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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A quantitative method of polymerase chain reaction (PCR) using both digoxigenin and radioactive labelled probes has been used for the detection of the c-erbB-2 proto-oncogene amplification in breast carcinomas with formalin-fixed paraffin-embedded tissue sections. c-erbB-2 proto-oncogene amplification has been demonstrated in 14 infiltrating ductal carcinomas. The technique consisted of the co-amplification of c-erbB-2 and IFN-gamma (interferon-gamma) genes. The latter was considered as a single copy gene per genome-equivalent. The aim of this study was to compare two quantitative PCR techniques based on the incorporation of either digoxigenin-11-dUTP or 32P-dCTP, during amplification. For the colorigenic method, using the Dig system, after electrophoresis and transfer, the specific bands were revealed with a chromogenic substrate of phosphatase. Their intensity estimated by scanning photometry following blot transparisation. After electrophoresis, the radioactive gel was submitted to radioautography and the band intensities evaluated by scanning spectrophotometry. For the 14 samples, a good agreement between both methods was noted. The colorigenic method is a valuable alternative to radiolabelling due to: i) time saving, ii) reagent conservation, iii) safe manipulation and iv) sensitivity of the same order for both methods.
Cell Mol Biol (Noisy-le-grand) 1995 Nov
PMID:Determination of amplification level of the c-erbB-2 proto-oncogene in human breast carcinomas: a comparative study between non-radioactive and radioactive labelling. 859 75

Glucocorticosteroids (GCS) are beneficial in allergic asthma. GCS therapy results in reduced mRNA expression of interleukin-4 (IL-4) and IL-5 in cells from bronchoalveolar lavage (BAL) but not of IFN-gamma. In vitro studies with blood-derived T cells, however, show inhibition of all three cytokines by GCS. We studied the effects of GCS on T cells from BAL in vitro, namely Th0-, Th1, and Th2-like clones; and we compared BAL- with blood-derived clones. Dexamethasone (DEX) inhibited the anti-CD3-induced production of IL-4, IL-5 and IFN-gamma in all 20 clones tested. IFN-gamma production was inhibited significantly less than IL-4 and IL-5. DEX enhanced the ratio IFN-gamma/IL-4 (mean +/- SEM: control, 28.7 +/- 17.6; with 10-7 M DEX, 55.0 +/- 27.5, P<0.005). Interestingly, two categories of clones were distinguished based on the effects of GCS on IL-2 production and IL-2R alpha expression and proliferation; 1) In low IL-2 producers DEX blocked IL-2 production and decreased IL-2R alpha expression and proliferation; 2) In high IL-2 producers DEX inhibited IL-2 production partially and enhanced IL-2R alpha expression and proliferation. Anti-IL-2 and anti-IL2R alpha blocked the DEX-induced increase in proliferation. High levels of added IL-2 induced the second type of response. In conclusion, the production of IL-4 and IL-5 by T-cell clones (derived either from BAL or blood) was more sensitive to inhibition by DEX than that of IFN-gamma, which may account for the therapeutic effects of glucocorticosteroids in patients with asthma. The differential effects of DEX on the proliferation of high and low IL-2 producers in vitro may implicate a selective outgrowth of Th1-like T cells in vivo in patients treated with steroids.
Am J Respir Cell Mol Biol 1996 Apr
PMID:Glucocorticosteroids affect functions of airway- and blood-derived human T-cell clones, favoring the Th1 profile through two mechanisms. 860 Sep 44

