Search PubMed⌕ Search

Biomedical subjects

C Baglioni

Publications and source records attributed to C Baglioni.

At least 109 records · Page 6Linked to original sources
PubMed1981

Inhibition of double-stranded ribonucleic acid activated protein kinase and 2',5'-oligo(adenylic acid) polymerase by ethidium bromide.

The activation of two enzymes induced by interferon, a protein kinase and the 2',5'-oligo(adenylic acid) polymerase [2'5'-oligo(A) polymerase], is inhibited by ethidium bromide. The activation of these enzymes requires double-stranded RNA (dsRNA), and binding of ethidium to dsRNA inhibits the activation process. This was shown by determining the concentration of ethidium inhibitory for poly(A) . poly(U)- and poly(I) . poly(C)-activated reactions. Activation of both protein kinase and 2',5'-oligo(A) polymerase is inhibited by much lower concentrations of ethidium with the former polymer as activator than with the latter polymer. Correspondingly, in the presence of magnesium, ethidium binds with much greater affinity to poly(A) . poly(U) than to poly(I) . poly(C). Synthesis of 2',5'-oligo(A) with poly(A) . poly(U) as activator is arrested by adding low ethidium concentrations, but it is resumed upon addition of poly(I) . poly(C). Kinase activity, however, is not inhibited when ethidium is added after the activating dsRNA. This suggests that the kinase interacts with dsRNA in a manner different from the 2',5'-oligo(A) polymerase interaction.

2',5'-Oligoadenylate Synthetase↗
PubMed1981

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Role of 2',5'-oligo(adenylic acid) polymerase in the degradation of ribonucleic acid linked to double-stranded ribonucleic acid by extracts of interferon-treated cells.

RNA covalently linked to double-stranded RNA (dsRNA) is preferentially degraded in extracts of interferon-treated HeLa cells [Nilsen, T. W., & Baglioni, C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2600-2604]. The size of the dsRNA required for this preferential degradation has been determined by annealing poly(I) of known length to the poly(C) tract of encephalomyocarditis virus (EMCV) RNA or by annealing poly(U) to poly(A) of known length of vesicular stomatitis virus mRNA. The dsRNA must be longer than about 60 base pairs to observe the preferential degradation of RNA. Moreover, triple-stranded regions that do not activate synthesis of 2',5'-oligo(A) and ethidium bromide, which intercalates in dsRNA and blocks 2',5'-olido(A) polymerase activation, prevent this degradation. Ethidium also blocks the degradation of the replicative intermediate of EMCV by extracts of interferon-treated cells. These experiments indicate that synthesis of 2',5'-oligo(A) is required for the degradation of RNA linked to dsRNA. The 2',5'-oligo(A)-dependent endonuclease does not cleave single- or double-stranded DNA, nor does it cleave homopolyribonucleotides. The potential role of the 2',5'-oligo(A) polymerase/endonuclease system in the inhibition of viral RNA replication is discussed.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Mechanisms of action of human interferons. Induction of 2'5'-oligo(A) polymerase.

Interferons induce an enzymatic activity that polymerizes ATP into 2'5'-oligoadenylate. This enzyme, designated 2'5'-oligo(A) polymerase, is induced in HeLa cells by type I interferon with a faster kinetics than by type II (immune) interferon. Cells treated with type I interferon in the presence of cycloheximide show elevated levels of polymerase when the inhibitor or protein synthesis is removed and actinomycin D is added to the cultures to prevent further synthesis of mRNA. This suggests that, in the presence of cycloheximide, interferon can activate transcription of mRNA for the 2'5'-oligo(A) polymerase, which is subsequently translated upon removal of this inhibitor. In contrast, cells treated with type II interferon in the same way do not show increased levels of polymerase. This indicates that type II interferon cannot activate transcription of a stable mRNA for the polymerase when protein synthesis is inhibited. This observation and the different kinetics of induction of 2'5'-oligo(A) polymerase suggest major differences in the mechanism of action of the different types of interferon, with type I being a "direct" inducer of the polymerase and type II an "indirect" inducer. The commitment of HeLa cells to the induction of 2'5'-oligo(A) polymerase declines if the cells are exposed to type I interferon in the presence of an inhibitor of RNA synthesis. This suggests that transcription of specific mRNAs is transiently activated by interferon and that repeated interaction of interferon with its receptor may be required for prolonged transcription of the mRNA for 2'5'-oligo(A) polymerase.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Virus-specific effects of interferon in embryonal carcinoma cells.

