RESUMEN
BACKGROUND: Pancreatic neuroendocrine tumours (Pan-NETs) are rare tumours that often present with or develop liver metastases. The aim of this retrospective study was to evaluate liver surgery and thermal hepatic ablation (THA) of Pan-NET liver metastases and to compare the outcomes with those of a control group. METHOD: Patients with Pan-NET treated in Uppsala University Hospital and Sahlgrenska University Hospital from 1995-2018 were included. Patient records were scrutinized for baseline parameters, survival, treatment and complications. RESULTS: Some 108 patients met the criteria for inclusion; 57 patients underwent treatment with liver surgery or THA and 51 constitute the control group. Median follow-up was 3.93 years. Five-year survival in the liver surgery/THA group was 70.6 (95 per cent c.i. 0.57 to 0.84) per cent versus 42.4 (95 per cent c.i. 40.7 to 59.1) per cent in the control group (P = 0.016) and median survival was 9.1 (95 per cent c.i. 6.5 to 11.7) versus 4.3 (95 per cent c.i. 3.4-5.2) years. In a multivariable analysis, surgery or THA was associated with a decreased death-years rate (hazard ratio 0.403 (95 per cent c.i. 0.208 to 0.782, P = 0.007). CONCLUSION: Liver surgery and/or THA was associated with longer overall survival in Pan-NET with acceptable mortality and morbidity rates. These treatments should thus be considered in Pan-NET patients with reasonable tumour burden in an intent to alleviate symptoms and to improve survival.
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Neoplasias Hepáticas , Neoplasias Pancreáticas , Hepatectomía , Humanos , Neoplasias Hepáticas/cirugía , Neoplasias Pancreáticas/cirugía , Estudios RetrospectivosRESUMEN
BACKGROUND: Traditionally, perforated diverticulitis with purulent peritonitis was treated with resection and colostomy (Hartmann's procedure), with inherent complications and risk of a permanent stoma. The DILALA (DIverticulitis - LAparoscopic LAvage versus resection (Hartmann's procedure) for acute diverticulitis with peritonitis) and other randomized trials found laparoscopic lavage to be a feasible and safe alternative. The medium-term follow-up results of DILALA are reported here. METHODS: Patients were randomized during surgery after being diagnosed with Hinchey grade III perforated diverticulitis at diagnostic laparoscopy. The primary outcome was the proportion of patients with one or more secondary operations from 0 to 24 months after the index procedure in the laparoscopic lavage versus Hartmann's procedure groups. The trial was registered as ISRCTN82208287. RESULTS: Forty-three patients were randomized to laparoscopic lavage and 40 to Hartmann's procedure. Patients in the lavage group had a 45 per cent reduced risk of undergoing one or more operations within 24 months (relative risk 0·55, 95 per cent c.i. 0·36 to 0·84; P = 0·012) and had fewer operations (ratio 0·51, 95 per cent c.i. 0·31 to 0·87; P = 0·024) compared with those in the Hartmann's group. No difference was found in mean number of readmissions (1·37 versus 1·50; P = 0·221) or mortality between patients randomized to laparoscopic lavage or Hartmann's procedure. Three patients in the lavage group and nine in the Hartmann's group had a colostomy at 24 months. CONCLUSION: Laparoscopic lavage is a better option for perforated diverticulitis with purulent peritonitis than open resection and colostomy.
