Immunogenic and antigenic properties of an HIV-1 gp120-derived multiple chain peptide.

An HIV-1 envelope protein gp120-derived monomeric peptide (amino acid residues 419-439) and its homologous multiple chain peptide (MCP) construct were compared for immunogenicity in mice. The Abs stimulated by the MCP recognized epitopes on the MCP that were not present on the homologous monomer. The anti-419-439 MCP sera recognized a conformational determinant on the native envelope glycoprotein, as indicated by: 1) detection of native but not denatured recombinant envelope glycoprotein by ELISA and dot blot and 2) reaction with infected cell lines expressing gp120 on their surface as detected by flow cytometry. In contrast, the anti-monomer sera were highly specific for the monomer and recognized the envelope glycoprotein at lower titers. The low reactivity of the anti-monomer sera with the envelope glycoprotein was not decreased by denaturation. Reciprocally, murine antiserum to HIV-1 envelope glycoprotein gp160 recognized the MCP construct but not the homologous monomeric peptide. The data indicate that the MCP construct forms additional antigenic determinants not present on the homologous monomer, and that the anti-419-439 MCP Abs recognize a conformational determinant on the envelope glycoprotein not recognized by Abs against the homologous monomer. Furthermore, antisera against another envelope-derived MCP (amino acid residues 105-117) also recognize conformational determinants on the envelope glycoprotein, whereas antisera against the homologous monomeric peptide do not.

Lymphocytes from some long-term seronegative heterosexual partners of HIV-infected individuals proliferate in response to HIV antigens.

A comparison of the proliferative responses of lymphocytes to human immunodeficiency virus (HIV) antigens from long-term, seronegative heterosexual partners of HIV-infected subjects, from normal unexposed controls and from healthy seropositive heterosexual partners or seropositive, asymptomatic men, reveals that lymphocytes from healthy seropositive individuals with strong proliferative responses to recall, microbial antigens respond only minimally to HIV proteins or envelope peptides, and that even these low responses do not occur in all individuals. If the frequency of responses to several HIV antigens are analyzed, lymphocytes from both HIV-exposed seropositive and seronegative partners of infected individuals proliferate to HIV antigens to a greater degree than lymphocytes from unexposed, normal control individuals. Although lymphocytes from seropositive partners proliferate to a greater degree than those from seronegative partners, the latter are more similar to seropositive partners than they are to normal controls. This observation suggests that these seronegative partners may have become sensitized to HIV antigens through sexual exposure but without infection, and/or that the presence or development of these small immune responses in some individuals might be associated with a failure to become infected.

Characterization of human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin.

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.

Three molecular weight forms of natural human interferon-gamma revealed by immunoprecipitation with monoclonal antibody.

Immune interferon (IFN-gamma), endogenously labeled with [35S]methionine, was produced in human peripheral blood lymphocyte cultures stimulated with 12-O-tetradecanoylphorbol-13-acetate and phytohemagglutinin. 35S-IFN-gamma, immunoprecipitated from the crude culture fluid with a monoclonal antibody, was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three monomeric forms with molecular weights of 25,000, 20,000, and 15,500, which we designate IFN-gamma I, II, and III, respectively. IFN-gamma I was the most, and IFN-gamma III the least, abundant in both immunoprecipitated 35S-IFN-gamma and chromatographically purified IFN-gamma preparations. Changes in the molecular size of the monomeric forms after glycosidase treatment suggested that IFN-gamma I contains more carbohydrate than IFN-gamma II, and that IFN-gamma III may not be glycosylated at all. Hence, the differences in the carbohydrate contents are likely to be the major cause of the molecular size heterogeneity of IFN-gamma I, II, and III.

Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin.

Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.

Natural and recombinant Escherichia coli-derived interferon-gamma differ in their reactivity with monoclonal antibody.

