Modulation of Aβ42 low-n oligomerization using a novel yeast reporter system

Background  

While traditional models of Alzheimer's disease focused on large fibrillar deposits of the Aβ₄₂ amyloid peptide in the brain, recent work suggests that the major pathogenic effects may be attributed to SDS-stable oligomers of Aβ₄₂. These Aβ₄₂ oligomers represent a rational target for therapeutic intervention, yet factors governing their assembly are poorly understood.     

Integration of an aberrant retrotransposon in Saccharomyces cerevisiae.

We describe an atypical composite Ty1 element that apparently resulted from the concurrent integration of two complete elements. A portion of the central region of one of these elements was inverted between two long terminal repeats. Inversions of this type have been detected among unintegrated retroviral circles. It now appears that such intermediates can be incorporated into the genome.     

Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases

Summary We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis that in functionally related macromolecules (i.e., a family of evolutionarily related enzymes), regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of observed specificity. It is the intent of our research to encapsulate such knowledge in a form which is capable of observing patterns which may serve as generalizable rules for macrostructural analysis (Liebman, M.N. 1986. Enzyme 36: 150–163), and to serve as the essential tools for the rational design of modified serine proteases and/or their natural inhibitors by the methods available through genetic engineering.     

Structural organization in the serine proteases. I. Macromolecular specificity in limited proteolysis

We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered about the serine proteases. The emphasis of these approaches is to compare and contrast macromolecules at the separate levels of secondary, tertiary, and quaternary structure. Our assumption is that in functionally related molecules, regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of specificity. Based on this assumption, the independent observations from studies of these enzymes in solution and in biological systems are combined with the structural observations from X-ray crystallographic analysis. A goal of the present research effort is to probe the biomolecular architecture of the serine proteases to evaluate the role of the three-dimensional structure beyond that of the active site in determining recognition and reactivity determinants for these enzymes, and to determine those principles that might be applied successfully to other enzyme systems as well. Of particular note has been our observation of a macromolecular recognition surface which occurs as a topographical feature outside of the active site and under evolutionary control to produce specificity towards macromolecular substrates and inhibitors. In addition we have established the important role of conformational changes that occur beyond the active site of an enzyme and differentiate between natural and synthetic inhibitor-enzyme interactions. This suggests that the specificity and reactivity determinants of a macromolecule are derived from its architecture and structural organization.     

A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA.

Recent evidence suggests that ribosomal RNAs have functional roles in translation. We describe here a new ribosomal RNA mutation that causes translational suppression and antibiotic resistance in eukaryotic cells. Using random mutagenesis of the cloned ribosomal RNA gene and in vivo selection, we isolated a C --> U mutation in the universally conserved sarcin/ricin domain in Saccharomyces cerevisiae 25S ribosomal RNA. This mutation changes the putative CG pair, which closes the GAGA tetraloop in the sarcin/ricin domain, into a weaker UG pair without eliminating ribosomal sensitivity to ricin. We show that suppression of several UGA, UAG, and frameshift mutations is evident when a portion of the cellular ribosomal RNA contains the C --> U mutation. Cells that contain essentially all mutant ribosomal RNA grow only 10% slower than the wild-type, but show increased suppression as well as resistance to paramomycin, G418, and hygromycin, and sensitivity to cycloheximide. Our results provide genetic evidence for the participation of the sarcin/ricin loop in maintaining translational accuracy and are discussed in terms of a hypothesis that this ribosomal RNA region normally undergoes a conformational change during translation.     

Isolation and Characterization of Amber Suppressors in Yeast

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome c in cyc1-179 strains suppressed with these efficient amber suppressors.     

CGS 10746B: an atypical antipsychotic candidate that selectively decreases dopamine release at behaviorally effective doses

CGS 10746B, a benzothiadiazepine, has a behavioral profile in mice and monkeys similar to the atypical antipsychotic clozapine. Unlike clozapine, CGS 10746B suppresses dopamine neuron firing rates and, when administered at behaviorally effective doses by the oral or intraperitoneal route, decreases neostriatal dopamine release without changing dopamine metabolism or occupying D2 receptors. CGS 10746B is the first atypical antipsychotic candidate that selectively decreases dopamine release.     

Nitrogen fertilization increases diversity and productivity of prairie communities used for bioenergy

Using prairie biomass as a renewable source of energy may constitute an important opportunity to improve the environmental sustainability of managed land. To date, assessments of the feasibility of using prairies for bioenergy production have focused on marginal areas with low yield potential. Growing prairies on more fertile soil or with moderate levels of fertilization may be an effective means of increasing yields, but increased fertility often reduces plant community diversity. At a fertile site in central Iowa with high production potential, we tested the hypothesis that nitrogen fertilization would increase aboveground biomass production but would decrease diversity of prairies sown and managed for bioenergy production. Over a 3 year period (years 2–4 after seeding), we measured aboveground biomass after plant senescence and species and functional‐group diversity in June and August for multispecies mixtures of prairie plants that received no fertilizer or 84 kg N ha^?1 year^?1. We found that nitrogen fertilization increased aboveground biomass production, but with or without fertilization, the prairies produced a substantial amount of biomass: averaging (±SE) 12.2 ± 1.3 and 9.1 ± 1.0 Mg ha^?1 in fertilized and unfertilized prairies, respectively. Unfertilized prairies had higher species diversity in June, whereas fertilized prairies had higher species diversity in August at the end of the study period. Functional‐group diversity was almost always higher in fertilized prairies. Composition of unfertilized prairies was characterized by native C₄ grasses and legumes, whereas fertilized prairies were characterized by native C₃ grasses and forbs. Although most research has found that nitrogen fertilization reduces prairie diversity, our results indicate that early‐spring nitrogen fertilization, when used with a postsenescence annual harvest, may increase prairie diversity. Managing prairies for bioenergy production, including the judicious use of fertilization, may be an effective means of increasing the amount of saleable products from managed lands while potentially increasing plant diversity.