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

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

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.
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PMID:Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking. 252 81

We analyzed the high affinity receptor for IFN-gamma of Raji cells and human placenta by combining Scatchard analysis, cross-linking experiments, and receptor purification. Only one high affinity binding site was found, Kd 2.1 X 10(-10). The receptor is a 90-kDa glycoprotein. However, multiple cross-linked products of 110 kDa to about 250 kDa could be generated and proteins of 90, 70, and 50 kDa could be obtained upon purification. These proteins all contained the same 90-kDa receptor, or part of it. We suggest that extensive cross-linking and/or proteolysis may explain many of the conflicting results published thus far. The extracellular domain of the 90-kDa receptor protein was highly resistant to digestion with trypsin or proteinase K. Trypsin digestion neither affected the number of binding sites per cell, nor the Kd for IFN-gamma. A cluster of sites for different proteases was found in the intracellular domain. The 50-kDa fragment created by trypsin digestion had the same characteristics as the isolated 50-kDa receptor fragment. It contained the IFN-gamma binding site and the receptor's extracellular and amino-terminal domain. N-linked glycosylation contributed about 15 kDa to its molecular mass, of which 4 kDa were attributable to sialic acid residues. O-Linked glycosylation was not detected. The number of binding sites per cell and the Kd for IFN-gamma were not affected by the presence or absence of N-linked glycosylation. The receptor contained at least one critical disulfide bridge and the reduced receptor could be reactivated in vitro.
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PMID:Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IFN-gamma. One single 90-kilodalton IFN-gamma receptor can lead to multiple cross-linked products and isolated proteins. 253 Feb 76

The supernatant of Mycoplasma arginini-infected murine L5178Y T lymphoma cell cultures (SN-L51) synergizes with small concentrations of IFN-gamma to activate murine peritoneal, thioglycollate-elicited macrophages (M phi) to exhibit cytostatic activity against tumor cells. Treatment of M phi with IFN-gamma and SN-L51 sequentially, but not in the reverse order, activates M phi, which indicates that SN-L51 contains a M phi-triggering factor (MTF). MTF activity could be inhibited by small concentrations of prostaglandin E2, but not by polymyxin B. M phi activated by IFN-gamma plus MTF produce cytostatic effects on tumor cells through a nitric oxide-dependent pathway. MTF activity in SN-L51 is associated with infection of L5178Y cells by M. arginini. Mycoplasma-free L5178Y cells do not produce MTF activity, infection of these L5178Y cells with M. arginini generates the activity, and supernatants of pure M. arginini cultures contain MTF activity. MTF activity is thermostable and resistant to acid, dilute alkali, proteases, and nucleases. MTF was partially purified by ammonium sulfate precipitation, chromatography, electrophoresis, and electroelution. On 12.5% SDS-urea gels, MTF activity migrated with a molecular mass of 2.5 to 4 kDa. MTF activity and the silver staining of this band was resistant to proteinase K; however, Coomassie staining of this band was abolished by proteinase K. The combined data suggest that MTF is either a stable peptide or a peptide linked to lipid or carbohydrate.
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PMID:Characterization and purification of a macrophage-triggering factor produced in Mycoplasma arginini-infected L5178Y cell cultures. 807 68

This paper reports the development of experimental procedures for the use of crude tissue section lysates, in conjunction with a non-radioactive polymerase chain reaction-high performance ion-exchange chromatographic (PCR/HPIEX) method, for the quantitative assessment of gene copy number in human biopsy samples. In particular, these methods have been established for the assessment of gene amplification with the FGF-2, FGF-3, FGF-4 and c-erb-B2 genes with the a single copy IFN-gamma gene used as an internal control. In principle, the same procedures could, in general, be simultaneously applied for monitoring the amplification or deletion of other oncogenes as well as normal genes in mammalian cells. Procedures to optimise the precision of the analysis have been examined, including the influence of the oligonucleotide primer concentrations, cycle number, extension temperature, and method of pre-treatment of the tissue sample with proteinase K. The results confirm that crude tissue lysates, rather than purified DNA samples, can be reliably employed, thus extending the scope of non-radioactive PCR/HPIEX methods for the assessment of aberrant gene copy number to biological samples such as tumour biopsy tissues.
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PMID:The use of crude tissue section lysates for PCR assessment of gene copy number with human tumour biopsy samples. 902 65

