RESUMEN
Pompe disease, caused by deficiency of acid alpha-glucosidase (GAA), leads to widespread glycogen accumulation and profound neuromuscular impairments. There has been controversy, however, regarding the role of central nervous system pathology in Pompe motor dysfunction. We hypothesized that absence of GAA protein causes progressive activation of neuropathological signaling, including pathways associated with cell death. To test this hypothesis, genomic data (Affymetrix Mouse Gene Array 2.0ST) from the midcervical spinal cord in 6 and 16 mo old Pompe (Gaa-/-) mice were evaluated (Broad Institute Molecular Signature Database), along with spinal cord histology. The midcervical cord was selected because it contains phrenic motoneurons, and phrenic-diaphragm dysfunction is prominent in Pompe disease. Several clinically important themes for the neurologic etiology of Pompe disease emerged from this unbiased genomic assessment. First, pathways associated with cell death were strongly upregulated as Gaa-/- mice aged, and motoneuron apoptosis was histologically verified. Second, proinflammatory signaling was dramatically upregulated in the Gaa-/- spinal cord. Third, many signal transduction pathways in the Gaa-/- cervical cord were altered in a manner suggestive of impaired synaptic function. Notably, glutamatergic signaling pathways were downregulated, as were "synaptic plasticity pathways" including genes related to neuroplasticity. Fourth, many genes and pathways related to cellular metabolism are dysregulated. Collectively, the data unequivocally confirm that systemic absence of GAA induces a complex neuropathological cascade in the spinal cord. Most importantly, the results indicate that Pompe is a neurodegenerative condition, and this underscores the need for early therapeutic intervention capable of targeting the central nervous system.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Médula Espinal/patología , Transcriptoma/genética , alfa-Glucosidasas/deficiencia , Animales , Muerte Celular , Vértebras Cervicales/patología , Perfilación de la Expresión Génica , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Inflamación/patología , Ratones , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , alfa-Glucosidasas/metabolismoRESUMEN
Pompe disease (glycogen storage disease type II) is caused by mutations in acid α-glucosidase (GAA) resulting in lysosomal pathology and impairment of the muscular and cardio-pulmonary systems. Enzyme replacement therapy (ERT), the only approved therapy for Pompe disease, improves muscle function by reducing glycogen accumulation but this approach entails several limitations including a short drug half-life and an antibody response that results in reduced efficacy. To address these limitations, new treatments such as gene therapy are under development to increase the intrinsic ability of the affected cells to produce GAA. Key components to gene therapy strategies include the choice of vector, promoter, and the route of administration. The efficacy of gene therapy depends on the ability of the vector to drive gene expression in the target tissue and also on the recipient's immune tolerance to the transgene protein. In this review, we discuss the preclinical and clinical studies that are paving the way for the development of a gene therapy strategy for patients with early and late onset Pompe disease as well as some of the challenges for advancing gene therapy.
Asunto(s)
Dependovirus , Terapia Genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Animales , HumanosRESUMEN
The highly organized array of intramembranous particles, the ciliary plaque, varies from the wild type in size and organization in two stocks of the Paramecium behavioral mutant, paranoiac. In one of these stocks, the alteration is dramatic.
Asunto(s)
Paramecium/genética , Animales , Membrana Celular/ultraestructura , Cilios/ultraestructura , Locomoción , Mutación , Paramecium/fisiología , Paramecium/ultraestructura , Sodio/farmacologíaRESUMEN
Following initial graft rejection, a second attempt at allogeneic immunotherapy is often contemplated, but data on the success is limited. We therefore report on 11 patients with hematologic malignancies, renal cell cancer or marrow failure who underwent a second reduced-intensity regimen for primary or secondary graft failure. Nine of the 11 patients initially engrafted with the second attempt including two of four who used the same donor. One of the patients engrafted after the third attempt using a different donor and conditioning regimen. There were two treatment-related deaths. Four patients died from progressive disease 1-9 months after the second transplant. Two patients are still in recovery phase less than 1 year from the second transplant. Long-term remission is possible and three patients are alive in complete remission.
