At high magnetic fields (7 and 8.4 T), water proton magnetic resonance images of brains of live mice and rats under pentobarbital anesthetization have been measured by a gradient echo pulse sequence with a spatial resolution of 65 x 65-microns pixel size and 700-microns slice thickness. The contrast in these images depicts anatomical details of the brain by numerous dark lines of various sizes. These lines are absent in the image taken by the usual spin echo sequence. They represent the blood vessels in the image slice and appear when the deoxyhemoglobin content in the red cells increases. This contrast is most pronounced in an anoxy brain but not present in a brain with diamagnetic oxy or carbon monoxide hemoglobin. The local field induced by the magnetic susceptibility change in the blood due to the paramagnetic deoxyhemoglobin causes the intra voxel dephasing of the water signals of the blood and the surrounding tissue. This oxygenation-dependent contrast is appreciable in high field images with high spatial resolution.

Eukaryotic cells control the levels of their major membrane lipid, phosphatidylcholine (PtdCho), by balancing synthesis with degradation via deacylation to glycerophosphocholine (GroPCho). Here we present evidence that in both yeast and mammalian cells this deacylation is catalyzed by neuropathy target esterase (NTE), a protein originally identified by its reaction with organophosphates, which cause nerve axon degeneration. YML059c, a Saccharomyces cerevisiae protein with sequence homology to NTE, had similar catalytic properties to the mammalian enzyme in assays of microsome preparations and, like NTE, was localized to the endoplasmic reticulum. Yeast lacking YML059c were viable under all conditions examined but, unlike the wild-type strain, did not convert PtdCho to GroPCho. Despite the absence of the deacylation pathway, the net rate of Levels of phosphatidylcholine (PtdCho), 1 the major membrane lipid of eukaryotic cells, are tightly regulated by coordination of its synthesis and degradation. In both yeast (1) and mammalian cells (2), PtdCho synthesized by the CDP-choline pathway (see Fig. 1) is deacylated by as yet unidentified phospholipases to form glycerophosphocholine (GroPCho). In principle, this deacylation at both sn-2 and sn-1 positions of PtdCho could be mediated either by a single enzyme with phospholipase B activity or by sequential action of a phospholipase A2 and a lysophospholipase. We have reported that when mixed micelles of PtdCho with detergent were incubated with the recombinant catalytic domain of neuropathy target esterase (NTE), fatty acid was liberated very slowly from the sn-2 position followed by rapid deacylation of the resulting lysophospholipid (3). Because the rates and selectivities of bond cleavage observed in phospholipase assays in vitro are profoundly affected by the physicochemical nature of the substrate (4 -6), it is possible that in vivo NTE could deacylate the sn-2 position of PtdCho more efficiently than observed in our experiments. Thus, NTE might be the phospholipase B responsible for converting PtdCho to GroPCho (see Fig. 1).NTE was originally identified as the target site for those organophosphates that cause a paralyzing delayed neuropathy with degeneration of long nerve axons (7). In adult animals NTE is present in the nervous system and a variety of nonneural tissues (8). NTE is also widely expressed during fetal development (9). Studies on green fluorescent protein (GFP)-tagged NTE constructs expressed in COS cells indicate that NTE is anchored to the cytoplasmic face of the endoplasmic reticulum (10).Definitive evidence that NTE can convert PtdCho to GroPCho could be obtained by comparing PtdCho metabolism in wild-type and NTE-null cells. However, mice lacking NTE die by mid-gestation (11, 12), and fibroblasts from day 8 embryos can be cultured for only limited periods (12). On the other hand, a gene, YML059c, in the yeast, Saccharomyces cerevisiae, encodes a putative protein with substantial sequence homology to NTE. The availability of a YML059c-null mutant...

The Drosophila Swiss cheese (sws) mutant is characterized by progressive degeneration of the adult nervous system, glial hyperwrapping, and neuronal apoptosis. The Swiss cheese protein (SWS) shares 39% sequence identity with human neuropathy target esterase (NTE), and a brain-specific deletion of SWS/NTE in mice causes a similar pattern of progressive neuronal degeneration. NTE reacts with organophosphate compounds that cause a paralyzing axonal degeneration in humans and has been shown to degrade endoplasmic reticulum-associated phosphatidylcholine (PtdCho) in cultured mammalian cells. However, its function within the nervous system has remained unknown. Here, we show that both the fly and mouse SWS proteins can rescue the defects that arise in sws mutant flies, whereas a point mutation in the proposed active site cannot restore SWS function. Overexpression of catalytically active SWS caused formation of abnormal intracellular membraneous structures and cell death. Cell-specific expression revealed that not only neurons but also glia depend autonomously on SWS. In wild-type flies, endogenous SWS was detected by immmunohistochemistry in the endoplasmic reticulum (the primary site of PtdCho processing) of neurons and in some glia. sws mutant flies lacked NTE-like esterase activity and had increased levels of PtdCho. Conversely, overexpression of SWS resulted in increased esterase activity and reduced PtdCho. We conclude that SWS is essential for membrane lipid homeostasis and cell survival in both neurons and glia of the adult Drosophila brain and that NTE may play an analogous role in vertebrates.

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A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727-1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A 2 (iPLA 2 ) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA 2 inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V max ϳ0.01 mol/min/mg and K m ϳ0.4 mM for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of 14 C-labeled products from two differentially labeled 14 C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V max ϳ20 mol/ min/mg and K m ϳ0.05 mM for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V max ϳ1 mol/min/mg and K m ϳ0.4 mM for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di-or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking. Neuropathy target esterase (NTE)1 is an integral membrane protein in neurons and some non-neural cell types; it was originally identified as the primary site of action for those organophosphates which, in humans and other vertebrates, cause a syndrome characterized by axonal degeneration (1). The NTE homologue in Drosophila, the swiss cheese protein, is essential for fly brain development (2). In the mouse, NTE is present in neurons from their earliest appearance in the nervous system and so is well placed to play a similar role in mammalian neural development (3). NTE also has a homologue in yeast (4), suggesting that it has functions beyond the nervous system and mediates a biochemical reaction highly conserved through evolution.In keeping with its reactivity with organophosphates, NTE belongs to the serine hydrolase class of enzymes. Since its discovery more than 30 years ago (5), NTE has been detected in vitro by assays with non-physiological ester substrates, most commonly with phenyl valerate (6). Clues to the cellular functions of NTE might be provided by identifying its natural substrate. Data on this point are sparse, but using artificial substrates, NTE activity in brain homogenates has been shown to catalyze hydrolysis of ester rather than ...