Embryonal carcinoma (EC) cells are susceptible to infection by a variety of viruses, but do not become resistant to infection by Semliki Forest virus or vesicular stomatitis virus (VSV) on treatment with interferon. These observations have led to the conclusion that interferon does not induce an antiviral state in EC cells. We report here, however, that EC cells treated with interferon become resistant to infection by two picornaviruses and two ts mutants of VSV, whereas they remain sensitive to wild-type VSV, Sindbis and influenza virus infectin. These results suggest that a partial antiviral state is induced in EC cells by interferon and that the induced antiviral protein(s) interferes with the replication of specific viruses. A significant common feature of these viruses is their replication through structures containing double-stranded RNA (dsRNA).

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells by 2'-O-methylated poly (inosinic acid) . poly(cytidylic acid), Correlations with interferon-inducing activity.

An oligonucleotide polymerase and a protein kinase which require double-stranded RNA (dsRNA) for activation are induced in HeLa cells by human fibroblast interferon. The polymerase synthesizes a series of oligonucleotides from ATP, whereas the kinase phosphorylates a polypeptide of Mr = 72,000 and the alpha subunit of initiation factor eIF-2. Partially or fully 2'-O-methylated derivatives of poly(inosinic acid) . poly(cytidylic acid) (rIn . rCn) were used to determine the structural requirements of dsRNA in the activation of these two enzymes. While fully methylated polymers failed to activate either enzyme, partially methylated polymers activated the enzymes in specific manners. The activation of the kinase by the rIn . rCn analogues was affected more severely by the level of methylation than was the activation of the polymerase. Moreover, fully methylated analogues blocked the activation of the kinase by rIn . rCn but not the activation of the polymerase. These observations are consistent with a biphasic model for enzyme activation similar to that proposed for interferon induction, which required the recognition of a relatively small region of rIn . rCn as the last step. Differences in the activation of the polymerase and kinase are explicable on the basis of the polymerase requirement for a smaller recognition region of the rIn . rCn duplex than the kinase. Dependence of polymerase activation on the level of methylation shows striking similarities with the interferon inducing activities of these analogues, suggesting a possible relationship between polymerase activation and interferon induction.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

2'5' oligo(A) polymerase activity in serum of mice infected with EMC virus or treated with interferon.

Interferon-treated cells show an increase in two double-stranded RNA (dsRNA)-dependent enzymatic activities involving an oligoadenylate polymerase and a protein kinase (ref. 1 and refs therein). The polymerase converts ATP into a series of oligonucleotides characterized by 2'5'-phosphodiester bonds, designated 2'5'-oligo(A) or 2-5A (ref. 1). These oligonucleotides activate an endoribonuclease that degrades RNA in extracts of control and interferon-treated cells. These observations have been made in tissue culture cells and no informatin is yet available on these enzymatic activities in animals with elevated interferon levels. We report here on 2-5A synthesis in tissue homogenates and serum of mice infected with encephalomyocarditis virus (EMCV); this virus induces interferon synthesis when injected intraperitoneally into mice. Significant synthesis of 2-5A was detected in extracts of spleen and lungs, but also, surprisingly, in the serum of these mice. Subsequent experiments showed synthesis of 2-5A in serum of mice treated with the interferon inducer poly(I) x poly(C) (ref. 3) or with mouse fibroblast interferon.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Mechanism of pppA(2'p5'A)n2'p5'AOH synthesis in extracts of interferon-treated HeLa cells.

