ARTICLESAnandamide, the naturally occurring amide of arachidonic acid with ethanolamine, meets all key criteria of an endogenous cannabinoid substance 1 : it is released on demand by stimulated neurons 2,3 ; it activates cannabinoid receptors with high affinity 1 ; and it is rapidly eliminated through a two-step process consisting of carrier-mediated transport followed by intracellular hydrolysis 2,4 . Anandamide hydrolysis is catalyzed by the enzyme fatty acid amide hydrolase (FAAH), a membrane-bound serine hydrolase 5,6 that also cleaves other bioactive fatty acid ethanolamides such as oleoylethanolamide 7 and palmitoylethanolamide 8 . Mutant mice lacking the gene encoding FAAH (Faah) cannot metabolize anandamide 9 and, although fertile and generally normal, show signs of enhanced anandamide activity at cannabinoid receptors such as reduced pain sensation 9 . This is suggestive that drugs targeting FAAH may heighten the tonic actions of anandamide, while possibly avoiding the multiple and often unwanted effects produced by ∆ 9 -tetrahydrocannabinol (∆ 9 -THC) and other direct-acting cannabinoid agonists 10,11 . To test this hypothesis, potent, selective and systemically active inhibitors of intracellular FAAH activity are needed. However, most current inhibitors of this enzyme lack the target selectivity and biological availability required for in vivo studies [12][13][14] , whereas newer compounds, though promising, have not yet been characterized 15,16 . Thus, the therapeutic potential of FAAH inhibition remains essentially unexplored. Lead identification and optimizationDespite its unusual catalytic mechanism 6 , FAAH is blocked by a variety of serine hydrolase inhibitors, including compounds with activated carbonyls 16 . Therefore we examined whether esters of carbamic acid such as the anti-cholinesterase agent carbaryl (compound 1; Table 1) might inhibit FAAH activity in rat brain membranes. Although compound 1 was ineffective, its positional isomer 2 produced a weak inhibition of FAAH (half-maximal inhibitory concentration (IC 50 ) = 18.6 ± 0.7 µM; mean ± s.e.m., n = 3), which was enhanced by replacing the N-methyl substituent with a cyclohexyl group (compound 3; IC 50 = 324 ± 31 nM). The aryl ester 4, the benzyloxyphenyl group of which can be regarded as an elongated bioisosteric variant of the naphthyl moiety of compound 2, inhibited the activity of FAAH with a potency (IC 50 = 396 ± 63 nM) equivalent to that of compound 3. A conformational analysis of compound 4 revealed families of accessible conformers differing mainly in the torsion angle around the O-CH 2 bond, with substituents in anti or gauche conformations (data not shown). As the latter conformations more closely resembled the shape of the naphthyl derivative 3, we hypothesized that they might be responsible for the interac-

The endogenous cannabinoids (endocannabinoids) are lipid molecules that may mediate retrograde signaling at central synapses and other forms of short-range neuronal communication. The monoglyceride 2-arachidonoylglycerol (2-AG) meets several criteria of an endocannabinoid substance: (i) it activates cannabinoid receptors; (ii) it is produced by neurons in an activity-dependent manner; and (iii) it is rapidly eliminated. 2-AG inactivation is only partially understood, but it may occur by transport into cells and enzymatic hydrolysis. Here we tested the hypothesis that monoglyceride lipase (MGL), a serine hydrolase that converts monoglycerides to fatty acid and glycerol, participates in 2-AG inactivation. We cloned MGL by homology from a rat brain cDNA library. Its cDNA sequence encoded for a 303-aa protein with a calculated molecular weight of 33,367 daltons. Northern blot and in situ hybridization analyses revealed that MGL mRNA is heterogeneously expressed in the rat brain, with highest levels in regions where CB1 cannabinoid receptors are also present (hippocampus, cortex, anterior thalamus, and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased MGL expression and attenuated N-methyl-D-aspartate/carbachol-induced 2-AG accumulation in these cells. No such effect was observed on the accumulation of anandamide, another endocannabinoid lipid. The results suggest that hydrolysis by means of MGL is a primary mechanism for 2-AG inactivation in intact neurons

To identify structural characteristics of the closely related cell surface receptors for insulin and IGF‐I that define their distinct physiological roles, we determined the complete primary structure of the human IGF‐I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30‐residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF‐I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg‐Lys‐Arg‐Arg sequence at position 707 of the IGF‐I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.

