Where Does All the Poison Go? Investigating Toxicokinetics of Newt (Taricha) Tetrodotoxin (TTX) in Garter Snakes (Thamnophis).

Animals that consume toxic diets provide models for understanding the molecular and physiological adaptations to ecological challenges. Garter snakes (Thamnophis) in western North America prey on Pacific newts (Taricha), which employ tetrodotoxin (TTX) as an antipredator defense. These snakes possess mutations in voltage-gated sodium channels (Nav), the molecular targets of TTX, that decrease the binding ability of TTX to sodium channels (target-site resistance). However, genetic variation at these loci that cannot explain all the phenotypic variation in TTX resistance in Thamnophis. We explored a separate means of resistance, toxin metabolism, to determine if TTX-resistant snakes either rapidly remove TTX or sequester TTX. We examined the metabolism and distribution of TTX in the body (toxicokinetics), to determine differences between TTX-resistant and TTX-sensitive snakes in the rates at which TTX is eliminated from organs and the whole body (using TTX half-life as our metric). We assayed TTX half-life in snakes from TTX-resistant and TTX-sensitive populations of three garter snake species with a coevolutionary history with newts (T. atratus, T. couchii, T. sirtalis), as well as two non-resistant "outgroup" species (T. elegans, Pituophis catenifer) that seldom (if ever) engage newts. We found TTX half-life varied across species, populations, and tissues. Interestingly, TTX half-life was shortest in T. elegans and P. catenifer compared to all other snakes. Furthermore, TTX-resistant populations of T. couchii and T. sirtalis eliminated TTX faster (shorter TTX half-life) than their TTX-sensitive counterparts, while populations of TTX-resistant and TTX-sensitive T. atratus showed no difference rates of TTX removal (same TTX half-life). The ability to rapidly eliminate TTX may have permitted increased prey consumption, which may have promoted the evolution of additional resistance mechanisms. Finally, snakes still retain substantial amounts of TTX, and we projected that snakes could be dangerous to their own predators days to weeks following the ingestion of a single newt. Thus, aspects of toxin metabolism may have been key in driving predator-prey relationships, and important in determining other ecological interactions.

Phenotypic and functional variation in venom and venom resistance of two sympatric rattlesnakes and their prey.

Predator-prey interactions often lead to the coevolution of adaptations associated with avoiding predation and, for predators, overcoming those defences. Antagonistic coevolutionary relationships are often not simple interactions between a single predator and prey but rather a complex web of interactions between multiple coexisting species. Coevolution between venomous rattlesnakes and small mammals has led to physiological venom resistance in several mammalian taxa. In general, viperid venoms contain large quantities of snake venom metalloproteinase toxins (SVMPs), which are inactivated by SVMP inhibitors expressed in resistant mammals. We explored variation in venom chemistry, SVMP expression, and SVMP resistance across four co-distributed species (California Ground Squirrels, Bryant's Woodrats, Southern Pacific Rattlesnakes, and Red Diamond Rattlesnakes) collected from four different populations in Southern California. Our aim was to understand phenotypic and functional variation in venom and venom resistance in order to compare coevolutionary dynamics of a system involving two sympatric predator-prey pairs to past studies that have focused on single pairs. Proteomic analysis of venoms indicated that these rattlesnakes express different phenotypes when in sympatry, with Red Diamonds expressing more typical viperid venom (with a diversity of SVMPs) and Southern Pacifics expressing a more atypical venom with a broader range of non-enzymatic toxins. We also found that although blood sera from both mammals were generally able to inhibit SVMPs from both rattlesnake species, inhibition depended strongly on the snake population, with snakes from one geographic site expressing SVMPs to which few mammals were resistant. Additionally, we found that Red Diamond venom, rather than woodrat resistance, was locally adapted. Our findings highlight the complexity of coevolutionary relationships between multiple predators and prey that exhibit similar offensive and defensive strategies in sympatry.

Therapeutic levels of human factor VIII in mice implanted with encapsulated cells: potential for gene therapy of haemophilia A.

BACKGROUND: A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein has been evaluated. The microcapsules are implanted intraperitoneally. In order to prevent cell immune rejection, cells are enclosed in non-antigenic biocompatible alginate microcapsules prior to their implantation into mice. It has been shown that encapsulated myoblasts can deliver therapeutic levels of Factor IX (FIX) in mice. The delivery of human Factor VIII (hFVIII) in mice using microcapsules was evaluated in this study. METHODS: Mouse C2C12 myoblasts and canine MDCK epithelial kidney cells were transduced with MFG-FVIII (B-domain deleted) vector. Selected recombinant clones were enclosed in alginate microcapsules. Encapsulated recombinant clones were subsequently implanted intraperitoneally into C57BL/6 and immunodeficient SCID mice. RESULTS: Plasma of mice receiving C2C12 and encapsulated MDCK cells had transient therapeutic levels of FVIII in immunocompetent C57BL/6 mice (up to 20% and 7% of physiological levels, respectively). In addition, FVIII delivery in SCID mice was also transient, suggesting that a non-immune mechanism must have contributed to the decline of hFVIII in plasma. Quantitative RT-PCR analysis confirmed directly that the decline of hFVIII is due to a reduction in steady-state hFVIII mRNA, consistent with transcriptional repression. Furthermore, encapsulated cells retrieved from implanted mice were viable, but secreted FVIII ex vivo at three-fold lower levels than the pre-implantation levels. In addition, antibodies to hFVIII were detected in immunocompetent C57BL/6 mice. CONCLUSIONS: Implantable microcapsules can deliver therapeutic levels of FVIII in mice, suggesting the potential of this gene therapy approach for haemophilia A. The findings suggest vector down-regulation in vivo.