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J Content

Publications and source records attributed to J Content.

At least 109 records · Page 6Linked to original sources
PubMed1988

The IFI-56K and IFI-54K interferon-inducible human genes belong to the same gene family.

The IFI-56K and IFI-54K human genes are coordinately regulated by interferon, double-stranded RNA and viruses in a number of cell lines. These genes encode polypeptides of 56 and 54 kDa, respectively, whose function remains to be determined. We analysed the possible structural relatedness between these syntenic and similarly regulated genes. We found that they are very closely related at the protein, mRNA and promoter levels. This suggests that the IFI-56K and IFI-54K genes are members of a gene family, which probably arose from duplication of an ancestor gene.

Amino Acid Sequence↗
PubMed1988

cDNA cloning and assignment to chromosome 21 of IFI-78K gene, the human equivalent of murine Mx gene.

Recently we have purified to homogeneity and characterized an interferon-induced human protein (p78 protein) which is the equivalent of the interferon-induced murine Mx protein responsible for a specific antiviral state against influenza virus infection. A cDNA library was constructed using mRNAs from interferon-induced human diploid fibroblasts. cDNA clones coding for the human p78 protein were identified and used to determine the chromosomal location of the corresponding gene (termed IFI-78K gene) by hybridization to DNA from a panel of human x rodent somatic cell hybrids. The newly identified gene is located on chromosome 21. This has been confirmed by the observation of a gene dosage effect using chromosome 21 trisomic cells (fibroblasts derived from Down's syndrome patients). Among all interferon-inducible genes mapped so far, the IFI-78K gene is the only one located on chromosome 21, together with the gene for the receptor of type I interferon. Our results also provide further evidence for homology between human chromosome 21 and mouse chromosome 16, since the gene encoding the mouse Mx protein (the presumed mouse homolog protein of human p78 protein) has been assigned to chromosome 16.

Animals↗
PubMed1988

Cloning and chromosomal location of human genes inducible by type I interferon.

When cells are treated with interferon several new proteins are induced. We have isolated by differential screening two cDNA clones corresponding to human genes inducible by IFN-alpha, termed IFI-4 and IFI-54K. The accumulation of the corresponding mRNA was followed as a function of either IFN dose or of time. The IFI-4 and IFI-54K genes, as well as two previously isolated IFN-inducible genes, namely the IFI-56K and low-molecular-weight 2-5A synthetase, were localized on the human chromosomes. Using cloned probes on Southern blots of DNA from a panel of rodent-human somatic cell hybrids, we have assigned the IFI-4 gene to chromosome 1 and the gene coding for the low-molecular-weight 2-5A synthetase to chromosome 12. We also showed that the IFI-54K and IFI-56K genes, unlike most of the IFN-inducible genes, are syntenic. They are both located on chromosome 10. In addition, evidence is given for the presence of a pseudogene homologous to IFI-56K on chromosome 13.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

Decreased production of IL-6 by peripheral blood mononuclear cells of patients with chronic lymphocytic leukemia and related disorders.

The present study has shown a decreased production of IL-6 by LPS-stimulated mononuclear cells of B-CLL blood. This decrease with attributed to an abnormality of either monocytes or T lymphocytes. A similar decrease was seen in HCL and NHL. Experiments with mixtures of normal and CLL cells suggest that the decreased IL-6 production in CLL is due neither to an inhibition of normal blood mononuclear cells IL-6 production by B-CLL lymphocytes nor to the absence in CLL blood of cell(s) or cytokine(s) needed for IL-6 production.

Humans↗
PubMed1987

New inducers revealed by the promoter sequence analysis of two interferon-activated human genes.

In order to investigate the molecular basis of the regulation of interferon-inducible genes, we isolated the promoter region of two such genes coding for the (2'-5')oligo(adenylate) synthetase and a 56-kDa protein (IFI-56K). The regions surrounding the cap site were sequenced and compared with the sequences of vertebrate and viral DNA present in the Genbank data bank. Small DNA segments were found in both genes which are homologous to part of the promoter region of other genes, such as those of interferon-beta, tumor necrosis factor beta, interleukin-2 and its receptor. Since these homologies were found located in functionally important regions of these genes, we tested whether their inducers also enhance the (2'-5')oligo(adenylate) synthetase and IFI-56K gene expression. We found that poly(rI).poly(rC) and interleukin-1, activators of the interferon-beta gene and of T lymphocytes respectively, are both able to enhance IFI-56K mRNA accumulation in all cell lines tested. Cycloheximide even superinduces this gene when added together with poly(rI).poly(rC) and interleukin-1 (but not when added with interferon). We showed that these inductions are direct and not mediated by interferon produced by cells in response to poly(rI).poly(rC) or interleukin-1. The promoter sequence analyses have thus led to the discovery of unexpected inducers, i.e. an interferon inducer such as poly(rI).poly(rC) is also able to directly induce a gene that is under the control of interferon.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

High-affinity binding sites for human 26-kDa protein (interleukin 6, B cell stimulatory factor-2, human hybridoma plasmacytoma growth factor, interferon-beta 2), different from those of type I interferon (alpha, beta), on lymphoblastoid cells.

