Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026764 (multiple myeloma)
 36,148 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CTL) by an enzyme release assay. We transfected P3/NS1-Ag4-1 (NS-1), a myeloma cell line derived from BALB/c mice with Escherichia coli beta-galactosidase (beta-Gal) gene, and isolated a stable transformant designated as NS-1/Z that expressed a high level of the enzyme activity intracellularly. The effector cells showing cytotoxicity against NS-1/Z were induced when the spleen cells of AKR or C3H mice were cultured with mitomycin C-treated BALB/c spleen cells for 4 days. When 2 x 10(4) NS-1/Z cells were incubated with varying numbers of effector cells, beta-Gal activity was released from the target cells depending on the number of effector cells and the time of incubation for up to 8 h. A highly sensitive enzyme assay was performed by using a fluorescent substrate, 4-methylumbelliferyl-beta-D-galactoside. The cytotoxicity was specific for H-2 haplotype of the stimulator cells, and was abolished by treating the effector cells with anti-Lyt 2 plus complement. The sensitivity of the enzyme release assay was comparable to that of 51Cr release assay. These results indicate that NS-1/Z can be used as a target cell line for the non-radioactive measurement of CTL activity.
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PMID:Establishment of an enzyme release assay for cytotoxic T lymphocyte activity. 154 35

To develop an anti-framework monoclonal antibody (mab) specific for the gamma (gamma)-chain of the T-cell antigen receptor (TCR), we expressed a part of the constant region of the gamma-chain (C gamma 2 gene segment) in E. coli using the pWR590 vector. This plasmid contains the E. coli lac promoter, operator, a truncated beta-galactosidase (beta-gal) gene (coding for the first 590 of the 1,007 amino acids of the beta-gal) and a polylinker region (at the 3' end of the beta-gal) containing nine restriction sites. These can be cleaved by any one of eight common restriction enzymes, permitting the introduction of the DNA fragment of interest. We employed the pT gamma 1 gamma-chain cDNA probe, which like the vast majority of the gamma-chain specific probes is aberrant and contains an in-frame stop codon at the junction of V and J regions. Computer analysis of the pT gamma 1 sequence revealed several MaeIII restriction sites that could result in a number of fragments. One of these fragments consisted of 245 base pairs (nucleotides 404-648) and contained most of the CI exon of the C gamma 2. Successful insertion of this fragment to the pWR590 vector was confirmed using restriction enzyme analysis. The C gamma insert was 12% of the construct. Expression of the pWR590-HpT gamma 1 recombinant plasmid in E. coli followed by SDS-PAGE analysis revealed a hybrid protein with a molecular weight of 85 kd which constituted at least 25% of the total E. coli insoluble protein. In contrast, cells transformed with the control pWR590 vector without insert expressed a 78 kd polypeptide chain. We developed several mabs against the pWR590-HpT gamma 1 hybrid protein by fusing spleen lymphocytes from BALB/c mice immunized with the pWR590-HpT gamma 1 protein, with cells of the NS1 mouse myeloma cell line. Screening of the mabs was carried out by ELISA against the pWR590-HpT gamma 1 hybrid protein and the control pWR590 beta-gal protein (beta-gal 590), derived by expressing in E. coli the pWR590 vector without gamma-chain insert. Two groups of mabs were obtained, those reacting with the pWR590-HpT gamma 1 hybrid protein only and those reacting with both the hybrid and the control beta-gal 590 proteins. The specificity of these mabs was further studied by Western blotting with similar results.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Development of a monoclonal antibody specific for the gamma chain of the T-cell antigen receptor using an open reading frame expression vector. 252 75

Hybridoma-produced monoclonal antibody (MoAb) against insulin is useful for insulin assays because of its specificity and plentiful supply. The spleen cells of male BALB/c mice immunized against monocomponent porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The resulting anti-insulin antibody (Ab) was purified and characterized by radioimmunoassay (RIA) using 125I-porcine insulin and sandwich-method enzyme immunoassay (EIA) using Ab-conjugated beads and beta-galactosidase. For reference, we used anti-insulin polyclonal antibody raised in guinea pigs (PoAb). Using the Ouchterlony technique, we identified the antibody as being of subclass IgG 1. We judged this antibody to be MoAb because it did not react at all with porcine insulin during EIA, in concentrations between 0 and 12.5 ng/ml; in contrast, PoAb reacted dose-dependently. During RIA, this Ab did not cross-react with glucagon, somatostatin or pancreatic polypeptide. It did cross-react with human and bovine insulins but not with rat insulin. The proper concentration of this MoAb for RIA proved to be 1:1,500,000 and the smallest detectable level of porcine insulin by RIA using this Ab was 0.5 ng/ml. These levels were similar to those obtained with PoAb. The binding activity of this MoAb to human insulin was quite similar to that of porcine insulin. RIA insulin determinations using our MoAb correlated well with those employing PoAb.
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PMID:Production of anti-insulin monoclonal antibody and its clinical application. 268 Mar 69

