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)

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

Two possible forms of tick-borne encephalitis virus (TBE) gene NS1 (called NS1' and NS1) were constructed using two overlapping cDNA-fragments of TBE genome and synthetic DNA fragments. This genes were expressed in E. coli cells in expression vector pUR290 as individual proteins or fusion with bacterial beta-galactosidase. The proteins NS1 (Mw. 39 kDa), beta-galactosidase-NS1' (Mw. 162 kDa) and beta-galactosidase-NS1 (Mw. 155 kDa) were effectively synthesized under the Plgc-promoter induction conditions. Expression of NS1' gene results in the formation of two virus-specific proteins (Mw. 46 and 44 kDa). All bacterial analogs of NS1 protein fixed monoclonal and polyclonal antibodies specific to viral NS1.
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PMID:[Expression of gene coding for the NS1 protein of the tick-borne encephalitis virus in Escherichia coli cell]. 215 Dec 83

The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome. JE protein coding sequences were expressed in E. coli by subcloning random fragments of cloned cDNA (P.C. McAda, P.W. Mason, C.S. Schmaljohn, J.M. Dalrymple, T.L. Mason, and M.J. Fournier, 1987, Virology 158, 348-360) into the bacteriophage lambda gt11 expression vector. Over 120 lambda gt11 recombinants expressing viral protein sequences as beta-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF). This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E. coli, providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the "ns2a" region of the JE genome.
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PMID:Expression of Japanese encephalitis virus antigens in Escherichia coli. 243 44

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

Part of a yellow fever virus-specified non-structural protein (NS1) was expressed in Escherichia coli as a fusion protein with beta-galactosidase. Immunization of mice with this partially purified NS1-beta-galactosidase fusion protein induced yellow fever virus-specific antibodies and provided some protection against intracerebral challenge with the virus.
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PMID:Reduction of yellow fever virus mouse neurovirulence by immunization with a bacterially synthesized non-structural protein (NS1) fragment. 313 49

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) on the growth of Streptococcus faecalis, and on the growth, beta-galactosidase synthesis, and various membrane-mediated processes, were studied in wild-type Escherichia coli JE1011 and its lipopolysaccharide-defective mutant NS1. DCCD (0.1 mM) completely inhibited the growth of S. faecalis and E. coli NS1 but had little effect on strain JE1011. The same amount of DCCD with E. coli NS1, but not with E. coli JE1011, inhibited the induction of beta-galactosidase, increased the permeability of the cells to o-nitrophenyl-beta-d-galactoside without causing extensive cell lysis or release of ultraviolet-absorbing materials, and inhibited the oxidation of certain intermediates of the tricarboxylic acid cycle. Inhibition of the oxidation of malate, fumarate, and alpha-ketoglutarate by DCCD appeared to be at the level of the transport system for these compounds. Inhibition of the membrane-bound adenosine triphosphatase by DCCD was not entirely responsible for these effects, since oxidation of these substances, and transport of [(14)C]succinate and [(14)C]fumarate, was inhibited by DCCD in a mutant, N(144), which lacked adenosine triphosphatase activity. It is concluded that lipopolysaccharide forms a barrier to DCCD in wild-type E. coli, and that DCCD can inhibit several processes in the cell.
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PMID:Effect of dicyclohexylcarbodiimide on growth and membrane-mediated processes in wild type and heptose-deficient mutants of Escherichia coli K-12. 427 56

We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding beta-galactosidase (beta-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the beta-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric beta-gal into nucleus. The recombinant AeDNV genomes (carrying the beta-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment.
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PMID:Densovirus of Aedes aegypti as an expression vector in mosquito cells. 795 54

To develop the immunochemical methods for determining 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in clinical samples, a variety of monoclonal anti-1,25(OH)2D3 antibodies have been generated. Two kinds of hapten-carrier conjugates, 25-hydroxyvitamin D3 3-hemisuccinate (hapten 3-HS) and 1 alpha-hydroxy-25,26,27-trinorvitamin D3 24-oic acid (hapten 24-OA) conjugated with bovine serum albumin, were used for immunization. Spleen cells from SD rats or BALB/c mice, each immunized with the conjugate of hapten 3-HS or 24-OA, were fused with P3/NS1/1-Ag4-1 myeloma cells. After screening by ELISA employing beta-galactosidase-labeled haptens, seven kinds of hybridomas secreting anti-1,25(OH)2D3 antibodies were established. Binding characteristics of these antibodies (Ka 0.73-20 x 10(9) M-1) were investigated by an RIA using tritium-labeled 1,25(OH)2D3. The data suggested that the rat monoclonal antibody 3R-1 derived from the hapten 3-HS and the mouse monoclonal antibody 24M-3 from the hapten 24-OA would be available for developing practical analytical systems.
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PMID:Production and characterization of monoclonal antibodies against two haptenic derivatives of 1 alpha,25-dihydroxyvitamin D3 conjugated with bovine serum albumin through the C-3 or C-24 position. 933 74

The nonstructural proteins NS1 and NS2 are thought to be expressed from the p7 promoter of Aedes densonucleosis virus (AeDNV). To study gene expression from the p7 promoter, eight different plasmids were constructed by fusing beta-galactosidase or beta-glucuronidase into the genome so that the reporter gene was in different open reading frames and under the transcriptional control of the p7 promoter. After transfection into C6/36 Aedes albopictus cells, constructs generated comparable amounts of RNA, but only the NS1 and NS2 fusion constructs produced appreciable levels of active enzyme. NS1 and NS2 fusion constructs contained wild-type AeDNV sequences from the p7 promoter downstream to nucleotide 458. The remaining constructs, with the exception of p7GUS.rf3, lacked some or all of these necessary sequences and inefficiently produced protein. These data suggest that sequences downstream of the p7 promoter play a role in translational regulation of gene expression from the p7 promoter of AeDNV.
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PMID:Gene expression and regulation from the p7 promoter of Aedes densonucleosis virus. 955 26

Ursodeoxycholic acid 7-N-acetylglucosaminides (UDCA 7-NAGs) are novel conjugated metabolites whose urine levels are expected to be a specific diagnostic index for primary biliary cirrhosis. To obtain a specific antibody which is useful for developing immunochemical analytical methods of UDCA 7-NAGs, a variety of monoclonal antibodies have been generated. Spleen cells from an A/J mouse, which had been immunized with a conjugate of nonamidated UDCA 7-NAG and bovine serum albumin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After screening by an enzyme-linked immunosorbent assay (ELISA) using a beta-galactosidase-labeled antigen, thirteen kinds of antibody-secreting hybridoma clones were established. Binding properties of these monoclonal antibodies were investigated in detail by ELISA. One of these antibodies, Ab-#8 (gamma1, kappa) had the most favorable characteristics for clinical application, which was group-specific to the 7-NAG conjugates of nonamidated, glycine- and taurine-amidated UDCAs providing a highly sensitive dose-response curve for each conjugate (midpoint 17 pg per assay for nonamidated UDCA 7-NAG). Cross-reactivities with eleven kinds of bile acids, including some potential interfering metabolites as UDCA 3-sulfate, were negligibly low. By using direct ELISA based on Ab-#8, daily urinary excretion rates of UDCA 7-NAGs of two healthy subjects were determined to be 1030 and 469 microg as GUDCA 7-NAG equivalent.
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PMID:Production and characterization of group-specific monoclonal antibodies recognizing nonamidated, glycine- and taurine-amidated ursodeoxycholic acid 7-N-acetylglucosaminides. 960 11


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