysine. Protein synthesis was not required for c-fos mRNA expression; however, kinase activity was necessary for c-fos induction. In cells plated onto fibronectin, c-fos mRNA levels were controlled by protein kinase C and phosphotyrosine kinase signaling pathways. In contrast, c-fos levels in collagen-adhering cells may involve protein kinase A. The signaling pathway involving the phosphorylation of focal adhesion kinase and mitogen-activated kinases was also shown to be transiently increased in osteoblasts on fibronectin and type I collagen, but not in cells on poly-L-lysine. These results demonstrate that osteoblast binding to the extracellular matrix through integrins induces c-fos and c-jun, and that both fibronectin and collagen affect these AP-1 transcription factors through protein kinase-sensitive pathways. Thus, osteoblast proliferation is modulated differentially by specific ECM components.
Asunto(s)
Matriz Extracelular/metabolismo , Integrinas/fisiología , Osteoblastos/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/fisiología , Animales , Anticuerpos/farmacología , Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular , División Celular , Células Cultivadas , Materiales Biocompatibles Revestidos , Colágeno/metabolismo , Medio de Cultivo Libre de Suero , Inhibidores Enzimáticos/farmacología , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Regulación de la Expresión Génica , Genes fos , Genes jun , Integrinas/inmunología , Sistema de Señalización de MAP Quinasas , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oligopéptidos/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteonectina/metabolismo , Fosforilación/efectos de los fármacos , Plásticos , Polilisina/metabolismo , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Tirosina Quinasas/fisiología , ARN Mensajero/biosíntesis , Ratas , Trombospondinas/metabolismo , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción AP-1/genéticaRESUMEN
The intracellular signaling pathway for osteoblast adhesion to the orthopedic implant material Ti6Al4V (TIV) was investigated and compared to integrin-mediated adhesion to extracellular matrix proteins. Primary osteoblasts from fetal rat calvaria were plated onto TIV, fibronectin (FN), and poly-L-lysine (PLL) and the levels of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), and AP-1 transcription factors, c-fos and c-jun, were compared by Western and Northern blots. Cells on all substrates showed maximum FAK phosphorylation within 60 min and then a decrease at 2 and 24 h. However, the subsequent signal transduction pathway differed on PLL compared to TIV and FN. MAPK was phosphorylated similarly in osteoblasts attached to FN and TIV, whereas cells on PLL demonstrated no MAPK phosphorylation. On TIV and FN, c-fos and c-jun mRNA levels were maximal within 1 h and then plateaued or declined by 2 h. On PLL, they increased at 2 h. Within 1 h, c-fos protein was stimulated in cells attached to TIV and FN and decreased in cells on PLL. c-jun protein increased on all substrates compared to unplated cells. Cytoskeletal changes visualized by phalloidin fluorescence microscopy at 4 h of culture were delayed on TIV compared to FN. In addition, approximately 50% fewer cells adhered to TIV compared to FN or PLL. By 24 h, a well-spread cytoskeleton with focal adhesion sites was apparent on TIV and FN, but cells on PLL were rounded with minimal cell spreading. During 6 days of culture, cells on FN and TIV proliferated, whereas the number of cells on PLL remained the same or decreased, depending on the initial plating density. We conclude that osteoblast adhesion to TIV implants is similar to osteoblast adhesion to FN and leads to osteoblast proliferation. These data provide evidence for the biocompatibility of TIV at a molecular level.