A double antibody sandwich immunoassay (EIA) was developed for the detection of Salmonella. The assay utilizes a beta-galactosidase-murine myeloma monoclonal antibody (M467) conjugate prepared with the heterobifunctional coupling reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and uses 4-methyl umbelliferyl beta-D-galactoside as a fluorogenic substrate for the enzyme. The EIA is sensitive and rapid, and compared favorably with the conventional cultural technique in the analysis of 60 feed samples naturally contaminated with Salmonella. Proteins or natural contaminants from the sample did not interfere in the assay.
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PMID:A fluorescent enzyme immunoassay for Salmonella detection. 328 71

Stable activated macrophage hybridomas were generated by somatic cell fusion between Propionibacterium acnes-induced peritoneal exudate cells and NS-1 myeloma cells. Five cell lines were obtained and each was cloned by limiting dilution; 59 clones were obtained. The cells of 2 clones (MP4-4 and MP4-8) which adhered to the culture dishes were selected for further analysis. These hybridomas exhibited non-specific esterase and beta-galactosidase intracellularly, and asialo GM1, Mac-1, Ia antigens and Fc-receptors on their cell surface. They did not, however, show phagocytic activity or secrete lysozyme. These hybridomas (MP4-4 and MP4-8) secreted the cytotoxic factor without any stimulation. Furthermore strong cytotoxic activity was found in ascites and sera from nude mice inoculated with these hybridomas. These activated macrophage hybridomas should be very useful in studies on cancer immunology and the physiology of macrophages.
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PMID:Activated macrophage hybridomas secreting a cytotoxic factor. 380 45

Spleen cells from Balb C mice immunized with purified Yu human myeloma IgE were fused with NS-1 mouse myeloma cells. After initial EIA screening for antibody-secreting cells, 20 hybrids were further characterized for cell growth, ascites production, antibody titer, specificity and affinity. Immunoglobulins purified from ascites fluid obtained from selected clones were labelled with beta-galactosidase. Combinations were made using either antibodies as capture and as conjugate against calibrated human IgE plasma samples. The combination of monoclonal anti IgE X b 10-22 as a capture antibody and X b 6-16 as a conjugate gave the best sensitivity and slope in EIA. It was successfully used in a sensitive two-step-enzyme-immunoassay for total IgE. The X b 6-16 conjugate was also assayed for the detection of allergen specific IgE antibodies. The results presented and discussed indicate that monoclonal antibodies could favourably substitute for polyclonal anti IgE antibodies in such assays.
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PMID:Monoclonal antibodies to human IgE: utilization for total IgE quantification and estimation of allergen specific IgE antibodies. 639 32

We have previously demonstrated that retrovirus-mediated genes were transferred to mouse glioma cells in a meningeal gliomatosis model (Yamada et al.: Japanese Journal of Cancer Research 83:1244-1247, 1992). This retrovirus vector contains the Escherichia coli. beta-galactosidase (beta-gal) gene as a marker for integration of the lacZ gene, which is controlled by the SV40 early promoter. We investigated whether lacZ genes could be specifically controlled in mouse glioma cells by glial-specific promoters, including the 2.5 kb 5' flanking region of the mouse glial fibrillary acidic protein (GFAP) gene, the 1.3 kb 5' flanking region of the myelin basic protein (MBP) gene, and the 1.5 kb 5' flanking region of the myelin proteolipid protein (PLP) gene. Psi-2 packaging cells were transfected with each retrovirus vector (GFAP promoter-, MBP promoter-, and PLP promoter-lacZ) and the infectious virus particles were recovered from the supernatants. Blue staining for beta-gal was detected in various fibroblast, myeloma, and glioma cell lines transduced with the retrovirus BAG vector. On the other hand, blue staining was only detected in glioma cells after transduction with the lacZ gene-bearing retrovirus controlled by glial-specific promoters. The strongest promoter activity was detected after transduction with the retrovirus in which the MBP promoter controlled the lacZ gene. Mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene were extremely sensitive to ganciclovir, while the parental cells and cells transduced with retrovirus containing the lacZ gene were not sensitive to ganciclovir.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Selective expression of foreign genes in glioma cells: use of the mouse myelin basic protein gene promoter to direct toxic gene expression. 750 43

