Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose CI 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.     

Offspring phenotype is shaped by the nonsperm fraction of semen

In a large majority of animal species, the only contribution of males to the next generation has been assumed to be their genes (sperm). However, along with sperm, seminal plasma contains a wide array of extracellular factors that have many important functions in reproduction. Yet, the potential intergenerational effects of these factors are virtually unknown. We investigated these effects in European whitefish (Coregonus lavaretus) by experimentally manipulating the presence and identity of seminal plasma and by fertilizing the eggs of multiple females with the manipulated and unmanipulated semen of several males in a full‐factorial breeding design. The presence of both own seminal plasma and foreign seminal plasma inhibited sperm motility, and the removal of own seminal plasma decreased embryo survival. Embryos hatched significantly earlier after both semen manipulations than in control fertilizations; foreign seminal plasma also increased offspring aerobic swimming performance. Given that our experimental design allowed us to control potentially confounding sperm‐mediated (sire) effects and maternal effects, our results indicate that seminal plasma may have direct intergenerational consequences for offspring phenotype and performance. This novel source of offspring phenotypic variance may provide new insights into the evolution of polyandry and mechanisms that maintain heritable variation in fitness and associated female mating preferences.     

Lakes as nitrous oxide sources in the boreal landscape

Estimates of regional and global freshwater N₂O emissions have remained inaccurate due to scarce data and complexity of the multiple processes driving N₂O fluxes the focus predominantly being on summer time measurements from emission hot spots, agricultural streams. Here, we present four‐season data of N₂O concentrations in the water columns of randomly selected boreal lakes covering a large variation in latitude, lake type, area, depth, water chemistry, and land use cover. Nitrate was the key driver for N₂O dynamics, explaining as much as 78% of the variation of the seasonal mean N₂O concentrations across all lakes. Nitrate concentrations varied among seasons being highest in winter and lowest in summer. Of the surface water samples, 71% were oversaturated with N₂O relative to the atmosphere. Largest oversaturation was measured in winter and lowest in summer stressing the importance to include full year N₂O measurements in annual emission estimates. Including winter data resulted in fourfold annual N₂O emission estimates compared to summer only measurements. Nutrient‐rich calcareous and large humic lakes had the highest annual N₂O emissions. Our emission estimates for Finnish and boreal lakes are 0.6 and 29 Gg N₂O‐N/year, respectively. The global warming potential of N₂O from lakes cannot be neglected in the boreal landscape, being 35% of that of diffusive CH₄ emission in Finnish lakes.     

The importance of hunting pressure, habitat preference and life history for population trends of breeding waterbirds in Finland

Populations of migratory species have undergone dramatic changes in recent decades, but little is known about the factors actually driving those changes. Of particular concern are quarry species such as migratory ducks (Anatidae), many of which have an unfavourable conservation status in Europe. By including both quarry and non-quarry species, as well as habitat preference and life history characteristics of the species, we investigated the relative importance of hunting pressure, both in Finland and at the European level, in explaining population changes of 16 species of migratory waterbirds in Finland during 1986–2011. Ban of lead shot in 1996 resulted in considerably lower annual hunting bags in Finland thereafter. Species which had the highest hunting pressure had the most negative slopes in population trends from 1986 up to 1997, suggesting that hunting probably limited those populations. However, in general population trends of the species were not strongly associated with hunting pressure in Finland or in Europe. Nor were basic life history characteristics (body mass and clutch size) associated with population trends of the species. In contrast, recent population declines were associated with habitat preferences of the species: those breeding mainly in eutrophic lakes had more negative population trends than those breeding in oligotrophic lakes or generalist species. Reasons for the relatively poor status of species preferring eutrophic lakes probably include over-eutrophication of nutrient-rich lakes, resulting in less abundant food resources, and increased nest depredation.     

Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of 'host range' mutants of E. coli K1 bacteriophages.     

The effect of land use change on the vascular plant species turnover in boreal lakes

The effects of different land use types on nutrient and chemical run-off have been widely researched, but the total effect of change in land use on the aquatic species community is not well-known. For this study we researched the effect of land use change on the vascular plant communities in lakes. The study was conducted with aerial photographs and GIS-techniques in the countryside of central Finland. We compared the change in vascular plant species community structure with the change in land use around 25 lakes, between the years 1933–1934 and 1996. The change in land use was analyzed in two different riparian zones. We found several indications of links between changes in land use and aquatic vascular plant species. According to our data a decrease in agricultural land use, in particular fields and meadows, appears to influence species turnover and increases the number of new vascular plant species. When the changes in fields and meadows were added together and compared to the change in species turnover, the correlation was even more evident. Changes in land use types in a zone 0–100 m from the lake shoreline correlated with a change in vascular plant species more often than changes in a larger, adjacent zone, 100–400 m from the shore. This indicates that changes in the few hundred meters nearest the lake have the most elemental consequences for aquatic vascular plant species, giving an indication of the breadth of the protection zones needed between freshwaters and agricultural lands.     

