RESUMEN
Obesity is a major risk factor for a plethora of metabolic disturbances including diabetes and cardiovascular disease. Accumulating evidence is showing that there is an adipose tissue depot-dependent relationship with obesity-induced metabolic dysfunction. While some adipose depots, such as subcutaneous fat, are generally metabolically innocuous, others such as visceral fat, are directly deleterious. A lesser known visceral adipose depot is the pericardial adipose tissue depot. We therefore set out to examine its transcriptional and morphological signature under chow and high-fat fed conditions, in comparison with other adipose depots, using a mouse model. Our results revealed that under chow conditions pericardial adipose tissue has uncoupling-protein 1 gene expression levels which are significantly higher than classical subcutaneous and visceral adipose depots. We also observed that under high-fat diet conditions, the pericardial adipose depot exhibits greatly upregulated transcript levels of inflammatory cytokines. Our results collectively indicate, for the first time, that the pericardial adipose tissue possesses a unique transcriptional and histological signature which has features of both a beige (brown fat-like) but also pro-inflammatory depot, such as visceral fat. This unique profile may be involved in metabolic dysfunction associated with obesity.
Asunto(s)
Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Expresión Génica , Obesidad/metabolismo , Pericardio/metabolismo , Pericardio/patología , Adipogénesis/genética , Tejido Adiposo Pardo/metabolismo , Animales , Dieta Alta en Grasa , Inflamación/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/patología , Grasa Subcutánea/metabolismo , Termogénesis/genética , Proteína Desacopladora 1/genéticaRESUMEN
As bone is used in a dynamic mechanical environment, understanding the structural origins of its time-dependent mechanical behaviour - and the alterations in metabolic bone disease - is of interest. However, at the scale of the mineralized fibrillar matrix (nanometre-level), the nature of the strain-rate dependent mechanics is incompletely understood. Here, we investigate the fibrillar- and mineral-deformation behaviour in a murine model of Cushing's syndrome, used to understand steroid induced osteoporosis, using synchrotron small- and wide-angle scattering/diffraction combined with in situ tensile testing at three strain rates ranging from 10-4 to 10-1 s-1. We find that the effective fibril- and mineral-modulus and fibrillar-reorientation show no significant increase with strain-rate in osteoporotic bone, but increase significantly in normal (wild-type) bone. By applying a fibril-lamellar two-level structural model of bone matrix deformation to fit the results, we obtain indications that altered collagen-mineral interactions at the nanoscale - along with altered fibrillar orientation distributions - may be the underlying reason for this altered strain-rate sensitivity. Our results suggest that an altered strain-rate sensitivity of the bone matrix in osteoporosis may be one of the contributing factors to reduced mechanical competence in such metabolic bone disorders, and that increasing this sensitivity may improve biomechanical performance.