Asunto(s)
Materiales Biocompatibles/química , Integrinas/fisiología , Prótesis e Implantes , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal , Titanio/química , Aleaciones , Animales , Adhesión Celular , División Celular , Células Cultivadas , Materiales Biocompatibles Revestidos , Citoesqueleto/ultraestructura , Fibronectinas , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Genes fos , Genes jun , Uniones Intercelulares/ultraestructura , Sistema de Señalización de MAP Quinasas , Ensayo de Materiales , Microscopía Fluorescente , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Fosforilación , Polilisina , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , Ratas , Factores de Tiempo , Factor de Transcripción AP-1/biosíntesisRESUMEN
Organosilicone molecules are important surfactant ingredients used in formulating pesticides. These methylated silicones are considered inert ingredients, but their superior surfactant properties allow them to wet, and either suffocate or disrupt important physiological processes in mites and insects. Aqueous solutions of the tri-siloxane surfactants Silwet L-77, Silwet 408, and Silwet 806 were bioassayed against adult female two-spotted spider mites, Tetranychus urticae Koch, with leaf dip methods to compare their toxicity with organosilicone molecules containing bulkier hydrophobic components. All three tri-siloxanes in aqueous solutions were equivalently toxic (LC50 = 5.5-8.9 ppm), whereas Silwet L-7607 solutions were less toxic (LC50 = 4,800 ppm) and Silwet L-7200 was nontoxic to mites. In another experiment, the toxicity of Silwet L-77 was affected by the wettability of leaf surfaces. The LC50 shifted from 22 to 84 ppm when mites were tested on bean and strawberry leaf disks, respectively. Droplet spreading on paraffin and surface tension were both related to the toxicity of surfactant solutions. Surface tensions of solutions below 23 mN/m caused > 90% mite mortality in leaf dip bioassays. A field test of Conserve SC and its formulation blank, with and without Dyne-Amic adjuvant (a vegetable oil-organosilicone surfactant mixture) revealed that Dyne-Amic had the greatest miticidal contribution, reducing mite populations by 70%, followed by formulation inactive ingredients. Spinosad, the listed active ingredient in Conserve, only contributed miticidal activity when synergized by Dyne-Amic. Researchers should include appropriate surfactant or formulation blank controls when testing insecticides or miticides, especially when using high spray volumes.
Asunto(s)
Ácaros , Compuestos de Organosilicio , Control de Plagas , Tensoactivos , Animales , Femenino , Control de Plagas/métodos , Hojas de la Planta , SolucionesRESUMEN
OBJECTIVE: Insulin-like growth factor-1 (IGF-l) levels are lower in older compared with younger subjects. We tested the hypothesis that the reduction in circulating IGF-l would be accompanied by upregulation in tissue IGF-l binding in at least some tissues. We tested erythrocyte IGF-l binding since blood is an accessible tissue in humans, and there is growing evidence to suggest that erythrocyte IGF-l binding is influenced by circulating IGF-l. DESIGN AND PATIENTS: We compared 9 healthy older males (61-68 years old) with 9 healthy younger males (15-19 years old). MEASUREMENTS: Standard techniques were used to assay circulating IGF-l and IGF binding proteins 1-5 (IGFBPs 1-5). Erythrocyte IGF-l binding was first measured by studies in which native [125l]-IGF-l was displaced with unlabelled native IGF-l. In order to determine a possible role for IGF binding proteins (IGFBP), native [125l]-IGF-l was displaced with des-(1-3)IGF-1, which binds with IGF receptors but not IGFBPs. RESULTS: As expected, circulating IGF-l was significantly lower in the older compared with the younger subjects. In addition, IGFBP-3 and 5 were significantly lower, and IGFBP-4 higher, in older compared with younger subjects. When native [125l]-IGF-l was displaced with unlabelled native IGF-l, the number of IGF-l binding sites per erythrocyte was higher in the older subjects (43 +/- 5 vs. 18 +/- 2, older vs. younger, respectively; P < 0.05). In contrast, when native [125l]-IGF-l was displaced with des-(1-3), IGF-l binding capacity was not different between the two age groups. CONCLUSIONS: Erythrocyte IGF binding was increased in older compared with younger subjects. Surprisingly, the mechanism of the increase may not be a simple up regulation of IGF-l receptors in response to reduced circulating IGF-l, but possibly by an increase in the levels of as yet unidentified erythrocyte membrane-associated IGF binding proteins.
