Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus

The question whether proteins fused to β1,4galactosyltransferase (β4GalT-1) influence the biocatalytic properties of the glycosyltransferase has not been addressed so far. In the present study we have chosen a novel approach to express the gene encoding for human β4GalT-1 from placenta in an N-terminal fusion with the pre-propeptide of the lipase from Staphylococcus hyicus. The pre-propeptide provides a secretion signal in Escherichia coli and was reported to protect fused proteins against proteolytic degradation. Expression of the fusion protein was challenged with an almost full-length version of human β4GalT-1 including parts of the signal anchor and the stem region (propeptide-natβ4GalT-1) and the full catalytic domain (His₆propeptide-catβ4GalT-1), respectively. We demonstrate that the fusion protein in propeptide-natβ4GalT-1 is cleaved off during purification using immobilized metal ion chromatography IMAC, most probably catalyzed by the immobilized Zn²⁺ ions. Cleavage can be avoided by deletion of five C-terminal amino acids of the propeptide and the stem region yielding His₆propeptide-catβ4GalT-1. Kinetic data reveal that both enzyme constructs possess specific biocatalytic characteristics when compared to a recombinant luminal β4GalT-1 construct. The catalytic efficiency towards more hydrophobic acceptor glycosides, e.g. benzyl 2-acetamido-2-deoxy-β-d-glucopyranoside and p-nitro phenyl 2-acetamido-2-deoxy-β-d-glucopyranoside, and the N-glycans of IgG from rat is significantly increased. In summary, the His₆propeptide-catβ4GalT-1 is very useful for biocatalytic applications involving hydrophobic acceptor glycosides and the propeptide-natβ4GalT-1 is able to glycosylate glycoproteins in an efficient way.     

Forest fire history in Norway: from fire-disturbed pine forests to fire-free spruce forests

I have used occurrence of macroscopic charcoal particles, pollen analyses and radiocarbon datings to examine local forest fire abundance in southern and central Norway. Peat cores, covering the last 1000 to 6000 yr, were sampled from 20 bog margin and swamp forest sites, and the charcoal records documented local fire occurrence in 10 of the sites. Forest fires have not occurred in the sites located in central Norway, whereas the fire occurrence in southern Norway showed large variation among the sites. However, forest fires ceased prior to the establishment of Norway spruce Picea abies in seven of the sites, whereas the establishment of spruce preceded the fire decline in three of the sites. Odds ratio calculations indicated that it is several hundred times more likely that fires occurred prior to, than after, the spruce establishment. Although time spans between fire decline and spruce establishment showed some variation, they did not increase along a gradient from east to west in Norway, suggesting that the establishment of spruce might have initiated a change from fire-prone to fire-free ecosystems.     

Multiple environmental parameters impact lipid cyclization in Sulfolobus acidocaldarius

Adaptation of lipid membrane composition is an important component of archaeal homeostatic response. Historically, the number of cyclopentyl and cyclohexyl rings in the glycerol dibiphytanyl glycerol tetraether (GDGT) Archaeal lipids has been linked to variation in environmental temperature. However, recent work with GDGT-making archaea highlight the roles of other factors, such as pH or energy availability, in influencing the degree of GDGT cyclization. To better understand the role of multiple variables in a consistent experimental framework and organism, we cultivated the model Crenarchaeon Sulfolobus acidocaldarius DSM639 at different combinations of temperature, pH, oxygen flux, or agitation speed. We quantified responses in growth rate, biomass yield, and core lipid compositions, specifically the degree of core GDGT cyclization. The degree of GDGT cyclization correlated with growth rate under most conditions. The results suggest the degree of cyclization in archaeal lipids records a universal response to energy availability at the cellular level, both in thermoacidophiles, and in other recent findings in the mesoneutrophilic Thaumarchaea. Although we isolated the effects of key individual parameters, there remains a need for multi-factor experiments (e.g., pH + temperature + redox) in order to more robustly establish a framework to better understand homeostatic membrane responses.     

Hibernation adaptation and eclosion synchrony in leaf-galling sawflies in subarctic Norway

The winter dormancy adaptation in gall-inducing sawflies is poorly known. Diapause termination and the following post-diapause quiescence enhance synchronous eclosion in spring. This is probably the most critical part in the life history in gall-inducing sawflies, as there is only a short phenological window of opportunity for mating and oviposition. In a 2?years’ study, diapause duration, termination, survival and eclosion synchrony were experimentally investigated for three gall-inducing sawfly species (Symphyta: Tenthredinidae: Pontania nivalis, P. glabrifrons and P. arcticornis). Field-collected galls, sampled from willows (Salix spp.) in early autumn, were kept under natural temperatures outdoors until next spring. Subsamples were successively transferred to the laboratory at 14-day intervals from October to April in a test of development time to eclosion and survival ratio. The time to eclosion decreased throughout the experiment, whereas the proportion successfully eclosed and eclosion synchronicity increased, all indicative of prepupae entering a diapause in early autumn. The diapause terminates midwinter, and the prepupae enter a post-diapause quiescence until the temperature in spring allows a direct development and contributes to a nearly synchronous eclosion. In all three species, males eclosed 1–2?days prior to females (protandry). We hypothesize that synchronous eclosion as well as protandry enhance mating and oviposition success. Our finding indicates that gall-inducing sawflies are well adapted to its harsh subarctic and arctic environment.     

Dendrochronological dating of four chests: A surprising result

Four grain chests, which cultural historians presumed were medieval, have been dendrochronologically dated by measuring the tree-ring widths on photographs. Only two chests proved to be clearly medieval. It is now planned to use dendrochronology to date 12 more grain chests that are known to be in private homes and museums.     

Immunoaffinity partitioning: synthesis and use of polyethylene glycol-oxirane for coupling to bovine serum albumin and monoclonal antibodies

Polyethylene glycol (PEG)-oxirane was synthesized by reacting aminated monomethoxy-PEG 5000 (NH2-MPEG 5000) with butanediol diglycidyl ether and used to derivatize bovine serum albumin (BSA) and monoclonal antibodies (mAb) against horseradish peroxidase (HRP) and porcine lactate dehydrogenase isoenzyme 5, respectively. Determination of oxirane end groups revealed a very high number, which arise from the chain breaks of the polymer. Covalent coupling of PEG-oxirane to BSA resulted in 30-50 times higher partition coefficients under optimized conditions. The mAb investigated could be modified with PEG-oxirane while retaining its binding properties and could be used as an affinity ligand for selective extraction of Ag in immunoaffinity partitioning. However, a high degree of modification results in a lower binding constant of mAb anti-HRP and higher [mAb]/[Ag] concentration ratios in immunoaffinity partition experiments.