Asunto(s)
Rechazo de Injerto , Trasplante de Células Madre Hematopoyéticas , Adulto , Anciano , Carcinoma de Células Renales/terapia , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Neoplasias Renales/terapia , Leucemia Mieloide Aguda/terapia , Persona de Mediana Edad , Síndromes Mielodisplásicos/terapia , Trasplante Homólogo , Resultado del TratamientoRESUMEN
Paranoiac and related mutants of Paramecium tetraurelia display altered membrane excitability. We describe an extension of behavioral characterizations of the paranoiac, fast-2, and tetraethylammonium-insensitive mutants, comparing in detail their reactions to sodium stimulation under standard culture conditions, when grown at various temperatures and when starved. We also use freeze-fracture electron microscopic techniques to analyze in these stocks the morphology of organized arrays of membrane particles, the ciliary plaques. This group of mutants is diverse, showing differences in behavior under standard culture conditions and different reactions to temperature and starvation stresses. Ciliary plaque morphology is altered in some, but not all, of the mutants. The possibility is discussed that these plaques may be sites of potassium or sodium transport.
Asunto(s)
Paramecium/genética , Animales , Movimiento Celular , Cilios/fisiología , Cruzamientos Genéticos , Canales Iónicos/metabolismo , Mutación , Paramecium/fisiología , FenotipoRESUMEN
Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken beta-actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1 alpha promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-beta-actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice. Neonatal intramuscular injection resulted in expression of hFX at 248 ng/ml (3.1% of normal), which derived from both liver and muscle. We conclude that neonatal gene therapy with an AAV vector with the CMV-beta-actin promoter might correct hemophilia due to hFX deficiency.
Asunto(s)
Actinas/genética , Citomegalovirus/genética , Dependovirus/genética , Factor X/genética , Hemofilia A/genética , Hemofilia A/terapia , Hígado/metabolismo , Factor 1 de Elongación Peptídica/genética , Regiones Promotoras Genéticas , Animales , Sitios de Unión , Southern Blotting , Pollos , ADN/metabolismo , ADN Complementario/metabolismo , Elementos de Facilitación Genéticos , Ensayo de Inmunoadsorción Enzimática , Femenino , Terapia Genética/métodos , Vectores Genéticos , Humanos , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción GenéticaRESUMEN
Pompe disease is a lethal cardioskeletal myopathy in infants and results from genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Genetic replacement of the cDNA for human GAA (hGAA) is one potential therapeutic approach. Three months after a single intramuscular injection of 10(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level. Direct transduction of a target muscle, however, may not correct all deficient cells. Therefore, the amount of enzyme that can be transferred to deficient cells from virally transduced cells was studied. Fibroblasts from an affected patient were transduced with AdhGAA, washed, and plated on transwell culture dishes to serve as donors of recombinant enzyme. Deficient fibroblasts were plated as acceptor cells, and were separated from the donor monolayer by a 22-microm pore size filter. Enzymatic and Western analyses demonstrate secretion of the 110-kDa precursor form of hGAA from the donor cells into the culture medium. This recombinant, 110-kDa species reaches the acceptor cells, where it can be taken up by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks to lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme. Patient fibroblasts receiving recombinant hGAA by this transfer mechanism reach levels of enzyme activity that are comparable to normal human fibroblasts. Skeletal muscle cell cultures from an affected patient were also transduced with Ad-hGAA. Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunocytochemistry, and enzyme activity is transferred to deficient skeletal muscle cells grown in coculture. Transfer of the precursor protein between muscle cells again occurs via mannose 6-phosphate receptors, as evidenced by competitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA-knockout mice demonstrate that hepatic transduction with adenovirus encoding either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism. Taken together, these data show that the mannose 6-phosphate receptor pathway provides a useful strategy for cell-to-cell distribution of virally derived recombinant GAA.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética/métodos , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , alfa-Glucosidasas/genética , Adenoviridae/genética , Animales , Western Blotting , Células Cultivadas , Técnicas de Cocultivo , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Lisosomas/metabolismo , Manosafosfatos/metabolismo , Ratones , Ratones Noqueados , Ratones Desnudos , Músculo Esquelético/citología , Miocardio/metabolismo , Placenta/metabolismo , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Transducción GenéticaRESUMEN
Delivery of genes or macromolecules to cardiovascular tissues provides new therapeutic opportunities for the treatment of many acquired and inherited diseases. To investigate electroporation as a delivery method in cardiac tissue, embryonic chick hearts were studied for uptake of propidium iodide (PI) or DNA encoding either green fluorescent protein (GFP) or luciferase following electrical shock. PI uptake increased monotonically from 6% of heart tissue after 3 shocks to 77% with 12 shocks. GFP and luciferase expression varied in proportion to shock number, with detectable levels in all electrically treated hearts. Thus, electroporation promotes uptake of PI and DNA in cardiac tissue, suggesting further application of this method for therapeutic genes.
