Structural and mechanistic characterization of leukocyte-type core 2 β1,6-N-acetylglucosaminyltransferase: a metal-ion-independent GT-A glycosyltransferase

Leukocyte-type core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-L) is an inverting, metal-ion-independent glycosyltransferase that catalyzes the formation of mucin-type core 2 O-glycans. C2GnT-L belongs to the GT-A fold, yet it lacks the metal ion binding DXD motif characteristic of other nucleoside disphosphate GT-A fold glycosyltransferases. To shed light on the basis for its metal ion independence, we have solved the X-ray crystal structure (2.3 Å resolution) of a mutant form of C2GnT-L (C217S) in complex with the nucleotide sugar product UDP and, using site-directed mutagenesis, examined the roles of R378 and K401 in both substrate binding and catalysis. The structure shows that C2GnT-L exists in an “open” conformation and a “closed” conformation and that, in the latter, R378 and K401 interact with the β-phosphate moiety of the bound UDP. The two conformations are likely to be important in catalysis, but the conformational changes that lead to their interconversion do not resemble the nucleotide-sugar-mediated loop ordering observed in other GT-A glycosyltransferases. R378 and K401 were found to be important in substrate binding and/or catalysis, an observation consistent with the suggestion that they serve the same role played by metal ion in all of the other GT-A glycosyltransferases studied to date. Notably, R378 and K401 appear to function in a manner similar to that of the arginine and lysine residues contained in the RX_4-5K motif found in the retaining GT-B glycosyltransferases.     

Categorization training results in shape- and category-selective human neural plasticity

Object category learning is a fundamental ability, requiring the combination of "bottom-up" stimulus-driven with "top-down" task-specific information. It therefore may be a fruitful domain for study of the general neural mechanisms underlying cortical plasticity. A simple model predicts that category learning involves the formation of a task-independent shape-selective representation that provides input to circuits learning the categorization task, with the computationally appealing prediction of facilitated learning of additional, novel tasks over the same stimuli. Using fMRI rapid-adaptation techniques, we find that categorization training (on morphed "cars") induced a significant release from adaptation for small shape changes in lateral occipital cortex irrespective of category membership, compatible with the sharpening of a representation coding for physical appearance. In contrast, an area in lateral prefrontal cortex, selectively activated during categorization, showed sensitivity posttraining to explicit changes in category membership. Further supporting the model, categorization training also improved discrimination performance on the trained stimuli.     

Insect Innate Immunity Database (IIID): An Annotation Tool for Identifying Immune Genes in Insect Genomes

The innate immune system is an ancient component of host defense. Since innate immunity pathways are well conserved throughout many eukaryotes, immune genes in model animals can be used to putatively identify homologous genes in newly sequenced genomes of non-model organisms. With the initiation of the “i5k” project, which aims to sequence 5,000 insect genomes by 2016, many novel insect genomes will soon become publicly available, yet few annotation resources are currently available for insects. Thus, we developed an online tool called the Insect Innate Immunity Database (IIID) to provide an open access resource for insect immunity and comparative biology research (http://www.vanderbilt.edu/IIID). The database provides users with simple exploratory tools to search the immune repertoires of five insect models (including Nasonia), spanning three orders, for specific immunity genes or genes within a particular immunity pathway. As a proof of principle, we used an initial database with only four insect models to annotate potential immune genes in the parasitoid wasp genus Nasonia. Results specify 306 putative immune genes in the genomes of N. vitripennis and its two sister species N. giraulti and N. longicornis. Of these genes, 146 were not found in previous annotations of Nasonia immunity genes. Combining these newly identified immune genes with those in previous annotations, Nasonia possess 489 putative immunity genes, the largest immune repertoire found in insects to date. While these computational predictions need to be complemented with functional studies, the IIID database can help initiate and augment annotations of the immune system in the plethora of insect genomes that will soon become available.     

