RESUMEN
Since there is an ethical need to minimise the experimental use of higher organisms such as fish, especially those used in acute toxicity testing, fish cells are considered to be useful surrogates for fish in toxicity screening. The use of fish cell lines in conventional bioassays such as neutral red retention assays is however labour intensive, lengthy and costly. The use of luminescent reporter genes has been explored in our laboratory. In this study, a transfected BF-2 cell line (BF-2/luc1) was used for rapid toxicity testing on selected chemicals and results were compared with those obtained with in vivo fish testing and in vitro fish cell neutral red retention assays. The effect of temperature on the sensitivity of BF-2/luc1 was also investigated. BF-2/luc1 cells were harvested and suspended in PBS at 2.5-3.0x10(6)cells/ml. Individual aliquots of the suspended cells (40 microl each) were incubated for either 0.5 or 6 h at room temperature (22 degrees C) in the presence or absence of the toxicants. Bioluminescence was assayed using 17.5 microl Brightglo luciferase reagent which lysed the cells and provided the substrate luciferin. Luminescence was measured in a luminometer (Turner TD 20/20). The EC50 values obtained from BF-2/luc1 cells (0.5-6 h) generally compared well with the LC50 values (24-96 h) obtained from the in vivo fish tests on a range of species. The present study also showed that BF-2/luc1 cell sensitivity increased significantly when incubation temperature during toxicant exposure increased from 15 to 35 degrees C. The use of luminescent reporter genes in monitoring fish cells offers the possible advantages of increased sensitivity over the neutral red retention assay and a more rapid test to replace stain based bioassays, and provides a rapid screening method that could reduce the need for acute fish toxicity testing.
Asunto(s)
Peces/fisiología , Genes Reporteros/genética , Luciferasas/genética , Pruebas de Toxicidad/métodos , Contaminantes Químicos del Agua/toxicidad , Animales , Línea Celular , Colorantes , Dosificación Letal Mediana , Mediciones Luminiscentes , Rojo Neutro , Perciformes , Plaguicidas/toxicidad , Temperatura , TransfecciónRESUMEN
BACKGROUND: Alterations in the insulin-like growth factor (IGF) system have been reported for different tumours. They are of particular interest in the search for new prognostic and therapeutic approaches in cancer. In childhood acute lymphoblastic leukaemia (ALL) the amount of "tumour mass" at diagnosis can exceed 1 kg. To understand the endocrine, paracrine, and autocrine potential of the malignant transformed progenitor cells, the ability of these cells to express components of the IGF system needs to be investigated. AIM: To characterise the expression pattern of genes of the IGF system in malignant lymphoblasts of children suffering from ALL. METHODS: Reverse transcription polymerase chain reaction of Ficoll separated mononuclear cells from 142 children with ALL, 127 cord blood samples, and 55 blood samples of age matched controls were studied. RESULTS: The expression of IGF-I, IGF-II, IGF binding protein 5 (IGFBP-5), and CTGF (IGFBP-rP2) was seen in a higher proportion of mononuclear cells of patients with ALL than in controls. Patients with ALL who were in continuous remission had a lower percentage of IGFBP-2 and IGFBP-3 expressing mononuclear cells at diagnosis than did those who developed a relapse. Only malignant lymphoblasts of B cell origin showed expression of CTGF (IGFBP-rP2). Malignant lymphoblasts of T cell origin more often expressed IGFBP-2 and IGFBP-5, whereas IGF-II and IGFBP-3 expression was seen more often in lymphoblasts of B cell origin. CONCLUSIONS: Malignant lymphoblasts of patients with ALL express components of the IGF system and therefore promote their own growth in an autocrine, paracrine, or endocrine manner. Whether these components will be useful as prognostic factors in the stratification of ALL treatment in children needs to be evaluated.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Somatomedinas/metabolismo , Biomarcadores de Tumor/genética , Niño , Expresión Génica , Humanos , Recién Nacido , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Modelos Logísticos , Proteínas de Neoplasias/genética , Estudios Prospectivos , Transducción de Señal , Somatomedinas/genéticaRESUMEN