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Colon/cirugía , Diverticulitis del Colon/cirugía , Perforación Intestinal/cirugía , Laparoscopía/métodos , Lavado Peritoneal/métodos , Peritonitis/terapia , Adulto , Anciano , Diverticulitis del Colon/complicaciones , Estudios de Factibilidad , Femenino , Estudios de Seguimiento , Humanos , Perforación Intestinal/complicaciones , Tiempo de Internación/tendencias , Masculino , Persona de Mediana Edad , Peritonitis/etiología , Estudios Prospectivos , Rotura Espontánea , Factores de Tiempo , Resultado del TratamientoRESUMEN
We have established a novel transgenic rat line carrying human microtubule-associated protein Tau-40 with mutation P301L. hTau-40/P301L transgenic male and female rats were followed up to 2 years of age. The hTau-40/P301L rats expressed human tau mRNA and protein in the limbic cortex and associated white matter, hippocampus and spinal cord. With increasing age, the staining density for phosphorylated tau increased in all these areas. Neither silver stains nor Fluoro-Jade staining indicated the presence of dying neurons, or axonal degeneration, and there was no evidence of increased gliosis or inflammation. However, some neurons did display dendritic abnormalities, and immunoblots revealed the presence of sarcosyl insoluble tau. A large test battery revealed no behavioral abnormalities in these rats, except a mild hyperactivity in the elevated plus maze. In conclusion, this transgenic tau rat may be a useful model for 'pretangle' pathology, although in this study conditions were not sufficient to induce significant neuronal loss or behavioral deficits.
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Química Encefálica/genética , Modelos Animales , Mutación/genética , Proteínas tau/química , Proteínas tau/genética , Animales , Femenino , Hipocampo/química , Hipocampo/metabolismo , Humanos , Sistema Límbico/química , Sistema Límbico/metabolismo , Masculino , Ratas , Ratas Transgénicas , Médula Espinal/química , Médula Espinal/metabolismoRESUMEN
OBJECTIVE: Our aim was to study how different SERMs modulate the inflammatory responses induced by lipopolysaccharide (LPS) or unmethylated CpG-oligonucleotides in mouse and rat microglial cells. MATERIALS AND METHODS: Inflammatory responses of mouse N9 microglial cells and rat primary hippocampal microglia to lipopolysaccharide (LPS) exposure were recorded by the secretion of nitric oxide (NO) and cytokine IL-6 in two models where SERM was added either 24 h before LPS addition or simultaneously or even after the LPS exposure. The responses of 17beta-estradiol, tamoxifen, raloxifene and ICI 182.780 were compared. Responses were recorded by ELISA, Northern and EMSA assays. RESULTS: SERMs but not 17beta-estradiol induced a significant, concentration-dependent anti-inflammatory response both in rat primary microglial cells and in mouse N9 microglial cells. The response was observed both in NO and IL-6 secretion as well as in total IL-6 mRNA expression. We have recently observed that histone deacetylase (HDAC) inhibitors can potentiate the LPS-induced inflammatory response. Raloxifene and tamoxifen inhibited the potentiation of LPS response induced by trichostatin A, an HDAC inhibitor, in N9 microglia. A SERM-induced anti-inflammatory response was observed in acute models where SERM was added simultaneously or even up to 6 h later than LPS exposure. In contrast, the pretreatment of N9 microglia with tamoxifen or raloxifene for 30 h before LPS exposure did not provide any protection against the LPS response. We also observed that the raloxifene-induced protection in N9 microglia was connected to a decline of LPS-induced DNA binding activity of AP-1 but not that of NF-kappaB transcription factors. CONCLUSIONS: Our results show that tamoxifen, raloxifene and ICI 182.780 induce an anti-inflammatory response in acute models of mouse and rat microglial cells. It seems that this response is not estrogen receptor-mediated but, probably, is attributable to some SERM-induced modulation of LPS-activated pro-inflammatory signalling cascades.
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Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Microglía/patología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Astrocitos/citología , Northern Blotting , Proliferación Celular , Células Cultivadas , Islas de CpG , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Estradiol/análogos & derivados , Estradiol/metabolismo , Estradiol/farmacología , Fulvestrant , Hipocampo/citología , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Interleucina-6/sangre , Interleucina-6/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Microglía/metabolismo , Óxido Nítrico/metabolismo , Oligonucleótidos/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Clorhidrato de Raloxifeno/farmacología , Ratas , Ratas Wistar , Tamoxifeno/farmacología , Factores de TiempoRESUMEN
BACKGROUND: Very little is known about attention-deficit hyperactivity disorder (ADHD) in African-American children, and although the familial transmission of ADHD has been well established in white samples, prior work has not evaluated this feature of ADHD in African-American families. METHOD: Subjects were 37 first-degree relatives of children with DSM-III-R-defined ADHD and 52 first-degree relatives of non-ADHD comparison children matched for ethnicity, age, and gender. DSM-III-R-based structured interviews (modified to include DSM-IV diagnoses) provided the basis for psychiatric diagnoses in relatives. RESULTS: The risks for both DSM-III-R and DSM-IV ADHD were significantly greater in first-degree relatives of ADHD probands than in relatives of controls. In addition, the relatives of ADHD probands also were at higher risk for oppositional defiant disorder, antisocial personality disorder, major depression, generalized anxiety, and substance use disorders. CONCLUSIONS: These results suggest that ADHD and related disorders are familial in African-Americans. Further work is needed to confirm the familial transmission of ADHD in African-American children and to explore the role of genetics as well as environmental factors in the transmission of the disorder.
