The origin and evolution of the surfactant system in fish: Insights into the evolution of lungs and swim bladders

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas-holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish ( the air-breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non-air-breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air-breathing organs of the other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP-B in tarpon and pirarucu lavage, and SPs ( A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

The chemistry and biology of human relaxin-3

A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin-3. Like human relaxin-1 and relaxin-2, relaxin-3 is predicted to consist of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin-1 and relaxin-2, however, relaxin-3 could not be successfully prepared by simple combination of the individual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S-protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin-3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin-3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin-3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin-1 or relaxin-2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three-dimensional structure of relaxin-3 and to identify key structural differences between the human relaxins.

Conformational changes involved in MscL channel gating measured using FRET spectroscopy

We demonstrate that fluorescence resonance energy transfer spectroscopy is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel of large conductance. Ensemble analysis is used to analyze the intensity of light emitted by AlexaFluor-labeled cysteine mutants reconstituted into artificial liposomes before and after acceptor photobleaching. The diameter of the protein is found to increase by 16 angstrom upon channel activation.

Versatile peptide dendrimers for nucleic acid delivery

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.

Efficiency, costs and trade-offs in marine reserve system design

With marine biodiversity conservation the primary goal for reserve planning initiatives, a site's conservation potential is typically evaluated on the basis of the biological and physical features it contains. By comparison, socio-economic information is seldom a formal consideration of the reserve system design problem and generally limited to an assessment of threats, vulnerability or compatibility with surrounding uses. This is perhaps surprising given broad recognition that the success of reserve establishment is highly dependent on widespread stakeholder and community support. Using information on the spatial distribution and intensity of commercial rock lobster catch in South Australia, we demonstrate the capacity of mathematical reserve selection procedures to integrate socio-economic and biophysical information for marine reserve system design. Analyses of trade-offs highlight the opportunities to design representative, efficient and practical marine reserve systems that minimise potential loss to commercial users. We found that the objective of minimising the areal extent of the reserve system was barely compromised by incorporating economic design constraints. With a small increase in area (< 3%) and boundary length (< 10%), the economic impact of marine reserves on the commercial rock lobster fishery was reduced by more than a third. We considered also how a reserve planner might prioritise conservation areas using information on a planning units selection frequency. We found that selection frequencies alone were not a reliable guide for the selection of marine reserve systems, but could be used with approaches such as summed irreplaceability to direct conservation effort for efficient marine reserve design.

Establishing representative no-take areas in the Great Barrier Reef : Large scale implementation of theory on marine protected areas

The Great Barrier Reef Marine Park, an area almost the size , of Japan, has a new network of no-take areas that significantly improves the protection of biodiversity. The new marine park zoning implements, in a quantitative manner, many of the theoretical design principles discussed in the literature. For example, the new network of no-take areas has at least 20% protection per bioregion, minimum levels of protection for all known habitats and special or unique features, and minimum sizes for no-take areas of at least 10 or 20 kat across at the smallest diameter Overall, more than 33% of the Great Barrier Reef Marine Park is now in no-take areas (previously 4.5%). The steps taken leading to this outcome were to clarify to the interested public why the existing level of protection wets inadequate; detail the conservation objectives of establishing new no-take areas; work with relevant and independent experts to define, and contribute to, the best scientific process to deliver on the objectives; describe the biodiversity (e.g., map bioregions); define operational principles needed to achieve the objectives; invite community input on all of The above; gather and layer the data gathered in round-table discussions; report the degree of achievement of principles for various options of no-take areas; and determine how to address negative impacts. Some of the key success factors in this case have global relevance and include focusing initial communication on the problem to be addressed; applying the precautionary principle; using independent experts; facilitating input to decision making; conducting extensive and participatory consultation; having an existing marine park that encompassed much of the ecosystem; having legislative power under federal law; developing high-level support; ensuring agency Priority and ownership; and being able to address the issue of displaced fishers.

Mapping water quality and substrate cover in optically complex coastal and reef waters: an integrated approach

Sustainable management of coastal and coral reef environments requires regular collection of accurate information on recognized ecosystem health indicators. Satellite image data and derived maps of water column and substrate biophysical properties provide an opportunity to develop baseline mapping and monitoring programs for coastal and coral reef ecosystem health indicators. A significant challenge for satellite image data in coastal and coral reef water bodies is the mixture of both clear and turbid waters. A new approach is presented in this paper to enable production of water quality and substrate cover type maps, linked to a field based coastal ecosystem health indicator monitoring program, for use in turbid to clear coastal and coral reef waters. An optimized optical domain method was applied to map selected water quality (Secchi depth, Kd PAR, tripton, CDOM) and substrate cover type (seagrass, algae, sand) parameters. The approach is demonstrated using commercially available Landsat 7 Enhanced Thematic Mapper image data over a coastal embayment exhibiting the range of substrate cover types and water quality conditions commonly found in sub-tropical and tropical coastal environments. Spatially extensive and quantitative maps of selected water quality and substrate cover parameters were produced for the study site. These map products were refined by interactions with management agencies to suit the information requirements of their monitoring and management programs. (c) 2004 Elsevier Ltd. All rights reserved.