Cell-surface sugar receptors may participate in interactions of lymphoid cells that influence their adhesive properties and proliferation. Their expression on cells of the pre-B line BLIN-I, the B-lymphoblastoid line Croco II, the myeloma line RPMI 8226 and the T-lymphoblastoid line CCRF-CEM was monitored with a panel of 14 types of chemically glycosylated E. coli beta-galactosidase at a non-saturating ligand concentration. Quantitative differences were determined for the capacity of the different cell types to bind constituents of the carbohydrate part of glycoconjugates. They were corroborated by analyses of binding for lactose-, beta-N-acetylgalactosamine-, beta-N-acetylglucosamine- and fucose-exposing neoglycoenzymes up to saturation levels. Values of dissociation constants of the tetrameric enzyme were in the range of 3-300 nM. Several types of sugar receptor led to carbohydrate-inhibitable adhesion of cells to 6 types of nitrocellulose-immobilized neoglycoprotein, their effectiveness being most obvious for the myeloma cells. Analyses of the carbohydrate-ligand-mediated adhesion of the other cell types revealed a comparatively decreased response. Only a few carbohydrates among the 7 types tested were effective in reducing cell adhesion to a far more complex ligand-bearing matrix than immobilized neoglycoproteins, namely bone-marrow stromal cell layers: sialic acid and N-acetylgalactosamine for B-lymphoblastoid cells and rhamnose for pre-B cells. These cellular interactions may encompass sugar receptors on the stromal cells and other types of molecular recognition in addition to the detected activities on the lymphoid cells.
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PMID:Adhesion of human lymphoid cell lines to immobilized carbohydrates and to bone-marrow stromal cell layers by surface sugar receptors. 839 77

In our previous study, gas-phase hydrazinolysis was used to analyze the glycoform of the O-linked oligosaccharide of human serum IgA1. All O-linked oligosaccharide chains are known to be present in the hinge portion. However, the number of O-linked oligosaccharide chains on IgA1 remained unclear. In order to determine the number of linked sugar chains, we applied matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the hinge glycopeptide prepared from human serum IgA1. MALDI-TOFMS did not show clear peaks, probably due to the microheterogeneity of the structure of each sugar chain. However, elimination of peripheral sialic acid and galactose residues by sequential treatment with neuraminidase and beta-galactosidase gave clear mass spectra with several sharp peaks. On the basis of these spectra, we conclude that IgA1 prepared from normal human serum carries different numbers of sugar chains. There are two major populations, one contains five GalNAc residues and the other four GalNAc residues. On the other hand, the hinge glycopeptide prepared from myeloma IgA1 was composed mainly of one population containing four GalNAc residues. Earlier, we reported incomplete glycosylation of IgA1 isolated from the serum of an IgA1 myeloma patient. In this experiment, the presence of four O-linked oligosaccharides per heavy chain of IgA1 from a myeloma patient was found. The reason why only four out of five sites on the hinge glycopeptide were fully glycosylated in the IgA1 from the IgA1 myeloma patient is not clear.
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PMID:Estimation of the number of O-linked oligosaccharides per heavy chain of human serum IgA1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the hinge glycopeptide. 888 26

Bcl-2 is an oncogene associated with prevention of apoptosis in a variety of cell types. Bcl-2 expression in B lymphoid cells prolongs antibody production, in vitro and in vivo. A line of transgenic mice (B6) has been developed that expresses human Bcl-2 in the B cells of SWR/SJL mice. B6 transgenic, nontransgenic littermates, and BALB/c mice were immunized with beta-galactosidase (B-gal) or sheep red blood cells (SRBC). The number of spleen cells recovered from immunized B6 mice was 3-4 times greater than syngeneic, nontransgenic littermates or BALB/c mice. Spleen cells from B-gal or SRBC immune B6, SWR/SJL, and BALB/c mice were fused with P3 myeloma cells to produce hybridomas. Forty-eight percent of the wells plated with fused B6 spleen cells produced B-gal-specific antibodies compared to 14% from BALB/c and 12% from SWR/SJL. Antibody-specific wells were subcloned, resulting in enhanced recovery of antigen-specific subclones with B6-derived fusions compared to controls. In the SRBC fusions, 17% of the wells plated with fused B6 spleen cells produced SRBC-specific antibodies compared to 6% for BALB/c and SWR/SJL spleens. After subcloning, B6-derived clones produced 8% positive subclones compared to 9.5% from SWR/SJL and 3.5% from BALB/c. Comparison of the isotype distribution of subclones showed a higher ratio of IgG antibodies compared to IgM from B6 mice in the B-gal fusions. IgA antibodies were recovered only from B6 mice. These data indicate that B6 transgenic mice that overexpress Bcl-2 in their B cells may be superior to other mouse strains for production of antigen-specific hybridomas.
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PMID:Evaluation of Bcl-2/B cell transgenic mice (B6) for hybridoma production. 891 86


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