RESUMEN
Pompe disease is a clinically and genetically heterogeneous autosomal recessive disorder caused by lysosomal acid α-glucosidase (GAA) deficiency. We report on two affected members of a non-consanguineous Caucasian family, including a classical infantile-onset patient with severe cardiomyopathy (IO) and his paternal grandmother with the adult-onset (AO) form. Two compound heterozygous sequence variants of the GAA gene were identified in each patient by mutation analyses (IO=c.1211A>G and c.1798C>T; AO=c.1211A>G and c.692+5G>T). For this study, the biochemical phenotype resulting from the missense mutation c.1211A>G in exon 8, which converts a highly conserved aspartate to glycine (p.Asp404Gly), was of specific interest because it had not been reported previously. Western blotting revealed a robust expression of all GAA isoforms in quadriceps muscle of both patients (fully CRIM positive), while enzymatic activity was 3.6% (IO) and 6.6% (AO) of normal controls. To further validate these findings, the c.1211A>G sequence variant was introduced in wild type GAA cDNA and over-expressed in HEK293T cells. Site-directed mutagenesis analyses confirmed that the mutation does not affect processing or expression of GAA protein, but rather impairs enzyme function. Similar results were reported for c.1798C>T (p.Arg600Cys), which further supports the biochemical phenotype observed in IO. The third mutation (c.692+5G>T, in intron 3) was predicted to affect normal splicing of the GAA mRNA, and qPCR indeed verified a 4-fold lower mRNA expression in AO. It is concluded that the novel sequence variant c.1211A>G results in full CRIM but significantly lower GAA activity, which in combination with c.1798C>T leads to infantile-onset Pompe disease. We surmise that the difference in disease severity between the two family members in this study is due to a milder effect of the intronic mutation c.692+5G>T (vs. c.1798C>T) on phenotype, partially preserving GAA activity and delaying onset in the proband (paternal grandmother).
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Mutación Missense , Edad de Inicio , Estudios de Casos y Controles , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo II/epidemiología , Células HEK293 , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación , Linaje , Fenotipo , Embarazo , Músculo Cuádriceps/enzimología , Empalme del ARN , Valores de ReferenciaRESUMEN
The widely used CHARMM additive all-atom force field includes parameters for proteins, nucleic acids, lipids, and carbohydrates. In the present article, an extension of the CHARMM force field to drug-like molecules is presented. The resulting CHARMM General Force Field (CGenFF) covers a wide range of chemical groups present in biomolecules and drug-like molecules, including a large number of heterocyclic scaffolds. The parametrization philosophy behind the force field focuses on quality at the expense of transferability, with the implementation concentrating on an extensible force field. Statistics related to the quality of the parametrization with a focus on experimental validation are presented. Additionally, the parametrization procedure, described fully in the present article in the context of the model systems, pyrrolidine, and 3-phenoxymethylpyrrolidine will allow users to readily extend the force field to chemical groups that are not explicitly covered in the force field as well as add functional groups to and link together molecules already available in the force field. CGenFF thus makes it possible to perform "all-CHARMM" simulations on drug-target interactions thereby extending the utility of CHARMM force fields to medicinally relevant systems.
Asunto(s)
Simulación por Computador , Modelos Químicos , Pirrolidinas/química , Modelos Moleculares , Simulación de Dinámica Molecular , Teoría Cuántica , Programas InformáticosRESUMEN
Cross-resistance presents an obstacle in cancer chemotherapy. Cadmium is a potential carcinogen whose exposure has been shown in epidemiological and laboratory experiments to cause lung cancer. Cadmium also induces various forms of resistance in human lung carcinoma cells. This resistance may be shared by antineoplastic agents, which should be a concern for chemotherapy of cadmium-induced lung cancer. In the present study, two subpopulations of human lung carcinoma A549 cells with a different magnitude of resistance to cadmium toxicity were shown to have a parallel resistance to the cytotoxic action of Adriamycin (ADR), an important anticancer drug. Several factors were examined to investigate the mechanism(s) for the cross-resistance, including cellular metallothionein and glutathione (GSH) concentrations, glutathione S-transferase activity, mdr1 expression, and antioxidant enzyme activities including superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Only cellular GSH content was elevated consistently in the cadmium/ADR-resistant cells relative to the cadmium/ADR-sensitive cells. Treatment with buthionine sulfoximine, a specific inhibitor of GSH synthesis sensitized both cell lines to ADR only when the cellular GSH levels were depleted to about 5% of control. This BSO treatment, however, did not affect cell viability. Further study revealed that the cadmium/ADR-resistant cells have a greater capacity in recovery of cellular GSH content following BSO treatment. The results demonstrate that cross-resistance to ADR exists in cadmium-resistant human lung carcinoma A549 cells, and enhanced GSH synthesis capacity, rather than elevated levels of cellular GSH, may be related to this resistance.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Cadmio/toxicidad , Carcinógenos Ambientales/toxicidad , Doxorrubicina/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Butionina Sulfoximina/farmacología , Resistencia a Medicamentos , Glutatión/análisis , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Células Tumorales CultivadasRESUMEN
