Bayesian Markov Random Field Analysis for Protein Function Prediction Based on Network Data

Inference of protein functions is one of the most important aims of modern biology. To fully exploit the large volumes of genomic data typically produced in modern-day genomic experiments, automated computational methods for protein function prediction are urgently needed. Established methods use sequence or structure similarity to infer functions but those types of data do not suffice to determine the biological context in which proteins act. Current high-throughput biological experiments produce large amounts of data on the interactions between proteins. Such data can be used to infer interaction networks and to predict the biological process that the protein is involved in. Here, we develop a probabilistic approach for protein function prediction using network data, such as protein-protein interaction measurements. We take a Bayesian approach to an existing Markov Random Field method by performing simultaneous estimation of the model parameters and prediction of protein functions. We use an adaptive Markov Chain Monte Carlo algorithm that leads to more accurate parameter estimates and consequently to improved prediction performance compared to the standard Markov Random Fields method. We tested our method using a high quality S.cereviciae validation network with 1622 proteins against 90 Gene Ontology terms of different levels of abstraction. Compared to three other protein function prediction methods, our approach shows very good prediction performance. Our method can be directly applied to protein-protein interaction or coexpression networks, but also can be extended to use multiple data sources. We apply our method to physical protein interaction data from S. cerevisiae and provide novel predictions, using 340 Gene Ontology terms, for 1170 unannotated proteins and we evaluate the predictions using the available literature.     

A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR

In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry.     

Proteinase Activated Receptor 1 Mediated Fibrosis in a Mouse Model of Liver Injury: A Role for Bone Marrow Derived Macrophages

Liver fibrosis results from the co-ordinated actions of myofibroblasts and macrophages, a proportion of which are of bone marrow origin. The functional effect of such bone marrow-derived cells on liver fibrosis is unclear. We examine whether changing bone marrow genotype can down-regulate the liver''s fibrotic response to injury and investigate mechanisms involved. Proteinase activated receptor 1 (PAR1) is up-regulated in fibrotic liver disease in humans, and deficiency of PAR1 is associated with reduced liver fibrosis in rodent models. In this study, recipient mice received bone marrow transplantation from PAR1-deficient or wild-type donors prior to carbon tetrachloride-induced liver fibrosis. Bone marrow transplantation alone from PAR1-deficient mice was able to confer significant reductions in hepatic collagen content and activated myofibroblast expansion on wild-type recipients. This effect was associated with a decrease in hepatic scar-associated macrophages and a reduction in macrophage recruitment from the bone marrow. In vitro, PAR1 signalling on bone marrow-derived macrophages directly induced their chemotaxis but did not stimulate proliferation. These data suggest that the bone marrow can modulate the fibrotic response of the liver to recurrent injury. PAR1 signalling can contribute to this response by mechanisms that include the regulation of macrophage recruitment.     

Mitochondria Contribute to NADPH Generation in Mouse Rod Photoreceptors

NADPH is the primary source of reducing equivalents in the cytosol. Its major source is considered to be the pentose phosphate pathway, but cytosolic NADP⁺-dependent dehydrogenases using intermediates of mitochondrial pathways for substrates have been known to contribute. Photoreceptors, a nonproliferating cell type, provide a unique model for measuring the functional utilization of NADPH at the single cell level. In these cells, NADPH availability can be monitored from the reduction of the all-trans-retinal generated by light to all-trans-retinol using single cell fluorescence imaging. We have used mouse rod photoreceptors to investigate the generation of NADPH by different metabolic pathways. In the absence of extracellular metabolic substrates, NADPH generation was severely compromised. Extracellular glutamine supported NADPH generation to levels comparable to those of glucose, but pyruvate and lactate were relatively ineffective. At low extracellular substrate concentrations, partial inhibition of ATP synthesis lowered, whereas suppression of ATP consumption augmented NADPH availability. Blocking pyruvate transport into mitochondria decreased NADPH availability, and addition of glutamine restored it. Our findings demonstrate that in a nonproliferating cell type, mitochondria-linked pathways can generate substantial amounts of NADPH and do so even when the pentose phosphate pathway is operational. Competing demands for ATP and NADPH at low metabolic substrate concentrations indicate a vulnerability to nutrient shortages. By supporting substantial NADPH generation, mitochondria provide alternative metabolic pathways that may support cell function and maintain viability under transient nutrient shortages. Such pathways may play an important role in protecting against retinal degeneration.     