Asunto(s)
Nanoestructuras , Osteoporosis , Animales , Matriz Ósea , Huesos , Ratones , Osteoporosis/inducido químicamente , Esteroides , Estrés MecánicoRESUMEN
Glucocorticoid-induced osteoporosis (GIOP) is a major secondary form of osteoporosis, with the fracture risk significantly elevated - at similar levels of bone mineral density - in patients taking glucocorticoids compared with non-users. The adverse bone structural changes at multiple hierarchical levels in GIOP, and their mechanistic consequences leading to reduced load-bearing capacity, are not clearly understood. Here we combine experimental X-ray nanoscale mechanical imaging with analytical modelling of the bone matrix mechanics to determine mechanisms causing bone material quality deterioration during development of GIOP. In situ synchrotron small-angle X-ray diffraction combined with tensile testing was used to measure nanoscale deformation mechanisms in a murine model of GIOP, due to a corticotrophin-releasing hormone promoter mutation, at multiple ages (8-, 12-, 24- and 36â¯weeks), complemented by quantitative micro-computed tomography and backscattered electron imaging to determine mineral concentrations. We develop a two-level hierarchical model of the bone matrix (mineralized fibril and lamella) to predict fibrillar mechanical response as a function of architectural parameters of the mineralized matrix. The fibrillar elastic modulus of GIOP-bone is lower than healthy bone throughout development, and nearly constant in time, in contrast to the progressively increasing stiffness in healthy bone. The lower mineral platelet aspect ratio value for GIOP compared to healthy bone in the multiscale model can explain the fibrillar deformation. Consistent with this result, independent measurement of mineral platelet lengths from wide-angle X-ray diffraction finds a shorter mineral platelet length in GIOP. Our results show how lowered mineralization combined with altered mineral nanostructure in GIOP leads to lowered mechanical competence. SIGNIFICANCE STATEMENT: Increased fragility in musculoskeletal disorders like osteoporosis are believed to arise due to alterations in bone structure at multiple length-scales from the organ down to the supramolecular-level, where collagen molecules and elongated mineral nanoparticles form stiff fibrils. However, the nature of these molecular-level alterations are not known. Here we used X-ray scattering to determine both how bone fibrils deform in secondary osteoporosis, as well as how the fibril orientation and mineral nanoparticle structure changes. We found that osteoporotic fibrils become less stiff both because the mineral nanoparticles became shorter and less efficient at transferring load from collagen, and because the fibrils are more randomly oriented. These results will help in the design of new composite musculoskeletal implants for bone repair.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Matriz Ósea/metabolismo , Glucocorticoides/efectos adversos , Osteoporosis , Animales , Matriz Ósea/patología , Modelos Animales de Enfermedad , Femenino , Glucocorticoides/farmacología , Humanos , Ratones , Ratones Transgénicos , Osteoporosis/inducido químicamente , Osteoporosis/metabolismo , Osteoporosis/patologíaRESUMEN
A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh(-120/+)) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh(-120/+) mice. We also find a much larger fibril strain/tissue strain ratio in Crh(-120/+) mice (~1.5) compared to the wild-type mice (~0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis.
Asunto(s)
Matriz Ósea/patología , Matriz Ósea/fisiopatología , Fracturas Óseas/fisiopatología , Osteoporosis/inducido químicamente , Osteoporosis/fisiopatología , Esteroides/efectos adversos , Animales , Matriz Ósea/diagnóstico por imagen , Femenino , Fémur/patología , Fémur/fisiopatología , Fémur/ultraestructura , Fracturas Óseas/diagnóstico por imagen , Fracturas Óseas/patología , Ratones Endogámicos C57BL , Osteoporosis/diagnóstico por imagen , Sincrotrones , Resistencia a la Tracción , Microtomografía por Rayos XRESUMEN
In metabolic bone diseases, the alterations in fibrillar level bone-material quality affecting macroscopic mechanical competence are not well-understood quantitatively. Here, we quantify the fibrillar level deformation in cantilever bending in a mouse model for hereditary rickets (Hpr). Microfocus in-situ synchrotron small-angle X-ray scattering (SAXS) combined with cantilever bending was used to resolve nanoscale fibril strain in tensile- and compressive tissue regions separately, with quantitative backscattered scanning electron microscopy used to measure microscale mineralization. Tissue-level flexural moduli for Hpr mice were significantly (p<0.01) smaller compared to wild-type (~5 to 10-fold reduction). At the fibrillar level, the fibril moduli within the tensile and compressive zones were significantly (p<0.05) lower by ~3- to 5-fold in Hpr mice compared to wild-type mice. Hpr mice have a lower mineral content (24.2±2.1Cawt.% versus 27.4±3.3Ca wt.%) and its distribution was more heterogeneous compared to wild-type animals. However, the average effective fibril modulus did not differ significantly (p>0.05) over ages (4, 7 and 10weeks) between tensile and compressive zones. Our results indicate that incompletely mineralized fibrils in Hpr mice have greater deformability and lower moduli in both compression and tension, and those compressive and tensile zones have similar moduli at the fibrillar level.
