RESUMEN
Familial juvenile nephronophthisis (NPH) is a chronic autosomal recessive kidney disease responsible for 15% of end stage renal failure in children. NPH is frequently (16% of cases) associated with Leber amaurosis (termed Senior-Løken syndrome, SLS). Linkage analyses, performed in 22 multiplex NPH families (18 without and 4 with ocular abnormalities), have localized the gene to a region between D2S48 and D2S51 on chromosome 2p. This was confirmed using adjacent microsatellite markers, one of which (AFM220ze3 at the D2S160 locus) gave a lod score of 4.78 at theta = 0.05 in the 18 families with isolated NPH, whereas the same marker excluded linkage with SLS. These results demonstrate linkage of the purely renal form of NPH to chromosome 2p, and suggest that there may be genetic heterogeneity between NPH and SLS.
Asunto(s)
Cromosomas Humanos Par 2 , Enfermedades Renales/genética , Secuencia de Bases , Niño , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , ADN Satélite/genética , Femenino , Genes Recesivos , Ligamiento Genético , Humanos , Fallo Renal Crónico/etiología , Fallo Renal Crónico/genética , Escala de Lod , Masculino , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: This study aimed to evaluate the potential impact of social inequalities on stage at diagnosis and long-term outcome of breast cancer patients attending the Institut Curie in Paris (France). METHODS: The study population included 14,610 breast cancer patients diagnosed and treated in the Institut Curie between 1981 and 2001. The socioeconomic status was determined from district of residence, median income for town of residence corrected by the consumption unit and body mass index. Logistic regression models adjusted on socioeconomic factors were used to evaluate clinical and pathologic features at diagnosis. Overall survival and distant metastasis were analysed with log-rank tests and Cox proportional hazards regression models. RESULTS: Patients living in lower income districts were more likely to be diagnosed with breast tumors size greater than 20 mm (P=0.01). Residents of high-income urban areas (>15,770 ) exhibited a significant overall survival and distant metastasis advantage (respectively HR=0.93 [0.86-0.99]; P=0.02 and HR=0.91 [0.85-0.98]; P=0.01). Breast cancer screening with mammography was independent of district of residence (P=0.61) or income (P=0.14). After adjusting for age at diagnosis and period, the risk of having breast cancer with unfavorable prognostic factors such as tumor size greater than 20 mm decreased with 1000 increase in district income (OR=0.986 [0.98-0.99]; P<0.001). Similarly, the risk of cancer death decreased for patients residing in districts with median income greater than 15,770 (HR=0.92 [0.86-0.98]; P=0.01). CONCLUSION: Despite the limitations of the study (aggregate data used to assess socioeconomic status, non representative cohort of French women), we observed that poorer breast cancer prognosis with advanced disease diagnosis and increased risk of breast cancer mortality was related to low socioeconomic status.
Asunto(s)
Neoplasias de la Mama/patología , Disparidades en el Estado de Salud , Clase Social , Neoplasias de la Mama/diagnóstico por imagen , Detección Precoz del Cáncer , Femenino , Francia , Humanos , Modelos Logísticos , Mamografía , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Factores de Riesgo , Análisis de SupervivenciaRESUMEN
The axonal transport of the diverse isotubulins in the motor axons of the rat sciatic nerve was studied by two-dimensional polyacrylamide gel electrophoresis after intraspinal injection of [35S]methionine. 3 wk after injection, the nerve segments carrying the labeled axonal proteins of the slow components a (SCa) and b (SCb) of axonal transport were homogenized in a cytoskeleton-stabilizing buffer and two distinct fractions, cytoskeletal (pellet, insoluble) and soluble (supernatant), were obtained by centrifugation. About two-thirds of the transported-labeled tubulin moved with SCa, the remainder with SCb. In both waves, tubulin was found to be associated mainly with the cytoskeletal fraction. The same isoforms of tubulin were transported with SCa and SCb; however, the level of a neuron-specific beta-tubulin subcomponent, termed beta', composed of two related isotubulins beta'1 and beta'2, was significantly greater in SCb than in SCa, relative to the other tubulin isoforms. In addition, certain specific isotubulins were unequally distributed between the cytoskeletal and the soluble fractions. In SCa as well as in SCb, alpha''-isotubulins were completely soluble in the motor axons. By contrast, alpha''' and beta'2-isotubulins, both posttranslationally modified isoforms, were always recovered in the cytoskeletal fraction and thus may represent isotubulins restricted to microtubule polymers. The different distribution of isotubulins suggests that a recruitment of tubulin isoforms, including specific posttranslational modifications of defined isoforms (such as, at least, phosphorylation of beta' and acetylation of alpha'), might be involved in the assembly of distinct subsets of axonal microtubules displaying differential properties of stability, velocity and perhaps of function.
