, Melissa Yssel, MB ChB, FC Path(SA) Chem 139, and Wendy M. Zakowicz, BS 79 Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.

In the autosomal recessive human disease, glutaric aciduria type I (GA-1), glutaryl-CoA dehydrogenase (GCDH) deficiency disrupts the mitochondrial catabolism of lysine and tryptophan. Affected individuals accumulate glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in the serum and often suffer acute striatal injury in childhood. Prior attempts to produce selective striatal vulnerability in an animal model have been unsuccessful. We hypothesized that acute striatal injury may be induced in GCDH-deficient (Gcdh-/-) mice by elevated dietary protein and lysine. Here, we show that high protein diets are lethal to 4-week-old and 8-week-old Gcdh-/- mice within 2-3 days and 7-8 days, respectively. High lysine alone resulted in vasogenic oedema and blood-brain barrier breakdown within the striatum, associated with serum and tissue GA accumulation, neuronal loss, haemorrhage, paralysis, seizures and death in 75% of 4-week-old Gcdh-/- mice after 3-12 days. In contrast, most 8-week-old Gcdh-/- mice survived on high lysine, but developed white matter lesions, reactive astrocytes and neuronal loss after 6 weeks. Thus, the Gcdh-/- mouse exposed to high protein or lysine may be a useful model of human GA-1 including developmentally dependent striatal vulnerability.

In the absence of the leucine biosynthetic precursor alpha-isopropylmalate (alpha-IPM), the yeast LEU3 protein (Leu3p) binds DNA and acts as a transcriptional repressor in an in vitro extract. Addition of alpha-IPM resulted in a dramatic increase in Leu3p-dependent transcription. The presence of alpha-IPM was also required for Leu3p to compete effectively with another transcriptional activator, GAL4/VP16, for limiting transcription factors. Therefore, the addition of alpha-IPM appears to convert a transcriptional repressor into an activator. This represents an example in eukaryotes of direct transcriptional regulation by a small effector molecule.

Glutaric acidemia type I (GA-I) is an inherited disorder of lysine and tryptophan metabolism presenting with striatal lesions anatomically and symptomatically similar to Huntington disease. Affected children commonly suffer acute brain injury in the context of a catabolic state associated with nonspecific illness. The mechanisms underlying injury and age-dependent susceptibility have been unknown, and lack of a diagnostic marker heralding brain injury has impeded intervention efforts. Using a mouse model of GA-I, we show that pathologic events began in the neuronal compartment while enhanced lysine accumulation in the immature brain allowed increased glutaric acid production resulting in age-dependent injury. Glutamate and GABA depletion correlated with brain glutaric acid accumulation and could be monitored in vivo by proton nuclear magnetic resonance ( 1 H NMR) spectroscopy as a diagnostic marker. Blocking brain lysine uptake reduced glutaric acid levels and brain injury. These findings provide what we believe are new monitoring and treatment strategies that may translate for use in human GA-I.

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.Despite intensive study, the actual mechanisms of transcriptional activation in eukaryotes remain largely speculative. Although activation can clearly affect the initiation of transcription (34), initiation is itself a complex process with several separable steps (13). Fractionation of HeLa cell transcription extracts has been critical to our understanding of initiation, providing a wealth of information and allowing identification of the binding of transcription factor IID (TFIID) to the TATA box as a central step in the initiation process (35). Recent studies support the idea that activators may interact directly with TFIID (33) or that they may act through one of the many other proteins involved in the preinitiation complex (19).Other fractionated transcription extracts have also been described, from rat liver (7), Drosophila embryos (38), and yeast nuclei (10). The fact that many of the fractions from these different species are interchangeable suggests that the fundamental transcription mechanisms are highly conserved (4,6,11,38). The yeast (Saccharomyces cerevisiae) system offers the unique opportunity to combine a genetic analysis with preparative biochemistry. All subunits of the yeast RNA polymerase II have been cloned and mutated (29), the yeast gene encoding TFIID has been extensively characterized (9,12,28), and the genes for many transcriptional activators have been isolated (18,32). The biochemical analysis of yeast transcription has progressed more slowly, in part because of the technical difficulties involved in preparing nuclei (14,16).We recently reported the preparation of selective in vitro transcription extracts from intact yeast cells (40). Here we report that small changes in our extraction and assay procedures greatly increase selective initiation by RNA polymerase II. We have also analyzed a range of reaction conditions that we find have dramatic effects on apparent transcriptional activation by the GAL4/VP16 fusion protein. Finally, we have proven the genera...