Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q9NRP7 (fused)
 58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hairy cell leukemia is a rare, B-cell malignancy uniquely sensitive to the antitumor effects of alpha and beta interferons (IFN). In order to further study the effects of IFN in this disease, we derived a cell line (HC1) from the peripheral blood mononuclear cells of a patient with hairy cell leukemia (HCL). Cells exhibited the typical morphological features of HCL, including the characteristic cytoplasmic projections by light, transmission, and scanning electron microscopy. HC1 cells were of B-cell lineage, as evidenced by immunophenotypic analysis. Although originally TRAP positive, HC1 cells lost this biochemical marker following 3 months in culture. Monoclonality of the cell line was confirmed by a clonal karyotypic abnormality characteristic of B-cell malignancies, and the presence of a single, distinctive fused terminal EBV fragment. The cells formed colonies in soft agar and were tumorigenic in irradiated nude mice. HC1 cells were sensitive to the antiproliferative effects of IFN-a and IFN-beta, but only moderately sensitive to the growth inhibitory effects of IFN-gamma. Incubating the cells in the presence of Type 1 IFN resulted in stabilization of cell numbers, without cellular proliferation or loss. Cell cycle analysis revealed that IFN-alpha resulted in a build-up of cells in the S phase of the cell cycle, suggesting a cytostatic effect of IFN on the growth of these cells. The HC1 cell line provides a model system which will be useful for in vitro studies of the biology and treatment of this disease.
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PMID:Establishment and characterization of an Epstein-Barr virus spontaneously transformed lymphocytic cell line derived from a hairy cell leukemia patient. 131 27

The efficacy of human peripheral blood monocytes (PBM) in killing of schistosomula is controversial. The purpose of this study was to determine the schistosomulacidal activity of human monocytes isolated by two different techniques. Peripheral blood monocytes were obtained either by venipuncture (PBMv) or plasmapheresis (PBMp), purified on Ficoll-Paque, and cultured briefly. The cells then were incubated with schistosomula (cell parasite ratio of 10(4):1) for 16 to 18 hours with or without interferon-gamma IFN-gamma (600 U/ml) or sera from patients with schistosomiasis as a source of antischistosomal antibodies (HASA). Freshly isolated PBMv treated with IFN-gamma or HASA did not kill schistosomula. Freshly isolated PBMp alone killed 22 +/- 13% (mean +/- standard deviation [SD]; n = 9) of worms over background and after incubation with IFN-gamma and HASA, 30 +/- 17%. PBMp cultured in vitro for 7 days killed 50 +/- 15% (mean +/- SD; n = 12) of the schistosomula. Pretreatment of the cells with IFN-gamma and incubation with HASA did not significantly enhance the parasite killing beyond this level. Electron microscopy showed that freshly isolated PBMp attached to the worms and fused occasionally with the outer tegumental membrane. Granules constituted 1.4% of the cytoplasmic volume. Degranulation onto the parasite surface was not observed. Peripheral blood monocytes obtained by plasmapheresis accumulated glycogen during in vitro culture with the parasite and released threefold more H2O2 than PBMv after exposure to phorbol myristate acetate. Thus plasmapheresis increases the schistosomulacidal activity of PBM, enhances the generation of H2O2 and promotes the accumulation of glycogen.
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PMID:Freshly isolated and cultured human monocytes obtained by plasmapheresis kill schistosomula of Schistosoma mansoni. 186 25

Balbc/c mice were immunized with purified recombinant E. coli-derived human gamma-interferon (HuIFN-gamma). Their spleen cells were fused with a mouse myeloma cell line (Sp2/0). Hybridomas producing antibodies reacting with HuIFN-gamma were screened by a soluble-phase radioimmunoassay using pure 125I-labeled cloned IFN-gamma as antigen, and tested for their ability to neutralize the antiviral activity of IFN. Three hybridomas S1-1, S1-2, and S1-3, were cloned and subcloned and remained stable. Although the antibodies produced by clones S1-1 and S1-2 were both able to neutralize specifically the antiviral activity of natural and recombinant HuIFN-gamma, they appeared to recognize different epitopes on the HuIFN-gamma molecule. The antibodies produced by the S1-3 clone failed to neutralize the antiviral activity of either interferon. The antibodies from all three clones were characterized as IgG1 subclass. Their affinity constants were determined from competitive inhibition curves and ranged from 1 to 4.3 X 10(8) M-1.
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PMID:Characterization of antibodies against recombinant HuIFN-gamma produced by hybridoma cells. 241 75

A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.
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PMID:Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma. 250 45

We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.
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PMID:Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway. 251 75