Asunto(s)
Electroporación/métodos , Técnicas de Transferencia de Gen , Miocardio/metabolismo , Animales , Embrión de Pollo , ADN/metabolismo , Marcadores Genéticos , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes , Indicadores y Reactivos , Luciferasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Propidio/metabolismoRESUMEN
BACKGROUND: The widely used non-volume-loaded abdominal heterotopic heart transplant (NL) in rats undergoes atrophy after transplantation. Various techniques have been designed to load the transplanted heart because of its potential immunological impact. Our aim was to create a volume-loaded heterotopic heart transplantation model (VL) capable of ejection and practical for routine studies. Using this model, we tested the hypothesis that VL isografts would retain myocardial performance comparable to native hearts (NH). METHODS: Heterotopic hearts were transplanted using and end-to-side anastomosis between the donor's superior vena cava and the recipient's abdominal inferior vena cava. The right ventricle loads the left ventricle (LV) via a direct anastomosis of the pulmonary artery to the left atrium. The LV ejects volume through an end-to-side anastomosis of the donor's aorta to the recipient's abdominal aorta. Hemodynamic data (systolic and diastolic LV pressures, dP/dt max and min, tau) were studied in-situ (at baseline and after adding volume) and in a Langendorff perfusion system (at baseline and after stimulation with isoproterenol) 2 weeks after transplantation. RESULTS: In situ systolic pressure and diastolic function of VL was superior to NL, and beta-adrenergic stimulated performance in the Langendorff perfusion of VL showed hemodynamic performance equivalent to NH, unlike NL which had a diminished response. CONCLUSION: This technique results in a volume-loaded ejecting heart transplant model that preserves anatomical structures. The VL can be evaluated in situ and after explantation in Langendorff perfusion system and may offer advantages if workload of the graft is of significance to the study performed.
Asunto(s)
Trasplante de Corazón/fisiología , Modelos Cardiovasculares , Trasplante Heterotópico/fisiología , Abdomen , Análisis de Varianza , Anastomosis Quirúrgica/métodos , Animales , Cardiotónicos/farmacología , Electrocardiografía/estadística & datos numéricos , Trasplante de Corazón/métodos , Trasplante de Corazón/estadística & datos numéricos , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Isoproterenol/farmacología , Masculino , Ratas , Ratas Endogámicas Lew , Técnicas de SuturaRESUMEN
The relative biological effectiveness was determined using sex-linked recessive lethals induced in Drosophila spermatozoa as the biological effect. The sex-linked recessive lethal test, a measure of mutations induced in germ cells and transmitted through successive generations, yields a linear dose-response curve in the range used in these experiments. A dose-response curve was determined from three exposures to tritiated water and three exposures to 60Co gamma radiation. The ratio of the slopes of these two response curves is 2.7 +/- 0.3, yielding a relative biological effectiveness that suggests the tritium beta particle is 2.7 times more effective per unit of energy absorbed in inducing gene mutations transmitted to successive generations than 60Co gamma radiation. The increase in relative biological effectiveness with higher linear energy transfer for tritium beta radiation strongly suggests that single-strand breaks are repaired by a nearly error-free repair mechanism. Ion tracks with a high density of ions (high linear energy transfer) are more efficient than tracks with a low ion density (low linear energy transfer) in inducing transmissible mutations, suggesting interaction among products of ionization. Since most transmitted mutations induced by ionizing radiation result from strand breakage, interaction probably occurs at this level with double-strand breaks being repaired by an error-prone mechanism yielding transmissible mutations.