Human cathepsin W (also called lymphopain) is a recently described papain-like cysteine protease of unknown function whose gene expression was found to be restricted to cytotoxic cells. Here we demonstrate that cathepsin W is expressed predominantly in NK cells and, to a lesser extent, in CTLs. Quantitative RT-PCR revealed that NK cells contained approximately 21 times more cathepsin W transcript than CTLs. The predominant expression of cathepsin W in NK cells was further confirmed by Western blot analysis and immunohistochemistry. IL-2-mediated stimulation of NK cells and CTLs revealed a stronger up-regulation of the cathepsin W gene and protein expression in NK cells (7-fold) than in CTLs (2-fold). Transfection experiments of HeLa cells and biochemical analyses revealed that cathepsin W is exclusively "high mannose-type" glycosylated and is mainly targeted to the endoplasmic reticulum (ER). Interestingly, the ER localization of cathepsin W was also found in NK cells, in which colocalization studies revealed an overlapping staining of cathepsin W and Con A, an ER-specific lectin. Furthermore, subcellular fractionation of cathepsin W-expressing cells confirmed the ER localization and showed that cathepsin W is membrane associated. Based on the results of this study, cathepsin W might represent a putative component of the ER-resident proteolytic machinery. The constitutive expression in NK cells and the stronger up-regulation of cathepsin W by IL-2 in NK cells than CTLs suggest that cathepsin W is not just a marker of cytotoxic cells but is, rather, specifically expressed in NK cells.
Asunto(s)
Catepsinas/biosíntesis , Cisteína Endopeptidasas/biosíntesis , Retículo Endoplásmico/enzimología , Células Asesinas Naturales/enzimología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Western Blotting , Catepsina W , Catepsinas/genética , Catepsinas/inmunología , Catepsinas/metabolismo , Células Clonales , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Inducción Enzimática/inmunología , Células HeLa , Humanos , Sueros Inmunes/biosíntesis , Sueros Inmunes/química , Interleucina-2/farmacología , Células Asesinas Naturales/metabolismo , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/metabolismo , TransfecciónAsunto(s)
Encéfalo/fisiología , Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/fisiología , Animales , Encéfalo/citología , Proteínas Portadoras/análisis , Proteínas del Citoesqueleto/metabolismo , Drosophila , Proteínas del Tejido Nervioso/análisis , Sinapsis/ultraestructura , Membranas Sinápticas/fisiología , Membranas Sinápticas/ultraestructuraRESUMEN
A human cDNA of 2166 bp encoding a novel cathepsin B-related protein was isolated and characterized. The amino acid sequence of the predicted protein of 467 aa was 46% identical with that of human tubulointerstitial nephritis antigen (TIN-ag), and therefore, the protein was tentatively designated as the TIN-ag-related protein (TIN-ag-RP). The amino acid sequence of TIN-ag-RP is composed of a 21 aa long signal sequence, a 181 aa long N-terminal domain containing two epidermal growth factor-like domains, a follistatin motif, and a 265 aa long cathepsin B-like domain. Interestingly, a serine residue has replaced the active site cysteine residue in the cathepsin B-like domain, resulting in a proteolytically inactive protein. Evolutionary analysis revealed that a distinct family of "TIN-ag-like" proteins had evolved in vertebrates. Northern blot analysis revealed a single TIN-ag-RP transcript of 2.4 kb in various tissues with the highest transcript levels detected in aorta, heart, placenta, skeletal muscle, kidney, and a colorectal adenocarcinoma cell line. Using a polyclonal anti-TIN-ag-RP antibody, TIN-ag-RP expression was predominantly seen in vascular smooth muscle (VSM) cells, but also in cardiac and skeletal muscle cells as well as in kidney. Interestingly, uterine smooth muscle cells completely lacked TIN-ag-RP expression, implying a regulated gene expression. Localization studies in HeLa cells stably transfected with TIN-ag-RP cDNA showed that TIN-ag-RP is glycosylated and actively secreted, a finding in line with its proposed function as a structural or regulatory protein similar to TIN-ag.