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Trastorno por Déficit de Atención con Hiperactividad/etnología , Negro o Afroamericano/psicología , Familia/psicología , Adolescente , Negro o Afroamericano/estadística & datos numéricos , Trastorno por Déficit de Atención con Hiperactividad/genética , Población Negra , Estudios de Casos y Controles , Niño , Femenino , Humanos , Modelos Logísticos , Masculino , Proyectos Piloto , Estados Unidos/epidemiologíaRESUMEN
OBJECTIVE: The authors' goal was to explore the nature of attention deficit hyperactivity disorder (ADHD) in African American children, which has received scant attention by psychiatric researchers. METHOD: Subjects were 19 African American children with DSM-III-R ADHD and 24 African American children without ADHD. Ethnically sensitive methods were used to evaluate the children comprehensively. The findings were compared with those from an earlier study of Caucasian children with ADHD. RESULTS: African American children with ADHD had higher levels of psychiatric disorders other than ADHD than did African American children who did not have ADHD. CONCLUSIONS: Among African American children, ADHD may be characterized by a narrower pattern of psychiatric comorbidity and dysfunction than has been observed in Caucasians. Given the small number of subjects studied, these findings are preliminary and must be replicated to confirm their validity.
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Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Negro o Afroamericano/estadística & datos numéricos , Adolescente , Factores de Edad , Trastorno por Déficit de Atención con Hiperactividad/epidemiología , Niño , Comorbilidad , Humanos , Trastornos Mentales/diagnóstico , Trastornos Mentales/epidemiología , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Reproducibilidad de los Resultados , Población Blanca/estadística & datos numéricosRESUMEN
A major issue is whether surface expression of the pre-TCR is necessary for signaling the development of immature thymocytes. To address this question, we generated transgenic mice expressing a TCRbeta chain that had a strong endoplasmic reticulum (ER) retrieval signal (TCRbetaER) and that was expressed intracellularly but failed to reach the cell surface. In TCRbetaER transgenic mice, there was a failure of allelic exclusion. Also, the transgene failed to rescue the developmental defects observed in TCRbeta-null mice. In contrast, TCRbeta transgenes with a mutant ER retrieval sequence or lacking this sequence signaled efficient allelic exclusion and suppressed the TCRbeta-/- defect. These data show that exit of the pre-TCR from the ER/cis-Golgi is required for progression through the double-negative thymocyte checkpoint.
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Retículo Endoplásmico Rugoso/metabolismo , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Aparato de Golgi/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/citología , Timo/citología , Alelos , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Diferenciación Celular , División Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
OBJECTIVE: Although a small literature of case reports suggests that mania co-occurs with pervasive developmental disorder (PDD), little is known about this overlap. The authors systematically investigated the overlap between mania and PDD in a consecutive sample of referred youths, examining its prevalence and correlates. It was hypothesized that children with PDD plus manic features have both disorders. METHOD: Subjects were consecutively referred children meeting diagnostic criteria on structured interview for PDD without mania (n = 52), the comorbid condition PDD + mania (n = 14), and mania without PDD (n = 114). All subjects were evaluated using a comprehensive diagnostic battery that included assessment of psychopathology (structured diagnostic interview and Child Behavior Checklist), cognition, and functioning. RESULTS: Of the 727 referred children, 52 met criteria for PDD, 114 met criteria for mania, and 14 met criteria for both. The 14 children with both PDD + mania represented 21% of the PDD subjects and 11% of all manic subjects. Clinical characteristics of PDD were similar in PDD subjects with and without mania, and manic features were similar in manic children with and without PDD. CONCLUSIONS: Children with PDD and mania may suffer from two disorders. Comorbid mania among patients with PDD may be more common than previously thought. Identification of the comorbid condition may have important therapeutic and scientific implications.
