A computational investigation of kinetoplastid trans-splicing

Trans-splicing is an unusual process in which two separate RNA strands are spliced together to yield a mature mRNA. We present a novel computational approach which has an overall accuracy of 82% and can predict 92% of known trans-splicing sites. We have applied our method to chromosomes 1 and 3 of Leishmania major, with high-confidence predictions for 85% and 88% of annotated genes respectively. We suggest some extensions of our method to other systems.     

Using co-occurrence network structure to extract synonymous gene and protein names from MEDLINE abstracts

Background  

Text-mining can assist biomedical researchers in reducing information overload by extracting useful knowledge from large collections of text. We developed a novel text-mining method based on analyzing the network structure created by symbol co-occurrences as a way to extend the capabilities of knowledge extraction. The method was applied to the task of automatic gene and protein name synonym extraction.     

A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics

Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.     

Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain

ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.     

Influence of interleukin-2 and interferon-gamma in murine schistosomiasis

Schistosoma mansoni-infected mice were administered at the time of parasite residency in the lung with recombinant murine interleukin (IL)-2 or interferon-gamma (IFN-gamma), to evaluate the impact of cytokines in host responses to primary schistosomiasis. S. mansoni lung-stage schistosomula did not affect plasma lipids levels in BALB/c, while elicited significant (p<0.05) increase in free fatty acids (FA) and decrease in cholesterol plasma levels in C57BL/6 and CD1 mice, and stimulated expression of mRNA for Th2 cytokines in BALB/c and Th1 cytokines in C57BL/6 and CD1 mice. Production of specific antibodies was negligible in the 3 strains. Interleukin-2 treatment elicited significant (p<0.001) decrease in triglycerides (TG) in CD1, and decrease in TG and cholesterol plasma levels and down-regulation of TNF-alpha mRNA expression in C57BL/6 mice. Induction of type 2 cytokines and/or IFN-gamma mRNA expression did not lead to increase in percentage of specific antibody responders in any mouse strain. Exogenous IL-2-related reduction in cholesterol plasma levels and TNF-alpha mRNA expression in C57BL/6 mice was associated with significant (p<0.05) decrease in adult worm recovery and egg count. Treatment with IFN-gamma elicited significant (p<0.05) free FA plasma levels increase in BALB/c and C57BL/6 and decrease in CD1 mice. Expression of type 2 cytokines mRNA was stimulated in BALB/c and CD1 mice, yet was not accompanied with increase in humoral responses. Exogenous IFN-gamma-related reduction in free FA plasma levels and IFN-gamma mRNA response, and up-regulation of TNF-alpha mRNA expression in CD1 mice were associated with significant increase in adult worm burden and egg load. The data were discussed in an attempt to define host factors predictive of resistance to schistosome infection.     

Ubiquitin crosstalk connecting cellular processes

Cullin 4 (Cul4), a member of the evolutionally conserved cullin protein family, serves as a scaffold to assemble multisubunit ubiquitin E3 ligase complexes. Cul4 interacts with the Ring finger-containing protein ROC1 through its C-terminal cullin domain and with substrate recruiting subunit(s) through its N-terminus. Previous studies have demonstrated that Cul4 E3 ligase ubiquitylates key regulators in cell cycle control and mediates their degradation through the proteasomal pathway, thus contributing to genome stability. Recent studies from several groups have revealed that Cul4 E3 ligase can target histones for ubiquitylation, and importantly, ubiquitylation of histones may facilitate the cellular response to DNA damage. Therefore, histone ubiquitylation by Cul4 E3 ligase constitutes a novel mechanism through which Cul4 regulates chromatin function and maintains genomic integrity. We outline these studies and suggest that histone ubiquitylation might play important roles in Cul4-regualted chromatin function including the cellular response to DNA damage and heterochromatin gene silencing.     

Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells

Background

Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.

Results

We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

Conclusion

The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells.     

Sensitivity of methods for estimating breeding values using genetic markers to the number of QTL and distribution of QTL variance

The objective of this simulation study was to compare the effect of the number of QTL and distribution of QTL variance on the accuracy of breeding values estimated with genomewide markers (MEBV). Three distinct methods were used to calculate MEBV: a Bayesian Method (BM), Least Angle Regression (LARS) and Partial Least Square Regression (PLSR). The accuracy of MEBV calculated with BM and LARS decreased when the number of simulated QTL increased. The accuracy decreased more when QTL had different variance values than when all QTL had an equal variance. The accuracy of MEBV calculated with PLSR was affected neither by the number of QTL nor by the distribution of QTL variance. Additional simulations and analyses showed that these conclusions were not affected by the number of individuals in the training population, by the number of markers and by the heritability of the trait. Results of this study show that the effect of the number of QTL and distribution of QTL variance on the accuracy of MEBV depends on the method that is used to calculate MEBV.     

Nucleotide-binding oligomerization domain-like receptors: intracellular pattern recognition molecules for pathogen detection and host defense

The nucleotide binding oligomerization domain-like receptor (NLR) family of pattern recognition molecules is involved in a diverse array of processes required for host immune responses against invading pathogens. Unlike TLRs that mediate extracellular recognition of microbes, several NLRs sense pathogens in the cytosol and upon activation induce host defense signaling pathways. Although TLRs and NLRs differ in their mode of pathogen recognition and function, they share similar domains for microbial sensing and cooperate to elicit immune responses against the pathogen. Genetic variation in several NLR genes is associated with the development of inflammatory disorders or increased susceptibility to microbial infection. Further understanding of NLRs should provide critical insight into the mechanisms of host defense and the pathogenesis of inflammatory diseases.     

First record of Renibacterium salmoninarum in the sea lamprey (Petromyzon marinus)

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a widespread problem with major implications for salmonid fish species. The mechanisms by which the bacterium has reached high levels of infection previously unrecorded in the Laurentian Great Lakes are presently unknown. Research involving reservoirs and mechanisms of R. salmoninarum transmission in fish is lacking because of the ecologic complexity of heterogeneous habitats and the lack of adequate funding. Herein, we report on the isolation of R. salmoninarum from the kidneys of the sea lamprey (Petromyzon marinus). The bacterium was cultured from kidneys of 16% and 4% of lampreys collected from two locations within the Lake Ontario watershed in 2003 and 2004, respectively. The identity of bacterial colonies was verified with the nested polymerase chain reaction and quantitative enzyme-linked immunosorbent assay.