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C Baglioni

Publications and source records attributed to C Baglioni.

At least 73 records · Page 4Linked to original sources
PubMed1985

Different mechanisms for activation of gene transcription by interferons alpha and gamma.

Treatment of HeLa cells with either recombinant interferon (rIFN) alpha 2 or gamma elevated the level of mRNA for 2',5'-oligo(A) synthetase and for a 56,000 Mr protein. Cycloheximide prevented the induction of these mRNAs when rIFN-gamma, but not rIFN-alpha 2, was the inducer. This suggested that these rIFNs activate gene transcription by different mechanisms. The level of induced mRNAs reached a maximum 8 or 12 hr, respectively, after addition of rIFN-alpha 2 or rIFN-gamma, and then slowly decreased. Inhibition of protein synthesis by cycloheximide 8 hr after the addition of rIFN-alpha 2 prevented the decline in mRNA for 2',5'-oligo(A) synthetase. Addition of an inhibitor of RNA synthesis or removal of IFN from the medium accelerated this decline. Although the IFN-alpha receptors were down-regulated in HeLa cells treated for 25 hr with rIFN-alpha 2, these cells responded to a second rIFN-alpha 2 treatment with a further increase in inducible mRNAs. These results suggest that the level of these mRNAs is regulated by two opposing processes. As long as IFN is present in the medium, it continues to activate transcription of inducible mRNAs. However, after 8-12 hr of treatment, an opposing process, which is inhibited by cycloheximide, causes a decline in the level of these mRNAs.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Kinetics of dephosphorylation of eIF-2(alpha P) and reutilization of mRNA.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) causes mRNA to accumulate in 48 S complexes containing Met-tRNAf and eIF-2(alpha P). When the eIF-2 alpha kinase is inhibited by 2-aminopurine, the mRNA is slowly transferred from 48 to 80 S initiation complexes after an initial lag. The cause of this lag was examined by investigating whether mRNA and Met-tRNAf dissociated from 48 S complexes before binding to 80 S. Both compounds were quantitatively transferred from 48 to 80 S complexes after addition of 2-aminopurine and the eIF-2(alpha P) bound to 48 S complexes was dephosphorylated after an initial lag more slowly than unbound eIF-2(alpha P), which was rapidly dephosphorylated. the eIF-2(alpha P) in isolated 48 S complexes was slowly dephosphorylated by partially purified lysate phosphatases, whereas free eIF-2(alpha P) was readily dephosphorylated. These results indicated that 48 S complexes could directly join to a 60 S ribosomal subunit after eIF-2(alpha P) dephosphorylation. The lag and slow kinetics of dephosphorylation of eIF-2(alpha P) bound to 48 S complexes accounted for the slow transfer of mRNA from 48 to 80 S complexes. Moreover, the mRNA bound to 48 S complexes was more susceptible to cleavage by an endonuclease than mRNA in polyribosomes, as shown by activating the (2'-5')oligo(A)-dependent endonuclease. This finding is discussed in view of the possible role of eIF-2 alpha kinase and endonuclease in the inhibition of viral mRNA translation in interferon-treated cells.

Endoribonucleases↗
PubMed1985

Binding of human recombinant 125I-interferon gamma to receptors on human cells.

