Temperature effects in hydrophobic interaction chromatography.

The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.

Isolation of an oxygen-sensitive FNR protein of Escherichia coli: interaction at activator and repressor sites of FNR-controlled genes.

The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state. To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis. In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth. The altered fur gene was overexpressed in E. coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures. An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein. The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer. In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription. The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site. A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.

Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.

Three-dimensional tracking of motile bacteria near a solid planar surface.

Knowing how motile bacteria move near and along a solid surface is crucial to understanding such diverse phenomena as the migration of infectious bacteria along a catheter, biofilm growth, and the movement of bacteria through the pore spaces of saturated soil, a critical step in the in situ bioremediation of contaminated aquifers. In this study, a tracking microscope is used to record the three-dimensional motion of Escherichia coli near a planar glass surface. Data from the tracking microscope are analyzed to quantify the effects of bacteria-surface interactions on the swimming behavior of bacteria. The speed of cells approaching the surface is found to decrease in agreement with the mathematical model of Ramia et al. [Ramia, M., Tullock, D. L. & Phan-Tien, N. (1993) Biophys J. 65,755-778], which represents the bacteria as spheres with a single polar flagellum rotating at a constant rate. The tendency of cells to swim adjacent to the surface is shown in computer-generated reproductions of cell traces. The attractive interaction potential between the cells and the solid surface is offered as one of several possible explanations for this tendency.

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.

Three-dimensional scroll waves of cAMP could direct cell movement and gene expression in Dictyostelium slugs.

Complex three-dimensional waves of excitation can explain the observed cell movement pattern in Dictyostelium slugs. Here we show that these three-dimensional waves can be produced by a realistic model for the cAMP relay system [Martiel, J. L. & Goldbeter, A. (1987) Biophys J. 52, 807-828]. The conversion of scroll waves in the prestalk zone of the slug into planar wave fronts in the prespore zone can result from a smaller fraction of relaying cells in the prespore zone. Further, we show that the cAMP concentrations to which cells in a slug are exposed over time display a simple pattern, despite the complex spatial geometry of the waves. This cAMP distribution agrees well with observed patterns of cAMP-regulated cell type-specific gene expression. The core of the spiral, which is a region of low cAMP concentration, might direct expression of stalk-specific genes during culmination.

Excitations in Bose-Einstein condensates: collective modes, quantum turbulence and matter wave statistics

In this thesis, we present the generation and studies of a 87Rb Bose-Einstein condensate (BEC) perturbed by an oscillatory excitation. The atoms are trapped in a harmonic magnetic trap where, after an evaporative cooling process, we produce the BEC. In order to study the effect caused by oscillatory excitations, a quadrupole magnetic field time oscillatory is superimposed to the trapping potential. Through this perturbation, collective modes were observed. The dipole mode is excited even for low excitation amplitudes. However, a minimum excitation energy is needed to excite the condensate quadrupole mode. Observing the excited cloud in TOF expansion, we note that for excitation amplitude in which the quadrupole mode is excited, the cloud expands without invert its aspect ratio. By looking these clouds, after long time-of-flight, it was possible to see vortices and, sometimes, a turbulent state in the condensed cloud. We calculated the momentum distribution of the perturbed BECs and a power law behavior, like the law to Kolmogorov turbulence, was observed. Furthermore, we show that using the method that we have developed to calculate the momentum distribution, the distribution curve (including the power law exponent) exhibits a dependence on the quadrupole mode oscillation of the cloud. The randomness distribution of peaks and depletions in density distribution image of an expanded turbulent BEC, remind us to the intensity profile of a speckle light beam. The analogy between matter-wave speckle and light speckle is justified by showing the similarities in the spatial propagation (or time expansion) of the waves. In addition, the second order correlation function is evaluated and the same dependence with distance was observed for the both waves. This creates the possibility to understand the properties of quantum matter in a disordered state. The propagation of a three-dimensional speckle field (as the matter-wave speckle described here) creates an opportunity to investigate the speckle phenomenon existing in dimensions higher than 2D (the case of light speckle).

