Molecular characterization of the translocation breakpoints in the Down syndrome mouse model Ts65Dn.

Ts65Dn is a mouse model of Down syndrome: a syndrome that results from chromosome (Chr) 21 trisomy and is associated with congenital defects, cognitive impairment, and ultimately Alzheimer's disease. Ts65Dn mice have segmental trisomy for distal mouse Chr 16, a region sharing conserved synteny with human Chr 21. As a result, this strain harbors three copies of over half of the human Chr 21 orthologs. The trisomic segment of Chr 16 is present as a translocation chromosome (Mmu17(16)), with breakpoints that have not been defined previously. To molecularly characterize the Chrs 16 and 17 breakpoints on the translocation chromosome in Ts65Dn mice, we used a selective enrichment and high-throughput paired-end sequencing approach. Analysis of paired-end reads flanking the Chr 16, Chr 17 junction on Mmu17(16) and de novo assembly of the reads directly spanning the junction provided the precise locations of the Chrs 16 and 17 breakpoints at 84,351,351 and 9,426,822 bp, respectively. These data provide the basis for low-cost, highly efficient genotyping of Ts65Dn mice. More importantly, these data provide, for the first time, complete characterization of gene dosage in Ts65Dn mice.

Noninvasive metabolic profiling using microfluidics for analysis of single preimplantation embryos.

Noninvasive analysis of metabolism at the single cell level will have many applications in evaluating cellular physiology. One clinically relevant application would be to determine the metabolic activities of embryos produced through assisted reproduction. There is increasing evidence that embryos with greater developmental capacity have distinct metabolic profiles. One of the standard techniques for evaluating embryonic metabolism has been to evaluate consumption and production of several key energetic substrates (glucose, pyruvate, and lactate) using microfluorometric enzymatic assays. These assays are performed manually using constriction pipets, which greatly limits the utility of this system. Through multilayer soft-lithography, we have designed a microfluidic device that can perform these assays in an automated fashion. Following manual loading of samples and enzyme cocktail reagents, this system performs sample and enzyme cocktail aliquotting, mixing of reagents, data acquisition, and data analysis without operator intervention. Optimization of design and operating regimens has resulted in the ability to perform serial measurements of glucose, pyruvate, and lactate in triplicate with submicroliter sample volumes within 5 min. The current architecture allows for automated analysis of 10 samples and intermittent calibration over a 3 h period. Standard curves generated for each metabolite have correlation coefficients that routinely exceed 0.99. With the use of a standard epifluorescent microscope and CCD camera, linearity is obtained with metabolite concentrations in the low micromolar range (low femtomoles of total analyte). This system is inherently flexible, being easily adapted for any NAD(P)H-based assay and scaled up in terms of sample ports. Open source JAVA-based software allows for simple alterations in routine algorithms. Furthermore, this device can be used as a standalone device in which media samples are loaded or be integrated into microfluidic culture systems for in line, real time metabolic evaluation. With the improved throughput and flexibility of this system, many barriers to evaluating metabolism of embryos and single cells are eliminated. As a proof of principle, metabolic activities of single murine embryos were evaluated using this device.

Oxidative metabolism of pyruvate is required for meiotic maturation of murine oocytes in vivo.

The requirement for oxidative metabolism of pyruvate during oogenesis in vivo was evaluated by inactivating Pdha1, a gene encoding an enzymatic subunit of pyruvate dehydrogenase complex, in murine oocytes at the beginning of the follicular growth phase. Immunohistochemical analysis revealed that Pdha1(-) oocytes have dramatically reduced amounts of pyruvate dehydrogenase enzyme by the secondary follicle stage. Despite this reduction, these oocytes grow to normal size, are ovulated, and can be fertilized. Pdha1(-) oocytes are, however, impaired in their ability to support embryonic development, as demonstrated by the failure of fertilized oocytes to develop beyond the one-cell zygote stage in vivo. Immunocytochemical evaluation showed that almost all (98.4%) ovulated Pdha1(-) oocytes have not completed meiotic maturation and/or have gross abnormalities of the meiotic spindle and chromatin. Meiotic maturation is even more compromised when these oocytes are matured in vitro in the absence of cumulus cells or in the presence of the gap junction inhibitor 18-alpha glycyrrhetinic acid, indicating that cumulus cells can partially compensate for this enzymatic deficiency through a gap junction-mediated mechanism. Ovulated Pdha1(-) oocytes were also shown to have reduced levels of total ATP content and NAD(P)H autofluorescence relative to oocytes without this enzymatic deficiency. These studies demonstrate that oxidative metabolism of pyruvate is essential for proper completion of oogenesis, serving as a vital source of energy during meiotic maturation. At earlier stages of oogenesis this metabolic pathway may not be necessary due to metabolic compensation by the granulosa cells.

