Putting into practice domain-linear motif interaction predictions for exploration of protein networks

PDZ domains recognise short sequence motifs at the extreme C-termini of proteins. A model based on microarray data has been recently published for predicting the binding preferences of PDZ domains to five residue long C-terminal sequences. Here we investigated the potential of this predictor for discovering novel protein interactions that involve PDZ domains. When tested on real negative data assembled from published literature, the predictor displayed a high false positive rate (FPR). We predicted and experimentally validated interactions between four PDZ domains derived from the human proteins MAGI1 and SCRIB and 19 peptides derived from human and viral C-termini of proteins. Measured binding intensities did not correlate with prediction scores, and the high FPR of the predictor was confirmed. Results indicate that limitations of the predictor may arise from an incomplete model definition and improper training of the model. Taking into account these limitations, we identified several novel putative interactions between PDZ domains of MAGI1 and SCRIB and the C-termini of the proteins FZD4, ARHGAP6, NET1, TANC1, GLUT7, MARCH3, MAS, ABC1, DLL1, TMEM215 and CYSLTR2. These proteins are localised to the membrane or suggested to act close to it and are often involved in G protein signalling. Furthermore, we showed that, while extension of minimal interacting domains or peptides toward tandem constructs or longer peptides never suppressed their ability to interact, the measured affinities and inferred specificity patterns often changed significantly. This suggests that if protein fragments interact, the full length proteins are also likely to interact, albeit possibly with altered affinities and specificities. Therefore, predictors dealing with protein fragments are promising tools for discovering protein interaction networks but their application to predict binding preferences within networks may be limited.     

Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.     

The effect of genetic variation on the lipid response to dietary change: recent findings

PURPOSE OF REVIEW: Dyslipidaemia is an important risk factor for cardiovascular disease, and can be modified by diet. However, the lipid response to dietary change may be influenced by genetic variation. This review examines recent research (published since August 2003) on the effect of genetic variation on the lipid response to dietary change. RECENT FINDINGS: In 10 reports describing intervention studies and seven reports describing observational studies, the lipid response to diet was modified by polymorphisms within the genes for apoE, apoB, apoCIII, lipoprotein lipase, hepatic lipase, endothelial lipase, the liver fatty acid-binding protein, the beta3-adrenergic receptor, adipsin and the peroxisome proliferator-activated receptor gamma. The studies varied widely in terms of the number and type of study participants, the composition and duration of the dietary interventions, the nutrients studied and dietary assessment methods used in the observational studies, and the polymorphisms analysed--some of which had not been studied before with regard to the lipid response to diet. SUMMARY: The lipid response to dietary change is highly complex. Future studies will have to be large in order to assess the effects of multiple polymorphisms, and will have to control for many factors other than diet. At present, it is premature to recommend the use of genotyping in the design of therapeutic diets. However, such studies may be useful in identifying the mechanisms by which dietary components influence lipid levels.     

Fatty acid composition of the amphipod Dikerogammarus villosus: feeding strategies and trophic links

Fatty acid (FA) compositions were determined for the invader amphipod Dikerogammarus villosus collected from July to September 2002, in an overheated, high-conductivity dammed reservoir in north-eastern France. Predominant fatty acids were the polyunsaturated fatty acids (PUFA): eicosapentaenoic acid (EPA), linoleic acid (LA), arachidonic acid (ARA), linolenic acid (LNA) together with the monounsaturated fatty acid 18:1omega9 and the saturated fatty acid 16:0. FA markers indicated that available food was constituted of incompletely degraded phytodetritus and terrestrial inputs, as well as animal remains. PUFA contents depended on the diet and the capacity of animals to desaturate and elongate LNA and LA in long chain PUFA as EPA and ARA respectively. Based on their FA compositions, we showed that gammarids represent naturally-occurring freshwater sources of essential PUFA, and could play a fundamental role in pelagic-benthic coupling and energy recycling in the ecosystem. The complexity of the feeding strategies of D. villosus--detritivorous, omnivorous, carnivorous--makes this species efficient at exploiting different components of the available food and may be a key factor in its high invasive success.     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed.     

Observing the confinement potential of bacterial pore-forming toxin receptors inside rafts with nonblinking Eu(3+)-doped oxide nanoparticles

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and ε-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 ± 0.05 μm(2). Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 μm(2)/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 ± 0.03 pN/μm at 37°C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family.     