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Trastorno Bipolar/epidemiología , Trastornos Generalizados del Desarrollo Infantil/epidemiología , Trastorno Bipolar/psicología , Niño , Trastornos Generalizados del Desarrollo Infantil/psicología , Comorbilidad , Femenino , Humanos , Masculino , PrevalenciaRESUMEN
We have generated Cbfa1-deficient mice. Homozygous mutants die of respiratory failure shortly after birth. Analysis of their skeletons revealed an absence of osteoblasts and bone. Heterozygous mice showed specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder, cleidocranial dysplasia (CCD). These defects are also observed in a mouse Ccd mutant for this disease. The Cbfa1 gene was shown to be deleted in the Ccd mutation. Analysis of embryonic Cbfa1 expression using a lacZ reporter gene revealed strong expression at sites of bone formation prior to the earliest stages of ossification. Thus, the Cbfa1 gene is essential for osteoblast differentiation and bone formation, and the Cbfa1 heterozygous mouse is a paradigm for a human skeletal disorder.
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Desarrollo Óseo/genética , Displasia Cleidocraneal/genética , Proteínas de Neoplasias , Osteoblastos/patología , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Eliminación de Gen , Marcación de Gen , Humanos , Ratones , Ratones Mutantes , SíndromeRESUMEN
OBJECTIVE: To evaluate whether attention-deficit hyperactivity disorder (ADHD) is a risk factor for psychoactive substance use disorders (PSUD), attending to issues of psychiatric comorbidity, family history, and adversity. METHOD: Using assessments from multiple domains, the authors examined 140 ADHD and 120 normal control subjects at baseline and 4 years later. Drug and alcohol abuse and dependence were operationally defined. RESULTS: No differences were detected in the rates of alcohol or drug abuse or dependence or in the rates of abuse of individual substances between the groups; both ADHD and control probands had a 15% rate of PSUD. Conduct and bipolar disorders predicted PSUD, independently of ADHD status. Family history of substance dependence and antisocial disorders was associated with PSUD in controls but less clearly so in ADHD probands. Family history of ADHD was not associated with risk for PSUD. ADHD probands had a significantly shorter time period between the onsets of abuse and dependence compared with controls (1.2 years versus 3 years, p < .01). CONCLUSIONS: Adolescents with and without ADHD had a similar risk for PSUD that was mediated by conduct and bipolar disorder. Since the risk for PSUD has been shown to be elevated in adults with ADHD when compared with controls, a sharp increase in PSUD is to be expected in grown-up ADHD children during the transition from adolescence to adulthood.
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Trastorno por Déficit de Atención con Hiperactividad/complicaciones , Trastornos Relacionados con Sustancias/epidemiología , Adolescente , Adulto , Trastorno Bipolar/complicaciones , Estudios de Casos y Controles , Niño , Trastornos de la Conducta Infantil/complicaciones , Estudios de Cohortes , Salud de la Familia , Humanos , Masculino , Análisis de Regresión , Estados Unidos/epidemiologíaRESUMEN
The ubiquitous Ca(2+)-binding protein calmodulin (CaM) is a key protein in Ca2+ homeostasis and activation of eukaryotic cells. CaM is the molecular link between free Ca2+ in the cell and the inhibition, or activation, of numerous enzymes. Many nuclear functions are under Ca2+/CaM control, and some transcriptional activators are known to be Ca2+ modulated indirectly through Ca2+/CaM-dependent protein kinases. But Ca2+/CaM has not yet been found to directly modulate any transcription factor or other DNA-binding protein. Transcription factors of the basic-helix-loop-helix (bHLH) group are important regulators in numerous systems. Here we report that binding of Ca(2+)-loaded CaM to the bHLH domains of several bHLH proteins directly inhibits their DNA binding. Other bHLH proteins are either less sensitive or resistant. Ca2+ ionophore selectively inhibits transcriptional activation by Ca2+/CaM-sensitive bHLH proteins in vivo, implying that Ca2+ can directly influence transcription through differential CaM inhibition of bHLH domains.
