DBParser: web-based software for shotgun proteomic data analyses

We describe a web-based program called 'DBParser' for rapidly culling, merging, and comparing sequence search engine results from multiple LC-MS/MS peptide analyses. DBParser employs the principle of parsimony to consolidate redundant protein assignments and derive the most concise set of proteins consistent with all of the assigned peptide sequences observed in an experiment or series of experiments. The resulting reports summarize peptide and protein identifications from multidimensional experiments that may contain a single data set or combine data from a group of data sets, all related to a single analytical sample. Additionally, the results of multiple experiments, each of which may contain several data sets, can be compared in reports that identify features that are common or different. DBParser actively links to the primary mass spectral data and to public online databases such as NCBI, GO, and Swiss-Prot in order to structure contextually specific reports for biologists and biochemists.     

Induction characteristics of reductive dehalogenation in the ortho-halophenol-respiring bacterium,Anaeromyxobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C dehalogenatesortho-substituted di- and mono-halogenated phenols and couples this activity to growth. Reductive dehalogenation activity has been reported to be inducible, however,this process has not been studied extensively. In this study, theinduction of reductive dehalogenation activity by strain 2CP-C is characterized. Constitutive 2-chlorophenoldechlorination activity occurs in non-induced fumarate-grown cells, with rates averaging 0.138 mol of Cl^- h^-1 mg of protein^-1. Once induced, these cultures dechlorinate 2-chlorophenol (2-CP) at rates as high as 116 mol of Cl^-1 h^-1 mg of protein^-1. Dechlorination of 2-CP is induced by phenol,2-chlorophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,6-dichlorophenol,and 2-bromophenol. Of the substrates tested, 2-bromophenol shows the highestinduction potential, yielding double the 2-chlorophenol dechlorination rate when compared to other inducing substrates. No induced dechlorination is observed at concentrations less than5 M 2-CP. When fumarate cultures were diluted 100-fold, fumarate reduction rates were reduced roughly according to the dilution factor, while dechlorination rates were similar in fumarate grown cells amended with 2-CP and cells diluted 100-fold prior to the addition of chlorophenol. This indicates that the majority of the fumarate-grown cells in late log phase were not induced when exposed to inducing substrates such as 2-CP. This observation may have ramifications on the success of bioaugmentation using halorespiring bacteria, which traditionally relies on growing culturesusing more readily utilized substrates. The rapid dechlorination rate and unique induction pattern also make strain 2CP-C a promising model organism for understanding the regulationof reductive dehalogenation at the enzymatic level.     

Bacterioplankton Dynamics in Estuarine Mesocosms: Effects of Tank Shape and Size

Mesocosms provide a powerful tool for investigating bacterial dynamics at small scales; however, even these controlled studies are not exempt from spatial influences. Differences in mesocosm shape and size may have profound effects on the enclosed community since these features may influence the behavior of the system. Studies were conducted in mesocosms of varying dimensions (narrow, deep and wide, shallow tanks) and volumes (0.1, 1, and 10 m3) in an attempt to decipher effects attributable to changes in container size and shape. Both mesocosm volume and shape affected the course of bacterial growth following containment. Bacterial abundance and production were high in both groups of 0.1 m3 tanks and in the large, wide, shallow tanks with the greatest light supply at depth. Differences in bacterial growth between differently shaped tanks (i.e., differing wall area to volume ratios) were observed among equal volume enclosures, with faster growth in the wide/shallow tanks. Light availability, phytoplankton growth, and primary production differed among tanks, and bacterial growth and production were correlated with these properties. During high nutrient conditions, mesocosm volume and shape influenced bacterial growth, possibly because of periphyton growth on walls in small tanks and elevated light levels in wide/shallow tanks. These results suggest the importance of considering container dimensions when designing and interpreting mesocosm experiments and may allow deeper understanding of the fundamental processes underlying patterns observed in the real world.     

Anaerobic Naphthalene Degradation by Microbial Pure Cultures under Nitrate-Reducing Conditions

Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added ¹⁴C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the ¹⁴C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.     

NTRK3 Is a Potential Tumor Suppressor Gene Commonly Inactivated by Epigenetic Mechanisms in Colorectal Cancer

NTRK3 is a member of the neurotrophin receptor family and regulates cell survival. It appears to be a dependence receptor, and thus has the potential to act as an oncogene or as a tumor suppressor gene. NTRK3 is a receptor for NT-3 and when bound to NT-3 it induces cell survival, but when NT-3 free, it induces apoptosis. We identified aberrantly methylated NTRK3 in colorectal cancers through a genome-wide screen for hypermethylated genes. This discovery led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. NTRK3 is methylated in 60% of colon adenomas and 67% of colon adenocarcinomas. NTRK3 methylation suppresses NTRK3 expression. Reconstitution of NTRK3 induces apoptosis in colorectal cancers, if NT-3 is absent. Furthermore, the loss of NTRK3 expression associates with neoplastic transformation in vitro and in vivo. We also found that a naturally occurring mutant NTRK3 found in human colorectal cancer inhibits the tumor suppressor activity of NTRK3. In summary, our findings suggest NTRK3 is a conditional tumor suppressor gene that is commonly inactivated in colorectal cancer by both epigenetic and genetic mechanisms whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.     

Characterization of anti-Z-DNA antibody binding sites on Z-DNA by nuclear magnetic resonance spectroscopy

Two monoclonal anti-Z-DNA antibodies, Z22 and Z44, were shown to bind to the oligonucleotides, d(CG)2 and d(CG)3, and to interact with different parts of the helix. 1H nuclear magnetic resonance spectroscopy showed that Fab fragments stabilize an ordered structure in the tetranucleotide d(CG)2. Nuclear Overhauser effects measured in the presence of Z22 Fab indicate a syn conformation of guanine residues of d(CG)2. Intermolecular transfer of saturation between the Fabs and bound d(CG)3 was detected by a saturation of the protein spectrum and observation of changes in the DNA spectrum. Antibodies with deuterated aromatic amino acids were prepared to eliminate the protein aromatic resonances and thereby allow a more detailed analysis of the transfer to the DNA base protons. The greatest transfer with Z44 was to the dC-5 protons although all of the base protons interact with this antibody. Little, if any, transfer to the DNA base protons was observed with Z22. These results are consistent with a Z44 binding site on the convex surface of the Z-helix (analogous to the major groove of B-DNA) and a Z22 binding site on the sugar-phosphate backbone.