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G Gribaudo

Publications and source records attributed to G Gribaudo.

At least 55 records · Page 3Linked to original sources
PubMed1987

Interferon-gamma inhibits cell replication, but not pp60src activity of RSV-transformed fibroblasts.

The present study analyzes the effect of murine IFN-gamma on DNA synthesis, ornithine decarboxylase activity and phosphorylation of pp60src of Rouse sarcoma virus transformed cells. When natural or recombinant IFN-gamma was added to quiescent SR-BALB or L1210 cells, stimulated with fetal calf serum, only the highest IFN-gamma concentrations (2000 U/ml) inhibited DNA synthesis of both tumor lines. By contrast, lower IFN-concentrations (20-200 U/ml) inhibited the DNA synthesis of L1210 but not of SR-BALB tumor cells. A similar pattern of inhibition was observed when ornithine decarboxylase activity was analyzed. Finally, when the levels of phosphorylation in SR-BALB cells were analyzed after IFN-gamma treatment, no difference with untreated controls was observed, even at the highest concentrations. These results suggest that SR-BALB tumor cells are insensitive to IFN-gamma and that src oncogene activity is not affected at IFN-concentrations inhibiting cell growth.

Animals↗
PubMed1985

Natural murine interferon-gamma. Evidence for post-translational proteolytic processing.

Murine interferon-gamma (MuIFN-gamma), produced by the T-cell lymphoma, L12-R4, and stimulated with phorbol myristic acetate, was purified by rat monoclonal antibodies AN-18.17.24. The purified product retained its biologic activity and consisted of two proteins (Mr = 17,500 and 16,500), as determined by sodium dodecyl sulfate-gel electrophoresis. Both species were found to be glycosylated, since their Mr values decreased to 14,500 and 13,500 when the producing cells were stimulated in the presence of tunicamycin. Analysis of intracellular and secreted forms in the presence of [35S] methionine revealed that MuIFN-gamma is synthetized as a single peptide of Mr = 17,500 and undergoes proteolytic cleavage during or after secretion. Peptide mapping by reverse phase high pressure liquid chromatography showed indeed that the peptide profile of both species is very similar, suggesting a high degree of homology in the primary structure. These results demonstrate that the molecular heterogeneity of the MuIFN-gamma is probably the outcome of proteolytic processing during or after secretion.

Animals↗
PubMed1985

Monoclonal antibodies to murine interferon-gamma: affinity purification and molecular characterization of murine interferon-gamma.

Monoclonal antibodies (MAb), AN-18.17.24, obtained by fusing the murine myeloma cell line Ag8.653 with spleen cells from rats immunized with murine interferon gamma (IFN-gamma) produced by the phorbol myristic acetate(PMA)-stimulated T-cell lymphoma L12-R4, were shown to be specific for MuIFN-gamma. An immunoadsorbent column was prepared and used for large-scale purification of MuIFN-gamma produced either by PMA-stimulated L12-R4 cells or by normal T-lymphocytes stimulated with Con A and interleukin 2. The purified product from L12-R4 cells, which retained biologic activity, was made up of two proteins (Mr = 16,500 and 17,500) as determined by sodium dodecyl sulfate gel electrophoresis. By contrast MuIFN-gamma produced by normal T-lymphocytes resulted in two proteins with Mr of 20,600 and 21,800 as determined by sodium dodecyl sulfate gel electrophoresis. These results indicate that MuIFN-gamma produced by L12-R4 tumor cells or by normal T-lymphocytes differ in their molecular weight, probably because of different degrees of glycosylation of the same molecule.

Animals↗
PubMed1985

Monoclonal antibodies against mouse gamma-interferon inhibit tumoricidal macrophage activation by T lymphocytes.

A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.

Animals↗
PubMed1984

Monoclonal antibodies against murine gamma interferon.

Monoclonal antibodies against murine immune interferon (IFN-gamma) were produced by fusing the murine nonsecreting myeloma cell line P3.X63.Ag8.653 with spleen cells from rats immunized with IFN-gamma-containing supernatants obtained by stimulating a T-cell lymphoma, L12-R4, with phorbol 12-myristate 13-acetate. Supernatants from a twice-cloned hybridoma (AN-18.17.24) were found to neutralize and to adsorb in depletion experiments up to 27 units of mouse IFN-gamma but not equivalent amounts of mouse leukocyte or fibroblast IFNs. The AN-18.17.24 monoclonal antibody neutralized to the same extent mouse IFN-gamma from different sources--namely, (i) concanavalin A-stimulated spleen cells, (ii) alloantigen-stimulated spleen cells, and (iii) monkey fibroblasts transfected with the cloned gene of murine IFN-gamma. Moreover, the monoclonal antibody displayed species specificity, since it did not neutralize IFN-gamma of human origin. Binding inhibition experiments with murine IFN-gamma preparations exposed to enzymatic or physicochemical degradation demonstrated that the protein moiety and not the carbohydrate residues were responsible for the binding to the AN-18.17.24 monoclonal antibody. Finally, this monoclonal antibody immunoprecipitated two molecular species of IFN-gamma of about 16.8 and 17.8 kilodaltons, respectively, from [35S]methionine- or [3H]glucosamine-labeled supernatants of stimulated L12-R4 cells.