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Calcio/fisiología , Calmodulina/fisiología , Secuencias Hélice-Asa-Hélice/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Bovinos , Línea Celular , Reactivos de Enlaces Cruzados/farmacología , ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Glutaral/farmacología , Humanos , Ionomicina/farmacología , Ratones , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Binding sites for SL3-3 enhancer factor 1 (SEF1) are important for the transcriptional activity in T lymphocytes and the tumorigenicity of SL3-3 murine leukemia virus. SEF1 is also implicated in the activity of many other leukemia, lymphoma, and sarcoma virus enhancers, and enhancers of genes for T cell receptor-CD3 subunits. We have purified several proteins binding to SEF1 sites from bovine thymus using a five-step purification procedure. The proteins migrated as 19 distinct bands representing molecular masses from 23 kDa to about 200 kDa in SDS-polyacrylamide gel electrophoresis. Ten DNA binding proteins, with molecular masses between 23 and 67 kDa, could be isolated after separation by SDS-polyacrylamide gel electrophoresis. The DNA binding specificities of these proteins were similar and corresponded to that of the SEF1 binding activity in nuclear extracts. Each of these isolated SEF1 proteins also bound to the essential delta-E3 element of the human T cell receptor delta enhancer. Antibodies against one SEF1 protein only reacted with the protein used for immunization, which indicates a limited homology between at least some SEF1 proteins. We also present data suggesting that SEF1 proteins exist in multiple forms with differences in their DNA binding specificity, and that high affinity DNA binding of the SEF1 proteins requires protein phosphorylation.
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Proteínas de Unión al ADN/aislamiento & purificación , Elementos de Facilitación Genéticos , Proteínas de Neoplasias , Linfocitos T/metabolismo , Factores de Transcripción/aislamiento & purificación , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Complejo CD3/metabolismo , Bovinos , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
A family of proteins, denoted SL3-3 enhancer factor 1 (SEF1), interacts with DNA sequences in the T cell specific enhancer of SL3-3 murine leukemia virus and in the enhancers of several other viruses. A putative SEF1 binding site was also identified in the T cell specific enhancer of the gene encoding the human T cell antigen receptor (TcR) associated CD3-epsilon polypeptide. In this study we show that the identified sequence is a strong SEF1 binding site, and that purified SEF1 proteins bind specifically to the sequence. We report also that the SEF1 binding site is important for T cell specific activation of transcription by the CD3-epsilon enhancer. We show that SEF1 binding sites are present also in the T cell specific enhancers of other subunits of the TcR-CD3 complex, and that SEF1 proteins appear to play a central role in the T cell specific expression of this set of enhancers.
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Complejo CD3/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Proteínas de Neoplasias , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Bovinos , Línea Celular , Núcleo Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Homología de Secuencia de Ácido Nucleico , Timo/inmunologíaRESUMEN
A family of nuclear proteins, designated SL3-3 enhancer factors 2 (SEF2), were found to interact with an Ephrussi box-like motif within the glucocorticoid response element in the enhancer of the murine leukemia virus SL3-3. Mutation of the DNA sequence decreased the basal enhancer activity in various cell lines. The important nucleotides for binding of SEF2 are conserved in most type C retroviruses. Various cell types displayed differences both in the sets of SEF2-DNA complexes formed and in their amounts. A cDNA which encoded a protein that interacted specifically with the SEF2-binding sequence was isolated from human thymocytes. The nucleotide sequence specificity of the recombinant protein, expressed in Escherichia coli, corresponded to that of at least one of the nuclear SEF2 proteins. Sequence analysis of the cDNA revealed that it belongs to the basic helix-loop-helix class of DNA-binding proteins. Several mRNA transcripts of different sizes were identified. Molecular analysis of cDNA clones revealed multiple related mRNA species containing alternative coding regions, which are most probably a result of differential splicing.
