A Combined Molecular Cloning and Mass Spectrometric Method to Identify, Characterize, and Design Frenatin Peptides from the Skin Secretion of Litoria infrafrenata

Amphibian skin secretions are unique sources of bioactive molecules, particularly bioactive peptides. In this study, the skin secretion of the white-lipped tree frog (Litoria infrafrenata) was obtained to identify peptides with putative therapeutic potential. By utilizing skin secretion-derived mRNA, a cDNA library was constructed, a frenatin gene was cloned and its encoded peptides were deduced and confirmed using RP-HPLC, MALDI-TOF and MS/MS. The deduced peptides were identified as frenatin 4.1 (GFLEKLKTGAKDFASAFVNSIKGT) and a post-translationally modified peptide, frenatin 4.2 (GFLEKLKTGAKDFASAFVNSIK.NH2). Antimicrobial activity of the peptides was assessed by determining their minimal inhibitory concentrations (MICs) using standard model microorganisms. Through studying structure–activity relationships, analogues of the two peptides were designed, resulting in synthesis of frenatin 4.1a (GFLEKLKKGAKDFASALVNSIKGT) and frenatin 4.2a (GFLLKLKLGAKLFASAFVNSIK.NH2). Both analogues exhibited improved antimicrobial activities, especially frenatin 4.2a, which displayed significant enhancement of broad spectrum antimicrobial efficiency. The peptide modifications applied in this study, may provide new ideas for the generation of leads for the design of antimicrobial peptides with therapeutic applications.

Comparison of mass transfer effects in the heterogeneously catalysed hydrogenation of phenyl acetylene in heptane and an ionic liquid

The selective heterogeneous catalytic reduction of phenyl acetylene to styrene over palladium supported on calcium carbonate is reported in both an ionic liquid and a molecular solvent. By using a rotating disc reactor in conjunction with results from a stirred tank reactor it is possible, for the first time, to disentangle the mass transfer contributions in the ionic liquid system. For both heptane and 1-butyl-3-methyl imidazolium bis{(trifluoromethyl)sulfonyl}imide, the reaction in the rotating disc reactor is dominated by reaction in the entrained film on the disc compared with very limited reaction in the bulk liquid. The lower reaction rate obtained in the ionic liquid compared with the organic solvent is shown to be due to the slow transport of the hydrogen dissolved in the liquid. It is clear from the results presented herein that, although the hydrodynamics of similar reactors used for biological treatment of wastewater are well understood, on using a more viscous fluid and higher rotation speeds necessary for fine chemical catalysis these simple relationships breakdown.

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8 - a novel prokineticin from Bombina orientalis

Prokineticins are small (8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr = 7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.

Molecular Hydrogen Emission from Protoplanetary Disks: Effects of X-ray Irradiation and Dust Evolution

Detailed models for the density and temperature profiles of gas and dust in protoplanetary disks are constructed by taking into account X-ray and UV irradiation from a central T Tauri star, as well as dust size growth and settling toward the disk midplane. The spatial and size distributions of dust grains are numerically computed by solving the coagulation equation for settling dust particles, with the result that the mass and total surface area of dust grains per unit volume of the gas in the disks are very small, except at the midplane. The H2 level populations and line emission are calculated using the derived physical structure of the disks. X-ray irradiation is the dominant heating source of the gas in the inner disk and in the surface layer, while the UV heating dominates otherwise. If the central star has strong X-ray and weak UV radiation, the H2 level populations are controlled by X-ray pumping, and the X-rayinduced transition lines could be observable. If the UV irradiation is strong, the level populations are controlled by thermal collisions or UV pumping, depending on the dust properties. As the dust particles evolve in the disks, the gas temperature at the disk surface drops because the grain photoelectric heating becomes less efficient. This makes the level populations change from LTE to non-LTE distributions, which results in changes to the line ratios. Our results suggest that dust evolution in protoplanetary disks could be observable through the H2 line ratios. The emission lines are strong from disks irradiated by strong UV and X-rays and possessing small dust grains; such disks will be good targets in which to observe H2 emission.