Animals↗
PubMed1984

Mouse gamma interferon produced by a cloned T-cell lymphoma. I. Purification and physicochemical characterization.

Mouse gamma interferon (IFN-gamma) was produced from a cloned T-cell lymphoma, L12-R4, stimulated with phorbol myristic acetate (PMA) at a concentration of 2 X 10(-7) M/ml. Absorption of crude IFN-gamma on silicic acid resulted in a 100-fold concentration and 10-fold purification. After application of this IFN-gamma to a Sephacryl S-200 column, 100% of the IFN activity was recovered in fractions containing molecular weights of 40 kD. When this material was applied to a Con A-Sepharose column, IFN activity was completely eluted with a linear gradient of alpha-methyl-mannoside. The specific activity of the recovered IFN-gamma was about 3.6 X 10(6) mg/ml of protein with 11,390-fold purification. Further investigations with Phenyl-Sepharose CL-4B and Chromatofocusing revealed that L12-R4 IFN-gamma is highly hydrophobic and has a pI around 5.4. Thus, by different types of chromatography procedures, L12-R4 IFN-gamma was found to be highly homogeneous with physicochemical properties similar to those produced by normal T-lymphocytes.

Animals↗
PubMed1984

Electrophoretic mobility of mouse immune (gamma) interferon produced by a cloned T-cell lymphoma.

In the present study we have analyzed the electrophoretic mobility of murine IFN-gamma produced by a T-cell lymphoma upon stimulation with phorbol myristic acetate. Two peaks of antiviral activity were recovered after gel electrophoresis of partially purified MuIFN-gamma, in the regions of 30 kd and 43 kd. Since MuIFN-gamma displays a molecular weight of about 16-17 kd in denaturating conditions, the above values represent oligomeric forms, probably composed of two or three units of lower molecular weight.

Animals↗
PubMed1982

[Structural characteristics of immune interferon in mice].

In the present study we have analyzed the biochemical characteristics of murine immune interferon (IFN-y). Ionic or non-ionic detergents inactivate IFN-y, while as far as organic solvents are concerned only those with high polarity inhibit antiviral activity. Finally when different kinds of enzymes were used, proteases but not lipases or neuraminidase inactivated IFN-y.

Animals↗
PubMedDate not supplied

The Ifi 200 genes: an emerging family of IFN-inducible genes.

The biological activities of interferons (IFNs) are mediated by IFN-induced proteins. One family is encoded by several structurally related genes located on murine chromosome 1 (Ifi 200 cluster) and three homologous genes (MNDA, IFI 16 and AIM2) located on human chromosome 1 as well, within a linkage group highly conserved between mouse and human. All the proteins of this family contain at least one copy of a conserved 200 amino acid domain, in addition to other regions that are different or missing among the various family members. Conservation of the 200 amino acid segment, therefore, may be responsible for a common function, while individually expressed domains may afford other tissue- or cell-specific functions. The data available demonstrate that at least two members of the Ifi 200 protein family, p202 and p204, inhibit cell proliferation in vitro. Moreover, high constitutive levels of p204 expression impair normal embryo development in transgenic animals. Here, we will review the principal features of murine and human proteins belonging to this family and their function in the cell growth-regulatory activities mediated by IFNs.

Animals↗
PubMedDate not supplied

Different mechanisms regulate the IFN-inducible expression of 40 kDa human 2'-5' oligo (A) synthetase in HeLa and Molt 4 cells.

Expression of 40 kDa 2'-5' oligo (A) synthetase can serve as a marker of interferon (IFN) activity on the biological target. The mechanisms of induction of human 40 kDa 2'-5' oligo (A) synthetase by IFNs were investigated in HeLa and Molt 4 cells. Using a combined treatment with cycloheximide and actinomycin D we observed that in HeLa cells IFN-alpha did not need ongoing protein synthesis to induce the enzyme, whereas the addition of cycloheximide prevented the induction by IFN-gamma. IFN-alpha induced the 40 kD enzyme in the T-cell line Molt 4 to a level comparable to that in HeLa cells, but only in the presence of active protein synthesis. These results suggest that an early response gene coding for a positive IFN-inducible protein may be needed in T cells, but not in HeLa cells to regulate the transcription of this 2'-5' oligo (A) synthetase gene.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Molecular cloning of interferon-gamma inducible genes from a murine pre-B cell leukemia.

In the present study, a pre-B cell leukemia L1210-C7, representing a very early stage of the B lineage, was used to characterize the molecular mechanisms exploited by IFN-gamma to modulate B cell activity. A cDNA library was prepared with poly (A)+ RNA from cells stimulated with IFN-gamma and three cDNAs clones complementary to IFN-gamma inducible mRNAs were isolated by differential screening. Of these, the 9.5 cDNA hybridized to a 2.4 kb mRNA not homologous with previously cloned IFN-gamma inducible mRNAs. Furthermore, when compared with RNAs obtained from cells of different origins (fibroblasts and T cells) the 9.5 mRNA appeared to be increased only in cells belonging to the B lineage. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the expression of a gene that could be employed as specific cell activation marker.

