A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology

Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.     

The multidrug transporters belonging to major facilitator superfamily in Mycobacterium tuberculosis

BACKGROUND: Both intrinsic and acquired multidrug resistance play an important role in the insurgence of tuberculosis. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors that block the multidrug transporter and allow traditional antibiotics to be effective. MATERIALS AND METHODS: We have undertaken the inventory of the drug transporters subfamily, included in the major facilitator superfamily (MFS), encoded by the complete genome of Mycobacterium tuberculosis (MTB). These proteins were identified on the basis of their characteristic stretches of amino acids and transmembrane segments (TMS) number. CONCLUSIONS: Genome analysis and searches of homology between the identified transporters and proteins characterized in other organisms revealed 16 open reading frames encoding putative drug efflux pumps belonging to MFS. In the case of two of them, we also have demonstrated that they function as drug efflux proteins.     

Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq

Embryonic stem cells (ESCs) can differentiate into any given cell type and therefore represent a versatile model to study the link between gene regulation and differentiation. To quantitatively assess the dynamics of enhancer activity during the early stages of murine ESC differentiation, we analyzed accessible genomic regions using STARR-seq, a massively parallel reporter assay. This resulted in a genome-wide quantitative map of active mESC enhancers, in pluripotency and during the early stages of differentiation. We find that only a minority of accessible regions is active and that such regions are enriched near promoters, characterized by specific chromatin marks, enriched for distinct sequence motifs, and modeling shows that active regions can be predicted from sequence alone. Regions that change their activity upon retinoic acid-induced differentiation are more prevalent at distal intergenic regions when compared to constitutively active enhancers. Further, analysis of differentially active enhancers verified the contribution of individual TF motifs toward activity and inducibility as well as their role in regulating endogenous genes. Notably, the activity of retinoic acid receptor alpha (RARα) occupied regions can either increase or decrease upon the addition of its ligand, retinoic acid, with the direction of the change correlating with spacing and orientation of the RARα consensus motif and the co-occurrence of additional sequence motifs. Together, our genome-wide enhancer activity map elucidates features associated with enhancer activity levels, identifies regulatory regions disregarded by computational prediction tools, and provides a resource for future studies into regulatory elements in mESCs.     

Relationships between Levels of Serum IgE,Cell-Bound IgE,and IgE-Receptors on Peripheral Blood Cells in a Pediatric Population

Background

Elevated serum immunoglobulin (Ig) E is a diagnostic marker of immediate-type allergic reactions. We hypothesize that serum IgE does not necessarily reflect total body IgE because in vivo IgE can be bound to cell surface receptors such as FcεRI and FcεRII (CD23). The aim of this study was to analyze the relationships between levels of serum IgE, cell-bound IgE, and IgE-receptors on peripheral blood cells in a pediatric population.

Methodology

Whole blood samples from 48 children (26 boys, 22 girls, mean age 10,3±5,4 years) were analyzed by flow cytometry for FcεRI, CD23, and cell-bound IgE on dendritic cells (CD11c+MHC class II+), monocytes (CD14+), basophils (CD123+MHC class II-) and neutrophils (myeloperoxidase+). Total serum IgE was measured by ELISA and converted into z-units to account for age-dependent normal ranges. Correlations were calculated using Spearman rank correlation test.

Principal Findings

Dendritic cells, monocytes, basophils, and neutrophils expressed the high affinity IgE-receptor FcεRI. Dendritic cells and monocytes also expressed the low affinity receptor CD23. The majority of IgE-receptor positive cells carried IgE on their surface. Expression of both IgE receptors was tightly correlated with cell-bound IgE. In general, cell-bound IgE on FcεRI+ cells correlated well with serum IgE. However, some patients carried high amounts of cell-bound IgE despite low total serum IgE levels.

Conclusion/Significance

In pediatric patients, levels of age-adjusted serum IgE, cell-bound IgE, and FcεRI correlate. Even in the absence of elevated levels of serum IgE, cell-bound IgE can be detected on peripheral blood cells in a subgroup of patients.     

The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability

The bovine seminal plasma protein PDC-109 exerts an essential influence on the sperm cell plasma membrane during capacitation. However, by any mechanism, it has to be ensured that this function of the protein on sperm cells is not initiated too early, that is, upon ejaculation when PDC-109 and sperm cells come into first contact, but rather at later stages of sperm genesis in the female genital tract. To answer the question of whether changes of the bovine sperm lipid composition can modulate the effect of PDC-109 on sperm membranes, we have investigated the influence of PDC-109 on the integrity of (i) differently composed lipid vesicles and of (ii) membranes from human red blood cells and bovine spermatozoa. PDC-109 most effectively disturbed lipid membranes composed of choline-containing phospholipids and in the absence of cholesterol. The impact of the protein on lipid vesicles was attenuated in the presence of cholesterol or of noncholine-containing phospholipids, such as phosphatidylethanolamine or phosphatidylserine. An extraction of cholesterol from lipid or biological membranes using methyl-beta-cyclodextrin caused an increased membrane perturbation by PDC-109. Our results argue for a oppositional effect of PDC-109 during sperm cell genesis. We hypothesize that the lipid composition of ejaculated bull sperm cells allows a binding of PDC-109 without leading to an impairment of the plasma membrane. At later stages of sperm cell genesis upon release of cholesterol from sperm membranes, PDC-109 triggers a destabilization of the cells.     

Cross-linked long-pitch actin dimer forms stoichiometric complexes with gelsolin segment 1 and/or deoxyribonuclease I that nonproductively interact with myosin subfragment 1

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.     

A novel,sequencing-free strategy for the functional characterization of Taenia solium proteomic fingerprint

The flatworm Taenia solium causes human and pig cysticercosis. When cysticerci are established in the human central nervous system, they cause neurocysticercosis, a potentially fatal disease. Neurocysticercosis is a persisting public health problem in rural regions of Mexico and other developing countries of Latin America, Asia, and Africa, where the infection is endemic. The great variability observed in the phenotypic and genotypic traits of cysticerci result in a great heterogeneity in the patterns of molecules secreted by them within their host.This work is aimed to identify and characterize cysticercal secretion proteins of T. solium cysticerci obtained from 5 naturally infected pigs from Guerrero, Mexico, using 2D-PAGE proteomic analysis. The isoelectric point (IP) and molecular weight (MW) of the spots were identified using the software ImageMaster 2D Platinum v.7.0. Since most secreted proteins are impossible to identify by mass spectrometry (MS) due to their low concentration in the sample, a novel strategy to predict their sequence was applied. In total, 108 conserved and 186 differential proteins were identified in five cysticercus cultures. Interestingly, we predicted the sequence of 14 proteins that were common in four out of five cysticercus cultures, which could be used to design vaccines or diagnostic methods for neurocysticercosis. A functional characterization of all sequences was performed using the algorithms SecretomeP, SignalP, and BlastKOALA. We found a possible link between signal transduction pathways in parasite cells and human cancer due to deregulation in signal transduction pathways. Bioinformatics analysis also demonstrated that the parasite release proteins by an exosome-like mechanism, which could be of biological interest.