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)

The Epstein-Barr nuclear antigen (EBNA)-3 and EBNA-4 proteins are thought to act as transcriptional transactivators. The yeast two-hybrid system and coimmunoprecipitation were used to demonstrate that EBNA-3 and -4 associate with the DNA-binding protein RBP-2N, an isoform of RBP-J kappa. A comparison between EBNA-3, EBNA-4, and EBNA-6 binding to RBP-2N indicated that EBNA-3 enhanced beta-galactosidase activity 4-fold more than EBNA-6 and 30-fold more than EBNA-4. Assay of RBP-2N deletion mutants demonstrated that EBNA-3 binds to regions of RBP-2N which are distinct from those to which EBNA-2 and -6 interact, whereas EBNA-4 binds to the same region of RBP-2N as EBNA-2 and -6 (amino acids 159-331 of RBP-2N). Interaction of both A- and B-type EBNA-3 with RBP-2N was also demonstrated by immunoprecipitation. RT-PCR analysis of a panel of B cell lymphomas and lymphoblastoid cell lines demonstrated that higher levels of RBP-2N were expressed, in comparison to RBP-J kappa, indicating that RBP-2N is a major isoform expressed in B cells. These results suggest that all the EBNA-3 family proteins lead to transcriptional regulation via interaction with RBP-2N.
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PMID:Epstein-Barr nuclear antigen-3 and -4 interact with RBP-2N, a major isoform of RBP-J kappa in B lymphocytes. 895 54

We have developed a miniviral vector, pH300, based on the human herpesviruses 1 and 4, herpes simplex virus type 1 (HSV-1), and Epstein-Barr virus (EBV), carrying EBV sequences for plasmid episomal maintenance and HSV-1 sequences for amplification and packaging in multimeric form into HSV-1 capsids in the presence of a helper virus and helper cell line. A reporter gene, the bacterial lacZ gene, which expressed beta-galactosidase, was inserted into the multiple cloning site of pH300 to make pH300-lac. The packaged pH300-lac DNA was very efficient in infecting human cells in tissue culture. The pH300-lac miniviral stock was used to infect in vitro various human cell types derived from breast cancer, lung cancer, and liver cancer. Up to 95% of cells were infected and expressed beta-galactosidase activity after exposure to viral stock at a multiplicity of infection of 3. There was essentially no apparent cytotoxicity after infection of cultured cells in vitro. To test in vivo gene delivery, human liver tumor cells preimplanted subcutaneously in nude mice and injected in situ with pH300-lac showed high efficiency of ectopic gene expression. The pH300 miniviral vector is a simple and effective gene transfer system which shows potential for gene therapy of cancer and inherited diseases.
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PMID:A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo. 897 Sep 63

Plasmids carrying the Epstein-Barr virus (EBV) latent gene EBNA1 and the EBV latent origin of replication (oriP) stay in transfected human cells as autonomously replicating extrachromosomal genetic units. They thus might represent a suitable tool for cytokine gene introduction into human tumor cells with the prospect of therapeutic antitumor vaccination. The aim of this study was to analyze whether such plasmids permit stable and efficient expression of cytokine genes in human non-Hodgkin lymphoma cells. We tested physical stability and expression levels of plasmids carrying EBNA1 and oriP for episomal maintenance, immunoglobulin light chain enhancer elements for augmentation of expression, and cytokine or marker genes after introduction into human NHL cell lines in vitro and in vivo after inoculation into nude mice. Data obtained with these EBV-based vectors were compared with another plasmid, not carrying EBNA1 and oriP. cDNAs coding for GM-CSF, IL6, TNF alpha, the chloramphenicolacetyltransferase (CAT) and the beta-galactosidase (lacZ) gene were transfected into the EBV-positive Burkitt's lymphoma cell line BL60 and the EBV-negative B cell lymphoma cell line BJA-B. EBV-derived vectors permitted a high, host cell independent transfection efficiency and high and host cell independent levels of expression. After removal of the selection pressure (hygromycin B) cytokine expression could be detected for several weeks in vitro and in vivo but, however, declined continuously. These experiments suggest that episomal BC-derived vectors represent an effective tool for cytokine gene transfer in human lymphoma cells.
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PMID:Suitability of Epstein-Barr virus-based episomal vectors for expression of cytokine genes in human lymphoma cells. 908 10

