Diversification of New Zealand weta (Orthoptera: Ensifera: Anostostomatidae) and their relationships in Australasia

New Zealand taxa from the Orthopteran family Anostostomatidae have been shown to consist of three broad groups, Hemiandrus (ground weta), Anisoura/Motuweta (tusked weta) and Hemideina-Deinacrida (tree-giant weta). The family is also present in Australia and New Caledonia, the nearest large land masses to New Zealand. All genera are endemic to their respective countries except Hemiandrus that occurs in New Zealand and Australia. We used nuclear and mitochondrial DNA sequence data to study within genera and among species-level genetic diversity within New Zealand and to examine phylogenetic relationships of taxa in Australasia. We found the Anostostomatidae to be monophyletic within Ensifera, and justifiably distinguished from the Stenopelmatidae among which they were formerly placed. However, the New Zealand Anostostomatidae are not monophyletic with respect to Australian and New Caledonian species in our analyses. Two of the New Zealand groups have closer allies in Australia and one in New Caledonia. We carried out maximum-likelihood and Bayesian analyses to reveal several well supported subgroupings. Our analysis included the most extensive sampling to date of Hemiandrus species and indicate that Australian and New Zealand Hemiandrus are not monophyletic. We used molecular dating approaches to test the plausibility of alternative biogeographic hypotheses for the origin of the New Zealand anostostomatid fauna and found support for divergence of the main clades at, or shortly after, Gondwanan break-up, and dispersal across the Tasman much more recently.     

Developing a Social,Cultural and Economic Report Card for a Regional Industrial Harbour

Report cards are increasingly used to provide ongoing snap-shots of progress towards specific ecosystem health goals, particularly in coastal regions where planners need to balance competing demands for coastal resources from a range of industries. While most previous report cards focus on the biophysical components of the system, there is a growing interest in including the social and economic implications of ecosystem management to provide a greater social-ecological system understanding. Such a report card was requested on the Gladstone Harbour area in central Queensland, Australia. Gladstone Harbour adjoins the southern Great Barrier Reef, and is also a major industrial and shipping port. Balancing social, economic and environmental interests is therefore of great concern to the regional managers. While environmental benchmarking procedures are well established within Australia (and elsewhere), a method for assessing social and economic performance of coastal management is generally lacking. The key aim of this study was to develop and pilot a system for the development of a report card relating to appropriate cultural, social and economic objectives. The approach developed uses a range of multicriteria decision analysis methods to assess and combine different qualitative and quantitative measures, including the use of Bayesian Belief Networks to combine the different measures and provide an overall quantitative score for each of the key management objectives. The approach developed is readily transferable for purposes of similar assessments in other regions.     

Extracellular Loops 2 and 4 of GLYT2 Are Required for N-Arachidonylglycine Inhibition of Glycine Transport

Concentrations of extracellular glycine in the central nervous system are regulated by Na⁺/Cl⁻-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-γ-aminobutyric acid and N-arachidonyl-d-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-l-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg⁵³¹, Lys⁵³², and Ile⁵⁴⁵ that contribute to the differences in NAGly sensitivity.     

Distinct conformational states mediate the transport and anion channel properties of the glutamate transporter EAAT-1

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+), 1 H(+), and the counter-transport of 1 K(+) ion. In addition to coupled ion fluxes, glutamate and Na(+) binding to the transporter activates a thermodynamically uncoupled anion conductance through the transporter. In this study, we have distinguished between these two conductance states of the EAAT-1 transporter using a [2-(trimethylammonium)ethyl]methanethiosulfonate-modified V452C mutant transporter. Glutamate binds to the modified mutant transporter and activates the uncoupled anion conductance but is not transported. The selective alteration of the transport function without altering the anion channel function of the V452C mutant transporter suggests that the two functions are generated by distinct conformational states of the transporter.     

Taking the cell by stealth or storm? Protein transduction domains (PTDs) as versatile vectors for delivery

A cell delivery system is increasing in use in many areas of cell and molecular biology and bio-medicine. This system is based on a number of naturally occurring protein motifs and/or sequences which show the remarkable ability to rapidly cross the mammalian cell membrane without compromising its structure or function. These so-called Protein Transduction Domains (PTDs) offer unprecedented advantages for intracellular delivery. These advantages include, but are not limited to, their applicability to all cell types (no cell type has yet been described which is not transduced by these PTDs), and the range of cargoes that can be transduced (including peptides, small proteins, full-length enzymes, DNA oligomers, peptide-nucleic acid oligomers, liposomes, and magnetic nanoparticles). Furthermore, the PTDs have been demonstrated to be suitable for in vivo delivery including delivery across the blood brain barrier, and have been shown to cross the plasma membrane rapidly and enter the cytoplasm and nuclear regions of the cell. In this review, the general properties of the most commonly used PTDs are described. The strategies currently being undertaken also highlight that improvements in membrane transduction are possible despite our lack of understanding of the exact biochemical and/or physical mechanisms of transduction. Recent examples of the range of potential applications are also discussed.     

