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Background: Radiation therapy (RT) plays an important role in management of pediatric central nervous system (CNS) malignancies. Centers are increasingly utilizing pencil beam scanning proton therapy (PBS-PT). However, the risk of brainstem necrosis has not yet been reported. In this study, we evaluate the rate of brainstem necrosis in pediatric patients with CNS malignancies treated with PBS-PT.Material and methods: Pediatric patients with non-hematologic CNS malignancies treated with PBS-PT who received dose to the brainstem were included. All procedures were approved by the institutional review board. Brainstem necrosis was defined as symptomatic toxicity. The actuarial rate was analyzed by the Kaplan Meier method.Results: One hundred and sixty-six consecutive patients were reviewed. Median age was 10 years (range 0.5-21 years). Four patients (2.4%) had prior radiation. Median maximum brainstem dose in the treated course was 55.4 Gy[RBE] (range 0.15-61.4 Gy[RBE]). In patients with prior RT, cumulative median maximum brainstem dose was 98.0 Gy [RBE] (range 17.0-111.0 Gy [RBE]). Median follow up was 19.6 months (range, 2.0-63.0). One patient who had previously been treated with twice-daily radiation therapy and intrathecal (IT) methotrexate experienced brainstem necrosis. The actuarial incidence of brainstem necrosis was 0.7% at 24 months (95% CI 0.1-5.1%).Conclusion: The rate of symptomatic brainstem necrosis was extremely low after treatment with PBS-PT in this study. Further work to clarify clinical and dosimetric parameters associated with risk of brainstem necrosis after PBS-PT is needed.
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Tronco Encefálico/efectos de la radiación , Neoplasias del Sistema Nervioso Central/radioterapia , Terapia de Protones/efectos adversos , Adolescente , Astrocitoma/radioterapia , Tronco Encefálico/patología , Niño , Preescolar , Ependimoma/radioterapia , Femenino , Humanos , Lactante , Estimación de Kaplan-Meier , Masculino , Meduloblastoma/radioterapia , Necrosis/epidemiología , Necrosis/etiología , Terapia de Protones/métodos , Dosis de Radiación , Traumatismos por Radiación/complicaciones , Reirradiación/efectos adversos , Adulto JovenRESUMEN
Recurrence of malignant brain tumors results in a poor prognosis with limited treatment options. High-dose chemotherapy with autologous hematopoietic cell rescue (AHCR) has been used in patients with recurrent malignant brain tumors and has shown improved outcomes compared with standard chemotherapy. Temozolomide is standard therapy for glioblastoma and has also shown activity in patients with medulloblastoma/primitive neuro-ectodermal tumor (PNET), particularly those with recurrent disease. Temozolomide was administered twice daily on days -10 to -6, followed by thiotepa 300 mg/m(2) per day and carboplatin dosed using the Calvert formula or body surface area on days -5 to -3, with AHCR day 0. Twenty-seven patients aged 3-46 years were enrolled. Diagnoses included high-grade glioma (n=12); medulloblastoma/PNET (n=9); central nervous system (CNS) germ cell tumor (n=4); ependymoma (n=1) and spinal cord PNET (n=1). Temozolomide doses ranged from 100 mg/m(2) per day to 400 mg/m(2) per day. There were no toxic deaths. Prolonged survival was noted in several patients including those with recurrent high-grade glioma, medulloblastoma and CNS germ cell tumor. Increased doses of temozolomide are feasible with AHCR. A phase II study using temozolomide, carboplatin and thiotepa with AHCR for children with recurrent malignant brain tumors is being conducted through the Pediatric Blood and Marrow Transplant Consortium.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/terapia , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Autoinjertos , Carboplatino/administración & dosificación , Niño , Preescolar , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Neoplasia Residual , Tasa de Supervivencia , Temozolomida , Tiotepa/administración & dosificaciónRESUMEN
Patients with neurofibromatosis type 1 (nf1) are at increased risk for both benign and malignant tumours, and distinguishing the malignant potential of an individual tumour is a common clinical problem in these patients. Here, we review two cases of uncommon malignancies (Hodgkin lymphoma and mediastinal germ-cell tumour) in patients with nf1. Although (18)F-fluorodeoxyglucose positron-emission tomography (fdg-pet) has been used to differentiate benign neurofibromas from malignant peripheral nerve sheath tumours, fdg-pet characteristics for more rare tumours have been poorly described in children with nf1. Here, we report the role of pet imaging in clinical decision-making in each case. In nf1, fdg-pet might be useful in the clinical management of unusual tumour presentations and might help to provide information about the malignant potential of uncommon tumours.
