RESUMEN
S100 protein is a calcium-binding protein found predominantly in the vertebrate nervous system. Genomic and complementary DNA probes were used in conjunction with a panel of rodent-human somatic cell hybrids to assign the gene for the beta subunit of S100 protein to the distal half of the long arm of human chromosome 21. This gene was identified as a candidate sequence which, when expressed in the trisomic state, may underlie the neurologic disturbances in Down syndrome.
Asunto(s)
Cromosomas Humanos Par 21 , Síndrome de Down/genética , Proteínas S100/genética , Mapeo Cromosómico , Clonación Molecular , Humanos , Sustancias Macromoleculares , Hibridación de Ácido NucleicoRESUMEN
The disulfide bonding characteristics of the pig lymph node plasma membrane (PM) proteins and glycoproteins have been examined by 1- and 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Reaction of the purified PM vesicles with N-ethyl maleimide (NEM) prior to detergent solubilization was found to markedly reduce the extent of intermolecular disulfide bonding subsequently observed. Thus the blocking of free sulfhydryl groups with NEM prevented the detergent-induced disulfide bonding of numerous components, including PM-bound actin. The extent of intermolecular disulfide bonding among the NEM-pretreated PM glycoproteins purified by lentil lectin affinity chromatography was found to be relatively limited, with only 3% of the total glycoprotein present as intermolecular disulfide-bonded complexes. In contrast, the degree of intramolecular disulfide bonding revealed by a modified 1-dimensional SDS-PAGE technique was quite striking. Among those polypeptides demonstrating a clearly altered mobility upon reduction was the heavy chain of class I and beta-chain of class II major histocompatibility complex (MHC) antigens. The class II alpha-chain, however, was much less affected. These changes have been compared with those observed for proteins containing intramolecular disulfide-bonded domains of known size and number, and considered in the light of recent information on the structure of MHC antigens.
Asunto(s)
Disulfuros/análisis , Glicoproteínas/análisis , Linfocitos/análisis , Proteínas de la Membrana/análisis , Animales , Electroforesis en Gel de Poliacrilamida , Etilmaleimida/farmacología , Técnicas In Vitro , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Sustancias Macromoleculares , Complejo Mayor de Histocompatibilidad , Proteínas de la Membrana/inmunología , Conformación Proteica , PorcinosRESUMEN
The actions of androgens in both peripheral and central tissues are linked in part to their ability to specifically bind and activate androgen receptors (ARs). ARs have been well studied in the rat hypothalamus and peripheral reproductive tissues, where they are directly involved in endocrine feedback mechanisms and reproduction. Previous studies revealed relatively high levels of AR and AR messenger RNA (mRNA) in the rat hippocampus; however, the action of androgen in this brain region remains unclear. To begin to address this issue, we used a multidisciplinary approach to quantitate hippocampal AR and AR mRNA levels and investigate their regulation after various hormonal manipulations. In vitro binding assays revealed a single, saturable, high affinity binding site for androgen in hippocampal cytosols. The expression of AR mRNA in the intact adult male rat hypothalamus and hippocampus was demonstrated using reverse transcription-polymerase chain reaction and quantified using a ribonuclease protection assay. Comparable levels of AR mRNA were found in the hippocampus and hypothalamus. In addition, in situ hybridization analysis revealed a unique distribution of AR mRNA in the hippocampus. AR mRNA was found predominately in the CA1 pyramidal cells, which form the major signal output of the hippocampal trisynaptic circuit. Reverse transcription-polymerase chain reaction of total RNA from microdissected hippocampal regions confirmed this distribution. Ribonuclease protection assay demonstrated a significant decrease in the AR mRNA content of the hippocampus in animals killed 4 days after castration or in intact rats after four daily injections of the AR antagonist, flutamide (15 mg/animal), compared to that in intact controls (P < 0.01). In contrast, a 35% increase (P < 0.05) in the hippocampal AR mRNA content was found in old (22-month-old) compared to young (5-month-old) male rats. In both cases, [3H]dihydrotestosterone binding to the cytosolic preparation did not parallel the changes observed in the AR mRNA content. Taken together, these data demonstrate that hippocampal cells containing AR can respond to circulating androgen to alter AR gene expression. Furthermore, AR mRNA autoregulation appears to be both age and tissue specific and does not directly follow the regulatory patterns described for other steroid hormone receptors found in the hippocampus.
Asunto(s)
Hipocampo/metabolismo , Hormonas/fisiología , ARN Mensajero/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Envejecimiento/metabolismo , Antagonistas de Receptores Androgénicos , Animales , Flutamida/farmacología , Masculino , Orquiectomía , Ratas , Ratas Endogámicas F344 , Factores de Tiempo , Distribución TisularRESUMEN
S100 protein is a low molecular weight calcium-binding protein widely distributed in the central nervous system of vertebrates. Recent evidence suggests that S100 protein may play a role in the regulation of glial proliferation and neuronal differentiation. The gene for S100 protein has been mapped to the 21q22 region, a chromosomal locus whose duplication has been implicated in the generation of Down Syndrome (DS). This raises the possibility that abnormalities in S100 protein gene dosage at a critical period during development may be responsible for some of the neurologic abnormalities associated with DS.
