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Research has been conducted on the kynurenine pathway catabolites. The reaction of these catabolites with iron and copper and interactions with alpha-crystallins have been investigated.

Studies were conducted to examine polyol osmolytes' balancing of low pH species with properties comparable to the A-state of ferricytochrome c and to determine the equivalence of the sugar-induced stability to the salt-induced A-state. The equine cytochrome c was purified and the circular dichroism spectra of the sugar-induced A-state was measured. The findings confirmed that the addition of sugars to acid-denatured cytochrome c yields a species nearly equivalent to sugar-induced A-state, the equilibrium model for notional kinetic folding intermediates.

The introduction of a disulfide cross-link into yeast iso-1-ferricytochrome c results in small native structural changes. The cross-link decreases local stability which is less than or equal to global stability. The local stability near the cross-link however, increases slightly. Changes in local stability for hydrophobic core residues are higher than changes in global stability. Circular dichroism studies suggest that the reduction in stability results from structure-induced stabilization of the denatured state.

Site-specific cross-linked peroxidase-cytochrome c complexes for probing interaction domains and the intramolecular electron transfer reaction are generated with the help of engineered cysteine residues in yeast cytochrome c peroxidase and yeast iso-1-cytochrome c. Stopped-flow methods are used to estimate the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to three peroxidase-cytochrome c noncovalent complexes. Results indicate significant differences in the rate of transfer between the complexes.

Screening of mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c indicates the presence of all residue combinations associated with naturally occurring cytochrome c sequences in the libraries. Enthalpic and entropic effects partially account for the charges in the Gibbs free energy of denaturation for the ferri form of the wild-type-protein and 13-interface variants. Thermodynamic interactions are observed in the helices. Thermodynamic data are compared with side-chain model data.

An aromatic ring on the invariant residue Phe-82 in cytochrome c was tested to function as an electron-transfer bridge between cytochrome c and cytochrome b5. The ruthenium complex on the surface of Ru-65-cyt b5 was shaped to induce speedy photo-induced electron transfer from ruthenium to the heme group. A straight 12-covalent-bond passage for electron transfer between the bipyridine ligand of the ruthenium complex and the imidazole group of His-63 was present. The experiments revealed that an aromatic side chain at residue 82 of cytochrome was not required for rapid electron transfer with cytochrome b5.

Abstract A number of studies have shown that increased APP levels, resulting from either a genomic locus duplication or alteration in APP regulatory sequences, can lead to development of early-onset dementias, including Alzheimer's disease (AD). Therefore, understanding how APP levels are regulated could provide valuable insight into the genetic basis of AD and illuminate novel therapeutic avenues for AD. Here we test the hypothesis that APP protein levels can be regulated by miRNAs, evolutionarily conserved small noncoding RNA molecules that play an important role in regulating gene expression. Utilizing human cell lines, we demonstrate that miRNAs hsa-mir-106a and hsa-mir-520c bind to their predicted target sequences in the APP 3'UTR and negatively regulate reporter gene expression. Over-expression of these miRNAs, but not control miRNAs, results in translational repression of APP mRNA and significantly reduces APP protein levels. These results are the first to demonstrate that levels of human APP can be regulated by miRNAs.

Abstract Background A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered. Results To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis. Conclusion Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.

The ruthenium-cytochrome c derivative is designed by labeling the sulfhydryl group on yeast cytochrome c. Kinetic activity reveals that ruthenium-cytochrome c derivative exhibts the same steady-state activity with yeast cytochrome bc(sub)1 complex. Data indicate that a flash photolysis of a 1:1 complex between reduced yeast cytochrome bc(sub)1 and the derivative rapidly photooxidizesheme c.