Nitrogen and Phosphorus in the Finnish Energy System, 1900–2003

In producing power, humans move the nutrients nitrogen (N) and phosphorus (P) from their long‐term geological and biological stocks and release or emit them in soil, water, and the atmosphere. In Finland, peat combustion is an important driver of N and P fluxes from the environment to human economy. The flows of N and P in the Finnish energy system were quantified with partial substance flow analysis, and the driving forces of emissions of nitrogen oxides (NOx) were analyzed using the ImPACT model. In the year 2000 in Finland, 140,000 tonnes of nitrogen entered the energy system, mainly in peat and hard coal. Combustion released an estimated 66,000 tonnes of N as nitrogen oxides (NOx) and nitrous oxides (N2O) and another 74,000 tonnes as elemental N2. Most of the emissions were borne in traffic. At the same time, 6,000 tonnes of P was estimated to enter the Finnish energy system, mostly in peat and wood. Ash was mainly used in earth construction and disposed in landfills; thus negligible levels of P were recycled back to nature. During the twentieth century, fuel‐borne input of N increased 20‐fold, and of P 8‐fold. In 1900–1950, the increasing use of hard coal slowly boosted N input, whereas wood fuels were the main carrier of P. Since 1970, the fluxes have been on the rise. NOx emissions leveled off in the 1980s, though, and then declined in conjunction with improvements in combustion technologies such as NOx removal (de‐NOx) technologies in energy production and catalytic converters in cars.     

Enzymes of the glucuronic acid pathway in Wistar and Gunn rats and their heterozygotes

A significant difference in UDP glucuronyltransferase activity (with p-nitrophenol as an acceptor) was found in the liver and kidneys of homozygous Wistar and Gunn rats. There was also a significant difference in hepatic UDP glucuronyltransferase activity between homozygous Wistar and heterozygous Gunn rats when the enzyme preparations were first activated by adding surfactants to the reaction mixture. This determination of surfactant-activated UDP glucuronyltransferase can be used to distinguish Wistar rats from heterozygous Gunn rats. Other enzymes of the glucuronic acid pathway were also studied in the liver and kidneys of homozygous Wistar and Gunn rats, but no differences were found.This study has been supported by grants from the U.S. Public Health Service (AM-06018-09 and the National Research Council for Natural Sciences, Finland.     

Mate Sampling and Assessment Procedures in Female Pied Flycatchers (Ficedula hypoleuca)

Models of mate sampling strategies predict that choosiness should decrease throughout the breeding season due to increasing costs of delaying mating. Therefore, individuals who start searching mates relatively late, should spend less time on sampling, and sample fewer candidates compared to early individuals. We observed mate searching behavior of female pied flycatchers (Ficedula hypoleuca) by radio-tracking in study areas with 6–12 unpaired males. Contrary to the prediction, the observed numbers of males sampled by the searching females increased with time, i.e. late arriving females visited more males than early arrivers. However, this seems to be due to more active sampling of males in short time by late-arriving females. The observed sampling pattern suggests some kind of comparison tactic, which seems, however, to be very variable among individual females. Mate-assessing females were characterized by a remarkably cryptic behavior, which may be 1) a way of gaining honest information about the male mating status or male/territory quality, or 2) a way of avoiding courtship costs.     

Determinants of the Mating Success of Polyterritorial Pied Flycatcher Males

Much attention has been paid to the polyterritorial mating system of some passerine birds. Here we report how a male's mating success is related to the behavioral traits of polyterritorial pied flycatcher (Ficedula hypoleuca) males. We found no evidence that the timing of polyterritoriality in relation to egg laying in the primary nest or the singing behavior of males have any influence on mating success. However, results show clearly that male mating success was improved with an increase in the distance between territories up to a distance of about 200–300 m whereupon there was no further enhancement of mating success. This finding is crucial for both the deception hypothesis and female-female aggression hypothesis which have been put forward to explain polyterritorial polygyny. Males who establish a distant second territory seemed to allocate more time to singing there. The association, although weak, between singing activity and distance between territories makes it more difficult for females to use song rate as a cue to discriminate males with a secondary territory far from the primary territory, and these are the males that are least likely to feed the young of a second female. Males who established a second territory late in relation to egg laying in the primary nest did not take over a close second territory as might be predicted from female-female aggression hypothesis.     

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.     