Recombinant human interferon gamma (rIFN-gamma) produced in Escherichia coli was labeled with 125I to study its binding to receptors of HeLa and lymphoblastoid cells. All the cell lines examined had receptors for rIFN-gamma, although the binding varied considerably among different cell lines. The binding of 125I-rIFN-gamma was competed up to 90% by the addition of unlabeled rIFN-gamma, although not by the addition of IFN-alpha or -beta. By adding increasing concentrations of unlabeled rIFN-gamma to binding assays containing a constant amount of 125I-rIFN-gamma, we determined a KD of 3.7 and 6.3 X 10(-10) M for its binding to Daudi and HeLa cells, respectively. About 13,000 receptors per cell were present in Daudi and 5,000 in HeLa cells. The Mr of the IFN-gamma/receptor complex was determined by cross-linking experiments to be about 125,000. This complex is smaller than the IFN-alpha/receptor complex that has a Mr of about 140,000. The rIFN-gamma receptor was down-regulated when HeLa cells were treated with this interferon, but not when these cells were treated with IFN-beta. These findings suggest that the receptors for IFN-alpha and -gamma differ in several characteristics. The turnover of the rIFN-gamma receptor was measured by inhibiting protein synthesis with cycloheximide and the half-life of this receptor was found to be 2 h. The unglycosylated rIFN-gamma was bound to cellular receptors with an affinity similar to that previously reported for natural IFN-gamma. The lymphoblastoid cell lines examined had high affinity receptors for rIFN-gamma, but did not respond to treatment with this IFN with an induction of the synthesis of the enzyme (2'-5')oligo(A) synthetase, whereas HeLa cells responded to rIFN-gamma. The reason for the lack of response of lymphoblastoid cells is presently unknown.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Single high affinity binding of interferon alpha 2 to receptors on human lymphoblastoid cells: internalization and inactivation of receptors.

The interaction of human recombinant interferon (rIFN) alpha 2 with its receptor on lymphoblastoid cells was studied using competitive displacement binding. The data were analysed with the LIGAND program, which tests their fit to one-site or multiple binding site models. The binding at 4 degrees and 37 degrees C fits a one-site model, with a similar KD for both IFN-sensitive and resistant cells. Binding at 37 degrees C to Daudi cells at high density fits artifactually a two-site model only when the receptor concentration is close to that of the KD. The binding of IFN to its receptor, therefore, follows a simple bimolecular interaction. Furthermore, IFN-sensitive and resistant cells internalize IFN at similar rates. We have examined whether IFN receptors are also internalized and whether they subsequently recycle to the cell surface. By measuring cell surface and total receptors, we have observed that after 2 h treatment with IFN total receptors remain constant whereas cell surface receptors decrease. After prolonged treatment with IFN, however, there is a loss of total receptors. By inactivating cell surface receptors with proteinase K, we have shown that a fraction of cell surface receptors becomes resistant to inactivation and is apparently internalized. Moreover, experiments which measure IFN receptors either during incubation in the presence of IFN or after IFN has been removed from the medium, show that receptors do not recycle to the cell surface after internalization. The addition of monensin, a drug which has been shown to inhibit receptor recycling, has no effect on the loss of IFN receptors.

Binding, Competitive↗
PubMed1985

Binding of unglycosylated and glycosylated human recombinant interferon-gamma to cellular receptors.

Recombinant human interferons (IFNs), either unglycosylated produced in E. coli (rIFN-gamma) or glycosylated produced in CHO cells (g-rIFN-gamma), were labeled with 125I to similar specific activities to study their interaction with cell-surface receptors. When analyzed by gel electrophoresis, rIFN-gamma run as a single polypeptide of Mr 15,000-17,000, whereas g-rIFN-gamma separated into three components of Mr 20,000, 22,000, and 43,000, which corresponded to the known size of the two monomeric and one dimeric forms of glycosylated natural IFN-gamma. The binding of the two species of 125I-IFN-gamma was competed equally by rIFN-gamma in competition displacement experiments with Daudi cells, indicating that these IFNs bind with similar high affinity to the same receptors. KD values of 1.25 X 10(-10) and 2.5 X 10(-10) M were determined for g-rIFN-gamma and rIFN-gamma, respectively. This relatively small difference in KD does not apparently result in a detectable difference in biological activity, as measured by the increase in 2',5'-oligo(A) synthetase activity in IFN-treated HeLa and A549 cells. These results indicate that glycosylation of IFN-gamma does not play a significant role in its interaction with cellular receptors and in the induction of a biological response.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Inhibition of binding to initiation complexes of nascent reovirus mRNA by double-stranded RNA-dependent protein kinase.

