No abstract

Myelin proteolipid protein (PLP) contains thioester-bound, long-chain fatty acids which are known to in¯uence the structure of the molecule. To gain further insights into the role of this post-translational modi®cation, we studied the effect that chemical deacylation of PLP had on the morphology of myelin and on the protein's ability to mediate the clustering of lipid vesicles. Incubation of rat optic nerves in isoosmotic solutions containing 100 mM hydroxylamine (HA) pH 7.4 led to deacylation of PLP and decompaction of myelin lamellae at the level of the intraperiod line. Incubation of nerves with milder nucleophilic agents (Tris and methylamine) or diluted HA, conditions that do not remove protein-bound fatty acids, caused no alterations in myelin structure. Other possible effects of HA which could have affected myelin compaction indirectly were ruled out. Incubation of optic nerves with 50 mM dithioerythritol (DTE) also led to the splitting of the myelin intraperiod line and this change again coincided with the removal of fatty acids. In addition, the apparently compacted CNS myelin in the PLP-less myelin-de®cient rat, like that in tissue containing deacylated PLP, was readily decompacted upon incubation in isoosmotic buffers, suggesting that the function of PLP as a stabilizer of the interlamellar attachment is, at least in part, mediated by fatty acylation. Furthermore, in contrast to the native protein, PLP deacylated with either HA or DTE failed to induce the clustering of phosphatidylcholine/cholesterol vesicles in vitro. This phenomenon is not due to side-effects of the deacylation procedure since, upon partial repalmitoylation, the protein recovered most of its original vesicle-clustering activity. Collectively, these ®ndings suggest that palmitoylation, by in¯uencing the adhesive properties of PLP, is important for stabilizing the multilamellar structure of myelin.

Nitric oxide (NO) has been implicated in the pathophysiology of both experimental autoimmune encephalomyelitis and multiple sclerosis (MS). NO-mediated protein damage in MS appears to be confined to large plaques where 3-nitrotyrosine has been detected. To determine whether nitrosative damage takes place beyond visible MS plaques, the occurrence of various NO-triggered protein modifications in normal-appearing white matter (NAWM) of eight MS brains was assessed and compared to that in white matter (WM) of four control brains. As determined by amino acid analysis and western blotting, no evidence of tyrosine nitration was found in the MS samples studied, suggesting that they did not contain appreciable amounts of plaque-derived material. The amino acid composition of total myelin proteins and proteolipid protein (PLP) was also unaltered in the diseased tissue, as was the fatty acid composition of PLP. In addition, we detected no changes in the number of protein free thiols suggesting that oxidation do not occur to any appreciable extent. However, the levels of nitrite in MS-NAWM were higher than those in control WM, while in the MS-gray matter (GM) the concentration of this ion was unaltered. Furthermore, five of the MS samples analyzed, and the same as those with high levels of glial fibrilary acidic protein, showed increased amounts of protein nitrosothiols as determined by the "biotin switch" method. S-nitrosation of GM proteins was again normal. There was no indication of N-nitrosation of tryptophan and N-terminal amino groups in both control and MS tissue. Overall, the data suggests that WM, but not GM, from MS brains is subjected to considerable nitrosative stress. This is the first report to present direct evidence of increased protein S-nitrosation and nitrite content in the brain parenchyma of MS patients.

