Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Patients with nephrotic syndrome frequently have suppressed immune responses. Previously, it has been determined that subjects with steroid-responsive nephrotic syndrome (SRNS) produce the lymphokine, soluble immune response suppressor (SIRS). In the present group of experiments, a potential pathway of suppressor cell activation was investigated. Sera from patients with SRNS stimulated normal CD8+ lymphocytes to produce SIRS. Serum SIRS-inducing activity was abrogated by treatment with proteinase K or boiling, but was not affected by dialysis, acidification to pH 2, or heating to 56 degrees C. This serum factor could be distinguished functionally and antigenically from SIRS and from interferon (IFN) alpha or IFN gamma. Supernatants of cultured patient lymphocytes enriched for CD4+ cells were also found to activate normal CD8+ lymphocytes to produce SIRS. The lymphocyte-derived activity showed similar characteristics to those of the serum factor. Molecular weight of both factors was estimated to be 13,000 to 18,000 daltons by gel filtration chromatography, and activity of serum and lymphocyte supernatant from the same patients eluted with similar patterns on reversed-phase HPLC. These data suggest that serum SIRS-inducing activity is derived from a suppressor-inducer lymphocyte, and indicate the presence of a regulatory mechanism for SIRS production in steroid-responsive nephrotic patients.
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PMID:A regulatory system for soluble immune response suppressor production in steroid-responsive nephrotic syndrome. 169 48

The interferon induced double-stranded-RNA-dependent eIF-2 alpha kinase has an established role in mediating part of interferons anti-viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3-F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3-F442A cells contains a regulatory RNA(s) capable of activating dsRNA-dependent eIF-2 alpha kinase. Fractionation of the cytoplasmic RNA by oligo(dT)-cellulose indicated that the regulatory RNA eluted with the poly(A)-rich RNA fraction. It bound tightly to the dsRNA-dependent eIF-2 alpha kinase and was immune-precipitated with its antibodies as a complex of regulatory RNA and dsRNA-dependent eIF-2 alpha kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA-dependent eIF-2 alpha kinase. The activity of the regulatory RNA was sensitive to the dsRNA-specific RNase VI but not to proteinase K, DNase I or ssRNA-specific RNase T1. The activation of the dsRNA-dependent eIF-2 alpha kinase by regulatory RNA was prevented by addition of a high concentration of poly(I).poly(C). The regulatory RNA was also shown to activate partially purified dsRNA-dependent eIF-2 alpha kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA-dependent eIF-2 alpha kinase, are consistent with a physiological role for the dsRNA-dependent eIF-2 alpha kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3-F442A cells is discussed.
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PMID:Activation of the double-stranded RNA-dependent eIF-2 alpha kinase by cellular RNA from 3T3-F442A cells. 170 58

Monocytes lysing a variety of tumor cells were isolated by adhesion to autologous serum-coated plastic surfaces. When the blood monocytes were co-cultured with K562 cells for 3-24 h, the supernatants contained soluble factors, termed monocyte cytotoxic factors (MCF), capable of lysing K562 and other tumor cells in a 48-h microcytotoxicity assay. The production of MCF was mediated by typical monocytes expressing a surface phenotype of CD11 (+), CD16 (-), LeuM1 (+). When target cells were pretreated with actinomycin D, they showed an increase in their susceptibility to lysis by MCF. Addition of the drug to MCF assays also resulted in an enhancement of MCF-mediated lysis. Thus, the lytic activity of MCF was detectable in an 18-h assay. The presence of interferon (IFN)-alpha or -gamma augmented the biological activity of MCF, while pretreatment of target cells with IFN did not enhance MCF activity. The absorption of MCF activity was not elevated by actinomycin D or IFN. MCF lysed target cells that were resistant to tumor necrosis factor (TNF). One result of importance is that MCF lysed autologous and allogeneic freshly isolated human tumor cells. The lysis of fresh human tumor cells by MCF was not inhibited by monoclonal antibodies directed against TNF, lymphotoxin (LT), IFN-alpha, IFN-gamma, or interleukin 1 (IL-1). Furthermore, TNF, LT, IFNs, and IL-1 did not kill fresh human tumor cells. MCF activity was stable at low temperatures but was destroyed by heating. The biological activity of MCF was reduced or abolished by serum, trypsin, chymotrypsin and proteinase K, indicating the proteinaceous nature of MCF. The lytic activity was resistant to protease inhibitors. These data indicate that MCF is a noble cytokine that acts on human fresh tumor cells.
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PMID:[Production and function of the monocyte cytotoxic factor (MCF)]. 244 Mar 86

