Seasonal variability of diurnal in-stream nitrate concentration oscillations under hydrologically stable conditions

Seasonal and diurnal variations of in-stream NO₃-N concentration oscillations were studied through high-frequency measurements of streamwater’s physical, chemical parameters (in-stream NO₃-N concentration, water temperature, dissolved oxygen, pH) and hydrometeorological variables (stream discharge, solar radiation) under hydrologically stable conditions. The study was carried out in 2006, within the 42 km² forested Pade? stream watershed in the southwestern part of Slovenia, which is characterized by distinctive hydrogeological settings (flysch) and climate conditions (transitional area between the Mediterranean and continental climate). Fine temporal data resolution (15-min interval) enabled identification of the factors responsible for seasonal variability in the diurnal pattern of the streamwater NO₃-N concentrations versus seasonal and diurnal behavior of meteorological and other water chemistry constituents. The observed seasonal variability of in-stream NO₃-N daily oscillations indicates the important role of primary production uptake, particularly during seasons when deciduous vegetation is dormant and light levels in the stream are high. Highest daily NO₃-N concentration amplitudes (0.3 mg/l-N) and daily changes in the NO₃-N flux (0.4–0.5 g/s-N) were observed in spring; the NO₃-N concentration oscillations in summer showed a considerably smaller effect of the in-stream uptake (maximum NO₃-N daily concentration amplitude 0.1 mg/l-N; daily change in the NO₃-N flux 0.02 g/s-N). Seasonal shifts in the timing of daily maximum (up to 6 h) and minimum NO₃-N concentrations (between 1 and 3 h) provided some additional indications of seasonal changes in the in-stream primary production uptake and its relation to the terrestrial component of the forested watershed.     

Niche partitioning in orbweaving spiders Meta menardi and Metellina merianae (Tetragnathidae)

Hypogean habitats are relatively simple exhibiting low diversity, low production and relative constancy of environmental factors, and are therefore appropriate for studying species coexistence in situ. We investigated the coexistence of two closely related, similarly sized orb-weaving spider species, Meta menardi and Metellina merianae, living syntopically in a Slovenian cave. We studied the annual dynamics of both species within a mixed population, and the impact of the ambient temperature, relative humidity, airflow and illumination, and compared their trophic niches to legacy data on prey of both species from 55 caves in Slovenia. We predicted a large overlap in their spatial niches and substantial differences in their temporal and trophic niches. We found that their spatial niches overlap greatly with few exceptions, mostly on the dates of notable meteorological changes in the cave but that their temporal niches differ significantly with r-strategy resembling epigean annual dynamic in M. merianae and a steady low abundance course in M. menardi within the cave. We also found that different predatory strategies significantly segregate their trophic niches: M. merianae uses a typical orb-weaving hunting strategy, while M. menardi combines web hunting with off-web hunting. Our findings suggest that both the diverse dynamics and trophic niches enable the coexistence of M. menardi and M. merianae despite their similar spatial niches, and that M. menardi, in particular, is optimally adapted to the epigean/hypogean ecotone.     

Influence of the acetylcholinesterase active site protonation on omega loop and active site dynamics

Existence of alternative entrances in acetylcholinesterase (AChE) could explain the contrast between the very high AChE catalytic efficiency and the narrow and long access path to the active site revealed by X-ray crystallography. Alternative entrances could facilitate diffusion of the reaction products or at least water and ions from the active site. Previous molecular dynamics simulations identified side door and back door as the most probable alternative entrances. The simulations of non-inhibited AChE suggested that the back door opening events occur only rarely (0.8% of the time in the 10ns trajectory). Here we present a molecular dynamics simulation of non-inhibited AChE, where the back door opening appears much more often (14% of the time in the 12ns trajectory) and where the side door opening was observed quite frequently (78% of trajectory time). We also present molecular dynamics, where the back door does not open at all, or where large conformational changes of the AChE omega loop occur together with alternative passage opening events. All these differences in AChE dynamical behavior are caused by different protonation states of two glutamate residues located on bottom of the active site gorge (Glu202 and G450 in Mus musculus AChE). Our results confirm the results of previous molecular dynamics simulations, expand the view and suggest the probable reasons for the overall conformational behavior of AChE omega loop.     

S3EPY: a Sparky extension for determination of small scalar couplings from spin-state-selective excitation NMR experiments

S3EPY is a Python extension to the program Sparky written to facilitate the assessment of coupling constants from in-phase/antiphase and spin-state-selective excitation (S³E) experiments. It enables the routine use of small scalar couplings by automating the coupling evaluation procedure. S3EPY provides an integrated graphical user interface to programs which outputs graphs and the table of determined couplings.     

Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1

The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16–31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 ϕ torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular α-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured ³J(H_N-H_Cα)-coupling constants, indicating that different conformers of GCN4p16–31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and ³J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and ³J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16–31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16–31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.     

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH₂) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O₂) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.     

