ÿþThe network root based on puma alife the outgroup Puma yagouaroundi indicates that the most ancestral lineage of current pumas is located primarily in central South America. When we considered M. trumani as the outgroup, once again the rooting was placed primarily in South America, reasserting the area of occurrence of the earliest ancestor of the current lineages and helping reveal the direction of evolution of pumas throughout their distribution.

Our next step was to assess the role of major rivers as historical barriers to female gene flow. Our sampling allowed a test of two riverine barriers proposed by Culver et al. (2000) , which postulated that the Amazon and Paraná rivers might have induced the differentiation of historical population units. Our results supported an important role for the Amazon River, corroborating the hypothesis previously raised for pumas ( Culver et al. , 2000 ), and showing a pattern consistent with inferences made for other Neotropical felids puma athletic shoes such as Leopardus pardalis , L. wiedii and Panthera onca ( Eizirik et al. , 1998 , 2001 ).

Such an observation has implications for the definition and delimitation of puma subspecies, two of which ( P. c. cabrerae and P. c. capricornensis ) have been suggested to be separated by puma basket black this river ( Culver et al. , 2000 ).The t MRCA estimates derived from both data sets were very similar, indicating that the age of the common ancestor for all samples was essentially the same as that for the South American subset. Taken together with the other results ( e.g. network, diversity indices), this finding supports the inference that most of the extant diversity of the puma mtDNA resides in South America, and that the deepest history of coalescence of its lineages is fully represented in this subcontinent.

The signal of demographic expansion on both subcontinents suggests that this process seems to have puma basket bow occurred simultaneously, after the last glacial maximum. This inference is consistent with the hypothesis raised by Culver et al. (2000) , of an extinction of North American pumas in the late Pleistocene, followed by re-colonization from South America. Our results corroborate and extend this hypothesis, showing evidence for a substantial and very recent demographic expansion in South America, which likely preceded or included the re-colonization of North America. Such a pattern was not clearly observed in previous studies and sheds light onto the historical demography of present-day puma lineages.

Scoring was performed by a pathologist. Histologic scores (H-Scores) were computed from both % positivity (A%, A=1-100) and intensity (B=0-3) using the equation, H Score=A × B, according to a well-established IHC scoring system [ 30 ].This was performed via mitochondrial fractionation using an assay kit from Pierce (Rockford, IL), according to manufacture's instructions, to yield mitochondrial and non-mitochondrial fractions. Both fractions were subjected to protein extraction using 1% SDS/0.1% NP-40 and sonication followed by centrifugation for 20 min at 15,000 xg at 4°C. In these studies, we subjected 25% of the mitochondrial proteins and 2.5% of the non-mitochondrial proteins to western blotting.

In contrast, these cells express low levels of Bad, Bim and Bok. To determine puma basket heart black whether EGFRvIII also co-expresses with proapoptotic proteins similar to EGFR, we examined three EGFRvIII-carrying GBM xenografts since EGFRvIII expression is not maintained in vitro [ 35 ]. The results ( Fig. 1b ) showed that these EGFRvIII-expressing GBM xenografts also express PUMA, Bax and Bmf. We also determined PUMA expression levels in GBM xenografts with high and low levels of EGFRvIII and the results ( Fig. S1 in Supplemental Data ) showed that D-317 MG xenograft co-expressed EGFRvIII and PUMA and that D-320 MG and D-456 MG xenografts with undetectable EGFR/EGFRvIII expression expressed PUMA at low and high levels, respectively.