A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters

We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.     

Functional behaviour of bone around dental implants

Achieving a long-term stable implant interface is a significant clinical issue when there is insufficient cortical bone stabilisation at implant placement. Clinical outcomes studies suggest that the higher risk implants are those placed in compromised cortical bone (thin, porous, etc.) in anatomical sites with minimal existing trabecular bone (characterised as type IV bone). In establishing and maintaining an implant interface in such an environment, one needs to consider the impact of masticatory forces, the response of bone to these forces and the impact of age on the adaptive capacity of bone. These forces, in turn, have the potential to create localised changes in interfacial stiffness through viscoelastic changes at the interface. Changes in bone as a function of age (e.g. localised hypermineralised osteopetrosis and localised areas of osteopenia) will alter the communication between osteocytes and osteoblasts creating the potential for differences in response of osteoblastic cells in the older population. A key to understanding the biomechanical and functional behaviour of implants in the older population is to control the anticipated modelling and remodelling behaviour through implant design that takes into account how tissues respond to the mechanically active environment.     

Meat-adaptive genes and the evolution of slower aging in humans

The chimpanzee life span is shorter than that of humans, which is consistent with a faster schedule of aging. We consider aspects of diet that may have selected for genes that allowed the evolution of longer human life spans with slower aging. Diet has changed remarkably during human evolution. All direct human ancestors are believed to have been largely herbivorous. Chimpanzees eat more meat than other great apes, but in captivity are sensitive to hypercholesterolemia and vascular disease. We argue that this dietary shift to increased regular consumption of fatty animal tissues in the course of hominid evolution was mediated by selection for "meat-adaptive" genes. This selection conferred resistance to disease risks associated with meat eating also increased life expectancy. One candidate gene is apolipoprotein E (apoE), with the E3 allele evolved in the genus Homo that reduces the risks for Alzheimer's and vascular disease, as well as influencing inflammation, infection, and neuronal growth. Other evolved genes mediate lipid metabolism and host defense. The timing of the evolution of apoE and other candidates for meat-adaptive genes is discussed in relation to key events in human evolution.     

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-fucosidase, a key step in the understanding of these important enzymes.     

Gene-trap mutagenesis: past, present and beyond

Although at least 35,000 human genes have been sequenced and mapped, adequate expression or functional information is available for only approximately 15% of them. Gene-trap mutagenesis is a technique that randomly generates loss-of-function mutations and reports the expression of many mouse genes. At present, several large-scale, gene-trap screens are being carried out with various new vectors, which aim to generate a public resource of mutagenized embryonic stem (ES) cells. This resource now includes more than 8,000 mutagenized ES-cell lines, which are freely available, making it an appropriate time to evaluate the recent advances in this area of genomic technology and the technical hurdles it has yet to overcome.     

An ontology driven architecture for derived representations of macromolecular structure

SUMMARY: An object metamodel based on a standard scientific ontology has been developed and used to generate a CORBA interface, an SQL schema and an XML representation for macromolecular structure (MMS) data. In addition to the interface and schema definitions, the metamodel was also used to generate the core elements of a CORBA reference server and a JDBC database loader. The Java source code which implements this metamodel, the CORBA server, database loader and XML converter along with detailed documentation and code examples are available as part of the OpenMMS toolkit. AVAILABILITY: http://openmms.sdsc.edu CONTACT: dsg@sdsc.edu     

Microenvironments and microbial community structure in sediments

The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments. An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000. There was a 0.2 mm overlap between the O2 and H2S profiles. pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface. The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm. Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S. The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment. Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm. Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.     

Use of conserved key amino acid positions to morph protein folds

By using three-dimensional (3D) structure alignments and a previously published method to determine Conserved Key Amino Acid Positions (CKAAPs) we propose a theoretical method to design mutations that can be used to morph the protein folds. The original Paracelsus challenge, met by several groups, called for the engineering of a stable but different structure by modifying less than 50% of the amino acid residues. We have used the sequences from the Protein Data Bank (PDB) identifiers 1ROP, and 2CRO, which were previously used in the Paracelsus challenge by those groups, and suggest mutation to CKAAPs to morph the protein fold. The total number of mutations suggested is less than 40% of the starting sequence theoretically improving the challenge results. From secondary structure prediction experiments of the proposed mutant sequence structures, we observe that each of the suggested mutant protein sequences likely folds to a different, non-native potentially stable target structure. These results are an early indicator that analyses using structure alignments leading to CKAAPs of a given structure are of value in protein engineering experiments.     

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.     

CKAAPs DB: a conserved key amino acid positions database

The Conserved Key Amino Acid Positions DataBase (CKAAPs DB) provides access to an analysis of structurally similar proteins with dissimilar sequences where key residues within a common fold are identified. The derivation and significance of CKAAPs starting from pairwise structure alignments is described fully in Reddy et al. [Reddy,B.V.B., Li,W.W., Shindyalov,I.N. and Bourne,P.E. (2000) PROTEINS:, in press]. The CKAAPs identified from this theoretical analysis are provided to experimentalists and theoreticians for potential use in protein engineering and modeling. It has been suggested that CKAAPs may be crucial features for protein folding, structural stability and function. Over 170 substructures, as defined by the Combinatorial Extension (CE) database, which are found in approximately 3000 representative polypeptide chains have been analyzed and are available in the CKAAPs DB. CKAAPs DB also provides CKAAPs of the representative set of proteins derived from the CE and FSSP databases. Thus the database contains over 5000 representative poly-peptide chains, covering all known structures in the PDB. A web interface to a relational database permits fast retrieval of structure-sequence alignments, CKAAPs and associated statistics. Users may query by PDB ID, protein name, function and Enzyme Classification number. Users may also submit protein alignments of their own to obtain CKAAPs. An interface to display CKAAPs on each structure from a web browser is also being implemented. CKAAPs DB is maintained by the San Diego Supercomputer Center and accessible at the URL http://ckaaps.sdsc.edu.