RESUMEN
Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.
Asunto(s)
Desulfovibrio vulgaris/metabolismo , Hidrógeno/metabolismo , Proteínas de la Membrana/metabolismo , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Periplasma/metabolismo , Sulfatos/metabolismo , Anaerobiosis , Desulfovibrio vulgaris/enzimología , Transporte de Electrón , Eliminación de Gen , Proteínas de la Membrana/genética , Viabilidad Microbiana , Oxidación-Reducción , Oxidorreductasas/genética , Periplasma/enzimologíaRESUMEN
OBJECTIVE: Analysis and evaluation of a multidisciplinary approach, postoperative results and survival of a group of patients with resected pancreatic cancer after a multimodal therapy. DESIGN: DESCRIPTIVE, prospective and observational study. PATIENTS: Between January 2004 and December 2004, 124 patients with pancreatic cancer were evaluated. In 30 patients pancreatic resection was performed, and they are the object of this study. Results of preoperative evaluation, postoperative morbidity and mortality, and long term survival were studied. RESULTS: Diagnostic evaluation was completed in ambulatory basis in 20% of the patients. In 63% of cases, admission was done in the same day of surgery. In 3 patients (9%), tumor resection was not achieved, therefore, concordance between radiological and surgical resectability rate was 91%. Resectability rate was 24.1%. Surgical Mortality was 3.3%, with a global morbidity rate of 56.6%. Survival at one, two, three and, four years was 76.2%, 56.3%, 43%, y 27.3% respectively. CONCLUSIONS: Technological development and coordination of efforts in multidisciplinary teams offer an accurate evaluation of tumor involvement, and may reduce the number of laparotomies without tumor resection. The application of a systematic and generalized multimodal treatment in pancreatic cancer is progressively showing a tendency of progressive increase in resectability and survival rates in pancreatic cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/terapia , Pancreatectomía/métodos , Neoplasias Pancreáticas/terapia , Grupo de Atención al Paciente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/diagnóstico , Carcinoma/diagnóstico por imagen , Carcinoma/patología , Carcinoma/terapia , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/secundario , Quimioterapia Adyuvante , Colangiopancreatografia Retrógrada Endoscópica , Colectomía , Terapia Combinada , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Diagnóstico por Imagen , Femenino , Hepatectomía/métodos , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Pancreatectomía/mortalidad , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/patología , Pancreaticoduodenectomía , Estudios Prospectivos , Stents , Tasa de Supervivencia , GemcitabinaRESUMEN
Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.
Asunto(s)
Elementos Transponibles de ADN , Regulación de la Expresión Génica , Genes Fúngicos , Genes Reguladores , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Genotipo , Datos de Secuencia Molecular , Mutación , Mapeo RestrictivoRESUMEN
One class of Ty insertion mutation in Saccharomyces cerevisiae activates expression of adjacent structural genes. The CYC7-H2 mutation, in which a Ty1 element is inserted 5' to the iso-2-cytochrome c coding region of CYC7, causes a 20-fold increase in CYC7 expression. Deletion analysis of CYC7-H2 has shown that distal regions of the Ty1 element are not essential for the transcriptional activation at CYC7. In this report, we have analyzed Ty1 and CYC7 RNA from two CYC7-H2 deletion derivative genes to determine whether a direct correlation exists between transcription of Ty1 and transcription of the adjacent gene. Assuming that all Ty1 elements in the genome are transcribed equally, amounts of CYC7-H2 deletion derivative Ty1 RNA were found to be at least fivefold lower than the amount estimated for the average Ty1 element. These same Ty1 deletion derivatives caused a 20-fold increase in adjacent CYC7 expression. This finding suggests that the mechanism by which Ty1 activates adjacent gene expression does not require normal levels of Ty1 transcription. Two inversion derivatives of the CYC7-H2 Ty1 have also been analyzed. These derivatives did not produce any iso-2-cytochrome c or any normal CYC7 mRNA. Instead they were found to produce a Tyl-CYC7 fusion RNA. Consistent with our findings on CYC7-H2 Ty1 transcription, the amount of the fusion RNA was very low. In addition, the Ty1 inversion derivatives produced a new RNA that mapped to sequences upstream from the inverted Ty1 segment. Similar to Ty1 insertions that activate transcription, the new RNA was found to be transcribed away from Ty1.