There are numerous histologically varied types of epithelial neoplasms of the pancreas with broad categorization as either exocrine or endocrine tumors according to dominant cell type and or elaboration of peptide hormones by neoplastic cells. Subclassification of these tumors is based in large part on whether they are grossly solid, cystic, or intraductal and by their histological differentiation. Exocrine tumors are by far the most common with invasive ductal adenocarcinoma representing 85% of all pancreatic neoplasms. 1 Rare histological variants such as adenosquamous and its subset of squamous-cell carcinoma are infrequently described in the literature with both having a reported incidence of 1-4 and 0.5% of all exocrine pancreatic neoplasms, respectively. 2,3 Other squamous lesions, such as benign lymphoepithelial cysts of the pancreas are also uncommon (less than 100 cases reported in the literature) and can be misdiagnosed as pancreatic masses or cystic neoplasms. [4][5][6] The normal pancreas is histologically devoid of native squamous elements. However, benign squamous metaplasia is reported in 9-64% of pancreases examined postmortem. Squamous metaplasia can be present in conditions associated with chronic inflammation such as chronic pancreatitis and pancreatic/biliary stent placement. It is also uncommonly identified in the adjacent ducts of 4% of surgical specimens resected for pancreatic adenocarcinoma 2 and rarely in benign pancreatic mucinous neoplasms. 7 Malignant squamous cells in the pancreas have been postulated to evolve through various mechanisms including malignant transformation of squamous metaplasia, squamous metaplastic change of a pre-existing adenocarcinoma, and differentiation with malignant transformation of primitive multipotent cells. 4,8 The rarity of pure squamous-cell carcinoma in the pancreas has led many investigators to question if this is truly a distinct pathologic entity, an under sampled primary pancreatic adenosquamous carcinoma, or most likely metastasis from an occult primary. 2,9 When diagnosed by fine-needle aspiration (FNA), the presence of squamous epithelium in a pancreatic lesion raises a broad differential including skin contaminants in percutaneous needle biopsies, atypical metaplasia, benign cysts with squamous differentiation, metastasis, or adenosquamous/squamous-cell carcinoma. Skin contaminants with bland cytologic features are easily distinguished from atypical metaplasia or carcinoma and rarely cause diagnostic confusion. Atypical squamous metaplasia can range in appearance from reactive to more atypical. Atypical squamous metaplastic features that have been reported demonstrate mild to moderate nuclear hyperchromasia, nuclear variation in size with enlargement up to four to five times normal, and nuclear membrane irregularity with relative preservation of nuclear/cytoplasmic ratios. Layfield et al. noted in their review of nine FNA cases containing squamous elements that atypical squamous metaplasia could be distinguished from adenosquamous/squamous-cel...

In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM-20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size-exclusion and ion-exchange chromatography in organic solvents. Matrix-assisted laser desorption ionizationtime of flight-mass spectrometry (MALDI-TOF-MS) of PLP and DM-20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester-bound fatty acids. Electrospray ionization-MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post-translational modification of PLP, and that the majority of PLP and DM-20 molecules in the CNS are fully acylated. A series of myelin-associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N-and C-terminal sequencing, tryptic digestion and peptide mapping by MALDI-TOF-MS. The results clearly showed that these polypeptides correspond to the N-terminal region (residues 1-105/112) and C-terminal region (residues 113/131-276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid-long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM-20, and are differentially distributed across the CNS. Keywords: mass spectrometry, myelin, palmitoylation, proteolipid protein, proteolysis. (Macklin et al. 1987;Nave et al. 1987). In addition to these species, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of brain proteolipids reveals the presence of minor low molecular weight forms (LMWPs, 12-18 kDa) (Lees and Bizzozero 1992). Two LMWPs have been isolated from bovine brain and appear to correspond to the first 113 amino acid of PLP plus an unknown N-terminal sequence, and to the last 160 residues with a blocked N-terminus (Lepage et al. 1986 Abbreviations used: DM-20, minor myelin proteolipid protein; ESI, electrospray-ionization; GLC, gas-liquid chromatography; IEC, ionexchange chromatography; LMWPs, low molecular weight (12-18 kDa) proteolipids; MALDI-TOF, matrix-assisted laser desorption ionizationtime of flight; MS, mass spectrometry; m/z, mass to charge ratio; PAGE, polyacrylamide gel electrophoresis; PLP, major myelin proteolipid protein; SDS, sodium dodecyl sulfate; SEC, size-exclusion chromatography.