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J Content

Publications and source records attributed to J Content.

At least 127 records · Page 7Linked to original sources
PubMed1984

Induction and regulation of the 26-kDa protein in the absence of synthesis of beta-interferon mRNA in human cells.

The expression of the gene coding for the 26-kDa protein coinduced with human beta-interferon (HuIFN-beta) in human fibroblasts has been measured by cytoplasmic dot hybridization in WISH cells. The production of the 26-kDa-protein mRNA is not induced by poly(I).poly(C) but maximally induced by cycloheximide alone. In contrast, HuIFN-beta is induced by poly(I).poly(C) and not by cycloheximide. WISH cells showed in addition a low constitutive level of 26-kDa-protein mRNA prior to induction. These results were confirmed by sizing the RNAs by Northern blot analysis. Pretreatment with partially purified or pure IFN-beta has only a slight effect on 26-kDa protein mRNA production. We have also determined the kinetics of induction and the amount of inducer required for an optimal induction of the 26-kDa-protein mRNA in WISH cells. This mRNA was thus maximally induced in WISH cells in the absence of detectable IFN-beta; it represents about 0.05% of poly(A)-rich mRNA in cycloheximide-induced WISH cells. We had already found that the 26-kDa-protein does not share the general characteristics of interferons. These results suggest that HuIFN-beta and the 26-kDa-protein genes are differently regulated.

Cell Line↗
PubMed1983

Effect of interferon on two human choriocarcinoma-derived cell lines.

Two cell lines derived from human choriocarcinomas (HCCM-5 and BeWo) are resistant to several biological effects of human interferon such as inhibition of VSV multiplication and inhibition of cell growth, but they develop a normal antiviral activity against EMCV. Nevertheless, in both cell lines, 2-5A synthetase and protein kinase are induced by IFN. 2-5A-dependent endonuclease can be measured by two independent methods and 2-5A itself is detected at least in poly(rI):poly(rC)- and IFN-treated BeWo cells. This is another example of two cell lines that are partially, with respect to the antiviral effect toward VSV, and totally, with respect to the anticellular effect, refractory to IFN treatment, although all the known elements of the 2-5A system are present.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Human fibroblast interferon RNA transcripts of different sizes in poly(I).poly(C) induced cells.

Northern blot analysis reveals that total RNA from human fibroblastoid cells (MG 63) induced with poly(I).poly(C) under conditions of IFN-beta production, contains predominantly a +/- 1,200 nucleotide long poly (A) mRNA (mRNA.M) which hybridizes with a Hu IFN-beta cDNA specific probe. But hybridization with this probe also enabled the detection of a polyadenylated RNA (RNA.I) with a length of between 3.5 kb-3.8 kb, representing 0.6% of the total hybridizable cellular RNA in superinduced cells. Mapping shows that the RNA.I contains all the sequence information present in mRNA.M. Furthermore, it also hybridizes to sequences, located downstream from the IFN-beta gene up to 2.5 kb beyond its poly A attachment site, while no hybridization to fragments located upstream of the IFN-beta mRNA cap site was observable. Hence this RNA.I corresponds to a transcript that starts at the same position as the major mRNA.M but which extends up to 2.5 kb beyond the 3'-end of mRNA.M where another polyadenylation signal is located.

Cell Line↗
PubMed1983

(2'-5')Oligoadenylate in rat liver: modulation after partial hepatectomy.

(2'-5')Oligoadenylate synthetase [(2'-5')A synthetase], which synthesizes a series of oligoadenylates ppp-(A2'p)n5'A [collectively referred to as (2'-5')A], has been described previously in rat liver cells, where its concentration varied with the growth status of this organ--i.e., it decreased during the early phase of rat liver regeneration after partial hepatectomy. Because double-stranded RNA, the only known activator of this enzyme, has been detected in rat liver nuclei, (2'-5')A synthesis could occur in this tissue in vivo. Analysis of rat liver cell extract after HPLC by the endonuclease-based radiobinding assay revealed several components with retention times similar to (2'-5')A trimer- and tetramer-like material. A further characterization of these compounds by their susceptibility to alkaline phosphatase and snake venom phosphodiesterase, their resistance to micrococcal nuclease, and their ability to activate an endonuclease indicated the natural occurrence of oligonucleotides indistinguishable from authentic (2'-5')A in rat liver cells. Using the combination of the radiobinding assay and a simplified (2'-5')A extraction procedure that does not involve HPLC, we further show that the early drop of (2'-5')A synthetase activity during rat liver regeneration was accompanied by a similar decrease in intracellular (2'-5')A concentration. The three characteristic phases of the (2'-5')A synthetase kinetics during the first 40 hr of liver regeneration were mimicked by the kinetics of the synthesis of the (2'-5')A oligonucleotides themselves: between 6 and 20 hr after hepatectomy, there was a sharp decrease in (2'-5')A concentration; between 20 and 24 hr, the concentration of (2'-5')A reached a minimum; at 36 hr or after the first wave of DNA synthesis (the major event of liver regeneration), the (2'-5')A concentration returned to normal. In this characterization of the (2'-5')A oligonucleotide family in a functional tissue of an animal that had not been previously treated with interferon or infected with virus, the data are compatible with a physiological role of the (2'-5')A system acting as an intracellular component of the regulatory mechanisms leading to cell proliferation or differentiation.

Adenine Nucleotides↗
PubMed1982

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells.

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.

