RESUMEN
The nusinersen development and approval process provide important lessons regarding the pathway to marketing approval for gene therapies. These lessons emphasize rigorous clinical trial design, flexibility in trial design and analysis, a collaborative effort with regular communications between the drug developer and the Food and Drug Administration (FDA), and use of FDA's expedited programs. These lessons are critical to the development of gene therapies for the treatment of serious or life-threatening rare diseases.
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Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos/administración & dosificación , Animales , Ensayos Clínicos como Asunto , Terapia Genética/efectos adversos , Terapia Genética/legislación & jurisprudencia , Humanos , Atrofia Muscular Espinal/genética , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Small for gestational age (SGA) children exhibiting catch-up (CU) growth have a greater risk of cardiometabolic diseases in later life compared with non-catch-up (NCU) SGA children. The aim of this study was to establish differences in metabolism and gene expression profiles between CU and NCU at age 4-9 years. CU children (n=22) had greater height, weight and body mass index standard deviation scores along with insulin-like growth factor-I (IGF-I) and fasting glucose levels but lower adiponectin values than NCU children (n=11; all P<0.05). Metabolic profiling demonstrated a fourfold decrease of urine myo-inositol in CU compared with NCU (P<0.05). There were 1558 genes differentially expressed in peripheral blood mononuclear cells between the groups (P<0.05). Integrated analysis of data identified myo-inositol related to gene clusters associated with an increase in insulin, growth factor and IGF-I signalling in CU children (P<0.05). Metabolic and transcriptomic profiles in CU SGA children showed changes that may relate to cardiometabolic risk.
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Recién Nacido Pequeño para la Edad Gestacional/crecimiento & desarrollo , Transcriptoma , Biomarcadores , Niño , Preescolar , Femenino , Humanos , Recién Nacido , Recién Nacido Pequeño para la Edad Gestacional/metabolismo , Masculino , MetabolómicaRESUMEN
Newell & Shanks (N&S) argue against the idea that any significant role for unconscious influences on decision making has been established by research to date. Inasmuch as this conclusion applies to the idea of an "intelligent cognitive unconscious," we would agree. Our concern is that the article could lead the unwary to conclude that there are no unconscious influences on decision making - and never could be. We give reasons why this may not be the case.
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Toma de Decisiones , Inconsciente en Psicología , HumanosRESUMEN
The power densities of microbial fuel cells with yeast cells as the anode catalyst were significantly increased by immobilizing the yeast in electrically conductive alginate electrodes. The peak power densities measured as a function of the electrical conductivity of the immobilized electrodes show that although power increases with rising electrical conductivity, it tends to saturate beyond a certain point. Changing the pH of the anode compartment at that point seems to further increase the power density, suggesting that proton transport limitations and not electrical conductivity will limit the power density from electrically conductive immobilized anodes.
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Alginatos/química , Fuentes de Energía Bioeléctrica/microbiología , Saccharomyces cerevisiae/citología , Conductividad Eléctrica , Electrodos , Diseño de Equipo , Ácido Glucurónico/química , Ácidos Hexurónicos/químicaRESUMEN
Human immunoglobulin M (IgM) rheumatoid factors (RFs) show primary direct enzyme-linked immunosorbent assay (ELISA) reactivity with Fab rather than Fc fragments of monoclonal antibody (mAb) II-481 directed against the Fc gamma-binding site of herpes simplex virus glycoprotein gE. This preferential anti-Fab specificity suggests that RFs react with antigen-binding portions of mAb II-481 as anti-idiotypic antibodies directed at the combining site regions of mAb reacting with the Fc gamma-binding region of gE. Analysis of this idiotype-anti-idiotype reaction employed polymerase chain reaction amplification and sequencing of the variable heavy and light (VH and VL) regions of mAb II-481. When VH and VL regions of mAb II-481 were synthesized as overlapping 7-mer peptides on polypropylene pins, a panel of 10 polyclonal and 6 monoclonal human IgM RFs reacted primarily with epitopes within the three solvent-exposed mAb II-481 complementarity determining regions (CDRs). Preincubation of single CDR heptamer peptides with IgM RFs in free solution, resulted in 63-100% inhibition of RF binding to mAb II-481 on the ELISA plate, confirming the antigenic importance of linear CDR regions for RF reactivity. Combinations of two or three CDR peptides frequently produced 94-100% inhibition of RF binding to whole mAb II-481. Control peptides, singly or in combination, showed no inhibition. Computer modeling suggested that the RF-reactive mAb II-481 Fv region and a previously demonstrated RF-reactive CH3 epitope displayed considerable three-dimensional similarities in conformation. These studies may provide insight into limited shape homologies possibly involved in an RF anti-idiotypic reaction.