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OBJECTIVE: To longitudinally analyze changes in plexiform neurofibroma (PN) volume in relation to age and body growth in children and young adults with neurofibromatosis type 1 and inoperable, symptomatic, or progressive PNs, using a sensitive, automated method of volumetric MRI analysis. METHODS: We included patients 25 years of age and younger with PNs entered in a natural history study or in treatment trials who had volumetric MRI over > or =16 months. RESULTS: We studied 49 patients (median age 8.3 years) with 61 PNs and a median evaluation period of 34 months (range 18 to 70). The PN growth rates varied among patients, but were constant within patients. Thirty-four patients (69%) experienced > or =20% increase in PN volume during the observation period. PN volume increased more rapidly than body weight over time (p = 0.026). Younger patients had the most rapid PN growth rate. CONCLUSIONS: Volume increase of plexiform neurofibromas is a realistic and meaningful trial endpoint. In most patients plexiform neurofibroma growth rate exceeded body growth rate. The youngest patients had the fastest plexiform neurofibroma growth rate, and clinical drug development should be directed toward this population. Age stratification for clinical trials for plexiform neurofibromas should be considered.
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Envejecimiento/patología , Peso Corporal , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Imagen por Resonancia Magnética/métodos , Neurofibroma Plexiforme/patología , Neurofibromatosis 1/patología , Niño , Femenino , Humanos , Masculino , Invasividad Neoplásica , Estadística como AsuntoRESUMEN
The labile SAUR transcripts from higher plants contain a conserved DST sequence in their 3'-untranslated regions. Two copies of a DST sequence from soybean are sufficient to destabilize reporter transcripts in cultured tobacco cells whereas variants bearing mutations in the conserved ATAGAT or GTA regions are inactive. To investigate the potential for conserved recognition components in mammalian and plant cells, we examined the function of this instability determinant in mouse NIH3T3 fibroblasts and tobacco BY2 cells. In fibroblasts, a tetrameric DST element from soybean accelerated deadenylation and decay of a reporter transcript. However, a version mutated in the ATAGAT region was equally effective in this regard, and a tetrameric DST element from Arabidopsis was inactive. In contrast, the soybean DST element was more active as an mRNA instability element than the mutant version and the Arabidopsis element, when tested as tetramers in tobacco cells. Hence, the plant DST element is not recognized in animal cells with the same sequence requirements as in plant cells. Therefore, its mode of recognition appears to be plant-specific.
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Nicotiana/genética , Plantas/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Células 3T3 , Animales , Arabidopsis/genética , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Células Vegetales , Poli A/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Glycine max/genética , Nicotiana/citologíaRESUMEN
RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable in vitro genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3' stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10(7) independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (K(d) as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B" demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins.
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Bacteriófago T7/genética , Selección Genética , Bacteriófago T7/metabolismo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Pulmón/metabolismo , Proteínas de Unión al ARN/metabolismo , Recombinación GenéticaRESUMEN
The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex. To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel shift analysis with crude E. coli extracts. Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly. Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein-protein interactions. The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE.
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Productos del Gen rev/química , Productos del Gen rev/metabolismo , VIH-1/metabolismo , Mutación/genética , ARN Viral/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Sitio Alostérico , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli , Productos del Gen rev/genética , VIH-1/genética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , ARN Viral/química , ARN Viral/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Termodinámica , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Rothmund-Thomson syndrome is an autosomal recessive disorder characterized by poikilodermatous skin changes that develop in infancy. Associated manifestations include juvenile cataracts, sparse hair, short stature, skeletal defects, dystrophic nails and teeth, and hypogonadism. An increased incidence of malignancy, including osteosarcoma, has been reported in patients with Rothmund-Thomson syndrome. The molecular basis of the disorder is not known. This report describes a patient with Rothmund-Thomson syndrome in whom two primary osteosarcomas developed 12 years apart. The presentation, diagnosis, and treatment of osteosarcoma in this patient with Rothmund-Thomson syndrome are described. Cytogenetic and molecular analysis of peripheral blood and skin fibroblasts had low level mosaicism for trisomy of chromosomes 2 and 8. Although several patients have been described with mosaic trisomy 8 and i(2q) (mosaic isochromosome for the long arm of chromosome 2), the patient described here is the first to have mosaic trisomy for the entire chromosomes 2 and 8. The cytogenetic findings in this patient are consistent with an underlying defect in chromosomal stability.