The interaction of human recombinant interferon (rIFN) alpha 2 with its receptor on lymphoblastoid cells was studied using competitive displacement binding. The data were analysed with the LIGAND program, which tests their fit to one-site or multiple binding site models. The binding at 4 degrees and 37 degrees C fits a one-site model, with a similar KD for both IFN-sensitive and resistant cells. Binding at 37 degrees C to Daudi cells at high density fits artifactually a two-site model only when the receptor concentration is close to that of the KD. The binding of IFN to its receptor, therefore, follows a simple bimolecular interaction. Furthermore, IFN-sensitive and resistant cells internalize IFN at similar rates. We have examined whether IFN receptors are also internalized and whether they subsequently recycle to the cell surface. By measuring cell surface and total receptors, we have observed that after 2 h treatment with IFN total receptors remain constant whereas cell surface receptors decrease. After prolonged treatment with IFN, however, there is a loss of total receptors. By inactivating cell surface receptors with proteinase K, we have shown that a fraction of cell surface receptors becomes resistant to inactivation and is apparently internalized. Moreover, experiments which measure IFN receptors either during incubation in the presence of IFN or after IFN has been removed from the medium, show that receptors do not recycle to the cell surface after internalization. The addition of monensin, a drug which has been shown to inhibit receptor recycling, has no effect on the loss of IFN receptors.
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PMID:Single high affinity binding of interferon alpha 2 to receptors on human lymphoblastoid cells: internalization and inactivation of receptors. 315 92

MxA is an interferon-induced 76-kDa GTPase that inhibits the multiplication of several RNA viruses. Deleting seven amino acids from the COOH terminus reduced the GTPase activity of purified MxA to 1.4%. MxA mutants with COOH-terminal deletions of 63 or more amino acids lost all ability to hydrolyze GTP and failed to bind guanine nucleotides. By contrast, an MxA deletion mutant consisting of 301 amino acids from the NH2 terminus and 87 amino acids from the COOH terminus retained about 9% of wild-type GTPase activity, underscoring the pivotal role of COOH-terminal sequences. Limited proteolysis of wild-type MxA with proteinase K resulted in two resistant polypeptides of 60 and 10 kDa, respectively, which copurified as a stable complex. The p60-p10 complex exhibited high GTPase activity, suggesting that it included all MxA domains required for this biochemical activity. Sequencing revealed that the NH2 terminus of the 60-kDa polypeptide mapped to leucine 41 and the NH2 terminus of the 10-kDa polypeptide to glutamine 564 of the MxA sequence. Based on these results we propose a model that suggests that the GTP-binding consensus element located in the NH2-terminal half of MxA is held in an active conformation by strong physical interactions with amino acids from the COOH-terminal region.
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PMID:Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein. 753 30

Blood lymphocytes of cancer patients lysed autologous, freshly isolated tumor cells. The autologous tumor-killing (ATK) activity is strongly associated with postoperative clinical course, indicating that ATK is a meaningful prognostic indicator and provides evidence for immunological control of tumor growth and metastasis. Large granular lymphocytes (LGL) with ATK activity released a soluble cytotoxic factor(s), termed LGL-CF (LGL-derived cytotoxic factor) during interaction with autologous tumor cells. The cytotoxic factor lysed autologous and allogeneic freshly isolated human tumor cells, while they were resistant to any of recombinant cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) alpha, IFN gamma, interleukin (IL)-1 alpha and IL-2. Biological activity of LGL-CF was not abrogated by monoclonal and polyclonal antibodies against these cytokines. LGL-CF also exhibited lysis of a variety of tumor cell lines, but not of nonmalignant cells. Actinomycin D augmented the lysis of LGL-CF. LGL-CF was stable at 56 degrees C, but was destroyed at 100 degrees C. Treatment of LGL-CF with trypsin or proteinase K reduced or abrogated the lytic effect, respectively, while it was resistant to papain, catalase, and superoxide dismutase. These results indicate that LGL produce a novel cytotoxic factor in response to autologous tumor cells that mediates lysis of fresh human tumor cells.
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PMID:Role of NK cell cytotoxic factor against fresh human tumors. 825 31