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Anticuerpos Monoclonales/química , Epítopos Inmunodominantes , Fragmentos de Inmunoglobulinas/química , Inmunoglobulina G/metabolismo , Inmunoglobulina M/inmunología , Factor Reumatoide/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Simulación por Computador , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Ratones , Datos de Secuencia Molecular , Conformación Proteica , Proteínas del Envoltorio Viral/metabolismoRESUMEN
BACKGROUND: Previous research indicates that individuals with seasonal depression (SD) do not exhibit the memory biases for negative self-referent information that characterize non-seasonal depression (NSD). The current study extended this work by examining processing of self-referent emotional information concerning potential future events in SD. METHOD: SD and NSD patients, along with never-depressed controls, completed a scenario-based measure of likelihood estimation for future positive and negative events happening either to the self or to another person. RESULTS: SD patients estimated future negative events as more likely to happen to both the self and others, relative to controls. In contrast, in the NSD sample this bias was specific to self-referred material. There were no group differences for positive events. CONCLUSIONS: These data provide further evidence that the self-referent bias for processing negative information that characterizes NSD can be absent in SD, this time in the domain of future event processing.
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Trastorno Depresivo/psicología , Emociones , Acontecimientos que Cambian la Vida , Trastorno Afectivo Estacional/psicología , Adolescente , Adulto , Cognición , Femenino , Desamparo Adquirido , Humanos , Funciones de Verosimilitud , Masculino , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Autoimagen , Disposición en Psicología , Adulto JovenRESUMEN
AIMS: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. METHODS AND RESULTS: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed-field gel electrophoresis blots, we analysed the copy number and localization of several maltose-related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. CONCLUSIONS: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.
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Fermentación , Maltosa/metabolismo , Saccharomyces/genética , Trisacáridos/metabolismo , Variaciones en el Número de Copia de ADN , Electroforesis en Gel de Campo Pulsado , Genes Fúngicos , Cariotipificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Saccharomyces/crecimiento & desarrollo , Saccharomyces/metabolismoRESUMEN
As a way of studying nucleosome assembly and maintenance in Saccharomyces cerevisiae, mutants bearing deletions or duplications of the genes encoding histones H2A and H2B were analyzed. Previous genetic analysis had shown that only one of these mutants exhibited dramatic and pleiotropic phenotypes. This mutant was also the only one that contained disrupted chromatin, suggesting that the original phenotypes were attributable to alterations in chromosome structure. The chromatin disruption in the mutant, however, did not extend over the entire genome, but rather was localized to specific regions. Thus, while the arrangement of nucleosomes over the HIS4 and GAL1 genes, the telomeres, and the long terminal repeats (delta sequences) of Ty retrotransposons appeared essentially normal, nucleosomes over the CYH2 and UBI4 genes and the centromere of chromosome III were dramatically disrupted. The observation that the mutant exhibited localized chromatin disruptions implies that the assembly or maintenance of nucleosomes differs over different parts of the yeast genome.
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Cromatina/ultraestructura , Histonas/genética , Saccharomyces cerevisiae/ultraestructura , Centrómero/ultraestructura , Cromatina/fisiología , Deleción Cromosómica , Elementos Transponibles de ADN , Galactosa , Regulación de la Expresión Génica , Genes Fúngicos , Histidina , Mutación , Fenotipo , ARN Mensajero/genética , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Novel sol-gel synthetic techniques were used to immobilize copper-zinc superoxide dismutase (CuZnSOD), cytochrome c, and myoglobin (Mb) by encapsulation in stable, optically transparent, porous silica glass matrices under mild conditions such that the biomolecules retained their characteristic reactivities and spectroscopic properties. The resulting glasses allowed transport of small molecules into and out of the glasses at reasonable rates but nevertheless retained the protein molecules within their pores. Chemical reactions of the immobilized proteins could be monitored by means of changes in their visible absorption spectra. Silica glasses containing the immobilized proteins were observed to have similar reactivities and spectroscopic properties to those found for the proteins in solution. For example, encapsulated CuZnSOD was demetallated and remetallated, encapsulated ferricytochrome c was reduced and then reoxidized, and encapsulated met Mb was reduced to deoxy Mb and then reacted either with dioxygen to make oxy Mb or with carbon monoxide to make carbonyl Mb.