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Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Virus de la Leucemia Murina/genética , Proteínas del Tejido Nervioso , Proteínas Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Transactivadores/metabolismo , Factores de Transcripción , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Northern Blotting , Línea Celular , Clonación Molecular/métodos , Escherichia coli/genética , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Poli A/análisis , Poli A/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción TCF , Factor de Transcripción 4 , Proteína 2 Similar al Factor de Transcripción 7 , TransfecciónRESUMEN
Interactions between SL3-3 enhancer factor 1 (SEF1) proteins and the enhancer of the murine leukemia virus SL3-3 were analyzed. SEF1 proteins were found to interact with two different DNA sequences within the DNA repeat region of the enhancer; these two motifs cooperated in enhancing initiation of transcription in T lymphocytes. Using an electrophoretic mobility shift assay, we identified nucleotides that are important for the SEF1 binding, and we deduced a sequence, 5'-TTTGCGGTTA/T-3' with highly improved binding of SEF1 proteins. We show that many different SEF1 binding sequences exist in the transcription control regions of different viral and cellular genes. The results indicate a general role of SEF1 proteins in T-cell gene expression.
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Cromosomas/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Genes Virales , Transactivadores/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Células HeLa/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Unión Proteica , Secuencias Repetitivas de Ácidos Nucleicos , Retroviridae/genética , Homología de Secuencia de Ácido Nucleico , Programas Informáticos , Transcripción GenéticaRESUMEN
Nuclear factor I (NFI) is shown to be of importance for the activity of the enhancer element of a T-cell leukemogenic murine retrovirus, SL3-3, and for the regulation of this element by glucocorticoid. Each nucleotide of the binding site of the NFI proteins was mutated, and the effects of the mutations were quantitated with an electrophoretic mobility shift assay. Mutations in the inverted repeat of the binding site have symmetric effects which strongly support the notion that NFI proteins preferentially bind to dyad symmetry sites. Such binding sites were shown to be more than 100 fold stronger than the corresponding single binding sites. We find dyad symmetry sequences which are much stronger NFI binding sites than NFI sites identified in different genes and also stronger than previously proposed consensus binding sequences for NFI.
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Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Virus de la Leucemia Murina/genética , Factores de Transcripción , Animales , Composición de Base , Unión Competitiva , Línea Celular , Deleción Cromosómica , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFI , Proteínas Nucleares , Conejos , Proteína 1 de Unión a la Caja YRESUMEN
An electrophoretic mobility shift assay was used to characterize interactions of nuclear proteins with a DNA segment in the enhancer element of the leukemogenic murine retrovirus SL3-3. Mutation of a DNA sequence of the 5'-TGTGG-3' type decreased transcription in vivo specifically in T-lymphocyte cell lines. Extracts of nuclei from different T-lymphocyte cell lines or cells from lymphoid organs resulted in much higher amounts of complexes in vitro with this DNA sequence than did extracts from other cell lines or organs tested. Differences were also found in the sets of complexes obtained with extracts from the different types of cells. The DNA sequence specificities of the different SL3-3 enhancer factor 1 (SEF1) protein complexes were found to be distinct from those of several other previously identified DNA motifs of the TGTGG type because of differences in several nucleotides critical for binding and because these other DNA motifs could not compete with the identified DNA sequence for binding of SEF1. Limited treatment with several different proteases cleaved the SEF1 proteins such that their DNA-binding domain(s) remained and created complexes with decreased and nondistinguishable electrophoretic mobility shifts and with new properties. These results indicate that the SEF1 proteins have a structure with a flexible and relatively vulnerable hinge region linking a DNA-binding domain(s) to a more variable domain(s) with other functions. We suggest that the binding of SEF1 is an essential factor for the T-cell tropism of SL3-3 and the ability of this virus to cause T-cell lymphomas.