LeIF, a gene homologue of the eukaryotic initiation factor 4A was first described as a leishmanial antigen that induced a Th1-type T cell response in peripheral blood mononuclear cells (PBMC) from leishmaniasis patients. Moreover, the interferon (IFN)-gamma production by PBMC was found to be interleukin (IL)-12 dependent. Herein, we characterize the effects of LeIF on cytokine production and expression of surface molecules by normal human monocytes as well as by monocyte-derived macrophages and dendritic cells (MoDC). LeIF was a strong inducer of IL-12 and, to a lesser extent, of IL-10 and tumor necrosis factor (TNF)-alpha in macrophages and MoDC. IL-12 production did not require CD40 triggering, confirming that the ability of LeIF to induce IL-12 was not mediated through an effect on T cells. However, addition of soluble CD40 ligand (L) synergistically augmented IL-12 production in macrophages and MoDC. The cytokine-inducing activity of LeIF is located in the N-terminal portion of the molecule and was both proteinase K sensitive and polymyxin B resistant. LeIF, lipopolysaccharide and fixed Staphylococcus aureus all induced comparable amounts of IL-12, validating the potent cytokine-inducing effects of LeIF. Moreover, of these stimuli, LeIF had the highest IL-12/IL-10 and IL-12/TNF-alpha ratio demonstrating the preference of LeIF for IL-12 induction. Studies investigating the expression of surface molecules showed that LeIF up-regulated B7-1 and CD54 (ICAM-1) on macrophages and MoDC. To our knowledge this is the first report describing IL-12 production, up-regulation of co-stimulatory and intercellular adhesion molecules by monocytic antigen-presenting cells in response to a protein from a pathogenic microorganism. These immunomodulatory characteristics of LeIF might be excellent properties for a Th1-type adjuvant.
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PMID:A Leishmania protein that modulates interleukin (IL)-12, IL-10 and tumor necrosis factor-alpha production and expression of B7-1 in human monocyte-derived antigen-presenting cells. 936 20

Ehrlichia chaffeensis, an obligatory intracellular bacterium of monocytes or macrophages, is the etiologic agent of human monocytic ehrlichiosis. Our previous study showed that gamma interferon (IFN-gamma) added prior to or at early stage of infection inhibited infection of human monocytes with E. chaffeensis; however, after 24 h of infection, IFN-gamma had no antiehrlichial effect. To test whether ehrlichial infection disrupts Janus kinase (Jak) and signal transducer and activator of transcription (Stat) signaling induced by IFN-gamma, tyrosine phosphorylation of Stat1, Jak1, and Jak2 in E. chaffeensis-infected THP-1 cells was examined by immunoprecipitation followed by immunoblot analysis. Viable E. chaffeensis organisms blocked tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-gamma within 30 min of infection. Similar results were obtained with human peripheral blood monocytes infected with E. chaffeensis. Heat or proteinase K treatment but not periodate treatment of E. chaffeensis abrogated the inhibitory effect, suggesting that protein factor(s) of E. chaffeensis is responsible for the inhibition of IFN-gamma-induced tyrosine phosphorylation. Preincubation of E. chaffeensis with the Fab fragment of dog anti-E. chaffeensis immunoglobulin G also abrogated the inhibitory effect. On the other hand, monodansylcadaverine, which does not block binding but blocks internalization of ehrlichiae into macrophages, did not have any influence on the tyrosine phosphorylation. These results indicate that ehrlichial binding to host cells is sufficient to inhibit Stat1 tyrosine phosphorylation induced by IFN-gamma. Protein kinase A (PKA) activity in THP-1 cells increased approximately 25-fold within 30 min of infection with E. chaffeensis. In THP-1 cells pretreated with a PKA inhibitor, Rp isomer of adenosine 3',5'-cyclic phosphorothioate, E. chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation was partially abrogated. These results suggest that E. chaffeensis blocks IFN-gamma-induced tyrosine phosphorylation of Jak and Stat through raising PKA activity in THP-1 cells, which may be an important survival mechanism of ehrlichiae within the host cell.
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PMID:Protein kinase A-mediated inhibition of gamma interferon-induced tyrosine phosphorylation of Janus kinases and latent cytoplasmic transcription factors in human monocytes by Ehrlichia chaffeensis. 959 10

To determine the relative contribution of lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild-type serogroup B meningococci (strain H44/76), and LPS-deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral-blood mononuclear cells with wild-type and LPS-deficient meningococci showed that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production and virtually all interferon (IFN)-gamma production. Based on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS in proteinase K-treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine-inducing capacity of isolated and purified LPS and LPS-containing meningococci. At concentrations of >10(7) bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non-LPS components was additive to that by LPS. Experiments with mice showed that non-LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non-LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF-alpha, IL-1beta, and IFN-gamma production.
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PMID:Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response. 1149 21

Gamma interferon (IFN-gamma) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-gamma-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-gamma and protein extracts were analyzed by immunoblotting. The data showed that IFN-gamma-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-gamma receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-gamma signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.
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PMID:Conditioned medium from enterohemorrhagic Escherichia coli-infected T84 cells inhibits signal transducer and activator of transcription 1 activation by gamma interferon. 1649 55


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