Asunto(s)
Axones/metabolismo , Neuronas Motoras/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Transporte Biológico , Citoesqueleto/metabolismo , Femenino , Microtúbulos/metabolismo , Ratas , Ratas Endogámicas , Nervio CiáticoRESUMEN
The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.
Asunto(s)
Encéfalo/metabolismo , Glutamatos/metabolismo , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/metabolismo , Aminoácidos/análisis , Animales , Cromatografía Líquida de Alta Presión , Ácido Glutámico , Espectrometría de Masas , Ratones , Fragmentos de Péptidos/análisisRESUMEN
The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome.
Asunto(s)
Colágeno/genética , Eliminación de Gen , Nefritis Hereditaria/genética , ARN Mensajero/análisis , Cromosoma X , Secuencia de Bases , Femenino , Ligamiento Genético , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , MutaciónRESUMEN
The efficacy of an inactivated vaccine containing the Eurasian isolate A/chicken/Italy/22A/98 H5N9 (H5N9-It) was compared with that of the fowlpox-vectored TROVACTM-AIV H5 (rFP-AIV-H5) vaccine against an H5N1 highly pathogenic avian influenza challenge. Five-week-old Muscovy ducks were vaccinated with either H5N9-It (0.5 ml) or rFP-AIV-H5 (5 log10 50% tissue culture infectious dose (TCID50)/dose), followed by a boost at 7 wk of age with the same vaccine (1.0 ml of H5N9-It or 5 log10 TCID50/dose rFP-AIV-H5), and a challenge at 9 wk of age with 10(7) egg infectious dose (lethality 50%) of A/crested eagle/ Belgium/01/2004 (H5N1). All unvaccinated challenged birds showed severe nervous signs (loss of balance, torticollis) starting 7 days postinfection (dpi). None of the vaccinated ducks showed these nervous signs. Shedding was measured in oropharyngeal and cloacal swabs, sampled from 3 to 19 dpi by titration in chicken embryo fibroblasts and by real-time reverse transcription-polymerase chain reaction. Virus shedding was significantly higher in oropharyngeal compared to cloacal swabs. Both vaccines reduced the percentage of positive swabs and the viral load in the swabs, but the reduction was higher with the H5N9-It vaccine. The inactivated vaccine induced hemagglutination inhibition (HI) titers (5.4 log2) that were boosted after the second administration (7.5 log2). rFP-AIV-H5-induced HI titers were lower (3 log2 only after the second administration), most probably because the fowlpox vector does not replicate in ducks. Altogether, these results indicate that significant protection from clinical signs and reduction in virus shedding may be achieved in ducks with conventional inactivated or fowlpox-vectored vaccine as compared with nonvaccinated challenged control birds.