The Trypanosoma cruzi HSP70 recombinant protein has the capacity to stimulate splenocytes or lymph node cells from naive mice in a non-haplotype-restricted way. The proliferative response is abolished by proteinase K digestion and by specific anti-HSP70 antibodies. The induced stimulation index was maximal after 24 h of incubation with the protein. This stimulation leads to cell death in a Fas-Fas ligand-independent way. The phenotype of the expanded cells was CD3(+) TCRalphass(+) CD4(+). HSP70-responsive cells express a broad range of cytokines including IFN-gamma, IL-2 and tumor necrosis factor-alpha. After 48 h of incubation with HSP70 there was a significant increase in relative intracellular levels of CD3 TCRalphass receptors. The expanded CD4(+) cell population expressed CD25; however, in contrast to concanavalin A-treated culture, delayed CD44 expression was observed.
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PMID:HSP70 from Trypanosoma cruzi is endowed with specific cell proliferation potential leading to apoptosis. 1109 8

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

The clinical benefits observed with probiotic use are mainly attributed to the antimicrobial substances produced by probiotic strains and to their immunomodulatory effects. Currently, the best-documented probiotic bacteria used in human therapy are lactic acid bacteria. In contrast, studies aiming to characterize the mechanisms responsible for the probiotic beneficial effects of Bacillus are rare. The current work seeks to contribute to such characterization by evaluating the antimicrobial and immunomodulatory activities of probiotic B. clausii strains. B. clausii strains release antimicrobial substances in the medium. Moreover, the release of these antimicrobial substances was observed during stationary growth phase and coincided with sporulation. These substances were active against Gram-positive bacteria, in particular against Staphylococcus aureus, Enterococcus faecium, and Clostridium difficile. The antimicrobial activity was resistant to subtilisin, proteinase K, and chymotrypsin treatment, whereas it was sensitive to pronase treatment. The evaluation of the immunomodulatory properties of probiotic B. clausii strains was performed in vitro on Swiss and C57 Bl/6j murine cells. The authors demonstrate that these strains, in their vegetative forms, are able to induce NOS II synthetase activity, IFN-gamma production, and CD4 T-cell proliferation.
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PMID:Bacillus clausii probiotic strains: antimicrobial and immunomodulatory activities. 1522 Jun 67

Membrane-associated Leishmania Ags (MLA) or soluble Leishmania Ags were used in vitro to stimulate cord blood or PBMC from healthy donors noninfected by Leishmania parasites. MLA, but not soluble Leishmania Ags, constantly induce strong proliferation of cord blood mononuclear cells and PBMC from noninfected individuals. Responding cells are CD3+, CD4+, TCRalphabeta+, CD45RO+, and CD45RA+ and secrete IFN-gamma and IL-10, but not IL-4. MLA do not activate NK cells nor NKT cells. Membrane Ags also induce purified macrophages from noninfected individuals to secrete IL-10 and TNF-alpha, but have no effect on IL-1alpha or IL-12 secretion. The effects of MLA are proteinase K-sensitive and resistant to lipid extraction. The lymphoproliferative responses are inhibited by anti-HLA-DR Abs and require Ag processing by APCs, excluding that the biological effect of MLA could be attributed to a superantigen. Finally, TCR repertoire analysis shows that the T cell expansion induced by MLA uses TCR with various variable beta segment rearrangements and CDR3 lengths, features much more characteristic to those observed with a polyclonal activator than with a conventional Ag. These results suggest a particular mechanism developed during the host's natural response to Leishmania parasites that allows direct activation of naive CD4 lymphocytes by parasite membrane-associated Ags.
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PMID:Mechanisms of the natural reactivity of lymphocytes from noninfected individuals to membrane-associated Leishmania infantum antigens. 1574 97

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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