Genetically engineered or chemically purified soluble monovalent major histocompatiblity complex (MHC)

Serologically distinct forms of H-2Kb are stabilized by loading cells expressing "empty" class I major histocompatibility complex (MHC) molecules with different H-2Kb binding peptides. The H-2Kb epitope recognized by monoclonal antibody (mAb) 28.8.6 was stabilized by ovalbumin (OVA) (257-264) and murine cytomegalovirus (MCMV) pp89 (168- 176) peptides, but not by vesicular stomatic virus nucleoprotein (VSV NP) (52-59) and influenza NP (Y345-360) peptides. The H-2Kb epitope recognized by mAb 34.4.20 was stabilized by VSV NP (52-59) peptide but not by OVA (257-264), MCMV pp89 (168-176), or influenza NP (Y345-360) peptides. Immunoprecipitation of H-2Kb molecules from normal cells showed that 28.8.6 and 34.4.20 epitopes were only present on a subset of all conformationally reactive H-2Kb molecules. Using alanine- substituted derivatives of the VSV peptide, the 28.8.6 epitope was completely stabilized by substitution of the first residue and partially stabilized by substitution of the third or the fifth residues in the peptides. These results indicate that distinct conformational MHC epitopes are dependent on the specific peptide that occupies the antigenic peptide binding groove on individual MHC molecules. The changes in MHC epitopes observed may also be important in understanding the diversity of T cell receptors used in an immune response and the influence of peptides on development of the T cell repertoire.

Fumonisin B(1) (FB(1)) is a worldwide corn contaminant and has been epidemiologically linked to the high incidence of human esophageal cancer in South Africa and China. FB(1) is hepatocarcinogenic in rats by an unknown mechanism. Inhibition of ceramide synthase and disruption of membrane phospholipids have been shown to be mechanisms of toxicity. Here we show overexpression of cyclin D1 protein in both preneoplastic and neoplastic liver specimens obtained from a long-term feeding study of FB(1) in rats. In rats fed FB(1) short-term, cyclin D1 protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in mRNA levels of cyclin D1. 2D electrophoresis of cyclin D1 protein in FB(1)-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3beta (GSK-3beta) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D1. In FB(1)-treated samples we detected GSK-3beta phosphorylated on serine 9; activated protein kinase B (Akt) appears to be responsible for this activity-inhibiting phosphorylation. These findings suggest that overexpression of cyclin D1 results from stabilization due to a lack of phosphorylation mediated by GSK-3beta. We also observed an increase in cyclin dependent kinase 4 (Cdk4) complexes with cyclin D1 in FB(1)-treated samples; additionally, elevated Cdk4 activity was shown by increased phosphorylation of the retinoblastoma protein. In summary, the activation of Akt leads to increased survival, inhibition of GSK-3beta activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle G(1)/S restriction point in hepatocytes. This is the first report suggesting the mechanism by which FB(1) acts as a carcinogen.

SummaryWe compared the conformation of empty and peptide-loaded class I major histocompatihility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the or2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to ~/2-microglobulin. No FRET was found between Fab fragments bound to empty H-2K b, but FRET was detected when empty H-2K b molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2K b molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.