Asunto(s)
Patos/virología , Virus de la Viruela de las Aves de Corral , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Aviar/prevención & control , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antivirales/sangre , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Aviar/virología , Vacunas de Productos Inactivados/inmunología , Esparcimiento de VirusRESUMEN
Interference by maternally derived antibody (MDA) is a major problem for the vaccination of young chickens against infectious bursal disease (IBD). The choice of the timing of vaccination and of the type (degree of attenuation) of modified-live vaccine (MLV) to use is often difficult. An IBD vectored vaccine (vHVT13), in which turkey herpesvirus (HVT) is used as the vector, was recently developed. This vaccine is administered once at the hatchery, either in ovo or by the subcutaneous route, to 1-day-old chicks at a time when MDA is maximal. In terms of safety, the vHVT13 vaccine had negligible impact on the bursa of Fabricius when compared with classical IBD MLV. Vaccination and challenge studies demonstrated that this vaccine is able to protect chickens against various IBD virus (IBDV) challenge strains including very virulent, classical, and USA variant IBDV, despite the presence of high-titred IBD MDA at the time of vaccination. These data show that the vector vaccine combines a safety and efficacy profile that cannot be achieved with classical IBD vaccines.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Inmunidad Materno-Adquirida/inmunología , Enfermedades de las Aves de Corral/prevención & control , Vacunas Virales/uso terapéutico , Animales , Animales Recién Nacidos/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/prevención & control , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Enfermedades de las Aves de Corral/inmunología , Vacunación/métodos , Vacunación/veterinariaRESUMEN
The relationship between microtubule dynamics and polyglutamylation of tubulin was investigated in young differentiating mouse brain neurons. Selective posttranslational labeling with [3H]glutamate and immunoblotting with a specific monoclonal antibody (GT335) enabled us to analyze polyglutamylation of both alpha and beta subunits. Nocodazole markedly inhibited incorporation of [3H]glutamate into alpha- and beta-tubulin, whereas taxol had no effect for alpha-tubulin and a stimulating effect for beta-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of alpha-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of alpha-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (1st-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated alpha-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated alpha-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of alpha- and beta-tubulin suggest that distinct enzymes are involved.
Asunto(s)
Encéfalo/citología , Glutamatos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Anticuerpos Monoclonales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Ácido Glutámico , Immunoblotting , Ratones , Microtúbulos/química , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Nocodazol/farmacología , Paclitaxel/farmacología , Polímeros , Estereoisomerismo , Tubulina (Proteína)/químicaRESUMEN
To gain new insight into the role of B-cell autophagy, we generated two novel mouse models deficient for the autophagy-related gene (Atg)5, one from the outset pro-B cell stage (Atg5(f/-) Mb1 cre) and the other in mature B cells only (Atg5(f/-) CD21 cre). We show that autophagy is dispensable for pro- to pre-B cell transition, but necessary at a basal level to maintain normal numbers of peripheral B cells. It appears non-essential for B-cell activation under B-cell receptor stimulation but required for their survival after lipopolysaccharide stimulation that drives plasmablast differentiation and for specific IgM production after immunization. Results obtained using Atg5(f/-) CD21 cre × C57BL/6(lpr/lpr) autoimmune-prone mice show that B-cell autophagy is involved in the maintenance of anti-nuclear antibody secretion, elevated number of long-lived plasma cells, and sustains IgG deposits in the kidneys. Thus, treatments specifically targeting autophagy might be beneficial in systemic autoimmune diseases.
Asunto(s)
Autoinmunidad/inmunología , Autofagia/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
Using clones N1E-115 and N1A-103 from mouse neuroblastoma C1300, a comparative analysis of c- and N-myc gene expression was undertaken both in proliferating cells and in cultures exposed to conditions which induce differentiation. Under the latter conditions, while N1E-115 cells extend abundant neurites and express many biochemical features of mature neurons, clone N1A-103 stops dividing and expresses certain neurospecific markers but is unable to differentiate morphologically. In both clones, chemical agents, i.e. 1-methyl cyclohexane carboxylic acid (CCA) or dimethyl sulfoxide (DMSO), induce a decrease in c-myc expression. Similar results were found for N-myc gene in N1E-115 cells, but in contrast, in clone N1A-103, N-myc expression is increased with CCA and not modified with DMSO. Globally, this study favours the hypothesis that changes in c-myc expression would correspond to cell division blockade and differentiation, while modulations in N-myc are more closely related to an early phase of terminal differentiation.
