Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclosporin (CsA) is an immunosuppressant which binds to cyclophilin (Cyp). The relationship between Cyp binding and immunosuppression has been questioned since one of the analogs of CsA, N-methyl-L-alanyl6 cyclosporin (methyl-alanyl CsA) binds to Cyp but is not immunosuppressive. We compared the immunosuppressive properties of CsA, methyl-alanyl CsA and o-acetyl-threonine2 cyclosporin (monoacetyl CyC), since monoacetyl CyC does not bind to Cyp when tested in cell-free assays and its immunosuppressive properties had not been tested. Cyp is a peptidyl-prolyl isomerase which is abundant in all human tissues, yet the activities of CsA are mostly confined to inhibition of T cell and thymocyte activation, and to neuro- and nephro-toxicity and are independent of inhibition of the isomerase. Activation of thymocytes and of T cells is regulated by the binding of a nuclear factor(s) (NFs) to the NF-AT region (-285 to -255) of the IL-2 promoter. We studied inhibition of binding to the NF-AT region of NFs derived from primary cultures of thymocytes treated with CsA or its analogs. In addition, we compared the effect of CsA and its analogs on the expression of the IL-2 gene in a stably transfected Jurkat-cell line (Fgl 5) which contains three copies of NF-AT and the reporter enzyme beta-galactosidase; and on inhibition of proliferation induced by concanavalin A (Con A) or IL-2. We found that monoacetyl CyC which does not bind to Cyp is immunosuppressive by our criteria when tested in cultured cells due to either a different mechanism of action or to metabolic activation.
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PMID:Comparison of the properties of the CsA analogs monoacetyl CyC (o-acetyl-threonine2 cyclosporin) and methyl-alanyl CsA (N-methyl-L-alanyl6 cyclosporin); monoacetyl cyclosporin is immunosuppressive without binding to cyclophilin. 162 22

Measles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable T cell clones, which displayed MHC-restricted MV Ag recognition, could be assessed by using purified MV proteins. Two fusion (F) protein-specific, two hemagglutinin-specific, and three nucleoprotein- or matrix protein-specific clones were shown to be established. The F protein-specific T cell clones together with a panel of previously generated F protein-specific T cell clones were characterized for their fine specificity by using beta-galactosidase fusion products, which contained different parts of the F protein. It was shown that at least two epitopes on the major part of the F protein (amino acid 2-513) can be recognized by mouse T cells. Functional characterization of three T cell clones showed that they were able to assist MV-specific B cells and bystander B cells for antibody production. Furthermore, they were shown to produce the lymphokines IL-2 and IFN-gamma. It was also shown that these T cell clones induced a MV-specific delayed type hypersensitivity response. These observations suggest that all of the T cell clones characterized belong to the TH1 helper subset.
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PMID:Measles virus-specific murine T cell clones: characterization of fine specificity and function. 252 70

cDNAs for human interleukin-2 receptor were recently cloned and sequenced (Leonard et al., 1984, Nature 311, 626-631; Nikaido et al., 1984, Nature 311, 631-635; Cosman et al., Nature 312, 768-771). In the studies reported here, we describe the expression of a cDNA clone for the human interleukin-2 receptor in E. coli using an "open reading frame" expression vector pMR100. The inserted cDNA was expressed in E. coli transformants as a tripartite fusion polypeptide fused to the lambda cI protein at its amino terminus and to beta-galactosidase at its carboxy terminus. We demonstrate that the bacterially produced IL-2 receptor protein can bind to IL-2.
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PMID:Expression of human interleukin-2 receptor cDNA in E. coli. 302 92

We have used the enhanced uptake by FcR-bearing cells observed when Ag is administered as an immune complex to investigate the possible impact of specific antibodies on processing and presentation of antigen by accessory cells. The Ag Escherichia coli beta-galactosidase alone or bound to different mAb was incubated with peritoneal macrophages. These were subsequently exposed to a battery of Ag-specific T hybridoma clones. The resulting production of IL-2 was taken as a measure of effective presentation. The results of 43 mAb-T clone combinations showed a potentiation of presentation of Ag at substimulatory concentration in the majority of the cases, indicating that each mAb is conducive to FcR-mediated uptake by macrophages, and that each T clone can be stimulated by properly presented Ag. In contrast, nine combinations yielded a lower response, two of them falling to baseline values. We attribute these results, which corroborate our previous evidence of directional help in the beta-galactosidase system, to a modulation in enzymatic processing of Ag and its subsequent presentation imposed by the paratope of the mAb binding to the relevant epitope.
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PMID:Differential activation of T cell clones stimulated by macrophages exposed to antigen complexed with monoclonal antibodies. A possible influence of paratope specificity on the mode of antigen processing. 312 2

