Increase in the immunogenicity of cancer cells exposed to microwaves

A suspension of 10(7) melanoma cells, submitted to microwave hyperthermia (2,450 MHz, 20 minutes, 44 degrees C) leads to a partial protection in mice inoculated 26 days afterwards with a suspension of 10(7) active cells of B16 melanoma (95% vitality). The period of 26 days between the two injections corresponds to the moment where the sera antibodies have an highest level. The kinetics of the primary response of the humoral immunity shows that B16 melanoma proliferation and number of deads can be related to an hypogammaglobulinemia.     

Covalent linkage of the type- and group-specific antigens to the peptide moiety of the peptidoglycan of serotype III group BStreptococcus

The attachment sites for the two major cell wall polysaccharides, the type-and group-specific antigens of a serotype III group B streptococcus (GBS) were investigated with [14C]lysine to label the peptide portion of the peptidoglycan and [3H]acetate to label both polysaccharide antigens as well as the glycan backbone of the peptidoglycan. Mutanolysin-treated cell walls were subjected to trypsin digestion, followed by exhaustive beta-elimination with 6N ammonium hydroxide at 37°C. The resulting products were purified by column chromatography prior to chemical, immunological, and high-voltage electrophoresis analyses. Data from these studies indicated that both cell wall polymers are covalently attached to the peptidoglycan via the peptide unit. Additionally, during synthesis and assembly both antigens attached only to nascent peptidoglycan.     

Characterization of the amino-terminal tryptic peptide of simian virus 40 small-t and large-T antigens.

Simian virus 40 small-t and large-T antigen were synthesized in vitro and labeled with methionine donated by initiator tRNA. Tryptic peptide fingerprinting was used to identify the amino-terminal peptide of the two proteins. Similar fingerprint analysis of small-t and large-T made in vitro in the absence of acetyl coenzyme A showed that the mobility of the amino-terminal peptide was changed under these conditions and suggested that it is acetylated. These data establish that the amino-terminal methionine residue of simian virus 40 small-t and large-T results from an initiation event, not post-translational cleavage, and provides additional evidence that the amino terminus of both proteins is acetylated. The identification of the amino-terminal peptide provides a useful marker for further studies on different forms of T-antigen from cells infected with and transformed by simian virus 40 and related viruses.     

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains.     

Metal binding to the HIV nucleocapsid peptide

 Co(II) and Zn(II) binding constants have been measured for binding to the HIV-1 nucleocapsid N-terminal metal binding domain (residues 1–18), using competition titration methods and monitoring Co(II) binding by visible absorbance spectroscopy. Enthalpies for binding were directly measured by isothermal titration colorimetry. The results are compared with recent studies of related systems, including a study of Zn(II) binding by the full length protein. Received: 1 December 1998 / Accepted: 31 December 1998     

Relation of the immunogenicity of artificial antigens to the number of polyelectrolyte-bound protein molecules

Complexes and covalent conjugates of protein antigens with polyelectrolytes of different molecular mass have been synthesised. The structure and composition of the resulting water-soluble complex particles were determined. Artificial antigen immunogenicity was shown to depend on the amount of protein molecules complexed with polyelectrolytes. Direct correlation between immunostimulating activity of the polymer-carrier, immunogenicity of complex antigens and size-dependent capacity of the polymer molecule to aggregate protein globules has been established.     

Control of electron transfer in DNA by peptide nucleic acids (PNA).

The one-electron oxidation of PNA-DNA hybrid containing G-triplet sequence was examined. In DNA duplex G-triplet was selectively cleaved by oxidation, whereas in PNA-DNA hybrid cleavage efficiency was extremely lowered. These result suggested that cleavage efficiency of PNA-DNA hybrid was different from that of B-form DNA duplex.     

Two-site binding of beta-cyclodextrin to the Alzheimer Abeta(1-40) peptide measured with combined PFG-NMR diffusion and induced chemical shifts

The interactions of Alzheimer's amyloid beta-peptide with cyclodextrins were studied by (1)H NMR: the translational diffusion coefficient of the peptide and chemical shift changes were studied by the presence of variable concentrations of cyclodextrins. For the full-length peptide, Abeta(1-40), the combined results of translational diffusion and chemical shift changes are consistent with a model where aromatic side chains interact with beta-cyclodextrin with dissociation constants in the millimolar range. The diffusion data were consistent with two beta-cyclodextrin molecules bound per peptide. The binding occurs at two sites, at F(19) and/or F(20) and at Y(10), with dissociation constants K(d)(F) = 4.7 mM and K(d)(Y) = 6.6 mM, respectively, in 10 mM sodium phosphate, pH 7.4 and 298 K. Shorter Alzheimer peptide fragments were studied to measure specific affinities for different binding sites. The N-terminal fragment Abeta(1-9) with a putative binding site at F(4) does not show measurable affinity for beta-cyclodextrin. The fragment Abeta(12-28) has similar apparent affinity (K(d) = 3.8 mM) to beta-cyclodextrin as the full-length peptide Abeta(1-40). Here, the diffusion data suggests a one-to-one stoichiometry, and the binding site is F(19) and/or F(20). Both diffusion results and chemical shift changes give the same affinity. A variant Abeta(12-28)G(19)G(20) without phenylalanines does not bind to beta-cyclodextrin. Other potential ligands, alpha-cyclodextrin, gamma-cyclodextrin, nicotine, and nornicotine do not bind to the Abeta(12-28) fragment. This study shows that combined (1)H NMR diffusion and chemical shift changes may be used to quantitatively determine affinities and stoichiometries of weak interactions, using unlabeled ligands and hosts of comparable sizes.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage

The alpha-anomeric Galbeta1-3GalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosylation. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-binding specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than Gal, since ABL recognizes GalNAc more than Gal in TFD. The synthesis was accomplished by C-6 oxidation of Gal from TFD or its alpha-benzyl derivative (BzlalphaTFD), followed by reductive amination between the C-6 aldehyde yielded and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or BzlalphaTFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens. Both immunogens raised anti-KLH antibody titers; however, TFD-KLH did not raise anti-TFD antibodies showing low TFD immunogenicity. In contrast, BzlalphaTFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps improve anti-carbohydrate immune response. When IgG and IgM anti-TFD antibodies were analyzed by competitive ELISA using TFD-related carbohydrates as inhibitors, a high specificity to TFD as well as an enhanced binding to GalNAc over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activity of GlcNAc in contrast to GalNAc. These findings indicate that the anti-TFD antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithelial cell lines.