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1.
J Musculoskelet Neuronal Interact ; 10(3): 207-19, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20811145

RESUMEN

Long-term bed-rest is used to simulate the effect of spaceflight on the human body and test different kinds of countermeasures. The 2nd Berlin BedRest Study (BBR2-2) tested the efficacy of whole-body vibration in addition to high-load resisitance exercise in preventing bone loss during bed-rest. Here we present the protocol of the study and discuss its implementation. Twenty-four male subjects underwent 60-days of six-degree head down tilt bed-rest and were randomised to an inactive control group (CTR), a high-load resistive exercise group (RE) or a high-load resistive exercise with whole-body vibration group (RVE). Subsequent to events in the course of the study (e.g. subject withdrawal), 9 subjects participated in the CTR-group, 7 in the RVE-group and 8 (7 beyond bed-rest day-30) in the RE-group. Fluid intake, urine output and axiallary temperature increased during bed-rest (p < .0001), though similarly in all groups (p > or = .17). Body weight changes differed between groups (p < .0001) with decreases in the CTR-group, marginal decreases in the RE-group and the RVE-group displaying significant decreases in body-weight beyond bed-rest day-51 only. In light of events and experiences of the current study, recommendations on various aspects of bed-rest methodology are also discussed.


Asunto(s)
Reposo en Cama/efectos adversos , Terapia por Ejercicio/métodos , Aptitud Física/fisiología , Simulación de Ingravidez/efectos adversos , Adulto , Berlin , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/etiología , Osteoporosis/fisiopatología , Osteoporosis/prevención & control , Resultado del Tratamiento , Vibración/uso terapéutico , Adulto Joven
2.
J Mol Biol ; 305(5): 1025-33, 2001 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-11162111

RESUMEN

The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Tris-acetate buffer, to test the hypothesis that site-specific binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A3T3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A5 tracts or phased runs of T3A3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1-20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the AnTn sequence motif.


Asunto(s)
Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Poli A/genética , Poli A/metabolismo , Cloruro de Sodio/metabolismo , Secuencia de Bases , Electroforesis Capilar , Iones/metabolismo , Iones/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Oligodesoxirribonucleótidos/química , Poli A/química , Cloruro de Sodio/farmacología , Soluciones
3.
Neurology ; 53(1): 26-33, 1999 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-10408532

RESUMEN

OBJECTIVE: To verify linkage to chromosome 19p13, to detect mutations in the CACNA1A gene, and to correlate genetic results to their clinical phenotypes in Italian families with familial hemiplegic migraine (FHM). BACKGROUND: FHM is an autosomal dominant disease, classified as a subtype of migraine with aura. Only a proportion of FHM patients have been associated with chromosome 19p13. Among these, four missense mutations within the CACNA1A gene in five unrelated families have been described. METHODS: A linkage study was performed in 19 patients affected by FHM from five families by studying microsatellite markers associated with the 19p13 region. All familial and seven additional sporadic patients with FHM were analyzed to search for mutations within the CACNA1A gene by applying the double gradient-denaturant gradient electrophoresis technique. RESULTS: Lod score values did not establish significantly linkage to chromosome 19. However, seven new genetic variants were detected: six were new polymorphisms. The seventh was a missense mutation present in family 1, and it was associated with a hemiplegic migraine phenotype without unconsciousness and cerebellar ataxia. Because this missense mutation is absent in the general population and cosegregates with the disease, it may be a pathologic mutation. CONCLUSIONS: Genetic heterogeneity of FHM has been shown in familial and sporadic FHM patients of Italian origin. The new missense mutation-G4644T-is associated with milder clinical features compared with typical FHM.


Asunto(s)
Canales de Calcio/genética , Cromosomas Humanos Par 19 , Hemiplejía/genética , Trastornos Migrañosos/genética , Mutación Missense , Adolescente , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ataxia Cerebelosa , Niño , Mapeo Cromosómico , ADN/sangre , Exones , Femenino , Ligamiento Genético , Marcadores Genéticos , Humanos , Intrones , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Linaje , Reacción en Cadena de la Polimerasa , Conejos , Ratas , Alineación de Secuencia
4.
Neurology ; 53(1): 38-43, 1999 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-10408534