Oleylethanolamide (OEA) is a natural analogue of the endogenous cannabinoid anandamide. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signalling 1. However, OEA does not activate cannabinoid receptors and its biological functions are still unknown 2. Here we show that, in rats, food deprivation markedly reduces OEA biosynthesis in the small intestine. Administration of OEA causes a potent and persistent decrease in food intake and gain in body mass. This anorexic effect is behaviourally selective and is associated with the discrete activation of brain regions (the paraventricular hypothalamic nucleus and the nucleus of the solitary tract) involved in the control of satiety. OEA does not affect food intake when injected into the brain ventricles, and its anorexic actions are prevented when peripheral sensory fibres are removed by treatment with capsaicin. These results indicate that OEA is a lipid mediator involved in the peripheral regulation of feeding

Fatty acid amide hydrolase (FAAH) is a promising target for modulating endocannabinoid and fatty acid ethanolamide signaling, which may have important therapeutic potential. We recently described a new class of O-arylcarbamate inhibitors of FAAH, including the cyclohexylcarbamic acid biphenyl-3-yl ester URB524 (half-maximal inhibitory concentration, IC(50) = 63 nM), which have significant anxiolytic-like properties in rats. In the present study, by introducing a selected group of substituents at the meta and para positions of the distal phenyl ring of URB524, we have characterized structure-activity profiles for this series of compounds and shown that introduction of small polar groups in the meta position greatly improves inhibitory potency. Most potent in the series was the m-carbamoyl derivative URB597 (4i, IC(50) = 4.6 nM). Furthermore, quantitative structure-activity relationship (QSAR) analysis of an extended set of meta-substituted derivatives revealed a negative correlation between potency and lipophilicity and suggested that small-sized substituents may undertake polar interactions with the binding pocket of the enzyme. Docking studies and molecular dynamics simulations, using the crystal structure of FAAH, indicated that the O-biphenyl scaffold of the carbamate inhibitors can be accommodated within a lipophilic region of the substrate-binding site, where their folded shape mimics the initial 10-12 carbon atoms of the arachidonyl moiety of anandamide (a natural FAAH substrate) and methyl arachidonyl fluorophosphonate (a nonselective FAAH inhibitor). Moreover, substituents at the meta position of the distal phenyl ring can form hydrogen bonds with atoms located on the polar section of a narrow channel pointing toward the membrane-associated side of the enzyme. The structure-activity characterization reported here should help optimize the pharmacodynamic and pharmacokinetic properties of this class of compounds.

The endogenous cannabinoid anandamide is removed from the synaptic space by a high-affinity transport system present in neurons and astrocytes, which is inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). After internalization, anandamide is hydrolyzed by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme that also cleaves AM404. Based on kinetic evidence, it has recently been suggested that anandamide internalization may be mediated by passive diffusion driven by FAAH activity. To test this possibility, in the present study, we have investigated anandamide internalization in wild-type and FAAH-deficient (FAAH ؊/؊ ) mice. Cortical neurons from either mouse strain internalized [ 3 H]anandamide through a similar mechanism, i.e., via a rapid temperature-sensitive and saturable process, which was blocked by AM404. Moreover, systemic administration of AM404 to either wild-type or FAAH ؊/؊ mice enhanced the hypothermic effects of exogenous anandamide, a response that was prevented by the CB 1 cannabinoid antagonist rimonabant (SR141716A). The results indicate that anandamide internalization in mouse brain neurons is independent of FAAH activity. In further support of this conclusion, the compound N-(5Z, 8Z, 11Z, 14Z eicosatetraenyl)-4-hydroxybenzamide (AM1172) blocked [ 3 H]anandamide internalization in rodent cortical neurons and human astrocytoma cells without acting as a FAAH substrate or inhibitor. AM1172 may serve as a prototype for novel anandamide transport inhibitors with increased metabolic stability.

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The marine cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in oligotrophic regions of the oceans. The inability to assimilate nitrate is considered an important factor underlying the distribution of Prochlorococcus, and thought to explain, in part, low abundance of Prochlorococcus in coastal, temperate, and upwelling zones. Here, we describe the widespread occurrence of a genomic island containing nitrite and nitrate assimilation genes in uncultured Prochlorococcus cells from marine surface waters. These genes are characterized by low GC content, form a separate phylogenetic clade most closely related to marine Synechococcus, and are located in a different genomic region compared with an orthologous cluster found in marine Synechococcus strains. This sequence distinction suggests that these genes were not transferred recently from Synechococcus. We demonstrate that the nitrogen assimilation genes encode functional proteins and are expressed in the ocean. Also, we find that their relative occurrence is higher in the Caribbean Sea and Indian Ocean compared with the Sargasso Sea and Eastern Pacific Ocean, which may be related to the nitrogen availability in each region. Our data suggest that the ability to assimilate nitrite and nitrate is associated with microdiverse lineages within high-and low-light (LL) adapted Prochlorococcus ecotypes. It challenges 2 long-held assumptions that (i) Prochlorococcus cannot assimilate nitrate, and (ii) only LL adapted ecotypes can use nitrite. The potential for previously unrecognized productivity by Prochlorococcus in the presence of oxidized nitrogen species has implications for understanding the biogeography of Prochlorococcus and its role in the oceanic carbon and nitrogen cycles.metagenomics ͉ cyanobacteria ͉ nitrogen cycle ͉ narB ͉ nirA