Rosemary L. Brown

Wild type huntingtin reduces the cellular toxicity of mutant huntingtin in mammalian cell models of Huntington's disease

OBJECTIVES Recent data suggest that wild type huntingtin can protect against apoptosis in the testis of mice expressing full length huntingtin transgenes with expanded CAG repeats. It is not clear if this protective effect was confined to particular cell types, or if wild type huntingtin exerted its protective effect in this model by simply reducing the formation of toxic proteolytic fragments from mutant huntingtin. METHODS We cotransfected neuronal (SK-N-SH, human neuroblastoma) and non-neuronal (COS-7, monkey kidney) cell lines with HD exon 1 (containing either 21 or 72 CAG repeats) construct DNA and either full length wild type huntingtin or pFLAG (control vector). RESULTS Full length wild type huntingtin significantly reduced cell death resulting from the mutantHD exon 1 fragments containing 72 CAG repeats in both cell lines. Wild type huntingtin did not significantly modulate cell death caused by transfection of HD exon 1 fragments containing 21 CAG repeats in either cell line. CONCLUSIONS Our results suggest that wild type huntingtin can significantly reduce the cellular toxicity of mutant HD exon 1 fragments in both neuronal and non-neuronal cell lines. This suggests that wild type huntingtin can be protective in different cell types and that it can act against the toxicity caused by a mutant huntingtin fragment as well as against a full length transgene.

Identification and Biochemical Characterization of a Novel Ryanodine Receptor Gene Mutation Associated with Malignant Hyperthermia

Background:Mutations in the skeletal muscle ryanodine receptor gene may result in altered calcium release from sarcoplasmic reticulum stores, giving rise to malignant hyperthermia (MH). MH is a pharmacogenetic skeletal muscle disorder triggered by volatile anesthetics and depolarizing muscle relaxants. Diagnosis of MH is by in vitro contracture testing of quadriceps muscle. DNA analysis of causative mutations is limited by the large number of mutations that cosegregate with MH and the relatively few that have been biochemically characterized. Methods:DNA sequence analysis was used to screen the skeletal muscle ryanodine receptor gene in MH-susceptible individuals. A diagnostic test using real-time polymerase chain reaction was developed to detect the mutation in individuals diagnosed as MH susceptible by in vitro contracture testing. The functional relevance of this mutation was examined in Epstein-Barr virus–immortalized B-lymphoblastoid cells. Results:A novel ryanodine receptor mutation (cytosine 14997 thymine resulting in a histidine 4833 tyrosine substitution) was identified in pathology specimens from two patients with fatal MH reactions. B lymphocytes from patients with this mutation were approximately twofold more sensitive than MH-negative cells to activation with 4-chloro-m-cresol. The amount of 45Ca2+ released from B lymphocytes of MH-susceptible patients was significantly greater than that released from cells of family members without this mutation. Haplotype analysis suggests that both families had a common ancestor. Conclusions:DNA analysis to detect mutations which cosegregate with MH as well as biochemical assays on cultured lymphocytes obtained from blood can serve as useful diagnostic tools for MH susceptibility and genotype–phenotype correlations.

A picomolar transition state analogue inhibitor of MTAN as a specific antibiotic for Helicobacter pylori.

Campylobacter and Helicobacter species express a 6-amino-6-deoxyfutalosine N-ribosylhydrolase (HpMTAN) proposed to function in menaquinone synthesis. BuT-DADMe-ImmA is a 36 pM transition state analogue of HpMTAN, and the crystal structure of the enzyme-inhibitor complex reveals the mechanism of inhibition. BuT-DADMe-ImmA has a MIC(90) value of <8 ng/mL for Helicobacter pylori growth but does not cause growth arrest in other common clinical pathogens, thus demonstrating potential as an H. pylori-specific antibiotic.

Syntheses of novel azasugar-containing mimics of heparan sulfate fragments as potential heparanase inhibitors

A series of eight novel, highly modified mono-, di- and trisaccharide derivatives, each containing an iminoalditol moiety, has been synthesized in the quest for potential heparanase inhibitors.

Crystallization of Sheep Liver Cytosolic Aldehyde Dehydrogenase in a form Suitable for High Resolution X-Ray Structural Analysis

Aldehyde dehydrogenase (AlDH) is an NAD+-dependent enzyme that is widely distributed through many different species, including both eukaryotes and prokaryotes. In animals, aldehyde dehydrogenases are found in a number of different bodily locations where they exist as several distinct isozymes. These enzymes share the general role of detoxification of aldehydes (Jakoby and Ziegler, 1990) but the individual isozymes show differences in specificity and reactivity that suggest they may have additional, more specialised, roles.

Crystallization and preliminary X-ray diffraction studies of a cobalt-substituted derivative of the iron-dependent alcohol dehydrogenase from Zymomonas mobilis.

The iron-dependent alcohol dehydrogenase from Zymomonas mobilis has been crystallized in a form suitable for X-ray diffraction studies. The crystals grew in hanging drops by vapor diffusion, equilibrating with a solution comprising 25-27% methoxypolyethylene glycol 5000 and 1 mM Co(2+) in a 0.2 M succinic acid/potassium hydroxide buffer at pH 5.5-5.7 at 281 K. Crystals are tetragonal, P4(1)22 (or P4(3)22), with unit-cell dimensions a = b = 125.7, c = 248.1 A. Four molecules comprise the asymmetric unit, and a self-rotation function indicates twofold local symmetry perpendicular to the unique axis and 15 degrees from a crystallographic twofold axis. Diffraction data to 3.0 A have been collected.