Despite a large number of studies on the Thl/Th2 balance during immune response to pathogens or protein antigens, little is known concerning the early events which regulate Thl/Th2 differentiation following a single injection of haptenic compounds. In this work, we studied how two mouse strains with different MHC haplotypes, SJL (H-2s) and Balb/c (H-2d), could develop different primary immune responses to subcutaneously injected benzylpenicillin coupled to tetanus toxoid (BPO-TT). The SJL mice showed a high BPO-specific IgG1 response that was maximum on day 10 and no BPO-specific IgG2a response. In contrast, Balb/c mice showed a high BPO-specific IgG2a response on days 15 and 22 and a weak IgG1 production. In SJL mice, the response to BPO-TT was characterized by a very early and high IL-4 mRNA expression. In Balb/c, a delayed and weaker expression of IL-4 mRNA was observed. Kinetics of IL-2 and IFN-gamma mRNA expression were comparable in both strains, but IFN-gamma mRNA expression was higher in SJL than in Balb/c. In vivo neutralization of IL-4 induced a significant BPO-specific IgG2a production and a two-fold reduction of IgG1 production in SJL mice while it accelerated production of BPO-specific IgG2a in Balb/c mice. In addition, studies of IL-12 p4O and IL-10 mRNA expression following immunization with BPO-TT showed a greater IL-12 p4O mRNA expression in Balb/c mice and a slightly higher IL-10 mRNA expression in SJL. Taken together, our data suggest that Th1 or Th2 differentiation in primary immune responses to haptenic compounds such as penicillin may be driven by the kinetics and the level of IL-4 production rather than by the level of IFN-gamma. Additional cytokines such as IL-10 and IL-12 are likely to contribute to the regulation of this response.
Mol Immunol 1996 Jan
PMID:Interleukin-4 plays a dominant role in Th1- or Th2-like responses during the primary immune response to the hapten penicillin. 860 26

Functionally active gamma interferon (IFN-gamma) receptors consist of an alpha subunit required for ligand binding and signal transduction and a beta subunit required primarily for signaling. Although the receptor alpha chain has been well characterized, little is known about the specific role of the receptor beta chain in IFN-gamma signaling. Expression of the wild-type human IFN-gamma receptor beta chain in murine L cells that stably express the human IFN-gamma receptor alpha chain (L.hgR) produced a murine cell line (L.hgR.myc beta) that responded to human IFN-gamma. Mutagenesis of the receptor beta-chain intracellular domain revealed that only two closely spaced, membrane-proximal sequences (P263PSIP267 and I270EEYL274) are required for function. Coprecipitation studies showed that these sequences are necessary for the specific and constitutive association of the receptor beta chain with the JAK-2 tyrosine kinase. These experiments also revealed that the IFN-gamma receptor alpha and beta chains are not preassociated on the surface of unstimulated cells but rather are induced to associate in an IFN-gamma-dependent fashion. A chimeric protein in which the intracellular domain of the beta chain was replaced by JAK-2 complemented human IFN-gamma signaling and biologic responsiveness in L.hgR. In contrast, a c-src-containing beta-chain chimera did not. These results indicate that the sole obligate role of the IFN-gamma receptor beta chain in signaling is to recruit JAK-2 into the ligand-assembled receptor complex.
Mol Cell Biol 1996 Jun
PMID:Ligand-induced assembly and activation of the gamma interferon receptor in intact cells. 864 32

Previous studies have indicated that the expression of viral oncoproteins, cell transformation, or phorbol ester treatment of cells can inhibit alpha/beta interferon (IFN-alpha/beta)-induced gene expression. The mechanisms by which these promoters of cell growth exert their inhibitory effects vary, but in most instances they involve a disruption of the IFN-alpha/beta-induced transcription complex ISGF3 such that the DNA-binding component of this complex (the 48-kDa ISGF3gamma protein) does not bind to the interferon-stimulated response element (ISRE). In this report, we demonstrated that phorbol ester treatment of human peripheral blood monocytes dramatically inhibits activation of IFN-alpha/B-stimulated early response genes but by a mechanism which does not involve abrogation of the ISRE binding of ISGF3gamma. Phorbol ester treatment of monocytes inhibited IFN alpha-stimulated tyrosine phosphorylation of the transcription factors Stat1alpha, Stat2, and Stat3 and of the tyrosine kinase Tyk2 but had no effect on IFN-gamma activation of Stat1alpha. IFNalpha-stimulated tyrosine phosphorylation of Jak1 and the alpha subunit of the IFN-alpha receptor were unaffected by phorbol 12-myristate 13-acetate (PMA). Moreover, PMA caused the dephosphorylation of Tyk2 but not of Jak1, which was activated by IFN. Pretreatment of cells with vanadate prevented the effects of PMA with regard to PMA-induced Tyk2 dephosphorylation. These observations suggest that PMA exerts its inhibitory effects by activation of a tyrosine phosphatase which selectively regulates Tyk2 but not Jak1 activity.
Mol Cell Biol 1996 Apr
PMID:Inhibition of alpha interferon but not gamma interferon signal transduction by phorbol esters is mediated by a tyrosine phosphatase. 865 15