Asunto(s)
Genes myc , Neuroblastoma/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Animales , Northern Blotting , Southern Blotting , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Ácidos Ciclohexanocarboxílicos/farmacología , ADN/análisis , Dimetilsulfóxido/farmacología , Relación Dosis-Respuesta a Droga , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Técnicas In Vitro , Ratones , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Plásmidos , ARN Mensajero/biosíntesis , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The ability of embryonic chicken muscle initiation factors to translate rabbit globin messenger RNA in an efficient, fractionated cell-free system has been examined. Although muscle factors stimulate leucine incorporation to only 15--35% the levels achieved with rabbit reticulocyte initiation factors, they synthesize more than one globin chain per mRNA molecule and both alpha and beta globin are produced. Increasing the ribosome concentration and adding the polyamine spermidine to the system produce stimulatory effects which are quantitatively and qualitatively similar for both factor preparations. The lower efficiency of synthesis of muscle factors relative to reticulocyte factors is also apparent when mRNA from encephalomyocarditis virus or embryonic chicken muscle polysomes are used in the cell-free system. These results do not support a specific restriction in the capacity of muscle factors to translate globin mRNA. Furthermore, the similarity of the effects of presumed non-specific components on the activity of muscle and reticulocyte factors suggests that globin synthesis in the cell-free system may be controlled in a similar fashion for both preparations.
Asunto(s)
Globinas/biosíntesis , Músculos/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Sistema Libre de Células , Embrión de Pollo , Virus de la Encefalomiocarditis/metabolismo , Cinética , Magnesio/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Viral/metabolismo , Conejos , Reticulocitos/metabolismo , Espermidina/farmacologíaRESUMEN
Previous studies from our laboratory have investigated messenger RNA metabolism in calf muscle cells in tissue culture. The analysis of mRNA was based on its poly(A) content. We have now examined directly the proportion of mRNA which contains poly(A) in these cells. After separation of poly(A)+ -and poly(A) - -RNA on oligo(dT) -cellulos, the two fractions were translated in a reconstituted, heterologous cell-free protein-synthesizing system and the products were compared with those from the translation of total RNA. The great majority of mRNA form either prefusion or postfusion cultures was poly(A)- containing; quantitative determinations show that about 70-90% of the actin mRNA is poly(A)-containing. In order to determine if a large fraction of the calf muscle mRNA can be translated by a heterologous cell-free system, [3H]-POLY(A)+ -RNA was added to reticulocyte lysates and the formation of initiation complexes was followed. These experiments suggest that the bulk of calf muscle cell mRNA would be utilized in such a system and justify the use of cell-free systems to examine the poly(A) content of total mRNA. Thus, differential polyadenylation does not seem to be an important aspect of mRNA metabolism in cultured muscle cells. The previous study of mRNA in these cells, based on poly(A) content, is apparently a valid measure of overall mRNA metabolism.
Asunto(s)
Proteínas Musculares/genética , Músculos/metabolismo , Poli A/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Línea Celular , Sistema Libre de Células , Histonas/genética , Proteínas Musculares/biosíntesis , ARN Mensajero/genéticaRESUMEN
A new procedure for the purification of rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) is presented. The enzyme solubilized in Lubrol PX was purified either by molecular sieving or by hydrophobic chromatography, followed by a preparative isoelectric focusing step. For this purpose, a new isoelectric focusing technique was developed which allows a good resolution of adenylate cyclase in a short period of time. When resolved by this procedure, the enzyme migrated as a single molecular species with a pI of 6.3. When isoelectric focusing was performed in the presence of EGTA, two distinct peaks of activity could be detected at pI 6.1 and 7.3. This suggests that adenylate cyclase consists of two subunits held together by divalent ions. It is shown that the purified adenylate cyclase has a smaller sedimentation coefficient and is less hydrophobic than the native one. We conclude that the adenylate cyclase containing complex was at least partially disaggregated by this procedure.