The human 26-kDa glycoprotein (26K) is a cytokine produced by lymphoid as well as nonlymphoid cells. So far it is active as (a) a potent hybridoma and plasmacytoma growth factor on mouse cells, (b) a B cell differentiating factor on human cells, and (c) (for some authors) an interferon (IFN). Internally labeled recombinant human 26K, obtained by translation of mRNA in Xenopus oocytes, was used to investigate the presence of specific receptors for this new cytokine and to analyze its binding to responsive cells. The results indicate that (a) the 26K-responsive human lymphoblastoid CESS cells express about 1500 high-affinity (Kd = 30 pM) binding sites for this cytokine, (b) this binding is not competed for by interleukin (IL)1, IL2, tumor necrosis factor (TNF), IFN-alpha 2, IFN-beta or IFN-gamma, and (c) these 26K-binding sites are different from the classical type I (alpha-beta) IFN receptors by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Binding Sites↗
PubMed1987

B cell growth modulating and differentiating activity of recombinant human 26-kd protein (BSF-2, HuIFN-beta 2, HPGF).

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in interleukin-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [35S]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an interleukin, and considering the confusing nomenclature now in use, this factor may better be renamed "interleukin 6'.

Animals↗
PubMed1987

Induction and regulation of mRNA encoding 26-kDa protein in human cell lines treated with recombinant human tumor necrosis factor.

A 26-kDa protein, originally described in human fibroblasts superinduced for interferon beta (IFN-beta) production, and termed IFN-beta 2 by other investigators, is induced by cycloheximide and by a 22-kDa, interleukin 1 (IL-1)-related factor. Although the structure and sequence of the corresponding gene show nonhomology with the IFN-beta gene, the gene is identical to that of B-cell stimulatory factor 2, a human interleukin, and displays a very potent growth and differentiation factor activity for B lymphocytes. In this work we show that IL-1 beta and tumor necrosis factor (TNF) strongly induce the 26-kDa protein in FS-4 fibroblasts and in some transformed cell lines. Addition of cycloheximide to recombinant (r)IL-1 beta and rTNF further enhances the level of 26-kDa-protein mRNA. We determined the kinetics of induction and the amounts of rTNF and rIL-1 beta required for optimal induction of this mRNA in FS-4 cells and in HeLa H21 cells and found that rIL-1 beta is a more efficient inducer of 26-kDa protein mRNA than is TNF. By analyzing the inducibility of the 26-kDa protein gene by rTNF and rIL-1 beta in a series of transformed cell lines that differ in their sensitivity to the cytotoxic action of TNF, we report a direct correlation between the 26-kDa protein mRNA expression and the resistance of these cells to the cytotoxic effect of TNF.

B-Lymphocytes↗
PubMed1987

The 3' untranslated region of the human interferon-beta mRNA has an inhibitory effect on translation.

In vitro-transcribed human interferon-beta (IFN-beta) mRNA, which contains all the sequence of the natural molecule, is poorly translated in a reticulocyte lysate or when injected in Xenopus oocytes. This low level of translation is due to an inhibition by the 5' and 3' untranslated regions (UTRs). Indeed, the replacement of these sequences by those of Xenopus beta-globin mRNA dramatically increases the translational efficiency of the mRNA, especially in oocytes. This phenomenon is not due to a difference in mRNA stability since both native and chimeric mRNAs remain undegraded, at least during the translation period considered. Construction of different chimeric molecules having various combinations of 5' and 3' UTRs from IFN-beta or Xenopus beta-globin mRNA or a small sequence of SP6 polylinker as 5' UTR has revealed that the 3' UTR of IFN-beta in itself has a pronounced inhibitory effect on translation in the two translation systems from animal cells. Indeed, the addition of this 3' UTR at the 3' end of the coding region of a chicken lysozyme mRNA also causes a large decrease of its translational capacity in both systems. However, the nature of the 5' noncoding sequence influences the degree of translation inhibition exerted by the 3' UTR. Remarkably, we observed no difference in translation level when the different mRNAs were tested in a wheat germ extract.

Animals↗
PubMed1986

Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts.

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.

Amino Acid Sequence↗
PubMed1986

Antiviral activity of a chemically stabilized 2-5A analog upon microinjection into HeLa cells.

2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.

Adenine Nucleotides↗
PubMed1986

Molecular cloning, full-length sequence and preliminary characterization of a 56-kDa protein induced by human interferons.