Asunto(s)
Catepsina B/química , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Factor de Crecimiento Epidérmico/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Nefritis Intersticial/metabolismo , Proteínas de Unión a Telómeros , Adulto , Secuencia de Aminoácidos , Animales , Antígenos de Superficie , Secuencia de Bases , Catálisis , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Línea Celular , ADN Complementario/química , Activación Enzimática , Proteínas de la Matriz Extracelular , Células HeLa , Humanos , Lipocalinas , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Especificidad de Órganos/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales CultivadasRESUMEN
Connective tissue growth factor (CTGF) is a major chemotactic and mitogenic factor for connective tissue cells. The amino acid sequence shares an overall 28-38% identity to IGFBPs and contains critical conserved sequences in the amino terminus. It has been demonstrated that human CTGF specifically binds IGFs with low affinity and is considered to be a member of the IGFBP superfamily (IGFBP-rP2). In the present study, the expression of CTGF (IGFBP-rP2) in human leukaemic lymphoblasts from children with acute lymphoblastic leukaemia (ALL) was investigated. RNA samples from tumour clones enriched by ficoll separation of bone marrow or peripheral blood mononuclear cells (MNC) from 107 patients with childhood ALL at diagnosis and 57 adult patients with chronic myeloid leukaemia (CML) were studied by RT-PCR. In addition MNC samples from children with IDDM and cord blood samples from healthy newborns were investigated as control groups. Sixty-one percent of the patients with ALL (65 of 107) were positive for CTGF (IGFBP-rP2) expression. In the control groups, no expression of CTGF (IGFBP-rP2) in peripheral MNC was detected, and in the group of adult CML patients only 3.5% (2 of 57) were positive for this gene. The role of CTGF (IGFBP-rP2) in lymphoblastic leukaemogenesis requires further evaluation, as does its potential utility as a tumour marker.
Asunto(s)
Biomarcadores de Tumor/análisis , Regulación Neoplásica de la Expresión Génica , Sustancias de Crecimiento/genética , Proteínas Inmediatas-Precoces/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Péptidos y Proteínas de Señalización Intercelular , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Factor de Crecimiento del Tejido Conjuntivo , Femenino , Sustancias de Crecimiento/análisis , Humanos , Proteínas Inmediatas-Precoces/análisis , Lactante , Recién Nacido , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Linfocitos/fisiología , Masculino , Persona de Mediana Edad , ARN Mensajero/análisisAsunto(s)
Catepsina B/clasificación , Catepsinas/química , Catepsinas/clasificación , Cisteína Endopeptidasas/clasificación , Endopeptidasas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Catepsina F , Catepsina L , Catepsina W , Catepsinas/genética , Mapeo Cromosómico , Cromosomas Humanos/genética , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Evolución Molecular , Biblioteca de Genes , Genes , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
The human gene encoding a novel tubulointerstitial nephritis antigen (TIN-ag)-related protein (TIN-ag-RP) was isolated, and its genomic organization was determined. BLAST searches revealed the highest degree of homology to several mammalian TIN-ag orthologues, and a weak homology to cathepsin B-like proteases. The 12 kb gene was mapped by fluorescence in situ hybridization to chromosome 1p34.2-3, a locus neither related to that of the human TIN-ag (6p11.2-12) nor to that of cathepsin B (8p22-23.1). The TIN-ag-RP is encoded in ten exons with introns ranging from 83 bp to 4 kb. In addition, the gene contained one exon in the 5'UTR, but none in the 3'UTR. Five of the 10 splice sites of the TIN-ag-RP gene were fully conserved when compared to a related gene of C. elegans, whereas only one splice site was identical to those found in cathepsin B genes. Furthermore, human TIN-ag-RP tagged with the T7-epitope, was expressed in HeLa cells, and was found to be localized in vesicular compartments as well as secreted into the medium suggesting the involvement of the endosomal trafficking pathway. Based on the high degree of homology of the amino acid sequences and genomic organization between TIN-ag-RP and TIN-ag, we suggest that both molecules may form a distinct group or family of TIN-ag-like proteins.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Matriz Extracelular/química , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de Unión a Telómeros , Secuencia de Aminoácidos , Antígenos de Superficie , Western Blotting , Catepsina B/química , Cromosomas Humanos Par 1 , Clonación Molecular , Exones , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.
