Use of reversed phase HP liquid chromatography to assay conversion of N-acylglycines to primary fatty acid amides by peptidylglycine-alpha-amidating monooxygenase

Primary fatty acid amides (R-CO-NH2) and N-acylglycines (R-CO-NH-CH2-COOH) are classes of compounds that have only recently been isolated and characterized from biological sources. Key questions remain regarding how these lipid amides are produced and degraded in biological systems. Relative to the fatty acids, little has been done to develop methods to separate and quantify the fatty acid amides and N-acylglycines. We describe reversed phase HPLC methods for the separation of C2-C12 primary fatty acid amides and N-acylglycines and also C12-C22 fatty acid amides. Separation within each class occurs primarily on the basis of simple interactions between the acyl chain and the chromatographic stationary phase, but the polar headgroups on these and related fatty acids and N-acylethanolamides modulate the absolute retention in reversed phase mode. We use these methods to measure the enzyme-mediated, two-step conversion of N-octanoylglycine to octanoamide.     

Characterization of cDNA encoding the human tRNA-guanine transglycosylase (TGT) catalytic subunit

Queuosine (Q) is a 7-deazaguanosine found in the first position of the anticodon of tRNAs that recognize NAU and NAC codons (Tyr, Asn, Asp and His). Eukaryotes synthesize Q by the base-for-base exchange of queuine (Q base) for guanine in the unmodified tRNA, a reaction catalyzed by TGT. A search of the human EST database for sequences with significant homology to the well studied TGT from Escherichia coli identified several candidates for full-length (1.3-1.4 kb) cDNA clones. Three candidate cDNA clones, available from IMAGE Consortium, LLNL, (Lennon et al., 1996, Genomics 33, 151-152) were obtained: IMAGE Clone Id Nos. 611146, 1422928, and 72154. Here we report the complete sequences of these clones. IMAGE:72154 contains an ORF encoding a 44 kDa polypeptide with high homology to bacterial TGTs and was subcloned into the mammalian expression vector pMAMneo-Cat. When this construct was transfected into the TGT-negative cell line, GC(3)/c1 (Gündüz et al., 1992, Biochim. Biophys. Acta 1139, 229-238), it restored the ability of the cells to form Q-containing tRNA. This TGT cDNA sequence is encoded in human chromosome 19 clone CTC-539A10 (GenBank accession no. AC011475), enabling determination of the exon-intron boundaries for the TGT gene. The sequence of IMAGE:611146 is 5'-truncated by 76 bp compared to that from IMAGE:72154 and, except for two differences in the 3'-non-coding region, the remainder of the sequence is identical to that of IMAGE:72154. IMAGE:1422928 is a 1390 bp chimera: the 5'-portion, bp 1-708, is identical to a genomic DNA sequence from chromosome 15 (GenBank accession no. AC067805, bp 148976-149683); the 3'-end, bp 726-1390, is identical to the 3'-end of the TGT cDNA sequence from IMAGE:611146.     

Herpetic stromal keratitis in the absence of viral antigen recognition

Herpetic stromal keratitis (HSK), resulting from ocular infection with herpes simplex virus (HSV), is thought to represent a T cell mediated immunopathologic lesion. Antigens recognized by the inflammatory T cells remain unresolved and non-TCR mediated activation of T cells (bystander activation) is considered as also involved. This report documents further evidence for the bystander activation mechanisms using three T cell transgenic RAG-/- mouse strains. Accordingly HSK occurred in PCC RAG-/-, P14 RAG-/-, and OT-1 RAG-/- mice. In none of the models could HSV specific T cell reactivity be demonstrated and animals were unprotected from lesion development by immunization prior to HSV ocular infection. The results support the role of bystander activation as a mechanism of T cell mediated immunopathology and show that CD8(+) as well as CD4(+) T cells can participate in HSK lesion development.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

N-(5-chloro-2-(hydroxymethyl)-N-alkyl-arylsulfonamides as gamma-secretase inhibitors

A series of N-alkylbenzenesulfonamides were developed from a high throughput screening hit. Classic and parallel synthesis strategies were employed to produce compounds with good in vitro and in vivo gamma-secretase activity.