While studies have addressed the diagnosis and progression of interproximal carious lesions within a primary tooth, few studies have addressed the development of proximal lesions in adjacent primary molars. The purpose of this study was to examine retrospectively the long term interproximal caries progression in primary molar teeth. Dental records of 150 children were retrospectively reviewed, 76 from a university pediatric dentistry clinic and 74 from a pediatric dentistry private practice. Out of the 387 teeth initially diagnosed with proximal caries, the combined university and private practice results for timing of the development of proximal lesions on adjacent tooth surfaces showed the following: simultaneous development-162 (41.9%); 1 to 24 months-65 (16.8%); 24 to 60 months-40 (10.3%); never-120 (31.0%). The combined results for formation of proximal caries in posterior quadrants showed that out of the 150 patients, the timing for development of additional quadrants with proximal caries was as follow: simultaneous development: 77 (51.3%); 1 to 24 months 31, (20.7%); 24 to 60 months 25, (16.7%); never 17 (11.3%). The conclusions of the study are that 69% of the primary molar teeth with proximal caries developed caries on the adjacent proximal surface and 89% of the patients who developed a proximal carious lesion on a primary molar tooth within one quadrant developed another primary molar proximal lesion in another quadrant.
Asunto(s)
Caries Dental/patología , Diente Molar/patología , Diente Primario/patología , Niño , Preescolar , Atención Dental para Niños , Caries Dental/diagnóstico , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Odontología Pediátrica , Estudios Retrospectivos , Factores de TiempoRESUMEN
Glutathione has been implicated to function in cytoprotection against cadmium toxicity. The mechanism by which glutathione plays this role has not been well understood. Because glutathione is an important antioxidant and several studies have shown that cadmium induces oxidative stress, this study was undertaken to determine whether development of cadmium resistance is linked to enhanced antioxidant activities. A cadmium-resistant subpopulation of human lung carcinoma A549 cells, which was developed by repeatedly exposing the cells to step-wise increased cadmium concentrations, was compared to a cadmium-sensitive one. The acquired cadmium resistance resulted from neither decreased cadmium uptake nor enhanced cellular metallothionein synthesis. Glutathione content, however, was markedly elevated in the cadmium-resistant cells. In contrast, the activities of the glutathione redox cycle related enzymes, glutathione peroxidase and reductase, were unchanged. Two other antioxidant enzymes, superoxide dismutase and catalase, were also not altered. The results suggest that the development of cadmium resistance in A549 cells unlikely results from enhanced antioxidant enzyme activities, although it is associated with elevated cellular glutathione levels. In addition, measurement of the mRNA and DNA levels for gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione biosynthesis, revealed that enhanced expression of the enzyme but not gene amplification is likely responsible for the elevation of cellular glutathione levels.
Asunto(s)
Antioxidantes/metabolismo , Cadmio/farmacología , Resistencia a Medicamentos , Glutatión/metabolismo , Neoplasias Pulmonares/metabolismo , Estrés Oxidativo , ADN/análisis , Glutamato-Cisteína Ligasa/genética , Humanos , Oxidación-Reducción , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Extracellular glutathione (GSH) is degraded by an external cell-surface enzyme, gamma-glutamyltranspeptidase (gamma-GT). The products are transported into cells to participate in important cellular processes. In the present study, we tested the hypothesis that extracellular GSH is a source of glutamic acid for cells that express gamma-GT. Under a glutamine-deficient culture condition, the extracellular GSH-supplemented glutamic acid would enhance intracellular glutamine synthesis, thereby stimulating cell proliferation. Human lung carcinoma A549 cells were cultured in glutamine-deficient Dulbecco's modified Eagle medium, and they did not proliferate unless glutamine was supplemented. Extracellular GSH, however, provoked a partial proliferation. The GSH effect correlated with a high level of gamma-GT activity and an increased intracellular level of glutamic acid. A constituent amino acid of GSH, glutamic acid but not cysteine, produced the same growth-stimulatory effect as GSH. Furthermore, neither oxothiazolidine-4-carboxylate (OTC), a cellular cysteine-delivery compound, nor cysteinylglycine, a dipeptide released from the gamma-GT reaction, stimulated cell proliferation. Moreover, buthionine sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, enhanced the GSH growth stimulatory effect, suggesting that increased cellular GSH synthesis does not correlate with cell growth stimulation. The results obtained demonstrated that glutamine is required for A549 cell proliferation and exogenous GSH partially substitutes for the growth stimulatory action of glutamine. It also suggests that the glutamic acid rather than the cysteine released from the GSH is responsible for the cell proliferation.
