The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 A in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain alpha/beta-fold with a central four-helix bundle connecting two seven-stranded parallel beta-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1'-carbonyl group near Tyr171 OH and NADP C4'N. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1' and C2' carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.     

T cell infiltration into class II MHC-disparate allografts and acute rejection is dependent on the IFN-gamma-induced chemokine Mig.

Direct evidence that cytokines with chemoattractant properties for leukocytes, chemokines, recruit alloantigen-primed T cells into transplanted allografts has been lacking. We present evidence that neutralization of a single chemokine inhibits T cell infiltration into class II MHC-disparate murine allografts and acute rejection. The chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma (Mig) are expressed in allogeneic skin grafts during the late stages of acute rejection. Survival of class II MHC-disparate B6.H-2bm12 allografts is prolonged from day 14 to day 55 posttransplant when C57BL/6 recipients are given a short course treatment with an antiserum to Mig. This treatment also inhibits T cell and macrophage infiltration into the allografts. B6.H-2bm12 allografts are also not rejected by IFN-gamma-/- C57BL/6 recipients. Injection of Mig directly into B6.H-2bm12 grafts on IFN-gamma-deficient recipients restores T cell infiltration and rejection. Therefore, the inability of IFN-gamma-deficient recipients to reject the class II MHC-disparate allografts is due to the lack of intraallograft Mig production and alloantigen-primed T cell recruitment to the graft. These results indicate for the first time the potential utility of chemokine neutralization strategies in preventing T cell infiltration into allografts and abrogating acute rejection.     

Phenotyping genetic diseases using an extension of mu-scores for multivariate data

As the field of genomics matures, more complex genotypes and phenotypes are being studied. Fanconi anemia (FA), for example, is an inherited chromosome instability syndrome with a complex array of variable disease phenotypes including congenital malformations, hematological manifestations, and cancer. To better understand specific aspects of the genetic etiology of FA and other rare diseases with complex phenotypes, it is often necessary to reduce the dimensions of the disease phenotype information. Towards this end, we extend a novel non-parametric approach to include information about a hierarchical structure among disease phenotypes. The proposed extension increases information content of the phenotype scores obtained and, thereby, the power of genotype-phenotype relationships studies.     

Hepatoselectivity of statins: design and synthesis of 4-sulfamoyl pyrroles as HMG-CoA reductase inhibitors

4-Sulfamoyl pyrroles were designed as novel hepatoselective HMG-CoA reductase inhibitors (statins) to reduce myalgia, a statin-induced adverse effect. The compounds were prepared via a [3+2] cycloaddition of a Münchnone with a sulfonamide-substituted alkyne. We identified compounds with greater selectivity for hepatocytes compared to L6-myocytes than rosuvastatin and atorvastatin. There was an inverse correlation of myocyte potencies and ClogP values. A number of analogs were effective at reducing cholesterol in acute and chronic in vivo models but they lacked sufficient chronic in vivo activity to warrant further development.     

Temperature dependence of acetylcholine receptor channels activated by different agonists

The temperature dependence of agonist binding and channel gating were measured for wild-type adult neuromuscular acetylcholine receptors activated by acetylcholine, carbamylcholine, or choline. With acetylcholine, temperature changed the gating rate constants (Q₁₀ ≈ 3.2) but had almost no effect on the equilibrium constant. The enthalpy change associated with gating was agonist-dependent, but for all three ligands it was approximately equal to the corresponding free-energy change. The equilibrium dissociation constant of the resting conformation (K_d), the slope of the rate-equilibrium free-energy relationship (Φ), and the acetylcholine association and dissociation rate constants were approximately temperature-independent. In the mutant αG153S, the choline association and dissociation rate constants were temperature-dependent (Q₁₀ ≈ 7.4) but K_d was not. By combining two independent mutations, we were able to compensate for the catalytic effect of temperature on the decay time constant of a synaptic current. At mouse body temperature, the channel-opening and -closing rate constants are ∼400 and 16 ms⁻¹. We hypothesize that the agonist dependence of the gating enthalpy change is associated with differences in ligand binding, specifically to the open-channel conformation of the protein.     

Structural basis of the ribosomal machinery for peptide bond formation,translocation, and nascent chain progression

Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.     

Spatial scale and the determinants of plant species richness

Traditional ecological theory has stressed the importance of competitive interactions in regulating species richness. Recent research has transcended this viewpoint by considering the role of stochastic processes, mosaic phenomena and nonequilibrium conditions in the regulation of richness. This growing body of work indicates that the determinants of plant species richness may vary predictably over different spatial scales.     

Genome-Wide Association Studies in an Isolated Founder Population from the Pacific Island of Kosrae

It has been argued that the limited genetic diversity and reduced allelic heterogeneity observed in isolated founder populations facilitates discovery of loci contributing to both Mendelian and complex disease. A strong founder effect, severe isolation, and substantial inbreeding have dramatically reduced genetic diversity in natives from the island of Kosrae, Federated States of Micronesia, who exhibit a high prevalence of obesity and other metabolic disorders. We hypothesized that genetic drift and possibly natural selection on Kosrae might have increased the frequency of previously rare genetic variants with relatively large effects, making these alleles readily detectable in genome-wide association analysis. However, mapping in large, inbred cohorts introduces analytic challenges, as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based. We performed genome-wide association analysis for 15 quantitative traits in 2,906 members of the Kosrae population, using novel approaches to manage the extreme relatedness in the sample. As positive controls, we observe association to known loci for plasma cholesterol, triglycerides, and C-reactive protein and to a compelling candidate loci for thyroid stimulating hormone and fasting plasma glucose. We show that our study is well powered to detect common alleles explaining ≥5% phenotypic variance. However, no such large effects were observed with genome-wide significance, arguing that even in such a severely inbred population, common alleles typically have modest effects. Finally, we show that a majority of common variants discovered in Caucasians have indistinguishable effect sizes on Kosrae, despite the major differences in population genetics and environment.