Asunto(s)
Radioisótopos de Cobalto , Mutación , Espermatozoides/efectos de la radiación , Tritio , Agua , Animales , Drosophila , Rayos gamma , Genes Letales , Genes Recesivos , Masculino , Efectividad Biológica RelativaRESUMEN
PURPOSE: To describe the evolution of a "collar-button" or mushroom-shaped choroidal metastasis. METHODS: Case report. RESULTS: A 52-year-old woman with known primary colon cancer and widespread extraocular metastases developed bilateral choroidal masses with neurosensory retinal detachments. Clinical and ultrasound evaluation demonstrated evolution of a choroidal metastasis with a "collar-button" configuration in the right eye. CONCLUSION: A choroidal metastasis may develop a mushroom-shaped configuration. This "collar-button" configuration is not pathognomonic for choroidal melanoma.
Asunto(s)
Neoplasias de la Coroides/diagnóstico por imagen , Neoplasias de la Coroides/secundario , Neoplasias del Colon/patología , Melanoma/diagnóstico por imagen , Melanoma/secundario , Femenino , Fondo de Ojo , Humanos , Persona de Mediana Edad , Desprendimiento de Retina/diagnóstico por imagen , UltrasonografíaRESUMEN
Two different mechanisms for mutagenesis following treatment with methyl methanesulfonate (MMS) are suggested from the dose-response curve that is best fit by the linear quadratic model where m = 0.130D + 0.038D2 (D = dose measured as alkylations per nucleotide X 10(3), APdN; m = percent sex-linked recessive lethals, SLRL). A predominant component of the dose-response curve at moderate to high dose is the quadratic component which is interpreted as the result of two single-strand breaks. The distribution of methyl adducts in vivo is consistent with the previously determined in vitro distribution of methyl adducts on DNA following treatment with MMS. With the use of HPLC, 82% of the 3H-labeled adducts are found on the N-7 of guanine. It has previously been shown by both in vitro studies and in vivo correlation with mutagenesis that the N-7 alkyl guanine is not itself a predominately genotoxic lesion; however, N-7 alkyl guanine destabilizes guanine resulting in an increased rate of hydrolysis producing apurinic sites. In data presented in this paper, the loss of labeled adducts is shown to be at a rate consistent with hydrolysis of the destabilized alkyl guanine. The apurinic site thus produced should be converted to single-strand breaks by AP endonucleases once sperm has fertilized the egg. Single-strand breaks are repaired by excision repair which is not error-prone; however, multiple breaks producing a proximity effect should lead to double-strand breaks that are repaired by an error-prone process. Mutations that are induced by a proximity effect would account for the quadratic term. It is hypothesized that a proximity effect is produced when two breaks are sufficiently close together to prevent using the complementary strand as a template. The linear component of the dose-response curve is probably due to alkylation of oxygens in the purine or pyrimidine ring leading to mispairing. However, due to the low frequency of ring-oxygen alkylation following treatment with MMS, this important genotoxic site is not the predominant one observed at experimental levels normally used in the laboratory. From the dose-response curve, it is calculated that at mutation frequencies of 10 times the spontaneous frequency or higher, the predominant mechanism is the multi-hit component; however, at mutation induced frequencies of 0.1 of the spontaneous frequency, which are levels more likely to be encountered in man's exposure to environmental mutagens, the dominant mechanism is the linear component.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
ADN/efectos de los fármacos , Genes Letales/efectos de los fármacos , Genes Recesivos/efectos de los fármacos , Metilmetanosulfonato/farmacología , Espermatozoides/efectos de los fármacos , Animales , ADN/genética , Relación Dosis-Respuesta a Droga , Drosophila melanogaster , Masculino , MetilaciónRESUMEN
