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Epidermoid cysts of the spinal cord are very rare tumors. We report a 31 year-old female who presented with a 5 months history of progressive lower extremity weakness and spasticity. Magnetic resonance imaging of the thoracic spine revealed a 2 cm intradural, extramedullary mass at the T4-5 level. A T4 and T5 osteoplastic laminotomy with complete removal of the intradural mass was performed. Intraoperative and final histological examination revealed an epidermoid cyst. Epidermoid cysts must be a consideration for intradural, extramedullary lesions of the spinal cord. Complete surgical resection offers the patient an opportunity for good neurologic outcome.

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.

Although the toxin 6-hydroxydopamine (6-OHDA) is utilized extensively in animal models of Parkinson's disease, the underlying mechanism of its toxic effects on dopaminergic neurons is not completely understood. We examined the effects of 6-OHDA on the CNS-derived tyrosine hydroxylase expressing B65 cell line, with particular attention to the regulation of the extracellular signal-regulated protein kinases (ERK). 6-OHDA elicited a dose-dependent cytotoxicity in B65 cells. Toxic doses of 6-OHDA also elicited a biphasic pattern of ERK phosphorylation with a prominent sustained phase, a pattern that differed from that observed with hydrogen peroxide (H(2)O(2)) treatment. 6-OHDA-elicited ERK phosphorylation was blocked by PD98059, an inhibitor of the upstream mitogen activated protein kinase kinase (MEK) that phosphorylates and activates ERK. PD98059 also conferred protection against 6-OHDA cytotoxicity, but did not affect H(2)O(2) toxicity in B65 cells. These results suggest that ERK activation plays a direct mechanistic role in 6-OHDA toxicity, rather than representing a protective compensatory response, and raise the possibility that abnormal patterns of ERK activation may contribute to dopaminergic neuronal cell death.

Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs). Site-directed mutagenesis of glutamic acids within the catalytic domain of ExoS (termed delta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381. The specific activity of E381A mutant was 0.02% of wild-type delta N222. Delta N222(E381A) retained the requirement of factor activating exoenzyme S (FAS) activation for the expression of ADP-ribosyltransferase activity. In contrast, E387A, E399A, and E414A mutants possessed ADP-ribosyltransferase activity similar to that of wild-type delta N222. Kinetic evaluation of E381A and two other mutants, E381D and E381S, showed that their primary defect was a lower kcat in the ADP-ribosylation of soybean trypsin inhibitor (SBTI). The Km for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to delta N222. In addition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as delta N222, which indicated that E381 mutants had retained their overall conformation. Together, these data identify E381 as contributing to the catalytic activity of exoenzyme S.

Recombinant exoenzyme S (rHisExoS) of Pseudomonas aeruginosa was expressed in Escherichia coli as a soluble, cytosolic His fusion protein. rHisExoS was purified by Ni(2+)-affinity chromatography in the presence of protease inhibitors without detectable degradation. rHisExoS possessed a specific activity (within twofold) for the factor-activating exoenzyme S-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) similar to that of native exoenzyme S. Analysis of several deletion peptides showed that delta N222, which encoded the carboxyl-terminal 222 amino acids of exoenzyme S, possessed factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity. delta N222 catalyzed the ADP-ribosylation of SBTI at a rate sixfold greater than rHisExoS. Relative to rHisExoS, delta N222 had a similar affinity for NAD, a threefold greater affinity for SBTI, and a four- to eightfold greater kcat for the ADP-ribosylation of SBTI. Like native exoenzyme S, rHisExoS chromatographed as an aggregate with an apparent molecular mass of > 300 kDa. In contrast, delta N222 did not chromatograph as an aggregate, which showed that the amino-terminal 99 amino acids of exoenzyme S were responsible for the aggregation phenotype.

