Kin Recognition in the largely Solitary Bee,Manuelia postica (Apidae: Xylocopinae)

The recognition of conspecifics is a central issue to social behaviour. In eusocial hymenopterans, kin recognition has been clearly demonstrated. Manuelia postica is a largely solitary bee species in which larvae develop inside individual cells within a nest and remain isolated from conspecifics until the destruction of partitions by adults. Nestmate recognition in M. postica has been previously demonstrated under experimental conditions. Isolation between individuals during development and nestmate recognition ability in adult females make M. postica an ideal species for testing the occurrence of kin recognition capacity in females. Kin recognition was demonstrated through cross‐fostering field experiments involving the single transfer of recently enclosed larvae, and subsequent laboratory recognition bioassays with emerging females. Results suggest kin recognition occurs through self‐referent phenotype matching. Given the basal position of Manuelia in the phylogeny of the Apidae, kin recognition may represent an ancestral recognition mechanism in Apidae species phylogenetically more derived than M. postica.     

Physiological approach to explain the ecological success of ‘superclones’ in aphids: Interplay between detoxification enzymes, metabolism and fitness

‘Superclones’ are predominant and time-persistent genotypes, exhibiting constant fitness across different environments. However, causes of this ecological success are still unknown. Therefore, we studied the physiological mechanisms that could explain this success, evaluating the effects of wheat chemical defences on detoxification enzymes [cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST), esterases (EST)], standard metabolic rate (SMR), and fitness-related traits [adult body mass and intrinsic rate of increase (r_m)] of two ‘superclones’ (Sa1 and Sa2) of the grain aphid, Sitobion avenae. Additionally, we compared ‘superclones’ with a less-frequent genotype (Sa46). Genotypes were reared on three wheat cultivars with different levels of hydroxamic acids (Hx; wheat chemical defences). Detoxification enzymes and SMR did not differ between wheat hosts. However, GST and EST were different between ‘superclones’ and Sa46, while Sa1 showed a higher SMR than Sa2 or Sa46 (p = 0.03). Differences between genotypes were found for r_m, which was higher for Sa1 than for Sa2 or Sa46. For all cases, genotype-host interactions were non-significant, except for aphid body mass. In conclusion, ‘superclones’ exhibit a broad host range, flat energetic costs for non-induced detoxification enzymes, and low variation in their reproductive performance on different defended hosts. However, physiological specialization of ‘superclones’ that could explain their ecological success was not evident in this study.     

Coilin phosphorylation mediates interaction with SMN and SmB′

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.     

Evaluation of uridine metabolism in human and animal spermatozoa

The objective of this study was to elucidate the role of uridine for spermatozoa, since this pyrimidine nucleoside was found in millimolar concentration in human seminal plasma. Here, the degradative activity of uridine-phosphorylase [EC 2.4.2.3] and the salvage activity of uridine kinase [EC 2.7.1.48] were detected in human spermatozoa. HPLC analysis depicted the uptake of exogeneous 14C-labelled adenine, but not of uridine and of hypoxanthine, into nucleotide pools of boar spermatozoa. On addition of uridine, the computer-assisted semen analysis (CASA) of human cells revealed a reduction of the percentage of motile spermatozoa in contrast to an elevation of some velocity parameters. It is concluded that exogeneous uridine could function as suppressor for early capacitation and as a substrate for phosphorolysis, if ribose is needed, rather than to satisfy a demand for intracellular pyrimidine nucleotides.     

Glycolipids from a colloid chemical point of view

Glycolipids are a group of compounds with a broad range of applications. Two types of glycolipids (alkylpolyglycosides and gangliosides) were examined with regard to their physicochemical properties. Despite their structural differences, they have in common that they are amphiphilic molecules and able to aggregate to form monolayers, bilayers, micelles, lyothropic mesophases or vesicles. The structures of glycolipid micelles were investigated by different experimental techniques in addition to molecular dynamic simulations. The knowledge of the physicochemical properties of gangliosides enables a better understanding of their biological functions. Structural features were obtained for the monosialogangliosides GM1, GM2 and GT1b from bovine brain by means of mass spectrometry. Further the aggregation behaviour was determined by small-angle neutron and dynamic light scattering experiments. Interaction studies of these compounds were carried out by means of surface plasmon resonance using gangliosides incorporated liposomes. They were used as model membranes that interact with the lectins WGA, RCA and HPA. The interaction of lectins immobilized to a modified silicon surface was investigated by in-situ ellipsometry.     

Early exposure to a nonhygienic environment alters pulmonary immunity and allergic responses

The hygiene hypothesis suggests that early life exposure to a nonhygienic environment that contains endotoxin reduces the risk of developing allergic diseases. The mechanisms underlying the hygiene hypothesis are unclear and may involve subtle immune system interactions that occur during maturation. Experimental objectives of this study were to use a novel animal model to test the hygiene hypothesis and to characterize early life immune system responses to a nonhygienic environment. Mice were reared in corn dust, a grain-processing byproduct with a high-endotoxin content and microbial products or in a low-endotoxin environment. The influence of early or later life exposure to corn dust on a subsequent allergen stimulus (ovalbumin) was assessed by bronchoalveolar lavage (BAL) cell analysis, lung histology, serum IgE, and BAL cytokine measurements. The influence of the corn dust environment on the developing pulmonary immune system was assessed by BAL cell analysis and immunostaining of lung tissue. The corn dust environment contained significantly more endotoxin (P < 0.001), and the dust exposures attenuated the cellular inflammatory response to ovalbumin in the adult mouse (P < 0.01) but did not reduce serum IgE levels or alter baseline BAL fluid proinflammatory cytokine levels. The corn dust environment did not induce significant neutrophilia in lavage fluid but significantly increased the number of antigen-presenting cells in alveolar walls early in life by approximately 37%. In conclusion, exposure to a nonhygienic environment did not induce significant airway neutrophilia, yet altered the population of immunologically active cells in the lung and reduced subsequent allergic inflammation.     

A generic building block for C- and N-terminal protein-labeling and protein-immobilization

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.     

Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K(d) of the ligand-sorbate complex is approximately 1 x 10(-6) and 0.4 x 10(-5)M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.     

Whole-genome expression profiling defines the HrpL regulon of Pseudomonas syringae pv. tomato DC3000, allows de novo reconstruction of the Hrp cis clement, and identifies novel coregulated genes

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.