Biochemical and Biophysical Characterization of Huntingtin

Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disease. HD has no cure, and patients pass away 10-20 years after the onset of symptoms. The causal mutation for HD is a trinucleotide repeat expansion in exon 1 of the huntingtin gene that leads to a polyglutamine (polyQ) repeat expansion in the N-terminal region of the huntingtin protein. Interestingly, there is a threshold of 37 polyQ repeats under which little or no disease exists; and above which, patients invariably show symptoms of HD. The huntingtin protein is a 350 kDa protein with unclear function. As the polyQ stretch expands, its propensity to aggregate increases with polyQ length. Models for polyQ toxicity include formation of aggregates that recruit and sequester essential cellular proteins, or altered function producing improper interactions between mutant huntingtin and other proteins. In both models, soluble expanded polyQ may be an intermediate state that can be targeted by potential therapeutics.

In the first study described herein, the conformation of soluble, expanded polyQ was determined to be linear and extended using equilibrium gel filtration and small-angle X-ray scattering. While attempts to purify and crystallize domains of the huntingtin protein were unsuccessful, the aggregation of huntingtin exon 1 was investigated using other biochemical techniques including dynamic light scattering, turbidity analysis, Congo red staining, and thioflavin T fluorescence. Chapter 4 describes crystallization experiments sent to the International Space Station and determination of the X-ray crystal structure of the anti-polyQ Fab MW1. In the final study, multimeric fibronectin type III (FN3) domain proteins were engineered to bind with high avidity to expanded polyQ tracts in mutant huntingtin exon 1. Surface plasmon resonance was used to observe binding of monomeric and multimeric FN3 proteins with huntingtin.

Genetic engineering of microorganisms: free release into the environment

Genetic engineering now makes possible the insertion of DNA from many organisms into other prokaryotic, eukaryotic and viral hosts. This technology has been used to construct a variety of such genetically engineered microorganisms (GEMs). The possibility of accidental or deliberate release of GEMs into the natural environment has recently raised much public concern. The prospect of deliberate release of these microorganisms has prompted an increased need to understand the processes of survival, expression, transfer and rearrangement of recombinant DNA molecules in microbial communities. The methodology which is being developed to investigate these processes will greatly enhance our ability to study microbial population ecology.

Some engineering issues of an FBG sensor with the dual gratings parallel matched interrogation method

The key issues of engineering application of the dual gratings parallel matched interrogation method are expanding the measurable range, improving the usability, and lowering the cost by adopting a compact and simple setup based on existing conditions and improving the precision of the data-processing scheme. A credible and effective data-processing scheme based on a novel divisional look-up table is proposed based on the advantages of other schemes. Any undetermined data is belonged to a certain section, which can be confirmed at first, then it can be looked up in the table to correspond to microstrain by the scheme. It not only solves inherent problems of the traditional one (double value and small measurable range) but also enhances the precision, which improves the performance of the system. From the experimental results, the measurable range of the system is 525 mu epsilon, and the precision is +/- 1 mu epsilon based on normal matched gratings. The system works in real time, which is competent for most engineering measurement requirements. (C) 2007 Elsevier GmbH. All rights reserved.