Trade-off between warning signal efficacy and mating success in the wood tiger moth

The coloration of species can have multiple functions, such as predator avoidance and sexual signalling, that directly affect fitness. As selection should favour traits that positively affect fitness, the genes underlying the trait should reach fixation, thereby preventing the evolution of polymorphisms. This is particularly true for aposematic species that rely on coloration as a warning signal to advertise their unprofitability to predators. Nonetheless, there are numerous examples of aposematic species showing remarkable colour polymorphisms. We examined whether colour polymorphism in the wood tiger moth is maintained by trade-offs between different functions of coloration. In Finland, males of this species have two distinct colour morphs: white and yellow. The efficacy of the warning signal of these morphs was tested by offering them to blue tits in the laboratory. Birds hesitated significantly longer to attack yellow than white males. In a field experiment, the survival of the yellow males was also higher than white males. However, mating experiments in the laboratory revealed that yellow males had lower mating success than white males. Our results offer an explanation for the maintenance of polymorphism via trade-off between survival selection and mating success.     

Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum

We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (alpha/beta)(8)-barrel fold enzymes to recognize beta-1,4-galactosylated and alpha-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, beta-1,4-galactosylated and alpha-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (-2-1-+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites -4 and -3 of Chit42, and at the subsite -4 of Chit33. Fucose units can be accepted as a branch at the putative -3 and -4 sites of Chit33 and as a branch point at -3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.     

Bi-antennary oligo-(N-acetyllactosamino)glycans of I-type are galactosylated preferentially at the GlcNAcβ1-6Gal linked arms by α1,3-galactosyltransferase of bovine thymus

1,3-Galactosylation of radiolabelled bi-antennary acceptors Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal-R (R=1-OH, 1-4GlcNAc or 1-4Glc) with bovine thymus 1,3-galactosyltransferase was studied. At all stages of the reactions the three acceptors reacted faster at the 1 6 linked arm than at the 1 3 linked branch. Hence, in addition to the doubly 1,3-galactosylated products, practically pure Gal1-4GlcNAc1-3(Gal1-3Gal1-4GlcNAc1-6)Gal-R could be obtained from the three acceptors in reactions that had proceeded to near completion. The isomeric mono-1,3-galactosylated products were identified by using exoglycosidases to remove the branches unprotected by 1,3-galactoses and by subsequently identifying the resulting linear glycans chromatographically.Abbreviations Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Lac lactose - LacNAc Gal1-4GlcNAc - MH maltoheptaose - MP maltopentaose - MT maltotriose - MTet maltotetraose - WGA wheat germ agglutinin - 3 position 3 of the galactose unit of LacNAc or Lac - 6 position 6 of the galactose unit of LacNAc or Lac     

Targeting of active rat {alpha}2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150{Delta}-carrier: toward synthesis of sLex-decorated L-selectin ligands

Interactions between selectins and their oligosaccharide-decoratedligands play a crucial role in the initiation of leukocyte extravasation.We have shown that synthetic multivalent sialyl Lewis x glycansinhibit strongly the adhesion of lymphocytes to endotheliumat sites of inflammation. However, enzyme-assisted synthesisof these oligosaccharides is hampered by the lack of sufficientamounts of specific glycosyltransferases. We report here theconstruction of Saccharomyces cerevisiae strains expressingthe soluble catalytic ectodomain of rat Galß1–3/4GlcNAc     

Predator presence may benefit: kestrels protect curlew nests against nest predators

We studied whether the presence of breeding kestrels (Falco tinnunculus) affected nest predation and breeding habitat selection of curlews (Numenius arquata) on an open flat farmland area in western Finland. We searched for nests of curlews from an area of 6 km² during 1985–1993. For each nest found, we recorded the fate of the nest, and the distance to the nearest kestrel nest and to the nearest perch. We measured the impact of breeding kestrels on nest predation by constructing artificial curlew nests in the vicinity of ten kestrel nests in 1993. Curlew nests were closer to kestrel nests than expected from random distribution, eventhough kestrels fed on average 5.5% of curlew chick production. Predation risk by kestrels was lower than predation risk by corvids and other generalist predators, which predated 9% of curlew nests surviving farming practices and an unknown proportion of chicks. Artificial nest experiment showed that nest predation was lower close to kestrel nests than further away suggesting that the breeding association of curlews and kestrels was a behavioural adaptation against nest predation. Thus, the presence of a predator may sometimes be beneficial to prey, and prey animals have behavioural adaptations to these situations.     