Asunto(s)
Envejecimiento/metabolismo , Eritrocitos/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Adolescente , Adulto , Anciano , Sitios de Unión , Fibroblastos/metabolismo , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/análisis , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/metabolismo , Unión Proteica , Regulación hacia ArribaRESUMEN
The in vivo expression of fibronectin, type I collagen, and several non-collagenous proteins was correlated with the development of bone in fetal and early neonatal rat calvariae. Fibronectin was the earliest matrix protein expressed in calvariae, with a peak expression in fetal 16- and 17-day (d) bones. Fibronectin expression coincided with the condensation of preosteoblasts prior to calcification and decreased once bone mineralization commenced. The expression of type I collagen, osteonectin, bone sialoprotein, and alkaline phosphatase mRNAs was found at 17 d. The increase in type I collagen mRNA levels was correlated with a 3.5-fold increase in calcium deposition at 19-20 d. Bone sialoprotein and alkaline phosphatase peaked on fetal 21 d while osteonectin remained at a low level and was localized to the osteoblast layer and the osteocyte lacunae. Osteopontin mRNA levels increased rapidly in neonatal calvariae. After birth, osteonectin and fibronectin were mainly associated with blood vessels. Thus, fibronectin is one of the earliest matrix proteins expressed in calvariae and is rapidly followed by type I collagen, bone sialoprotein, and alkaline phosphatase. Osteocalcin, osteonectin, and osteopontin mRNAs have similar patterns of expression in the developing fetal calvaria, and their synthesis coincided with mineralization.
Asunto(s)
Desarrollo Óseo/genética , Desarrollo Óseo/fisiología , Calcificación Fisiológica/fisiología , Proteínas de la Matriz Extracelular/genética , Animales , Animales Recién Nacidos , Densidad Ósea/fisiología , Calcio/metabolismo , Feto/anatomía & histología , Feto/fisiología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Edad Gestacional , Hibridación in Situ , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Cráneo/anatomía & histología , Cráneo/fisiología , Factores de TiempoRESUMEN
Bacillus thuringiensis toxins are insecticidal to a variety of insect species. The selectivity of the toxins produced by these bacteria is dependent on both the toxin structure and the receptor sites that are present in different insect species. One of these toxins, CryIAc, is highly insecticidal to the noctuid pest Heliothis virescens. Using toxin overlay assay, a 120-kDa glycoprotein was identified as a toxin-binding protein. This protein was partially purified, its N-terminal sequence was determined, and the full-length cDNA encoding this protein was isolated from a H. virescens midgut library. The B. thuringiensis toxin-binding protein, BTBP1, has high homology to aminopeptidase N from eukaryotes and prokaryotes.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas , Endotoxinas/genética , Endotoxinas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Secuencia de Bases , Antígenos CD13/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Complementario/genética , Proteínas Hemolisinas , Humanos , Datos de Secuencia Molecular , Mariposas Nocturnas , Homología de Secuencia de AminoácidoRESUMEN
The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut.
Asunto(s)
Bacillus thuringiensis/patogenicidad , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Portadoras/aislamiento & purificación , Endotoxinas/metabolismo , Intestinos/química , Lepidópteros/química , Animales , Toxinas de Bacillus thuringiensis , Proteínas Portadoras/metabolismo , Cromatografía por Intercambio Iónico , Proteínas Hemolisinas , Microvellosidades/químicaRESUMEN
Cotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp C. congregata. CcPDV virions are present in large numbers in the oviducts of C. congregata and injected with eggs into the hemocoel of M. sexta larvae during parasitization. Injection of sucrose density purified virions into nonparasitized larvae causes several of the parasitism-induced alterations in the physiology of host larvae that occur as a result of natural parasitism, including the synthesis of novel hemolymph proteins and abrogation of the host's immune response against the developing parasites. One of these proteins, early-expressed protein 1 (EP1), is a 190-kDa molecule which constitutes up to 5% of the total hemolymph protein by 24 hr following oviposition by the wasp. Using N-terminal sequence data for EP1 to construct primers for use in the polymerase chain reaction, we amplified and cloned a cDNA corresponding to the gene encoding EP1. This cDNA hybridized to DNA of the CcPDV genome, but not to DNA isolated from M. sexta larvae, suggesting that EP1 is a CcPDV gene product. A cDNA clone was isolated from an expression library generated from RNA extracted from newly parasitized M. sexta larvae. Sequence analysis of the cDNA clone revealed the presence of an open reading frame of 819 bp encoding a protein of 30.7 kDa. In vitro transcription/translation of the cDNA clone produced a protein of approximately 31 kDa, which was immunoprecipitated by EP1-specific polyclonal antiserum generated against purified deglycosylated EP1. EP1-like sequences also were amplified from male wasp genomic DNA, suggestive of integration of EP1-like sequences in the genome. This report constitutes the first evidence that a specific protein isolated from a parasitized host insect is a wasp polydnavirus gene product.