Galactic Edge Clouds I. Molecular Line Observations and Chemical Modelling of Edge Cloud 2

Edge Cloud 2 (EC2) is a molecular cloud, about 35 pc in size, with one of the largest galactocentric distances known to exist in the Milky Way. We present observations of a peak CO emission region in the cloud and use these to determine its physical characteristics. We calculate a gas temperature of 20 K and a density of n(H2)~10^4 cm-3. Based on our CO maps, we estimate the mass of EC2 at around 10^4 Msolar and continuum observations suggest a dust-to-gas mass ratio as low as 0.001. Chemical models have been developed to reproduce the abundances in EC2, and they indicate that heavy element abundances may be reduced by a factor of 5 relative to the solar neighborhood (similar to dwarf irregular galaxies and damped Lya systems), very low extinction (A_V <4 mag) due to a very low dust-to-gas mass ratio, an enhanced cosmic-ray ionization rate, and a higher UV field compared to local interstellar values. The reduced abundances may be attributed to the low level of star formation in this region and are probably also related to the continuing infall of primordial (or low-metallicity) halo gas since the Milky Way formed. Finally, we note that shocks from the old supernova remnant GSH 138-01-94 may have determined the morphology and dynamics of EC2.

Bradykinin-related peptides, including a novel structural variant, (Val1)-bradykinin, from the skin secretion of Guenther's frog, Hylarana guentheri and their molecular precursors.

Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val1)-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.

Liquid structure of the ionic liquid, 1-methyl-4-cyanopyridinium bis{(trifluoromethyl)sulfonyl}imide determined from neutron scattering and molecular dynamics simulations

The liquid structure of 1-methyl-4-cyanopyridinium bis {(trifluoromethyl)sulfonyl}imide, a prototypical ionic liquid containing an electron-withdrawing group on the cation, has been investigated at 368 K. Experimental neutron scattering combined with empirical potential structure refinement analysis of the data and classical molecular dynamics simulations have been used to probe the liquid structure in detail. Both techniques generated highly consistent results that provide valuable validation of the force fields and refinement approaches. A significant degree of apparent charge ordering is found in the liquid structure, although the nonspherical shape of the ions results in interpenetration of cations into the first shell of adjacent cations, with much shorter closest contact distances than the averaged center-of-mass cation-cation and cation-anion separations.

Measurements of the XUV mass absorption coefficient of an overdense liquid metal

The increase in the XUV mass absorption coefficient of liquid aluminium, produced by high-power-laser shock-compression, is measured using XUV laser radiography. At a photon energy of 63 eV a change in the mass absorption coefficient by up to a factor of similar to2.2 is determined at densities close to twice that of solid and electron temperatures of the order of 1 eV. Comparison with hydrodynamic simulations indicate that the absorption coefficient scales with density as rho (1.3 +/-0.2).

SiO in G34.26: Outflows and shocks in a high mass star forming region

We have looked for SiO emission as evidence of shocks in the high mass star formation region G34.26+0.15. JCMT, VLA and FCRAO observations show that SiO emission is widespread across the region. The SiO emission highlights a massive, collimated out ow and other regions where stellar winds are interacting with molecular clumps. As in other star forming regions, there is also SiO at ambient velocities which is related to the out ow activity. No strong SiO abundance enhancement was measured in either the out ow or the low velocity gas, though abundances up to 10(-8) are possible if the SiO is locally enhanced in clumps and optically thick. SiO emission is not detected from the hot core itself, indicating either that SiO is not strongly enhanced in the hot core or that column densities in the region where grain mantle evaporation has taken place are low. In line of sight spiral arm clouds, we measure a SiO abundance of 0.4-2 x 10(-10), consistent with previous estimates for quiescent clouds.

Macroecological patterns and niche structure in a new marine food web

The integration of detailed information on feeding interactions with measures of abundance and body mass of individuals provides a powerful platform for understanding ecosystem organisation. Metabolism and, by proxy, body mass constrain the flux, turnover and storage of energy and biomass in food webs. Here, we present the first food web data for Lough Hyne, a species rich Irish Sea Lough. Through the application of individual-and size-based analysis of the abundance-body mass relationship, we tested predictions derived from the metabolic theory of ecology. We found that individual body mass constrained the flux of biomass and determined its distribution within the food web. Body mass was also an important determinant of diet width and niche overlap, and predator diets were nested hierarchically, such that diet width increased with body mass. We applied a novel measure of predator-prey biomass flux which revealed that most interactions in Lough Hyne were weak, whereas only a few were strong. Further, the patterning of interaction strength between prey sharing a common predator revealed that strong interactions were nearly always coupled with weak interactions. Our findings illustrate that important insights into the organisation, structure and stability of ecosystems can be achieved through the theoretical exploration of detailed empirical data.