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Vidrio , Proteínas/química , Animales , Bovinos , Grupo Citocromo c/química , Geles , Caballos , Mioglobina/química , Soluciones , Análisis Espectral , Superóxido Dismutasa/químicaRESUMEN
We present a case of fulminant leptospirosis that was acquired in the suburban area by a 48-year-old male renal transplant recipient. He developed acute renal and hepatic failure with profound jaundice. Spirochetes were identified on liver biopsy. Weil's disease was suspected, and the diagnosis was further supported by a positive serum Leptospira interrogans icterohaemorrhagiae antibody titer. Unfortunately, he suffered from recurrent lower gastrointestinal bleeding, had a prolonged hospital course, and eventually succumbed to overwhelming sepsis. This case is the third report to our knowledge of leptospirosis in a renal transplant recipient in the English literature.
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Trasplante de Riñón/efectos adversos , Leptospira interrogans serovar icterohaemorrhagiae , Leptospirosis/complicaciones , Lesión Renal Aguda/etiología , Biopsia , Resultado Fatal , Humanos , Leptospira interrogans serovar icterohaemorrhagiae/clasificación , Leptospira interrogans serovar icterohaemorrhagiae/aislamiento & purificación , Leptospirosis/microbiología , Hígado/microbiología , Fallo Hepático Agudo/etiología , Masculino , Persona de Mediana Edad , Sepsis/etiologíaRESUMEN
Two groups of adult male Munich-Wistar rats and a third group of nondiabetic age-matched and weight-matched normal control rats underwent micropuncture study 1 mo, and morphologic studies 14 mo, after induction of streptozotocin diabetes or sham treatment. All animals were fed standard rat chow. Diabetic rats received daily ultralente insulin to maintain stable moderate hyperglycemia (approximately 350 mg/dl). In addition, one group of diabetic rats was treated with the angiotensin I converting enzyme inhibitor, enalapril, 15 mg/liter of drinking water. Average kidney weight, whole kidney and single-nephron glomerular filtration rate, and glomerular plasma flow rate were elevated to similar values in both groups of diabetic rats, relative to normal control rats. Non-enalapril-treated diabetic rats exhibited significant elevations in mean glomerular capillary hydraulic pressure and transcapillary hydraulic pressure gradient, compared with the other groups studied, and only this group eventually developed marked and progressive albuminuria. Likewise, histological examination of the kidneys at 14 mo disclosed a high incidence of glomerular structural abnormalities only in non-enalapril-treated diabetic rats. These findings indicate that prevention of glomerular capillary hypertension in rats with diabetes mellitus effectively protects against the subsequent development of glomerular structural injury and proteinuria. This protection is afforded despite pronounced hyperglycemia and elevated levels of glucosylated hemoglobin, further supporting our view that hemodynamic rather than metabolic factors predominate in the pathogenesis of diabetic glomerulopathy.
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Nefropatías Diabéticas/prevención & control , Hipertensión Renal/tratamiento farmacológico , Glomérulos Renales/irrigación sanguínea , Inhibidores de la Enzima Convertidora de Angiotensina , Animales , Enalapril/uso terapéutico , Tasa de Filtración Glomerular/efectos de los fármacos , Hemoglobina Glucada/análisis , Hemodinámica , Riñón/patología , Masculino , Microcirculación , Ratas , Flujo Sanguíneo Regional , Factores de TiempoRESUMEN
The presence of H2A-H2B dimers in nucleosomes can inhibit the binding of transcription factors to chromatin templates. To study the roles of histones H2A and H2B in transcriptional repression in vivo, mutant forms of these histones were analyzed in two different assay systems. Two repression domains were identified in H2A. One domain includes residues that fall in the beginning of the H2A-H2B dimerization region, and the second is in the H2A N terminus, a region of potential interactions with nonhistone proteins. The function of H2A and H2B in one repression assay was found to be dependent on three SPT (suppressor of Ty) genes whose products are important for chromatin-mediated repression. These results suggest that repressive chromatin structure may be established through the interactions of the Spt proteins with these histones. In contrast, other proteins, the products of the HIR (histone regulation) genes, may function to direct H2A and H2B to specific promoters.
