EVOLUTIONARY RELATIONSHIPS AMONG COLONIES OF COLUMBIAN GROUND SQUIRRELS AS SHOWN BY MITOCHONDRIAL DNA

Mitochondrial DNA (mtDNA) from 71 Columbian ground squirrels (Spermophilus columbianus) collected in 12 locations in western Canada were assayed for restriction-site variation with 10 endonucleases. Five of these endonucleases revealed variant patterns, and the composite genotypes were used to develop a linear transformation series among the mtDNA genotypes. Two of the four clones had a wide distribution, while the remaining two clones were geographically restricted. The mtDNA of Columbian ground squirrels was also compared to two other species of Sciuridae: Richardson's ground squirrels (S. richardsonii) and Arctic ground squirrels (S. parryii). Calculation of divergences from fragment length and restriction-site data indicated that Arctic ground squirrels and Richardson's ground squirrels were more closely related to each other than either was to Columbian ground squirrels. The transformation series among clones within the Columbian ground squirrels was rooted using Richardson's and Arctic ground squirrels as out-groups. From these data, we conclude that the colonization by female founders of Columbian ground squirrel populations occurred after deglaciation along the eastern ranges of the Rocky Mountains, while colonies on the western ranges may have been present before extensive deglaciation occurred, having existed in refugia in northwestern Alberta.     

Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.     

Molecular analysis of a new splice variant of the human melanocortin-1 receptor.

The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin-1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin-1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C-terminal tail of the melanocortin-1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin-1 receptor (melanocortin-1 receptor B) appears pharmacologically similar to the non-spliced melanocortin-1 receptor. Melanocortin-1 receptor B is expressed in testis, fetal heart and melanomas.     

Replacement of a native freshwater macroinvertebrate species by an invader: implications for biological water quality monitoring

The rate of freshwater invasions may be increasing, and macroinvertebrate invaders can have significant impacts on native macroinvertebrate assemblage structure through biotic interactions. More pollution-tolerant invaders can often replace native species. We examined implications of a species replacement for accurate biological monitoring of river systems using biotic indices. Our study uses Northern Ireland and the Isle of Man as examples of countries that possess river networks with many riverine macroinvertebrate assemblages subject to invasion. The introduced amphipod crustacean Gammarus pulex has replaced the native species G. duebeni celticus in many rivers in N. Ireland and the Isle of Man. Extensive seasonal data sets (119 sites) from three river networks, Lough Neagh and the Lagan in N. Ireland, and island-wide in the Isle of Man, were used to investigate the assumed equivalence of the native and invader in biotic indices concerned with the water quality monitoring system. Based on the derivation of the Biological Monitoring Working Party (BMWP) score, the Average Score Per Taxon (ASPT), as an example of a commonly used biotic index of water quality, we found index scores were lower in G. pulex sites compared to G. d. celticus-only sites. This indicated that assemblages were dominated by taxa more tolerant of organic pollution in the invader sites and more sensitive in the native sites. Inclusion of the invader in generation of the ASPT index, overinflated the ASPT values obtained compared to those with the native’s inclusion. This questions the accuracy of the ASPT and similar indices in rivers where the invader had replaced the native. We argue that with invasion pressures increasing, the validity of water quality indices such as the BMWP/ASPT needs to be re-examined in catchments where invaders have replaced natives. Indices such as the BMWP/ASPT are based on family level taxa and are inevitably coarse in their resolution given the wide range of water qualities tolerated by different genera within families. We argue that this resolution is even more compromised by the presence of very pollution-tolerant invaders, who may have replaced natives in disturbed or degraded river systems. The whole structure of water quality indices such as the BMWP/ASPT may need revising to take into account the presence of invasive species within monitored assemblages.     

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.     

Invader–invader interactions in relation to environmental heterogeneity leads to zonation of two invasive amphipods, Dikerogammarus villosus (Sowinsky) and Gammarus tigrinus Sexton: amphipod pilot species project (AMPIS) report 6

As biological invasions continue, interactions occur not only between invaders and natives, but increasingly new invaders come into contact with previous invaders. Whilst this can lead to species replacements, co-existence may occur, but we lack knowledge of processes driving such patterns. Since environmental heterogeneity can determine species richness and co-existence, the present study examines habitat use and its mediation of the predatory interaction between invasive aquatic amphipods, the Ponto-Caspian Dikerogammarus villosus and the N. American Gammarus tigrinus. In the Dutch Lake IJsselmeer, we found broad segregation of D. villosus and G. tigrinus by habitat type, the former predominating in the boulder zone and the latter in the soft sediment. However, the two species co-exist in the boulder zone, both on the short and longer terms. We used an experimental simulation of habitat heterogeneity and show that both species utilize crevices, different sized holes in a plastic grid, non-randomly. These amphipods appear to optimise the use of holes with respect to their ‘C-shape’ body size. When placed together, D. villosus adults preyed on G. tigrinus adults and juveniles, while G. tigrinus adults preyed on D. villosus juveniles. Juveniles were also predators and both species were cannibalistic. However, the impact on G. tigrinus of the superior intraguild predator, D. villosus, was significantly reduced where experimental grids were present as compared to absent. This mitigation of intraguild predation between the two species in complex habitats may explain the co-existence of these two invasive species.     

Exploring Hypotheses of the Actions of TGF-β1 in Epidermal Wound Healing Using a 3D Computational Multiscale Model of the Human Epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-β1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-β1 literature–derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units (keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-β1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged (by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-β1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-β1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing.     

The biosensor based on the pyruvate oxidase modified conducting polymer for phosphate ions determinations

An enzymatic biosensor was fabricated by the covalent immobilization of pyruvate oxidase (PyO) onto the nano-particle comprised poly-5,2':5',2'-terthiophene-3'-carboxylic acid, poly-TTCA (nano-CP) layers on a glassy carbon electrode (GCE) for the amperometric detection of the phosphate ions. The direct electron transfer reaction of the immobilized PyO onto the nano-CP layers was investigated and the electron transfer rate constant was determined to be 0.65 s(-1). The electrochemically prepared nano-CP lowered the oxidation potential (+0.40 V versus Ag/AgCl) of an enzymatically generated H(2)O(2) by PyO in a phosphate solution. Experimental parameters affecting the sensitivity of the biosensors, such as amounts of the cofactors, the pH, the applied potential, and the temperature were optimized. A linear response for the detection of the phosphate ion was observed between 1.0 microM and 100 microM and the detection limit was determined to be about 0.3 microM. The response time of the biosensors was about 6s. The biosensor showed good selectivity towards other interfering anions. The long-term storage stability of the phosphate biosensor was studied and the sensor was applied in a human serum sample for the phosphate ions detection.