The relative importance of different sites of alkylation on DNA was determined by comparing two ethylating agents. 1-Ethyl-1-nitrosourea (ENU) ethylates DNA with a higher proportion of total adducts on ring oxygens than ethyl methanesulfonate, which ethylates with a higher proportion of total adducts on the N-7 of guanine. Research with somatic cells in culture and prokaryotes strongly suggests that O6-guanine (O6-G) is the principal genotoxic site. To determine the importance in germ-line mutagenesis of the O6-G site relative to the N-7 of guanine, dose-response curves were constructed for both ENU and EMS, where dose was measured as total adducts per deoxynucleotide (APdN) and response as sex-linked recessive lethals (SLRL) induced in Drosophila melanogaster spermatozoa. For both mutagens the dose response curve was linear and extrapolated to the origin. The dose-response curve for ENU was fit to an equation m = 6.2D, and the dose response curve for EMS, from this and previous experiments, was m = 3.2D where m = %SLRL and D = APdN X 10(-3). Therefore, ENU is 1.9 times more efficient per adduct in inducing SLRL mutations than EMS. In vitro studies showed that ENU induced 9.5% of its total adducts on O6-G while EMS induced 2.0% of its adducts on O6-G. If O6-G was the sole genotoxic site, then ENU should be 4.8 times more efficient per adduct than EMS. In contrast, if N-7 G was the sole genotoxic site, ENU would be only 0.19 as effective as EMS. It was concluded that while O6-G was the principal genotoxic site, N-7 G made a significant contribution to germ-line mutagenesis.
Asunto(s)
Drosophila melanogaster/genética , Metanosulfonato de Etilo/farmacología , Etilnitrosourea/farmacología , Mutación , Espermatozoides/efectos de los fármacos , Animales , Radioisótopos de Carbono , Cruzamientos Genéticos , ADN/efectos de los fármacos , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Drosophila melanogaster/efectos de los fármacos , Metanosulfonato de Etilo/metabolismo , Etilnitrosourea/metabolismo , Femenino , Masculino , Pruebas de Mutagenicidad/métodos , Técnica de Dilución de Radioisótopos , Timidina/metabolismo , TritioRESUMEN
Tritium beta radiation (3H beta-radiation) in the form of tritiated water was used to induce mutations at the alcohol dehydrogenase (Adh) locus in male Drosophila melanogaster post-meiotic germ cells. All 23 Adh null mutations were large deletions (> 20 kb), determined by genetic complementation and Southern blot analyses. 27 Adh null mutations have been induced by 100-kVp X-rays (Aaron, 1979) and have been genetically and molecularly characterized (Ashburner et al., 1982; Chia et al., 1985; LoMonaco et al., 1987; Mahmoud et al., 1991). In contrast to 3H beta-radiation, 100-kVp X-rays induced a bimodal distribution of Adh null mutations, intragenic mutations, < or = 250 bp, and large deletions, > 100 kb. A statistically significant difference was observed between the frequency of large deletions (23/23 or 1.0) induced by 3H beta-radiation and the frequency of large deletions (19/27 or 0.7) induced by 100-kVp X-rays. However, a statistical difference was not observed between the size distribution of the large deletions induced by 3H beta-radiation and X-rays. The relative deletion frequency (RDF) induced by 3H beta-radiation and 100-kVp X-rays was (1.0/0.7 = 1.4). The relative biological effectiveness (RBE) of these two radiation sources was 1.4, determined from the ratio of the regression coefficients of the respective 3H beta-radiation and X-ray sex-linked recessive lethal (SLRL) dose-response data. The large difference in size between the two classes of X-ray-induced Adh null mutations and the increase in mutation frequency and deletion frequency for 3H beta-radiation with respect to X-rays may indicate that the relative deletion frequency (RDF) is the molecular biological basis for the increase in the RBE for radiation sources with a mean LET value < or = 10 keV/microns.
