The Human Metabolome Database (HMDB, http://www.hmdb.ca) is a richly annotated resource that is designed to address the broad needs of biochemists, clinical chemists, physicians, medical geneticists, nutritionists and members of the metabolomics community. Since its first release in 2007, the HMDB has been used to facilitate the research for nearly 100 published studies in metabolomics, clinical biochemistry and systems biology. The most recent release of HMDB (version 2.0) has been significantly expanded and enhanced over the previous release (version 1.0). In particular, the number of fully annotated metabolite entries has grown from 2180 to more than 6800 (a 300% increase), while the number of metabolites with biofluid or tissue concentration data has grown by a factor of five (from 883 to 4413). Similarly, the number of purified compounds with reference to NMR, LC-MS and GC-MS spectra has more than doubled (from 380 to more than 790 compounds). In addition to this significant expansion in database size, many new database searching tools and new data content has been added or enhanced. These include better algorithms for spectral searching and matching, more powerful chemical substructure searches, faster text searching software, as well as dedicated pathway searching tools and customized, clickable metabolic maps. Changes to the user-interface have also been implemented to accommodate future expansion and to make database navigation much easier. These improvements should make the HMDB much more useful to a much wider community of users.

Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBDCenA). Related CBDs occur in at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDCenA. The adsorption of CBDCenA to crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.

PlasMapper is a comprehensive web server that automatically generates and annotates high-quality circular plasmid maps. Taking only the plasmid/vector DNA sequence as input, PlasMapper uses sequence pattern matching and BLAST alignment to automatically identify and label common promoters, terminators, cloning sites, restriction sites, reporter genes, affinity tags, selectable marker genes, replication origins and open reading frames. PlasMapper then presents the identified features in textual form and as high-resolution, multicolored graphical output. The appearance and contents of the output can be customized in numerous ways using several supplied options. Further, PlasMapper images can be rendered in both rasterized (PNG and JPG) and vector graphics (SVG) formats to accommodate a variety of user needs or preferences. The images and textual output are of sufficient quality that they may be used directly in publications or presentations. The PlasMapper web server is freely accessible at http://wishart.biology.ualberta.ca/PlasMapper.

The Human Metabolome Database (HMDB) is a Web‐based bioinformatic/cheminformatic resource with detailed information about human metabolites and metabolic enzymes. It can be used for fields of study including metabolomics, biochemistry, clinical chemistry, biomarker discovery, medicine, nutrition, and general education. In addition to its comprehensive literature‐derived data, the HMDB contains an extensive collection of experimental metabolite concentration data for plasma, urine, CSF, and/or other biofluids The HMDB is fully searchable, with many tools for viewing, sorting and extracting metabolite names, chemical structures, biofluid concentrations, enzymes, genes, NMR or MS spectra, and disease information. Each metabolite entry in the HMDB contains an average of 90 separate data fields including a comprehensive compound description, names and synonyms, chemical structure information, physico‐chemical data, reference NMR and MS spectra, normal and abnormal biofluid concentrations, tissue locations, disease associations, pathway information, enzyme data, gene sequence data, and SNP and mutation data, as well as extensive links to images, references and other public databases. Curr. Protoc. Bioinform. 25:14.8.1‐14.8.45. © 2009 by John Wiley & Sons, Inc.

Approximately 1% to 2% of persons in the general population are homozygous for a lipoprotein receptor-binding defective form of apoE (apoE2/2). However, only a small percentage (2% to 5%) of all apoE2/2 homozygotes develop type III hyperlipoproteinemia. Interaction with other genetic and environmental factors are required for the expression of this lipid abnormality. We sought to investigate the possible role of LPL gene mutations in the development of hyperlipoproteinemia in apoE2/2 homozygotes and in apoE2 heterozygotes. As a first step, we performed DNA sequence analysis of all 10 LPL coding exons in 2 patients with the apoE2/2 genotype who had type III hyperlipoproteinemia and identified a single missense mutation (Asn 291-->Ser) in exon 6 of the LPL gene. The mutation was then found in 5 of 18 patients with type III hyperlipoproteinemia who had the apoE2/2 genotype (allele frequency = 13.9%; P < or = 7.4 x 10(-5)) and 6 of 22 hyperlipidemic E2 heterozygous patients with the apoE3/2 and E4/2 genotype (allele frequency = 13.6%; P = 2.2 x 10(-5)). In contrast, this mutation was found in only 3 of 230 normolipidemic controls (allele frequency = 0.7%). In vitro mutagenesis studies revealed that the Asn 291-->Ser mutant LPL had approximately 60% of LPL catalytic activity and approximately 70% of specific activity compared with wild-type LPL. The heparin-binding affinity of the mutant LPL was not impaired. Our data suggest that the Asn 291-->Ser substitution is likely to be a significant predisposing factor contributing to the expression of different forms of hyperlipidemia when associated with other genetic factors such as the presence of apoE2.

