Separation of amino acids and peptides by temperature induced phase partitioning. Theoretical model for partitioning and experimental data

There is a strong interest in use of ‘smart polymers’ in separation systems. These are polymers which can react on external influence, such as temperature or pH change. With such polymers it is possible from the outside to affect the properties of a separation system. Amphiphilic copolymers show drastic changes in solubility properties, such as self-association and phase separation, at e.g. temperature increase. The random copolymers of ethylene oxide and propylene oxide units (EOPO-polymers) can form aqueous two-phase systems above the copolymer cloud point temperature. Two phases are formed, one consisting of 40–60% polymer in water and the other of almost 100% water. Amino acids and peptides can be partitioned in the thermoseparating systems. The partitioning strongly depends on the solute hydrophobicity, where aromatic amino acids and peptides are partitioned to the polymer phase and hydrophilic to the water phase. Salt effects can be used to enhance the partitioning of charged molecules. The thermodynamic driving forces which govern the partitioning of molecules in a thermoseparated aqueous phase system is described with use of the Flory-Huggins theory for polymer solutions. Expressions are derived which show the entropic and enthalpic effects on solute partitioning. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ab initio study of phenyl benzoate: structure,conformational analysis,dipole moment,IR and Raman vibrational spectra

The molecular structure (bond distances and angles), conformational properties, dipole moment and vibrational spectroscopic data (vibrational frequencies, IR and Raman intensities) of phenyl benzoate were calculated using Hartree–Fock (HF), density functional (DFT), and second order Møller–Plesset perturbation theory (MP2) with basis sets ranging from 6-31G* to 6-311++G**. The theoretical results are discussed mainly in terms of comparisons with available experimental data. For geometric data, good agreement between theory and experiment is obtained for the MP2, B3LYP and B3PW91 levels with basis sets including diffuse functions. The B3LYP/6-31+G* theory level estimates the shape of the experimental functions for phenyl torsion around the Ph–O and Ph–C bonds well, but reproduces the height of the rotational barriers poorly. The B3LYP/6-31+G* harmonic force constants were scaled by applying the scaled quantum mechanical force field (SQM) technique. The calculated vibrational spectra were interpreted and band assignments were reported. They are in excellent agreement with experimental IR and Raman spectra.Figure Calculated and experimental (GED) potential energy functions for torsional motion of phenyl benzoate relative to the minimum value. a The potential function for torsion about the O₃–C₄ bond. b The potential function for torsion about the C₂–C₁₀ bond.     

Field release of genetically marked Azospirillum brasilense in association with Sorghum bicolor L.

The agronomic impact of genetically tagged azospirilla (Azospirillum brasilense)was assessed in open field and their fluctuation were monitored in the soil/rhizosphere. Strain performance, upon inoculation of sorghum, was evaluated over a two-years period; agronomic treatments included nitrogen application (0, 80, 160 kg ha^–1), and types of inoculant (Sp245 lacZ, Sp6 gusA, Sp6 IAA⁺⁺ gusA). Grain yield was higher for inoculated seed plots than in non-inoculated ones, whereas nitrogen content, biomass of plant residues and nitrogen in plant residues gave values that were not statistically different. Root length density (RLD) of sorghum at the end of the stem elongation stage was affected only by the indole-3-acetic acid (IAA) overproducer Azospirillum strain (A. brasilense Sp6 IAA⁺⁺ gusA) with respect to the normal IAA producer (A. brasilense Sp6 gusA), being higher in the first 40 cm of depth, notwithstanding the level of nitrogen fertilization. The traceability of the released genetically modified strains enabled to monitor their ability to colonise soil and roots. Moreover, the genetic modification per se vs. the non-modified counterpart, did not affect the culturable aerobic population in soil, microfungi, streptomycetes, fluorescent pseudomonads, soil microbial biomass, or some microbial activities, all selected as important indicators.     

Canny's compensating pressure theory fails a test

Canny's compensating pressure theory for water transport (American Journal of Botany 85: 897–909) has evolved from the premise that cavitation pressures are only a few tenths of a megapascal negative (approximately −0.3 MPa). In contradiction, “vulnerability curves” indicate that xylem pressures can drop below −3 MPa in some species without causing a loss of hydraulic conductivity. Canny claims these curves do not measure the limits to negative pressure by cavitation, but rather the limits to the compensating tissue pressure that otherwise quickly refills cavitated conduits. Compensating pressure is derived from the turgor pressure of the living cells in the tissue. To test this claim, we compared vulnerability curves of Betula nigra stems given three treatments: (1) living control, (2) killed in a microwave oven, and (3) perfused with a −1.5 MPa (10% w/w) mannitol solution. According to Canny's theory, the microwaved and mannitol curves should show cavitation and loss of conductance beginning at approximately −0.3 MPa because in both cases, the turgor pressure would be eliminated or substantially reduced compared to controls. We also tested the refilling capability of nonstressed stems where compensating pressure would be in full operation and compared this with dead stems with no compensating pressure. According to Canny's interpretation of vulnerability curves, the living stems should refill within 5 min. Results failed to support the compensating tissue theory because (a) all vulnerability curves were identical, reaching a −1.5 MPa threshold before substantial loss of conductance occurred, and (b) killed or living stems had equally slow refilling rates showing no significant increase in conductivity after 30 min. In consequence, the cohesion theory remains the most parsimonious explanation of xylem sap ascent in plants.     

Phototropism in hypocotyls of radish. VI. No exchange of endogenous indole-3-acetic acid between peripheral and central cell layers during first and second positive phototropic curvatures

Using a physicochemical method, the distribution of endogenous indole-3-acetic acid (IAA) was measured in the peripheral and central cell layers, as well as in the illuminated and shaded sides of phototropically stimulated radish hypocotyls (Raphanus sativus var. hortensis f. gigantissimus Makino). The IAA was evenly distributed over the illuminated and shaded sides in the first and second positive phototropic curvatures induced by a pulse or a continuous unilateral illumination with blue light. Also, no net exchange of the IAA between the peripheral and central cell layers was observed during these curvatures. These results suggest that phototropism of radish hypocotyls cannot be described by the Cholodny-Went theory.     

Evolutionary Landscapes for the Acquisition of New Ligand Recognition by RNA Aptamers

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro—unconstrained by confounding biological factors—is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences—defined as the sets functional tolerance for point mutations—and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.     

Identifying ‘firebreaks’ to fragment dispersal networks of a marine parasite

Marine ecosystems are beset by disease outbreaks, and efficient strategies to control dispersal of pathogens are scarce. We tested whether introducing no-farming areas or ‘firebreaks’ could disconnect dispersal networks of a parasitic disease affecting the world’s largest marine fish farming industry (～1000 farms). Larval salmon lice (Lepeophtheirus salmonis) are released from and transported among salmon farms by ocean currents, creating inter-farm networks of louse dispersal. We used a state-of-the-art biophysical model to predict louse movement along the Norwegian coastline and network analysis to identify firebreaks to dispersal. At least one firebreak that fragmented the network into two large unconnected groups of farms was identified for all seasons. During spring, when wild salmon migrate out into the ocean, and louse levels per fish at farms must be minimised, two effective firebreaks were created by removing 13 and 21 farms (1.3% and 2.2% of all farms in the system) at ～61°N and 67°N, respectively. We have demonstrated that dispersal models coupled with network analysis can identify no-farming zones that fragment dispersal networks. Reduced dispersal pathways should lower infection pressure at farms, slow the evolution of resistance to parasite control measures, and alleviate infection pressure on wild salmon populations.     

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.