letters to nature 434 NATURE | VOL 403 | 27 JANUARY 2000 | www.nature.com ®xed to the skull using dental acrylic. About a week after surgery, animals were implanted with morphine pellets and behavioural testing began 4 days later. Infusions of 0.5 ml per side were made through 28-gauge injector cannulae over 1 min, and cannulae were left in place for 1 min. All drugs were made fresh each day and were dissolved in ACSF. Dye was injected after the experiment to mark the injection site in all animals. Conditioned place-aversion procedureA balanced place-conditioning procedure was used to measure aversion in a chamber with two distinct sides 10 . On the ®rst day (preconditioning day), rats were allowed free access to both sides of the chamber for 15 min. Animals that spent more than 80% of the time on one side were eliminated. On the next two days (pairing days), the animals were given an intraperitoneal injection of naltrexone (1 mg per kg) or saline, and were con®ned to one side for 30 min. Animals given naltrexone on pairing day 1 were given saline on pairing day 2 and con®ned to the opposite side, and vice versa. All adrenergic drugs were microinjected on each of the two pairing days 5 min before naltrexone or saline; controls were similarly injected with ACSF. During pairing, an observer scored each occurrence of somatic withdrawal signs. On day 4 (test day), animals were given no drug injections and were returned to the test apparatus for 15 min with free access to both compartments, and the time spent in each compartment was measured. For shock training, place conditioning was carried out in drug-naive animals as above, except that, on pairing day 1, animals received a 0.8 mA foot shock (randomly given for 1 s every 3 min through the chamber oor) over the course of the 30-min session; on pairing day 2, they received no foot shock and were con®ned to the opposite side.

Nogo-A is a potent neurite growth inhibitor in vitro and plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

rMAL, the rat myelin and lymphocyte protein, is a small hydrophobic protein of 17 kDa with four putative transmembrane domains and is expressed in oligodendrocytes and Schwann cells, the myelinating cells of the nervous system. In addition, transcript expression has been found in kidney, spleen, and intestine. Confocal microscopy and immunoelectron microscopy with an affinity-purified antibody localized rMAL to compact myelin in a pattern similar to the structural myelin proteins: myelin basic protein and proteolipid protein. In kidney and stomach epithelia, rMAL is located almost exclusively on the apical (luminal) membranes of the cells lining distal tubuli in kidney and the glandular part of the stomach. Biochemical analysis of plasma membranes isolated from spinal cord and kidney demonstrated that rMAL is a proteolipid that is present in detergent insoluble complexes typical for proteins associated with glycosphingolipids. Lipid and protein analysis showed a co-enrichment of glycosphingolipids and rMAL protein within these complexes, indicating a close association of rMAL to glycosphingolipids in myelin and in kidney in vivo. We conclude that specific rMAL-glycosphingolipid interactions may lead to the formation and maintenance of stable protein-lipid microdomains in myelin and apical epithelial membranes. They may contribute to specific properties of these highly specialized plasma membranes.

Within the CNS, the normal form of cellular prion protein (PrP C ) is expressed on neurons, oligodendrocytes, and astrocytes. The contribution of these cell types to prion replication and pathogenesis is unclear. To assess the role of oligodendrocytes, we expressed PrP

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By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolyticaily active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtUis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb Hinfl-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. stearothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8°C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostabiity was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B.caldolyticus.Bacterial species belonging to the genus Bacillus secrete a variety of enzymes, some of which are of economical importance, in particular the ax-amylases and proteinases (5). The various Bacillus species show large variations in optimum growth temperatures, which are often, but not invariably, reflected in the thermostabilities of their extracellular enzymes (23). By comparing thermostable and thermolabile variants of homologous enzymes, information on the mechanisms involved in thermostability of enzymes can be obtained (22,31,37).We selected the neutral proteases from bacilli as a model system in such an approach. Neutral proteases (Npr) are metalloendoproteinases which show optimum activity at neutral pH. These enzymes contain one zinc atom per molecule and may be stabilized by calcium binding (17). Several npr genes have been cloned and expressed in Bacillus subtilis, including the genes from B. subtilis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, and their nucleotide sequences have been determined (8,12,18,33,38,40,44). Recently, the npr gene from Bacillus brevis was also cloned and sequenced (la). Additionally, the primary and tertiary structures of the neutral proteases from Bacillus cereus and Bacillus thermoproteolyticus have been determined (21,28,34,35). The neutral proteases from B. subtilis and B. amyloliquefaciens are rather thermolabile, whereas the enzymes from B. stearothermophilus and ...

Primary cultures of rat oligodendrocytes were incubated with a fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoylceramide+ ++ (C6-NBD-ceramide). This compound is known to stain the Golgi complex specifically. Within 30 min of incubation at 37 degrees C most of the C6-NBD-ceramide was incorporated into the perinuclear Golgi system, as revealed by conventional and confocal laser fluorescence microscopy. Interestingly, C6-NBD-ceramide was found to accumulate also in smaller, oval-shaped structures in many of the processes, at distances up to 30 microns from the nucleus. This implies the possibility that these structures are Golgi (-derived) complexes. Indeed, after incubation of oligodendrocytes with C6-NBD-ceramide and rhodamine-labeled transferrin both fluorescent labels colocalized in the Golgi system of the cell body as well as in the structures in the processes. Additional support for the Golgi character of these structures was obtained by transmission electron microscopy. Particularly in oligodendrocytes cocultured with neurons, many Golgi structures were present all over the processes. The results lead us to conclude that, in the oligodendrocyte, the Golgi complex does not only reside in the perikaryon, but also in the processes. One can speculate that a polarized biosynthetic activity, involving the presence of the Golgi near the site of myelin synthesis, may be advantageous to the oligodendrocyte for assembly and/or repair of the myelin membrane at the distal end of the processes.

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