Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in E. coli each of the woodrat proteins gave the characteristic maximum at 450 nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.

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; cGKI L2/L2 mice and cGKI-deficient bone marrow-chimeras. Accordingly, antibody-mediated blockade of interleukin-6 normalized platelet counts in cGKI-SM mice. Conclusions-Abnormal cGMP/cGKI signaling in nonhematopoietic cells affects thrombopoiesis via elevated interleukin-6production and results in thrombocytosis in vivo. Dysfunction of cGMP/cGKI signaling in nonhematopoietic cells contributes to a high platelet count, which is potentially associated with thrombosis. Zhang et al Thrombocytosis in cGKI-Deficient Mice 1821membrane system, and the synthesis of platelet-specific proteins and granules. 11 Together, these changes give rise to the platelet production machinery of mature megakaryocytes that release thrombocytes into the bone marrow (BM)-sinusoid vessels via long cytoplasmic processes, termed proplatelets. 12Fundamental functions of cAMP/cGMP signaling in mature platelets have been recognized for many years [13][14][15] ; however, the role of cAMP or cGMP in the maturation of megakaryocytes and the production of platelets in vivo are much less known. Recent in vitro findings indicate that platelet release from fetal liver cells-derived megakaryocytes is enhanced by high cGMP, but blocked by high cAMP levels in response to prostaglandin I 2 or agonists that directly activate adenylyl cyclase. 16 As in many other cell types, the rate of cGMP production through soluble guanylyl cyclase activity and its degradation via cGMP-hydrolyzing phospodiesterases (PDEs) determine the amplitude, duration, and spatiotemporal distribution of an intraplatelet cGMP signal. 17 Of the 3 known platelet PDE isoforms, 2 isoforms, PDE-2 and PDE-3, may maintain an intensive cross-talk between nitric oxide (NO)/soluble guanylyl cyclase/cGMP and prostaglandin I 2 /adenylyl cyclase/ cAMP because these PDEs limit, with different affinities for their substrates and with different catalytic rates, the levels of both cyclic nucleotides, producing either inactive 5´-AMP or 5´-GMP. 18 Most effects of high intraplatelet cGMP are directly mediated by cGMP-dependent protein kinase type I (cGKI), which is presumably the major effector of NO/cGMP in many cells. 13,14 Interestingly, mature platelets express only the cGKIβ isozyme, whereas in numerous other cell types, such as hippocampal neurons and vascular or visceral smooth muscle (SM) cells, 2 cGKI isoforms, cGKIα and cGKIβ, act downstream of NO and cGMP. 19 In platelets, NO/cGMP signaling via cGKIβ efficiently opposes most agonist-induced and Ca 2+-dependent activation/aggregation steps. 13,20,21 However, the effect of cGKI on megakaryopoiesis and platelet biogenesis in vivo is not completely known.To study the role of cGMP/cGKI signaling in megakaryopoiesis and platelet production in vivo, we took advantage of several established or recently generated transgenic mouse lines. 22Interestingly, we detected high platelet counts in conventional cGKI-null mutants (cGKI L1/L1 ) and in adult cGKI-SM mice with cGKI expression specifically restored in SM, but not in other cell types, 22 whe...