Asunto(s)
Resorción Ósea/prevención & control , Catepsinas/metabolismo , Osteoclastos/enzimología , Sulfonas/farmacología , Animales , Secuencia de Bases , Catepsina K , Catepsinas/antagonistas & inhibidores , Compartimento Celular , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Cartilla de ADN , Inmunohistoquímica , Ratones , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/metabolismo , Envejecimiento/metabolismo , Secuencia de Aminoácidos/genética , Animales , Animales Recién Nacidos/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/metabolismo , Ratas , Distribución Tisular/fisiologíaRESUMEN
Human cathepsin F is a recently described papain-like cysteine protease of unknown function. To investigate the evolutionary relatedness to other human cathepsins, we determined the genomic organization and the chromosomal localization of cathepsin F and isolated its putative promoter region. The gene of human cathepsin F (CTSF) is composed of twelve exons and eleven introns and was found to be similar to that of cathepsin W but different from the cathepsins K, S, L, O, B, and C. The splice sites of nine out of the eleven introns were identical to those determined in the cathepsin W gene (CTSW), whereas introns one and ten were unique for CTSF. The 4. 7 kb gene was mapped to the long arm of chromosome 11 at position q13.1-3, a locus shared with CTSW. Phylogenetic analysis of human cathepsin protein sequences demonstrated that (i) cathepsins F and W are evolutionarily separated from other human cathepsins, and (ii) cysteine proteases closely related to human cathepsin W and F are also expressed in parasites and mammals. Based on these phylogenetic findings, on the presence of a particular protein motif ("ERFNAQ") in the propeptides of cathepsins F and W as well as the genomic organization and chromosomal localization of their genes, we concluded that F and W form a novel subgroup of cathepsin proteases. We suggest the naming "cathepsin F-like" proteases distinct from the previously described cathepsins "L- and B-like" subgroups.
Asunto(s)
Catepsinas/genética , Cisteína Endopeptidasas/genética , Catepsina F , Catepsina W , Catepsinas/clasificación , Mapeo Cromosómico , Cromosomas Humanos Par 11/genética , Clonación Molecular , Cartilla de ADN , Exones , Humanos , Intrones , FilogeniaRESUMEN
Human cathepsin F is a novel papain-like cysteine protease of unknown function. Here, we describe the complete human cathepsin F (CTSF) gene which is composed of 13 exons. In addition to a previous report, two novel upstream located exons whose splice sites interrupted the propeptide of cathepsin F within the 'cystatin-like' domain, recently described by Nagler et al. (Biochem. Biophys. Res. Comm. 257, 313-318, 1999) were identified. A comparison of the genomic structures between this novel part of the cathepsin F gene and those of several cystatin genes revealed striking similarities, supporting the hypothesis that the cathepsin F gene resulted from a gene fusion between an ancestral cystatin and cathepsin gene.
Asunto(s)
Catepsinas/genética , Cistatinas/genética , Secuencia de Aminoácidos , Catepsina F , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Insulin-like growth factor-binding proteins (IGFBPs) are important modulators of IGF action. In 50 children suffering from acute lymphoblastic leukaemia (ALL), we studied the serum levels of IGFBP-1,-2 and-3. The mean standard deviation score (SDS) values were estimated to be 0.7, 3.1 and -1.7 for the IGFBP-1,-2 and-3, respectively, compared with the normal range defined by a SDS from -2 to +2. IGFBP-1 and-3 were normal, but for IGFBP-2 we found a significantly elevated serum level compared with control groups (P < 0.05). However, during chemotherapy this increased serum IGFBP-2 normalized. In addition, we found a correlation between higher serum levels and the detection rate of the IGFBP-2 transcript in corresponding cells. In patients with ALL, the detection rates of IGFBP-2 mRNA were estimated to be 72% and 35% at the time of diagnosis and at day 33 of chemotherapy respectively; in the control groups (healthy children and children at their initial presentation of diabetes mellitus), the values were 28% and 33% respectively. Based on the correlation between IGFBP-2 serum levels and the corresponding gene expression as well as the normalization of IGFBP-2 levels during chemotherapy, we concluded that the increased serum level mainly originated from the tumour clone itself. Furthermore, possible functional consequences of elevated IGFBP-2 were outlined.