The proliferation of mouse mammary epithelial cells in response to specific mitogens is modulated by the mammary fat pad in vitro

Summary The ability of the murine mammary fat pad to directly stimulate the growth of mammary epithelial cells and to modulate the effects of various mammogenic agents has been investigated in a newly described, hormone- and serum-free coculture system. COMMA-1D mouse mammary epithelial cells were cultured for 5 or 7 d with various supplements in the absence or presence of epithelium-free mammary fat pad explants from virgin female BALB/c mice. Cocultured fat pad stimulated increases in the DNA content of COMMA-1D cultures by two- to threefold or six-to eightfold after 5 or 7 d, respectively. The mitogenic effect was additive to that of 10% fetal calf serum and could not be attributed to the release of prostaglandin E₂ or synthesis of prostaglandins by epithelial cells. In addition, bovine serum albumin attenuated (P<0.05) the mitogenic effect of cocultured mammary fat pad. Added alone, insulinlike growth factor-I, epidermal growth factor, and insulin increased (P<0.05) total DNA of COMMA-1D cultures by 2.5-, 3.7-, and 2.3-fold, respectively. Cocultured mammary fat pad markedly interacted (P<0.01) with these mitogens to yield final DNA values that were 21.2-, 13.3-, and 22.1-fold greater than in basal medium only. Associated with this proliferation was the formation of numerous domes above the COMMA-1D monolayer. There was no proliferative response to growth hormone or prolactin in the absence or presence of cocultured fat pad (P>0.05). Whereas hydrocortisone did not alter cell number, it attenuated (P<0.05) the mitogenic effect of cocultured mammary fat pad. These results indicate that the murine mammary fat pad is not only a direct source of mitogenic activity, but also modulates the response of mammary epithelial cells to certain mammogens.     

Identification of a 3rd Na+ Binding Site of the Glycine Transporter,GlyT2

The Na⁺/Cl^- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na⁺ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na⁺ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na⁺-dependent leucine transporter, LeuT_Aa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na⁺ binding sites in GlyT2, but the physical location of the third Na⁺ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuT_Aa. Although betaine transport by BetP is coupled to the co-transport of two Na⁺ ions, the first Na⁺ site is not conserved between BetP and LeuT_Aa, the so called Na1'' site. We hypothesized that the third Na⁺ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1'' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na⁺ placed at the location consistent with the Na1'' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na⁺ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.     

Multiple plant traits influence community composition of insect herbivores: a comparison of two understorey shrubs

Structural and nutritional plant traits influence the ability of insect herbivores to locate, consume and persist on their hosts yet it is uncommon for ecologists to consider how multiple plant traits influence insect community composition. We sampled herbivorous insects on two understorey shrub species common to eucalypt forests of south-eastern Australia, namely Cassinia arcuata (Asteraceae) and Daviesia ulicifolia (Fabaceae). Regression analyses were used to assess the relative influence of plant structure (canopy volume), nutritional quality (macronutrients and total phenolics) and plant productivity (leaf litter) on insect abundance and species richness. Total N content of D. ulicifolia was significantly higher than C. arcuata, while the concentrations of P, K, Ca and Mg were higher in C. arcuata. Total phenolics and leaf litter were significantly lower in D. ulicifolia compared to C. arcuata. Insect composition was similar between the two shrubs but C. arcuata supported greater abundances. Canopy volume and the macronutrients P and Ca were important predictors of insect abundance on C. arcuata, whereas canopy volume alone, but neither plant productivity nor macronutrients, influenced the abundance of insects on D. ulicifolia. Ca was an important predictor of insect species richness on C. arcuata and P was an important predictor on D. ulicifolia. By quantifying a range of plant traits, we have provided an understanding of factors likely to influence the composition of herbivorous insects inhabiting these two shrubs. Traits including leaf architecture, foliar morphology and volatile terpenoids may yet explain the greater number of insects on C. arcuata since they influence the availability of microhabitats and apparency of host plants.     

Cryptic lineage diversity,body size divergence,and sympatry in a species complex of Australian lizards (Gehyra)

Understanding the joint evolutionary and ecological underpinnings of sympatry among close relatives remains a key challenge in biology. This problem can be addressed through joint phylogenomic and phenotypic analysis of complexes of closely related lineages within, and across, species and hence representing the speciation continuum. For a complex of tropical geckos from northern Australia—Gehyra nana and close relatives—we combine mtDNA phylogeography, exon‐capture sequencing, and morphological data to resolve independently evolving lineages and infer their divergence history and patterns of morphological evolution. Gehyra nana is found to include nine divergent lineages and is paraphyletic with four other species from the Kimberley region of north‐west Australia. Across these 13 taxa, 12 of which are restricted to rocky habitats, several lineages overlap geographically, including on the diverse Kimberley islands. Morphological evolution is dominated by body size shifts, and both body size and shape have evolved gradually across the group. However, larger body size shifts are observed among overlapping taxa than among closely related parapatric lineages of G. nana, and sympatric lineages are more divergent than expected at random. Whether elevated body size differences among sympatric lineages are due to ecological sorting or character displacement remains to be determined.     

Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer

The aspartate transporter from Pyrococcus horikoshii (Glt_Ph) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of Glt_Ph provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting Glt_Ph into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of Glt_Ph were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of Glt_Ph revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.