Asunto(s)
Glicoproteínas/genética , Hemolinfa/química , Proteínas Inmediatas-Precoces/genética , Manduca/parasitología , Polydnaviridae/genética , Proteínas Virales , Avispas/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Glicoproteínas/biosíntesis , Glicoproteínas/aislamiento & purificación , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/aislamiento & purificación , Larva/parasitología , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Biosíntesis de Proteínas , Factores de Tiempo , Transcripción GenéticaRESUMEN
A series of rat caries experiments was carried out to test the relative cariogenic potential and to identify the major carcinogenic elements of 22 popular snack foods. Parameters that were measured included rat caries, number of cariogenic bacteria in plaque, salivary parameters including flow rate, buffering capacity, total protein, lysozyme and amylase content, and composition of test foods including protein, fat, phosphorus, calcium, fluoride, galactose, glucose, total reducing sugar, sucrose, and starch. Many interesting relationships were observed between food components, numbers of plaque bacteria, salivary components, and specific types of carious lesions. Protein, fat, and phosphorus in foods were all associated with inhibition of both sulcal and buccolingual (smooth-surface) caries. Food fluoride was associated with inhibition of buccolingual caries, whereas calcium was related to inhibition of sulcal caries. Glucose, reducing sugar, and sucrose in foods were all related to promotion of both sulcal and smooth-surface caries. The numbers of Streptococcus sobrinus in plaque were associated with promotion of smooth-surface caries only, whereas lactobacilli, non-mutans bacteria, and total viable flora were related to promotion of both smooth-surface and sulcal caries. The salivary flow rate was associated with inhibition of both buccolingual and sulcal caries. Salivary buffering capacity (at pH 7) and salivary lysozyme delivery were associated with inhibition of number and severity of sulcal caries, while the salivary amylase content was related to the promotion of the number of sulcal lesions.
Asunto(s)
Bacterias/aislamiento & purificación , Cariogénicos , Caries Dental/etiología , Caries Dental/microbiología , Placa Dental/microbiología , Análisis de los Alimentos , Alimentos , Saliva/fisiología , Amilasas/análisis , Animales , Fenómenos Fisiológicos Bacterianos , Tampones (Química) , Calcio/análisis , Recuento de Colonia Microbiana , Carbohidratos de la Dieta/análisis , Grasas de la Dieta/análisis , Proteínas en la Dieta/análisis , Lactobacillus/aislamiento & purificación , Lactobacillus/fisiología , Muramidasa/análisis , Fósforo/análisis , Ratas , Ratas Sprague-Dawley , Saliva/enzimología , Proteínas y Péptidos Salivales/análisis , Tasa de Secreción , Streptococcus sobrinus/aislamiento & purificación , Streptococcus sobrinus/fisiologíaAsunto(s)
Bacillus thuringiensis , Proteínas Bacterianas , Toxinas Bacterianas , Endotoxinas , Insecticidas , Control Biológico de Vectores , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas , Insecticidas/química , Insecticidas/toxicidadRESUMEN
We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to approximately 18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (pI8.7) protein, as well as the phosphorylated (pI8.2) derivative. In contrast, older (passage 27-35, 55-68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (pI 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.
Asunto(s)
Antígenos de Diferenciación/genética , Proteínas Portadoras/genética , Fibroblastos/metabolismo , Hemaglutininas/genética , ARN Mensajero/metabolismo , Proteínas Sanguíneas/farmacología , Northern Blotting , Células Cultivadas , Replicación del ADN , Electroforesis en Gel Bidimensional , Fibroblastos/citología , Técnica del Anticuerpo Fluorescente , Galectina 3 , Expresión Génica/efectos de los fármacos , Humanos , Focalización Isoeléctrica , ARN Mensajero/genéticaRESUMEN
Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7. When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2. The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group. This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment. The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei. Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35. Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells.
