This review describes (from both the animal and human literature) the biological consequences of losses in nervous system docosahexaenoate (DHA). It then concentrates on biological mechanisms that may serve to explain changes in brain and retinal function. Brief consideration is given to actions of DHA as a nonesterified fatty acid and as a docosanoid or other bioactive molecule. The role of DHA-phospholipids in regulating G-protein signaling is presented in the context of studies with rhodopsin. It is clear that the visual pigment responds to the degree of unsaturation of the membrane lipids. At the cell biological level, DHA is shown to have a protective role in a cell culture model of apoptosis in relation to its effects in increasing cellular phosphatidylserine (PS); also, the loss of DHA leads to a loss in PS. Thus, through its effects on PS, DHA may play an important role in the regulation of cell signaling and in cell proliferation. Finally, progress has been made recently in nuclear magnetic resonance studies to delineate differences in molecular structure and order in biomembranes due to subtle changes in the degree of phospholipid unsaturation.Paper no. L8776 in Lipids 36, 945-959 (September 2001)

Serum albumin is the principal transporter of fatty acids that are otherwise insoluble in circulating plasma. While the crystal structure of human serum albumin (HSA) as well as its binding with fatty acids has been characterized, the three dimensional structure of bovine serum albumin (BSA) has not been determined although both albumins share 76% sequence homology. In this study we used mass spectrometry coupled with chemical cross-linking, to probe the tertiary structure of BSA. BSA was modified with lysine specific cross-linkers, bis(sulfosuccinimidyl) suberate (BS 3 ), disuccinimidyl suberate (DSS) or disuccinimidyl glutarate (DSG), digested with trypsin and analyzed by tandem mass spectrometry. With O-18 labeling during the digestion, through-space cross-linked peptides were readily identified in mass spectra by a characteristic 8 Da shift. From the cross-linked peptides identified in this study, we found that 12 pairs of lysine residues were separated within 20 Å, while 5 pairs were spaced between 20 and 24 Å. The spatial distance constraints generated from five K-K pairs in BSA were consistent with the corresponding distance obtained from the crystal structure of HSA, although only six equivalent K-K pairs could be compared. According to our data, the distance between K235 of IIA and K374 of IIB domain in BSA was farther by 7-11 Å than that expected from the crystal structure of HSA, suggesting structural differences between BSA and HSA in this region. The distance constraints obtained for lysine residues using various cross-linkers should be valuable in assisting the determination of the 3-D structure of

The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released ''on demand'' by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC͞phosphatase pathway, which is up-regulated by LPS, whereas NAPE-PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC͞phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC͞phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target.biosynthesis ͉ phosphatase ͉ phospholipase C ͉ phosphoanandamide T he endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) is a lipid transmitter synthesized and released ''on demand'' by neurons in the brain (1). AEA is also generated by macrophages (2), where its bacterial endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock (3, 4) and liver cirrhosis (5, 6). Macrophage-derived AEA has been also implicated in antiinflammatory effects both in the periphery (7) and in the central nervous system (8, 9). AEA is thought to be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE), through cleavage by a phospholipase D (NAPE-PLD) (10, 11), upregulation of which can result in increased tissue levels of AEA (12). We earlier reported that LPS potently stimulates AEA synthesis in RAW264.7 mouse macrophages, in which it increases both the generation of NAPE from [ 14 C]diarachidonoyl phosphatidylcholine and the conversion of NAPE to AEA (4). Because these effects could be prevented by inhibitors of RNA transcription or protein synthesis (4), we hypothesized that LPS induces the expression of proteins involved in the biosynthesis of AEA, and a subtraction cloning strategy using resting and LPS-treated macrophages may help identifying such proteins. Although a specific N-acyltransferase (NAT) involved in the generation of NAPE has not yet been discovered, a NAPEspecific PLD has been identified and its ability to generate AEA from NAPE has been established (11). The results presented here indicate that, unexpectedly, NAPE-PLD is not involved in the stimulated synthesis of AEA in RAW264.7 macrophages. Instead, we identified the lipid phosphoanandamide (pAEA), which is also presen...

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