Cholesterol and Clioquinol modulation of Aβ(1-42) interaction with phospholipid bilayers and metals

The β-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-β peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, Aβ(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state ³¹P and ²H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in β-sheet, were observed using specifically ¹³C-labelled Aβ(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely ¹⁵N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric β-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.     

Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.     

DNA bending by charged peptides: electrophoretic and spectroscopic analyses

We are testing the idea that placement of fixed charges near one face of the DNA double helix can induce DNA bending by a purely electrostatic mechanism. If stretching forces between DNA phosphates are significant, fixed charges should induce DNA bending by asymmetrically modulating these forces. We have previously tested this hypothesis by adding charged residues to small bZIP DNA binding peptides and monitoring DNA bending using electrophoretic phasing assays. Our results were consistent with an electrostatic model of DNA bending in predicted directions. We now confirm these observations with fluorescence resonance energy transfer (FRET). Using a "U"-shaped DNA probe, we report that DNA bending by charged bZIP peptides is readily detected by FRET. We further show that charged bZIP peptides cause DNA bending rather than DNA twisting.     

Ninety-six haploid yeast strains with individual disruptions of open reading frames between YOR097C and YOR192C, constructed for the Saccharomyces genome deletion project, have an additional mutation in the mismatch repair gene MSH3

As part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploid strains that differed only by single gene deletions were constructed. We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene.     

Dominant-subordinate relationships in hamsters: sex differences in reactions to familiar opponents

In the majority of mammalian species, males are dominant over and more aggressive than females. In contrast, some reports suggest that female golden hamsters are more aggressive than males but systematic comparisons using the same methods for both sexes are rare. We observed same-sexed pairs of hamsters over repeated trials to assess whether sex differences existed in the level of agonistic behavior and in the development and maintenance of dominant-subordinate relationships with familiar partners. There were no sex differences in measures of agonistic behavior or fear responses (fleeing) during the initial series of three trials on the first day of testing. Following a four-day interval, males that had lost in session 1 showed fearful responses to a familiar dominant male and were not likely to engage in a fight with him. In contrast, females that lost the initial fights were not fearful and fought vigorously with the familiar winner in subsequent encounters. Although the amount of agonistic behavior engaged in by females did decrease over the course of the three sessions, females that lost did not demonstrate an increase in fear, as measured by the latency to flee. Males that lost fights did show increased fear during later trials and sessions. These results suggest that female hamsters are less affected by losing fights than males are and thus that females are less likely than males to develop highly polarized dominant-subordinate relationships. Further work is needed to understand the mechanisms underlying these sex differences.     

Low functional diversity and no redundancy in British avian assemblages

1. Spatial and temporal patterns in functional diversity can reveal the patterns and processes behind community assembly and whether ecological redundancy exists. Here, we analyse functional diversity in British avian assemblages over a period of about 20 years. 2. Functional diversity is generally lower than expected by chance, indicating that assemblages contain species with relatively similar functional traits. One potential explanation is filtering for traits suitable to particular habitats, though other explanations exist. 3. There was no evidence of ecological redundancy over the 20 years. In fact, changes in functional diversity were almost exactly proportional to changes in species richness. 4. The absence of functional redundancy results from little redundancy intrinsic to the species' functional relationships and also because compositional change was nonrandom. Observed extinction and colonization events caused greater changes in functional diversity than if these events were random. 5. Our findings suggest that community assembly is influenced by the traits of species and that observed changes in functional diversity provide no reason to believe that the functioning of natural systems is buffered against change by ecological redundancy.     

Development of a pharmacodynamic model of murine malaria and antimalarial treatment with dihydroartemisinin

Antimalarial treatment strategies based on in vitro studies are limited by the paucity of pharmacodynamic information for dosage regimen design. We postulated that a murine model could be used for pre-clinical stages of drug development, especially in dose–response studies and evaluation of combination therapies. Swiss mice infected with Plasmodium berghei parasites (2–5% starting parasitaemia) were given dihydroartemisinin (0–100 mg/kg single dose). Parasite density was regularly determined from thin blood films. A parasite population growth model comprising parasite multiplication, decline in erythrocyte count with increasing parasitaemia and parasite clearance after drug administration was developed. This model described the rise in parasitaemia following inoculation, the nadir following dihydroartemisinin administration, and the subsequent resurgence of parasitaemia (analogous to ‘recrudescence’). At doses of 10, 30 and 100 mg/kg dihydroartemisinin, there was a graded response with 2.5 ± 1, 5 ± 1 and 12 ± 4-fold decreases in parasitaemia, respectively. The nadir parasitaemia (at 21–27 h) was also dose-dependent. This study demonstrates that a murine malaria pharmacodynamic model is a valuable tool for understanding how single drugs and their dosing schedules alter the time course and level of infection.     

Germ-line DNA copy number variation frequencies in a large North American population

Genomic copy number variation (CNV) is a recently identified form of global genetic variation in the human genome. The Affymetrix GeneChip 100 and 500 K SNP genotyping platforms were used to perform a large-scale population-based study of CNV frequency. We constructed a genomic map of 578 CNV regions, covering approximately 220 Mb (7.3%) of the human genome, identifying 183 previously unknown intervals. Copy number changes were observed to occur infrequently (<1%) in the majority (>93%) of these genomic regions, but encompass hundreds of genes and disease loci. This North American population-based map will be a useful resource for future genetic studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A polyvalent vaccine for high-risk prostate patients: “are more antigens better?”

We have shown the immunogenicity and safety of synthetic carbohydrate vaccines when conjugated to the carrier keyhole limpet hemocyanin (KLH) and given with the adjuvant, QS-21, in patients with biochemically relapsed prostate cancer. To determine whether immune response could be further enhanced with stimulation by multiple antigens, a hexavalent vaccine was prepared using previously determined doses and administered in a Phase II setting to 30 high-risk patients. The hexavalent vaccine included GM2, Globo H, Lewis^y, glycosylated MUC-1-32mer and Tn and TF in a clustered formation, conjugated to KLH and mixed with QS-21. Eight vaccinations were administered over 13 months. All 30 patients had significant elevations in antibody titers to at least two of the six antigens; 22 patients had increased reactivity with FACS. These serologic responses were lower than that seen previously in patients treated with the respective monovalent vaccines. The reciprocal median combined IgM and IgG antibody titers with ELISA against MUC1, Tn, TF, globo H and GM2 for these 30 patients were 640, 80, 120, 40 and 0, compared to 1280, 640, 1280, 320 and 160 seen in patients receiving individual monovalent vaccines. This hexavalent vaccine of synthetic “self” antigens broke immunologic tolerance against two or more antigens in all 30 vaccinated patients, was safe, but antibody titers against several of the antigens were lower than those seen in individual monovalent trials. No impact on PSA slope was detected. We address the relevance of the multivalent approach for prostate cancer treatment. Supported by the Prostate Cancer Foundation, The PepsiCo Foundation, The Sharon Hels and Brad Reed Fund, Swim Across America, The Sara Chait Foundation. Dr. Philip Livingston is a consultant for and shareholder in Progenics Pharmaceuticals, Inc.