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Huesos/patología , Huesos/fisiopatología , Calcificación Fisiológica/fisiología , Fuerza Compresiva/fisiología , Minerales/metabolismo , Raquitismo/fisiopatología , Resistencia a la Tracción/fisiología , Animales , Fenómenos Biomecánicos/fisiología , Modelos Animales de Enfermedad , Módulo de Elasticidad , Húmero/diagnóstico por imagen , Húmero/patología , Húmero/fisiopatología , Ratones , Modelos Biológicos , Radiografía , Raquitismo/patologíaRESUMEN
Metabolic bone disorders such as rickets are associated with altered in vivo muscular force distributions on the skeletal system. During development, these altered forces can potentially affect the spatial and temporal dynamics of mineralised tissue formation, but the exact mechanisms are not known. Here we have used a murine model of hypophosphatemic rickets (Hpr) to study the development of the mineralised nanostructure in the intramembranously ossifying scapulae (shoulder bone). Using position-resolved scanning small angle X-ray scattering (SAXS), we quantified the degree and direction of mineral nanocrystallite alignment over the width of the scapulae, from the load bearing lateral border (LB) regions to the intermediate infraspinous fossa (IF) tissue. These measurements revealed a significant (p<0.05) increase in mineral nanocrystallite alignment in the LB when compared to the IF region, with increased tissue maturation in wild-type mice; this was absent in mice with rickets. The crystallites were more closely aligned to the macroscopic bone boundary in the LB when compared to the IF region in both wild type and Hpr mice, but the degree of alignment was reduced in Hpr mice. These findings are consistent with a correlation between the nanocrystallites within fibrils and in vivo muscular forces. Thus our results indicate a relevant mechanism for the observed increased macroscopic deformability in rickets, via a significant alteration in the mineral particle alignment, which is mediated by an altered spatial distribution of muscle forces.
Asunto(s)
Envejecimiento/patología , Raquitismo Hipofosfatémico Familiar/patología , Minerales/metabolismo , Nanopartículas/química , Escápula/crecimiento & desarrollo , Escápula/patología , Animales , Cristalización , Ratones , Escápula/anomalías , Dispersión del Ángulo Pequeño , Sincrotrones , Difracción de Rayos XRESUMEN
AIMS/HYPOTHESIS: We identified a mouse with a point mutation (Y12STOP) in the Kcnj11 subunit of the K(ATP) channel. This point mutation is identical to that found in a patient with congenital hyperinsulinism of infancy (HI). We aimed to characterise the phenotype arising from this loss-of-function mutation and to compare it with that of other mouse models and patients with HI. METHODS: We phenotyped an N-ethyl-N-nitrosourea-induced mutation on a C3H/HeH background (Kcnj11 ( Y12STOP )) using intraperitoneal glucose tolerance testing to measure glucose and insulin plasma concentrations. Insulin secretion and response to incretins were measured on isolated islets. RESULTS: Homozygous male and female adult Kcnj11 ( Y12STOP ) mice exhibited impaired glucose tolerance and a defect in insulin secretion as measured in vivo and in vitro. Islets had an impaired incretin response and reduced insulin content. CONCLUSIONS/INTERPRETATION: The phenotype of homozygous Kcnj11 ( Y12STOP ) mice is consistent with that of other Kcnj11-knockout mouse models. In contrast to the patient carrying this mutation homozygously, the mice studied did not have hyperinsulinaemia or hypoglycaemia. It has been reported that HI patients may develop diabetes and our mouse model may reflect this clinical feature. The Kcnj11 ( Y12STOP ) model may thus be useful in further studies of K(ATP) channel function in various cell types and in investigation of the development of hyperglycaemia in HI patients.