Beneficial effects of enhanced UV-B radiation under field conditions: improvement of needle water relations and survival capacity of Pinus pinea L. seedlings during the dry Mediterranean summer

The possible mechanism(s) by which supplemental UV-B radiation alleviates the adverse effects of summer drought in Mediterranean pines (Petropoulou et al. 1995) were investigated with seedlings of Pinus pinea. Plants received ambient or ambient plus supplemental UV-B radiation (biologically equivalent to a 15% ozone depletion over Patras, 38.3° N, 29.1° E) and natural precipitation or additional irrigation. Treatments started on 1 February, 1994 and lasted up to the end of the dry period (29 September). In well-watered plants, UV-B radiation had no influence on photosystem II photochemical efficiency and biomass accumulation. Water stressed plants suffered from needle loss and reduced photosystem II photochemical efficiency during the summer. These symptoms, however, were less pronounced in plants receiving supplemental UV-B radiation, resulting in higher total biomass at plant harvest. Laboratory tests showed that enhanced UV-B radiation did not improve the tolerance of photosystem II against drought, high light, high temperature and oxidative stress. Enhanced UV-B radiation, however, improved the water economy of water stressed plants, as judged by measurements of needle relative water content. In addition, it caused an almost two-fold increase of cuticle thickness. No such UV-B radiation effects were observed in well-watered pines. The results indicate that the combination of water stress and UV-B radiation may trigger specific responses, enabling the plants to avoid excessive water loss and, thereby, maintain a more efficient photosynthetic apparatus during the summer. The extent of this apparently positive UV-B radiation effect would depend on the amount of summer precipitation. Abbreviations: DW – dry weight, F_v/F_m – ratio of variable to maximum fluorescence, A ₃₀₀ – absorbance at 300 nm, PAR – photosynthetically active radiation, PS II – photosystem II, RWC – relative water content, TCA – trichloroacetic acid, UV-B_BE – biologically effective ultraviolet-B radiation     

Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.     

Retrocyclins kill bacilli and germinating spores of Bacillus anthracis and inactivate anthrax lethal toxin

Theta-defensins are cyclic octadecapeptides encoded by the modified alpha-defensin genes of certain nonhuman primates. The recent demonstration that human alpha-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of theta-defensins on Bacillus anthracis (Sterne). We tested rhesus theta-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of theta-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of theta-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that theta-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.     

How can a beta-sheet peptide be both a potent antimicrobial and harmfully toxic? Molecular dynamics simulations of protegrin-1 in micelles

In this work, the naturally occurring beta-hairpin antimicrobial peptide protegrin-1 (PG-1) is studied by molecular dynamics simulation in all-atom sodium dodecylsulfate and dodecylphosphocholine micelles. These simulations provide a high-resolution picture of the interactions between the peptide and simple models of bacterial and mammalian membranes. Both micelles show significant disruption, as is expected for a peptide that is both active against bacteria and toxic to host cells. There is, however, clear differentiation between the behavior in SDS versus DPC, which suggests different mechanisms of interaction for PG-1 with mammalian and bacterial membranes. Specifically, the equilibrium orientation of the peptide relative to SDS is a mirror image of its position relative to DPC. In both systems, the arginine residues of PG-1 strongly interact with the head groups of the micelles. In DPC, the peptide prefers a location closer to the core of the micelle with Phe12, Val14, and Val16 imbedded in the core and the other side of the hairpin, which includes Leu5 and Tyr7, located closer to the surface of the micelle. In SDS, the peptide prefers a location at the micelle-water interface. The peptide position is reversed, with Leu5 and Cys6 imbedded furthest in the micelle core and Phe12, Val14, and Val16 on the surface of the micelle. We discuss the implications of these results with respect to activity and toxicity.     

The photoprotective role of epidermal anthocyanins and surface pubescence in young leaves of grapevine (Vitis vinifera)

BACKGROUND AND AIMS: Depending on cultivar, surfaces of young leaves of Vitis vinifera may be glabrous-green ('Soultanina') or transiently have anthocyanins ('Siriki') or pubescence ('Athiri'). A test is made of the hypothesis that anthocyanins and pubescence act as light screens affording a photoprotective advantage to the corresponding leaves, and an assessment is made of the magnitude of their effect. METHODS: Measurements were made on young leaves of the three cultivars in spring under field conditions. Photosynthetic gas-exchange and in vivo chlorophyll fluorescence were measured. Photosynthetic and photoprotective pigments were analysed by HPLC. KEY RESULTS: Compared with glabrous-green leaves, both anthocyanic and pubescent leaves had greater dark-adapted PSII photochemical efficiency and net photosynthesis. In leaves possessing either anthocyanins or pubescence, the ratio of xanthophyll cycle components to total chlorophyll, and mid-day de-epoxidation state of the xanthophyll cycle were considerably smaller, than in glabrous-green leaves. These differences were more evident in pubescent leaves, probably indicating that trichomes were more effective in decreasing light stress than anthocyanins in the epidermis. CONCLUSIONS: Light screens, especially in the form of pubescence, decrease the risk of photoinhibition whilst allowing leaves to maintain a smaller content of xanthophyll cycle components and depend less on xanthophyll cycle energy dissipation. This combination of photoprotective features, i.e. decreased photon flux to the photosynthetic apparatus and lower xanthophyll cycle utilization rates may be particularly advantageous under stressful conditions.