Analysis of the hypoxia-activated dinitrobenzamide mustard phosphate pre-prodrug PR-104 and its alcohol metabolite PR-104A in plasma and tissues by liquid chromatography-mass spectrometry

PR-104 is a dinitrobenzamide mustard pre-prodrug that is activated by reduction to a cytotoxic hydroxylamine metabolite in hypoxic tumour cells; it has recently commenced Phase I clinical trial. Here, we report two validated methods for the determination of PR-104 and its alcohol hydrolysis product, PR-104A in plasma and tissues across species. A high pH LC/MS/MS method was optimised for rapid and sensitive analysis of both analytes in rat, dog and human plasma. This assay was linear over the concentration range 0.005-2.5 microg/ml for PR-104 and 0.05-25 microg/ml for PR-104A (0.005-2.5 microg/ml for rat). A second method, using a low pH LC separation, was designed to provide higher chromatographic resolution, facilitating identification of metabolites. Both methods were successfully applied to the plasma pharmacokinetics of PR-104 and PR-104A in rats. In addition, cytotoxic reduced metabolites of PR-104A were identified in human tumour xenografts by the higher chromatographic resolution method.     

Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L

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A biogeographic reversal in sexual size dimorphism along a continental temperature gradient

The magnitude and direction of sexual size dimorphism (SSD) varies greatly across the animal kingdom, reflecting differential selection pressures on the reproductive and/or ecological roles of males and females. If the selection pressures and constraints imposed on body size change along environmental gradients, then SSD will vary geographically in a predictable way. Here, we uncover a biogeographical reversal in SSD of lizards from Central and North America: in warm, low latitude environments, males are larger than females, but at colder, high latitudes, females are larger than males. Comparisons to expectations under a Brownian motion model of SSD evolution indicate that this pattern reflects differences in the evolutionary rates and/or trajectories of sex‐specific body sizes. The SSD gradient we found is strongly related to mean annual temperature, but is independent of species richness and body size differences among species within grid cells, suggesting that the biogeography of SSD reflects gradients in sexual and/or fecundity selection, rather than intersexual niche divergence to minimize intraspecific competition. We demonstrate that the SSD gradient is driven by stronger variation in male size than in female size and is independent of clutch mass. This suggests that gradients in sexual selection and male–male competition, rather than fecundity selection to maximize reproductive output by females in seasonal environments, are predominantly responsible for the gradient.     

Host diversity and behavior determine patterns of interspecies transmission and geographic diffusion of avian influenza A subtypes among North American wild reservoir species

Wild birds can carry avian influenza viruses (AIV), including those with pandemic or panzootic potential, long distances. Even though AIV has a broad host range, few studies account for host diversity when estimating AIV spread. We analyzed AIV genomic sequences from North American wild birds, including 303 newly sequenced isolates, to estimate interspecies and geographic viral transition patterns among multiple co-circulating subtypes. Our results show high transition rates within Anseriformes and Charadriiformes, but limited transitions between these orders. Patterns of transition between species were positively associated with breeding habitat range overlap, and negatively associated with host genetic distance. Distance between regions (negative correlation) and summer temperature at origin (positive correlation) were strong predictors of transition between locations. Taken together, this study demonstrates that host diversity and ecology can determine evolutionary processes that underlie AIV natural history and spread. Understanding these processes can provide important insights for effective control of AIV.     

Differential effects of the novel non-peptidic opioid 4-tyrosylamido-6-benzyl-1,2,3,4 tetrahydroquinoline (CGPM-9) on in vitro rat t lymphocyte and macrophage functions

Opioid receptors have been reported on immune cells of several species and shown to subserve effector functions of these cell types. Mu-selective opioid agonists such as morphine are immunosuppressive, whereas certain delta-opioid receptor-selective agonists have been associated with immunopotentiation. We have previously shown that intracerebroventricular administration of the non-peptidic delta-opioid receptor agonists did not alter certain parameters of immunocompetence. In this study, we evaluated the in vitro effects of the novel non-peptidic opioid 4-tyrosylamido-6-benzyl-1,2,3,4 tetrahydroquinoline (CGPM-9) on lymphocyte and macrophage functions. We demonstrated that CGPM-9 enhanced rat thymic lymphocyte proliferative response to concanavalin A (2.85- to 5.5-fold increases), and suppressed LPS-induced nitric oxide (67 to 72 percent reduction) and TNF-alpha production (46 percent reduction) by peritoneal macrophages, compared with untreated control. The mu-opioid receptor selective antagonist CTOP used at equimolar doses, significantly suppressed the effect of CGPM-9 on lymphocyte and macrophage functions (CTOP alone did not show any effect on lymphocyte or macrophage functions). In summary, CGPM-9 activated thymic lymphocyte proliferation and suppressed macrophage functions by acting at mu-opioid receptors. This suggests that opioid receptors on immunocytes may be coupled to different signaling pathways depending on the cell type and effector function being analyzed. The mechanism (s) associated with the differential effect of CGPM-9 on these immune cells remains to be elucidated. The pharmacotherapeutic potential for compounds such as CGPM-9 which potentiate T lymphocyte proliferation and suppress production of macrophage-derived inflammatory cytokines is substantial in research and clinical medicine.     

Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.