Differential metabolic consequences of fumarate hydratase and respiratory chain defects

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.     

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.     

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.     

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.     

Effect of habitat area and isolation on plant trait distribution in European forests and grasslands

A number of studies show contrasting results in how plant species with specific life‐history strategies respond to fragmentation, but a general analysis on whether traits affect plant species occurrences in relation to habitat area and isolation has not been performed. We used published data from forests and grasslands in north‐central Europe to analyse if there are general patterns of sensitivity to isolation and dependency of area for species using three traits: life‐span, clonality, and seed weight. We show that a larger share of all forest species was affected by habitat isolation and area as compared to grassland species. Persistence‐related traits, life‐span and clonality, were associated to habitat area and the dispersal and recruitment related trait, seed weight, to isolation in both forest and grassland patches. Occurrence of clonal plant species decreased with habitat area, opposite to non‐clonal plant species, and long‐lived plant species decreased with grassland area. The directions of these responses partly challenge some earlier views, suggesting that further decrease in habitat area will lead to a change in plant species community composition, towards relatively fewer clonal and long‐lived plants with large seeds in small forest patches and fewer clonal plants with small seeds in small grassland patches. It is likely that this altered community has been reached in many fragmented European landscapes consisting of small and isolated natural and semi‐natural patches, where many non‐clonal and short‐lived species have already disappeared. Our study based on a large‐scale dataset reveals general and useful insights concerning area and isolation effects on plant species composition that can improve the outcome of conservation and restoration efforts of plant communities in rural landscapes.     

Tree roots as self-similar branching structures: axis differentiation and segment tapering in coarse roots of three boreal forest tree species

We applied a fractal root model to the 3D architecture of the coarse root systems of Betula pendula Roth, Picea abies (L.) H. Karst., and Pinus sylvestris L. in mixed boreal forests. Our dataset consisted of 60 root systems excavated in five different mixed forest stands. We analyzed the variability of the model parameters with respect to species, site type, and different root axes. According to our results, the cross-sectional area of root segments (i.e. second power of diameter) was a suitable variable for analyzing the values of parameters of the fractal model. The parameter values varied with generation and order of root segments; the roots thus did not follow the simple fractal branching. The variation of parameters along the root axes showed the existence of a zone of rapid tapering in all tree species. The model was, with parameter values analyzed from the data, moderately capable of accounting for the main coarse root characteristics. It was important for model predictions to take into account the tapering of root segments. We conclude that, in boreal forests, tree root systems are the output of the axis-specific morphogenetic branching rules and functional adaptation to spatial heterogeneity in the soil.     

Variation in Immune Parameters and Disease Prevalence among Lesser Black-Backed Gulls (Larus fuscus sp.) with Different Migratory Strategies

The ability to control infections is a key trait for migrants that must be balanced against other costly features of the migratory life. In this study we explored the links between migration and disease ecology by examining natural variation in parasite exposure and immunity in several populations of Lesser Black-backed Gulls (Larus fuscus) with different migratory strategies. We found higher activity of natural antibodies in long distance migrants from the nominate subspecies L.f.fuscus. Circulating levels of IgY showed large variation at the population level, while immune parameters associated with antimicrobial activity showed extensive variation at the individual level irrespective of population or migratory strategy. Pathogen prevalence showed large geographical variation. However, the seroprevalence of one of the gull-specific subtypes of avian influenza (H16) was associated to the migratory strategy, with lower prevalence among the long-distance migrants, suggesting that migration may play a role in disease dynamics of certain pathogens at the population level.