Asunto(s)
Cromosomas Humanos Par 21 , Síndrome de Down/genética , Proteínas S100/genética , Mapeo Cromosómico , Clonación Molecular , Genes , Humanos , Proteínas S100/análisisRESUMEN
The reproducibility of preparation, stability at 4 degrees C, and detergent solubilization characteristics of plasma membrane vesicles purified from domestic pig mesenteric lymph node tissue have been examined. It was found that 2% (w/v) Nonidet P-40 solubilized 50-60% and 2% (w/v) sodium deoxycholate solubilized 60-70% of the total membrane protein. As judged by 125I-labelled lentil lectin staining of the sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns, 2% (w/v) Nonidet p-40 solubilized approximately 73%, and 2% (w/v) sodium deoxycholate approximately 82% of the total glycoprotein. Actin and a myosin-like component were identified as major constituents of both the Nonidet P-40 and the sodium deoxycholate insoluble fractions, suggesting the possibility that the detergent-insoluble fraction may represent a membrane-associated cytoskeletal network analogous to that which has been demonstrated for the erythrocyte membrane. Consistent with such an intimate association between actin and the plasma membrane, it was found that very little actin could be eluted from the intact membrane vesicles by dialysis against low ionic strength ATP solutions, 0.6 M KCl, or by incubation with DNase I.
Asunto(s)
Ganglios Linfáticos/ultraestructura , Actinas/fisiología , Animales , Membrana Celular/ultraestructura , Citoesqueleto/ultraestructura , Desoxirribonucleasas , Detergentes , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/aislamiento & purificación , Solubilidad , PorcinosRESUMEN
A simple modification of the conventional one-dimensional sodium dodecyl sulfate--polyacrylamide gel electrophoresis technique has been used to visualize inter- and intramolecular disulfide bonding in proteins. The gradient of reducing agent established between adjacent slab gel tracks by electrophoresing identical protein samples next to one another, one containing and the other not containing 2-mercaptoethanol, has been used to visualize the change in mobility of disulfide bond-containing proteins throughout the transition from a reducing to a nonreducing environment. As illustrated by an analysis of immunoglobulin heavy and light chains, the method particularly facilitates the positive identification of proteins containing intrachain disulfide bonds.
Asunto(s)
Disulfuros/análisis , Proteínas/análisis , Animales , Sitios de Unión , Membrana Celular/análisis , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/sangre , Humanos , Linfocitos/análisis , Dodecil Sulfato de Sodio , PorcinosRESUMEN
The receptiveness of the brain to monocyte infiltration was studied in rats that had been injected intracerebrally with Corynebacterium parvum. At 0-17 days after intracerebral injection and 18 h after intravenous injection of diI-labeled isogenous mononuclear cells, host rats were sacrificed and cells from the vicinity of the injection site and from the contralateral cerebral hemisphere were dissociated and analyzed by flow cytometry. In rats sacrificed 4-11 days postinjection of C. parvum, diI-labeled mononuclear cells were detected in cell preparations from the hemisphere ipsilateral and, to a lesser extent, contralateral to the injection site. No extravasation of cells from the blood to the brain was detected in rats injected intracerebrally with saline. By immunohistochemistry, many macrophages were detected in the hemisphere ipsilateral to injection of C. parvum. In additional experiments, the dissociated CNS cell population was labeled with OX-42 antibodies to the type 3 complement receptor, which is present on monocytes but not lymphocytes. Some cells in the brain were labeled with both diI and OX-42 and therefore were identified as monocytes that had entered the brain from the blood. In conclusion, monocytes can home to both sides of the brain after unilateral injection of a strong inflammatory agent but monocyte infiltration into the brain is delayed in comparison to monocyte inflammatory responses that have been reported in nonneural tissues.
Asunto(s)
Antígenos CD , Antígenos de Neoplasias , Proteínas Aviares , Proteínas Sanguíneas , Encéfalo/patología , Infecciones por Bacterias Grampositivas/patología , Leucocitos Mononucleares/patología , Propionibacterium acnes , Traslado Adoptivo , Animales , Antígenos de Superficie/análisis , Basigina , Encéfalo/inmunología , Carbocianinas , Citometría de Flujo , Colorantes Fluorescentes , Infecciones por Bacterias Grampositivas/inmunología , Inflamación , Leucocitos Mononucleares/inmunología , Masculino , Glicoproteínas de Membrana/análisis , Monocitos/inmunología , Monocitos/patología , Ratas , Ratas Endogámicas Lew , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The gene which encodes the beta subunit of the S100 protein was mapped to 21q22.2----q22.3 by in situ hybridization. Concurrently, a subtle translocation involving this region of chromosome 21 and 9q34 was confirmed.
Asunto(s)
Cromosomas Humanos Par 21 , Cromosomas Humanos Par 9 , Proteínas S100/genética , Translocación Genética , Mapeo Cromosómico , Clonación Molecular , ADN , Síndrome de Down/genética , Humanos , Lactante , Cariotipificación , Masculino , Hibridación de Ácido NucleicoRESUMEN
S100 protein is a calcium-binding protein found in vertebrate nervous tissue. Synthesis of S100 protein in the rat glioma cell line, C6, is inhibited by the addition of anti-microtubular drugs. We have cloned a cDNA for the beta subunit of S100 protein from rat brain in a lambda gt 11 expression vector and used this cDNA to measure the amounts of S100 beta subunit mRNA in C6 cells after treatment with anti-microtubular drugs. Levels of alpha-tubulin and beta-actin mRNAs were also measured. All measurements were performed using RNA-RNA hybridization techniques at high stringency with rat mRNA-specific probes. After 24 h of treatment, the S100 beta subunit mRNA was reduced to levels of 25% by colchicine and 32% by vinblastine when compared to untreated controls. In contrast, the levels of tubulin and actin mRNAs were only slightly changed by these treatments. These studies demonstrate that disruption of the microtubular cytoskeleton causes a specific reduction in the level of S100 protein mRNA in C6 cells.
Asunto(s)
Colchicina/farmacología , ARN Mensajero/metabolismo , Proteínas S100/genética , Transcripción Genética/efectos de los fármacos , Vinblastina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN/metabolismo , Glioma/metabolismo , Sustancias Macromoleculares , Microtúbulos/efectos de los fármacos , Hibridación de Ácido Nucleico , ARN Mensajero/genética , RatasRESUMEN
S100 protein is a low molecular weight, EF-hand, Ca2(+)-binding protein widely distributed and conserved in the central nervous system of vertebrates. The gene coding for the beta subunit of human S100 protein (S100 beta) has been recently mapped to chromosome 21. In order to study the expression of this gene in normal and abnormal brain development, we have isolated and characterized overlapping genomic clones spanning the region coding for human S100 beta and its flanking sequences. The intron-exon organization of the human S100 beta gene is similar to that of the genes coding for several other members of the S100 protein subfamily of EF-hand proteins. The human S100 beta gene is composed of 3 exons, the first of which specifies the 5'-untranslated region, while the second and third each encode a single EF-hand, Ca2(+)-binding domain. The promoter region contains several potential regulatory transcription elements including the cAMP-responsive elements CRE and AP-2. A novel sequence motif, the S100 protein element, situated in close proximity to the TATA box of the genes of several members of the S100 protein subfamily, has been identified. In addition, multiple repeats with similar nucleotide sequence and location to the recently reported beta globin direct repeat elements have been also found in the human S100 beta promoter. A full length (17.3 kilobases) copy of the human S100 beta gene was constructed and transfected into rat glioma C6 cells. Stable transfectants were shown to express correctly initiated transcripts of the human S100 beta gene, indicating that the cloned sequences contain functional regulatory transcription elements.
Asunto(s)
Genes , Proteínas S100/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Biblioteca Genómica , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
The beta-subunit of S100 protein (S100 beta) is highly conserved in the mammalian brain. The gene coding for human S100 beta has been mapped to chromosome 21. In order to study the consequences of overexpression of the S100 beta gene, transgenic mice were generated by microinjection of a 17.3 kilobase human genomic fragment containing the three exons and the transcription control elements of the human S100 beta gene. Mice from four transgenic lines carried approximately 10-100 transgene copies. Northern blotting demonstrated a tissue-specific and gene dose-dependent expression of human S100 beta mRNA in mouse brain. Increased expression of S100 beta mRNA was correlated with an increased production of S100 beta protein. Examination of brain sections by in situ hybridization and immunocytochemistry indicated that S100 beta was localized globally to astrocytes, as well as to discrete neurons in the mesencephalic and motor trigeminal, facial, and lemniscus nuclei in both normal and transgenic mice. In peripheral tissues, human S100 beta was expressed at 10-50-fold lower levels than in brain. The strict gene dosage dependence and cell specificity of transgene expression suggest the presence of a locus control region (LCR) in the human S100 beta gene. The mice tolerated 10-100-fold higher than normal levels of S100 beta gene expression in brain without any gross physical or behavioral abnormalities. The high-level expression and cell specificity of the S100 beta promoter/LCR suggest that it may provide a valuable tool to direct the expression of other transgenic products to specific cell types in the CNS.