Chemical and structural disorder in eumelanins: A possible explanation for broadband absorbance

We report the results of an experimental and theoretical study of the electronic and structural properties of a key eumelanin precursor-5,6,-dihydroxyindole-2-carboxylic acid ( DHICA) - and its dimeric forms. We have used optical spectroscopy to follow the oxidative polymerization of DHICA to eumelanin and observe red shifting and broadening of the absorption spectrum as the reaction proceeds. First principles density functional theory calculations indicate that DHICA oligomers ( possible reaction products of oxidative polymerization) have the gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital red-shifted gaps with respect to the monomer. Furthermore, different bonding configurations ( leading to oligomers with different structures) produce a range of gaps. These experimental and theoretical results lend support to the chemical disorder model where the broadband monotonic absorption characteristic of all melanins is a consequence of the superposition of a large number of nonhomogeneously broadened Gaussian transitions associated with each of the components of a melanin ensemble. These results suggest that the traditional model of eumelanin as an amorphous organic semiconductor is not required to explain its optical properties and should be thoroughly reexamined. These results have significant implications for our understanding of the physics, chemistry, and biological function of these important biological macromolecules. Indeed, one may speculate that the robust functionality of melanins in vitro is a direct consequence of its heterogeneity, i.e., chemical disorder is a "low cost" natural resource in these systems

C-terminal charged cluster of MscL, RKKEE, functions as a pH sensor

The RKKEE cluster of charged residues located within the cytoplasmic helix of the bacterial mechanosensitive channel, MscL, is essential for the channel function. The structure of MscL determined by x-ray crystallography and electron paramagnetic resonance spectroscopy has revealed discrepancies toward the C-terminus suggesting that the structure of the C-terminal helical bundle differs depending on the pH of the cytoplasm. In this study we examined the effect of pH as well as charge reversal and residue substitution within the RKKEE cluster on the mechanosensitivity of Escherichia coli MscL reconstituted into liposomes using the patch-clamp technique. Protonation of either positively or negatively charged residues within the cluster, achieved by changing the experimental pH or residue substitution within the RKKEE cluster, significantly increased the free energy of activation for the MscL channel due to an increase in activation pressure. Our data suggest that the orientation of the C-terminal helices relative to the aqueous medium is pH dependent, indicating that the RKKEE cluster functions as a proton sensor by adjusting the channel sensitivity to membrane tension in a pH-dependent fashion. A possible implication of our results for the physiology of bacterial cells is briefly discussed.

Effect of rainfall as a component of climate change on estuarine fish production in Queensland, Australia

The speculation that climate change may impact on sustainable fish production suggests a need to understand how these effects influence fish catch on a broad scale. With a gross annual value of A$ 2.2 billion, the fishing industry is a significant primary industry in Australia. Many commercially important fish species use estuarine habitats such as mangroves, tidal flats and seagrass beds as nurseries or breeding grounds and have lifecycles correlated to rainfall and temperature patterns. Correlation of catches of mullet (e.g. Mugil cephalus) and barramundi (Lates calcarifer) with rainfall suggests that fisheries may be sensitive to effects of climate change. This work reviews key commercial fish and crustacean species and their link to estuaries and climate parameters. A conceptual model demonstrates ecological and biophysical links of estuarine habitats that influences capture fisheries production. The difficulty involved in explaining the effect of climate change on fisheries arising from the lack of ecological knowledge may be overcome by relating climate parameters with long-term fish catch data. Catch per unit effort (CPUE), rainfall, the Southern Oscillation Index (SOI) and catch time series for specific combinations of climate seasons and regions have been explored and surplus production models applied to Queensland's commercial fish catch data with the program CLIMPROD. Results indicate that up to 30% of Queensland's total fish catch and up to 80% of the barramundi catch variation for specific regions can be explained by rainfall often with a lagged response to rainfall events. Our approach allows an evaluation of the economic consequences of climate parameters on estuarine fisheries. thus highlighting the need to develop forecast models and manage estuaries for future climate chan e impact by adjusting the quota for climate change sensitive species. Different modelling approaches are discussed with respect to their forecast ability. (c) 2006 Elsevier Ltd. All rights reserved.