Intrafamilial variability of noncompaction of the ventricular myocardium.

BACKGROUND: Noncompaction of the ventricular myocardium (NVM) is a relatively uncommon form of cardiomyopathy characterized by a highly trabeculated myocardium. This report describes the clinical and genetic evaluation of a 3-generation kindred. METHODS: Family members were initially evaluated by 2-dimensional echocardiography. Most family members with signs of NVM were further evaluated by magnetic resonance imaging. Genetic analyses included mutational screening of the taffazin (TAZ) and alpha-dystrobrevin (DTNA) genes. RESULTS: Eight family members had signs of NVM. Considerable interindividual variation was noted in terms of spatial distribution and severity of affected regions and ventricular dysfunction. Depending on which of 2 previously proposed quantitative diagnostic criteria were used and where ventricular myocardial measurements were taken, between 4 and 7 of these individuals had findings that were considered diagnostic. Magnetic resonance imaging served as a useful adjunct for confirming or establishing diagnoses in all 8 individuals. No mutation was found in TAZ or DTNA. CONCLUSIONS: This kindred demonstrates the remarkably wide phenotypic spectrum that can be seen in familial cases of NVM, ranging from prenatal/neonatal lethality to a complete lack of symptoms. The fact that all 8 affected individuals either have shown improvement in ventricular function or symptoms during childhood or have been asymptomatic indicates that NVM can have a relatively benign course. The degree and nature of cardiac involvement are also quite varied, and there is a weak correlation with ventricular function and symptoms. Evaluation of families with NVM requires careful assessment that uses a combination of imaging techniques and diagnostic criteria.

Phosphoenolpyruvate carboxykinase and the critical role of cataplerosis in the control of hepatic metabolism.

BACKGROUND: The metabolic function of PEPCK-C is not fully understood; deletion of the gene for the enzyme in mice provides an opportunity to fully assess its function. METHODS: The gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C) was deleted in mice by homologous recombination (PEPCK-C-/- mice) and the metabolic consequences assessed. RESULTS: PEPCK-C-/- mice became severely hypoglycemic by day two after birth and then died with profound hypoglycemia (12 mg/dl). The mice had milk in their stomachs at day two after birth and the administration of glucose raised the concentration of blood glucose in the mice but did not result in an increased survival. PEPCK-C-/- mice have two to three times the hepatic triglyceride content as control littermates on the second day after birth. These mice also had an elevation of lactate (2.5 times), beta-hydroxybutyrate (3 times) and triglyceride (50%) in their blood, as compared to control animals. On day two after birth, alanine, glycine, glutamine, glutamate, aspartate and asparagine were elevated in the blood of the PEPCK-C-/- mice and the blood urea nitrogen concentration was increased by 2-fold. The rate of oxidation of [2-14C]-acetate, and [5-14C]-glutamate to 14CO2 by liver slices from PEPCK-C-/- mice at two days of age was greatly reduced, as was the rate of fatty acid synthesis from acetate and glucose. As predicted by the lack of PEPCK-C, the concentration of malate in the livers of the PEPCK-C-/- mice was 10 times that of controls. CONCLUSION: We conclude that PEPCK-C is required not only for gluconeogenesis and glyceroneogenesis but also for cataplerosis (i.e. the removal of citric acid cycle anions) and that the failure of this process in the livers of PEPCK-C-/- mice results in a marked reduction in citric acid cycle flux and the shunting of hepatic lipid into triglyceride, resulting in a fatty liver.

Biochemical and structural brain alterations in female mice with cerebral pyruvate dehydrogenase deficiency.

Pyruvate dehydrogenase complex (PDC) deficiency is an inborn metabolic disorder associated with a variety of neurologic abnormalities. This report describes the development and initial characterization of a novel murine model system in which PDC deficiency has been introduced specifically into the developing nervous system. The absence of liveborn male and a roughly 50% reduction in female offspring following induction of the X-linked mutation indicate that extensive deficiency of PDC in the nervous system leads to pre-natal lethality. Brain tissue from surviving females at post-natal days 15 and 35 was shown to have approximately 75% of wild-type PDC activity, suggesting that a threshold of enzyme activity exists for post-natal survival. Detailed histological analyses of brain tissue revealed structural defects such as disordered neuronal cytoarchitecture and neuropil fibers in grey matter, and reduced size of bundles and disorganization of fibers in white matter. Many of the histologic abnormalities resemble those found in human female patients who carry mutations in the X-linked ortholog. These findings demonstrate a requirement for PDC activity within the nervous system for survival in utero and suggest that impaired pyruvate metabolism in the developing brain can affect neuronal migration, axonal growth and cell-cell interactions.