Trio Haploinsufficiency Causes Neurodevelopmental Disease-Associated Deficits

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Impact of seasonal variations and nutrient inputs on nitrogen cycling and degradation of hexadecane by replicated river biofilms

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both alkB and nirK were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for nirK from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.     

The Protein Data Bank: unifying the archive

The Protein Data Bank (PDB; http://www.pdb.org/) is the single worldwide archive of structural data of biological macromolecules. This paper describes the progress that has been made in validating all data in the PDB archive and in releasing a uniform archive for the community. We have now produced a collection of mmCIF data files for the PDB archive (ftp://beta.rcsb.org/pub/pdb/uniformity/data/mmCIF/). A utility application that converts the mmCIF data files to the PDB format (called CIFTr) has also been released to provide support for existing software.     

Molecular vehicles for targeted drug delivery

Targeted drug delivery by cell-specific cytokines and antibodies promises greater drug efficacy and reduced side effects. We describe a novel strategy for assembly of drug delivery vehicles that does not require chemical modification of targeting proteins. The strategy relies on a noncovalent binding of standardized "payload" modules to targeting proteins expressed with a "docking" tag. The payload modules are constructed by linking drug carriers to an adapter protein capable of binding to a docking tag. Using fragments of bovine ribonuclease A as an adapter protein and a docking tag, we have constructed vascular endothelial growth factor (VEGF) based vehicles for gene delivery and for liposome delivery. Assembled vehicles displayed remarkable selectivity in drug delivery to cells overexpressing VEGF receptors. We expect that our strategy can be employed for targeted delivery of many therapeutic or imaging agents by different recombinant targeting proteins.     

Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.     

Peroxynitrite mediates TNF-alpha-induced endothelial barrier dysfunction and nitration of actin

We tested the hypothesis that tumor necrosis factor (TNF)-alpha induces a peroxynitrite (ONOO(-))-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated beta-actin (NO(2)-beta-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. beta-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted beta-actin was quantified in terms of its nitrotyrosine/beta-actin ratio with anti-nitrotyrosine and anti-beta-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO(2)-beta-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with beta-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/beta-actin ratio at 0.5 h with minimal association of the NO(2)-beta-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/beta-actin ratio and permeability were prevented by the anti-ONOO(-) agent Urate. The data indicate that TNF induces an ONOO(-)-dependent barrier dysfunction, which is associated with the generation of NO(2)-beta-actin.     

Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.