A coupled, cell-free system for the transcription and translation of reovirus mRNA was developed. Activated reovirions were incubated with reticulocyte lysate and an appropriate energy mix. Active transcription was obtained, but protein synthesis was inhibited after a short lag even by low concentrations of reovirions. This inhibition was abolished by the addition of the kinase inhibitor 2-aminopurine. With this addition, the synthesis of viral proteins could be detected in reaction mixtures containing nuclease-treated reticulocyte lysate. The binding of nascent reovirus mRNA to 80S initiation complexes measured after 2 min of incubation was greatly inhibited, whereas the binding of cellular mRNA added to the same reaction mixtures for the next 2 min was not inhibited. The inhibition of reovirus mRNA binding could not be explained by the synthesis of defective templates, since most of the mRNA could be bound to 80S complexes after the addition of 2-aminopurine. These results indicate that the binding of nascent reovirus mRNA was preferentially inhibited by a protein kinase. Reovirions preincubated with reticulocyte lysate could phosphorylate initiation factor eIF-2. This phosphorylation was inhibited by the addition of high concentrations of double-stranded RNA, which are inhibitory for the eIF-2 kinase present in elevated levels in reticulocyte lysate and in interferon-treated cells. These results indicate that the translation of viral mRNA may be preferentially inhibited in interferon-treated cells by the eIF-2 kinase activated by viral transcriptional complexes containing double-stranded RNA.

Animals↗
PubMed1985

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.

Animals↗
PubMed1984

Treatment of lymphoblastoid cells with interferon decreases insulin binding.

Lymphoblastoid Daudi cells, which are highly sensitive to growth inhibition by interferon (IFN), can be grown in a defined serum-free medium containing insulin, transferrin, and albumin as the only proteins. We examined whether the growth inhibition by IFN could be in part due to a change in receptors for insulin or transferrin. Cells treated for at least 2 days with 100 units/ml of IFN-alpha 2 bound less 125I-insulin and after 3 days of treatment this binding was reduced by more than 50%. No change in the binding of 125I-transferrin was observed. Treatment with IFN of Raji cells, which are resistant to growth inhibition by IFN, resulted in a similar decrease in 125I-insulin binding. Growth inhibition of Daudi cells by serum deprivation had no effect on 125I-insulin binding. Therefore, the IFN-induced loss of insulin binding sites is not a consequence of growth inhibition.

Burkitt Lymphoma↗
PubMed1984

Antibodies to chromosome 21 coded cell surface components block binding of human alpha interferon but not gamma interferon to human cells.

Antisera raised against a human X mouse hybrid cell line containing human chromosome 21 as its only human chromosome, block induction of an antiviral state by human alpha interferon (IFN-alpha), block induction of (2'-5')oligoisoadenylate synthetase [2'-5')A synthetase), and block binding of 125I-labeled and 35S-labeled recombinant, human IFN-alpha A, but not 125I-labeled IFN-gamma, to cell surface receptors. The data presented clearly demonstrate that the cell surface receptors for IFN-alpha and IFN-gamma are different, and provide independent evidence of the role of a chromosome 21 coded cell surface molecule in the pathway to the generation of the antiviral state.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Lack of correlation between thymidine kinase activity and the antiviral or antiproliferative response to interferon.

Murine L cells lacking thymidine kinase activity (Ltk-) do not respond to the antiviral or antiproliferative activity of interferon, whereas human tk- cells show a normal response. Furthermore, a clone of tk+ cells derived from Ltk- cells by DNA-mediated transfer of the tk herpes gene does not respond to interferon. Cells of this clone and Ltk- cells do not produce interferon when infected with Newcastle disease virus or treated with poly(I) X poly(C) and DEAE-dextran. Possible reasons for this inability to produce interferon and the lack of response to interferon are discussed.

Animals↗
PubMed1984

Cleavage of nascent reovirus mRNA by localized activation of the 2'-5'-oligoadenylate-dependent endoribonuclease.