Asunto(s)
Deleción Cromosómica , Citocromos c , Genes Fúngicos , Genes , Saccharomyces cerevisiae/genética , Transcripción Genética , Alelos , Grupo Citocromo c/genética , Mutación , Hibridación de Ácido NucleicoRESUMEN
Ty transposable element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent gene expression. Several cis-acting regulatory regions within Ty1 that are responsible for these effects were identified. A 211-base-pair (bp) region functions as an activator. This region includes the so-called U5 domain of delta and 145 bp of adjacent epsilon sequences. Unlike activation by the intact Ty1, activation by the 211-bp Ty1 subfragment is cell-type independent. The presence of a 112-bp fragment from a more distal region of Ty1 confers cell-type specificity to the activator. The 112-bp fragment includes sequences with homology to mammalian enhancers and to a yeast a/alpha control site. In addition, Ty1 regions that exert negative effects on gene expression were identified. These results demonstrate that the Ty1 transcriptional control region consists of multiple components with distinct regulatory functions.
Asunto(s)
Grupo Citocromo c/genética , Citocromos c , Elementos Transponibles de ADN , Genes Fúngicos , Genes Reguladores , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Diferenciación Celular , Deleción Cromosómica , ADN Recombinante , Regulación de la Expresión Génica , ARN de Hongos/genética , ARN Mensajero/genética , Activación TranscripcionalRESUMEN
We have determined the nucleotide sequence of both delta elements of a Ty1 transposon inserted near the CYC7 gene in the Saccharomyces cerevisiae CYC7-H2 mutant. The upstream delta element in this Ty1 has an unusual inverted repeat structure that may have been formed by an error during reverse transcription.
Asunto(s)
Elementos Transponibles de ADN , Genes Fúngicos , Mutación , Saccharomyces cerevisiae/genética , Transcripción Genética , Composición de Base , Secuencia de BasesRESUMEN
Ty1 activation of gene expression observed in haploid cell types of Saccharomyces cerevisiae requires the STE7 and STE12 gene products. An activator sequence within Ty1 that is responsive to these two regulators has been defined. Complex formation between a factor in whole-cell extracts and the DNA regulatory element showed the same dependence on the STE7 and STE12 gene products as did reporter gene expression. Base pair substitutions within the binding site abolished the ability to form the factor-DNA complex and to activate gene expression. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for gene activation. Because the predicted protein for the STE7 gene product is homologous to protein kinases, we suggest that protein phosphorylation may directly or indirectly regulate formation of this DNA-protein complex.
Asunto(s)
Regulación de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Secuencia de Bases , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Haploidia , Metilación , Transcripción Genética , Activación TranscripcionalRESUMEN
Some insertion mutations in Saccharomyces cerevisiae activate the expression of adjacent structural genes. The CYC7-H2 mutation is a Ty1 insertion 5' to the iso-2-cytochrome c coding region of CYC7. The Ty1 insertion causes a 20-fold increase in CYC7 expression in a and alpha haploid cell types of S. cerevisiae. This activation is repressed in the a/alpha diploid cell type. Previous computer analysis of the CYC7-H2 Ty1 activator region identified two related sequences with homology both to mammalian enhancers and to a yeast a/alpha control site. A 112-base-pair (bp) DNA fragment encompassing one of these blocks of homology functioned as one component of the Ty1 activator. A 28-bp synthetic oligonucleotide with the wild-type homology block sequence was also functional. A single base pair mutation within the enhancer core of the synthetic 28-bp regulatory element reduced its activation ability to near background amounts. In addition, the 112-bp Ty1 fragment by itself functioned as a target for repression of adjacent gene expression in a/alpha diploid cells.
