Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, we examined the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice. We found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures approximately 0.5 microm across, whereas the expanded ataxin-1 localizes to a single approximately 2-microm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1. Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic leukaemia protein. Nuclear matrix preparations demonstrate that ataxin-1 associates with the nuclear matrix in Purkinje and COS cells. We therefore propose that a critical aspect of SCA1 pathogenesis involves the disruption of a nuclear matrix-associated domain.
Transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene, a polyglutamine neurodegenerative disorder, develop ataxia with ataxin-1 localized to aggregates within cerebellar Purkinje cells nuclei. To examine the importance of nuclear localization and aggregation in pathogenesis, mice expressing ataxin-1[82] with a mutated NLS were established. These mice did not develop disease, demonstrating that nuclear localization is critical for pathogenesis. In a second series of transgenic mice, ataxin-1[77] containing a deletion within the self-association region was expressed within Purkinje cells nuclei. These mice developed ataxia and Purkinje cell pathology similar to the original SCA1 mice. However, no evidence of nuclear ataxin-1 aggregates was found. Thus, although nuclear localization of ataxin-1 is necessary, nuclear aggregation of ataxin-1 is not required to initiate pathogenesis in transgenic mice.
Stereotactic radiation treatment provided effective local control of "aggressive" Grade I and Grade II meningiomas, whereas Grade III lesions were associated with poor outcome. The outcome of cases in the malignant progression group was intermediate between that of the Grade II and Grade III groups, with the lesions showing a tendency toward malignancy.
The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate dicarboxylic amino acids with similar rate constants. However, eTATase exhibits -102-104-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to a-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L -A s~ and L-GIu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidin0 group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino acid substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acids.Keywords: aspartate aminotransferase; kinetics; substrate specificity; tyrosine aminotransferase Escherichia coli aspartate aminotransferase (eAATase; EC 2.6.1.1) and tyrosine aminotransferase (eTATase; EC 2.6.1.5) are PLP-containing transaminases that catalyze the reversible interconversion of amino acids and their corresponding a-keto acids. The bound cofactor acts as a transient amino group carrier, shuttling between the pyridoxal phosphate and pyridoxamine phosphate forms. The transamination reaction is described by a ping-pong bi-bi mechanism (Equation lA,B): with the side-chain carboxylate group of aspartate or glutamate (Kirsch et al., 1984;Malashkevich et al., 1993). Mutational stud-
2071 Background: EGFRvIII is a mutant version of EGF receptor resulting from the genomic deletion of exons 2 through 7 and is expressed only in certain tumors. AMG 595 is an experimental therapeutic specifically targeting EGFRvIII and consists of an EGFRvIII-specific antibody conjugated to the maytansinoid antimicrotubule agent DM-1. Due to its specificity, mechanism of action, and pre-clinical activity, AMG 595 is expected to have clinical effect only in tumors expressing EGFRvIII. Since the reported prevalence of EGFRvIII in glioblastoma multiforme (GBM) is ~30%, prospective selection of patients with EGFRvIII positive tumors was desired for clinical development. An immunohistochemical (IHC) assay developed with Dako using a novel EGFRvIII antibody is currently being employed for patient selection for the AMG 595 phase I study in recurrent GBM (NCT01475006). Methods: An appropriate IHC reagent for human tissue was created using the variable region of a novel EGFRvIII-specific antibody developed using Xenomouse technology. Staining conditions were optimized using Dako pharmDx reagents, the Dako Link 48 Autostainer, and FFPE tissue sections. Confirmatory transcript analyses of adjacent sections were conducted using the NanoString platform. Results: Robust and reproducible staining for EGFRvIII was observed using archived GBM resections. Percentage of stained cells correlated with levels of EGFRvIII transcripts, and tumors without staining did not express EGFRvIII transcripts. Although some tumors exhibited homogenous staining, most were heterogeneous with varying distribution and percentages of stained tumor cells similar to literature reports of IHC analysis utilizing other EGFRvIII-specific antibodies. Tumor samples from patients entering into the AMG 595 Phase 1 study analyzed with this IHC test have displayed the predicted staining prevalence. Conclusions: The developed EGFRvIII IHC assay, approved under an Investigational Device Exemption, is currently being successfully employed to prospectively select patients in an ongoing phase I trial. Use of this well characterized IHC test will enable correlation of clinical outcome and staining characteristics to inform subsequent studies.
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