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLex and sLea respectively) decorated ligands. Endothelial cells have been shown to express sLex epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLex on sialylated N-acetyllactosamine via the action of α(1,3)fucosyltransferase(s), endothelial cells can also degrade sLex to Lewis x through the action of α(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLex, which facilitates their adhesion to endothelial E- and P-selectin. This revised version was published online in November 2006 with corrections to the Cover Date.     

Breeding salmonids for feed efficiency in current fishmeal and future plant-based diet environments

The aquaculture industry is increasingly replacing fishmeal in feeds for carnivorous fish with soybean meal (SBM). This diet change presents a potential for genotype-environment (G × E) interactions. We tested whether current salmonid breeding programmes that evaluate and select within fishmeal diets also improve growth and efficiency on potential future SBM diets. A total of 1680 European whitefish from 70 families were reared with either fishmeal- or SBM-based diets in a split-family design. Individual daily gain (DG), daily feed intake (DFI) and feed efficiency (FE) were recorded. Traits displayed only weak G × E interactions as variances and heritabilities did not differ substantially between the diets, and cross-diet genetic correlations were near unity. In both diets, DFI exhibited moderate heritability and had very high genetic correlation with DG whereas FE had low heritability. Predicted genetic responses demonstrated that selection to increase DG and FE on the fishmeal diet lead to favourable responses on the SBM diet. Selection for FE based on an index including DG and DFI achieved at least double FE gain versus selection on DG alone. Therefore, current breeding programmes are improving the biological ability of salmonids to use novel plant-based diets, and aiding the aquaculture industry to reduce fishmeal use.     

Development and validation of a molecular method for the diagnosis of medically important fungal infections

The increasing incidence of severe fungal infections highlights the need for rapid and precise identification methods in clinical mycology. The aim of this study was to develop and validate a culture-indipendent molecular approach that could allow the detection of fungal pathogens in clinical samples, with particular attention to the identification of drug-resistant Candida and Aspergillus species. A real-time multiplex PCR assay was developed using TaqMan probes specific for highly discriminating ITS sequences. In its multiplex format the assay showed a high specificity, clearly discriminating among different species, as well as a high sensitivity (20 CFU/1 mL sample), making it a potentially useful starting point for the development of a more complete molecular diagnostic assay.     

Effect of Glycosylation and Additional Domains on the Thermostability of a Family 10 Xylanase Produced by Thermopolyspora flexuosa