Molecular abundances in the magellanic clouds .1. A multiline study of five cloud cores

Nine H II regions of the LMC were mapped in (CO)-C-13(1-0) and three in (CO)-C-12(1-0) to study the physical properties of the interstellar medium in the Magellanic Clouds. For N113 the molecular core is found to have a peak position which differs from that of the associated H II region by 20 ''. Toward this molecular core the (CO)-C-12 and (CO)-C-13 peak T-MB line temperatures of 7.3 K and 1.2 K are the highest so far found in the Magellanic Clouds. The molecular concentrations associated with N113, N44BC, N159HW, and N214DE in the LMC and LIRS 36 in the SMC were investigated in a variety of molecular species to study the chemical properties of the interstellar medium. I(HCO+)/I(HCN) and I(HCN)/I(HNC) intensity ratios as well as lower limits to the I((CO)-C-13)/I((CO)-O-18) ratio were derived for the rotational 1-0 transitions. Generally, HCO+ is stronger than HCN, and HCN is stronger than HNC. The high relative HCO+ intensities are consistent with a high ionization flux from supernovae remnants and young stars, possibly coupled with a large extent of the HCO+ emission region. The bulk of the HCN arises from relatively compact dense cloud cores. Warm or shocked gas enhances HCN relative to HNC. From chemical model calculations it is predicted that I(HCN)/I(HNC) close to one should be obtained with higher angular resolution (less than or similar to 30 '') toward the cloud cores. Comparing virial masses with those obtained from the integrated CO intensity provides an H-2 mass-to-CO luminosity conversion factor of 1.8 x 10(20) mol cm(-2) (K km s(-1))(-1) for N113 and 2.4 x 10(20) mol cm(-2) (K km s(-1))(-1) for N44BC. This is consistent with values derived for the Galactic disk.

Molecular abundances in the Magellanic Clouds .2. Deuterated species in the LMC

The first definite discoveries of extragalactic deuterium are reported. DCO+ has been detected in three and DCN has been measured in one star-forming region of the Large Magellanic Cloud (LMC). While the HCO+/DCO+ abundance ratios are found to be 19 +/- 3, 24 +/- 4, and 67 +/- 18 for N113, N44BC and N159HW, respectively, a HCN/DCN abundance ratio of 23 +/- 5 is obtained for N113. These results are consistent with a gas temperature of about 20 K and a D/H ratio of about 1.5 x 10(-5), consistent with that observed in the Galaxy. If the cloud temperature is closer to 30 K, then a D/H ratio is required to be up to an order of magnitude larger. Because this ratio provides a lower limit to the primordial D/H ratio, it indicates that the baryon mass density alone is unable to close the universe.

Energy distributions of copper ions and atoms sputtered by atomic and molecular ions

Non-resonant multiphoton ionization combined with quadrupole and time-of-flight analysis has been used to study sputtering by both atomic and molecular ion beams. The mass spectra and energy distributions of both sputtered atoms and secondary ions produced by 3.6 keV Ar+, N+, N-2(+), CF2+ and CF3+ ion bombardment at 45 degrees to a polycrystalline copper target have been measured. The energy distributions of the copper ions and atoms are found to be different and quite complex. The ion distributions can generally be described by a linear collision cascade model, with possible evidence for a knock-on contribution. The sputtered atom distributions are partially described by a combination of linear collision cascade and dense cascade (thermal spike) models. This is interpreted as support for a time-evolving sputtering mechanism.

Molecular line emission from a protoplanetary disk irradiated externally by a nearby massive star

Star formation often occurs within or nearby stellar clusters. Irradiation by nearby massive stars can photoevaporate protoplanetary disks around young stars (so-called proplyds) which raises questions regarding the ability of planet formation to take place in these environments. We investigate the two-dimensional physical and chemical structure of a protoplanetary disk surrounding a low-mass (T Tauri) star which is irradiated by a nearby massive O-type star to determine the survivability and observability of molecules in proplyds. Compared with an isolated star-disk system, the gas temperature ranges from a factor of a few (in the disk midplane) to around two orders of magnitude (in the disk surface) higher in the irradiated disk. Although the UV flux in the outer disk, in particular, is several orders of magnitude higher, the surface density of the disk is sufficient for effective shielding of the disk midplane so that the disk remains predominantly molecular in nature. We also find that non-volatile molecules, such as HCN and H2O, are able to freeze out onto dust grains in the disk midplane so that the formation of icy planetesimals, e.g., comets, may also be possible in proplyds. We have calculated the molecular line emission from the disk assuming LTE and determined that multiple transitions of atomic carbon, CO (and isotopologues, 13CO and C18O), HCO+, CN, and HCN may be observable with ALMA, allowing characterization of the gas column density, temperature, and optical depth in proplyds at the distance of Orion (˜400 pc).