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Histonas/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cartilla de ADN/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Histonas/química , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Supresión Genética , Transcripción GenéticaRESUMEN
OBJECTIVE: To search for studies on tongue-lip adhesion and tongue repositioning used as isolated treatments for obstructive sleep apnoea in children with Pierre Robin sequence. METHODS: A systematic literature search of PubMed/Medline and three additional databases, from inception through to 8 July 2016, was performed by two authors. RESULTS: Seven studies with 90 patients (59 tongue-lip adhesion and 31 tongue repositioning patients) met the inclusion criteria. Tongue-lip adhesion reduced the mean (± standard deviation) apnoea/hypopnoea index from 30.8 ± 22.3 to 15.4 ± 18.9 events per hour (50 per cent reduction). The apnoea/hypopnoea index mean difference for tongue-lip adhesion was -15.28 events per hour (95 per cent confidence interval = -30.70 to 0.15; p = 0.05). Tongue-lip adhesion improved the lowest oxygen saturation from 75.8 ± 6.8 to 84.4 ± 7.3 per cent. Tongue repositioning reduced the apnoea/hypopnoea index from 46.5 to 17.4 events per hour (62.6 per cent reduction). Tongue repositioning improved the mean oxygen saturation from 90.8 ± 1.2 to 95.0 ± 0.5 per cent. CONCLUSION: Tongue-lip adhesion and tongue repositioning can improve apnoea/hypopnoea index and oxygenation parameters in children with Pierre Robin sequence and obstructive sleep apnoea.
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Labio/cirugía , Síndrome de Pierre Robin/complicaciones , Apnea Obstructiva del Sueño/cirugía , Lengua/cirugía , Niño , Humanos , Consumo de Oxígeno , Índice de Severidad de la Enfermedad , Apnea Obstructiva del Sueño/congénito , Apnea Obstructiva del Sueño/fisiopatología , Adherencias Tisulares , Resultado del TratamientoRESUMEN
At a meeting of the EU/US/Clinical Trials in Alzheimer's Disease (CTAD) Task Force in December 2016, an international group of investigators from industry, academia, and regulatory agencies reviewed lessons learned from ongoing and planned prevention trials, which will help guide future clinical trials of AD treatments, particularly in the pre-clinical space. The Task Force discussed challenges that need to be addressed across all aspects of clinical trials, calling for innovation in recruitment and retention, infrastructure development, and the selection of outcome measures. While cognitive change provides a marker of disease progression across the disease continuum, there remains a need to identify the optimal assessment tools that provide clinically meaningful endpoints. Patient- and informant-reported assessments of cognition and function may be useful but present additional challenges. Imaging and other biomarkers are also essential to maximize the efficiency of and the information learned from clinical trials.
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Enfermedad de Alzheimer/prevención & control , Ensayos Clínicos como Asunto , Nootrópicos/uso terapéutico , Comités Consultivos , Enfermedad de Alzheimer/diagnóstico , Unión Europea , Humanos , Evaluación de Resultado en la Atención de Salud , Estados UnidosRESUMEN
Trisomy 18 is usually a lethal chromosomal abnormality and is the second most common autosomal trisomy in humans, with an incidence of 1:8000 live births. It is commonly associated with abnormalities of the lower and upper extremities, having the frequency of 95% and 65%, respectively. A newborn female olive baboon (Papio hamadryas anubis) was diagnosed with intrauterine growth retardation and severe arthrogryposis-like congenital joint deformities. Cytogenetic analysis including G-banding and fluorescence in situ hybridization (FISH) revealed that the congenital abnormalities were associated with chromosomal mosaicism for trisomy 18. Genetic analysis with microsatellites from chromosome 18 confirmed the maternal origin of the extra chromosome 18. This is the first report of trisomy 18 in the baboon, which may be a promising animal model of human disease.
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Anomalías Múltiples/veterinaria , Enfermedades de los Monos/genética , Papio hamadryas/genética , Trisomía , Anomalías Múltiples/genética , Animales , Animales Recién Nacidos , Aberraciones Cromosómicas , Mapeo Cromosómico , Cromosomas Humanos , Cromosomas de los Mamíferos , Femenino , Humanos , Masculino , LinajeRESUMEN
The binding of p.o. benzo(a)pyrene (BP) to the DNA of mice was investigated. With a single dose of 1 microgram, levels of DNA binding were highest in the liver, followed by the intestine, colon, and stomach. In all organs, the majority of DNA-associated radioactivity was in the form of adducts which did not release ethyl acetate-soluble BP tetrols on acid hydrolysis. In both stomach and liver, the formation of acid-hydrolyzable and non-acid-hydrolyzable BP-DNA adducts was linearly related to dose, over a carcinogen dosage range of 10(-8) to 10(-3) g (liver) or 10(-7) to 10(-3) g (stomach). Repair or removal via cell turnover of liver BP-DNA adducts over a period of 7 days proceeded with the same efficiency when the dose of the administered carcinogen was varied over a range of 100,000-fold. These results suggest that in vivo the initial interaction between DNA and ingested BP takes place in the same manner both at high doses typical of laboratory carcinogenesis experiments and at low doses typical of human exposure.