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Neoplasias Óseas/complicaciones , Neoplasias Femorales/complicaciones , Húmero , Neoplasias Primarias Múltiples , Osteosarcoma/complicaciones , Síndrome Rothmund-Thomson/complicaciones , Neoplasias Óseas/patología , Niño , Cromosomas Humanos Par 2/genética , Cromosomas Humanos Par 8/genética , Humanos , Cariotipificación , Imagen por Resonancia Magnética , Masculino , Mosaicismo , Osteosarcoma/patología , Síndrome Rothmund-Thomson/genéticaAsunto(s)
Biosíntesis de Proteínas , Proteínas/metabolismo , ARN/metabolismo , Secuencia de Bases , Técnicas de Cultivo de Célula , Escherichia coli/genética , Escherichia coli/metabolismo , Productos del Gen rev/genética , Genes Reporteros , Técnicas Genéticas , Cinética , Operón Lac/genética , Modelos Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fosfoproteínas/genética , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Proteínas/química , ARN/química , Proteínas de Unión al ARN/genética , Supresión Genética , Transformación Genética , NucleolinaRESUMEN
RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5' UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5' UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5' UTRs are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5' UTRs are multiple RNA secondary structure elements, two of which function in cis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem-loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5' UTR. Our data show that this stem-loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate of rne mRNA in response to changes in RNase E activity.
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Regiones no Traducidas 5'/química , Secuencia Conservada/genética , Endorribonucleasas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Conformación de Ácido Nucleico , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , Alquilación , Secuencia de Aminoácidos , Emparejamiento Base/genética , Secuencia de Bases , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Retroalimentación , Semivida , Modelos Genéticos , Datos de Secuencia Molecular , Mutación/genética , Filogenia , Estabilidad del ARN/genética , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
BACKGROUND: Despite improvements in the treatment of pediatric acute lymphoblastic leukemia, approximately one in five patients will develop recurrent disease. The majority of these patients do not survive. This limited institution study sought to improve event-free survival (EFS) by intensification of chemotherapy. PROCEDURE: Twenty-one patients with either an isolated marrow (n = 16) or a combined marrow and central nervous system relapse (n = 5) received treatment according to Children's Hospital of Philadelphia protocol CHP-540. Six patients had an initial remission of <36 months, and five patients had relapsed within 1 year of completion of phase III therapy. Induction and reinduction therapy consisted of idarubicin, vincristine, dexamethasone, asparaginase, and triple intrathecal chemotherapy. Consolidation and reconsolidation therapy employed high-dose cytarabine, etoposide, and asparaginase given in a sequential manner. Maintenance therapy included courses of high- or low-dose cytarabine followed by sequential etoposide and asparaginase pulse, moderate-dose methotrexate with delayed leukovorin rescue, and vincristine/dexamethasone pulses. Therapy continued for 2 years from the start of interim maintenance in the 16 patients who did not receive a bone marrow transplant (BMT). Two patients underwent an HLA-identical sibling BMT specified by protocol. Four received a nonprotocol-prescribed alternative donor BMT. RESULTS: The complete remission induction rate was 95%. With a median follow-up from date of relapse of 49 months in survivors, the actuarial EFS based on intent to treat is 75%. There were three toxic deaths in patients in CR and two deaths from relapse. CONCLUSIONS: This regimen is toxic but effective and deserves study in a larger setting.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/efectos de los fármacos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Adolescente , Antibióticos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Antineoplásicos/administración & dosificación , Antineoplásicos Hormonales/administración & dosificación , Antineoplásicos Fitogénicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Asparaginasa/administración & dosificación , Trasplante de Médula Ósea , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Niño , Preescolar , Citarabina/administración & dosificación , Dexametasona/administración & dosificación , Supervivencia sin Enfermedad , Etopósido/administración & dosificación , Femenino , Estudios de Seguimiento , Humanos , Idarrubicina/administración & dosificación , Lactante , Inyecciones Espinales , Masculino , Inducción de Remisión , Vincristina/administración & dosificaciónRESUMEN
RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end. Here we show that the preference of E. coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.