Monoclonal antibody GIF-1 was found to neutralize human natural immune interferon (IFN-gamma), but not Escherichia coli-derived recombinant IFN-gamma. In addition, GIF-1 antibody failed to immunoprecipitate 125I-labeled recombinant IFN-gamma, whereas it precipitated natural IFN-gamma in a concentration-dependent manner. The lack of recognition of recombinant IFN-gamma by antibody GIF-1 may not be due to the absence of the oligosaccharide moiety in the molecules of recombinant IFN-gamma alone, because removal of carbohydrate from natural IFN-gamma by treatment with a mixture of glycosidases did not alter the selective binding of antibody, i.e., deglycosylated and untreated natural IFN-gamma were equally neutralized and immunoprecipitated by GIF-1 antibody. In addition, a minor monomeric component of natural IFN-gamma with the m.w. of 15,500, which apparently lacks carbohydrate, was also recognized by antibody GIF-1. These results suggest that the discriminative recognition of natural and recombinant IFN-gamma by monoclonal antibody GIF-1 may be due to a conformational difference at or near the active regions of natural and recombinant human IFN-gamma molecules.

Stimulation of lymphokine production by teleocidin, aplysiatoxin, and debromoaplysiatoxin.

Previous studies showed that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-gamma) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-gamma and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-gamma during a multistep purification procedure.

Effects of glycosidase treatment on the physicochemical properties and biological activity of human interferon-gamma.

Highly purified human interferon (IFN)-gamma was treated with a preparation of mixed glycosidases in order to evaluate the effect of carbohydrate depletion on its biological activity, isoelectric point, and molecular size. Glycosidase treatment did not reduce the antiviral activity of IFN-gamma in cultures of human fibroblasts and in bat lung cells. No antiviral activity was observed before or after treatment with glycosidases in pig, mink, bovine, murine, and monkey cells. The degree of neutralization of IFN-gamma activity with specific antibody was also not significantly affected by glycosidase treatment. Several components of IFN-gamma activity were resolved by nonequilibrium pH gradient electrophoresis, with major peaks of activity at pI 8.5 and 8.7. Glycosidase treatment of IFN-gamma resulted in a reduced charge heterogeneity and a higher pI of 9.3. 125I-labeled IFN-gamma was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two bands with molecular weights of 25,000 and 20,000. Glycosidase treatment reduced the apparent molecular weight of these bands to 18,500 and 16,000, respectively. The results suggest that both the Mr = 25,000 and 20,000 bands, thought to be monomeric forms of IFN-gamma, are glycosylated.

N-acetylation of drugs. Observations on the properties of partially purified N-acetyltransferase from peripheral blood of rabbit.

A method is described for the partial purification of N-acetyltransferase (EC 2.3.1.5) from peripheral blood of rabbit. This enzyme purified from both rapid- and slow-ioniazid-acetylator phenotype rabbits was examined with regard to stability, substrate specificity, cation effect, rate of inactivation, temperature and pH optimum. Data show that in the biochemical characteristics examined the enzyme is independent of acetylator phenotype. The possible significance of these findings to the evolutionary origin of drug-acetylating enzymes in rabbit blood and liver is discussed.

The stringent and relaxed phenomena in Saccharomyces cerevisiae.

We present here a detailed analysis of the effect of amino acid starvation and the addition of cycloheximide on RNA metabolism of yeast cells and spheroplasts. These effects have been studied at the level of uridine phosphorylation, methylation of rRNA, and biosynthesis of 35 S, 4 S, and 5 S RNA species. Amino acid starvation inhibits the phosphorylation of uridine assigned for RNA synthesis more than that for other metabolic processes. This implies that a salvage pathway for the synthesis of UMP and CMP is regulated by the rate of transcription and perhaps is localized in the nucleus. The rate of rRNA methylation is not coupled with the rate of transcription; therefore, quantitation of 35 S RNA synthesis (yeast rRNA primary transcript by [methyl-3H]methionine labeling is unreliable. Biosynthesis of 35 S RNA ceases immediately after the cells are transferred to an amino-acid-deficient medium; at a later time 4 S and 5 S RNAs are also inhibited. Therefore, coordination and noncoordination in the stringent response of these RNA species depend upon the time of starvation. Although addition of a small dose (less than 1.0 microgram/ml) of cycloheximide to starved yeast spheroplasts does not alter the rate of uridine phosphorylation, it increases the rate of entrance of uridine into total RNA. This effect is of greater magnitude in 4 S and 5 S than in 35 S RNA. Since the drug does not alter the rate of decay of 35 S RNA that takes place in starvation, it has a selective effect on transcription. A similar small dose, however, produces inhibition of transcription of all these RNA species in nonstarved conditions. This opposite effect of the drug appears to be a characteristic feature of RNA metabolism in eukaryotes.