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Benzopirenos/metabolismo , ADN/metabolismo , Administración Oral , Animales , Benzo(a)pireno , Benzopirenos/administración & dosificación , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/metabolismo , Hígado/metabolismo , Masculino , Ratones , Compuestos Policíclicos/metabolismoRESUMEN
Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P1 dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using 32P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to 32P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and 32P-postlabeling were 70.1 +/- 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.
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ADN/análisis , Peces/genética , Contaminantes Químicos del Agua/toxicidad , Contaminantes del Agua/toxicidad , Animales , Cromatografía Líquida de Alta Presión , Hígado/análisis , Neoplasias Hepáticas/inducido químicamenteRESUMEN
Max protein forms specific DNA-binding dimeric complexes with itself and with proteins of the c-myc gene family. A large volume of data has accumulated on the role of the c-myc proto-oncogene in cell proliferation, differentiation and tumorigenesis. To elucidate the role of max in regulating c-myc functions and the effect of both proteins on cell proliferation and differentiation, we transfected murine erythroleukemia (MEL) cells with a full-length wild-type (wt) human max gene and a mutant containing a double point mutation in the basic region (bm), which abolishes specific DNA binding. All clones expressing wt-max grow slowly, and the process of inducer-mediated differentiation is delayed. Furthermore, cells transfected with the mutated max exhibit growth retardation, accumulation in the G0/G1 phase of the cell cycle and spontaneous differentiation. Our findings are consistent with a model in which a large excess of wt-Max in the cells enhances the formation of Max-Max growth-suppressor complexes, while elevated bm-Max deprives the cell of growth-promoting Myc-Max heterodimers in a dominant-negative manner, presumably by inactivating endogenous Myc and Max.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Genes myc , Leucemia Eritroblástica Aguda/patología , Factores de Transcripción , Acetamidas/farmacología , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Ciclo Celular , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Expresión Génica , Ratones , Datos de Secuencia Molecular , Mutación , Proto-Oncogenes Mas , Transfección , Células Tumorales Cultivadas , Zinc/farmacologíaRESUMEN
Like most proteolytic enzymes, the aspartic proteinases bind substrates and most inhibitors within an extended active site cleft. Bound ligands typically adopt a beta-strand conformation. Interactions with groups on both sides of the cleft determine the primary as well as secondary specificity of the enzymes. We have pursued the discovery of the sometimes subtle distinctions between members of the aspartic proteinase family by two routes. In the first case, we have constructed sets of oligopeptide substrates with systematic variation in each position to assess interactions at one position at a time. In the second type of experiment, we have altered residues of the enzymes in order to test theories of selectivity. The combination of the two approaches has provided a better understanding of the forces involved in determining specificity of enzyme action.
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Ácido Aspártico Endopeptidasas/metabolismo , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión , Catepsina D/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Oligopéptidos/síntesis química , Pepsina A/metabolismo , Proteínas Protozoarias , Especificidad por SustratoRESUMEN
The tissue specificity of a chromosomal protein fraction, extractable from chromatin with 5 M urea at low ionic strength, has been examined in HeLa, A549 and HT 29 cells. Electrophoresis in polyacrylamide gels indicates that each cell type has a different content of 5 M urea soluble proteins which are distinguishable from the histones, from the tight DNA-binding proteins and from the high-mobility-group chromosomal proteins. Antibodies against 5 M urea soluble proteins extracted from HeLa cells were produced in mice. Although each of the mice tested prior to immunization contained a detectable amount of antibodies against both the 5 M urea soluble proteins and tight DNA-binding proteins, immunization elevated the level of the antibodies in the serum over 100-fold. The antibodies do not distinguish between the 5 M urea extracts obtained from different sources because most of the antibodies are directed against antigens shared by the cells studied. Immunofluorescence studies reveal that components which cross-react with 5 M urea soluble chromosomal proteins are also present in the cytoplasm. We conclude the following. (1) 5 M urea extracts from chromatin a group of proteins which differs among cells. (2) Mice contain detectable amounts of autoantibodies against these chromosomal proteins. (3) Immunization with the 5 M urea extractable fraction elicits antibodies against a restricted number of antigenic components which are shared among the cells studied. (4) 5 M urea extractable proteins are found both in the nucleus and cytoplasm; part of these may be cytoskeletal elements. Because the antisera do not react with histones, high-mobility-group proteins and tight DNA-binding proteins, they may be used for various functional studies on the 5 M urea extractable chromosomal protein fraction.