Asunto(s)
Citocromos c , Elementos Transponibles de ADN , Elementos de Facilitación Genéticos , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Genes Reguladores , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Grupo Citocromo c/genética , Diploidia , Genes , Mutación , Hibridación de Ácido Nucleico , Plásmidos , Transcripción GenéticaRESUMEN
Diaphragm disease (DD) induced by non-steroidal anti-inflammatory drugs (NSAIDs) and neuromuscular and vascular hamartoma (NMVH) are rare entities that can be difficult to recognize clinically, radiologically and pathologically. Both may cause small bowel obstruction and both reveal annular constrictions macroscopically. However, the 2 entities differ in that the constrictions are purely fibrous in DD whereas they have a hamartomatous histopathology in NMVH. We describe the case of a 76-year-old woman with a history of prolonged NSAID use who developed clinical features and gross pathology consistent with DD and histopathology of NMVH.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Hamartoma/inducido químicamente , Enfermedades del Íleon/inducido químicamente , Obstrucción Intestinal/etiología , Anciano , Femenino , Hamartoma/patología , Humanos , Enfermedades del Íleon/patologíaRESUMEN
An increase in bisphosphoglycerate mutase (BPGM) and a decrease in pyruvate kinase (PK), i.e. a decrease in PK/BPGM ratio, was observed in red cell populations from anemic rats containing 95% down to 3% reticulocytes in blood. Such a ratio has been used to study the fractionation of recticulocytes, according to their degree of maturation, after counter-current distribution of those cell populations in dextranpoly (ethylene glycol) two-phase systems. When applying this procedure to the fractionation according to age of erythrocytes from normal rats, the decrease of PK with cellular age was observed without a significant variation in BPGM activity.
Asunto(s)
Bisfosfoglicerato Mutasa/sangre , Envejecimiento Eritrocítico , Eritrocitos/enzimología , Fosfotransferasas/sangre , Piruvato Quinasa/sangre , Reticulocitos/enzimología , Anemia/enzimología , Animales , Eritropoyesis , RatasRESUMEN
The kinetics of the reaction of the thiol residue in Zn(2+)-dependent beta-lactamase II with 5,5'-dithiobis[2-nitrobenzoic acid], and the concomitant inactivation revealed that both events take place at the same rate. The inactivation could not be reverted by incubation with Zn2+ or by using a substrate concentration about eight times the Km of the enzyme. EDTA incubation also produced inactivation of the enzyme, although it was reverted by increasing the substrate concentration in the assay. A dual role is proposed for Zn2+ in beta-lactamase. The kinetic analysis of the thiol modification and the concomitant inactivation is in agreement with previous reports on the implication of the metal ion in catalysis. A role in stabilizing the native structure of the enzyme is also suggested.
Asunto(s)
Cefalosporinasa/química , Ácido Ditionitrobenzoico/química , Zinc/química , Bacillus/enzimología , Cefalosporinasa/metabolismo , Ácido Ditionitrobenzoico/metabolismo , Activación Enzimática , Cinética , Zinc/metabolismoRESUMEN
The system composed of 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) and Zn2+ catalyses the degradation of cephalosporins. The beta-lactam opening fits to a first-order process, with a constant directly proportional to the zinc ion concentration. The pH and Tris concentration dependency displayed by the first-order constant, as well as the nature of the degradation products point to a mechanism that can be considered as an extension of that proposed for the benzylpenicillin degradation. The mechanism proposed here, and the values of the kinetic constants calculated, as compared with those of beta-lactamases, lead to the conclusion that the Tris-Zn2+ system simulates the catalytic action of the serine beta-lactamases rather than the action of the Zn(2+)-dependent type of enzymes.
Asunto(s)
Antibacterianos/química , Trometamina , Zinc , beta-Lactamasas/metabolismo , Antibacterianos/metabolismo , Cefaloridina/química , Cinética , Matemática , Modelos Químicos , Modelos Teóricos , Relación Estructura-ActividadRESUMEN
Infectious acute gastroenteritis (AGE) is a major health problem worldwide. Salmonella is a leading cause of AGE outbreaks, but viruses may be responsible for up to 80% of cases. We compared the frequency and characteristics of AGE out breaks in Catalonia due to norovirus and Salmonella and the changes in these outbreaks from 2000 through 2010. In 2006 through 2010, we also investigated the distribution by season, setting, and implicated food, the incidence rates of cases associated, and the hospitalization rates. Differences in proportions were estimated by Pearson's chi-square test, and the odds ratio (OR) and 95% confidence interval (CI) were calculated. In 2000 through 2010, the number of AGE outbreaks caused by Salmonella decreased and those caused by norovirus significantly increased. From 2006 onward, norovirus was the most common etiology in AGE outbreaks, but in foodborne outbreaks, Salmonella was the more common cause until 2010. The incidence rate per 10(5) inhabitants was greater for norovirus (20.81 versus 3.97, P < 0.001), and the hospitalization rate was lower for norovirus (0.84 versus 4.69, P < 0.001). Salmonella infections occurred more frequently in the warmer months, and norovirus infections were more common in the colder months, both in terms of total outbreaks (OR = 4.50; 95% CI, 2.85 to 7.11; P < 0.001) and foodborne outbreaks (OR = 4.38; 95% CI, 2.42 to 7.95; P < 0.001). Norovirus infections were less common in private homes (OR = 0.08; 95% CI, 0.04 to 0.14; P < 0.001) and more common in nursing homes (P < 0.001) and hospitals or long-term care facilities (OR = 14.09; 95% CI, 3.35 to 59.33; P < 0.001). Foods most frequently implicated in norovirus infection outbreaks were seafood (22% ; OR = 7.89; 95% CI, 2.59 to 24.3; P < 0.001), and those most common in Salmonella infection outbreaks were mayonnaise and similar items (30.2%; OR = 0.05; 95% CI, 0.01 to 0.22; P < 0.001). Foodborne outbreaks in which the vehicle was not identified were more frequent in cases of norovirus infection (OR = 4.59; 95% CI, 2.54 to 8.30; P < 0.001). Our results indicate that norovirus rather than Salmonella is the most common cause of AGE outbreaks in Catalonia. Foodborne AGE outbreaks were more commonly caused by norovirus than by Salmonella only in 2010, the last year of the study.