Infectious Chikungunya Virus in the Saliva of Mice,Monkeys and Humans

Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7^-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7^-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7^-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7^-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities.     

The mechanism of the angiotensin-converting enzyme inhibitor quinapril is not related to bradykinin level in heart tissue

In order to examine the effect of the angiotensin-converting enzyme inhibitor (ACEi) quinapril, we performed a sensitive and specific radioimmunoassay (RIA) to quantify bradykinin, BK-(1-9), in heart and kidney tissues. The BK-(1-9) level was unaffected in the heart of sham and water-deprived rats treated for 2h with quinapril (10mg/kg), but was significantly higher in the kidneys in the two groups. In these conditions, circulating and tissue angiotensin II (Ang II) levels were significantly decreased by quinapril. Moreover, our results indicated that acute treatment with this dose of quinapril induced kinin-mediated effects which were not related to its action on bradykinin degradation in rat hearts.     

Diel vertical and horizontal distribution of crustacean zooplankton and young of the year fish in a sub-alpine lake: an approach based on high frequency sampling

Understanding the spatial dynamics of predators and their preyis one of the most important goals in aquatic ecology. We studiedspatial and temporal onshore–offshore distribution patternsin young of the year (YOY) Eurasian perch (Perca fluviatilis)and crustacean zooplankton (Daphnia hyalina, Cyclops prealpinus)along a transect in Lake Annecy (France). Our study representsa first attempt at coupling hydroacoustic fish survey and highfrequency zooplankton recording to assess simultaneously thelarge-scale distribution patterns of YOY fish and their zooplanktonprey over a diel cycle (day, dusk and night sampling). We hypothesizedthat the spatial distribution of zooplankton could be shapedby both anti-predator behaviour (horizontal and vertical migrations)and predation losses. Fish biomass, size structure and dietwere assessed from split-beam echosounding and net trawlingsamples, whereas crustacean abundances were estimated with asmall modified Longhurst–Hardy continuous plankton recorder.We evaluated the diel changes in the spatial distribution patternsof fish and zooplankton and determined the overlap between theirdistributions. Fish biomass was dominated by YOY perch in upperwarmer layers and salmonids (Coregonus lavaretus and Salvelinusalpinus) in the colder and oxygenated deep layers. YOY perchwere aggregated in dense schools in the epilimnion during theday and dispersed at night. Fish biomass was distributed alonga strong increasing onshore–offshore gradient at night,whereas crustacean prey showed a decreasing gradient. This onshore–offshorenegative gradient in crustacean distribution, expressed on ashorter scale during the day, shifted toward the surface watersat night. A distinct kinetic of diel vertical migration (DVM)patterns was exhibited by daphnid and cyclopoid populationsand resulted in distinct vulnerability to perch predation. Spatio-temporaldistribution of crustaceans in Lake Annecy during the diel cyclestudy was probably shaped both by predation loss to YOY perchand by anti-predator behaviour (DVM, DHM) by zooplankton. Theimplications for fine-scale studies of fish-zooplankton interactionsare discussed.     

The microRNA pathway controls germ cell proliferation and differentiation in C. elegans

The discovery of the miRNA pathway revealed a new layer of molecular control of biological processes. To uncover new functions of this gene regulatory pathway, we undertook the characterization of the two miRNA-specific Argonaute proteins in Caenorhabditis elegans, ALG-1 and ALG-2. We first observed that the loss-of-function of alg-1 and alg-2 genes resulted in reduced progeny number. An extensive analysis of the germline of these mutants revealed a reduced mitotic region, indicating fewer proliferating germ cells. We also observed an early entry into meiosis in alg-1 and alg-2 mutant animals. We detected ALG-1 and ALG-2 protein expressions in the distal tip cell (DTC), a specialized cell located at the tip of both C. elegans gonadal arms that regulates mitosis-meiosis transition. Re-establishing the expression of alg-1 specifically in the DTC of mutant animals partially rescued the observed germline defects. Further analyses also support the implication of the miRNA pathway in gametogenesis. Interestingly, we observed that disruption of five miRNAs expressed in the DTC led to similar phenotypes. Finally, gene expression analysis of alg-1 mutant gonads suggests that the miRNA pathway is involved in the regulation of different pathways important for germline proliferation and differentiation. Collectively, our data indicate that the miRNA pathway plays a crucial role in the control of germ cell biogenesis in C. elegans.