The DNase of Epstein-Barr virus (EBV) is a 470-amino-acid protein which possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA and single-stranded DNA as substrates. It has been reported that this protein may be found in the nucleus and/or cytoplasm of infected cells. In this study, using cell fractionation and immunoblotting to determine the distribution of EBV DNase in Akata cells stimulated with anti-human immunoglobulin G antibody (anti-IgG), the DNase was found to be located predominantly in the nucleus. To map the signals in DNase which mediate its nuclear localization, we monitored the nuclear transport of fusion proteins consisting of various fragments of EBV DNase linked to a cytoplasmic protein, beta-galactosidase (beta-Gal). The results demonstrated that two regions of the DNase with nuclear localization signal (NLS) activity, designated NLS-A (amino acids 239-266) and NLS-B (amino acids 291-306), were able independently to localize the beta-Gal to the nuclei of HEp-2 and HeLa cells. Five basic residues (R or K) were found in each NLS and distributed differently in primary structure. The basic domains and flanking residues of NLS-A and NLS-B are 250YKRPCKRSFIRFI262 and 294LKDVRKRKLGPGH306, respectively. Further examination of these sequences revealed that NLS-A contains bulky aromatic amino acids (Y and F) which may diminish its capacity to act as a strong NLS and lacks the typical proline and glycine helix-breakers. However, NLS-B contains typical proline and glycine helix-breakers and the histidine residue at amino acid 306 is required for NLS activity. In addition, two hydrophobic regions within the DNase were found to inhibit the function of NLS-A but not NLS-B, suggesting that these two domains are different types of NLSs and differ in their sensitivity to hydrophobic regions in the context of protein structure.
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PMID:Epstein-Barr virus DNase contains two nuclear localization signals, which are different in sensitivity to the hydrophobic regions. 968 72

Mice harboring random gene-trap insertions of a lacZ (beta-galactosidase)-neomycin resistance fusion cassette (beta-geo) were analyzed for expression in the hippocampus. In 4 of 15 lines reporter gene activity was observed in the hippocampal formation. In the obn line, enzyme activity was detected in the CA1-3 hippocampal subfields, in hpk expression was restricted to CA1, but in both lines reporter activity was also present in other brain regions. In the third line, kin, reporter activity was robustly expressed throughout the stratum pyrimidale of CA1-3, with only low-level expression elsewhere. The final line (glnC) displayed ubiquitous expression of the reporter and was not analyzed further. Fusion transcripts for the first three lines were characterized; all encode polypeptides with features of membrane-associated signalling proteins. The obn fusion identified a human cDNA (B2-1) encoding a pleckstrin homology (PH) domain, while hpk sequences matched the Epstein-Barr Virus (EBV) inducible G-protein coupled receptor, EBI-1. kin identified an alternative form of the abl-related nonreceptor tyrosine kinase c-arg. Electrophysiological studies on mice homozygous for the insertions revealed normal synaptic transmission, paired pulse facilitation and paired-pulse depression at Schaffer collateral-commissural CA1 synapses, and normal long-term potentiation (LTP) in obn and kin. hpk mice displayed an increase in hippocampal CA1 long-term potentiation (LTP), suggesting a role for this receptor in synaptic plasticity.
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PMID:Gene-trapping to identify and analyze genes expressed in the mouse hippocampus. 982 57

Thioredoxin (TRX) has disulfide reducing activity and is reported to be involved in various cellular functions including the promotion of cell growth and apoptosis. To help understand the molecular mechanism through which TRX is involved in immunological systems, we screened a cDNA library derived from a B-cell population of Epstein-Barr virus-transformed human peripheral blood lymhocyte for TRX binding proteins by use of a yeast two-hybrid system. Among plasmids from positive clones, a plasmid contained an insert which has homology with human p40phox, a cytosolic component of phagocyte oxidase. This insert sequence extended from the base + 181 to the stop codon of p40phox. The entire coding region of p40phox was shown to interact with TRX both in assays of histidine prototrophy and beta-galactosidase activity; in contrast, no interaction was observed with substituted mutant TRX (C32S/C35S), which lacks reducing activity. These results showed that p40phox interacts with TRX and indicated the possibility of TRX-dependent regulation of phagocyte oxidase activity.
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PMID:Demonstration of the interaction of thioredoxin with p40phox, a phagocyte oxidase component, using a yeast two-hybrid system. 1039 71

The present study was aimed at devising an efficient nonviral strategy for suicide gene therapy of hepatocellular carcinoma (HCC). To improve the efficiency of DNA delivery and expression, we applied Epstein-Barr virus (EBV)-based plasmid vectors instead of conventional plasmid vectors and combined them with cationic liposome (EBV/lipoplex) or polyamidoamine dendrimer (PAAD) (EBV/polyplex). When the beta-galactosidase gene was transferred to HuH7, PLC/PRF/5, or HLE cells, < or =50-fold higher beta-galactosidase activities were demonstrated in the cells transfected with EBV vector compared with those transfected with conventional plasmid vectors. PAAD-mediated transfection of HCC with pSES.Tk (an EBV-based vector carrying the herpes simplex virus-1 thymidine kinase gene) resulted in a marked reduction in viable cell number by the addition of ganciclovir (GCV). The HCC cells transfected with pSES.Tk/PAAD showed 100- to 1000-fold higher susceptibilities to GCV than those transfected with pS.Tk (a conventional plasmid vector carrying herpes simplex virus-1 thymidine kinase gene)/PAAD. The pSES.Tk-transfected HCC cells were effectively killed by day 9 in culture with a clinically feasible concentration of GCV (25 microM), whereas the pS.Tk-transfected cells survived the culture. These results demonstrate highly efficient suicide gene transfer into various HCC cells by EBV-based plasmid vectors in vitro, suggesting the possible application of this nonviral vector system to gene therapy of HCC.
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PMID:Highly efficient suicide gene expression in hepatocellular carcinoma cells by epstein-barr virus-based plasmid vectors combined with polyamidoamine dendrimer. 1067 53

