RESUMEN
Brain injury, including that due to stroke, leaves individuals with cognitive deficits that can disrupt daily aspect of living. As of now there are few treatments that shown limited amounts of success in improving functional outcome. The use of stimulants such as amphetamine have shown some success in improving outcome following brain injury. While the pharmacological mechanisms for amphetamine are known; the specific processes responsible for improving behavioral outcome following injury remain unknown. Understanding these mechanisms can help to refine the use of amphetamine as a potential treatment or lead to the use of other methods that share the same pharmacological properties. One proposed mechanism is amphetamine's impact upon noradrenaline (NA). In the current, study noradrenergic antagonists were administered prior to amphetamine to pharmacologically block α- and ß-adrenergic receptors. The results demonstrated that the blockade of these receptors disrupted amphetamines ability to induce recovery from hemispatial neglect using an established aspiration lesion model. This suggests that amphetamine's ability to ameliorate neglect deficits may be due in part to noradrenaline. These results further support the role of noradrenaline in functional recovery. Finally, the development of polytherapies and combined therapeutics, while promising, may need to consider the possibility that drug interactions can negate the effectiveness of treatment.
Asunto(s)
Antagonistas Adrenérgicos/farmacología , Anfetamina/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Norepinefrina/antagonistas & inhibidores , Trastornos de la Percepción/tratamiento farmacológico , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Trastornos de la Percepción/metabolismo , Trastornos de la Percepción/patología , Fenoxibenzamina/farmacología , Propranolol/farmacología , Ratas Long-Evans , Receptores Adrenérgicos/metabolismo , Recuperación de la Función/efectos de los fármacosRESUMEN
XRCC3 was originally identified as a human gene able to complement the DNA damage sensitivity, chromosomal instability and impaired growth of the mutant hamster cell line irs1SF. More recently, it has been cloned, sequenced and found to bear sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51. The phenotype of irs1SF and the identification of XRCC3 as a member of the RAD51 gene family have suggested a role for XRCC3 in repair of DNA damage by homologous recombination. Homologous recombinational repair (HRR) of a specifically induced chromosomal double-strand break (DSB) was assayed in irs1SF cells with and without transient complementation by human XRCC3. Complementation with XRCC3 increased the frequencies of repair by 34- to 260-fold. The results confirm a role for XRCC3 in HRR of DNA DSB, and the importance of this repair pathway for the maintenance of chromosomal integrity in mammalian cells.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/fisiología , Recombinación Genética , Animales , Bovinos , Línea Celular , Cricetinae , Daño del ADN , Proteínas de Unión al ADN/genética , Prueba de Complementación Genética , HumanosRESUMEN
The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.
Asunto(s)
Aberraciones Cromosómicas , Cromosomas Humanos Par 4/genética , Cromosomas Humanos Par 7/genética , Cromosomas/ultraestructura , Reparación del ADN/genética , Proteínas de Unión al ADN/fisiología , Animales , Células CHO/efectos de los fármacos , Células CHO/metabolismo , Células CHO/efectos de la radiación , Células CHO/ultraestructura , Cromosomas/genética , Cricetinae , Cricetulus/genética , Proteínas de Unión al ADN/genética , Resistencia a Medicamentos/genética , Prueba de Complementación Genética , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Mitomicina/farmacología , Tolerancia a Radiación/genética , Especificidad de la EspecieRESUMEN
Recently, human and rodent homologs of yeast repair genes Rad51 and Rad52 have been identified and proposed to play roles in DNA double-strand break (DSB) repair. In this study, cell cycle-dependent expression of human and rodent RAD51 and RAD52 proteins was monitored using two approaches. First, flow cytometric measurements of DNA content and immunofluorescence were used to determine the phase-specific levels of RAD51 and RAD52 protein expression in irradiated and control populations. The expression of both proteins was lowest in G0/G1, increased in S and reached a maximum in G2/M. No difference was found in the whole-cell level of RAD51 or RAD52 protein expression between gamma-irradiated and control cell populations. Second, cell cycle-dependent protein expression was confirmed by Western analysis of populations synchronized in G0, G1 and G2 phases. Analysis of V3, a hamster equivalent of SCID, indicates that the protein level increases of RAD51 and RAD52 from G0 to G1/S/G2 do not require DNA-PK.
Asunto(s)
Ciclo Celular/genética , Reparación del ADN , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Células CHO , Ciclo Celular/efectos de la radiación , Células Cultivadas , Radioisótopos de Cesio , Cricetinae , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/efectos de la radiación , Fibroblastos , Proteínas Fúngicas/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Datos de Secuencia Molecular , Recombinasa Rad51 , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , PielRESUMEN
High-resolution orientational constraints from solid-state NMR spectroscopy of uniformly aligned biological macromolecules provide a great structural analysis problem. Several approaches to this problem have been made in the past. Here a vector algebra method is developed that provides analytical solutions for the torsion angles and a concise and simple view of the structural possibilities. Numerical instabilities in this approach are easily predicted. Insight into how the structural ambiguities arise in the first place and how they can be reduced in number is demonstrated with this new approach.