Asunto(s)
Partículas beta , Mutación , Eliminación de Secuencia , Espermatozoides/efectos de la radiación , Tritio , Rayos X , Alcohol Deshidrogenasa/genética , Animales , Drosophila melanogaster , Prueba de Complementación Genética , Masculino , Agua/químicaRESUMEN
The 2-chloroethyl methanesulfonate (2CIEMS)-induced alcohol dehydrogenase (Adh) null germline mutation frequency in treated Drosophila melanogaster second instar larval gonia was two orders of magnitude greater than the spontaneous mutation frequency. DNA sequence analysis of 83 Adh null mutations showed that 40 mutations of independent origin were at 23 sites in the Adh gene. The mutation spectrum contained only GC --> AT transitions with 35 mutations (87.5%) at the middle or 3' guanine. In addition, characteristics of glutathione (GSH)-mediated bioactivation were determined for 2CIEMS in vitro. Rates of GSH-mediated conjugation, catalyzed by purified rat liver glutathione-S-transferase (GST), and binding of [35S]GSH-mediated conjugation products to calf thymus DNA were determined for 2CIEMS, 1,2-dichloroethane (EDC) and 1,2-dibromoethane (EDB). The relative rates of GSH-mediated conjugation were the following: 5 mM EDB > 40 mM 2CIEMS > 40 mM EDC. A similar trend was observed for DNA binding of the [35S]GSH-mediated conjugation products when differences in mutagen concentration were considered: EDB > 2CIEMS > EDC. The ratios of DNA binding to GSH conjugation calculated for EDB, EDC and 2CIEMS were 6.8 x 10(-5), 9.3 x 10(-5) and 19.1 x 10(-5), respectively. A narrow range, less than a 3-fold difference, in the ratios of DNA binding to GSH conjugation indicates that the bioactivation of 2CIEMS is mediated by the same mechanism as EDB and EDC. Consequently, 2CIEMS, EDC and EDB may induce a specific mutation in premeiotic germ cells.
Asunto(s)
Células Germinativas/efectos de los fármacos , Mutación de Línea Germinal , Mesilatos/toxicidad , Mutágenos/toxicidad , Animales , Secuencia de Bases , Drosophila melanogaster , Femenino , Masculino , Datos de Secuencia Molecular , RatasRESUMEN
The 2-chloroethyl methanesulfonate (2ClEMS)-induced alcohol dehydrogenase (Adh) null germline mutation frequency in treated Drosophila melanogaster second instar larval gonia was two orders of magnitude greater than the spontaneous mutation frequency. DNA sequence analysis of 83 Adh null mutations showed that 40 mutations of independent origin were at 23 sites in the Adh gene. The mutation spectrum contained only GC-->AT transitions with 35 mutations (87.5%) at the middle or 3' guanine. In addition, characteristics of glutathione (GSH)-mediated bioactivation were determined for 2ClEMS in vitro. Rates of GSH-mediated conjugation, catalyzed by purified rat liver glutathione-S-transferase (GST), and binding of [35S]GSH-mediated conjugation products to calf thymus DNA were determined for 2ClEMS, 1,2-dichloroethane (EDC) and 1,2-dibromoethane (EDB). The relative rates of GSH-mediated conjugation were the following: 5 mM EDB > 40 mM 2ClEMS > 40 mM EDC. A similar trend was observed for DNA binding of the [35S]GSH-mediated conjugation products when differences in mutagen concentration were considered: EDB > 2ClEMS > EDC. The ratios of DNA binding to GSH conjugation calculated for EDB, EDC and 2ClEMS were 6.8 x 10(-5), 9.3 x 10(-5) and 19.1 x 10(-5), respectively. A narrow range, less than a 3-fold difference, in the ratios of DNA binding to GSH conjugation indicates that the bioactivation of 2ClEMS is mediated by the same mechanism as EDB and EDC. Consequently, 2ClEMS, EDC and EDB may induce a specific mutation in premeiotic germ cells.
