The occupancy of two distinct conformations by active-site histidine-119 in crystals of ribonuclease is modulated by pH

AbstractStructures of a semisynthetic RNase have been obtained to a resolution of 2.0 Å at pH values of 5.2, 6.5, 7.5, and 8.8, respectively. The principle structural transformation occurring over this pH range is the conversion of the side chain of active site residue His-119 from one conformation (X1 = −43° to −57°) at low pH to another (X1 = + 159° to + 168°) at higher pH values. On the basis of this observation, a model is proposed that reconciles the disparate pK values for His-119 in the enzyme-substrate complex that have been deduced from kinetic studies and from proton NMR measurements in the presence of pseudosubstrates

Urinary Metabolic Phenotyping Reveals Differences in the Metabolic Status of Healthy and Inflammatory Bowel Disease (IBD) Children in Relation to Growth and Disease Activity.

Growth failure and delayed puberty are well known features of children and adolescents with inflammatory bowel disease (IBD), in addition to the chronic course of the disease. Urinary metabonomics was applied in order to better understand metabolic changes between healthy and IBD children. 21 Pediatric patients with IBD (mean age 14.8 years, 8 males) were enrolled from the Pediatric Gastroenterology Outpatient Clinic over two years. Clinical and biological data were collected at baseline, 6, and 12 months. 27 healthy children (mean age 12.9 years, 16 males) were assessed at baseline. Urine samples were collected at each visit and subjected to ¹H Nuclear Magnetic Resonance (NMR) spectroscopy. Using ¹H NMR metabonomics, we determined that urine metabolic profiles of IBD children differ significantly from healthy controls. Metabolic differences include central energy metabolism, amino acid, and gut microbial metabolic pathways. The analysis described that combined urinary urea and phenylacetylglutamine-two readouts of nitrogen metabolism-may be relevant to monitor metabolic status in the course of disease. Non-invasive sampling of urine followed by metabonomic profiling can elucidate and monitor the metabolic status of children in relation to disease status. Further developments of omic-approaches in pediatric research might deliver novel nutritional and metabolic hypotheses

Structure of a Bovine Thrombin-Hirudin\u3csub\u3e51-65\u3c/sub\u3e Complex Determined by a Combination of Molecular Replacement and Graphics. Incorporation of Known Structural Information in Molecular Replacement

Crystals of the bovine thrombin-hirudins51-65 complex have space group P6122 with cell constants a = 116.4, and c = 200.6 Å and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 Å. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in the interactions of the C-terminal peptide of hirudin with thrombin. The hirudin peptide in complex 2 has a higher temperature factor as compared with peptide 1 which may be correlated partly with a larger number of short-range electrostatic interactions between peptide 1 and thrombin and partly with the fact that thrombin 2 is -thrombin which is cleaved at Thr149A near the peptide binding site. Later, using this structure as a test case, it was shown that the position for the second thrombin could also be determined by a novel modification of the molecular-replacement method in which the contribution of the known molecule is subtracted from the structure factors. This approach is facile and applicable to any crystal containing two or more macromolecules in the asymmetric unit in which some but not all of the molecules can be determined by molecular replacement

Crystallization of Hemoglobins II and III of the Symbiont-Harboring Clam Lucina pectinata

Diffraction data to 2.7 A resolution were measured on crystals of the homotetramers of components II and III of the cytoplacmic hemoglobin of the symbiont-harboring clam Lucina pectinata. Even though the crystallization conditions are different and the sequence homology of the two hemoglobins is only 63%, the crystals are isomorphous to each other and to the heterotetramer Hb II/III, implying that the residues primarily involved in the intermolecular interactions and responsible for crystal cohesion may be invariant

The Structure of a Complex of Bovine &-Thrombin and Recombinant Hirudin at 2.8-A Resolution

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme

The Structure of a Complex of Bovine &-Thrombin and Recombinant Hirudin at 2.8-A Resolution

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme

Circadian and Feeding Rhythms Orchestrate the Diurnal Liver Acetylome.

