N-glycoproteomics - an automated workflow approach

Glycan decorations dictate protein functions and thus have crucialimportance in life sciences. Previously glycoprotein analysiswas mainly focused on the analysis of the liberated glycansallowing detailed structural, but lacking positional information.Analysis of intact glycopeptides required purified glycoproteinsand manual interpretation of spectra. We developed an approachwhere mixtures of native glycopeptides were analyzed with tandemmass spectrometry and the spectra were analyzed with automatedin silico workflows. The latter included combination of theoriginal spectra, generation of a human N-glycopeptide library,matching the glycopeptide spectra to the theoretical peptidefragments, scoring the observations, predicting the glycan composition,which were then matched against the observed spectra, statisticalvalidation of the results with target–decoy filtering,and finally the calculation of glycan structures. We verifiedthis approach with the 150 serotransferrin glycopeptide spectra,where we automatically generated 10⁵ putative interpretationsfrom >10⁹ theoretical glycopeptides. After scoring 62 glycopeptidespectra obtained validated interpretation with concomitant aminoacid sequences, glycan compositions, and structures. When applyingthis method to an unknown mixture of human plasma glycoproteinswe identified 80 glycopeptides with their glycan compositionsor structures. Instead of weeks and months of interpretationwork of mass spectrometry files our automated workflow can beexecuted in few hours and provide information concomitantlyfrom both the amino acid and glycan moieties of intact glycopeptidesin mixtures. No advanced computational skills were needed touse these preformed and tested workflows. In case users wantto add complexity to the analysis they are allowed to alterall parameters and rebuild the workflows.     

Species traits are associated with the quality of bioclimatic models

Aim To analyse the effects of nine species trait variables on the accuracy of bioclimatic envelope models built for 98 butterfly species. Location Finland, northern Europe. Methods Data from a national butterfly atlas monitoring scheme (NAFI) collected from 1991–2003 with a resolution of 10 × 10 km were used in the analyses. Generalized additive models (GAMs) were constructed for 98 butterfly species to predict their occurrence as a function of climatic variables. Modelling accuracy was measured as the cross‐validation area under the curve (AUC) of the receiver–operating characteristic plot. Observed variation in modelling accuracy was related to species traits using multiple GAMs. The effects of phylogenetic relatedness among butterflies were accounted for by using generalized estimation equations. Results The values of the cross‐validation AUC for the 98 species varied between 0.56 and 1.00 with a mean of 0.79. Five species trait variables were included in the GAM that explained 71.4% of the observed variation in modelling accuracy. Four variables remained significant after accounting for phylogenetic relatedness. Species with high mobility and a long flight period were modelled less accurately than species with low mobility and a short flight period. Large species (>50 mm in wing span) were modelled more accurately than small ones. Species inhabiting mires had especially poor models, whereas the models for species inhabiting rocky outcrops, field verges and open fells were more accurate compared with other habitats. Main conclusions These results draw attention to the importance of species traits variables for species–climate impact models. Most importantly, species traits may have a strong impact on the performance of bioclimatic envelope models, and certain trait groups can be inherently difficult to model reliably. These uncertainties should be taken into account by downweighting or excluding species with such traits in studies applying bioclimatic modelling and making assessments of the impacts of climate change.     

Enzyme-assisted synthesis and structure characterization of glucuronide conjugates of eleven anabolic steroid metabolites

Enzyme-assisted in vitro synthesis of eleven glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites was performed using biphenyl-induced rat liver microsomal enzymes. The substrates within the study were the main compounds and metabolites detected in human urine after dosing of, e.g. metandienone, metenolone, methyltestosterone, nandrolone, and testosterone. Yields of glucuronidation reactions were 13-28% for most compounds, but significantly higher (77-78%) for the substrates with 4-ene-3-one double bond system of the steroid A-ring. Characterization of glucuronide-conjugated AAS structures was based on nuclear magnetic resonance spectroscopy ((1)H NMR) and on liquid chromatographic-mass spectrometric (LC-MS) and tandem mass spectrometric (LC-MS/MS) analyses in positive and negative ion mode electrospray ionization (ESI). Only minor differences were observed in optimal synthesis conditions between various substrates, which offer a potential to apply this in vitro assay as a default method for glucuronidation of new AAS substrates. The method allowed for a rapid production pathway of stereochemically pure AAS glucuronides in milligram amount, such as needed, e.g. in the development of analytical methods in forensic or pharmaceutical sciences, as well as in doping control.     

Enzyme-assisted synthesis and structure characterization of glucuronic acid conjugates of losartan, candesartan, and zolarsartan

Three angiotensin II receptor antagonists—losartan, candesartan, and zolarsartan—were investigated. All the compounds, which are structural analogues, are metabolized via conjugation to glucuronic acid. Interestingly, both O- and N-glucuronidation take place, so that regioisomers are formed. One ether O-glucuronide, two acyl O-glucuronides, and five tetrazole-N-glucuronides were biosynthesized, in milligram scale, from the three sartan aglycones. Liver microsomes from bovine, moose, rat, and pig and recombinant human UDP-glucuronosyltransferases were used as catalysts. The synthesized compounds were identified as sartan glucuronides by mass spectrometry, while the sites of glucuronidation were determined by nuclear magnetic resonance spectroscopy. Drug metabolites are needed as standards for pharmaceutical research and, as the present study shows, they can easily be produced with enzymes as catalyst.     