A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-gamma was fused through its 5'-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-gamma gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-gamma cell lines were obtained that produce 1.5-2 million units of IFN-gamma per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-gamma. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-gamma with 1-2 X 10(8) units/mg protein. Like IFN-gamma from human white blood cells, the IFN-gamma from CHO-gamma cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-gamma/ml culture per day.
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PMID:Efficient constitutive production of human IFN-gamma in Chinese hamster ovary cells. 301 45

Sezary's syndrome is a leukemic proliferation of OKT4+ lymphocytes. Sezary cells were isolated by differential centrifugation and fused to CEM.8azar.C, and HGPRTase-lacking clone of CEM. The hybrid cells were studied for their ability to produce soluble mediators of human monocyte cytotoxicity. The product of a single clone, FtF3, which bore the surface phenotype of Sezary cells, was characterized. Monocyte cytotoxicity-inducing factor (MCF) was found to be stable at pH 2 for 1 hr, unlike IFN-gamma, and was found to be more heat stable as well. Moreover, treatment of MCF with antisera to IFN-gamma, IFN-alpha or a combination of IFN-gamma and IFN-alpha failed to neutralize its biologic activity. MCF binds to matrix gel Red A. MCF eluted from this dye-ligand was found to have an apparent m.w. of 11,500 by gel filtration and 14,700 by SDS-polyacrylamide gel electrophoresis. MCF produced by hybridized Sezary cells appear to be neither IFN-gamma nor an altered molecular form of IFN-gamma, yet is a potent inducer of human monocyte cytotoxicity.
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PMID:Identification of a human monocyte cytotoxicity-inducing factor from T cell hybridomas produced from Sezary's cells. 308 3

We have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN-gamma, IFN-alpha, and IFN-beta 2 (IL-6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased approximately 25-fold above barely detectable levels in unstimulated promyelocytic HL-60 cells, in response to IFN-gamma. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN-gamma-inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN-alpha, and there was no response to IL-6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL-60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D3, agents that stimulate the differentiation of HL-60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S-transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85-95 kDa in the nuclear extracts of IFN-gamma-treated HL-60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL-60 cells treated with IFN-gamma, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN-inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, 124PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double-stranded DNA in vitro and exhibited a similar elution profile from DNA-cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA-binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents.
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PMID:IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines. 753 52

We have characterized the transcriptional response to IFN-gamma in two maturationally distinct macrophage populations: the mature RAW 264.7 cell line, phenotypically identical to thioglycollate-elicited peritoneal macrophages, and the less mature WEHI-3 cell line. We first investigated the use of two IFN-gamma-responsive regulatory elements, the interferon-stimulated response element (ISRE) and the gamma-activated sequence (GAS), in these cells. Transient transfection assays revealed that synthetic promoter constructs containing either the ISRE or GAS regulatory motif fused to a luciferase reporter gene were transcriptionally inactive in the WEHI-3 cell line. We then analyzed the expression in the two cell lines of a panel of known IFN-gamma-responsive genes that are transcriptionally controlled by different regulatory elements. RT-PCR analysis revealed that both cell lines responded to IFN-gamma treatment by up-regulating genes that are transcriptionally controlled by kappa B or W box DNA binding motifs. However, genes regulated by ISRE or GAS elements were induced by IFN-gamma only in the RAW 264.7 cell line. Kinetic analysis of the transcriptional activity of synthetic promoter constructs in the RAW 264.7 cell line showed rapid IFN-gamma induction through both the ISRE and GAS motifs, indicating that both elements are utilized early after IFN-gamma stimulation in mature macrophages. These results suggest that cis-acting DNA response element utilization, and the subsequent profiles of IFN-gamma-induced gene expression, differ in macrophages at different stages of maturation.
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PMID:Differential utilization of IFN-gamma-responsive elements in two maturationally distinct macrophage cell lines. 759 98

Recombinant DNA techniques were used to clone, construct and express the bifunctional molecule FV/IFN-gamma. The FV/IFN-gamma is a single-chain 42KD fusion protein expressed in E. coli under control of the strong T7 bacteriophage promoter in the expression vector pT7-7-FV-IFN-gamma. The fused gene fragment FV-IFN-gamma containing a single-chain anti-TAG72 FV gene fragment as well as the human recombinant cDNA fragment of IFN-gamma molecule. The renatured soluble form of FV/IFN-gamma was purified from E. coli inclusion bodies using HTPT chromatography. The yield of this fusion protein was estimated at 10mg/L. Our data showed that the FV/IFN-gamma molecule retained the TAG72 antigen-binding specificity and the IFN-gamma activity as measured in ELISA, Western blotting and up-regulation of CEA expression by IFN-gamma. Therefore, it may prove to be useful in targeting the biological effect of IFN-gamma to tumor cells and stimulating its immune destruction.
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PMID:Recombinant bifunctional molecule FV/IFN-gamma possesses the anti-tumor FV as well as the gamma interferon activities. 780 74


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