Biomedical subjects

P Poupart

Publications and source records attributed to P Poupart.

10 recordsLinked to original sources

PubMed1994

Paratuberculosis.

Paratuberculosis (Johne's disease) is a chronic, wasting, widespread mycobacteriosis of ruminants. It involves extensive mycobacterial shedding, which accounts for the high contagiousness, and ends with a fatal enteritis. Decreases in weight, milk production, and fertility produce severe economic loss. The DNA of the etiological agent (Mycobacterium paratuberculosis) has a base composition (66 to 67% G+C) within the range of that of mycobacteria (62 to 70% G+C), a size (4.4 x 10(6) to 4.7 x 10(6) bp) larger than that of most pathogenic mycobacteria (2.0 x 10(6) to 4.2 x 10(6) bp), and a high relatedness (> 90%) to Mycobacterium avium DNA. However, the DNAs of the two organisms can be distinguished by restriction fragment length polymorphism analysis. M. paratuberculosis genes coding for a transposase, a cell wall-associated protein (P34), and two heat shock proteins have been cloned and sequenced. Nucleic acid probes (two of which are species specific) are used, after PCR amplification, for M. paratuberculosis identification in stools and milk. As in leprosy, with disease progression, cellular immune reactions decrease and humoral immune reactions increase. Cutaneous testing with sensitins, lymphocyte proliferation assays, and cytokine tests are used to monitor cellular immune reactions in paratuberculosis, but these tests lack specificity. Complement fixation, immunodiffusion, and enzymometric tests based on antibodies to M. paratuberculosis extracts, to mycobacterial antigen complex A36, to glycolipids, and to proteins help identify affected cattle but are not species specific. The carboxyl-terminal portion of the 34-kDa cell wall-associated A36 protein (P34) carries species-specific B-cell epitopes and is the basis for an enzyme-linked immunosorbent assay. Diagnostic tests for paratuberculosis are also used in Crohn's disease, a chronic human ileitis mimicking Johne's disease, in which isolates identified as M. paratuberculosis have been found.

Amino Acid Sequence↗

PubMed1993

Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne's disease.

A species-specific recombinant clone (F57) was obtained from a genomic library of Mycobacterium paratuberculosis in the transcription vector pGem 3Z. This clone proved to be specific for all mycobacteria tested, including M. avium, and was able to recognize all of the tested M. paratuberculosis strains isolated from animals and humans (patients with Crohn's disease). The F57 insert was sequenced and a segment of 620 bp with a G + C content of 58.9% was identified. Comparison of the sequence with sequences in the EMBL and UGEN data banks revealed the uniqueness of the F57 sequence, which had no resemblance to other known genes.

Animals↗

PubMed1988

Genes for IFN-beta-2 (IL-6), tumor necrosis factor, and IL-1 are expressed at high levels in the organs of normal individuals.

The gene of a cytokine designated IFN-beta-2, or IL-6, and recently identified as identical to the B cell-stimulatory factor 2, is transcribed at high levels in the spleen, liver, kidney, and peripheral blood leukocytes of normal individuals. The number of IFN-beta-2/IL-6 transcripts present endogenously in normal human tissues (0.6 to 16 copies/cell) is comparable to that present in normal cells induced in vitro with human rTNF. This is in marked contrast to the absence of detectable IFN-beta-1 transcripts (less than 0.0003 copy/cell) in the same samples of human tissue. The expression of the IFN-beta-2/IL-6 gene is closely associated with that of two other cytokines TNF, and IL-1. Thus, significant levels of IFN-beta-2/IL-6, TNF, IL-1 alpha, and IL-1 beta, mRNA were detected in all the samples of normal tissue tested and those samples which contained high levels of IFN-beta/IL-6 mRNA also contained high levels of TNF, and IL-1 beta mRNA. These results suggest that these cytokines may function in consort as regulators of cellular growth and function in normal tissues.

Gene Expression Regulation↗

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↗

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↗

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↗