Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2–15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on β-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1–1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.

Influence of elastase on alanine-rich peptide hydrogels

The self-assembly of the alanine-rich amphiphilic peptides Lys(Ala)6Lys (KA6K) and Lys(Ala)6Glu (KA6E)with homotelechelic or heterotelechelic charged termini respectively has been investigated in aqueous solution. These peptides contain hexa-alanine sequences designed to serve as substrates for the enzyme elastase. Electrostatic repulsion of the lysine termini in KA6K prevents self-assembly, whereas in contrast KA6E is observed, through electron microscopy, to form tape-like fibrils, which based on X-ray scattering contain layers of thickness equal to the molecular length. The alanine residues enable efficient packing of the side-chains in a beta-sheet structure, as revealed by circular dichroism, FTIR and X-ray diffraction experiments. In buffer, KA6E is able to form hydrogels at sufficiently high concentration. These were used as substrates for elastase, and enzyme-induced de-gelation was observed due to the disruption of the beta-sheet fibrillar network. We propose that hydrogels of the simple designed amphiphilic peptide KA6E may serve as model substrates for elastase and this could ultimately lead to applications in biomedicine and regenerative medicine.

Self-assembly of a Model Peptide incorporating a Hexa-Histidine sequence attached to an Oligo-Alanine sequence, and binding to Gold NTA/Nickel nanoparticles

Amyloid fibrils are formed by a model surfactant-like peptide (Ala)10-(His)6 containing a hexahistidine tag. This peptide undergoes a remarkable two-step self-assembly process with two distinct critical aggregation concentrations (cac’s), probed by fluorescence techniques. A micromolar range cac is ascribed to the formation of prefibrillar structures, whereas a millimolar range cac is associated with the formation of well-defined but more compact fibrils. We examine the labeling of these model tagged amyloid fibrils using Ni-NTA functionalized gold nanoparticles (Nanogold). Successful labeling is demonstrated via electron microscopy imaging. The specificity of tagging does not disrupt the β-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.

Secondary cyclogenesis along an occluded front leading to damaging wind gusts: windstorm Kyrill, January 2007

Windstorm Kyrill affected large parts of Europe in January 2007 and caused widespread havoc and loss of life. In this study the formation of a secondary cyclone, Kyill II, along the occluded front of the mature cyclone Kyrill and the occurrence of severe wind gusts as Kyrill II passed over Germany are investigated with the help of high-resolution regional climate model simulations. Kyrill underwent an explosive cyclogenesis south of Greenland as the storm crossed polewards of an intense upper-level jet stream. Later in its life cycle secondary cyclogenesis occurred just west of the British Isles. The formation of Kyrill II along the occluded front was associated (a) with frontolytic strain and (b) with strong diabatic heating in combination with a developing upper-level shortwave trough. Sensitivity studies with reduced latent heat release feature a similar development but a weaker secondary cyclone, revealing the importance of diabatic processes during the formation of Kyrill II. Kyrill II moved further towards Europe and its development was favored by a split jet structure aloft, which maintained the cyclone’s exceptionally deep core pressure (below 965 hPa) for at least 36 hours. The occurrence of hurricane force winds related to the strong cold front over North and Central Germany is analyzed using convection-permitting simulations. The lower troposphere exhibits conditional instability, a turbulent flow and evaporative cooling. Simulation at high spatio-temporal resolution suggests that the downward mixing of high momentum (the wind speed at 875 hPa widely exceeded 45 m s-1) accounts for widespread severe surface wind gusts, which is in agreement with observed widespread losses.

New insights into the structure of exotic nuclei using the RISING active stopper

This conference paper outlines the operation and some of the preliminary physics results using the GSI RISING active stopper. Data are presented from an experiment using combined isomer and beta‐delayed gamma‐ray spectroscopy to study low‐lying spectral and decay properties of heavy‐neutron‐rich nuclei around A∼190 produced following the relativistic projectile fragmentation of 208Pb primary beam. The response of the RISING active stopper detector is demonstrated for both the implantation of heavy secondary fragments and in‐situ decay of beta‐particles. Beta‐delayed gamma‐ray spectroscopy following decays of the neutron‐rich nucleus 194Re is presented to demonstrate the experimental performance of the set‐up. The resulting information inferred from excited states in the W and Os daughter nuclei is compared with results from Skyrme Hartree‐Fock predictions of the evolution of nuclear shape.