We have achieved efficient transduction of tumour metastases in vivo by the vascular delivery of retroviral producer cells. Experimental liver metastases in mice were created by intrasplenic injection of tumour cells into the portal venous circulation. Following the establishment of micrometastases, delivery of retroviral producer cells by the same route with a vector containing the Escherichia coli beta-galactosidase (lacZ) gene demonstrated selective in vivo gene transfer to tumour deposits. By this approach, two retroviral producer cell lines encoding cytokines (IL-4 and IL-2) directed tumoricidal inflammatory responses to established metastases. Cytokine gene targeting inhibited metastasis formation and caused significant overall reduction in tumour burden. These results suggest a novel therapeutic approach for the treatment of disseminated cancer.
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PMID:Gene therapy of metastatic cancer by in vivo retroviral gene targeting. 767 Apr 93

IL-2-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the IL-2 promoter is essential for IL-2 transcription. Using a cell line that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated beta-galactosidase activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited beta-galactosidase mRNA levels. Similar inhibition of NFAT-1-mediated gene expression was observed in a second cell line, which is stably transfected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 microM, both compounds inhibited IL-2 mRNA and protein levels in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhibition was reversed by exogenous IL-2. WIN 53071 inhibited IL-2 production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, calcium-independent anti-CD28 Ab and PMA-induced IL-2 production was resistant. Both compounds altered the NFAT-1 transcriptional complex, causing its retarded mobility on gels. By these functional criteria, we believe we have identified two structurally distinct, novel inhibitors of NFAT-1-mediated transcription.
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PMID:Functional characterization of novel IL-2 transcriptional inhibitors. 770 10

Neoplastic cells are generally poor immunogens. Transfection of the murine tumor CT-26 with beta-galactosidase (beta-gal), a protein from Escherichia coli, did not alter its growth rate in vivo, or its lethality, and did not elicit a measurable anti-beta-gal immune response. Immunization with beta-gal-expressing recombinant vaccinia viruses (rVV) elicited specific anti-beta-gal cytolytic T lymphocytes, but rVV-beta-gal was only marginally therapeutic when given to tumor-bearing mice. With the aim of expanding the immune response against beta-gal, used here as a model tumor Ag, we gave mice exogenous IL-2 starting 12 h after the poxvirus. The therapeutic effectiveness of the combination of poxvirus and IL-2 was far greater than either of these treatments alone. When the cDNA for IL-2 was inserted into the viral genome of the rVV construct to make a double recombinant (drVV), antitumor activity was further augmented. One mechanism of action may be the enhanced activation or expansion of cytotoxic T cells, because a marked increase in primary cytotoxic responses against vaccinia determinants was observed. Interestingly, other cytokines (mGM-CSF, mTNF-alpha, and mIFN-gamma) inserted into the rVV genome did not modify the efficacy of the rVV constructs. The increase in specific CTL responses against beta-gal by drVV expressing the tumor-associated Ags (TAA) and IL-2 was more pronounced in mice bearing the lacZ-transduced tumor than in those bearing the parental cell line, suggesting that the TAA presented by growing tumor cells can either pre-activate or otherwise amplify the immune response induced by the rVV. Unfortunately, in several long-term surviving mice, tumor recurred that no longer expressed beta-gal. These results indicate that treatment of disseminated tumors by using recombinant viruses expressing TAA can be enhanced by IL-2 provided exogenously, or encoded within the recombinant virus.
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PMID:IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases. 773 Jun 32