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Intolerancia a la Glucosa/genética , Hiperinsulinismo/genética , Mutación/genética , Fenotipo , Canales de Potasio de Rectificación Interna/genética , Animales , Femenino , Genotipo , Prueba de Tolerancia a la Glucosa , Humanos , Masculino , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: The Gnas transcription unit located within an imprinting region encodes several proteins, including the G-protein alpha-subunit, Gsalpha, its isoform XLalphas and their variant truncated neural forms GsalphaN1 and XLN1. Gsalpha and GsalphaN1 are expressed predominantly from the maternally derived allele in some tissues, whereas XLalphas and XLN1 are expressed exclusively from the paternally derived allele. The relative contribution of full-length Gsalpha and XLalphas, and truncated forms GsalphaN1 and XLN1 to phenotype is unknown. The edematous-small point mutation (Oed-Sml) in exon 6 of Gnas lies downstream of GsalphaN1 and XLN1, but affects full-length Gsalpha and XLalphas, allowing us to address the role of full-length Gsalpha and XLalphas. The aim of this study was therefore to determine the metabolic phenotypes of Oed and Sml mice, and to correlate phenotypes with affected transcripts. METHODS: Mice were fed standard or high-fat diets and weighed regularly. Fat mass was determined by DEXA analysis. Indirect calorimetry was used to measure metabolic rate. Glucose was measured in tolerance tests and biochemical parameters in fasted plasma samples. Histological analysis of fat and liver was carried out post mortem. RESULTS: Oed mice are obese on either diet and have a reduced metabolic rate. Sml mice are lean and are resistant to a high-fat diet and have an increased metabolic rate. CONCLUSION: Adult Oed and Sml mice have opposite metabolic phenotypes. On maternal inheritance, the obese Oed phenotype can be attributed to non-functional full-length Gsalpha. In contrast, on paternal inheritance, Sml mice were small and resistant to the development of obesity on a high-fat diet, effects that can be attributed to mutant XLalphas. Thus, the neural isoforms, GsalphaN1 and XLN1, do not appear to play a role in these metabolic phenotypes.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Hiperglucemia/genética , Mutación Missense/genética , Obesidad/genética , Animales , Biomarcadores/sangre , Composición Corporal , Cromograninas , Grasas de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Masculino , Ratones , Mutación Puntual/genética , Isoformas de ProteínasRESUMEN
Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.
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Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/orina , Orina/química , Animales , Diabetes Mellitus Tipo 2/genética , Femenino , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Metilaminas/metabolismo , Metilaminas/orina , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/orina , Nucleótidos/metabolismo , Nucleótidos/orina , Ratas , Ratas Zucker , Receptores de Superficie Celular/genética , Receptores de Leptina , Especificidad de la EspecieRESUMEN
This paper reviews recent studies on the role of Nnt (nicotinamide nucleotide transhydrogenase) in insulin secretion and detoxification of ROS (reactive oxygen species). Glucose-stimulated insulin release from pancreatic beta-cells is mediated by increased metabolism. This elevates intracellular [ATP], thereby closing KATP channels (ATP-sensitive potassium channels) and producing membrane depolarization, activation of voltage-gated Ca2+ channels, Ca2+ influx and, consequently, insulin secretion. The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion, which results from a naturally occurring deletion in the Nnt gene. Transgenic expression of the wild-type Nnt gene in C57BL/6J mice rescues the phenotype. Knockdown of Nnt in the insulin-secreting cell line MIN6 with small interfering RNA dramatically reduced Ca2+ influx and insulin secretion. Similarly, mice carrying ENU (N-ethyl-N-nitrosourea)-induced loss-of-function mutations in Nnt were glucose intolerant and secreted less insulin during a glucose tolerance test. Islets isolated from these mice showed impaired insulin secretion in response to glucose, but not to the KATP channel blocker tolbutamide. This is explained by the fact that glucose failed to elevate ATP in Nnt mutant islets. Nnt is a nuclear-encoded mitochondrial protein involved in detoxification of ROS. beta-Cells isolated from Nnt mutant mice showed increased ROS production on glucose stimulation. We hypothesize that Nnt mutations enhance glucose-dependent ROS production and thereby impair beta-cell mitochondrial metabolism, possibly via activation of uncoupling proteins. This reduces ATP production and lowers KATP channel activity. Consequently, glucose-dependent electrical activity and insulin secretion are impaired.
Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , NADP Transhidrogenasas/metabolismo , Estrés Oxidativo/fisiología , Animales , Secreción de Insulina , Ratones , Ratones Noqueados , Membranas Mitocondriales/fisiología , NADP Transhidrogenasas/deficiencia , NADP Transhidrogenasas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Diabetes is one of the most challenging health problems of the 21st century with an alarming increase in the prevalence of type-2 diabetes mellitus (T2DM) and associated conditions such as hypertension, dyslipidemias and obesity. T2DM is a complex genetic disease comprised of many metabolic disorders with a common phenotype of glucose intolerance. Patients with T2DM would have inherited a variety of different genetic factors that together with environmental factors combine as the primary cause. This complicates the genetic study of the disease and means that different methodological approaches are needed if we hope to identify susceptibility genes and genetic variants. The biochemical and physiological processes that underpin T2DM are still unclear although most certainly involve impairment in insulin secretion and insulin action. In this review, we will discuss the most exciting advances in understanding the genetics of T2DM by looking at recent discoveries employing human association studies and candidate genes arising from animal models.
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Diabetes Mellitus Tipo 2/genética , Animales , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Femenino , Genética de Población , Humanos , Resistencia a la Insulina/genética , Masculino , Ratones , Modelos Biológicos , Modelos Genéticos , Obesidad/genética , Factores de RiesgoRESUMEN
Gamete competition models were used to explore the relationships between 13 ACE gene polymorphisms and plasma ACE concentration in a set of Nigerian families. Several markers in the 5' and 3' regions of the gene were significantly associated with ACE concentration (P < 10(-4)). Multi-locus genotypes comprising different combinations of markers from the 5' UTR and the 3' region of the gene were also analysed; in addition to G2350A, in the 3' region, two markers from the 5' UTR (A-5466C and A-240T) were found to be associated with ACE concentration. These results are consistent with reports that have suggested the presence of at least two ACE-linked QTLs, and demonstrate the utility of gamete competition models in the exploratory investigation of the relationship between a quantitative trait and multiple variants in a small genomic region.
Asunto(s)
Células Germinativas , Haplotipos , Modelos Biológicos , Peptidil-Dipeptidasa A/genética , Polimorfismo de Nucleótido Simple , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , NigeriaRESUMEN
AIMS/HYPOTHESIS: C57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes. METHODS: Glucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis. RESULTS: C57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close KATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver. CONCLUSIONS/INTERPRETATION: The C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.
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Glucemia/metabolismo , Intolerancia a la Glucosa/genética , NADP Transhidrogenasas/genética , Animales , Señalización del Calcio/efectos de los fármacos , Cruzamientos Genéticos , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Ayuno , Femenino , Expresión Génica/genética , Genotipo , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Intolerancia a la Glucosa/sangre , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Mutación , Fenotipo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Sitios de Carácter Cuantitativo/genética , Análisis de Regresión , Factores Sexuales , Tolbutamida/farmacologíaRESUMEN
Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done. Species for which radiation hybrid-based transcript maps have been established include human, rat, mouse, dog, cat and zebrafish. We have established a comprehensive first-generation-placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.
