Pharmacophore Modeling for Anti-Chagas Drug Design Using the Fragment Molecular Orbital Method

BackgroundChagas disease, caused by the parasite Trypanosoma cruzi, is a neglected tropical disease that causes severe human health problems. To develop a new chemotherapeutic agent for the treatment of Chagas disease, we predicted a pharmacophore model for T. cruzi dihydroorotate dehydrogenase (TcDHODH) by fragment molecular orbital (FMO) calculation for orotate, oxonate, and 43 orotate derivatives.Conclusions/SignificanceFMO-based interaction energy analyses revealed a pharmacophore model for TcDHODH inhibitor. Hydrogen bond acceptor pharmacophores correspond to Lys43 and Lys214, hydrogen bond donor and acceptor pharmacophores correspond to Asn67 and Asn194, and the aromatic ring pharmacophore corresponds to FMN, which shows important characteristics of compounds that inhibit TcDHODH. In addition, the Lys214 residue is not conserved between TcDHODH and human DHODH. Our analysis suggests that these orotate derivatives should preferentially bind to TcDHODH, increasing their selectivity. Our results obtained by pharmacophore modeling provides insight into the structural requirements for the design of TcDHODH inhibitors and their development as new anti-Chagas drugs.     

Effects of Acute Endurance Exercise Performed in the Morning and Evening on Inflammatory Cytokine and Metabolic Hormone Responses

Purpose

To compare the effects of endurance exercise performed in the morning and evening on inflammatory cytokine responses in young men.

Methods

Fourteen healthy male participants aged 24.3 ± 0.8 years (mean ± standard error) performed endurance exercise in the morning (0900–1000 h) on one day and then in the evening (1700–1800 h) on another day with an interval of at least 1 week between each trial. In both the morning and evening trials, the participants walked for 60 minutes at approximately 60% of the maximal oxygen uptake (V·O2max) on a treadmill. Blood samples were collected to determine hormones and inflammatory cytokines at pre-exercise, immediately post exercise, and 2 h post exercise.

Results

Plasma interleukin (IL)-6 and adrenaline concentrations were significantly higher immediately after exercise in the evening trial than in the morning trial (P < 0.01, both). Serum free fatty acids concentrations were significantly higher in the evening trial than in the morning trial at 2 h after exercise (P < 0.05). Furthermore, a significant correlation was observed between the levels of IL-6 immediately post-exercise and free fatty acids 2 h post-exercise in the evening (r = 0.68, P < 0.01).

Conclusions

These findings suggest that the effect of acute endurance exercise in the evening enhances the plasma IL-6 and adrenaline concentrations compared to that in the morning. In addition, IL-6 was involved in increasing free fatty acids, suggesting that the evening is more effective for exercise-induced lipolysis compared with the morning.     

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K_m of 338 μM and V_max of 601 μmol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.     

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.     

Phenosite: a web database integrating the mouse phenotyping platform and the experimental procedures in mice

Recently, a number of collaborative large-scale mouse mutagenesis programs have been launched. These programs aim for a better understanding of the roles of all individual coding genes and the biological systems in which these genes participate. In international efforts to share phenotypic data among facilities/institutes, it is desirable to integrate information obtained from different phenotypic platforms reliably. Since the definitions of specific phenotypes often depend on a tacit understanding of concepts that tends to vary among different facilities, it is necessary to define phenotypes based on the explicit evidence of assay results. We have developed a website termed PhenoSITE (Phenome Semantics Information with Terminology of Experiments: http://www.gsc.riken.jp/Mouse/), in which we are trying to integrate phenotype-related information using an experimental-evidence-based approach. The site's features include (1) a baseline database for our phenotyping platform; (2) an ontology associating international phenotypic definitions with experimental terminologies used in our phenotyping platform; (3) a database for standardized operation procedures of the phenotyping platform; and (4) a database for mouse mutants using data produced from the large-scale mutagenesis program at RIKEN GSC. We have developed two types of integrated viewers to enhance the accessibility to mutant resource information. One viewer depicts a matrix view of the ontology-based classification and chromosomal location of each gene; the other depicts ontology-mediated integration of experimental protocols, baseline data, and mutant information. These approaches rely entirely upon experiment-based evidence, ensuring the reliability of the integrated data from different phenotyping platforms.     

Purification, identification, characterization, and cDNA cloning of a high molecular weight extracellular superoxide dismutase of hamster that transiently increases in plasma during arousal from hibernation

We previously studied antioxidant profiles in the plasma of hibernating Syrian hamsters and found a transient increase of a superoxide radical-scavenging activity during the arousal phase. In this report, we purified and identified the high molecular weight superoxide dismutase (SOD)-like factor from the plasma of arousing hamsters. The cyanide-sensitive 240 kDa SOD-like factor showed a significant homology to mammalian extracellular SOD (EC-SOD) reported, although the molecular mass of EC-SOD was 135 kDa. The cDNA cloning revealed that the 240 kDa SOD-like factor was identical to the hamster ortholog of EC-SOD. It consisted of 245 amino acid residues including a signal sequence of 20 amino acid residues. Five cysteine residues that would participate in inner- and inter-subunit bonds were well conserved among species. Interestingly, there were four potential N-glycosylation sites in hamster EC-SOD, whereas there is only one site in other species. The amino acid sequence analysis indicated that three of the four sites were modified. These results suggest that the anomalistically high molecular weight of hamster EC-SOD is ascribed, at least in part, to the addition of extra sugar chains. Furthermore, results obtained here also propose the involvement of EC-SOD in the antioxidative defense of hibernating hamsters.     

Molecular phylogenetic position of the genera Stephanonympha and Caduceia (Parabasalia) inferred from nuclear small subunit rRNA gene sequences

Nuclear small subunit (SSU) rRNA gene sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the Devescovinidae (Caduceia versatilis) and polymastigont Calonymphidae (Stephanonympha nelumbium). The unidentified SSU rRNA sequence Nk3, previously obtained from the termite Neotermes koshunensis, has also been shown to derive from a Stephanonympha sp. by in situ hybridization. These sequences were analysed in a broad phylogeny including nearly all identified parabasalid sequences available in the databases, and some as yet unidentified sequences likely deriving from the new order Cristamonadida (Devescovinidae, Calonymphidae, and hypermastigids Lophomonadida). A global phylogeny of parabasalids reveals a partial agreement between the clades identified in this work and the last classification of this phylum into four orders. However, this classification is still incongruent with our data and new taxonomic considerations are proposed. The analysis confirms the monophyly of the Cristamonadida and separates this order into two groups: the first unites nearly all the Devescovinidae including Caduceia and the Calonymphidae Coronympha and Metacoronympha, whereas the second group is composed of a few Devescovinidae, Lophomonadida, and Calonymphidae such as Stephanonympha. Caduceia is closely related to Devescovina, corroborating the marked morphological similarity between these two genera whereas Stephanonympha groups together with the Calonymphidae Snyderella and Calonympha. These data also confirm the polyphyly of the families Devescovinidae and Calonymphidae and support the arrangement of the axostyle-pelta complexes as a valuable character for taxonomic considerations within the Calonymphidae.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.