Asunto(s)
Adenilil Ciclasas/aislamiento & purificación , Encéfalo/enzimología , Adenilil Ciclasas/metabolismo , Animales , Cromatografía en Gel , Ácido Egtácico , Focalización Isoeléctrica/métodos , Sustancias Macromoleculares , Masculino , Peso Molecular , Ratas , Solubilidad , TemperaturaRESUMEN
We investigated the effects of the diencephalon and mesencephalon on the central respiratory drive originating from ponto-medullary regions in normoxic and hypoxic conditions, using central nervous system preparations from newborn rats. We used two approaches: 1) electrophysiological analysis of respiratory frequency and the amplitude of inspiratory C4 activity and 2) immunohistochemical detection of Fos protein, an activity-dependent neuronal marker. We found that, in normoxic conditions, the mesencephalon moderated respiratory frequency, probably by means of an inhibitory effect on ventral medullary respiratory neurons. Diencephalic inputs restored respiratory frequency. Moreover, O(2)-sensing areas in the diencephalon (caudal lateral and posterior hypothalamic areas) and mesencephalon (ventrolateral and dorsolateral periaqueductal gray) seem to increase the amplitude of respiratory bursts during adaptation of the central respiratory drive to hypoxia. In contrast, decrease in respiratory frequency during hypoxia is thought to be mediated by a cluster of ventral hypothalamic neurons.
Asunto(s)
Diencéfalo/fisiopatología , Hipoxia/fisiopatología , Mesencéfalo/fisiopatología , Puente/fisiopatología , Respiración , Centro Respiratorio/fisiología , Animales , Animales Recién Nacidos , Diencéfalo/anatomía & histología , Electrofisiología/métodos , Inmunohistoquímica/métodos , Técnicas In Vitro , Mesencéfalo/anatomía & histología , Redes Neurales de la Computación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
We define the spatial and temporal patterns of expression of the gene encoding the glycolytic enzyme, beta-enolase, during mouse ontogenesis. Transcripts were detected by in situ hybridization using 35S labelled cRNA probes. The beta-enolase gene is expressed only in striated muscles. It is first detected in the embryo, in the cardiac tube and in newly formed myotomes. In the muscle masses of the limb, beta gene expression occurs at a low level in primary fibers, and subsequently greatly increases at a time which corresponds to the onset of innervation and secondary fiber formation. Later in development, it becomes undetectable in slow-twitch fibers. Our results demonstrate the multistep regulation of the beta-enolase gene. The regulation of this muscle-specific gene in somites is discussed in terms of the myogenic sequences of the MyoD family shown to be present when it is activated.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Código Genético/genética , Glucólisis/genética , Músculos/embriología , Fosfopiruvato Hidratasa/genética , Animales , Desarrollo Embrionario y Fetal/genética , Extremidades , Edad Gestacional , Ratones , Transcripción Genética/genéticaRESUMEN
A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.
