Lipoxygenase interactions with natural flavonoid, quercetin, reveal a complex with protocatechuic acid in its X-ray structure at 2.1 A resolution

PUFA metabolites have a profound effect on inflammatory diseases and cancer progression. Blocking their production by inhibiting PUFA metabolizing enzymes (dioxygenases: cyclooxygenases and LOXs) might be a successful way to control and relieve such problems, if we learn to better understand their actions at a molecular level. Compounds with strong antioxidative and free radical scavenging properties, such as polyphenols, could be effective in blocking PUFA activities, and natural flavonoids possess such qualities. Quercetin belongs to the group of natural catecholic compounds and is known as a potent, competitive inhibitor of LOX. Structural analysis reveals that quercetin entrapped within LOX undergoes degradation, and the resulting compound has been identified by X-ray analysis as protocatechuic acid (3,4-dihydroxybenzoic acid) positioned near the iron site. Its C3-OH group points toward His523, C4-OH forms a hydrogen bond with O=C from the enzyme's C-terminus, and the carboxylic group is incorporated into the hydrogen bonding network of the active-site neighborhood via Gln514. This unexpected result, together with our previous observations concerning other polyphenols, yields new evidence about the metabolism of natural flavonoids. These compounds might be vulnerable to the co-oxidase activity of LOX, leading to enzyme-stimulated oxidative degradation, which results in an inhibitor of a lower molecular weight.     

Measurements of protein sequence-structure correlations

Correlations between protein structures and amino acid sequences are widely used for protein structure prediction. For example, secondary structure predictors generally use correlations between a secondary structure sequence and corresponding primary structure sequence, whereas threading algorithms and similar tertiary structure predictors typically incorporate interresidue contact potentials. To investigate the relative importance of these sequence-structure interactions, we measured the mutual information among the primary structure, secondary structure and side-chain surface exposure, both for adjacent residues along the amino acid sequence and for tertiary structure contacts between residues distantly separated along the backbone. We found that local interactions along the amino acid chain are far more important than non-local contacts and that correlations between proximate amino acids are essentially uninformative. This suggests that knowledge-based contact potentials may be less important for structure predication than is generally believed.     

Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse

We have developed a genotyping system for detecting genetic contamination in the laboratory mouse based on assaying single-nucleotide polymorphism (SNP) markers positioned on all autosomes and the X chromosome. This system provides a fast, reliable, and cost-effective way for genetic monitoring, while maintaining a very high degree of confidence. We describe the allelic distribution of 235 SNPs in 48 mouse strains, thereby creating a database of polymorphisms useful for genotyping purposes. The SNP markers used in this study were chosen from publicly available SNP databases. Four genotyping methods were evaluated, and dynamic two-tube allele-specific PCR assays were developed for each marker and tested on a set of 48 inbred mouse strains. The minimal number of assays sufficient to distinguish groups consisting of different numbers of mouse strains was estimated, and a panel of 28 SNPs sufficient to distinguish virtually all of the inbred strains tested was selected. Amplifluor SNP detection assays were developed for these markers and tested on an extended list of 96 strains. This panel was used as a genetic quality control approach to monitor the genotypes of nearly 300 inbred, wild-derived, congenic, consomic, and recombinant inbred strains maintained at The Jackson Laboratory. We have concluded that this marker panel is sufficient for genetic contamination monitoring in colonies containing a large number of genetically diverse mouse strains and that reduced versions of the panel could be implemented in facilities housing a lower number of strains.     

The impact of carboxy nitroxide antioxidants on irradiated ataxia telangiectasia cells

Three water-soluble carboxy nitroxide antioxidants, 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl, 4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl, and 3-carboxy-2,2,5,5-tetramethylpyrrolidin-1-yloxyl, show significant impact on the postirradiation survival rates of ataxia telangiectasia (A-T) cells compared to normal cells, an assay which represents a model for understanding the impact of ROS damage on the A-T phenotype. The effects of these antioxidants are much more significant than those of vitamin E or Trolox (a water-soluble vitamin E analog), studied using the same cell survival model.     

Retinal ganglion cell type, size, and spacing can be specified independent of homotypic dendritic contacts

In Brn3b(-/-) mice, where 80% of retinal ganglion cells degenerate early in development, the remaining 20% include most or all ganglion cell types. Cells of the same type cover the retinal surface evenly but tile it incompletely, indicating that a regular mosaic and normal dendritic field size can be maintained in the absence of contact among homotypic cells. In Math5(-/-) mice, where only approximately 5% of ganglion cells are formed, the dendritic arbors of at least two types among the residual ganglion cells are indistinguishable from normal in shape and size, even though throughout development they are separated by millimeters from the nearest neighboring ganglion cell of the same type. It appears that the primary phenotype of retinal ganglion cells can develop without homotypic contact; dendritic repulsion may be an end-stage mechanism that fine-tunes the dendritic arbors for more efficient coverage of the retinal surface.     

Feeling by sight or seeing by touch?

We have addressed the role of occipital and somatosensory cortex in a tactile discrimination task. Sight-ed and congenitally blind subjects rated the roughness and distance spacing for a series of raised dot patterns. When judging roughness, intermediate dot spacings were perceived as being the most rough, while distance judgments generated a linear relation. Low-frequency rTMS applied to somatosensory cortex disrupted roughness without affecting distance judgments, while rTMS to occipital cortex disrupted distance but not roughness judgments. We also tested an early blind patient with bilateral occipital cortex damage. Her performance on the roughness determination task was normal; however, she was greatly impaired with distance judgments. The findings suggest a double-dissociation effect in which roughness and distance are primarily processed in somatosensory and occipital cortex, respectively. The differential effect of rTMS on task performance and corroborative clinical evidence suggest that occipital cortex is engaged in tactile tasks requiring fine spatial discrimination.     

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.     

ApoE and clusterin cooperatively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo

Apolipoprotein E (apoE) and clusterin can influence structure, toxicity, and accumulation of the amyloid-beta (Abeta) peptide in brain. Both molecules may also be involved in Abeta metabolism prior to its deposition. To assess this possibility, we compared PDAPP transgenic mice that develop age-dependent Abeta accumulation in the absence of apoE or clusterin as well as in the absence of both proteins. apoE(-/-) and clusterin(-/-) mice accumulated similar Abeta levels but much less fibrillar Abeta. In contrast, apoE(-/-)/clusterin(-/-) mice had both earlier onset and markedly increased Abeta and amyloid deposition. Both apoE(-/-) and apoE(-/-)/clusterin(-/-) mice had elevated CSF and brain interstitial fluid Abeta, as well as significant differences in the elimination half-life of interstitial fluid Abeta measured by in vivo microdialysis. These findings demonstrate additive effects of apoE and clusterin on influencing Abeta deposition and that apoE plays an important role in regulating extracellular CNS Abeta metabolism independent of Abeta synthesis.