X-ray crystal structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in complex with donor substrate analogues

The Golgi-resident glycosyltransferase, UDP-N-acetyl-d-glucosamine:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I), initiates the conversion of high-mannose oligosaccharides to complex and hybrid structures in the biosynthesis of N-linked glycans. Reported here are the X-ray crystal structures of GnT I in complex with UDP-CH2-GlcNAc (a non-hydrolyzable C-glycosidic phosphonate), UDP-2-deoxy-2-fluoro-glucose, UDP-glucose and UDP. Collectively, these structures provide evidence for the importance of the GlcNAc moiety and its N-acetyl group in donor substrate binding, as well as insight into the role played by the flexible 318-330 loop in substrate binding and product release. In addition, the UDP-CH2-GlcNAc complex reveals a well-defined glycerol molecule poised for nucleophilic attack on the C1 atom of the donor substrate analogue. The position and orientation of this glycerol molecule have allowed us to model the binding of the Manalpha1,3Manbeta1 moiety of the acceptor substrate and, based on the model, to suggest a rationalization for the main determinants of GnT I acceptor specificity.     

IGF2BP1: a novel binding protein of p38 MAPK

Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1β (IL-1β) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1β enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.     

Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein

Plasma phospholipid transfer protein (PLTP) is thought to be involved in the remodeling of high density lipoproteins (HDL), which are atheroprotective. It is also involved in the metabolism of very low density lipoproteins (VLDL). Hence, PLTP is thought to be an important factor in lipoprotein metabolism and the development of atherosclerosis. We have overexpressed PLTP in mice heterozygous for the low density lipoprotein (LDL) receptor, a model for atherosclerosis. We show that increased PLTP activity results in a dose-dependent decrease in HDL, and a moderate stimulation of VLDL secretion (相似文献   

X-ray crystal structure of a galactose-specific C-type lectin possessing a novel decameric quaternary structure

Rattlesnake venom lectin (RSL) from the western diamondback rattlesnake (Crotalus atrox) is an oligomeric galactose-specific C-type lectin. The X-ray crystal structure of RSL, in complex with lactose and thiodigalactoside, at 2.2 and 2.3 A resolution, respectively, reveals a decameric protein composed of two 5-fold symmetric pentamers arranged in a staggered, back-to-back orientation. Each monomer corresponds to a single canonical C-type lectin carbohydrate recognition domain devoid of accessory domains and is disulfide-bonded to a monomer in the other pentamer. The structure is the first example of that of a carbohydrate complex of a vertebrate galactose-specific C-type lectin. The 10 carbohydrate-binding sites, located on the rim of the decamer, suggest a role for multivalent interactions and a mechanism for RSL's ability to promote receptor cross-linking and cell aggregation.     

Collaboration or Contention? Decentralised Marine Governance in Berau

Conservation of marine space is a new frontier in environmentalists’ involvement with resource governance in Indonesia. The coastal and marine area of Berau was established as a District Marine Conservation Area (MCA) based on District Head Regulation No. 31/2005. The total MCA of 1.27 million ha was expected to become part of the wider MCA networks of the East Borneo Seascape and the Sulu Sulawesi Marine Ecoregion, as well as of the Coral Triangle international network of conservation areas in Southeast Asia and the Pacific. The Berau MCA was developed in collaboration between the Berau district government and international environmental non-governmental organisations (NGOs) such as The Nature Conservancy (TNC) and the World Wide Fund for Nature (WWF). They claimed to use a new concept of partnership with the decentralised district government as the principal institutional partner. However, the governance framework is full of legal disconnects due to decentralisation of resource management, and its implementation faces the real-life challenges of political-economic and historical valuations of the marine environment producing conflicts of interest between the multi-scalar actors involved. Following the very process of establishing the Berau MCA over a five-year period (2005–2010), this paper shows why it is necessary to understand how collaboration and contention are constructed and in turn construct actors' perceptions and perspectives on marine conservation and resource extraction in decentralised coastal governance.