Parent-Child Interaction and Children's Self-Concept

This study is designed to investigate the relationships between marital communication, the quality of parents' ability to assist their children in joint problem-solving, and children's independent mastery attempts and perceived competence at problem-solving, and behavioral indicators of self-esteem. Couples' skill at regulating their own and their children's negative affect within the marital and parent-child family subsystems is hypothesized to predict the quality of their assistance, or scaffolding behavior, to their children during joint problem-solving. Further, the quality of parental scaffolding behavior is expected to predict children's independent mastery attempts, levels of perceived competence at problemsolving, and behavioral indicators of self-esteem. Families for the study will be those with children between 3 1/2 to six years of age recruited from subjects participating in a longitudinal study of communication in marriage being conducted at the Denver Center for Marital and Family Studies. Families will participate in three interaction tasks designed to tap parental scaffolding behavior during problemsolving with their children. Children will be administered self-report measures to tap their perceived competence at such problem-solving as those in the interaction tasks and parents will complete a questionnaire tapping the behavioral indicators of their child's self-esteem. Family interaction data will be coded with the use of a microanalytic coding system developed by this study, the Parent-Child Interaction Coding System. Marital communication data at three time points, premaritally, during the transition to parenthood , and concurrently, will be obtained from couples' interactions from the longitudinal study. The clinical significance of this study includes implications for training couples how to effectively regulate negative affect and offer their children sensitive assistance during joint problem-solving.

Elysia timida (Risso, 1818) three decades of research

During the last 30 years, studies on Elysia timida (Risso, 1818) have addressed various aspects related to food sources, photosynthetic efficiency of kleptoplasts, population genetics, chemical ecology and reproductive biology, both in the Mediterranean Sea and in the Mar Menor coastal lagoon. E. timida shows a strong specific interaction with Acetabularia acetabulum, retaining functional chloroplasts for at least 45 days and obtaining extra energy in periods when food resources are scarce. It shows control of parapodia, avoiding pigment photodestruction under oversaturated light conditions. The chemical ecological relationships established between E. timida and its potential predator fish, Thalassoma pavo, have also been evaluated, and it has been found that that the extracts of the mollusc contain repellent and unpalatable polypropionate compounds. Population genetics has demonstrated the genetic divergence between populations showing high and significant values of FST and genetic distances, and at least six privative alleles that are not shared with Mediterranean populations have been detected in lagoon populations. This sacoglossan is a poecilogonic species, and its lagoon populations show a greater reproductive output than Mediterranean populations; they produce a greater number of egg masses and embyros per individual, and the capsules have a wider diameter.

Shear wave velocity estimation in the metropolitan area of Málaga (S Spain)

We carry out a seismic noise study based on array measurements at three sites in the Málaga basin, South Spain, for the further estimation of shear wave velocity profiles. For this purpose, we use both the H/V method and the f–k technique in order to characterize the different materials present in the zone, i.e., Quaternary sediments and Pliocene sedimentary rocks above the bedrock. The H/V analysis shows frequency peaks going from 1 Hz, in areas close to the border of the basin, to 0.3 Hz in places located toward the center of the formation. The f–k analysis allows obtaining the dispersion curves associated with each site and subsequently, estimating the Vs profiles by inversion of the respective group velocities. In this way, the basin basement can be characterized by S-wave velocities greater than 2000 m/s. Regarding the basin fill, it is divided into three layers defined by different wave velocity intervals. The shallowest one is featured by velocities ranging from 150 to 400 m/s and comprises the Quaternary sediments, while velocities going from 550–700 to1200–1600 m/s characterize the two underlying layers composed by Pliocene sediments. Finally, the information provided by the three Vs profiles is integrated in a 2D cross-section of the basin to have a spatial view of its sedimentary structure. The results obtained here, in addition to providing useful information about the infill of the basin near the metropolitan area of Málaga, will be very helpful for future seismic zonation studies in the region.