Asunto(s)
Antígenos de Diferenciación/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Hemaglutininas/aislamiento & purificación , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel Bidimensional , Escherichia coli/genética , Galectina 3 , Hemaglutininas/genética , Hemaglutininas/metabolismo , Focalización Isoeléctrica , Cinética , Ratones , Datos de Secuencia Molecular , Fosforilación , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Carbohydrate Binding Protein 35 (CBP35) is a galactose-specific lectin found in the nucleus and cytoplasm of mouse 3T3 fibroblasts. In these cultures, the level of expression and nuclear localization of CBP35 was correlated with the proliferative state of the cells. CBP35 is also found in human fibroblasts. We have compared the expression and localization of CBP35 in human fibroblasts of different replicative capacities: young (passage 11), intermediate (passage 19), and old (passage 33) SL66 cells, and fibroblasts derived from a patient with Werner's syndrome. The results indicate that the expression of CBP35 in cells with either age-acquired or congenital replicative deficiencies was unresponsive to serum stimulation, in contrast with that found in young normal human fibroblasts and in 3T3 cells.
Asunto(s)
División Celular/fisiología , Fibroblastos/fisiología , Expresión Génica/fisiología , Hemaglutininas/genética , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Galectinas , Humanos , Immunoblotting , Técnicas In VitroRESUMEN
Fibroblasts derived from patients with Werner's syndrome (WS) were incubated with radioactive sulfate to study the incorporation of 35S into glycosaminoglycans (GAGs). The accumulation of cell-associated 35S radioactivity in the GAGs of WS fibroblasts was consistently higher than parallel accumulation in normal human fibroblasts, but was substantially less than in fibroblasts derived from patients with Hurler's syndrome (HS). However, when fibroblasts were labeled with 35SO4(2-), trypsinized to remove extracellular and pericellular radioactive GAGs, replated, and chased to follow the fate of the intracellular radioactivity, both WS and normal cells showed a rapid release of the intracellular 35S, while HS cells showed little or no loss of intracellular radioactivity. The radioactivity released from WS and normal cells was of low molecular weight (LMW), eluting from gel filtration columns at the same position as free sulfate. These results establish that WS cells degrade intracellular sulfated GAGs and argue against the hypothesis that a defect in GAG degradation pathways is the basis for the increased level of cell-associated GAGs. Other possible explanations for the increased cell-associated [35S]GAGs in WS cells as compared with normal cells were also considered: increased GAG sulfation; an increase in GAG chain length; an increased rate of GAG synthesis; and a decreased rate of shedding of cell surface proteoglycan into the medium. No difference between normal and WS fibroblasts in any of the above parameters was observed. These results strongly imply that the primary biochemical defect in WS fibroblasts does not involve sulfated GAG metabolism.
Asunto(s)
Glicosaminoglicanos/metabolismo , Síndrome de Werner/metabolismo , Línea Celular , Fibroblastos/metabolismo , Glicosaminoglicanos/aislamiento & purificación , Humanos , Cinética , Mucopolisacaridosis I/metabolismo , Valores de Referencia , Piel/metabolismo , Sulfatos/metabolismo , Radioisótopos de AzufreRESUMEN
Chicken muscle cell cultures were incubated at 41 degrees C, the physiological chicken body temperature, and compared with cultures incubated at 37 degrees C, the typical cell culture incubation temperature. The cultures incubated at 41 degrees C show not only an increase in creatine kinase (CK)-specific activity but also a marked increase in the percentage of adult muscle CK isozyme (MM-CK) in 7-day muscle cultures. Muscle cell cultures incubated in the presence of cytosine arabinoside (ara-C), a cell proliferation inhibitor, do not have the mononucleated cell overgrowth seen at 41 degrees C and thus exhibit a further increase in creatine kinase-specific activity compared with cultures incubated at 41 degrees C in the absence of ara-C. These results suggest that muscle cell cultures incubated at 41 degrees C are more highly differentiated than those incubated at 37 degrees C.