Asunto(s)
Células Germinativas/efectos de los fármacos , Mutación de Línea Germinal , Mesilatos/toxicidad , Mutágenos/toxicidad , Alcohol Deshidrogenasa/genética , Animales , Secuencia de Bases , ADN/metabolismo , Aductos de ADN/metabolismo , Análisis Mutacional de ADN , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Dibromuro de Etileno/química , Dibromuro de Etileno/toxicidad , Dicloruros de Etileno/química , Dicloruros de Etileno/toxicidad , Femenino , Glutatión/química , Glutatión Transferasa/metabolismo , Larva/efectos de los fármacos , Masculino , Mesilatos/química , Datos de Secuencia Molecular , Mutágenos/química , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: Various sampling techniques of the cervix have established false negative rates ranging from 18% to 45%. A number of studies suggest that this false negative rate can be reduced by sampling techniques that are associated with higher yields of endocervical cells. METHOD: This study enrolled 301 women, each of whom had a Pap smear obtained using three different sampling instruments (a cervical brush, a plastic spatula, and a cotton swab) in random order. RESULTS: The cervical brush yielded a better endocervical sample than either of the other instruments (P < .001), and there was no difference between the swab and spatula. In addition, the recovery of endocervical cells increased with each additional sample taken, regardless of instrument used (P < .001), although the difference in yield between the second and third samples was not significant. CONCLUSIONS: To enhance the yield of endocervical cells in Pap smear sampling, consideration should be given to using the cervical brush routinely as a sampling instrument and to taking more than one sample per screening.
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Prueba de Papanicolaou , Frotis Vaginal/instrumentación , Adolescente , Adulto , Reacciones Falso Negativas , Femenino , Humanos , Persona de Mediana Edad , Frotis Vaginal/normasRESUMEN
Pediatric orbital pseudotumor may be associated with iritis, unlike the adult form of this disorder. However, orbital pseudotumor is seldom considered in the differential diagnosis of childhood uveitis. We report two cases of children with uveitis who were ultimately diagnosed as having orbital pseudotumor. No proptosis was noted in either child. For those pediatric patients with a persistent or recurrent uveitis and a previous negative diagnostic workup, the possibility of an orbital pseudotumor should be considered and ultrasonography, magnetic resonance imaging, and/or computed tomography performed.
Asunto(s)
Seudotumor Orbitario/diagnóstico por imagen , Uveítis/diagnóstico por imagen , Niño , Diagnóstico Diferencial , Femenino , Fondo de Ojo , Humanos , Imagen por Resonancia Magnética , Masculino , Prednisona/uso terapéutico , Tomografía Computarizada por Rayos X , UltrasonografíaRESUMEN
Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-alpha (G6Pase-alpha), a nine-transmembrane domain, endoplasmic reticulum-associated protein expressed primarily in the liver and kidney. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase-alpha (AAV2-G6Pase-alpha) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. We now show that neonatal infusion of GSD-Ia mice with an AAV serotype 1-G6Pase-alpha (AAV1-G6Pase-alpha) or AAV serotype 8-G6Pase-alpha (AAV8-G6Pase-alpha) results in hepatic expression of the G6Pase-alpha transgene and markedly improves the survival of the mice. However, only AAV1-G6Pase-alpha can achieve significant renal transgene expression. A more effective strategy, in which a neonatal AAV1-G6Pase-alpha infusion is followed by a second infusion at age one week, provides sustained expression of a complete, functional, G6Pase-alpha system in both the liver and kidney and corrects the metabolic abnormalities in GSD-Ia mice for the 57 week length of the study. This effective use of gene therapy to correct metabolic imbalances and disease progression in GSD-Ia mice holds promise for the future of gene therapy in humans.