Treatment of HeLa cells with interferon results in the induction of an enzymatic activity designated 2'5'oligo(A) polymerase. The polymerase requires continuous presence of double-stranded RNA (dsRNA) for activity, since degradation of dsRNA abolishes synthesis of the oligomeric series pppA(2'p5'A)n. These oligonucleotides are formed initially at a constant rate with dimer synthesized faster than trimer, and the latter faster than tetramer. After 45 min, accumulation of the dimer declines whereas that of other oligomers still proceeds at a linear rate. These results suggest that an oligomer remains associated with the enzyme for possible consecutive additions of adenylate, since no significant accumulation of dimer precedes synthesis of trimer. The relative amounts of the different oligomers found at the end of a reaction may reflect an increasing probability of release as the oligomers are elongated. The accumulation of dimer, however, decreases when it becomes a substrate for adenylate addition; incorporation of isolated dimer into 2'5'-oligo(A) was directly shown. Other nucleotides with a blocked p5'A terminus, like A5'ppppp5'A and NADH, can serve as adenylate acceptors in the presence of dsRNA. The adenosine triphosphates 2'-dATP and 3'-dATP are not incorporated efficiently into 2'5'-oligo(A) and inhibit its synthesis.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Increased levels of (2'-5')oligo(A) polymerase activity in human lymphoblastoid cells treated with glucocorticoids.

An enzymatic activity that synthesizes (2'-5')-oligo(A) from ATP is induced in animal cells treated with interferon. This activity, designated (2'-5')A polymerase, is also elevated in human lymphoblastoid Daudi and Raji cells treated with hydrocortisone. The polymerase activity increases significantly after 24 hr of treatment and declines when hydrocortisone is removed from the culture medium. The product of the enzyme prepared from hydrocortisone-treated cells is indistinguishable from (2'-5')oligo(A) synthesized with polymerase of interferon-treated cells either by an endonuclease activation assay or by chromatographic analysis. The increase in (2'-5')A polymerase is not mediated by secretion of interferon by hydrocortisone-treated cells; less than 1 unit of interferon per ml is present in the culture medium during treatment with this glucocorticoid hormone. Moreover, this increase is related to the concentration of hydrocortisone in the culture medium and is inhibited by the addition of cortexolone. This steroid interferes with the interaction between glucocorticoid hormones and their receptor. Cortexolone has no effect, however, on the induction of (2'-5')A polymerase by interferon. The synthetic glucocorticoid dexamethasone also increases the polymerase activity. Experiments with inhibitors show that such an increase requires RNA and protein synthesis.

2',5'-Oligoadenylate Synthetase↗
PubMed1980

Inhibition of RNA and protein synthesis in interferon-treated HeLa cells infected with vesicular stomatitis virus.

Synthesis of vesicular stomatitis virus RNA and protein is almost completely inhibited in HeLa cells treated with relatively high doses of human fibroblast interferon. With lower concentrations of interferon virus replication is inhibited, but near normal amounts of virus RNA are found in cells infected at a m.o.i. of 10. All the virus RNA species are found in these cells with the exception of genomic size RNA. In contrast, synthesis of all the virus proteins is equally inhibited in proportion to the interferon concentration used to treat the cells. This inhibition is due to a decline in the rate of protein synthesis, which occurs in interferon-treated cells sooner after infection than in untreated cells. The decreased rate of protein synthesis is accompanied by a change in the polysome pattern of infected cells, characterized by polysome run-off and increase in 80S ribosomes. At the same time, a larger proportion of the virus poly(A)-containing RNA is not associated with polysomes in interferon-treated cells than in control cells. The non-polysomal virus RNA has a sedimentation rate identical with that of polysomal virus RNA. Possible causes for the decline in the rate of protein synthesis observed in interferon-treated cells are discussed.

HeLa Cells↗