Shaping dots and lines: adding modularity into protein interaction networks using structural information

Determining protein interaction networks and generating models to simulate network changes in time and space are crucial for understanding a biological system and for predicting the effect of mutants found in diseases. In this review we discuss the great potential of using structural information together with computational tools towards reaching this goal: the prediction of new protein interactions, the estimation of affinities and kinetic rate constants between protein complexes, and finally the determination of which interactions are compatible with each other and which interactions are exclusive. The latter one will be important to reorganize large scale networks into functional modular networks.     

ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera

Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33?kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.     

In vitro fermentation of various carbohydrate-rich feed ingredients combined with chyme from pigs

Increased carbohydrate fermentation, compared with protein fermentation, could benefit gut health. In two in vitro experiments, the effect of carbohydrate-rich feed ingredients on fermentation characteristics of ileal chyme from pigs was assessed, using the cumulative gas production technique. Ingredients of the first experiment included gums, inulins, pectins, transgalacto-oligosaccharides, lactose and xylan. In the second experiment, a gum, pectin and transgalacto-oligosaccharides were added at different starting weights, to determine their effects on fermentation characteristics of chyme, in relation to differences in the carbohydrate concentrations. In comparison to fermentation of chyme alone, added carbohydrates led to higher total gas production (p < 0.05), faster maximum rate of gas production (except for xylan) (p < 0.05), and a decreased branched-chain fatty acids to straight chain fatty acids ratio (BCR) (p < 0.05). In the second experiment, for all carbohydrate ingredients, the BCR decreased with increasing starting weights (p < 0.05). If these supplemented dietary carbohydrates were to reach the terminal ileum of the living animal, carbohydrate fermentation in the large intestine could be stimulated, which is known to have beneficial effects on host health.     

Nodular fasciitis: diagnosis by fine needle aspiration biopsy

OBJECTIVE: To describe the cytomorphologic features of nodular fasciitis that differentiate it from schwannoma. STUDY DESIGN: The cytomorphologic features of 10 cases of nodular fasciitis were compared to those of 4 cases of biopsy-proven schwannoma. Aspirate smears were evaluated for cellular cohesion, cell type and stroma. Immunoperoxidase stains were utilized in select cases. RESULTS: The cases of nodular fasciitis exhibited cohesive clusters of epithelioid to spindle-shaped cells in a background of single, intact mesenchymal cells; inflammatory cells; and myxoid stroma. In contrast, schwannomas lacked single, intact cells and inflammation. Schwannoma stroma was also myxoid but appeared more finely fibrillar, and cell clusters were notable for alternating areas of hypercellularity and hypocellularity. Immunoperoxidase stains demonstrated smooth muscle actin reactivity in 5 cases of nodular fasciitis and S-100 in 2 cases of schwannoma. CONCLUSION: Nodular fasciitis can be distinguished from schwannomas on the basis of cytomorphologic features and immunocytochemical profile. Cytologic diagnosis of nodular fasciitis is important since it obviates the need for surgical excision.     

Ancestral gene fusion in cellobiose dehydrogenases reflects a specific evolution of GMC oxidoreductases in fungi

Cellobiose dehydrogenases (CDHs) are extracellular hemoflavoenzymes that are thought to be involved in the degradation of two of the most abundant biopolymers in the biosphere, cellulose and lignin. To date, these enzymes, consisting of a cytochrome domain and a flavin domain, have been detected and sequenced exclusively in the kingdom of fungi. Independent phylogenetic analyses of two distinct domains of CDH genes reveal that they evolved in parallel as fused genes. Whereas the cytochrome domains are unique sequence motifs, the flavin domains clearly belong to the glucose-methanol-choline (GMC) oxidoreductase family--an evolution line of widespread flavoproteins extending from the Archae to higher eukaryotes. The most probable unrooted phylogenetic tree obtained from our analysis of 52 selected GMC members reveals five principal evolutionary branches: cellobiose dehydrogenase, cholesterol oxidase (COX), hydroxynitrile lyase, alcohol oxidase (AOX)/glucose oxidase (GOX)/choline dehydrogenase, and a branch of dehydrogenases with various specificities containing also an Archaeon open reading frame (ORF). Cellobiose dehydrogenases cluster with cholesterol oxidases and the clade of various specificities, whereas hydroxynitrile lyases are closely related to glucose oxidases, alcohol oxidases, and choline dehydrogenases. The results indicate that the evolutionary line from a primordial GMC flavoprotein to extant cellobiose dehydrogenases was augmented after an early acquisition of the cytochrome domain to form two distinct branches for basidiomycetes and ascomycetes. One ascomycetous evolutionary line of CDHs has acquired a carbohydrate-binding module (CBM) of type 1, the sequence of which is similar to that of corresponding domains in several glycosidases. This is the first attempt towards a comprehensive phylogenetic analysis of cellobiose dehydrogenases.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

Tagging quantitative trait loci for maturity-corrected late blight resistance in tetraploid potato with PCR-based candidate gene markers

Late blight caused by the oomycete Phytophthora infestans is the economically most important and destructive disease in potato cultivation. Quantitative resistance to late blight available in tetraploid cultivars is correlated with late maturity in temperate climates, which is an undesirable characteristic. A total of 30 DNA-based markers known to be linked to loci for pathogen resistance in diploid potato were selected and tested as polymerase chain reaction-based markers for linkage with quantitative trait loci (QTL) for late blight resistance and plant maturity in two half-sib families of tetraploid potatoes. Most markers originated from within or were physically closely linked to candidate genes for quantitative resistance factors. The families were repeatedly evaluated in the field for quantitative resistance to late blight and maturity. Resistance was corrected for the maturity effect. Nine of eleven different map segments tagged by the markers harbored QTL affecting maturity-corrected resistance. Interactions were found between unlinked resistance QTL, providing testable strategies for marker-assisted selection in tetraploid potato. Based on the linkage observed between QTL for resistance and plant maturity and based on the genetic interactions observed between candidate genes tagging resistance QTL, we discuss models for the molecular basis of quantitative resistance and maturity.