Association Patterns in Saproxylic Insect Networks in Three Iberian Mediterranean Woodlands and Their Resistance to Microhabitat Loss

The assessment of the relationship between species diversity, species interactions and environmental characteristics is indispensable for understanding network architecture and ecological distribution in complex networks. Saproxylic insect communities inhabiting tree hollow microhabitats within Mediterranean woodlands are highly dependent on woodland configuration and on microhabitat supply they harbor, so can be studied under the network analysis perspective. We assessed the differences in interacting patterns according to woodland site, and analysed the importance of functional species in modelling network architecture. We then evaluated their implications for saproxylic assemblages’ persistence, through simulations of three possible scenarios of loss of tree hollow microhabitat. Tree hollow-saproxylic insect networks per woodland site presented a significant nested pattern. Those woodlands with higher complexity of tree individuals and tree hollow microhabitats also housed higher species/interactions diversity and complexity of saproxylic networks, and exhibited a higher degree of nestedness, suggesting that a higher woodland complexity positively influences saproxylic diversity and interaction complexity, thus determining higher degree of nestedness. Moreover, the number of insects acting as key interconnectors (nodes falling into the core region, using core/periphery tests) was similar among woodland sites, but the species identity varied on each. Such differences in insect core composition among woodland sites suggest the functional role they depict at woodland scale. Tree hollows acting as core corresponded with large tree hollows near the ground and simultaneously housing various breeding microsites, whereas core insects were species mediating relevant ecological interactions within saproxylic communities, e.g. predation, competitive or facilitation interactions. Differences in network patterns and tree hollow characteristics among woodland sites clearly defined different sensitivity to microhabitat loss, and higher saproxylic diversity and woodland complexity showed positive relation with robustness. These results highlight that woodland complexity goes hand in hand with biotic and ecological complexity of saproxylic networks, and together exhibited positive effects on network robustness.

Sub-Cellular Localization and Complex Formation by Aminoacyl-tRNA Synthetases in Cyanobacteria: Evidence for Interaction of Membrane-Anchored ValRS with ATP Synthase

tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries.

(Table 1) Ice floe size and other characteristics during aerial observations in Prydz Bay, Antarctica

On the basis of aerial photographs of sea ice floes in the marginal ice zone (MIZ) of Prydz Bay acquired from December 2004 to February 2005 during the 21st Chinese National Antarctic Research Expedition, image processing techniques are employed to extract some geometric parameters of floes from two merged transects covering the whole MIZ. Variations of these parameters with the distance into the MIZ are then obtained. Different parameters of floe size, namely area, perimeter, and mean caliper diameter (MCD), follow three similar stages of increasing, flat and increasing again, with distance from the open ocean. Floe shape parameters (roundness and the ratio of perimeter to MCD), however, have less significant variations than that of floe size. Then, to modify the deviation of the cumulative floe size distribution from the ideal power law, an upper truncated power-law function and a Weibull function are used, and four calculated parameters of the above functions are found to be important descriptors of the evolution of floe size distribution in the MIZ. Among them, Lr of the upper truncated power-law function indicates the upper limit of floe size and roughly equals the maximum floe size in each square sample area. L0 in the Weibull distribution shows an increasing proportion of larger floes in squares farther from the open ocean and roughly equals the mean floe size. D in the upper truncated power-law function is closely associated with the degree of confinement during ice breakup. Its decrease with the distance into MIZ indicates the weakening of confinement conditions on floes owing to wave attenuation. The gamma of the Weibull distribution characterizes the degree of homogeneity in a data set. It also decreases with distance into MIZ, implying that floe size distributes increase in range. Finally, a statistical test on floe size is performed to divide the whole MIZ into three distinct zones made up of floes of quite different characteristics. This zonal structure of floe size also agrees well with the trends of floe shape and floe size distribution, and is believed to be a straightforward result of wave-ice interaction in the MIZ.

Interaction of waves and currents /

This report presents an overview of wave-current interacion, including comprehensive review of references to significant U.S. and foreign literature available through December 1981. Specific topics under review are the effects of horizontally and vertically varying currents on waves, wave refraction by currents, dissipation and turbulence, small- and medium-scale currents, caustics and focusing, and wave breaking. The results of the review are then examined for engineering applications. The most appropriate general-purpose computer program to include wave-current interaction is the Dutch Rijkswaterstaat program CREDIZ, which is based on a parabolic wave equation. Further applications include wave and current forces on structures and possibly sediment transport. The report concludes with a brief state-of-the-art review of wave-current interaction and a list of topics needing further research and development.