Lysine acetylation is involved in various biological processes and is considered a key reversible post-translational modification in the regulation of gene expression, enzyme activity, and subcellular localization. This post-translational modification is therefore highly relevant in the context of circadian biology, but its characterization on the proteome-wide scale and its circadian clock dependence are still poorly described. Here, we provide a comprehensive and rhythmic acetylome map of the mouse liver. Rhythmic acetylated proteins showed subcellular localization-specific phases that correlated with the related metabolites in the regulated pathways. Mitochondrial proteins were over-represented among the rhythmically acetylated proteins and were highly correlated with SIRT3-dependent deacetylation. SIRT3 activity being nicotinamide adenine dinucleotide (NAD) <sup>+</sup> level-dependent, we show that NAD <sup>+</sup> is orchestrated by both feeding rhythms and the circadian clock through the NAD <sup>+</sup> salvage pathway but also via the nicotinamide riboside pathway. Hence, the diurnal acetylome relies on a functional circadian clock and affects important diurnal metabolic pathways in the mouse liver

MRI atlas of a lizard brain

Magnetic resonance imaging (MRI) is an established technique for neuroanatomical analysis, being particularly useful in the medical sciences. However, the application of MRI to evolutionary neuroscience is still in its infancy. Few magnetic resonance brain atlases exist outside the standard model organisms in neuroscience and no magnetic resonance atlas has been produced for any reptile brain. A detailed understanding of reptilian brain anatomy is necessary to elucidate the evolutionary origin of enigmatic brain structures such as the cerebral cortex. Here, we present a magnetic resonance atlas for the brain of a representative squamate reptile, the Australian tawny dragon (Agamidae: Ctenophorus decresii), which has been the subject of numerous ecological and behavioral studies. We used a high-field 11.74T magnet, a paramagnetic contrasting-enhancing agent and minimum-deformation modeling of the brains of thirteen adult male individuals. From this, we created a high-resolution three-dimensional model of a lizard brain. The 3D-MRI model can be freely downloaded and allows a better comprehension of brain areas, nuclei, and fiber tracts, facilitating comparison with other species and setting the basis for future comparative evolution imaging studies. The MRI model and atlas of a tawny dragon brain (Ctenophorus decresii) can be viewed online and downloaded using the Wiley Biolucida Server at wiley.biolucida.net.Government of Australia, Grant/Award Numbers: APA#31/2011, IPRS#1182/2010; National Science and Engineering Research Council of Canada, Grant/Award Number: PGSD3-415253-2012; Quebec Nature and Technology Research Fund, Grant/AwardNumber: 208332; National Imaging Facility of Australia; Spanish Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional, Grant/Award Number:BFU2015-68537-

Energy Resolution studies for NEXT

This work aims to present the current state of simulations of electroluminescence (EL) produced in gas-based detectors with special interest for NEXT --- Neutrino Experiment with a Xenon TPC. NEXT is a neutrinoless double beta decay experiment, thus needs outstanding energy resolution which can be achieved by using electroluminescence. The process of light production is reviewed and properties such as EL yield and associated fluctuations, excitation and electroluminescence efficiencies, and energy resolution, are calculated. An EL production region with a 5 mm width gap between two infinite parallel planes is considered, where a uniform electric field is produced. The pressure and temperature considered are 10 bar and 293 K, respectively. The results show that, even for low values of VUV photon detection efficiency, good energy resolution can be achieved: below 0.4 % (FWHM) at $Q_{\beta\beta}=$2.458 MeV

Urinary metabolic insights into host-gut microbial interactions in healthy and IBD children.

To identify metabolic signatures in urine samples from healthy and inflammatory bowel disease (IBD) children. We applied liquid chromatography and gas chromatography coupled to targeted mass spectrometry (MS)-based metabolite profiling to identify and quantify bile acids and host-gut microbial metabolites in urine samples collected from 21 pediatric IBD patients monitored three times over one year (baseline, 6 and 12 mo), and 27 age- and gender-matched healthy children. urinary metabolic profiles of IBD children differ significantly from healthy controls. Such metabolic differences encompass central energy metabolism, amino acids, bile acids and gut microbial metabolites. In particular, levels of pyroglutamic acid, glutamic acid, glycine and cysteine, were significantly higher in IBD children in the course of the study. This suggests that glutathione cannot be optimally synthesized and replenished. Whilst alterations of the enterohepatic circulation of bile acids in pediatric IBD patients is known, we show here that non-invasive urinary bile acid profiling can assess those altered hepatic and intestinal barrier dysfunctions. The present study shows how non-invasive sampling of urine followed by targeted MS-based metabonomic analysis can elucidate and monitor the metabolic status of children with different GI health/disease status