A central factor in the pathogenesis of inflammatory and fibrotic lung disease (adult respiratory distress syndrome, sarcoidosis, idiopathic pulmonary fibrosis) is the locally elevated number of alveolar macrophages (AM). An elevation in the production rate of AM, chemoattraction and differentiation of monocytes, or a diminution in the death rate might be underlying mechanisms. The aim of the present study was to investigate the modulatory role of endotoxin and cytokines on the death rate of human AM. Lipopolysaccharide (LPS) treatment resulted in a 4-fold increase (7.6 to 30.2%) of AM death. AM death was apoptotic as assessed by in situ DNA end labeling (ISDE), transmission electron microscopy, DNA gel electrophoresis, fluorometry of fragmented DNA, and an ELISA specific for histone-associated DNA fragments. Among the different bacterial cell wall components tested, LPS was the only inducer of apoptosis in human AM. None of the tested cytokines (interleukin-1 beta [IL-1 beta], IL-4, IL-6, IL-10, tumor necrosis factor-alpha [TNF-alpha], transforming growth factor-beta 2 [TGF-beta 2], interferon-gamma [IFN-gamma], macrophage colony-stimulating factor [M-CSF], granulocyte colony-stimulating factor [G-CSF], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) was capable of enhancing the spontaneous rate of apoptosis. However, LPS-induced apoptosis was significantly enhanced by the macrophage-activating cytokine IFN-gamma, and reduced by the macrophage-deactivating cytokines IL-4, IL-10, and TGF-beta.
Am J Respir Cell Mol Biol 1996 Jul
PMID:Apoptosis in human alveolar macrophages is induced by endotoxin and is modulated by cytokines. 867 23

A fast purification method for recombinant human interferon-gamma, produced in E. coli, was elaborated. Human IFN-gamma accumulated in the cytoplasm of E. coli cells as inclusion bodies (IB). After lysis, the IB were isolated from the cell debris by means of a density gradient ultracentrifugation, and solubilized in 6 M guanidine hydrochloride. The subsequent refolding step was optimized for a maximal recovery of the biologically active dimer. Refolded IFN-gamma was then purified to homogeneity in a single cation exchange chromatographic step, yielding 12.5 mg protein per liter E. coli culture. The dimeric nature of the refolded protein was visualized by means of interchain cross-linking. In a subsequent Western blot the resulting derivative was recognized by a panel of five monoclonal antibodies, indicating that those epitopes on the protein surface remained unaffected upon cross-linking.
Biochem Mol Biol Int 1995 Dec
PMID:A rapid single-step purification method for human interferon-gamma from isolated Escherichia coli inclusion bodies. 874 47