Asunto(s)
Anticuerpos Monoclonales , Glutamatos/metabolismo , Tubulina (Proteína)/análisis , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Astrocitos/química , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Pulmón/química , Masculino , Ratones , Datos de Secuencia Molecular , Neuronas/química , Bazo/química , Testículo/químicaRESUMEN
The expression of alpha- and beta-tubulin mRNAs has been studied during mouse brain development by using in vitro translation and specific cDNA clone hybridization techniques. The results presented here indicate that about half of the isotubulins expressed in vivo by brain cells are directly encoded by mRNA, the other isotypes being of post-translational origin. Among the six brain beta-tubulin mRNAs detected after RNA size fractionation followed by in vitro translation, four (1.8a, 1.8c, 2.4 and 3.5 kb) appear to be uniquely expressed in brain, and very likely in neurons. These four beta-tubulin RNA transcripts are differently regulated during development. The 1.8a-kb mRNA, which codes for the neurospecific beta' 1-isotubulin, is expressed very early, whereas the 1.8c, 2.4 and 3.5-kb mRNAs, which all code for a beta 4-tubulin isotype, are all expressed later. We isolated a beta-tubulin cDNA clone which specifically hybridizes to the large, neural 3.5-kb mRNA. The two other ubiquitous tubulin mRNAs, 1.8b and 2.9 kb in length, both direct the synthesis of an electrophoretically distinct beta 3 isotype. These results support the idea that within the tubulin multigene family, distinct beta-tubulin isogenes could be specifically expressed at various steps of the neuronal differentiation, thus contributing to the isotubulin diversity and specificity observed in this cell type and possibly involved in the developmental changes of its structure and function.
Asunto(s)
Encéfalo/embriología , Tubulina (Proteína)/genética , Animales , Encéfalo/fisiología , Diferenciación Celular , Clonación Molecular , Regulación de la Expresión Génica , Punto Isoeléctrico , Ratones , Peso Molecular , Familia de Multigenes , Polimorfismo Genético , ARN Mensajero/genéticaRESUMEN
Specific gene transcription from chromatin has been examined in two different systems by using direct measurement of specific sequences by hybridization with excess DNA probe. An improved procedure has been used for hybridization of DNA immobilized on filters. This procedure is sensitive to less than one part in 10(5). Polyoma RNA sequences were detected a level of 0.004% in the overall transcript from chromatin of ts-a-transformed 3T3 fibroblasts. By the use of constructed plasmid DNA carrying rabbit globin sequences, globin RNA was titrated in the RNA transcribed from chromatin of bone marrow cells. Its relative frequency was 0.01%. Results obtained with this new approach confirm that DNA in chromatin is not randomly transcribed. They further illustrate that a reproducible assay system is now available for studying control elements active on gene expression at the transcriptional level, taking advantage of the constructed plasmids containing eukaryotic gene DNA fragments.
Asunto(s)
Cromatina , ADN Recombinante , ARN Viral , Transcripción Genética , Animales , Médula Ósea/metabolismo , Transformación Celular Viral , ADN , ADN Bacteriano , Hibridación de Ácido Nucleico , Poliomavirus/genética , ARN/metabolismo , ConejosRESUMEN
Various mitochondrial inhibitors are tested in neuroblastoma cells. Their effects on the mit-proteins and some cytoskeletal proteins are compared to those of CCA, a differentiation inducer. This comparison favours the hypothesis that the primary effect of CCA induction is an alteration of the electrochemical gradient.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Ácidos Ciclohexanocarboxílicos/farmacología , Mitocondrias/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Actinas/biosíntesis , Animales , Células Cultivadas , Proteínas de Filamentos Intermediarios/biosíntesis , Potenciales de la Membrana/efectos de los fármacos , Ratones , Neuroblastoma/patología , Tubulina (Proteína)/biosíntesis , VimentinaRESUMEN
The mouse monoclonal antibody ME 101 raised against human peripherin, an intermediate filament protein (IFP) specific to well defined neuronal populations, recognizes all the major classes of vertebrate IFP in immunoblotting assays. Desmin, GFAP, vimentin, peripherin and the lightest neurofilament protein (NF-L) were cleaved into carboxy- and amino-terminal halves by N-chlorosuccinimide at their unique trytophan residue. Whereas the antibody directed against the epitope common to every IFP (intermediate filament antigen or IFA) and located on the carboxy-terminal end of the rod domain recognizes the carboxy-terminal half, the ME 101 antibody, as the present study illustrates, recognizes specifically the amino-terminal half. From the amino acid sequence data of IFP, it is deduced that the cognate epitope is localized on the amino-terminal part of coil la.