Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches

Shearzyme (GH10 endo-1,4-β-d-xylanase) and two different α-l-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with α-l-Araf (1→2)-monosubstituted β-d-Xylp residues or α-l-Araf (1→2)- and (1→3) doubly substituted β-d-Xylp residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp, d-II: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-d-Xylp, m-I: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp and m-II: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-d-Xylp. To our knowledge, this is the first report on structural ¹H and ¹³C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly α-l-Araf (1→2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.     

Spatiotemporal and long-term variation in phytoplankton communities in the oligotrophic Lake Pyhäjärvi on the Finnish-Russian border

As part of the joint Finnish-Russian research and development project “Assessment of the ecological state of the transboundary waters”, seasonal and spatial variations in the phytoplankton communities of Lake Pyhäjärvi were studied in order to identify possible long-term (1963–2002) changes and to present recent data (since 2002) on phytoplankton biomass and species composition. Some changes in both phytoplankton biomass and species composition, particularly in the littoral zone and northern basins of the lake, were obvious at the end of the 1980s; in particular, the density of blue-greens increased during that period. Biomasses increased five-fold in 1980, decreased after 1990 and have remained low (0.14 mg l^?l) to the present time. The effective water protection measures applied, especially reducing the phosphorus load from municipal wastewaters and airborne pollution, can now be seen to have improved the water quality. Although no significant temporal differences were found in phytoplankton biomass during the intensive biweekly sampling in 2002, considerable spatial variation was seen within the lake. An obvious change in the species composition of phytoplankton and an increase in biomass were seen even in the loaded parts of the lake. Despite the spatial differences in phytoplankton biomass and community structure, the changed species composition in the northern part of the lake indicated a clear recovery from the blue-green and Chrysophyceae dominated high biomass of the 1980s.     

Documenting Freedom From Disease and Re-Establishing a Free Status After a Breakdown Rabies

Rabies reappeared in Finland in the spring of 1988 after a 29-year absence. This time rabies occurred in sylvatic form and the major species involved was the raccoon dog. During the outbreak 1988–89 66 animals were diagnosed rabid. Vaccination of cats, cattle and horses was strongly recommended and vaccination of dogs was compulsory in the outbreak area. A field trial was started on oral immunisation of raccoon dogs and foxes against rabies using baits containing rabies vaccine strain. The outbreak area and a wide buffer zone were baited three times. Finland was declared free of rabies again in 1991. Oral vaccination campaign with vaccine baits has been organised along the southeastern border once a year since the beginning of 90s. Continuous surveillance and epidemiological screening is necessary to detect any new outbreaks of rabies at an early stage.     

A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.     

Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.     

Chronic nicotine modifies the effects of morphine on extracellular striatal dopamine and ventral tegmental GABA

Previously, we have shown that 7-week oral nicotine treatment enhances morphine-induced behaviors and dopaminergic activity in the mouse brain. In this study, we further characterized the nicotine-morphine interaction in the mesolimbic and nigrostriatal dopaminergic systems, as well as in the GABAergic control of these systems. In nicotine-pretreated mice, morphine-induced dopamine release in the caudate putamen and nucleus accumbens was significantly augmented, as measured by microdialysis. Chronic nicotine treatment did not change basal extracellular concentrations of dopamine and its metabolites in the caudate putamen and nucleus accumbens, nor did it affect the rate of dopamine synthesis, as assessed by 3-hydroxybenzylhydrazine dihydrochloride-induced DOPA accumulation. GABAergic control of dopaminergic activity was studied by measuring extracellular GABA in the presence of nipecotic acid, an inhibitor of GABA uptake. Acute (0.3 mg/kg or 0.5 mg/kg i.p.) and chronic nicotine, as well as morphine (15 mg/kg s.c.) in control mice decreased nipecotic acid-induced increase in extracellular GABA in the ventral tegmental area/substantia nigra (VTA/SN). In contrast, in nicotine-treated mice, morphine increased GABA levels in the presence of nipecotic acid. We did not find any alterations in GABA(B)-receptor function after chronic nicotine treatment. Thus, our data show that chronic nicotine treatment sensitizes dopaminergic systems to morphine and affects GABAergic systems in the VTA/SN.     

Internalization of Echovirus 1 in Caveolae

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor alpha 2 beta 1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, alpha 2 beta 1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.     

Climate drivers of diatom distribution in shallow subarctic lakes

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Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) differ in their suitability as hosts for the endangered freshwater pearl mussel (Margaritifera margaritifera) in northern Fennoscandian rivers

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