Asunto(s)
División Celular/efectos de los fármacos , Glutatión/farmacología , Butionina Sulfoximina , Cisteína/farmacología , Glutamatos/metabolismo , Glutamina/deficiencia , Glutatión/metabolismo , Humanos , Técnicas In Vitro , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Células Tumorales Cultivadas , gamma-Glutamiltransferasa/metabolismoAsunto(s)
Psicoanálisis , Sociedades Médicas , Adulto , Desarrollo Humano , Humanos , Apego a Objetos , ViolenciaRESUMEN
The purpose of this study was to document whether there was a significant difference in the number and severity of generalized fears and dental fears between patients who did and patients who did not experience hand-over-mouth and/or restraint as children. Patient records in a dental school children's clinic and a private pediatric dental practice were examined to identify patients who had experienced hand-over-mouth and/or restraint. A set of verbal questions was designed, tested, and used to ascertain the differences between the HOM/restraint group and the comparison group. One hundred twenty-two subjects were interviewed, 61 who had experienced HOM/restraint and 61 who had not. When compared for generalized fears and specific dental fears, the two groups showed no statistically significant differences (P = 0.86 and P = 0.36 respectively). No statistically significant difference appeared between the two groups when asked how they felt about visiting the dental office (P = 0.41). When three different formats were used to question the subjects relative to their early dental memories, the two groups showed no statistical difference in negative or positive responses (P = 0.38, 0.75, and 0.25 respectively). More than two times as many HOM/restraint subjects as comparison subjects described negative experiences in a physician's office or hospital. This difference was statistically significant (P < 0.01).
Asunto(s)
Terapia Aversiva , Ansiedad al Tratamiento Odontológico/etiología , Atención Odontológica/psicología , Relaciones Dentista-Paciente , Memoria , Restricción Física/efectos adversos , Adolescente , Adulto , Distribución de Chi-Cuadrado , Niño , Femenino , Humanos , Masculino , Estudios RetrospectivosRESUMEN
The purpose of this project was to survey Indiana dentists concerning dental care for Indiana infants and toddlers. Dental care issues covered were the age for a child's first dental visit, frequency of rampant or nursing caries cases examined, and dental referral sources for infants and toddlers. The survey also helped determine the dentist's perception of parents' attitudes toward services recommended by the dentist. A twelve question survey was mailed to 2006 general and pediatric dentists in the State of Indiana. The results of the survey were: 1. A large percentage of Indiana dental practitioners do not recommend the first dental visit or examine children for their dental visit until the child is more than one year of age. 2. A majority of Indiana dental practitioners see children with nursing caries or rampant caries on a monthly basis. 3. The majority of Indiana dental practitioners refer children with nursing caries to pediatric dentists for treatment. 4. Although the full treatment plan may undergo some modification based on various parental concerns, a majority of practitioners do not experience difficulty in obtaining treatment plan acceptance for nursing or rampant caries cases.
Asunto(s)
Preescolar , Atención Odontológica/estadística & datos numéricos , Caries Dental/epidemiología , Lactante , Factores de Edad , Alimentación con Biberón/efectos adversos , Lactancia Materna , Distribución de Chi-Cuadrado , Caries Dental/etiología , Humanos , Indiana , Padres , Aceptación de la Atención de Salud , Derivación y Consulta , Encuestas y CuestionariosRESUMEN
The author observes a resemblance between (1) the "polysemous" technique of imputing meaning to reality practiced in medieval biblical studies and in Dante's writing and (2) the technique of interpretation in contemporary psychoanalysis. She explores the roots of this resemblance in the development of intellectual history and provides examples of polysemous meanings in Dante's Divine Comedy, which is in part an autobiographical journey of self-reflection and self-realization (like psychoanalysis). She then suggests some implications of this resemblance for contemporary psychiatry.
Asunto(s)
Literatura Medieval , Poesía como Asunto , Interpretación Psicoanalítica , Terapia Psicoanalítica , Conducta Sexual , Teoría Freudiana , HumanosRESUMEN
Compared with nonalcoholic controls, women alcoholics displayed cognitive deficits on verbal and nonverbal tasks. Although menstruating and nonmenstruating alcoholics performed similarly on verbal and nonverbal tasks, menstruating controls performed better than nonmenstruating controls on both tasks.