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Plasma orotate was measured using liquid chromatography mass spectrometry according to a previously published method [ 15 ]. For amino acid analysis of plasma and tissues, ion exchange chromatography with ninhydrin detection was performed using a Biochrom 30 Amino Acid Analyzer Biochrom, Cambridge, UK. For tissue analysis, citrulline was removed due to tissue interference with detection. Ornithine transcarbamylase enzyme activity was measured by a colorimetric assay which detects the formation of L-citrulline [ 16 ].

Liver tissue was homogenized in mitochondrial lysis buffer: 0.


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The gels were run at V for approximately 1. Louis, MO. The membranes were washed three times for 10 min each with TBS containing 0. Summary statistics were calculated for all data. P-values less than 0. To quantify the difference in poly I:C and baseline amounts of each amino acid, we used a t-statistic for the difference between poly I:C and baseline, and plotted these t-statistics.

T-statistics were computed R v3. To compute the p-value of the significance in differences of change in spf-j versus WT, we used a form of permutation test[ 17 ] as follows. For each amino acid, let D spf-j represent the mean change in amino acids from baseline to spf-j. Therefore, D spf-j is defined as the difference between mean amount of spf-j polyI:C amino acid and the mean amount of spf-j amino acid. D therefore represents our statistic for difference in change from baseline to polyIC.

Random permuted values of D perm are computed by permuting the labels of spf-j , WT, baseline and polyI:C for each value of amino acid, and re-calculating D. The p-value is computed as the mean number of D perm less than the original, unpermuted D. We performed , permutations for each p-value computation.

OTC was considered the likely candidate for the spf-J phenotype based on the results of a non-complementation test with spf performed at the Jackson Laboratory [ 18 ]. PCR products amplified from both mutant and WT mice were of the expected size data not shown. However, the gDNA sequence for spf-J contained a c.

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Interestingly, a single patient with late-onset OTC deficiency in adolescence displaying the same mutation as spf-j has been described [ 19 ]. A PCR amplification of gDNA from spf-J revealed a transversion mutation in exon 3 leading to an asparagine replacement of a conserved lysine residue at position 80 K80N. Hem — hemizygote, Het — heterozygote.

With the identification of the mutation in exon 3, we next sought to develop a genotyping assay for rapid identification of progeny Fig. Scanning exon 3, we identified an allele specific EcoRI site. Fragments amplified by PCR of genomic DNA were digested with EcoRI to yield fragments of , and 79 base pairs bp , allowing us to easily identify WT , 79 bp , female heterozygotes , , 79 bp and male hemizygotes , 79 bp.

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This genotyping assay was then used for subsequent identification of all male experimental animals. We were able to identify spf-J animals by coat and skin abnormalities Fig. Despite these early differences, by 12 weeks of age, the size discrepancy disappeared and the coat color and density became similar to WT, making visual identification difficult Figs.

Consistent with the improvement in coat phenotype and weight, suggesting a milder phenotype, spf-J also showed comparable survival when compared to WT Fig. In addition to increased survival, spf-J also display fecundity. Each hemizygous male is capable of producing litters that are comparable for the B6 background data not shown. Overall, these data suggest that although spf-J shares some early visible phenotypic characteristics with spf-ash, spf-J mice show improved survival and a recovery of linear growth, weigh and coat texture. A Photos of spf-J at 4 weeks top and 12 weeks bottom.

WT — wild-type. Given the longer survival, we hypothesized that spf-J would display milder enzymatic findings. Livers from 8-week-old spf-J were harvested after euthanasia, and snap frozen. In contrast, the amount of OTC protein in spf-J was drastically reduced to below the detectable limit by immunoblot Fig.

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Due to this drastic reduction in hepatic OTC protein, we next measured enzyme activity in liver homogenates. This enzyme activity level is similar to the previously reported case of late-onset OTCD mentioned above [ 19 ]. Overall, our protein and enzyme findings in the spf-J are consistent with the milder phenotype we observed. B Immunoblot of OTC protein. Spf-ash , another model of OTCD, display features of hyperammonemia and orotic aciduria at baseline [ 21 ]. Since the observable phenotype and reduction in enzyme activity was milder in spf-J , we hypothesized that the biochemical hallmarks of OTCD would be milder or absent at baseline.

Blood was collected and analyzed for plasma ammonia, plasma orotate and plasma amino acids Fig. On normal chow, spf-J failed to show differences in plasma ammonia or plasma orotate Figs. When plasma amino acids were measured, some significant differences were seen Table 1. In spf-J , glutamine, a metabolic sink for ammonia [ 22 ], was mildly increased over WT Urea cycle intermediates ornithine In the spf-j liver Table 2 , only a few amino acid perturbations were seen: decreased aspartate Interestingly, in the cerebrum Table 3 , 15 of the 20 amino acids measured were increased in spf-j including two well-established neuroactive amino acids, glycine Plasma was collected in 8-week-old spf-j and WT and snap frozen until analysis.

WT — wild type, SD — standard deviation. Liver tissue was collected in 8-week-old spf-j and WT and snap frozen until analysis. Cerebral tissue was collected in 8-week-old spf-j and WT and snap frozen until analysis. With the plasma amino acid profile suggesting perturbations in the biochemical phenotype at baseline, we next tried to provoke hyperammonemia and orotic aciduria.

Spf-J and WT littermates were maintained on a high protein diet for 2 weeks Figs. At the end of 2 weeks the animals were bled retro-orbitally and plasma ammonia and orotate were determined. Although we were not able to increase plasma ammonia significantly in spf-J , plasma orotate increased 2. Therefore, spf-J , while displaying mild plasma biochemical abnormalities at baseline, may be provoked by protein challenge to display one of the biochemical hallmarks of OTCD, orotate accumulation.

C Plasma ammonia. D Liver transaminases. Patients with urea cycle disorders may experience episodes of metabolic instability termed acute metabolic decompensation. To simulate viral infection with a systemic inflammatory response in spf-j mice, intraperitoneal poly I:C was given once a day for 3 days [ 23 ]. Following injection with poly I:C, spf-j mice fail to show differences in plasma ammonia Fig. To explore amino acid perturbations, plasma, liver and cerebral amino acids were measured.

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Heat maps were constructed showing the change from baseline for each tissue Fig. In plasma, there was a similar general decrease in amino acids from baseline in both spf-j and WT Fig. Similar to plasma, most of the changes from baseline in liver were not statistically different Fig. Unlike plasma and liver, the effect of poly I:C treatment in the brain was more pronounced Fig.

Heat maps were constructed as described in Methods to represent the change in amino acid concentrations from baseline for plasma left panel , liver middle panel and cerebrum right panel. Due to the inheritance of OTCD, the phenotypic expression of the disease severity in patients is dependent upon the nature of the mutation, genetic background and in females, X-inactivation in the liver.


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  5. In males, this disorder classically manifests with symptomatic hyperammonemia in infancy, although milder alleles have been described [ 12 , 13 , 19 ]. In addition, phenotypic variability can be seen in males within the same family with the same mutation suggesting modifier alleles may play a role [ 24 ]. Galloway et al.

    The precipitant of this episode was unknown. This amino acid substitution in the OTC enzyme is identical to the mutation described here in the spf-J mouse Fig. The mutation lies outside the substrate binding and catalytic regions of the enzyme [ 13 ] and may be related to homo-oligomerization or stability of the enzyme.

    These findings lead us to suggest that the K80N mutation may destabilize the enzyme, leading to its early degradation.