Our group has previously reported that the nuclear factor Yin-Yang 1 (YY1), a ubiquitous DNA-binding protein, is able to interact with a silencer element (BE) in the gamma interferon (IFN-gamma) promoter region. In this study, we demonstrated that YY1 can directly inhibit the activity of the IFN-gamma promoter by interacting with multiple sites in the promoter. In cotransfection assays, a YY1 expression vector significantly inhibited IFN-gamma promoter activity. Mutation of the YY1 binding site in the native IFN-gamma promoter was associated with an increase in the IFN-gamma promoter activity. Analysis of the DNA sequences of the IFN-gamma promoter revealed a second functional YY1 binding site (BED) that overlaps with an AP1 binding site. In this element, AP1 enhancer activity was suppressed by YY1. Since the nuclear level of YY1 does not change upon cell activation, our data support a model that the nuclear factor YY1 acts to suppress basal IFN-gamma transcription by interacting with the promoter at multiple DNA binding sites. This repression can occur through two mechanisms: (i) cooperation with an as-yet-unidentified AP2-like repressor protein and (ii) competition for DNA binding with the transactivating factor AP1.
Mol Cell Biol 1996 Sep
PMID:The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element. 875 32

Gamma interferon (IFN-gamma) signals to the nucleus through the activation, by tyrosine phosphorylation, of the latent cytoplasmic transcription factor Stat1 (signal transducer and activator of transcription). It has been demonstrated that the activity of Stat1 is dependent on tyrosine phosphorylation which is regulated by Jak tyrosine kinases as well as by the as-yet-unidentified protein tyrosine phosphatase. We report that the N-terminal domain of Stat1, which is highly conserved among all STAT family members, is required for its tyrosine dephosphorylation. A single amino acid substitution (Arg-31 to Ala) in the Stat1 N-terminal domain inhibited Stat1 tyrosine dephosphorylation. The deletion of the Stat1 N-terminal domain resulted in a mutant Stat1 protein which was constitutively phosphorylated on Tyr-701. Upon IFN-gamma stimulation, the tyrosine phosphorylation of this mutant protein was further enhanced but was not down-regulated by protein tyrosine phosphatase in vivo. When expressed in NIH 3T3 cells, this mutant protein greatly enhanced the antiproliferative activity of IFN-gamma. We suggest that the N-terminal domains of STATs are crucial for modulating STAT activities through regulating the tyrosine dephosphorylation of STATs.
Mol Cell Biol 1996 Sep
PMID:Enhancement of antiproliferative activity of gamma interferon by the specific inhibition of tyrosine dephosphorylation of Stat1. 875 52

Peptides eluted from murine Major Histocompatibility Complex (MHC) class II molecules are predominantly fragments of self proteins, which include apolipoprotein E, cystatin-c, transferrin receptor, MHC class II and Ii chains. These naturally processed self peptides are expected to be presented during ontogeny. Therefore, immune responses to these peptides in syngeneic hosts may be under physiological control so as to modulate auto-reactivity. As would be expected from our current understanding, T cells reactive to such antigens should be deleted or clonally anergized. To explore this possibility, we investigated the immunogenicity of a number of these self peptides in mice that express MHC class II, from which these peptides were eluted. T cell and antibody responses were measured following immunization of mice with the appropriate peptide. Surprisingly, many of these peptides were highly immunogenic in normal mice. T cells reactive to these self peptides are restricted by syngeneic MHC class II and were blocked by alpha CD4 antibodies. T cells primed with the native protein in vivo could be challenged with the appropriate self peptide in vitro. Some of the self epitopes induce Th1 cells as indicated by IFN-gamma but not IL-4 production and others induce Th2 cells. Antipeptide antibodies were detected only at higher doses of antigen. Our results suggest that T cells specific for many of the naturally processed self peptides are not deleted but tolerance to these peptides is still maintained in vivo. Presumably the high-affinity self-reactive T cells are deleted in the thymus and the low-affinity self peptide reactive T cells remain unresponsive to antigen challenge in vitro. Upon antigen priming in vivo, many of these self-reactive T cells become activated and readily respond to antigen challenge in vitro. These results point to the physiological control of the maintenance of tolerance to naturally processed self peptides.
Mol Immunol
PMID:Immune responses to self peptides naturally presented by murine class II major histocompatibility complex molecules. 876 Feb 74


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