Replication-defective adenoviruses are arousing growing interest as both gene therapy and vaccine vectors. In a phase I clinical trial designed to evaluate the feasibility and tolerance of recombinant adenovirus (rAd)mediated gene transfer, we previously demonstrated that a single intratumoral injection of 10(9) PFU of rAd encoding the beta-galactosidase protein (Ad-beta-Gal) induced strong short-term (1-3 months) humoral, helper (Th1 type) and cytotoxic T cell responses specific for the transgene product in patients with advanced lung cancer. The purpose of the present study was to evaluate the persistence of long-lasting immunity to the transgene protein and in parallel, to assess patient immunocompetence revealed by responses to recall antigens (tetanus toxoid, purified protein derivative), viral pathogens (Epstein-Barr virus, influenza virus), and allogeneic antigens in mixed lymphocytic reactions. The beta-Gal-specific proliferative response declined rapidly in patients with progressive disease, as did responses to the other antigens. In contrast, a long-lasting proliferative response to beta-gal was maintained in an immunocompetent patient in complete remission 2 years after an injection of 108 PFU of Ad-beta-Gal. Anti-beta-Gal humoral (IgG and IgA) responses persisted notably, as did responses to TT and poliomyelytic antigens. While T cell effector cytotoxic responses specific for the viral peptides plummeted, the frequency of anti-beta-Gal CTL precursors remained particularly high, thus attesting to major immunization. Despite the impact of both advanced disease and chemotherapy on immunocompetence, we show the long-term persistence of immunity to the transgene protein vectorized by rAd.
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PMID:Longitudinal follow-up of cellular and humoral immunity induced by recombinant adenovirus-mediated gene therapy in cancer patients. 1098 63

The present study reports a novel nonviral method to efficiently and specifically target carcinoembryonic antigen (CEA)-producing cholangiocarcinoma (CC) cells in vitro. Epstein-Barr virus (EBV)-based and conventional plasmid vectors were constructed that possess the beta-galactosidase (beta-gal) or herpes simplex virus-1 (HSV-1) thymidine kinase (Tk) genes as well as tandem repeats of the human genomic sequence -82 to -42 bp from the transcriptional start site of the CEA gene. The plasmids were transfected by means of polyamidoamine dendrimer into CEA-positive (HuCC-T1) or -negative cell lines. Transfection of the conventional plasmid vector with the CEA promoter and beta-gal gene resulted in a very low or undetectable level of marker gene expression even in the CEA-positive cell line. Transferring the HSV-1 Tk gene by conventional plasmid did not affect the susceptibility of HuCC-T1 cells to ganciclovir. In marked contrast, strong beta-gal expression was specifically obtained in HuCC-T1 cells by transfecting the EBV-based plasmid in which the CEA promoter and a ubiquitous promoter (SRalpha) are employed to drive the EBV-encoded nuclear antigen 1 (EBNA1) and beta-gal genes, respectively (pTES.beta). Furthermore, CEA-positive but not -negative tumor cells were rendered highly susceptible to ganciclovir when transfected with the EBV-based vector that carries the CEA promoter-EBNA1 and SRalpha-HSV-1 Tk genes (pTES.Tk). These results strongly suggest that the EBV-based plasmid vector/cationic polymer system (EBV/polyplex) equipped with the CEA promoter provides an efficient nonviral method for the targeted gene therapy of CEA-producing malignancies.
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PMID:Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by polyamidoamine dendrimer-mediated transfer of an Epstein-Barr virus (EBV)-based plasmid vector carrying the CEA promoter. 1102 96

Electroporation has been applied to introducing DNA into several organs; however, gene expression was localized around the injected area. Examined was the efficiency of intrarenal injection of DNA followed by in vivo electroporation, using FITC-labeled oligodeoxynucleotides (FITC-ODN) and plasmid DNA expressing beta-galactosidase or luciferase. FITC-ODN or expression vectors were injected into the left renal artery; thereafter, the left kidney was electroporated between a pair of tweezer-type electrodes. FITC-ODN were transferred into all glomeruli, and transfected cells were identified as mesangial cells. Four d after transfection of the pCAGGS-LacZ gene, beta-galactosidase expression was observed in 75% of glomeruli. To compare the transfection efficacy by electroporation with that by the hemagglutinating virus of Japan (HVJ) liposome method, a luciferase reporter gene, pActLuc, was transferred into glomeruli by either electroporation or the HVJ liposome method. On day 4, electroporation resulted in higher glomerular luciferase activity than did the HVJ liposome method. We also observed that co-transfection of pcEBNA, an expression vector for Epstein-Barr virus nuclear antigen, and poriP-cLuc, oriP-harboring vector, resulted in an eightfold higher luciferase gene expression than simple poriP-cLUC: No histologic damages were seen in glomeruli or tubular epithelial cells. In conclusion, gene transfer into renal artery followed by electroporation was an effective and simple strategy for gene transfer that targets glomerular mesangial cells.
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PMID:Electroporation-mediated gene transfer that targets glomeruli. 1131 53


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