Asunto(s)
Genoma , Células Híbridas/efectos de la radiación , ARN Mensajero/genética , Animales , Mapeo Cromosómico , Etiquetas de Secuencia Expresada , Hibridación in Situ , RatonesRESUMEN
EXT1 and EXT2 are two genes responsible for the majority of cases of hereditary multiple exostoses (HME), a dominantly inherited bone disorder. In order to develop an efficient screening strategy for mutations in these genes, we performed two independent blind screens of EXT1 and EXT2 in 34 unrelated patients with HME, using denaturing high-performance liquid chromatography (DHPLC) and fluorescent single-strand conformation polymorphism analysis (F-SSCP). The mutation likely to cause HME was found in 29 (85%) of the 34 probands: in 22 of these (76%), the mutation was in EXT1; seven patients (24%) had EXT2 mutations. Nineteen of these disease mutations have not been previously reported. Of the 42 different amplicon variants identified in total in the cohort, 40 were detected by DHPLC and 39 by F-SSCP. This corresponds to mutation detection efficiencies of 95% and 93% respectively. We have also found that we can confidently distinguish between different sequence variants in the same fragment using F-SSCP but not DHPLC. In light of this, and the similarly high sensitivities of the two techniques, we propose to continue screening with F-SSCP.
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Exostosis Múltiple Hereditaria/genética , Pruebas Genéticas/métodos , N-Acetilglucosaminiltransferasas , Proteínas/genética , Cromatografía Líquida de Alta Presión , ADN/análisis , ADN/sangre , Análisis Mutacional de ADN/métodos , Femenino , Colorantes Fluorescentes , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
To understand the cellular and molecular mechanisms that underlie generalized absence seizures sufficiently well to design rational, efficacious new therapies for patients, it is necessary to turn to animal models to gain insights into these mechanisms. The lethargic (lh/lh) mutant mouse expresses spontaneous absence seizures that share behavioral, electrographic, and anticonvulsant profiles with absence seizures in patients. This validates its use to study the mechanisms that underlie absence seizures. This chapter discusses two scientific approaches that involve the use of lh/lh mice. The first part of the chapter discusses neurobiologic approaches used to investigate critical mechanisms that regulate the synchronized burst firing within the thalamocortical network that generates absence seizures. Two of these critical mechanisms have been studied in detail with lh/lh mice. The first critical mechanism involves the required activation of gamma-aminobutyric acid B (GABAB) receptors to generate absence seizures. Because the numbers of GABAB receptors are increased in thalamocortical populations among lh/lh mice compared with littermates without epilepsy, these receptors appear to play a pathophysiologic role in the expression of absence seizures among lh/lh mice. Moreover, there may be a role for GABAB receptors in the generation of absence seizures among humans, because administration of compounds that activate GABAB receptors can produce absence seizures among humans. These findings suggest that GABAB receptor antagonists may represent a new class of antiabsence compounds that will be efficacious against absence seizures among patients. A second critical mechanism that regulates generation of absence seizures involves GABAA receptors in the nucleus reticularis thalami (NRT), a nucleus that sends GABA-ergic afferents to thalamic relay nuclei. Activation of GABAA receptors in the NRT appears to suppress the generation of absence seizures among lh/lh mice and in other models. Moreover, clonazepam may exert its antiabsence actions through this mechanism. Together, these findings suggest that compounds that selectively activate GABAA receptor isoforms expressed in NRT may represent a class of antiabsence drugs that could have fewer side effects than compounds currently used to treat patients. The second part of the chapter discusses a molecular genetic approach to delineation of the mechanisms that underlie absence seizures. Absence seizures among lh/lh mice are caused by a single-gene defect on chromosome 2. If positional cloning and gene isolation techniques are successful, it will be possible to identify the lh disease gene. Subsequent studies of the lh gene product should greatly increase not only our understanding of the pathophysiologic basis for absence seizures among lh/lh mice but also our ability to seek similar mutations in homologous genes in human families that express absence seizures. Accordingly, strategies and progress in cloning and identifying the lh disease gene are presented.