Among the various proteins which are induced when human cells are treated with interferon, a predominant protein of unknown function, with molecular mass 56 kDa, has been observed. With the aim of exploring the molecular basis of the regulation of this protein and of its mRNA, in order to understand its biological function and its possible contribution to the various antiviral and non-antiviral actions exerted by interferons, we cloned a full-length cDNA copy of the 1.8-1.9 X 10(3)-base 56-kDa-protein mRNA and determined its sequence. The cDNA contains 1662 nucleotides derived from the 56-kDa-protein mRNA, including a poly(A) tail of 20 residues. Primer extension experiments indicate that the 5' end of this cDNA clone is probably located only 13 nucleotides downstream of the actual cap site of the 56-kDa-protein mRNA. It consists of a 64-nucleotide-long 5'-non-coding segment, a coding segment of 1434 nucleotides terminated by a TAG triplet and a 141-nucleotide 3'-non-coding segment. The encoded protein of 478 amino acid residues has a molecular mass of 55335 Da and a single potential site for N-glycosylation. The protein contains an excess of basic amino acids and most of them are localized in the carboxy-terminal half of the molecule. A single [35S]methionine-labeled 56-kDa protein was obtained using an SP64 construction to allow the cell-free transcription and translation of the cloned cDNA. Microinjection of this labeled protein in Xenopus oocytes indicates that the 56-kDa protein is cytoplasmic.

Amino Acid Sequence↗
PubMed1986

Full-length sequence and expression of the 42 kDa 2-5A synthetase induced by human interferon.

Interferon-induced 2-5A synthetases are probably involved in some antiviral actions of interferon. In human cells two different mRNAs (1.6, 1.8 kb long) coding for this protein are transcribed from the same gene and are produced by differential splicing. The relationship between the two mRNAs of different size and the active enzyme is not clear, nor is the cellular localization of the latter known. We have cloned a cDNA corresponding to the 1.6 kb RNA. This cDNA was sequenced and its complete coding region was subcloned into pSP64. The resulting plasmid was used to direct the synthesis of micrograms of capped RNA transcript after linearization in the 3'-non-coding region. A 39 kDa protein was synthesized when this RNA was translated in rabbit reticulocyte lysate. When this capped RNA was introduced by microinjection into Xenopus oocytes, production of 2-5A synthetase was clearly observed in the cytoplasm and 10-30% of the enzyme accumulated with time in the nucleoplasm. Analysis of cytoplasmic homogenates of these oocytes on a glycerol gradient revealed that the enzyme is fully active in the monomeric form.

2',5'-Oligoadenylate Synthetase↗
PubMed1986

Interleukin 1 as a cytokine inducer.

Human interleukin 1 (IL 1) exerts an interferon-like antiviral activity in certain strains of human diploid fibroblasts. It is shown that this antiviral effect is indirect, in that it is mediated by production of interferon-beta. IL 1 also induces synthesis of the mRNA of another secreted protein, 26K, whose biological function is still controversial. Finally, IL 1 has bone marrow colony stimulating activity which is shown to be due to induction of colony stimulating factor in adherent phagocytic cells present in the bone marrow cultures. These findings emphasize the concept that several of the varied biological effects of IL 1 may be indirect.

Bone Marrow Cells↗
PubMed1985

Induction of a 26-kDa-protein mRNA in human cells treated with an interleukin-1-related, leukocyte-derived factor.

A human-leukocyte-derived antiviral protein (22-kDa factor), known to be an inducer of interferon-beta (IFN-beta) in fibroblastoid cells, and to be closely related to interleukin-1 (IL-1), was shown to likewise act as inducer of the mRNA of a 26-kDa secreted protein. This protein was first described as the gene product of an mRNA that is co-induced with the mRNA of IFN-beta by superinduction of fibroblasts (treatment with dsRNA and cycloheximide). Subsequently it was shown to be induced by treatment with cycloheximide only. The 22-kDa factor induced high levels of the 26-kDa-protein mRNA and low levels of IFN-beta mRNA. Addition of cycloheximide to the 22-kDa factor resulted in further significant increases in mRNA levels for both the 26-kDa-protein and IFN-beta. These observations add to the evidence already available that transcription of the genes for IFN-beta and the 26-kDa-protein are differently regulated. The observation that a factor that belongs to the IL-1 family induces the 26-kDa-protein suggest that the latter plays a role as an intermediary or effector molecule in inflammatory or immunoregulatory processes.

Cell Line↗
PubMed1985

Stimulation of fibroblast interferon production by a 22K protein from human leukocytes.

We have studied the appearance of human interferon-beta (HuIFN-beta) as well as its mRNA in cells treated with a protein, 22K factor, isolated from the culture supernatant of mitogen-stimulated human peripheral blood leukocytes. By itself 22K was found to be unable to induce production of significant amounts of HuIFN-beta protein. However, when aided by treatment with cycloheximide or cycloheximide and actinomycin D (superinduction), 22K caused increases in production ranging from 3- to 20-fold, depending on the cells (diploid or MG-63 osteosarcoma) and the induction schedule. Cells treated with 22K alone produced small amounts of HuIFN-beta mRNA, which was only detectable with a highly sensitive method. In combination with cycloheximide, 22K induced levels of mRNA detectable with less sensitive methods as well. These experiments provide further support for the concept that the antiviral activity of 22K is mediated by its ability to stimulate transcription of the HuIFN-beta gene in cells.

Blood Proteins↗