Addition of cell extracts prepared from untreated or interferon-treated cells reduced the yield of reovirus RNA synthesized in transcription reactions with purified virions. The reduction observed with the extract of interferon-treated cells was greater than that observed with the extract of untreated cells. The yield of reovirus RNA could be restored to the level obtained without addition of cell extracts by carrying out transcription either in the presence of an inhibitor of 2'-5'-oligoadenylate (2-5A) synthesis, 2'-dATP, or in the presence of the 3'-methyl analog of 5'-monophosphate 2-5A, which inhibits the 2-5A-dependent endoribonuclease. This enzyme was activated in a localized way since neither mRNA added to transcription reactions containing the extract of interferon-treated cells nor rRNA was appreciably degraded, whereas accumulation of reovirus mRNA was drastically inhibited. These findings provide evidence for an antiviral role of the 2-5A synthetase/endoribonuclease system, which preferentially cleaves nascent viral RNA.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Interferon is a polypeptide hormone.

Interferons (IFNs) bind to specific high-affinity receptors on the cell surface. The human IFN-alpha receptor is a highly hydrophobic membrane protein (molecular weight about 100,000). After binding to the receptor, IFN is internalized and degraded by lysosomal proteases. The receptor is also internalized, does not recycle to the cell surface and is gradually inactivated. An unidentified mechanism transduces a signal from the interferon/receptor complex to induce the transcription of specific genes. There are striking similarities between IFN and polypeptide hormones in their interaction with cell surface receptors, internalization of polypeptide/receptor complexes and degradation of the polypeptides in lysosomes.

Hormones↗
PubMed1984

Correlation between growth inhibition and presence of 5'-methylthioadenosine in cells treated with interferon.

Treatment with interferon of several lines of human and murine cells resulted in a modest increase of the S- adenyosylmethionine level and in a greater increase of the S-adenosylhomocysteine level, with a consequent decrease in the S- adenyosylmethionine /S-adenosylhomocysteine ratio. These results confirmed a previous report (F. de Ferra and C. Baglioni , J. Biol. Chem., 258: 2118-2121, 1983) of such an effect of interferon on HeLa cells. The increase in S- adenyosylmethionine and S-adenosylhomocysteine was not observed in lymphoblastoid Raji cells, which are not growth inhibited by interferon. When Daudi cells and two other cell lines sensitive to growth inhibition by interferon were labeled with [35S]methionine, two novel labeled compounds were detected by high-pressure liquid chromatography analysis of cell extracts. These compounds were not present in untreated cells, and were either absent or present in reduced amount in extracts of cells resistant to the antiproliferative effect of interferon. One of the labeled compounds was tentatively identified with 5'-methylthioadenosine (MTA), a naturally occurring nucleoside, which was previously shown to inhibit murine lymphoid cell proliferation. Addition of MTA to cultures of different cell lines resulted in inhibition of cell growth. When the MTA concentration was measured in Daudi cells treated in this way, a relatively low concentration of MTA was detected, which was comparable to that found in interferon-treated cells. It seems possible that the presence of MTA in interferon-treated cells contributes to the inhibition of cell growth.

Adenosine↗
PubMed1983

Phosphorylation of initiation factor eIF-2 alpha, binding of mRNA to 48 S complexes, and its reutilization in initiation of protein synthesis.

