Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM*

The kynurenine pathway is the major route of l-tryptophan (l-Trp) catabolism in biology, leading ultimately to the formation of NAD⁺. The initial and rate-limiting step of the kynurenine pathway involves oxidation of l-Trp to N-formylkynurenine. This is an O₂-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237–244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of l-Trp, 1-methyl-l-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.     

The Carboxy-Terminal Tails of Septins Cdc11 and Shs1 Recruit Myosin-II Binding Factor Bni5 to the Bud Neck in Saccharomyces cerevisiae

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT–qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT–qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).     

Automatic methods for characterization of sexual dimorphism of adult femora: distal femur

Quantifying sex differences in femoral size and shape has extensive applications in forensics and prosthesis design. By applying strong statistical techniques such as principal component analysis (PCA), certain three-dimensional (3D) morphological variations of adult femora can be quantified over various femoral sizes. Coupling this statistical approach with a novel feature generation and extraction technique, localization of statistically significant (p<0.05) features are automatically defined and measured. Also, predefined anatomical landmarks and surgical axes have been calculated automatically. In all methods, femoral scale is controlled as a possible parameter of shape. By extensively comparing measurements across 92 male and 74 female femora, the dimorphic characteristics of the distal femur are shown. These differences have not been accounted for in many prosthetic systems and consequently these systems have limited sizing accuracy.     

Human Salivary Amylase Gene Copy Number Impacts Oral and Gut Microbiomes

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Is hepatitis C virus genotype 4 predominant in Saudi Arabia?

Hepatitis C virus (HCV) genotype 4 is believed to be predominant in the Middle East including Saudi Arabia (SA). We attempted to genotype 80 HCV isolates from different parts of SA by direct sequencing of a variable 222bp fragment from the NS5B region. The phylogenetic analysis of the NS5B sequences was complemented by direct sequence analysis of the conserved 5'-NCR region for HCV type-specific polymorphism. All 80 NS5B sequences separated into 3 clades which comprised 6 type 1b variants, 30 type 4 variants (24 of type 4a and 6 of type 4c or d) and 44 type 3 variants. Apart from two definitive type 3b variants the other 42 type 3 NS5B sequences formed 4 clusters with low similarity to type 3a-f HCV sequences from the database. The precise subtyping of these 42 type 3 variants awaits sequencing of longer HCV RNA stretches. Our results indicate that HCV type 4 may not be the only dominant genotype in SA.     

Three-Dimensional Structure of Aleutian Mink Disease Parvovirus: Implications for Disease Pathogenicity

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.     

Shrub Densities on Pre-1985 Reclaimed Mine Lands in Wyoming

The reestablishment of native shrubs is part of the mandate under which mining companies extract mineral resources in Wyoming and other western states. Post-mining shrub density and species mixtures have been topics debated by various mine reclamation stake holders. By law, coal-mined lands in Wyoming must now meet a post-mining shrub density of 1 shrub/m² on 20% of the affected area. To better understand the long-term results of shrub reclamation methods, we measured shrub density by species in 14 pre-1985 seedings at eight mines in three geographic regions of Wyoming. The sites studied were selected as Atriplex canescens (Pursh) Nutt. (fourwing saltbush) and grass or as Artemisia tridentata Nutt. ssp. wyomingensis (Beetle and Young) (Wyoming big sagebrush), fourwing saltbush, and grass post-mining communities. Shrub density and composition varied by site but typically reflected the seed mixtures used. Seedings that used a diversity of shrub species generally had greater 1994 shrub densities. Seeding rates between 60 and 1000 shrub seeds/m² had a positive, linear relationship with shrub density up to 0.6 shrubs/m² when sagebrush was part of the shrub seed mixture. We conclude that the likelihood of meeting the shrub standard can be enhanced by seeding diverse shrub mixtures at high seeding rates.     

Focal adhesion proteins FAK and paxillin increase in hypertrophied skeletal muscle

Components of signaling pathways for mechanotransduction duringload-induced enlargement of skeletal muscle have not been completelydefined. We hypothesized that loading of skeletal muscle would resultin an adaptive increase in the expression of two focal adhesion complex(FAC)-related proteins, focal adhesion kinase (FAK) and paxillin, aswell as increased FAK activity. FAK protein was immunolocalized to thesarcolemmal region of rooster anterior latissimus dorsi (ALD) myofibersin the middle of the ALD muscle. FAK (77 and 81%) and paxillin (206 and 202%) protein concentrations per unit of total protein in Westernblots increased significantly after 1.5 and 7 days, but not after 13 days, of stretch-induced hypertrophy-hyperplasia of the ALD muscle. FAK autokinase activity in immunoprecipitates was increased after 1.5, 7, and 13 days in stretched ALD muscles. To determine whether increasedFAK and paxillin protein concentrations are associated with hypertrophyand/or new fiber formation, two additional experiments were performed.First, during formation of primary chicken myotubes (a model of newfiber formation), FAK protein concentration (63%), FAK activity(157%), and paxillin protein concentration (97%) increased comparedwith myoblasts. Second, FAK (112% and 611%) and paxillin (87% and431%) protein concentrations per unit of total protein in the soleusmuscle increased at 1 and 8 days after surgical ablation of thesynergistic gastrocnemius muscle (a model of hypertrophy withouthyperplasia). Thus increases in components of the FAC occur inhypertrophying muscle of animals and in newly formed muscle fibers inculture. Furthermore, increased FAK activity suggests a possibleconvergence of signaling at the FAC in load-induced growth of skeletal muscle.