Self-assembled arginine-capped peptide bolaamphiphile nanosheets for cell culture and controlled wettability surfaces

The spontaneous assembly of a peptide bolaamphiphile in water, namely, RFL4FR (R, arginine; F, phenylalanine; L, leucine) is investigated, along with its novel properties in surface modification and usage as substrates for cell culture. RFL4FR self-assembles into nanosheets through lateral association of the peptide backbone. The L4 sequence is located within the core of the nanosheets, whereas the R moieties are exposed to the water at the surface of the nanosheets. Kinetic assays indicate that the self-assembly is driven by a remarkable two-step process, where a nucleation phase is followed by fast growth of nanosheets with an autocatalysis process. The internal structure of the nanosheets is formed from ultrathin bolaamphiphile monolayers with a crystalline orthorhombic symmetry with cross-β organization. We show that human corneal stromal fibroblast (hCSF) cells can grow on polystyrene films coated with films dried from RFL4FR solutions. For the first time, this type of amphiphilic peptide is used as a substrate to modulate the wettability of solid surfaces for cell culture applications.

Genetic algorithm inversion of the average 1D crustal structure using local and regional earthquakes

Knowing the best 1D model of the crustal and upper mantle structure is useful not only for routine hypocenter determination, but also for linearized joint inversions of hypocenters and 3D crustal structure, where a good choice of the initial model can be very important. Here, we tested the combination of a simple GA inversion with the widely used HYPO71 program to find the best three-layer model (upper crust, lower crust, and upper mantle) by minimizing the overall P- and S-arrival residuals, using local and regional earthquakes in two areas of the Brazilian shield. Results from the Tocantins Province (Central Brazil) and the southern border of the Sao Francisco craton (SE Brazil) indicated an average crustal thickness of 38 and 43 km, respectively, consistent with previous estimates from receiver functions and seismic refraction lines. The GA + HYPO71 inversion produced correct Vp/Vs ratios (1.73 and 1.71, respectively), as expected from Wadati diagrams. Tests with synthetic data showed that the method is robust for the crustal thickness, Pn velocity, and Vp/Vs ratio when using events with distance up to about 400 km, despite the small number of events available (7 and 22, respectively). The velocities of the upper and lower crusts, however, are less well constrained. Interestingly, in the Tocantins Province, the GA + HYPO71 inversion showed a secondary solution (local minimum) for the average crustal thickness, besides the global minimum solution, which was caused by the existence of two distinct domains in the Central Brazil with very different crustal thicknesses. (C) 2010 Elsevier Ltd. All rights reserved.

THE ENDOPARASITE PILOSTYLES ULEI (APODANTHACEAE - CUCURBITALES) INFLUENCES WOOD STRUCTURE IN THREE HOST SPECIES OF MIMOSA

Pilostyles species (Apodanthaceae) are endoparasites in stems of the plant family Fabaceae. The body comprises masses of parenchyma in the host bark and cortex, with sinkers, comprising groups of twisted tracheal elements surrounded by parenchyma that enter the secondary xylem of the host plant. Here we report for the first time the effects of Pilostyles parasitism on host secondary xylem. We obtained healthy and parasitized stems from Mimosa foliolosa, M. maguirei and M. setosa and compared vessel element length, fiber length, vessel diameter and vessel frequency, measured through digital imaging. Also, tree height and girth were compared between healthy and parasitized M. setosa. When parasitized, plant size, vessel diameter, vessel element length and fiber length are all less than in healthy plants. Also, vessel frequency is greater and vessels are narrower in parasitized stems. These responses to parasitism are similar to those observed in stressed plants. Thus, hosts respond to the parasite by changing its wood micromorphology in favour of increased hydraulic safety.

Edge effects as the principal cause of area effects on birds in fragmented secondary forest

Bird communities in tropical forests are strongly affected by both patch area and habitat edges. The fact that both effects are intrinsically confounded in space raises questions about how these two widely reported ecological patterns interact, and whether they are independent or simply different spatial manifestations of the same phenomenon. Moreover, do small patches of secondary forest, in landscapes where the most sensitive species have gone locally extinct, exhibit similar patterns to those previously observed in fragmented and continuous primary forests? We addressed these questions by testing edge-related differences in vegetation structure and bird community composition at 31 sites in fragmented and continuous landscapes in the imperilled Atlantic forest of Brazil. Over a two-year period, birds were captured with mist nets to a standardized effort of 680 net-hours at each site (similar to 22 000 net-hours resulting in 3381 captures from 114 species). We found that the bird community in patches of secondary forest was degraded in species composition compared to primary continuous forest, but still exhibited a strong response to edge effects. In fragmented secondary forests, edge and area effects also interacted, such that the magnitude of edge to interior differences on bird community composition declined markedly with patch size. The change in bird species composition between forest interiors and edges was similar to the change in community composition between large and small patches (because species had congruent responses to edge and area), but after controlling for edge effects community composition was no longer affected by patch area. Our results show that although secondary forests hold an impoverished bird community, ecological patterns such as area and edge effects are similar to those reported for primary forests. Our data provide further evidence that edge effects are the main drivers of area effects in fragmented landscapes.