The effects of different structural features on the thermostability of Thermopolyspora flexuosa xylanase XYN10A were investigated. A C-terminal carbohydrate binding module had only a slight effect, whereas a polyhistidine tag increased the thermostability of XYN10A xylanase. In contrast, glycosylation at Asn26, located in an exposed loop, decreased the thermostability of the xylanase. The presence of a substrate increased stability mainly at low pH.The thermophilic actinomycete Thermopolyspora flexuosa, previously named Nonomuraea flexuosa and before that Actinomadura flexuosa or Microtetraspora flexuosa (15), produces family 11 and family 10 xylanases, which show high thermostability (16, 17, 22). T. flexuosa xylanase XYN10A has a C-terminal family 13 carbohydrate binding module (CBM) (22). Many xylanases have an additional CBM, which can be a cellulose binding domain (CBD) or a xylan binding domain (XBD) (1, 5, 7, 22, 25, 28). XBD typically increases activity against insoluble xylan (1, 5, 24), although some XBDs also bind soluble xylans (21, 25).We studied the thermostability of T. flexuosa xylanase XYN10A and how CBM and other additional groups affect its thermostability. In addition to confirming the previously described importance of terminal regions, our study identified a loop that is important for the thermostability of T. flexuosa XYN10A. In general, identification of sites important for protein stability is necessary for targeted mutagenesis attempts to increase thermostability.The T. flexuosa xyn10A gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AJ508953","term_id":"57897980","term_text":"AJ508953"}}AJ508953) (22), which encodes the full-length XYN10A xylanase (1-AAST… SYNA-448) containing the catalytic domain and CBM, and a truncated gene, which encodes the catalytic domain only (1-AAST… DALN-301) were expressed in Trichoderma reesei as 3′ fusions to a sequence that encodes the Cel6A CBD (A+B) carrier polypeptide and a Kex2 cleavage site (RDKR) (27). In this article, the catalytic domain and the full-length enzyme are referred to as XYN10A and XYN10A-CBM, respectively. The catalytic domain was also produced in Escherichia coli. For production in E. coli, the sequence encoding the catalytic domain was cloned into a pKKtac vector (33) with and without an additional 3′ sequence encoding a 6×His tag at the protein C terminus (… DALNHHHHHH).The proteins were purified by hydrophobic interaction chromatography using a Phenyl Sepharose column and by ion-exchange chromatography using a DEAE Sepharose FF column (Amersham Pharmacia Biotech). The 6×His-tagged XYN10A xylanase produced in E. coli was purified by affinity chromatography using Ni-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen).Mass spectrometric (MS) analyses were performed on a high-resolution 4.7-T hybrid quadrupole-Fourier transform ion cyclotron resonance (FT-ICR) instrument (APEX-Qe; Bruker Daltonics), which employs electrospray ionization (ESI) (see supplemental material for details).Xylanase activity was measured with a 3,5-dinitrosalicylic acid assay by using 1% solubilized birchwood xylan as a substrate (33). The optimum temperature, residual activity, and half-life assays were performed as described earlier (36). SWISS-MODEL (4) was used to automatically model T. flexuosa XYN10A and XYN10A-CBM (PDB codes for the modeling templates are 1v6w and 1e0w, respectively [12, 14]).The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the masses of XYN10A xylanase and XYN10A-CBM produced in Trichoderma reesei were ∼37 kDa and ∼50 kDa, respectively (Fig. ​(Fig.1A).1A). MS analysis of the 6×His-tagged XYN10A produced in E. coli (SDS-PAGE not shown) indicated the presence of a single protein form (Fig. ​(Fig.1B),1B), with a measured mass of 34,943.25 Da. This is consistent with the theoretical mass of 6×His-tagged XYN10A (34,942.93 Da). In contrast, XYN10A produced in T. reesei was heterogeneously modified, and six protein forms (numbered 1 to 6) were detected (Fig. ​(Fig.1B).1B). The mass of form 1 (34,120.76 Da) is in excellent agreement with the calculated mass of XYN10A (34120.73 Da). The masses of forms 2 and 3, with mass increments of ∼203 and ∼162 Da, respectively, suggested protein glycosylation (+203 Da = GlcNAc; +162 Da = Man). There are two potential sites for N-glycosylation in XYN10A, Asn26 and Asn95. These six protein forms were resolved only by the high-resolution FT-ICR MS technique, not by SDS-PAGE (eluted as a single band [Fig. ​[Fig.1A1A]).Open in a separate windowFIG. 1.(A) SDS-PAGE of purified XYN10A and XYN10A-CBM produced in Trichoderma reesei. Lane 1, molecular weight markers; lane 2, catalytic domain (XYN10A); lane 3, full-length enzyme (XYN10A-CBM). (B) ESI FT-ICR mass spectra of XYN10A with a 6×His tag produced in E. coli (bottom) and XYN10A produced in T. reesei (top). Only the expanded view at m/z 1260 to 1300, with the signals representing the most abundant protein ion charge state z = 27+, is presented. For the measured and calculated masses of the protein forms identified, see the supplemental material.In order to locate the glycosylation site or sites, XYN10A proteins produced in E. coli and T. reesei were subjected to on-line pepsin digestion (see supplemental material for details). The sequence coverage for XYN10A xylanase produced in E. coli was 62%. For XYN10A produced in T. reesei, a lower sequence coverage was obtained, but three glycopeptides (residues 20 to 44, 20 to 46, and 20 to 59), carrying one GlcNAc residue, were detected (glycopeptides A to C in Fig. S1B in the supplemental material). A triply charged glycopeptide A was further analyzed by collision-induced dissociation (CID) measurement (see inset in Fig. S1B in the supplemental material). A ladder of b-type fragment ions further identified this peptide and verified Asn26 as the N-glycosylation site in XYN10A, carrying GlcNAc(Man) as a glycan core structure.The additional sequences attached to the catalytic domain affected the thermostability of XYN10A xylanase. The deletion of the native C-terminal CBM domain (XYN10A produced in T. reesei) slightly decreased (∼2°C) the apparent temperature optimum in the region of 70 to 75°C (Table ​(Table11 and Fig. ​Fig.2A).2A). However, at 80°C, the deletion of the CBM domain increased the activity (Fig. ​(Fig.2A).2A). Furthermore, the half-life in the presence of the substrate at 80°C was lower when the CBM was present (Table ​(Table22).Open in a separate windowFIG. 2.Enzyme activity and stability profiles. (A) Enzyme activity as a function of temperature. The enzymes were incubated for 30 min at each temperature at pH 7. (B) Enzyme inactivation as a function of temperature. The enzyme samples were incubated without the substrate for 30 min at each temperature (pH 7), and the residual activity was measured at 70°C. Values are means ± standard deviations (error bars) for three experiments. Symbols: ⧫, XYN10A xylanase produced in T. reesei; ⋄, XYN10A-CBM produced in T. reesei; ▪, XYN10A produced in E. coli; □, XYN10A-6×His produced in E. coli.

TABLE 1.

Peaks of the optimum temperatures (30-min assay)^a