Insulin‐ and Exercise‐Stimulated Skeletal Muscle Blood Flow and Glucose Uptake in Obese Men

Objective: Insulin resistance in obese subjects results in the impaired use of glucose by insulin‐sensitive tissues, e.g., skeletal muscle. In the present study, we determined whether insulin resistance in obesity is associated with an impaired ability of exercise to stimulate muscle blood flow, oxygen delivery, or glucose uptake. Research Methods and Procedures: Nine obese (body mass index = 36 ± 2 kg/m²) and 11 age‐matched nonobese men (body mass index = 22 ± 1 kg/m²) performed one‐legged isometric exercise during hyperinsulinemia. Rates of femoral muscle blood flow, oxygen consumption, and glucose uptake were measured simultaneously in both legs using [¹⁵O]H₂O, [¹⁵O]O₂, [¹⁸F]fluoro‐deoxy‐glucose, and positron emission tomography. Results: The obese subjects exhibited resistance to insulin stimulation of glucose uptake in resting muscle, regardless of whether glucose uptake was expressed per kilogram of femoral muscle mass (p = 0.001) or per the total mass of quadriceps femoris muscle. At similar workloads, oxygen consumption, blood flow, and glucose uptake were lower in the obese than the nonobese subjects when expressed per kilogram of muscle, but similar when expressed per quadriceps femoris muscle mass. Discussion: We conclude that obesity is characterized by insulin resistance of glucose uptake in resting skeletal muscle regardless of how glucose uptake is expressed. When compared with nonobese individuals at similar absolute workloads and under identical hyperinsulinemic conditions, the ability of exercise to increase muscle oxygen uptake, blood flow, and glucose uptake per muscle mass is blunted in obese insulin‐resistant subjects. However, these defects are compensated for by an increase in muscle mass.     

Proliferation, oxidative stress and cell death in cells exposed to 872 MHz radiofrequency radiation and oxidants

Human SH-SY5Y neuroblastoma and mouse L929 fibroblast cells were exposed to 872 MHz radiofrequency (RF) radiation using continuous waves (CW) or a modulated signal similar to that emitted by GSM mobile phones at a specific absorption rate (SAR) of 5 W/kg in isothermal conditions. To investigate possible combined effects with other agents, menadione was used to induce reactive oxygen species, and tert-butylhydroperoxide (t-BOOH) was used to induce lipid peroxidation. After 1 or 24 h of exposure, reduced cellular glutathione levels, lipid peroxidation, proliferation, caspase 3 activity, DNA fragmentation and viability were measured. Two statistically significant differences related to RF radiation were observed: Lipid peroxidation induced by t-BOOH was increased in SH-SY5Y (but not in L929) cells, and menadione-induced caspase 3 activity was increased in L929 (but not in SH-SY5Y) cells. Both differences were statistically significant only for the GSM-modulated signal. The other end points were not significantly affected in any of the experimental conditions, and no effects were observed from exposure to RF radiation alone. The positive findings may be due to chance, but they may also reflect effects that occur only in cells sensitized by chemical stress. Further studies are required to investigate the reproducibility and dose response of the possible effects.     

Effects of 45-Hz magnetic fields on the functional state of the human brain

The influence of sinusoidal 45-Hz magnetic fields on the brain functions of 20 volunteers was investigated in a double-blind study using spectral analysis of EEG and measurements of Omega potentials and reaction time (RT). The field strength was 1,000 A/m (1.26 mT) and the duration of exposure was 1 h. Ten volunteers were exposed to a continuous field and ten received an intermittent exposure (1 s on/1 s off). Each person received one real and one sham exposure. One half of the volunteers got the real exposure first and the sham treatment after at least 24 h. For the rest, the sequence was inverse. The measurements of EEG, omega potentials and RT were performed before and after each exposure. Several statistically significant changes were observed, most of them after intermittent exposure. In the EEG, an increase of alpha (7.6–13.9 Hz) activity and a decrease of delta (1.5–3.9 Hz) activity were observed. β waves (14.2–20 Hz) increased in the frontal derivations as did the total power in occipital derivations. The mean and peak frequencies of EEG increased mainly in the frontal derivations. No direct effects on RT were seen. Learning to perform the RT test (decrease of RT in repeated trials), however, seemed to be affected by the exposure. The persons who received real exposure first learned more slowly than those who got sham exposure first. Further experiments are necessary to confirm the findings and for understanding the mechanisms of the effects. © 1993 Wiley-Liss. Inc.     

Evolution of heterospecific attraction: using other species as cues in habitat selection

We analyzed the ecological conditions that may favor a habitat selection process in which later arriving individuals (colonists) use the presence of earlier established species (residents) as a cue to profitable breeding sites (heterospecific attraction). In our model, colonists assessing potential breeding patches could select between high-quality source and low-quality sink patches. A proportion of the source patches were occupied by residents. Colonists could either directly sample the relative quality of the patches (termed samplers) or, alternatively, they could also use residents as a cue of patch quality (cue-users). Cue-users gained benefit from lowered costs when assessing occupied source patches. The cue-using strategy is an efficient way to choose the best possible patch not only when interspecific competition is intense, but also when benefits from social aggregation exceed the effects of competition. High relative cost of sampling empty patches increases the fitness of the cue-using strategy relative to samplers. The strongest attraction to heterospecifics was predicted when the benefit from aggregating with residents exceeded the effects of competition, and approximately half of the landscape consisted of occupied, high-quality source patches.