RESUMEN

OBJECTIVE: To search for mutations in the calcium channel gene CACNA1A and to study the genotype-phenotype correlation in a family with a severe familial hemiplegic migraine (FHM) phenotype and a slowly progressive cerebellar ataxia. BACKGROUND: CACNA1A gene mutations on chromosome 19 are involved in approximately 50% of FHM families. The association of FHM and cerebellar ataxia has been reported in a small number of FHM families, all linked to chromosome 19. METHODS: The proband, in addition to typical hemiplegic migraine attacks, experienced severe episodes during which hemiplegia was associated with acutely altered consciousness and fever lasting several days. She, as well as her affected sister, developed a permanent, late-onset cerebellar ataxia and cerebellar atrophy evident on MRI. Linkage analysis was performed and the whole CACNA1A gene, 47 exon-intron boundaries, was analyzed by double gradient-denaturing gradient gel electrophoresis (DG-DGGE). RESULTS: Genetic studies suggested linkage to chromosome 19p13, and DG-DGGE analysis detected a heteroduplex fragment in exon 13 of the CACNA1A gene. By direct sequencing, a G-to-A substitution resulting in an arginine to glutamine change at codon 583 in the second putative voltage sensor domain of the channel alpha1A-subunit, was identified, possibly representing the disease-causing mutation. The proband and her affected sister were treated with acetazolamide, reporting freedom from new FHM attacks but no benefit in the progression of ataxia. CONCLUSIONS: The combination of episodic dysfunction and permanent deficit could depend on the variety of functions of calcium channels and their distribution in the nervous system.


Asunto(s)
Acetazolamida/uso terapéutico , Canales de Calcio/genética , Ataxia Cerebelosa/genética , Convulsivantes/uso terapéutico , Hemiplejía/genética , Trastornos Migrañosos/genética , Mutación Puntual , Adulto , Anciano , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Encéfalo/patología , Canales de Calcio/química , Ataxia Cerebelosa/tratamiento farmacológico , Ataxia Cerebelosa/patología , Exones , Femenino , Hemiplejía/tratamiento farmacológico , Hemiplejía/patología , Humanos , Intrones , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/patología , Datos de Secuencia Molecular , Linaje , Conejos , Alineación de Secuencia , Homología de Secuencia de Aminoácido
5.
Biotechniques ; 22(2): 326-30, 1997 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9043706

RESUMEN

A novel technique is reported for screening point mutations is genomic DNA: double gradient, denaturing gradient gel electrophoresis (DG-DGGE). Unlike conventional DGGE, which exploits a single gradient of denaturing chemicals (typically urea and formamide) along the migration path to force the two hetero- and two homo-duplexes to partially unwind and separate, DG-DGGE superimposes a second (porous) gradient over the denaturing one. With the help of the sieving gradient, molecules such as the hetero-duplexes, which often produce curtains and smears instead of sharp zones, due to lack of a sharp melting transition, are re-compacted into remarkably narrow bands. Even homo-duplexes with minute melting temperature differences, giving a single band in DGGE, are resolved into two zones in DG-DGGE. The technique has been applied to the analysis of a number of point mutations in several exons of the cystic fibrosis transmembrane conductance regulator gene.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN/análisis , Electroforesis/métodos , Mutación Puntual , Fibrosis Quística/genética , ADN/química , Exones , Humanos , Desnaturalización de Ácido Nucleico , Dodecil Sulfato de Sodio
6.
Biotechniques ; 21(5): 926-28, 930, 932, 1996 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8922636

RESUMEN

A method for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is reported here. The method is based on the principle of temperature gradient gel electrophoresis (TGGE), a variant of denaturing gradient gel electrophoresis (DGGE), and exploits the differential melting of mutant and wild-type PCR-amplified DNA fragments during electrophoresis through a temperature gradient. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the melting points (Tms) of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The temperature increment applied is typically minute (1 degree -1.5 degrees C) and the sweep speed is rather shallow (e.g., 0.05 degree C/min). Additionally, the denaturing thermal gradient is not controlled externally, but generated internally by Joule heat produced by voltage ramps. Point mutants are fully resolved into a spectrum of four bands, with a dynamic range extending from 45 degrees C (low melters) up to 70 degrees C for high melters. The present method can thus be universally applied to any type of point mutation.


