Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.     

Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase

It has been well established that phosphorylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H(+)/K(+)-ATPase, reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H(+)/K(+)-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation, and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H(+)/K(+)-ATPase.     

NFkappaB regulates plasma apolipoprotein A-I and high density lipoprotein cholesterol through inhibition of peroxisome proliferator-activated receptor alpha

The levels of plasma HDL cholesterol and apoA-I in NFkappaB p50 subunit-deficient mice were significantly higher than those in wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To examine the role of NFkappaBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NFkappaB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of IkappaBalpha super-repressor ameliorated the reduction. This IkappaBalpha-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor alpha (PPARalpha), suggesting that NFkappaB inactivation induces apoA-I through activation of PPARalpha. To further support this idea, the expression of IkappaBalpha increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARalpha-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFkappaB increases apoA-I and HDL cholesterol through activation of PPARalpha in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic disease.     

Effect of some constituents of chicken egg yolk lipoprotein on the growth and IgM production of human-human hybridoma cells and other human-derived cells

Chicken egg yolk lipoprotein (YLP) was partially fractionated into some constituents, and the effect of constituents of YLP were examined on the growth and immunoglobulin (IgM and IgG) secretion of a HB4C5 human-human hybridoma cell line cultured in serum-free medium. Among the fractions, YP-1 and YP-2 fractions (LDL-rich fractions) were found to enhance the growth and IgM secretion of HB4C5 cells. The promoting activity was found in the commercial LDL. The lipid fraction in YP-2 fraction conjugated with 2-maltosyl-a-cyclodextrin was found to enhance the growth and IgM secretion of HB4C5 cells. Livetin-rich YP-3 and YP-4 fractions had no significant promoting activity. Commercial -livetin and phosphatidyl choline possessed no growth-promoting activity. Phosphatidyl choline enhanced the IgM secretion of HB4C5 cells.     

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.     

Isolation and characterization of a mutant ColE1 plasmid that allows constitutive colicin E1 synthesis.

It has been possible to isolate a ColE1 mutant which synthesizes colicin E1 constitutively. This result shows that there must be a gene(s) responsible for the regulation of colicin E1 synthesis.