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Clonación Molecular , Epilepsia Tipo Ausencia/genética , Regulación de la Expresión Génica , Ratones Mutantes Neurológicos/genética , Ratones Mutantes Neurológicos/fisiología , Receptores de GABA/genética , Animales , Mapeo Cromosómico , RatonesRESUMEN
X-linked dystonia-parkinsonism (XDP) is a recessive disorder characterized by generalized dystonia with some patients exhibiting parkinsonism. The disease gene, DYT3, is located between DXS453 (DXS993) and DXS559, and strongest linkage disequilibrium is found distal to DXS7117 and proximal to DXS559. We have isolated and analyzed four novel polymorphic markers between DXS7117 and DXS559 and, by haplotype analysis, have narrowed the candidate interval to <350 kb. A sequence-ready contig of 700 kb has been constructed spanning DXS7117 to DXS559 and is composed of 35 PACs, BACs, and cosmids. Nine genes and novel ESTs have been mapped into this contig, and mutations in the coding regions and intron-exon borders of two genes have been excluded as the cause of XDP. Several of the other genes and ESTs located within the contig code for proteins implicated in normal brain development and function and are candidates for DYT3.
Asunto(s)
Mapeo Cromosómico , Distonía/genética , Trastornos Parkinsonianos/genética , Cromosoma X/genética , Mapeo Contig , Cósmidos , Etiquetas de Secuencia Expresada , Ligamiento Genético , Marcadores Genéticos , Humanos , Desequilibrio de Ligamiento , Mapeo Físico de Cromosoma , Síndrome , Secuencias Repetidas en TándemRESUMEN
Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.
Asunto(s)
Etilnitrosourea/farmacología , Genes Letales , Mutágenos/farmacología , Proteínas de Unión al ARN/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario , Ratones , Ratones Endogámicos C57BL , Ratones Quaking , Datos de Secuencia Molecular , Mutagénesis , Proteínas de Unión al ARN/efectos de los fármacosRESUMEN
A health level 7 (HL7)-conformant data link to exchange information between the mainframe hospital information system (HIS) of our hospital and our home-grown picture archiving and communications system (PACS) is a result of a collaborative effort between the HIS department and the PACS development team. Based of the ability to link examination requisitions and image studies, applications have been generated to optimise workflow and to improve the reliability and distribution of radiology information. Now, images can be routed to individual radiologists and clinicians; worklists facilitate radiology reporting; applications exist to create, edit, and view reports and images via the internet; and automated quality control now limits the incidence of "lost" cases and errors in image routing. By following the HL7 standard to develop the gateway to the legacy system, the development of a radiology information system for booking, reading, reporting, and billing remains universal and does not preclude the option to integrate off-the-shelf commercial products.
Asunto(s)
Redes de Comunicación de Computadores , Sistemas de Información en Hospital , Sistemas de Información Radiológica , Sistemas de Administración de Bases de Datos , Diagnóstico por Imagen , Sistemas de Información en Hospital/organización & administración , Humanos , Almacenamiento y Recuperación de la Información , Internet , Control de Calidad , Sistemas de Información Radiológica/instrumentación , Sistemas de Información Radiológica/organización & administración , Integración de Sistemas , Telerradiología , Carga de TrabajoRESUMEN
A distributed design is the most cost-effective system for small-to medium-scale picture archiving and communications systems (PACS) implementations. However, the design presents an interesting challenge to developers and implementers: to make stored image data, distributed throughout the PACS network, appear to be centralized with a single access point for users. A key component for the distributed system is a central or master database, containing all the studies that have been scanned into the PACS. Each study includes a list of one or more locations for that particular dataset so that applications can easily find it. Non-Digital Imaging and Communications in Medicine (DICOM) clients, such as our worldwide web (WWW)-based PACS browser, query the master database directly to find the images, then jump to the most appropriate location via a distributed web-based viewing system. The Master Database Broker provides DICOM clients with the same functionality by translating DICOM queries to master database searches and distributing retrieval requests transparently to the appropriate source. The Broker also acts as a storage service class provider, allowing users to store selected image subsets and reformatted images with the original study, without having to know on which server the original data are stored.