Asunto(s)
Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Adolescente , Niño , Preescolar , Expresión Génica , Humanos , Lactante , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , ARN Mensajero/análisisRESUMEN
The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Terminales Presinápticos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , Exones , Hipocampo/metabolismo , Hipocampo/ultraestructura , Humanos , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Péptidos/química , Terminales Presinápticos/ultraestructura , Ratas , Homología de Secuencia de Aminoácido , Repeticiones de Trinucleótidos , Dedos de Zinc/genéticaRESUMEN
Antisera against a rat brain synaptic protein preparation, the postsynaptic density (PSD) fraction, were used to isolate cDNA clones by expression screening of a rat brain cDNA library. About one fifth of more than 200 analyzed cDNAs encoding potential synapse-associated proteins were previously unknown. Identifiable proteins include, among others, components of the pre- and postsynaptic cytoskeleton, synaptic vesicle proteins and several protein kinases and kinase substrates. This demonstrates that both pre- and postsynaptic elements purify with the PSD fraction.
Asunto(s)
Química Encefálica/fisiología , Conexinas/análisis , Proteínas del Tejido Nervioso/análisis , Sinapsis/química , Animales , Clonación Molecular , Proteínas del Citoesqueleto/análisis , ADN Complementario/aislamiento & purificación , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Membrana/análisis , Datos de Secuencia Molecular , Proteínas Quinasas/análisis , RatasRESUMEN
Studies on the identification and characterization of constituents of rat brain synaptic junctions have lead to the isolation of cDNA clones encoding segments of alpha-adducin. These and other studies suggest that adducin, a protein involved in promoting the assembly of actin and spectrin filaments at the plasma membrane, may play a role in dynamic assembly-disassembly processes underlying synaptic plasticity. In order to verify that brain alpha-adducin is indeed a constituent of synaptic structures, we have generated monoclonal antibodies against epitopes in the C-terminal region of alpha-adducin and have determined its spatial and sub-cellular distribution in postnatal day-30 rat brain. Alpha-adducin is found to be highly enriched in regions with high synapse densities of the hippocampus, corpus striatum, cerebral cortex and cerebellum. Immuno-electron microscopic analysis of peroxidase stained sections of the hippocampus and the cerebellum revealed that alpha-adducin is localized at distinct sub-cellular structures. In the CA1 and CA3 regions of the hippocampus alpha-adducin immunoreactivity is found in a distinct subset of dendrites and dendritic spines. In the molecular layer of the cerebellum, a distinct fraction of pre-synaptic terminals of parallel fiber terminals is labeled. In both cases the majority of synaptic structures does not contain adducin. Significant immunoreactivity is also detected in processes of glial cells both in the hippocampus and the cerebellum.
Asunto(s)
Química Encefálica/fisiología , Proteínas de Unión a Calmodulina/análisis , Proteínas del Citoesqueleto/análisis , Proteínas de la Membrana/análisis , Proteínas del Tejido Nervioso/análisis , Fracciones Subcelulares/química , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Cerebelo/química , Hipocampo/química , Inmunohistoquímica , Datos de Secuencia Molecular , Ratas , Ratas WistarRESUMEN
The aminopeptidase N (CD13, EC 3.4.11.2) is a well-characterized surface molecule expressed in a variety of cell types and species. Recent data indicate an expression of the APN mRNA and the corresponding aminopeptidase activity in human peripheral T cells and related cell lines as well. Here, the sensitive method of competitive PCR was used to quantify low amounts of APN mRNA in T cell lines. An APN cDNA fragment enshortened by a deletion of 87 bp was used as an internal APN-specific standard. The myelo-monocytic cell line U937 and the lymphoid T cell lines HuT78 and H9 contain 2.3 x 10(7), 5.9 x 10(6) and 5.6 x 10(6) copies/micrograms total RNA, corresponding to 160, 70 and 50 copies/cell, respectively. These data have been confirmed by determination of the APN activity, that represents a fraction only of the total cellular neutral aminopeptidase activity in hematopoetic cells. In the case of the CD13-positive cell line U937, approximately 60-70% of the total neutral aminopeptidase activity could be attributed to APN. In contrast, only a minor fraction (5-20%) of the cellular neutral aminopeptidase activity in the T cell lines H9 and HuT78 represents APN. The results suggest that APN gene expression within the hematopoetic system is not restricted to myelo-monocytic cells, instead a low APN expression may be a common feature of lymphocytes, at least of T cells, too.