Asunto(s)
ADN/análisis , Mutación Puntual , Electroforesis Capilar , Temperatura
7.
Biotechniques ; 19(2): 254-8, 260-3, 1995 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8527147

RESUMEN

Duchenne (DMD) and Becker (BMD) muscular dystrophies are the two most common myopathies described so far. In the late 80s, Chamberlain et al. and Beggs et al. proposed two PCR assays allowing detection of over 98% DMD/BMD deletions. Since each of them is based on specific co-amplification of 9 dystrophin gene exons, a method attempting simultaneous analysis of DMD/BMD should offer unambiguous resolution and identification of 18 DNA fragments ranging in size from approximately 100 to 500 bp. We have developed a novel capillary electrophoresis method that allows simultaneous analysis of the two PCR sets with full diagnostic value. It consists of (a) an ultrastable inner capillary coating based on a novel acrylamide monomer (N-acryloyl amino ethoxy ethanol); (b) a very low viscosity (barely 70 mPa) sieving polymer solution, formed by short-chain (average mol wt of 230,000, 55,000 Mn) polyacrylamides; (c) substitution of four fragments in the classical multiplex reaction (181 and 535 bp in the Beggs, 416 and 459 bp in the Chamberlain) with four new fragments of different lengths (170, 313, 154 and 88 bp, respectively). These new conditions allow resolution and unambiguous identification of all 18 PCR-amplified fragments in a single electrophoretic run. The set of 18 fragments comprises the following: 88, 113, 139, 154, 170, 196, 202, 238, 268, 271, 313, 331, 357, 360, 388, 410, 506 and 547 bp.


Asunto(s)
Distrofina/genética , Electroforesis Capilar/métodos , Distrofias Musculares/genética , Reacción en Cadena de la Polimerasa/métodos , Resinas Acrílicas/química , Electroforesis en Gel de Poliacrilamida/métodos , Exones , Humanos , Recién Nacido , Datos de Secuencia Molecular , Distrofias Musculares/diagnóstico , Tamizaje Neonatal , Diagnóstico Prenatal
8.
Thromb Haemost ; 62(2): 708-14, 1989 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-2530647

RESUMEN

Four mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/l CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate adsorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/l CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Anticuerpos Monoclonales/inmunología , Glicoproteínas/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Reacciones Antígeno-Anticuerpo , Unión Competitiva , Calcio/sangre , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/aislamiento & purificación , Humanos , Inmunoensayo/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína C/aislamiento & purificación , Conformación Proteica , Proteína S , Radioinmunoensayo
9.
Thromb Haemost ; 55(2): 246-9, 1986 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-3520937

RESUMEN

A new electrophoretic method is described for rapid screening of abnormalities of the multimeric structure of von Willebrand factor in von Willebrand's disease. The method is based on the transfer of the separated proteins from agarose gels onto nitrocellulose foils followed by immunoperoxidase staining. It has the advantage of not requiring radio-iodinated antibodies and reduces the working time for the entire procedure from 5-6 days to 3 days. Electroblotting followed by immunoperoxidase staining differentiates patients with intact multimeric structure from those without intermediate and/or large multimers. The more subtle defects of the inner structure of the smallest multimers found in patients with type II von Willebrand's disease can also be identified. A potential disadvantage of electroblotting and immunoperoxidase staining is the lesser sensitivity of this technique, which results in the detection of a smaller number of multimers (11-12 bands) than by autoradiography without transfer onto nitrocellulose (16-17 bands).


Asunto(s)
Electroforesis en Gel de Agar/métodos , Electroforesis/métodos , Técnicas para Inmunoenzimas , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/análisis , Autorradiografía , Carbazoles , Fenómenos Químicos , Química , Colodión , Electroforesis en Gel de Poliacrilamida , Humanos , Radioisótopos de Yodo
10.
Thromb Haemost ; 52(3): 263-6, 1984 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-6549416

RESUMEN

Protein C, an antithrombotic protein, was measured immunologically in 299 patients with clinical conditions associated with a high frequency of venous or arterial thromboembolism. The mean protein C antigen (PC:Ag) level was high for 48 patients with ischemic heart disease and, to a lesser extent, for 95 diabetics. In 28 patients with thrombotic strokes, 48 patients with proximal deep-vein thrombosis and in 80 patients with localized or metastatic tumors, mean PC:Ag was normal. Comparison of the pattern of changes of PC:Ag levels with those of fibrinogen, orosomucoid and prothrombin in 21 patients during the postoperative period and in 20 patients with active rheumatoid arthritis ruled out the possibility that high PC:Ag is non-specific, acute-phase reaction to inflammation, tissue injury or neoplastic growth. Therefore, high PC:Ag might be specifically related to the thrombotic tendency of these patients, but the mechanism of such a relationship remains to be clarified.


Asunto(s)
Enfermedad Coronaria/sangre , Diabetes Mellitus/sangre , Glicoproteínas/sangre , Adolescente , Adulto , Anciano , Antígenos/análisis , Femenino , Glicoproteínas/inmunología , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Proteína C , Trombosis/sangre
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