Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.     

Evidence for a Novel Marine Harmful Algal Bloom: Cyanotoxin (Microcystin) Transfer from Land to Sea Otters

“Super-blooms” of cyanobacteria that produce potent and environmentally persistent biotoxins (microcystins) are an emerging global health issue in freshwater habitats. Monitoring of the marine environment for secondary impacts has been minimal, although microcystin-contaminated freshwater is known to be entering marine ecosystems. Here we confirm deaths of marine mammals from microcystin intoxication and provide evidence implicating land-sea flow with trophic transfer through marine invertebrates as the most likely route of exposure. This hypothesis was evaluated through environmental detection of potential freshwater and marine microcystin sources, sea otter necropsy with biochemical analysis of tissues and evaluation of bioaccumulation of freshwater microcystins by marine invertebrates. Ocean discharge of freshwater microcystins was confirmed for three nutrient-impaired rivers flowing into the Monterey Bay National Marine Sanctuary, and microcystin concentrations up to 2,900 ppm (2.9 million ppb) were detected in a freshwater lake and downstream tributaries to within 1 km of the ocean. Deaths of 21 southern sea otters, a federally listed threatened species, were linked to microcystin intoxication. Finally, farmed and free-living marine clams, mussels and oysters of species that are often consumed by sea otters and humans exhibited significant biomagnification (to 107 times ambient water levels) and slow depuration of freshwater cyanotoxins, suggesting a potentially serious environmental and public health threat that extends from the lowest trophic levels of nutrient-impaired freshwater habitat to apex marine predators. Microcystin-poisoned sea otters were commonly recovered near river mouths and harbors and contaminated marine bivalves were implicated as the most likely source of this potent hepatotoxin for wild otters. This is the first report of deaths of marine mammals due to cyanotoxins and confirms the existence of a novel class of marine “harmful algal bloom” in the Pacific coastal environment; that of hepatotoxic shellfish poisoning (HSP), suggesting that animals and humans are at risk from microcystin poisoning when consuming shellfish harvested at the land-sea interface.     

Using Genotyping-By-Sequencing (GBS) for Genomic Discovery in Cultivated Oat

Advances in next-generation sequencing offer high-throughput and cost-effective genotyping alternatives, including genotyping-by-sequencing (GBS). Results have shown that this methodology is efficient for genotyping a variety of species, including those with complex genomes. To assess the utility of GBS in cultivated hexaploid oat (Avena sativa L.), seven bi-parental mapping populations and diverse inbred lines from breeding programs around the world were studied. We examined technical factors that influence GBS SNP calls, established a workflow that combines two bioinformatics pipelines for GBS SNP calling, and provided a nomenclature for oat GBS loci. The high-throughput GBS system enabled us to place 45,117 loci on an oat consensus map, thus establishing a positional reference for further genomic studies. Using the diversity lines, we estimated that a minimum density of one marker per 2 to 2.8 cM would be required for genome-wide association studies (GWAS), and GBS markers met this density requirement in most chromosome regions. We also demonstrated the utility of GBS in additional diagnostic applications related to oat breeding. We conclude that GBS is a powerful and useful approach, which will have many additional applications in oat breeding and genomic studies.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

A comparative cytogenetic study of 17 <Emphasis Type="Italic">Avena</Emphasis> species using Am1 and (GAA)<Subscript>6</Subscript> oligonucleotide FISH probes

Fluorescence in situ hybridization (FISH) was performed to explore the genomic and species relationships among 17 taxa using Am1 (oligo-Am1) and (GAA)₆ oligonucleotide probes. Oligo-Am1 (51 bp) hybridized strongly over almost the entire length of all chromosomes in the C genome. Six translocations between the A and C genomes were found in AACC tetraploids and AACCDD hexaploids, four minor translocations between the C and D genomes were found in AACCDD hexaploids, and two large translocations between the C and D genomes were found in A. sativa. In the 17 Avena species, (GAA)₆ regions mainly appear as sharp, thin bands at pericentromeric positions in the A, B, and C genomes and at termini in the B genome. However, no (GAA)₆ signal loci were observed in the D genome. The (GAA)₆ signal number was constant in both AA and CC diploids, though with different signal intensities. The (GAA)₆ signal pattern was diverse in AABB, AACC, and AACCDD polyploids, with each species exhibiting one signal pattern. The (GAA)₆ signal number was consistent in diploids and varied in polyploids, describing an intragenomic relationship among Avena species. This study is the first to test these two oligonucleotides, which are based on synthesized repeat units (18–51 bp), in the genus Avena. Our approach paves the way for future studies in which FISH probes can be used to assign other landmark genomic sequence oligonucleotides to physical chromosomes.     

Lysolecithin-cholesterol interaction. A spin-resonance and electron-micrographic study.

Another publication (rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. O.(1975) Can. j. Biochem. 53, 189-195) has established that lysolecithin and cholesterol interact to form an equimolar complex. We have investigated this complex using the techniques of electron spin resonance (e.s.r) and electronmicroscopy. By varying the cholesterol concentration with lysolecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithin and equimolar complex below 50 mol % cholesterol, and between crystalline cholesterol and equimolar complex above 50 mol % cholesterol. We have observed an interesting alteration in morphology by electron microscopy, and an isotropic to anisotropic spectral change using 3-dosylcholestane and 12-doxylstearic acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol %. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits no phase changes in the physiological temperature range. Implications for membrane structure are discussed.