Animals↗
PubMedDate not supplied

[Isolation of enterococci species causative of infections and sensitivity to antimicrobial drugs].

Between April 1, 1999 and June 30, 2000, 144 isolates of enterococci (one per patient) from cultures of several anatomic sites were collected. One hundred and nineteen (82.6%) E. faecalis, 11 (7.6%) E. faecium and 14 (9.7%) of other species (5 E. raffinosus, 4 E. avium, 3 E. casseliflavus, 1 E. pseudoavium, and 1 E. dispar) were associated with clinical infections. The most common sites of isolation were: the urinary tract 54.9%, abdominal cavity 12.5%, surgical wounds 12.5%, abscesses 6.9% and diabetic foot 6.2%. High-level resistance to gentamicin or streptomycin or both was detected in 48.6% of the isolates. E. faecium and E. raffinosus were significantly more resistant than E. faecalis to ampicilin and imipenem. None of the strains exhibited beta-lactamase activity. One strain of E. faecium (0.7%) was resistant to vancomicin and teicoplanin (Van A phenotype) and two strains of E. casseliflavus (1.4%) showed low level of resistance to vancomicin (Van C phenotype). Because of these diverse antimicrobial resistance mechanisms, successful treatment and control of enterococcal infections with current antimicrobial agents are becoming increasingly difficult.

Abscess↗
PubMedDate not supplied

Characterization of the nuclear factors involved in 202 gene induction by IFN-alpha in NIH-3T3 cells.

When treated with IFN-alpha, NIH-3T3 cells express after a few hours high levels of the mouse 202 gene mRNA. This activation takes place at the transcriptional level as shown by nuclear "run on" assay. For this purpose a fragment of 806 base-pairs (the b fragment), spanning the 5'-flanking region of the 202 gene, was linked to the reporter CAT gene and transiently transfected into mouse NIH 3T3. The data suggest that the b fragment is sufficient to confer transcriptional inducibility upon IFN stimulation and can account in large part for the response of the 202 gene. Binding assays, using a 40-bp probe derived from the IFN-stimulated response element (ISRE) comprised in the b fragment, demonstrated the presence of two DNA-binding proteins. One of these, defined as complex A, was inducible upon IFN treatment, whereas the other, defined as complex B, was constitutively present regardless of IFN treatment. The IFN-alpha-induced complex A appears to have the necessary characteristics to be the transcriptional activator of the 202 gene: it requires the same nucleotides for binding as are required for IFN-dependent gene activation and is dependent on IFN-alpha treatment.

Animals↗
PubMedDate not supplied

Production of sheep polyclonal antibodies against the 2'-5' oligoadenylate synthetases.

A 20-amino acid peptide corresponding to a common part of the 40 and 46 kD forms of human 2'-5' oligoadenylate synthetase was coupled to keyole lymphet haemocyanin (KLH) and used as immunogen in sheep. After a cycle of four immunizations, immunoglobulins able to recognize the 20-amino acid peptide as evaluated in ELISA assays were purified by an immunoadsorbent with the peptide immobilized on Sepharose CL-4B and used in Western blot employing a secondary anti-sheep antibodies and iodinated protein A as indicator system. Results obtained using extracts from human and mouse cells treated for 15 hr with IFN-alpha as antigen demonstrated that the anti-peptide antibodies recognize several forms of the 2'-5' oligoadenylate synthetase enzyme complex. These antibodies therefore represent a useful tool for monitoring the induction of the above enzymes.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Anti-interferon-gamma antibodies in sera from HIV infected patients.

High serum levels of antibodies to interferon-gamma (IFN-gamma) have been found in patients infected with human immunodeficiency virus (HIV). A radioimmunoassay (RIA) with a recombinant IFN-gamma protein or an affinity purified IFN-gamma preparation as antigens, was developed to detect the specific anti-IFN-gamma antibodies. Reactivity of sera to IFN-gamma was confirmed by Western blot analysis. These antibodies, however, do not seem to recognize the active site of the molecule, since they do not neutralize the antiviral IFN-gamma activity in a biological assay. These results enforce the hypothesis of the role of autoimmunization during the course of the disease.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[Biological activity of gamma interferon on various cell types in human and murine systems].

The effects of interferon (IFN gamma) and of IFN alpha/beta on normal T and B lymphocytes and various T and non-T human and murine cell lines have been investigated. IFN gamma, unlike IFN alpha/beta, did not promote the antiviral state in T cells. The lack of antiviral activity was confirmed at the biochemical level by the finding that 2',5' oligoadenylate synthetase activity is not induced in T cells by IFN gamma only.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Interferon gamma does not induce antiviral resistance in T lymphocytes.

We compared the activity of human recombinant alpha and gamma interferons (IFNs) on normal T lymphocytes and various T cell lines. IFN gamma, unlike IFN alpha, did not promote the antiviral state in these cells, or induce the activity of 2'-5' oligoadenylate synthetase. The lack of antiviral effect was observed using an RNA virus (VSV) and a DNA virus (HSV, type 1) as challenger viruses.

2',5'-Oligoadenylate Synthetase↗