The linearization of the stromal extracellular matrix (ECM) by cancer associated fibroblasts (CAFs) facilitates tumor cell growth and metastasis. However, the mechanism by which the ECM is remodeled is not fully understood. Hic-5 (TGFβ1i1), a focal adhesion scaffold protein, has previously been reported to be crucial for stromal ECM deposition and remodeling in vivo. Herein we show that CAFs lacking Hic-5 exhibit a significant reduction in the ability to form fibrillar adhesions, a specialized form of focal adhesion that promote fibronectin fibrillogenesis. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1. Furthermore, Src dependent phosphorylation of Hic-5 facilitated the interaction with tensin1 to prevent β1 integrin internalization and trafficking to the lysosome. The interaction between Hic-5 and tensin1 was mechanosensitive, promoting fibrillar adhesion formation and fibronectin fibrillogenesis in a rigidity dependent fashion. Importantly, this Src dependent mechanism was conserved in three-dimensional (3D) ECM environments. Immunohistochemistry of tensin1 showed enrichment in CAFs in vivo, which was abrogated upon deletion of Hic-5. Interestingly, elevated Hic-5 expression correlates with reduced distant metastasis free survival in patients with basal-like, HER2+ and grade 3 tumors. Thus, we have identified Hic-5 as a crucial regulator of ECM remodeling in CAFs by promoting fibrillar adhesion formation through a novel interaction with tensin1.

This study presents a detailed analysis of the acidic N-terminal region of the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain. Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype acidic activation domain from VP16 and the acid-rich region of Autographa californica multicapsid NPV (AcMNPV) IE1. These chimeric constructs were fully capable of transactivation in transient assays. The chimeric OpMNPV IE1s containing the herpes simplex virus VP16 and AcMNPV IE1 acidic activation domains consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 acidic activation domain. Transactivation by the chimeric constructs is enhanced synergistically when cotransfected with IE2 into Lymantria dispar and Spodoptera frugiperda cells. Both N- to C-terminal and C- to N-terminal deletions of the OpMNPV acidic activation domain were constructed to define functional domains within the OpMNPV IE1 acidic activation domain. At least two potential activation domains were identified. Within each of these domains, two core regions at amino acids 28-43 and amino acids 113-124 were identified that were similar to core regions of VP16 and GAL4, which contain predominately acidic and bulky hydrophobic amino acids.

Ie0 is the only gene of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) that is known to be spliced. In this study, cDNAs of ie0 were isolated, cloned, and sequenced. It was observed that IE0 contains 35 amino acids (aa) added to the N-terminus of IE1. In addition, it was found that the leader sequence of ie0 contains a 4-aa minicistron. To functionally characterize IE0, ie0 cDNAs were expressed under control of either the ie1 or the ie0 promoter. Unexpectedly, examination of ie0 translation products revealed that the predominant product from ie0 mRNAs was not IE0, but IE1. Mutation analysis showed that IE1 translation was preferentially initiated from either of two AUGs found in the first 15 nucleotides (nt) of the ie1 ORF that are internal to the ie0 ORF. It is unknown whether the internal translation initiation occurs via a leaky scanning mechanism or by an internal ribosomal entry site. Transactivation analysis with constructs that had point mutations in the ie1 AUGs and were translated only as IE0 revealed that OpMNPV IE0 is a 14- to 15-fold stronger transactivator than IE1. IE0 was also shown to be autoregulatory and to transactivate early genes in an enhancer-independent or -dependent manner. These results suggest that differential expression of baculovirus early genes can be obtained by coexpression of IE0 and IE1 in infected cells, which may permit subtle regulation of specific sets of viral genes.