Protein subunit interfaces: a statistical analysis of hot spots in Sm proteins

The distinguishing property of Sm protein associations is very high stability. In order to understand this property, we analyzed the interfaces and compared the properties of Sm protein interfaces with those of a test set, the Binding Interface Database (BID). The comparison revealed that the main differences between the interfaces of Sm proteins and those of the BID set are the content of charged residues, the coordination numbers of the residues, knowledge-based pair potentials, and the conservation scores of hot spots. In Sm proteins, the interfaces have more hydrophobic and fewer charged residues than the surfaces, which is also the case for the BID test set and other proteins. However, in the interfaces, the content of charged residues in Sm proteins (26%) is substantially larger than that in the BID set (22%). Hot spots are residues that make up a small fraction of the interfaces, but they contribute most of the binding energy. These residues are critical to protein–protein interactions. Analyses of knowledge-based pair potentials of hot spot and non-hot spot residues in Sm proteins show that they are significantly different; their mean values are 31.5 and 11.3, respectively. In the BID set, this difference is smaller; in this case, the mean values for hot spot and non-hot spot residues are 20.7 and 12.4, respectively. Hence, the pair potentials of hot spots differ significantly for the Sm and BID data sets. In the interfaces of Sm proteins, the amino acids are tightly packed, and the coordination numbers are larger in Sm proteins than in the BID set for both hot spots and non-hot spots. At the same time, the coordination numbers are higher for hot spots; the average coordination number of the hot spot residues in Sm proteins is 7.7, while it is 6.1 for the non-hot spot residues. The difference in the calculated average conservation score for hot spots and non-hot spots in Sm proteins is significantly larger than it is in the BID set. In Sm proteins, the average conservation score for the hot spots is 7.4. Hot spots are surrounded by residues that are moderately conserved (5.9). The average conservation score for the other interface residues is 5.6. The conservation scores in the BID set do not show a significant distinction between hot and non-hot spots: the mean values for hot and non-hot spot residues are 5.5 and 5.2, respectively. These data show that structurally conserved residues and hot spots are significantly correlated in Sm proteins.     

Chemical composition and antimicrobial activity of volatiles from Degenia velebitica, a European stenoendemic plant of the Brassicaceae family

Free and glucosidic bound leaf volatiles of Degenia velebitica were isolated and fractionated simultaneously into H₂O‐soluble, H₂O‐insoluble, and highly volatile compounds by hydrodistillation–adsorption (HDA) and analyzed by GC/MS. Among the 24 constituents identified, the main compounds obtained by the HDA method were S‐ and/or N‐atom containing compounds, i.e., 6‐(methylsulfanyl)hexanenitrile ( 10 ; 26.78%), dimethyl trisulfide ( 6 ; 26.35%), 3,4,5‐trimethylpyrazole ( 17 ; 13.33%), hex‐5‐enenitrile ( 2 ; 10.11%), dimethyl tetrasulfide ( 8 ; 4.93%), and pent‐4‐enyl isothiocyanate ( 7 ; 4.45%). In addition, O‐glycosidically bound volatiles and free volatiles were isolated by solvent extraction. Sixteen volatile O‐aglycones and twelve free volatile components were identified. The main O‐aglycones were eugenol ( 19 ; 24.15%), 2‐methoxy‐4‐vinylphenol ( 11 ; 11.50%), and benzyl alcohol ( 20 ; 9.49%), and the main free volatiles were (9Z,12Z)‐octa‐9,12‐dienic acid (38.35%), hexadecanoic acid (22.64%), and phytol (5.80%). The H₂O‐soluble volatile fraction obtained by HDA, containing mostly glucosinolate degradation products and 3,4,5‐trimethylpyrazole ( 17 ), was evaluated for antimicrobial activity by determining inhibition zones with the diffusion method as well as minimal inhibitory concentrations (MIC) and minimal microbicidal concentrations (MMC) with the micro‐dilution method. The fraction expressed activity against the tested Gram‐positive and Gram‐negative bacteria as well as against yeast, with MIC values equal to or lower than 16.7 μg/ml.     

A survey of entodiniomorphid ciliates in chimpanzees and bonobos

Intestinal entodiniomorphid ciliates are commonly diagnosed in the feces of wild apes of the genera Pan and Gorilla. Although some authors previously considered entodiniomorphid ciliates as possible pathogens, a symbiotic function within the intestinal ecosystem and their participation in fiber fermentation has been proposed. Previous studies have suggested that these ciliates gradually disappear under captive conditions. We studied entodiniomorphid ciliates in 23 captive groups of chimpanzees, three groups of captive bonobos and six populations of wild chimpanzees. Fecal samples were examined using Sheather's flotation and Merthiolate‐Iodine‐Formaldehyde Concentration (MIFC) methods. We quantified the number of ciliates per gram of feces. The MIFC method was more sensitive for ciliate detection than the flotation method. Ciliates of genus Troglodytella were detected in 13 groups of captive chimpanzees, two groups of bonobos and in all wild chimpanzee populations studied. The absence of entodiniomorphids in some captive groups might be because of the extensive administration of chemotherapeutics in the past or a side‐effect of the causative or prophylactic administration of antiparasitic or antibiotic drugs. The infection intensities of ciliates in captive chimpanzees were higher than in wild ones. We suppose that the over‐supply of starch, typical in captive primate diets, might induce an increase in the number of ciliates. In vitro studies on metabolism and biochemical activities of entodiniomorphids are needed to clarify their role in ape digestion. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.