The formation of 80 S initiation complexes containing labeled viral mRNA was drastically inhibited when mRNA binding assays were carried out with reticulocyte lysate preincubated with double-stranded RNA (dsRNA). When the assays were analyzed by centrifugation on sucrose gradients, the mRNA incubated with lysate pretreated with dsRNA sedimented as a 48 S complex. Met-tRNA, GDP, and phosphorylated initiation factor eIF-2(alpha P) were shown to co-sediment with the 48 S complex. Therefore, the formation of this complex was attributed to the phosphorylation of eIF-2 alpha by a dsRNA-activated protein kinase. These observations suggested that mRNA could bind to a 40 S ribosomal subunit containing Met-tRNAf, GDP, and eIF-2(alpha P), but the joining of a 60 S ribosomal subunit was inhibited. When the 48 S complex was isolated and incubated with lysate without added dsRNA, the mRNA could form 80 S initiation complexes. The shift of mRNA from 48 S to 80 S complexes was also observed when the eIF-2 alpha kinase activity was inhibited by the addition of 2-aminopurine. This shift was quite slow, however, when compared to the rate of binding of free mRNA to 80 S initiation complexes. The 2-aminopurine was effective in reversing the inhibition of protein synthesis by dsRNA and in maintaining a linear rate of protein synthesis for 3 h in lysates. Without added 2-aminopurine, protein synthesis was inhibited after 90 min even in lysates supplemented with hemin and eIF-2(alpha P) was detected in these lysates. This finding indicated that eIF-2 alpha phosphorylation could be in part responsible for limiting the duration of protein synthesis in mammalian cell-free systems.

Adenosine Triphosphate↗
PubMed1983

Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA.

A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C. (1981) Virology 112, 426-435). To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon. An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon. The level of these compounds increased gradually and in proportion to the interferon concentration used. With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio. An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques. When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed. Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap.

Chromatography, High Pressure Liquid↗
PubMed1983

Treatment of human cells and interferon has no effect on the glycosylation of viral and cellular proteins.

The effect of interferon on the glycosylation of viral and cellular proteins was examined in human cells. Vesicular stomatitis virus released from control and interferon-treated HeLa cells was found to be equally glycosylated. Likewise, interferon treatment of RPMI 8226 cells, a human cell line secreting immunoglobulin G, had no effect on protein glycosylation. In addition, treatment with interferon of RPMI 8226 cells and of lymphoblastoid Daudi cells had no effect on the incorporation of N-acetylglucosamine into dolichol derivatives in a cell-free assay. Similar treatment of HeLa and murine L cells showed an apparent inhibition of this glycosyltransferase activity. This effect of interferon in HeLa cells could at least in part by accounted for by a high nucleotide pyrophosphatase activity, which degraded the sugar nucleotide substrate. These results indicate that interferon does not inhibit protein glycosylation in human cells.

Cells, Cultured↗
PubMed1983

Characterization of an interferon receptor on human lymphoblastoid cells.

A cell-free assay was developed to measure the binding of iodinated human interferon-alpha 2 to membranes prepared from lymphoblastoid Daudi cells. The kinetics of binding were similar at 0 degrees C and 30 degrees C, with 1.3-fold more interferon bound at the higher temperature. Membrane preparations treated with Triton X-100 proved to be a convenient source of solubilized receptor. An assay was developed to measure the binding of 125I-labeled interferon (125I-interferon) to solubilized receptors, based on the precipitation of interferon-receptor complexes with polyethylene glycol. Optimal binding with this assay was obtained at 0 degrees C. The solubilized receptor was analyzed by zonal sedimentation centrifugation and gel filtration. Sedimentation analysis in H2O and 2H2O gradients provided the sedimentation coefficient and the partial specific volume of the receptor-Triton X-100 complex. Gel filtration chromatography provided the Stokes radius of this complex. From these data we calculated several physical parameters, including Mr = 95,000 for the protein portion of the complex. The receptor is a highly asymmetric and hydrophobic membrane protein. 125I-Interferon could be crosslinked to receptors of intact Daudi cells or of isolated membranes by use of disuccinimidyl suberate. The covalently linked 125I-interferon-receptor complexes were analyzed by gel electrophoresis. A single band with Mr = 140,000 was detected in gel autoradiographs. If one molecule of interferon is present in this complex, the Mr of the receptor is close to 120,000. Possible reasons for the different Mr values obtained with the two analytical procedures used are discussed.

Burkitt Lymphoma↗