Forest structure in a mosaic of rainforest sites: The effect of fragmentation and recovery after clear cut

A variety of human-induced disturbances such as forest fragmentation and recovery after deforestation for pasture or agricultural activities have resulted in a complex landscape mosaic in the Una region of northeastern Brazil. Using a set of vegetation descriptors, we investigated the main structural changes observed in forest categories that comprise the major components of the regional landscape and searched for potential key descriptors that could be used to discriminate among different forest categories. We assessed the forest structure of five habitat categories defined as (I) interiors and (2) edges of large fragments of old-growth forest (>1000 ha), (3) interiors and (4) edges of small forest fragments (<100 ha), and (5) early secondary forests. Forest descriptors used here were: frequency of herbaceous lianas and woody climbers, number of standing dead trees, number of fallen trunks, litter depth, number of pioneer plants (early secondary and shade-intolerant species), vertical foliage stratification profile and distribution Of trees in different diameter classes. Edges and interiors of forest fragments were significantly different only in the number of standing dead trees. Secondary forests and edges of fragments showed differences in litter depth, fallen trunks and number of pioneer trees, and secondary forests were significantly different from fragment interiors in the number of standing dead trees and the number of pioneer trees. Horizontal and vertical structure evaluated via ordination analysis showed that fragment interiors, compared to secondary forests, were characterized by a greater number of medium (25-35 cm) and large (35-50 cm) trees and smaller numbers of thin trees (5-10 cm). There was great heterogeneity at the edges of small and large fragments, as these sites were distributed along almost the entire gradient. Most interiors of large and small fragments presented higher values of foliage densities at higher strata ( 15-20 m and at 20-25 m height), and lower densities at 1-5 m. All secondary forests and some fragment edge sites showed an opposite tendency. A discriminant function highlighted differences among forest categories, with transects of large fragment interiors and secondary forests representing two extremes along a disturbance gradient determined by foliage structure (densities at 15-20 m and 20-25 m), with the edges of both large and small fragments and the interiors of small fragments scattered across the gradient. The major underlying processes determining patterns of forest disturbance in the study region are discussed, highlighting the importance of forest fragments, independently of its size, as forests recovery after clear cut show a greatly distinct structure, with profound implications on fauna movements. (C) 2009 Elsevier BY. All rights reserved.

(1)H, (15)N and (13)C assignments of the Rhipicephalus (Boophilus) microplus anti-microbial peptide microplusin

Microplusin, a Rhipicephalus (Boophilus) microplus anti-microbial peptide (AMP) is the first member of a new family of cysteine-rich AMPs with histidine-rich regions at the N- and C-termini, which is being fully characterized by biophysical and biochemical methods. Here we report the NMR resonance assignments for (1)H, (15)N, and (13)C nuclei in the backbone and side chains of the microplusin as basis for further studies of structure, backbone dynamics and interactions mapping.

Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds

Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85 angstrom. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. (C) 2009 Elsevier Inc. All rights reserved.

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

Polyaniline/layered zirconium phosphate nanocomposites: Secondary-like doped polyaniline obtained by the layer-by-layer technique

In the present work, nanocomposites of polyaniline (PANI) and layered alpha-Zr(HPO4)(2).H2O (alpha-ZrP) were prepared using two different approaches: (i) the in situ aniline polymerization in the presence of the layered inorganic material and (ii) the layer-by-layer (LBL) assembly using an aqueous solution of the polycation emeraldine salt (ES-PANI) and a dispersion of exfoliated negative slabs of alpha-ZrP. These materials were characterized spectroscopically using mainly resonance Raman scattering at four exciting radiations and electronic absorption in the UV-VIS-NIR region. Structural and textural characterizations were carried out using powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). The polymer obtained by the in situ aniline polymerization is located primarily in the external surface of the inorganic material although aniline monomers were intercalated between alpha-ZrP interlayer regions before oxidative polymerization. Through resonance Raman spectroscopy, it was observed that the formed polymer has semiquinone units (ES-PANI) and also azo bonds (-N = N-), showing that this method results in a polymer with a different structure from the usual ""head-to-tail"" ES-PANI. The LBL assembly of pre-formed ES-PANI and exfoliated alpha-ZrP particles produces homogeneous films with reproducible deposition from layer to layer, up to 20 bilayers. Resonance Raman (lambda(0) = 632.8 nm) spectrum of PANI/ZrP LBL film shows an enhancement in the intensity of the polaronic band at 1333 cm(-1) (nu C-N center dot+) and the decrease of the band intensity at 1485 cm(-1) compared to bulk ES-PANI. Its UV-VIS-NIR spectrum presents an absorption tail in the NIR region assigned to delocalized free charge carrier. These spectroscopic features are characteristic of highly conductive secondary doped PANI suggesting that polymeric chains in PANI/ZrP LBL film have a more extended conformation than in bulk ES-PANI.