A soluble domain of the interleukin(IL)-2 receptor, the alpha chain synthesized in Escherichia coli, was employed to study expression and refolding of the protein. The results showed that it is possible to obtain biologically active synthetic methionine-free IL-2 receptor alpha chain (synIL-2R alpha) after BrCN cleavage and renaturation of the crude cleavage material, although the alpha chain is expressed as a deglycosylated, methionine-free protein. The soluble receptor comprises amino acids 1-219 and forms 5 disulfide bonds in its biologically active state. Biological activity has been analysed by affinity chromatography and ELISA with mutant [Ala125]IL-2 and monoclonal antibodies as ligands. Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein. Aggregation could be limited by varying the oxidation conditions. The deletion of a non-bridging cysteine at position 192 in the synIL-2R alpha did not affect the renaturation yield of the receptor protein. Additionally a cysteine-free and methionine-free beta-galactosidase derivative was fused to the soluble synIL-2R alpha derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN-cleavage material. It is suggested that cysteine impurities from cyanogen-bromide-cleaved peptides might interfere seriously with the refolding process of the synthetic IL-2 receptor alpha-subunit.
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PMID:Isolation and refolding of a mutant methionine-free interleukin-2-receptor alpha chain synthesized as a fusion protein in Escherichia coli. 803 3

A transgenic mouse system has been established to follow the pattern of IL-2 expression at the level of single T cells. This was achieved by introducing a human IL-2 promoter-driven reporter gene (Escherichia coli lacZ) into the germline of mice and monitoring its product, beta-galactosidase (beta-gal), by FACS analysis. Ex vivo experiments confirmed that the regulated expression of the transgene is comparable with that of the endogenous IL-2 gene. Transgene expression is inducible by mitogens, restricted to T cells, and diminished by immunosuppressive agents, such as cyclosporin A, at concentrations known to suppress IL-2 transcription. Depending on the mitogens used, 30-50% of peripheral T cells produced IL-2 with an asynchronous induction pattern, as measured by transgenic beta-gal activity. Both helper (CD4+CD8-) and cytotoxic T cells (CD4-CD8+) respond with comparable heterogenous expression levels but they show different frequencies of beta-gal production. Transgenic beta-gal-producing T cells were detectable as early as 2 h after mitogen stimulation. These cells represent a transitional IL-2 secreting, IL-2 receptor alpha-chain negative T cell population, which occurs in the autocrine process of T cell activation. Administration of staphylococcal enterotoxin A (SEA), a bacterial superantigen, resulted in a T cell specific (Thy-1.2) increase (2.5-fold) of reporter gene expression in vivo. In summary, we could demonstrate that IL-2 promoter-driven reporter gene expression in transgenic mice is a sensitive tool to characterize IL-2 expressing cells phenotypically.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:IL-2 promoter-driven lacZ expression as a monitoring tool for IL-2 expression in primary T cells of transgenic mice. 815 96

CD28 is a 44 kDa Ig superfamily cell surface molecule expressed on most mature T cells. Through its interaction with the recently identified B7/BB1 counter-receptor, it is believed to play an important role as a co-stimulator of T cells along with the TCR-CD3 complex. Activation of T cells with CD28 mAbs synergizes with TCR-CD3 and CD2 stimulation, resulting in long term T cell proliferation, differentiation of cytotoxic T cells and production of large amounts of cytokines. In order to further delineate the role of CD28 in signal transduction and T cell activation, human CD28 was transfected into CD3+ murine T cell hybridomas. High levels of cell surface CD28 expression was achieved by protoplast fusion. The transfected molecule retained all the native CD28 mAb epitopes found on human T cells. In these transfectants, CD28 mAbs, similarly to CD3 mAbs, were able to induce Ca2+ mobilization, IL-2 promoter induction (measured as beta-galactosidase activity in T cells hybridomas pre-transfected with the IL-2-lac Z reporter gene), IL-2 secretion, TNF alpha production and apoptosis (observed as growth arrest and genome fragmentation). The parental host cells, or cells transfected with vector alone, responded only to mAbs to CD3. IL-2 secretion in the transfectants was obtained using either an IgM mAb to CD28 or IgG mAbs presented on the surface of IgG-FcR+ B lymphoma cells. Optimal activation via CD28 was inhibited by suboptimal concentrations of soluble CD3 mAb, suggesting an interaction between the two pathways. The immunosuppressive drugs Cyclosporin A and FK506 completely blocked CD28 and CD3 mediated IL-2 production in these transfectants whereas rapamycin had only a partial inhibitory effect. Finally, since the transfected human CD28 molecule confers full functional responsiveness to the murine T cell hybridomas without the need for costimulators such as PMA, this model is ideal for studying the structure-function relationships of the CD28 molecule as well as the transmembrane and cytoplasmic associations implied in CD28 signaling.
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PMID:Functional expression of human CD28 in murine T cell hybridomas. 830 98


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