Asunto(s)
Gramicidina/química , Modelos Químicos , Conformación Proteica , Espectroscopía de Resonancia Magnética , Anomalía TorsionalRESUMEN
In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.
Asunto(s)
Reparación del ADN/genética , Recombinación Genética/genética , Adenina Fosforribosiltransferasa/genética , Animales , Secuencia de Bases , Células CHO , Cromosomas/genética , Cricetinae , Daño del ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Reordenamiento Génico/genética , Marcación de Gen , Datos de Secuencia Molecular , Mutación Puntual/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae , Análisis de Secuencia de ADNRESUMEN
Functional regulation of proteins is central to living organisms. Here it is shown that a nonfunctional conformational state of a polypeptide can be kinetically trapped in a lipid bilayer environment. This state is a metastable structure that is stable for weeks just above the phase transition temperature of the lipid. When the samples are incubated for several days at 68 degrees C, 50% of the trapped conformation converts to the minimum-energy functional state. This result suggests the possibility that another mechanism for functional regulation of protein activity may be available for membrane proteins: that cells may insert proteins into membranes in inactive states pending the biological demand for protein function.
Asunto(s)
Proteínas/química , Espectroscopía de Resonancia Magnética , Conformación Proteica , Proteínas/fisiologíaRESUMEN
In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.
Asunto(s)
Cromosomas Humanos/genética , Electroporación/métodos , Endonucleasas/administración & dosificación , Recombinación Genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , ADN/genética , Marcación de Gen , Vectores Genéticos , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Datos de Secuencia MolecularRESUMEN
Tyrosine hydroxylase activity was assayed in microdissected substantia nigra and striata from seven strains of mice (BALB, CBA, YBR, WB, IS, MOLG, and CAST). In the substantia nigra where tyrosine hydroxylase activity is thought to be proportional to dopaminergic neuron number, only CBA had a different (lower) enzyme activity compared with BALB. However in the striatum, tyrosine hydroxylase activity was larger for IS, MOLG and CAST compared with BALB. Further investigation of the CAST striatum showed that dopamine content and dopamine uptake activity were also higher in comparison with BALB. All three dopaminergic parameters were larger because of lower protein levels in the CAST striatum. A lower absolute amount of glutamic acid decarboxylase activity in CAST versus BALB striatum was consistent with the possibility of a smaller CAST striatum. In contrast to dopamine, the serotonin content in CAST striatum was reduced in proportion to the decrease in protein content. We suggest that the CAST striatum is smaller than BALB striatum and is innervated by proportionally fewer serotoninergic terminals, but the amount of dopaminergic innervation of the CAST striatum is not altered by the size of the target.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/fisiología , Ratones Endogámicos BALB C/metabolismo , Muridae/metabolismo , Sustancia Negra/metabolismo , Animales , Glutamato Descarboxilasa/metabolismo , Ratones , Serotonina/metabolismo , Especificidad de la Especie , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.
Asunto(s)
Gramicidina , Canales Iónicos/fisiología , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética/métodos , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Targeted recombination in murine embryonic stem cells promises to be a powerful tool for introducing specific mutations into target genes to study development in mice and to create animal models of human disease. Gene targeting also holds potential for correcting genetic defects as an approach to human gene therapy. To precisely modify target genes, homologous recombination must proceed with high fidelity. However, several results have suggested that targeted recombination may be highly mutagenic. To test the accuracy of gene targeting we analyzed 44 independent targeted recombinants at the hypoxanthine phosphoribosyltransferase (HPRT) locus in a human fibroblast cell line and in mouse embryonic stem cells. We surveyed 80 kilobases around the sites of recombination by using chemical cleavage of mismatches. Only two mutations were found: a T----G transversion and a thymidine deletion. Thus, gene targeting in mammalian cells can be extremely accurate. These results demonstrate the feasibility of generating precise modifications of mammalian genomes by gene targeting.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Recombinación Genética , Animales , Secuencia de Bases , Fibroblastos , Vectores Genéticos , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , Células MadreRESUMEN
Immunization of adults has been deficient in the United States. According to interviews conducted during their visits to an emergency room, only 20.1% of 350 patients who fit into high-risk categories for immunization had heard of pneumococcal vaccine, whereas 82.7% had heard of influenza vaccine. Only 8.6% and 47.8%, respectively, had ever been given pneumococcal or influenza vaccine. Previous pneumococcal vaccination was six times more common (10.3% vs 1.6%) and prior influenza vaccination twice as common (52.7% vs 25.4%) in the respondents who could identify a primary care provider or clinic than in those who could not. Of the patients who had not received a specific vaccine, about 60% indicated that they would take pneumococcal or influenza vaccine if it was offered while they were in the emergency room setting. Offering vaccine in an emergency room setting promises to complement other approaches to immunizing adults at high risk for complications of influenza and pneumococcal infections.