Mechanics of cellular adhesion to artificial artery templates

We are using polymer templates to grow artificial artery grafts in vivo for the replacement of diseased blood vessels. We have previously shown that adhesion of macrophages to the template starts the graft formation. We present a study of the mechanics of macrophage adhesion to these templates on a single cell and single bond level with optical tweezers. For whole cells, in vitro cell adhesion densities decreased significantly from polymer templates polyethylene to silicone to Tygon (167, 135, and 65 cells/mm(2)). These cell densities were correlated with the graft formation success rate (50%, 25%, and 0%). Single-bond rupture forces at a loading rate of 450 pN/s were quantified by adhesion of trapped 2-mm spheres to macrophages. Rupture force distributions were dominated by nonspecific adhesion (forces, < 40 pN). On polystyrene, preadsorption of fibronectin or presence of serum proteins in the cell medium significantly enhanced adhesion strength from a mean rupture force of 20 pN to 28 pN or 33 pN, respectively. The enhancement of adhesion by fibronectin and serum is additive (mean rupture force of 43 pN). The fraction of specific binding forces in the presence of serum was similar for polystyrene and polymethyl-methacrylate, but specific binding forces were not observed for silica. Again, we found correlation to in vivo experiments, where the density of adherent cells is higher on polystyrene than on silica templates, and can be further enhanced by fibronectin adsorption. These findings show that in vitro adhesion testing can be used for template optimization and to substitute for in-vivo experiments.

Neuronal voltage-gated sodium channel subtypes: Key roles in inflammatory and neuropathic pain

Voltage-gated sodium channels (VGSCs) play an important role in neuronal excitability. Regulation of VGSC activity is a complex phenomenon that occurs at multiple levels in the cell, including transcriptional regulation, post-translational modification and membrane insertion and retrieval. Multiple VGSC subtypes exist that vary in their biophysical and pharmacological properties and tissue distribution. Any alteration of the VGSC subtype profile of a neuron or the mechanisms that regulate VGSC activity can cause significant changes in neuronal excitability. Inflammatory and neuropathic pain states are characterised by alterations in VGSC subtype composition and activity in sensory neurons. This review focuses on the VGSC subtypes involved in such pain states. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular dynamics study of MscL interactions with a curved lipid bilayer

Mechanosensitivity is a ubiquitous sensory mechanism found in living organisms. The simplest known mechanotransducing mechanism is found in bacteria in the form of the mechanosensitive membrane channel of large conductance, MscL. This channel has been studied extensively using a variety of methods at a functional and structural level. The channel is gated by membrane tension in the lipid bilayer alone. It serves as a safety valve protecting bacterial cells against hypoosmotic shock. MscL of Escherichia coli embedded in bilayers composed of asymmetric amounts of single-tailed and double-tailed lipids has been shown to gate spontaneously, even in the absence of membrane tension. To gain insight into the effect of the lipid membrane composition and geometry on MscL structure, a fully solvated, all-atom model of MscL in a stress-free curved bilayer composed of double- and single-tailed lipids was studied using a 9.5-ns molecular dynamics simulation. The bilayer was modeled as a domed structure accommodating the asymmetric composition of the monolayers. During the course of the simulation a spontaneous restructuring of the periplasmic loops occurred, leading to interactions between one of the loops and phospholipid headgroups. Previous experimental studies of the role of the loops agree with the observation that opening starts with a restructuring of the periplasmic loop, suggesting an effect of the curved bilayer. Because of limited resources, only one simulation of the large system was performed. However, the results obtained suggest that through the geometry and composition of the bilayer the protein structure can be affected even on short timescales.

Regional patterns of agricultural land use and deforestation in Colombia

An expanding human population and associated demands for goods and services continues to exert an increasing pressure on ecological systems. Although the rate of expansion of agricultural lands has slowed since 1960, rapid deforestation still occurs in many tropical countries, including Colombia. However, the location and extent of deforestation and associated ecological impacts within tropical countries is often not well known. The primary aim of this study was to obtain an understanding of the spatial patterns of forest conversion for agricultural land uses in Colombia. We modeled native forest conversion in Colombia at regional and national-levels using logistic regression and classification trees. We investigated the impact of ignoring the regional variability of model parameters, and identified biophysical and socioeconomic factors that best explain the current spatial pattern and inter-regional variation in forest cover. We validated our predictions for the Amazon region using MODIS satellite imagery. The regional-level classification tree that accounted for regional heterogeneity had the greatest discrimination ability. Factors related to accessibility (distance to roads and towns) were related to the presence of forest cover, although this relationship varied regionally. In order to identify areas with a high risk of deforestation, we used predictions from the best model, refined by areas with rural population growth rates of > 2%. We ranked forest ecosystem types in terms of levels of threat of conversion. Our results provide useful inputs to planning for biodiversity conservation in Colombia, by identifying areas and ecosystem types that are vulnerable to deforestation. Several of the predicted deforestation hotspots coincide with areas that are outstanding in terms of biodiversity value.