Asunto(s)
Infecciones por Caliciviridae/epidemiología , Gastroenteritis/epidemiología , Norovirus/aislamiento & purificación , Intoxicación Alimentaria por Salmonella/epidemiología , Enfermedad Aguda , Adulto , Infecciones por Caliciviridae/transmisión , Preescolar , Infección Hospitalaria , Brotes de Enfermedades , Gastroenteritis/microbiología , Gastroenteritis/virología , Hospitalización , Hospitales , Humanos , Masculino , Norovirus/patogenicidad , Casas de Salud , Salmonella/aislamiento & purificación , Salmonella/patogenicidad , Intoxicación Alimentaria por Salmonella/transmisión , Estaciones del Año , España/epidemiologíaRESUMEN
INTRODUCTION: Autoimmune hepatitis is a necroinflammatory disorder of unknown etiology characterized by the presence of circulating antibodies, hypergammaglobulinemia, and response to immunosuppression. It has the histological features of chronic hepatitis. The onset is usually insidious, but in some patients the presentation may be acute and occasionally severe. Certain drugs can induce chronic hepatitis mimicking autoimmune hepatitis. Different autoantibodies have been associated with this process but they are not detectable after drug withdrawal and clinical resolution. CASE PRESENTATION: We describe a case of drug-induced acute hepatitis associated with antinuclear, antisoluble liver-pancreas and anti-smooth muscle autoantibodies in a 66-year-old woman. Abnormal clinical and biochemical parameters resolved after drug withdrawal, but six months later anti-soluble liver-pancreas antibodies remained positive and liver biopsy showed chronic hepatitis and septal fibrosis. Furthermore, our patient has a HLA genotype associated with autoimmune hepatitis. CONCLUSION: Patient follow-up will disclose whether our patient suffers from an autoimmune disease and if the presence of anti-soluble liver antigens could precede the development of an autoimmune hepatitis, as the presence of antimitochondrial antibodies can precede primary biliary cirrhosis.
RESUMEN
A large outbreak of gastroenteritis occurred in Catalonia in June 2002 with 1435 cases and 117 hospitalizations. Consumption of a hard pastry with vanilla cream was strongly associated with illness. Stool samples from cases and food-handlers were analysed. The premises of the food manufacturer were inspected and food samples were taken for microbiological analysis. Salmonella serotype Enteriditis was isolated from 154 cases, three food-handlers and nine food samples. Outbreak-associated strains showed a coincident phage type, antibiotype and pulse-field gel electrophoresis pattern. Inadequate handling of foods containing eggs occurred because the establishment exceeded its safe food production capacity to meet demand for the pastry, which was consumed on the day of a traditional festival. Excessive production of foods for holidays or special events represents a potential public health threat.
Asunto(s)
Brotes de Enfermedades , Microbiología de Alimentos , Intoxicación Alimentaria por Salmonella/epidemiología , Salmonella enteritidis/aislamiento & purificación , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Productos Lácteos/microbiología , Heces/microbiología , Femenino , Manipulación de Alimentos , Vacaciones y Feriados , Humanos , Lactante , Masculino , Persona de Mediana Edad , Intoxicación Alimentaria por Salmonella/microbiología , Salmonella enteritidis/clasificación , España/epidemiologíaRESUMEN
A yeast-Escherichia coli shuttle vector containing the M13 origin of replication has been constructed. This vector allows selection and replication in both Saccharomyces cerevisiae and E. coli, as well as single-stranded packaging from E. coli upon infection with a helper phage. The presence of a polylinker with various unique restriction sites facilitates the cloning of desired genes.