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Cianobacterias/enzimología , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , ARN/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Cianobacterias/genética , Endorribonucleasas/química , Endorribonucleasas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Prueba de Complementación Genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
BACKGROUND: We assessed efficacy and morbidity of chemotherapy and 1, 800 cGy of hypofractionated craniospinal irradiation (CSI) in children with central nervous system (CNS) relapse following first remisssion of acute lymphoblastic leukemia (ALL). PROCEDURE: Nineteen patients with isolated CNS relapse and 4 with combined CNS/marrow or CNS/testicular relapse received treatment according to Children's Hospital of Philadelphia (CHOP) protocols CHP-449 and CHP-497. CNS treatment included intrathecal methotrexate, cytarabine, and hydrocortisone and 1,800 cGy CSI in 16 fractions over 12 months. Systemic therapy consisted of reinductions with vincristine, prednisone, and daunorubicin and reconsolidations with cytarabine, etoposide, and L-asparaginase every 56 days for 2 years. Outcome measures were event-free survival (EFS), survival, growth, and neuropsychologic assessment or school performance. RESULTS: Follow-up of survivors from first relapse ranges from 52 to 133 months(median 91 months). Actuarial survival and EFSat 10 years are 58% (CI95 = 38-78%) and 54% (CI95 = 32-76%). Events include 2 second CNS, 4 marrow, 1 testicular, and 2 testicular/marrow relapses and 1 secondary leukemia. EFS is 100% (CI95 = 93-100%) in 9 patients with recurrence more than 26 months from diagnosis. Three patients have significant treatment-related reduction in stature. Median full-scale IQs of 6 patients tested were 112 pretreatment and 111 posttreatment among surviving patients. All 17 survivors attend regular school, but 2 receive supplementary special services. CONCLUSIONS: Lower dose, hypofractionated CSI, intrathecal chemotherapy, and moderately intensive systemic chemotherapy provide excellent disease control for patients with late isolated CNS or combined marrow and CNS relapse. Children with brief first remissions remain at substantial risk of subsequent relapse with this therapy, especially in the marrow and testes.
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Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Neoplasias del Sistema Nervioso Central/radioterapia , Recurrencia Local de Neoplasia/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/radioterapia , Adolescente , Adulto , Neoplasias del Sistema Nervioso Central/mortalidad , Niño , Preescolar , Terapia Combinada , Femenino , Humanos , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Dosificación Radioterapéutica , Tasa de SupervivenciaRESUMEN
High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5' untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5' untranslated region, as evidenced by the significant acceleration in papA mRNA decay that results from its removal.
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Regiones no Traducidas 5'/química , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Conformación de Ácido Nucleico , ARN Mensajero/metabolismo , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , Alquilación , Secuencia de Bases , CME-Carbodiimida/análogos & derivados , Proteínas Fimbrias , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Semivida , ARN/química , ARN/genética , ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Eliminación de Secuencia , Ésteres del Ácido SulfúricoRESUMEN
BACKGROUND: Follicular lymphoma in childhood is rare. The authors present four unusual primary follicular lymphomas of the testis in children. METHODS: Tumor tissue was evaluated using light microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) and bcl-2 gene rearrangements. Southern blot and immunohistochemical analyses were used to detect bcl-6 gene rearrangements and protein expression, respectively. RESULTS: Four young boys ranging in age from 3 to 10 years were diagnosed with Stage IE follicular large cell lymphoma (Grade 3). A B-cell phenotype was documented in all four cases; monoclonality was confirmed in three cases by demonstration of light chain restriction or clonal IgH gene rearrangement. None of the lymphomas expressed Bcl-2 or p53 protein, and bcl-2 gene rearrangements were not found in the three lymphomas studied. In contrast, Bcl-6 protein was expressed by all three lymphomas studied, and a bcl-6 gene rearrangement was detected in the one case analyzed by Southern blot. All four boys were treated by orchiectomy and combination chemotherapy and are alive with no evidence of disease 18-44 months following their initial diagnoses. CONCLUSIONS: Follicular lymphomas may rarely occur as primary testicular tumors in prepubertal boys and, when localized, appear to be associated with a favorable prognosis. In contrast to follicular lymphoma in adults, pediatric follicular lymphomas of the testis are usually of large cell type (Grade 3) and lack bcl-2 or p53 abnormalities. The identification, in one case, of a bcl-6 gene rearrangement suggests an alternate molecular pathogenesis for pediatric follicular lymphoma.