The transcriptional regulation of the Pseudomonas aeruginosa exoS gene was investigated. Expression of exoS in P. aeruginosa PA103 was dependent upon growth in a low-cation environment and the presence of a functional exsA gene. Promoter fusion analysis indicated that a 285-bp PstI-NsiI fragment, located 5' of the exoS coding region, contained a functional promoter for exoS. Expression of the reporter gene was inducible in a low-cation growth environment and required a functional copy of exsA. Divergent promoters, coordinately regulated with exoS transcription, were identified within the PstI-NsiI fragment. A fusion derivative of ExsA, MALA3A2, was shown to bind directly to the PstI-NsiI probe. DNase I protection analysis demonstrated that MALA3A2 bound to the intergenic region between the postulated -35 boxes of each promoter region. Northern (RNA) blot analysis with probes internal to and upstream of exoS demonstrated that separate, coordinately regulated mRNAs were expressed in P. aeruginosa. These data suggested that a locus, coregulated with exoS transcription, was located upstream of exoS. DNA sequence analysis of the exoS upstream region revealed three open reading frames, ORF 1, ORF 2, and ORF 3. ORF 1 demonstrated significant homology to the SycE/YerA protein of Yersinia sp. SycE/YerA is postulated to function as a chaperone for the YopE cytotoxin. The loci encoding YopE and ExoS show similarities in genetic organization, protein composition, and regulation.

The structural gene for the 49-kDa form of exoenzyme S (exoS) isolated from Pseudomonas aeruginosa 388 was expressed in both Escherichia coli and P. aeruginosa PA103. Expression of exoS in E. coli under the transcriptional regulation of the T7 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 49 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Expression of exoS in P. aeruginosa PA103 under the transcriptional regulation of the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS yielded a nitrilotriacetic acid-inducible extracellular protein with an apparent molecular mass of 49 kDa. Recombinant ExoS (rExoS) reacted with the anti-49-kDa form of exoenzyme S immunoglobulin G, existed as an aggregate as determined by gel filtration chromatography, and ADP-ribosylated soybean trypsin inhibitor at a specific activity that was similar (within twofold) to that of native exoenzyme S. Allelic exchange of exoS with a tetracycline gene cartridge yielded a strain of P. aeruginosa 388 that did not express detectable amounts of either ExoS in an immunoblot analysis using the anti-49-kDa form of exoenzyme S immunoglobulin G or ADP-ribosyltransferase activity under standard enzyme assay conditions. Expression of catalytically active rExoS in E. coli demonstrated that exoS was necessary and sufficient for the factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity of exoenzyme S. Expression of nitrilotriacetic acid-inducible rExoS in P. aeruginosa PA103 demonstrated that the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS encoded a functional exoenzyme S promoter. Expression analysis and allelic exchange experiments suggest that the 49- and 53-kDa forms of exoenzyme S are encoded by separate genes.

We report the purification and proteolytic characterization of the 49-kDa form of exoenzyme S and the cloning of the structural gene for the 49-kDa form of exoenzyme S (exoS). The 49-kDa form of exoenzyme S was purified from SDS-polyacrylamide gels. Conditions were established that allowed efficient trypsin digestion of the 49-kDa form of exoenzyme S. Amino acid sequence determination of the amino terminus and tryptic peptides of the 49-kDa form of exoenzyme S allowed the generation of degenerate oligonucleotides, which were used to amplify DNA encoding an amino-terminal sequence and an internal sequence of the 49-kDa form of exoenzyme S. These DNA fragments were used to clone the entire structural gene for the 49-kDa form of exoenzyme S (exoS) from a cosmid library of Pseudomonas aeruginosa strain 388. The 49-kDa form of exoenzyme S (ExoS) is predicted to be a 453 amino acid protein. The predicted amino acid sequence indicates that ExoS is secreted from Pseudomonas without cleavage of an amino-terminal sequence. BESTFIT analysis identified three regions of alignment between ExoS and the active site of Escherichia coli heat-labile enterotoxin. One region of homology appears to be shared among several members of the family of bacterial ADP-ribosyltransferases.

Exoenzyme S was purified > 1,500-fold from the culture supernatant fluid of Pseudomonas aeruginosa 388 at high yield without utilization of solvents or detergents. Two proteins, with apparent molecular sizes of 53 and 49 kDa, cofractionated with exoenzyme S activity. Rabbit anti-49-kDa-protein immunoglobulin G was prepared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified 49-kDa protein as immunogen. Anti-49-kDa-protein IgG inhibited the ADP-ribosyltransferase activity of purified exoenzyme S in a dose-dependent manner, which indicated a role for the 49-kDa protein in the ADP-ribosylation reaction. Analysis by ultrafiltration showed that exoenzyme S activity and the 53- and 49-kDa proteins cofractionated and that exoenzyme S was apparently > 300 kDa in size. Urea (8 M) and 1.0% Triton X-100 reversibly decreased the apparent molecular sizes of exoenzyme S activity and the 53- and 49-kDa proteins to between 30 and 100 kDa.