Asunto(s)
Colifagos/genética , Replicación del ADN , Escherichia coli/genética , Vectores Genéticos , Plásmidos , Saccharomyces cerevisiae/genética , Secuencia de Bases , Clonación Molecular , Escherichia coli/crecimiento & desarrollo , Mitosis , Datos de Secuencia Molecular , Mapeo Restrictivo , Saccharomyces cerevisiae/citologíaRESUMEN
In this study we report the isolation of temperature-sensitive mutants that affect pre-mRNA splicing. A bank of approximately 1000 temperature-sensitive Saccharomyces cerevisiae strains was generated and screened on RNA gel blots by hybridization with an actin intron probe. We isolated 16 mutants defining 11 new complementation groups prp(rna)17-prp(rna)27 with four phenotypic classes of mutants and 21 mutants in the prp2-prp11 complementation groups (formerly rna2-rna11). The majority of the complementation groups share a phenotype of pre-mRNA accumulation, seen in all of the prp(rna)2-prp(rna)11 mutants. Three novel classes of mutants were isolated in this study. One class, consisting of two complementation groups, exhibits an accumulation of the lariat intermediate of splicing, with no change in the levels of pre-mRNA. The second class, also represented by two complementation groups, shows an accumulation of the intron released after splicing. The third novel class, comprising one complementation group, accumulates both pre-mRNA and the released intron. All mutants isolated were recessive for the splicing phenotype. Only 2 of the 11 complementation groups, although recessive, were not temperature sensitive. This study, together with previous isolation of the prp(rna)2-prp(rna)11 groups and the spliceosomal snRNAs, puts at least 26 gene products involved directly or indirectly in pre-mRNA splicing.
Asunto(s)
Precursores del ARN/genética , Empalme del ARN , Saccharomyces cerevisiae/genética , Alelos , Genes Fúngicos , Genes Letales , Prueba de Complementación Genética , Intrones , Mutación , Fenotipo , ARN de Hongos/genética , TemperaturaRESUMEN
The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas Fúngicas/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ARN Helicasas DEAD-box , Prueba de Complementación Genética , Calor , Sustancias Macromoleculares , Datos de Secuencia Molecular , Familia de Multigenes , ARN Helicasas , ARN Nucleotidiltransferasas/genética , Factores de Empalme de ARN , Mapeo Restrictivo , Saccharomyces cerevisiaeRESUMEN
Prp24 was previously isolated as a suppressor of a cold-sensitive U4 mutation and is required for at least the first step of splicing in vitro. Our investigation of the in vitro RNA binding properties of the purified Prp24 protein shows that it binds preferentially to the U4/U6 hybrid snRNAs compared to other snRNAs. The interaction between Prp24 and the U4/U6 hybrid appears to involve two regions in the RNA: the 39-57 region of U6 and stem II of the U4/U6 hybrid. Interestingly, some U4 mutations, which destabilize stem II, increase the affinity of Prp24 for the U4/U6 RNAs compared to the wild type. This suggests that the binding of Prp24 to the U4/U6 RNAs may involve some destabilization of the RNA duplex. We also found that Prp24 can stimulate the annealing of U4 and U6, suggesting that Prp24 participates in both the formation and disassembly of the U4/U6 hybrid during splicing.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Secuencia de Bases , Radical Hidroxilo , Ligandos , Datos de Secuencia Molecular , Mutación , Sondas de Oligonucleótidos , ARN/metabolismo , Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Empalmosomas/metabolismo , Relación Estructura-Actividad , TemperaturaRESUMEN
Glycogen phosphorylase (EC 2.4.1.1) may be assayed in the glycogen degradation direction by a continuous spectrophotometric method. The formation of glucose 1-phosphate from glycogen and phosphate produces a controlled change of pH which can be measured by the changes in absorbance of phenol red added to the system. The procedure may be conveniently applied to a stopped-flow spectrophotometer to measure the rate of the reaction. Therefore the activity of the enzyme may be determined at low conventional concentrations and, by the same technique, at high enzyme concentrations approaching those supposed to exist in vivo.