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Linfoma Folicular/genética , Neoplasias Testiculares/genética , Niño , Preescolar , Reordenamiento Génico , Humanos , Inmunofenotipificación , Linfoma de Células B/genética , Linfoma Folicular/patología , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Neoplasias Testiculares/patología , Proteína p53 Supresora de Tumor/análisisRESUMEN
The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".
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Leucina/metabolismo , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/fisiología , Empalmosomas/metabolismo , Secuencia de Aminoácidos , Autoantígenos , Secuencia de Bases , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Fenotipo , ARN Nuclear Pequeño/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/biosíntesis , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U2/biosíntesis , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/farmacología , Ribonucleoproteínas Nucleares Pequeñas , Termodinámica , Proteínas Nucleares snRNPRESUMEN
Novel RNA-binding proteins with customized specificities can be isolated by genetic selection from combinatorial libraries. Such proteins have great potential as agents for targeted manipulation of gene expression.
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Regulación de la Expresión Génica , Proteínas de Unión al ARN/genética , Sitios de Unión , Biblioteca de Genes , Variación Genética , Biblioteca de Péptidos , Unión Proteica , Selección Genética , Dedos de ZincRESUMEN
The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused. Previous studies have identified two domains of this 5' UTR that together are responsible for its stabilizing effect. One is a 5'-terminal stem-loop. The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA. Here we report a detailed investigation of the function of these two stabilizing elements. Our data indicate that mRNA protection by a 5' stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation. Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5' UTR segment by bound ribosomes. Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5' UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region. Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5' stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent. These findings imply that the ompA 5' UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/metabolismo , Secuencia de Bases , Endorribonucleasas/metabolismo , Activación Enzimática , Escherichia coli/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
PURPOSE: We reviewed all pediatric cases referred for emergent/urgent therapy (requiring treatment within 48 hours) to identify frequency, patterns of presentation, and efficacy of therapy. We defined five categories of emergent/urgent therapy based on irradiated site and/or signs: Group I, spinal cord compression; Group II, respiratory compromise; Group III, infradiaphagmatic distress; Group IV, intracranial signs; Group V, pain. MATERIALS AND METHODS: From 2/1/88-3/1/ 94, 104 children with 115 problems were referred by specialists at the Children's Hospital of Philadelphia. Diagnosis, nature of the emergency, and response were examined. Responses were categorized as complete resolution, improvement or stabilization, and progression. RESULTS: The 104 children represented 12% of referrals during the study period. The most common tumors were CNS PNET and gliomas (20%); and neuroblastoma (20%). Forty-five problems occurred with newly diagnosed tumors and 70 after progression. Ninety-one episodes were managed with radiation therapy and 24 with other modalities. Patients with spinal cord/cauda equina (n = 33) compression improved (55%) or stabilized (30%). Patients with respiratory compromise from thoracic (n = 14) or abdominal (n = 5) disease had a response rate of 72%. Eight patients in group III had a 66% response. In Group IV (n = 16), 63% had complete responses and 19% had stabilization. Group V (n = 15) patients had a complete or partial response of 93%. CONCLUSION: Approximately 10% of children referred for radiation therapy required emergent/urgent treatment. Eighty percent of patients achieved stabilization or showed improvement in signs and symptoms, indicating that radiotherapy is a valuable and reliable component of multimodal care in such situations.