Altering substrate specificity of phosphatidylcholine-preferring phospholipase C of Bacillus cereus by random mutagenesis of the headgroup binding site

PLC(Bc) is a 28.5 kDa monomeric enzyme that catalyzes the hydrolysis of the phosphodiester bond of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to provide a diacylglycerol and the corresponding phosphorylated headgroup. Because single replacements of Glu4, Tyr56, and Phe66 in the headgroup binding pocket led to changes in substrate specificity [Martin et al. (2000) Biochemistry 39, 3410-3415], a combinatorial library of approximately 6000 maltose binding protein-PLC(Bc) fusion protein mutants containing random permutations of these three residues was generated to identify PLC(Bc) mutants with altered specificity profiles and high catalytic activities. Members of this library were screened for hydrolytic activity toward the water soluble substrates C6PC, C6PE, and C6PS using a novel protocol that was conducted in a 96-well format and featured the in situ cleavage of the fusion protein to release the mutant PLC(Bc)s. Ten mutant enzymes that exhibited significant preferences toward C6PE or C6PS were selected and analyzed by steady-state kinetics to determine their specificity constants, k(cat)/K(M). The C6PS selective clones E4G, E4Q/Y56T/F66Y, and E4K/Y56V exhibited higher specificity constants toward C6PS than wt, whereas Y56T, F66Y, and Y56T/F66Y were C6PE selective and had comparable or higher specificity constants than wt for C6PE. The corresponding wt residues were singly reinserted back into the E4Q/Y56T/F66Y and E4K/Y56V mutants via site-directed mutagenesis, and the E4Q/F66Y mutant thus obtained exhibited a 10-fold higher specificity constant toward C6PS than wt, a value significantly higher than other PLC(Bc) mutants. On the basis of available data, an aromatic residue at position 66 appears important for significant catalytic activity toward all three substrates, especially C6PC and C6PE. The charge of residue 4 also appears to be a determinant of enzyme specificity as a negatively charged residue at this position endows the enzyme with C6PC and C6PE preference, whereas a polar neutral or positively charged residue results in C6PS selectivity. Replacing Tyr56 with Val, Ala, Thr, or Ser greatly reduces activity toward C6PC. Thus, the substrate specificity of PLC(Bc) can be modulated by varying three of the amino acid residues that constitute the headgroup binding pocket, and it is now apparent that this enzyme is not evolutionarily optimized to hydrolyze phospholipids with ethanolamine or serine headgroups.     

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.     

Application of Crustacean Chitin as a Co-diluent in Direct Compression of Tablets

A “simplex-centroid mixture design” was used to study the direct-compression properties of binary and ternary mixtures of chitin and two cellulosic direct-compression diluents. Native milled and fractioned (125–250 μm) crustacean chitin of lobster origin was blended with microcrystalline cellulose, MCC (Avicel® PH 102) and spray-dried lactose–cellulose, SDLC Cellactose® (composed of a spray-dried mixture of alpha-lactose monohydrate 75% and cellulose powder 25%). An instrumented single-punch tablet machine was used for tablet compactions. The flowability of the powder mixtures composed of a high percentage of chitin and SDLC was clearly improved. The fractioned pure chitin powder was easily compressed into tablets by using a magnesium stearate level of 0.1% (w/w) but, as the die lubricant level was 0.5% (w/w), the tablet strength collapsed dramatically. The tablets compressed from the binary mixtures of MCC and SDLC exhibited elevated mechanical strengths (>100 N) independent of the die lubricant level applied. In conclusion, fractioned chitin of crustacean origin can be used as an abundant direct-compression co-diluent with the established cellulosic excipients to modify the mechanical strength and, consequently, the disintegration of the tablets. Chitin of crustacean origin, however, is a lubrication-sensitive material, and this should be taken into account in formulating direct-compression tablets of it.     

Enolases from Gram-positive bacterial pathogens and commensal lactobacilli share functional similarity in virulence-associated traits

Enolase occurs as a cytoplasmic and a surface-associated protein in bacteria. Enolases of the bacterial pathogens Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus, as well as of the commensal lactic acid bacteria, Lactobacillus crispatus and Lactobacillus johnsonii, were purified as His(6)-fusion proteins from recombinant Escherichia coli. The fusion proteins were compared for putative virulence-associated functions, i.e., binding of human plasminogen, enhancement of plasminogen activation by human plasminogen activators, as well as binding to immobilized laminin, fibronectin and collagens. The individual enolases showed varying efficiencies in these functions. In particular, highly and equally effective interactions with plasminogen and laminin were seen with lactobacillar and staphylococcal enolases.