Flaviviral helicase: insights into the mechanism of action of a motor protein

Motor proteins are involved in crucial cell activities, such as cargo transport or nucleic acid remodeling, by converting the free energy of ATP hydrolysis into motion or mechanical work. Flavivirus helicase is a motor protein involved in dsRNA separation during viral replication, thus essential for virus infection. Since a clear vision of the protein activity, in particular of the relationship between ATP cycling and dynamics, is missing, we carried over a molecular dynamics study on Dengue virus helicase in its ATP bound and unbound states. Our simulations show different opening levels of the ssRNA access site to the helicase core. Specifically, we show that ATP induces a closed state into the ssRNA access site, likely involved in the helicase unwinding activity.     

Enzymatic synthesis of substituted epicatechins for bioactivity studies in neurological disorders

Glucuronidated and/or methylated metabolites of the proanthocyanidin (PA) monomer (-)-epicatechin are detected in both blood and brain following feeding of rodents with a monomeric grape seed PA extract shown to reduce symptoms in a mouse model of Alzheimer's disease. To generate metabolites for future mechanistic studies, we investigated the ability of recombinant human glucuronosyl transferases of the UGT1A and UGT2B families to glucuronidate epicatechin or 3'-O-methyl epicatechin in vitro. Of twelve enzymes tested, UGT1A9 was the most efficient, producing epicatechin 3'-O-glucuronide as the major product. Incubation of UGT1A9 with 3'-O-methyl-epicatechin resulted in two major products, one of which was identified as 3'-O-methyl-epicatechin 5-O-glucuronide, a major metabolite found in blood plasma and brain tissue of the rodents following feeding with a grape seed extract. We also investigated in vitro methylation of epicatechin and epicatechin glucuronides by human catechol O-methyltransferase. Enzymatic production of 3'-O-methyl-epicatechin 5-O-glucuronide was optimized to 50% overall yield. These studies form a basis for generation of mg quantities of pure epicatechin (methyl) glucuronides of biological significance, and provide clarification of structure of previously identified epicatechin metabolites.     

Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.     

Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8(+) and Th1 CD4(+) T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3(+) cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4(+) T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3(+) primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.     

Soil Nematode Responses to Crop Management and Conversion to Native Grasses

Soil nematode community response to treatments of three, four-year crop rotations (spring wheat-pea-spring wheat-flax, spring wheat-green manure-spring wheat-flax, and spring wheat-alfalfa-alfalfa-flax) under conventional and organic management, and native tall grass restoration (restored prairie) were assessed in June 2003, and July and August 2004. The research site was the Glenlea Long-term Rotation and Crop Management Study, in the Red River Valley, Manitoba, established in 1992. The nematode community varied more with sample occasion than management and rotation. The restored prairie favored high colonizer-persister (c-p) value omnivores and carnivores, and fungivores but less bacterivores. The restored prairie soil food web was highly structured, mature and low-to-moderately enriched as indicated by structure (SI), maturity (MI) and enrichment (EI) index values, respectively. Higher abundance of fungivores and channel index (CI) values suggested fungal-dominated decomposition. Nematode diversity was low even after more than a decade of restoration. A longer time may be required to attain higher diversity for this restored fragmented prairie site distant from native prairies. No consistent differences were found between organic and conventional management for nematode trophic abundance, with the exception of enrichment opportunists of the c-p 1 group which were favored by conventional management. Although EI was lower and SI was higher for organic than conventional their absolute values suggested decomposition channels to be primarily bacterial, and fewer trophic links with both management scenarios. A high abundance of fungivores in the rotation including the green manure indicates greater fungal decomposition.     

Regulation of the glucocorticoid response to stress-related disorders by the Hsp90-binding immunophilin FKBP51

Immunophilin is the collective name given to a family of proteins that bind immunosuppressive drugs: Some immunophilins are Hsp90-binding cochaperones that affect steroid receptor function. Mood and anxiety disorders are stress-related diseases characterized by an impaired function of the mineralocorticoid and glucocorticoid receptors, two of the major regulatory elements of the hypothalamus-pituitary-adrenocortical axis. Genetic variations of the FK506-binding protein of 51-kDa, FKBP51, one of the immunophilins bound to those steroid receptor complexes, were associated with the effectiveness of treatments against depression and with a major risk-factor for the development of post-traumatic stress disorders. Interestingly, immunophilins show polymorphisms and some polymorphic isoforms of FKBP51 correlate with a greater impairment of steroid receptor functions. In this review, we discuss different aspects of the role of FKBP51 in such steroid receptor function and the impact of genetic variants of the immunophilin on the dysregulation of the stress response.     

Reflections on Plant and Soil Nematode Ecology: Past,Present and Future

The purpose of this review is to highlight key developments in nematode ecology from its beginnings to where it stands today as a discipline within nematology. Emerging areas of research appear to be driven by crop production constraints, environmental health concerns, and advances in technology. In contrast to past ecological studies which mainly focused on management of plant-parasitic nematodes, current studies reflect differential sensitivity of nematode faunae. These differences, identified in both aquatic and terrestrial environments include response to stressors, environmental conditions, and management practices. Methodological advances will continue to influence the role nematodes have in addressing the nature of interactions between organisms, and of organisms with their environments. In particular, the C. elegans genetic model, nematode faunal analysis and nematode metagenetic analysis can be used by ecologists generally and not restricted to nematologists.     

Helminth community structure in birds of prey (Accipitriformes and Falconiformes) in southern Italy

We compared helminth communities in 6 species of birds of prey from the Calabria region of southern Italy. In total, 31 helminth taxa, including 17 nematodes, 9 digeneans, 3 acanthocephalans, and 2 cestodes, were found. All helminth species were observed in the gastrointestinal tract, except for 3 spirurid nematodes. Most of the parasite species were detected in at least 2 hosts, but 13 helminth species were found in only 1 host. At the infracommunity level, the overall species richness and Brillouin's index of diversity varied by host, with the highest values in a generalist feeder, the Eurasian buzzard (Buteo buteo), and the lowest in a specialist, the western honey buzzard (Pernis apivorus). Species richness was gender dependent only in the sparrow hawk (Accipiter nisus). The helminth communities were characterized by different dominant species, namely, Centrorhynchus spp. (Acanthocephala) in the Eurasian buzzard and common kestrel (Falco tinnunculus), Parastrigea intermedia (Digenea) in the marsh harrier (Circus aeruginosus), Physaloptera alata (Nematoda) in the sparrow hawk, Serratospiculum tendo (Nematoda) in the peregrine falcon (Falco peregrinus), and Strigea falconis (Digenea) in the western honey buzzard. Statistical analyses confirmed a highly significant difference of helminth infracommunity structure among host species. We conclude that in the Calabria region of southern Italy, each of the raptor species studied is distinct in terms of its helminth communities, and more diverse feeding habits of the host correspond with richer helminth communities.