RESUMEN
Infections with genital human papillomaviruses (HPV) are likely to be neutralised more efficiently if a mucosal immune response can be elicited at the viral entry site. Local IgA antibodies are highly induced when antigens are co-administered with mucosal adjuvants, such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) which, however, are not expected to have wide application because of their pronounced toxicity. We have immunised mice intranasally with HPV-6b virus-like particles (VLPs) and a genetically modified LT-derived molecule with only residual toxicity, LTR72, and compared the humoral responses with those obtained following systemic immunisation with VLPs and the MF59 adjuvant. Titration of anti-HPV antibodies in sera and vaginal secretions established that LTR72 was able to elicit higher serum and mucosal IgA titers, in addition to IgG serum levels, comparable to those obtained by parenteral immunisation. These results confirm the potential of toxin-derived adjuvants and extend their use in combination with HPV antigens.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Anticuerpos Antivirales/biosíntesis , Toxinas Bacterianas/farmacología , Enterotoxinas/farmacología , Proteínas de Escherichia coli , Papillomaviridae/inmunología , Polisorbatos/farmacología , Escualeno/farmacología , Vacunas Virales/inmunología , Virión/inmunología , Administración Intranasal , Animales , Femenino , Inmunización , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Ratones , Ratones Endogámicos BALB C , Vacunas Virales/administración & dosificaciónRESUMEN
By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor.
Asunto(s)
Interleucina-1/metabolismo , Mutagénesis Sitio-Dirigida , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Dinoprostona/metabolismo , Humanos , Interleucina-1/química , Interleucina-6/genética , Ratones , Datos de Secuencia Molecular , ARN/biosíntesis , ARN/metabolismo , Receptores de Interleucina-1/metabolismo , Células Tumorales CultivadasRESUMEN
A simple and rapid method for analysis of the core of 2',5'-oligoadenylates, mainly based on the use of high performance liquid chromatography (HPLC), is described. Perchloric acid extracts of tissues or cells were first treated with nuclease P1. Portions of the extracts were then digested with alkaline phosphatase. HPLC analysis of the extracts was performed on a column system composed of an Ultrasphere ODS precolumn (4.6 x 45 mm) and an Ultrasphere Octyl column (4.6 x 250 mm) by stepwise elution using a 50 mM ammonium phosphate buffer, pH 7, containing 3.5 and 7% methanol. Three species of the core of 2',5'-oligoadenylates (dimer, trimer and tetramer) from a number of samples were eluted separately with 7% methanol, and the concentration of each core was directly estimated using constant values calculated with the standard core. The level of the core of 2',5'-oligoadenylates in tissues and cells determined by our method is similar to that reported by other authors who used biological, radiobinding or radioimmunological assays.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Oligorribonucleótidos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Hígado/metabolismo , Masculino , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas , Ratas Endogámicas , Venenos de Serpiente/metabolismoRESUMEN
This paper describes regulatory sequences responsible for the control of human interleukin 1 beta gene expression in cells of the monocytic lineage. Hybrid plasmids containing different regions of the interleukin 1 beta gene and the bacterial chloramphenicol acetyltransferase gene were transfected into human monocytic THP-1 or U937 cells. After treatment with 12-O-tetradecanoylphorbol-13-acetate, which triggers cell differentiation, we have identified an enhancer sequence which mediates the induction of gene activity by 12-O-tetradecanoylphorbol-13-acetate. The enhancer, approximately 180 base pairs long, is located between positions -2982 and -2795 upstream from the transcriptional start site. Further analysis has shown that 132 base pairs immediately upstream from the start site of transcription are sufficient to promote constitutive expression of the gene. Additional promoter elements, located within the first intron, maximize the level of gene expression. Although activated monocytes and macrophages are the main producers of interleukin 1 beta, both the enhancer and the constitutive promoter can function in monocytic cells as well as in nonmonocytic HeLa cells.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/fisiología , Genes Reguladores/genética , Interleucina-1/genética , Secuencia de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Monocitos/fisiología , Plásmidos/genética , Mapeo Restrictivo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Células Tumorales CultivadasRESUMEN
Down regulation of poly(ADP-ribose)polymerase (ADPRP) activity was observed in mouse LW-cells after treatment with 2'-5'oligoadenylates or with fibroblast interferon and poly(rI) poly(rC). The poly(rI) poly(rC)-induced inhibition of the enzymatic activity correlates with the observed increase of endogenous 2',5'-oligoadenylate cores which were reported to be potent inhibitors of ADPRP in vitro.
Asunto(s)
Nucleótidos de Adenina/farmacología , Células L/enzimología , Oligorribonucleótidos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Nucleótidos de Adenina/análisis , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Interferones/farmacología , Ratones , Oligorribonucleótidos/análisisRESUMEN
The effects of analogs of diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) were examined on the ADP-ribosylation reaction of histone H1 catalysed by purified bovine thymus poly(ADP-ribose)transferase. Among the compounds tested, epsilon Ap4A and ApCH2pppA were shown to be the most efficient inhibitors of the enzyme. From kinetic studies of their action, it appears that epsilon Ap4A and ApCH2pppA might be 'mixed type' inhibitors.