Amino Acid Sequence↗
PubMed1982

2'-Phosphodiesterase activity in human cell lines treated or untreated with human interferon.

2'-Phosphodiesterase activity was investigated, by measuring either the disappearance of (2',5')oligo(adenylate) or the release of 5'AMP, in several human cell lines (RSa, IFr, HEC-1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2'-5')oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2'-phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1-25000 nM). The activity of 2'-phosphodiesterase was dependent on Mg(OAc)2. Our results indicate that in various human cell lines the modulation of (2'-5')oligo(adenylate) metabolism by interferon does not involve an increase of 2'-phosphodiesterase activity.

Adenine Nucleotides↗
PubMed1982

Secretory proteins induced in human fibroblasts under conditions used for the production of interferon beta.

Human fibroblast cells treated with a combination of inhibitors of protein and RNA synthesis [cycloheximide and actinomycin D as used to superinduce interferon beta (IFN-beta)] secrete two proteins with molecular masses of 22000 and 27000 kilodaltons (called 22-kDal and 27-kDal) that are precipitable with an antiserum raised against impure IFN-beta but are antigenically distinct from IFN-beta 1. Translation in vitro of mRNA extracted from human fibroblast cells induced for the production of IFN-beta leads to the synthesis of a 26-kDal protein that is structurally closely related to the 22- and 27-kDal proteins. This 26-kDal protein mRNA is relatively abundant and also appears in human fibroblasts induced only with cycloheximide. It has been partially purified by sucrose gradient centrifugation and more extensively by diazobenzyloxymethyl-cellulose hybridization to plasmid DNA from a bacterial cDNA clone. When translated in an in vitro reticulocyte system supplemented with dog pancreas microsomes, the 26-kDal protein and two other intermediates corresponding presumably to its signal-cleaved (19-kDal) and partially glycosylated (24-kDal) forms were observed. Crude, partially purified, and highly purified 26-kDal mRNA failed to program the synthesis of antiviral or ppp(A2'p5')nA synthetase-inducing activity when translated in Xenopus laevis oocytes. Moreover, partially purified 22-kDal and 27-kDal (i.e., the in vivo equivalents of the 26-kDal protein) are also devoid of antiviral or ppp(A2'p5')nA synthetase-inducing activity. Hence, this 26-kDal mRNA, although presumably identical to the human IFN-beta 2 mRNA described by Weissenbach et al. [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, K., Soreq, H., Nir. U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl. Acad. Sci. USA 77, 7152-7156], cannot be considered to be a fibroblast interferon mRNA.

Cells, Cultured↗
PubMed1982

Cell-free coupling of Newcastle disease virus RNA transcription, translation and Co-translational processing.

A cell-free coupled system for transcription, translation and glycoprotein processing of the Newcastle disease virus genome is described. The system consists of a rabbit reticulocyte lysate preincubated with micrococcal nuclease and of detergent-disrupted purified Newcastle disease virions. [35S]methionine incorporation was linear for 2 h. Polypeptides NP and M, the presumably unglycosylated analogues of glycoproteins HN and possibly F, were identified as translation products. When in vitro synthesis was carried out in the presence of dog pancreas microsomes the HN analogue (pre-HN) was converted to an 80K (approx.) protein which comigrated on polyacrylamide gels with HN synthesized in vivo and which, except for a small fragment, was protected from proteolytic degradation. In immunoprecipitation studies, antiserum against HN purified from virions reacted with both the processed and the unprocessed form of HN synthesized in vitro.

Animals↗
PubMed1982

The human fibroblast interferon gene(s) and their expression in heterologous cells.

A clone has been identified which contains the genetic information specifying human fibroblast interferon (beta 1) and which was derived from a messenger RNA population. On the basis of the nucleotide sequence the complete amino acid sequence (166 residues) of human fibroblast interferon was deduced. It is preceded by a 21 amino acids long signal sequence. Also, a genomic clone was isolated and characterized in detail; on this basis we can conclude that the interferon beta 1 gene does not contain introns. The nucleotide sequence of the putative promoter region was deduced and compared with other eukaryotic promoters. So far, no evidence has been obtained for other human interferons of the fibroblast type. The interferon beta 1 was expressed in the mature form of Escherichia coli without glycosylation. Fully processed interferon was obtained by transfection of monkey CV1-cells with an SV40-derivative, in which the interferon gene replaced the major structural protein VP1.

Amino Acid Sequence↗
PubMed1981

Effects of interferon on sensitive and resistant L1210 cell lines.

L1210 cells resistant to the antiviral and anticellular effects of interferon are not inducible for the 2,5A synthetase and for the protein kinase activities. Cloning of one resistant L1210 strain has revealed heterogeneity of the cell population with respect to their antiviral and anticellular response as well as protein kinase and 2,5A synthetase inducibility. The defect in the response of truly interferon-resistant L1210 cells appears to reside at an early step of interferon action.

2',5'-Oligoadenylate Synthetase↗
PubMed1981

Resistance to interferon of a human adenocarcinoma cell line, HEC-1, and its sensitivity to natural killer cell action.

A human endometrial cancer cell line, HEC-1, was found to be resistant to the antiviral and anticellular action of interferon. However, HEC-1 cells were susceptible to the cytotoxicity of natural killer (NK) cells, and interferon enhanced such NK activity. When HEC-1 cells were treated with interferon, sensitivity of HEC cells to the cytotoxicity of NK cells was not suppressed.

Adenocarcinoma↗