Proceedings of the National Academy of Sciences of the United States of America
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Proc. Natl. Acad. Sci. U.S.A. · Apr 2009
Identification of a fluorescent general anesthetic, 1-aminoanthracene.
We identified a fluorophore, 1-aminoanthracene (1-AMA), that is anesthetic, potentiates GABAergic transmission, and gives an appropriate dissociation constant, K(d) approximately 0.1 mM, for binding to the general anesthetic site in horse spleen apoferritin (HSAF). 1-AMA fluorescence is enhanced when bound to HSAF. Thus, displacement of 1-AMA from HSAF by other anesthetics attenuates the fluorescence signal and allows determination of K(d), as validated by isothermal titration calorimetry. ⋯ Furthermore, 1-AMA reversibly immobilizes stage 45-50 Xenopus laevis tadpoles (EC(50) = 16 microM) and fluorescence micrographs show 1-AMA localized to brain and olfactory regions. Thus, 1-AMA provides an unprecedented opportunity for studying general anesthetic distribution in vivo at the cellular and subcellular levels.
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Proc. Natl. Acad. Sci. U.S.A. · Apr 2009
Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps.
Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. ⋯ Immunoneutralization of histone during Leishmania-neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site.
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Proc. Natl. Acad. Sci. U.S.A. · Apr 2009
Identification of intracellular carriers for the endocannabinoid anandamide.
The endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) is an uncharged neuromodulatory lipid that, similar to many neurotransmitters, is inactivated through its cellular uptake and subsequent catabolism. AEA is hydrolyzed by fatty acid amide hydrolase (FAAH), an enzyme localized on the endoplasmic reticulum. In contrast to most neuromodulators, the hydrophilic cytosol poses a diffusional barrier for the efficient delivery of AEA to its site of catabolism. ⋯ Similar results were observed in COS-7 cells stably expressing FAAH. Consistent with the roles of FABP as AEA carriers, administration of the competitive FABP ligand oleic acid or the selective non-lipid FABP inhibitor BMS309403 attenuated AEA uptake and hydrolysis by approximately 50% in N18TG2 and COS-7 cells. Taken together, FABPs represent the first proteins known to transport AEA from the plasma membrane to FAAH for inactivation and may therefore be novel pharmacological targets.
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Proc. Natl. Acad. Sci. U.S.A. · Apr 2009
PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis.
Sepsis, a leading cause of death worldwide, involves concomitant expression of an overzealous inflammatory response and inefficient bacterial clearance. Macrophage function is pivotal to the development of these two aspects during sepsis; however, the mechanisms underlying these changes remain unclear. ⋯ To the extent that this is a macrophage-specific aspect of the effects of PD-1, we found the following: first, peritoneal macrophages expressed significantly higher levels of PD-1 during sepsis, which was associated with their development of cellular dysfunction; second, when peritoneal macrophages were depleted (using clodronate liposomes) from PD-1(-/-) mice, the animals' bactericidal capacity was lowered, their inflammatory cytokine levels were elevated, and protection from septic lethality was diminished; and third, blood monocytes from both septic mice and patients with septic shock shared markedly increased PD-1 levels. Together, these data suggest that PD-1 may not only be a dysfunctional marker/effector of macrophages/monocytes, but may also be a potential therapeutic target for designing measures to modulate the innate immune response, thereby preventing the detrimental effects of sepsis.
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Proc. Natl. Acad. Sci. U.S.A. · Mar 2009
Endocannabinoids in the rat basolateral amygdala enhance memory consolidation and enable glucocorticoid modulation of memory.
Extensive evidence indicates that the basolateral complex of the amygdala (BLA) modulates the consolidation of memories for emotionally arousing experiences, an effect that involves the activation of the glucocorticoid system. Because the BLA expresses high densities of cannabinoid CB1 receptors, the present experiments investigated whether the endocannabinoid system in the BLA influences memory consolidation and whether glucocorticoids interact with this system. The CB1 receptor agonist WIN55,212-2 (5-50 ng per 0.2 microL per side), infused bilaterally into the BLA of male Sprague-Dawley rats immediately after inhibitory avoidance training, induced dose-dependent enhancement of 48-h retention. ⋯ Delayed infusions of WIN55,212-2 or AM251 administered into the BLA 3 h after training or immediate posttraining infusions of these drugs into the adjacent central amygdala did not significantly alter retention performance. Last, intra-BLA infusions of a low and otherwise nonimpairing dose of AM251 (0.14 ng per 0.2 microL per side) blocked the memory-enhancing effect induced by systemic administration of corticosterone (3 mg/kg, s.c.). These findings indicate that endocannabinoids in the BLA enhance memory consolidation and suggest that CB1 activity within this brain region is required for enabling glucocorticoid effects on memory consolidation enhancement.