Apparent survival,territory turnover and site fidelity rates in Northern Goshawk Accipiter gentilis populations close to the northern range limit

Capsule: Mark–recapture data suggest low apparent survival and sex- and population-specific site fidelity and territory turnover in adult Northern Goshawks Accipiter gentilis breeding in northern Europe.

Aims: To understand how species cope with global environmental change requires knowledge of variation in population demographic rates, especially from populations close to the species’ northern range limit and from keystone species such as raptors. We analyse apparent survival and breeding dispersal propensity of adult Northern Goshawks breeding in northern Europe.

Methods: We used long-term mark–recapture data from two populations in Finland, northern Europe, and Cormack–Jolly–Seber models and binomial generalized linear models to investigate sex- and population-specific variation in apparent survival, territory turnover and site fidelity.

Results: We report low apparent survival (53–72%) of breeding adult Goshawks. Breeding dispersal propensity was higher in females than males, especially in northern Finland, contrasting with previous studies that suggest high site fidelity in both sexes.

Conclusion: Low apparent survival in females may be mainly due to permanent emigration outside the study areas, whereas in males the survival rate may truly be low. Both demographic aspects may be driven by the combination of sex-specific roles related to breeding and difficult environmental conditions prevailing in northern latitudes during the non-breeding season.     

Complementarity and other key criteria in the conservation of herb-rich forests in Finland

Complementarity of the nature reserve network in southernmost Finlandwas examined using a simple heuristic algorithm and occurrence data of 75characteristic herb-rich forest vascular plant species in 126 protected and 120non-protected sites. Three different minimum sets were selected to represent 1,5, or 10 occurrences of each species. In each minimum set there weresignificantly more protected than non-protected sites. Thus, although efficiencyis not maximal in the existing reserve network, the network does provide arepresentative basis for the conservation of herb-rich forest plants. However,some deficiencies were also noticed, particularly new reserves in floristicallydiverse herb-rich forests along watercourses would supplement the existingreserve network. On the other hand, the growing concern over the shortcomings ofreserve selection studies using presence/absence data is echoed here, becausemany of the protected forests not included in the minimum sets harbour importantnature conservation values. With regard to the five biological reserve selectioncriteria (e.g. occurrences of threatened species) considered here, theperformance of the existing reserve network is rather good. However, examinationof five reserve design criteria revealed some deficiencies, particularlyvulnerability of many reserves to potential edge effects.     

Community structure, survival and mortality factors in arctic populations of Eupontania leaf gallers

We studied survival, mortality factors, and community structure of nine species of leaf-galling sawflies, Eupontania spp., living on ten willow species (Salix spp.) at six sites on the Russian arctic tundra. The sawfly species represented two different gall types: the viminalis-type, which forms pea-shaped galls on the underside of leaf blades, and the vesicator-type, which forms bean-shaped galls on both sides of the leaf blade. Gall communities in the northernmost site had only one parasitoid species, but up to six parasitoids were found at the southernmost site. Inquiline parasitoids were encountered only in the two southern sites. Survival of the larvae varied between 20.0 and 82.8% among galler species at different sites. Parasitoids were the most important mortality factor for the sawflies. They caused mortality of 7.8-65.4%, depending on galler species and site, and it was highest in the northernmost site. Plant-specific mortality varied from 1.7 to 28.4% by galler species and it tended to decrease towards the north. Mortality from parasitoids was greater in the vesicator-type gallers than in the viminalis-type gallers. The total mortality caused by parasitoids in the arctic communities does not appear to differ from that in the diverse southern communities of Eupontania in Middle Europe, Scandinavia and North America, despite the assemblage having only a few members in the Arctic. The largest difference between the southern and the northern communities was the lack of inquiline parasitoids in the north. Our data do not support the hypothesis that abiotic, rather than biotic, factors would be more important in determining the abundance of populations of herbivorous insects in the harsh arctic environment.     

Diet variation of common buzzards in Finland supports the alternative prey hypothesis

The regional synchrony of short-term population fluctuations of small rodents and small game has usually been explained by varying impacts of generalist predators subsisting on both voles and small game (the "alternative prey hypothesis" APH). APH says that densities of predators increase as a response to increasing vole densities and then these predators shift their diet from the main prey to the alternative prey when the main prey decline and vice versa. We studied the diet composition of breeding common buzzards Buteo buteo during 1985-92 in western Finland. Microtus voles were the main prey and water voles, shrews, forest grouse, hares and small birds the most important alternative prey. Our data from the between-year variation in the diet composition of buzzards fulfilled the main predictions of APH. The yearly proportion of main prey (Microtus voles) in the diet was higher in years of high than low vole abundance. The proportion of grouse in the diet of buzzards was negatively related to the abundance of Microtus voles in the field and was nearly independent of grouse abundance in the field. In addition, buzzards mainly took grouse chicks and young hares which is consistent with the prediction of APH. Therefore, we conclude that buzzards are able to shift their diet in the way predicted by the APH and that buzzards, together with other generalist predators, may reduce the breeding success of small game in the decline phase of the vole cycle, and thus substantially contribute to the existence of short-term population cycles of small game.     

Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H NMR spectroscopy in vivo,ex vivo,and in vitro

Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1H NMR visible lipids increased. HRMAS 1H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1H and 31P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo.