We have previously identified a self-reactive y8 T-cell clone (KN6) specific for the H-2T region gene product We have previously demonstrated that the product encoded by the H-2T22 gene is recognized by the T-cell receptor (TCR) derived from the mouse KN6 y6 T-cell hybridoma (7). Here, we present a mutational analysis of the 122 gene. The results demonstrate that mutations in the putative antigenbinding groove ofT22 affect recognition by the KN6 'y6 TCR.We also demonstrate that the KN6 cells can be activated by the TAP2-deficient cell line RMA-S or by splenocytes from TAP1 mutant mice. TAP1/TAP2 is a peptide transporter that lies in the major route for the assembly and cell surface expression of the MHC class I-peptide complex. Our results suggest that peptide is involved in the interaction of KN6 y8 TCR with T22 molecules and that this putative peptide is loaded to a T22 molecule by a TAP1/TAP2-independent mechanism.
Many constituents of crude drugs in Japanese Kampo formulas are thought to function as pro-drugs, whose pharmacological activity is manifested after oral administration. Proteins and peptides in crude drugs may be digested and metabolized in the digestive tract and liver. However, few studies have reported the pharmacological activity of peptides in crude drugs. Here, we applied an analysis using LC-tandem mass spectrometry (LC-MS/MS) to identify the compounds derived from six crude drugs that are assumed to have anti-inflammatory effects. To simulate in vivo protease digestion, each water-soluble fraction of the crude drug extracts was treated with proteases, including endoproteinases and exopeptidases. Amines in the resultant digests were modified by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and analyzed using LC-MS/MS, which demonstrated the presence of four decarboxylated amino acids (primary amines). In the digest of the hydrophilic fraction of the fruit of Ziziphus jujuba Miller var. inermis Rehder (Taiso), isobutylamine, isoamylamine, and 2-methylbutylamine were identified, which may be derived from valinyl, leucinyl, and isoleucinyl residues, respectively. Additionally, tyramine possibly derived from tyrosyl residues was identified in the digests of all the crude drugs. In primary cultured rat hepatocytes treated with interleukin-1β, all these decarboxylated amino acids suppressed the production of nitric oxide, a proinflammatory mediator. Our approach, i.e., in vitro protease digestion and LC-MS/MS analysis, suggests that decarboxylated amino acids may be formed in vivo from peptides and may be responsible for the anti-inflammatory effect of crude drugs included in Kampo medicine.
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