Interleukin 1 (IL-1) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A 2 (PLA 2 ) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFB consensus site. Immunoblot analysis identified NFB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFB consensus motif. An NFB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE 2 production (IC 50 ؍ ϳ2 M), indicating a role of NFB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1-stimulated PGE 2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA 2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 B motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA 2 gene induction and support the IL-1 activation and participation of distinct NFB protein dimers in RSF prostanoid and IL-8 formation.Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation and hyperproliferation of the synovial lining (1). Enhanced levels of the cytokine, interleukin (IL)-1, 1 perpetuate the disease process through up-regulation of a multitude of factors leading to eicosanoid formation, matrix degradation, bone resorption, and proliferation in the joint (2-6). We and others have demonstrated that human rheumatoid synovial fibroblast (RSF) prostaglandin (PG) E 2 accumulation in response to IL-1 is a direct result of the coordinate up-regulation of 85-kDa phospholipase A 2 (PLA 2 ) and the induction of COX II (6 -8). Indeed, we reported that depletion of IL-1-induced 85-kDa PLA 2 to basal levels by antisense severely compromised the ability of RSF to make PGE 2 . However, the mechanism(s) by which IL-1 regulates 85-kDa PLA 2 and COX II gene induction in this system have not been elucidated.IL-1 is a potent activator of nuclear factor B (NFB) (1, 9, 10) in other cell systems, and this transcription factor in turn regulates a wide variety of inflammatory and immunoregulatory genes (10 -16). 5Ј-flanking regulatory regions for both the human 85-kDa PLA 2 and COX II genes have recently been isolated (17, 18), and sequence analysis has identified a number of possible transcription factor consensus binding motifs, including NFB. The putative NFB motif in the 85-kDa PLA 2 promoter is located at Ϫ1099 base pairs (17), whereas the NFB consensus site in the human COX II promoter is located at Ϫ233 base pairs (18).NFB is a dimeric DNA binding protein comprised of members of the NFB/Rel/dorsal family of protei...
Cyanobacteria from a diversity of marine and freshwater habitats are known to produce neurotoxic secondary metabolites. 1 Herein, we describe the complete stereostructure, synthesis, and biological properties of kalkitoxin (1), a novel neurotoxic lipopeptide from a Caribbean collection of Lyngbya majuscula.The organic extract of this L. majuscula exhibited potent brine shrimp and fish toxicity. 2 Using these assays, the toxic metabolite kalkitoxin (1), was isolated by sequential silica gel VLC, CC, and normal-phase HPLC (12.8 mg, 0.3% of extract). Subsequently, bioassay-guided fractionation using a primary cell culture of rat neurons in a microphysiometer 3 or inhibition of IL-1 stimulation of sPLA 2 in hepatocarcinoma cells 4 led to re-isolation of 1 in small yield from various Caribbean collections of L. majuscula.Kalkitoxin (1) analyzed for C 21 H 38 N 2 OS indicated four degrees of unsaturation; from 13 C NMR analysis in DMSO-d 6 two were due to double bonds, one to a carbonyl group, and the remaining one to a ring system. 5 Data from E.COSY, HSQC, and a modified HSQMBC 6 experiments in benzene-d 6 allowed deduction of six partial structures for 1 (Supporting Information). One partial structure was composed of a sec-butyl group in which the methine component was deshielded to a chemical shift (δ 2.28) consistent with its being adjacent to a carbonyl. A second partial structure was composed of a methylated tertiary amide group which existed in two conformations (Supporting Information). A third partial structure possessed a deshielded methylene (δ 3.35) that could be sequentially connected by E.COSY to a second methylene group, and by HSQMBC to a methine and high-field methyl group. By E.COSY, an additional high-field methylene group (δ 1.10, 1.02) was adjacent to a methine which also bore a methyl group. The fifth partial structure was composed of a similar -CH 2 -CH-CH 3 grouping; however, in this case, the methylene group protons were deshielded to δ2 .31 and δ 2.55. The final partial structure, based on E.COSY correlations, HSQMBC, and chemical shift models, 7 was composed of a thiazoline ring with an ethylene appendage; this was further substantiated by EIMS fragmentations (Supporting Information). HSQMBC data were used to connect these partial structures and gave the full planar structural assignment of 1.The C3 stereochemistry of kalkitoxin was determined by Marfey's analysis. Kalkitoxin was ozonized and then hydrolyzed in 6 N HCl to obtain cysteic acid. Marfey's analysis of this hydrolysate yielded L-cysteic acid, defining C3 as R. The limited amount of kalkitoxin precluded determination of the C2′ stereochemistry. The relative stereochemistry of the three chiral centers within the aliphatic chain of kalkitoxin (C7, C8, C10) was determined using the J-based configuration analysis method. 8 The 3 J CH values were measured by a modification of the recently reported HSQMBC pulse sequence, 6 and the 3 J HH values were determined utilizing the E.COSY pulse sequence. 9 To overcome the limited sample size...
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