The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Abstract. Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys TM and Ser 322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif. EUKOCYTE localization to sites of inflammation is a precisely coordinated multistep process involving distinct families of adhesion receptors and chemokines (9, 56). Regulation of the neutrophil adhesive state is critical for leukocyte function. One major neutrophil adhesion molecule, the Mac-1/3~ integrin (CDIlb/CD18), is both quantitatively upregulated (59) and assumes an active conformation upon neutrophil exposure to chemotactic factors (10). In contrast, another major neutrophil adhesion molecule, L-selectin, is rapidly downregulated upon neutrophil activation (5,17,26,28,29). However, antibodies against either Mac-1 or L-selectin are effective in blocking neutrophil recruitment to sites of inflammation in vivo (28,36,62) and neutrophil adhesion to stimulated endothelial cells in vitro Address all correspondence to Takashi Kei Kishimoto, Ph.D., Boehringer Ingelheim Pharmaceuticals, Department of Immunology 1-5, 900 Ridgebury Road, Ridgefield, CT 06877. (18,20,31,51,5...

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase-and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene. Soluble epoxide hydrolase (sEH)3 is a ubiquitous enzyme found in many tissues such as liver, kidney, heart, and ovary (1). sEH catalyzes the degradation of endogenous epoxy lipids such as epoxyeicosatrienoic acids (EETs) to their less active diols (dihydroxyeicosatrienoic acids, DHETs) and hence plays a critical role in the control of EET levels (2). These epoxy lipids are potent vasodilators, regulating cerebral and renal homodynamic and blood pressure (3-5). In addition, EETs inhibit platelet aggregation (6), promote fibrinolysis (7) and have antiinflammatory properties (8, 9). Whereas deletion of the sEH gene, Ephx2, has been reported to reduce blood pressure in male mice (10), the inhibition of endogenous EET hydrolysis may provide pharmacological benefit in hypertension and acute inflammation (11).The human Ephx2 gene encodes sEH and consists of 19 exons encoding 555 amino acids (12). There is 73% homology between the human and mouse sEH protein sequences (13), with 100% conservation in the catalytic residues (14). Each monomer of the homodimeric mouse sEH has two distinct domains (14,15). The N-terminal domain exhibits phosphatase activity, and the C-terminal domain is responsible for the epoxide hydrolase activities (...

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2> TRAF3 >> TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.