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)

A new method for mammalian cell transformation is proposed which is based on incorporation of plasmids into interpolyelectrolyte complexes (IPECs) with carbon chain polycations. The method is illustrated by examples of pRSV CAT and p beta-Gal plasmid IPECs with poly(N-ethyl-4-vinylpyridinium bromide) (C2PVP) and poly(N-ethyl-4-vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium+ ++) bromides random copolymer (C16PVP). These IPECs are produced spontaneously due to formation of a cooperative system of interchain electrostatic bonds after mixing DNA and polycation solutions. The interaction of IPEC with normal mouse fibroblasts NIH 3T3, human T-lymphoma "Jurkat", and Mardin Darby canine kidney cells has been studied. The data obtained has revealed that plasmid incorporation into IPECs significantly enhances both DNA adsorption on the plasma membrane and DNA uptake into a cell. The in vitro transformation of NIH 3T3 cells was monitored by a standard cloramphenicol acetyltransferase (CAT) assay (pRSV CAT plasmid) and by detection of beta-galactosidase (beta-Gal) expression using 4-methylumbeliferril beta-D-galactopyranoside as a substrate (p beta-Gal plasmid). In both cases it has been proved that IPEC-incorporated plasmids possess an ability for efficient cell transformation. The transforming activity of IPECs depends on their composition and polycation chemical structure. Under optimal conditions the efficiency of cell transformation with IPECs is several fold higher than that observed during standard calcium phosphate precipitation. The mechanism of the phenomenon observed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Efficient transformation of mammalian cells using DNA interpolyelectrolyte complexes with carbon chain polycations. 830 14

A thioic O-acid ester-containing sulfolipid (thionsulfolipid) was isolated from cells of picoplankton cyanobacterium, Synechococcus sp. The lipid accounted for about 0.2% of the lyophilized cells. The lipid was subjected to mild alkaline hydrolysis, and the structures of the hydrolysis products were identified by infrared spectra, mass and nuclear magnetic resonance spectrometries, as fatty acids and hexadecane-, hexadecene- and tetradecanethioic S-acids. Thioic S-acid was further confirmed by the synthesis of hexadecanethioic S-acid from palmitoylchloride and hydrogen sulfide. The positional distribution of the thioic acid ester in the lipid was determined by beta-galactosidase, sulphur-oxygen exchange reaction using silver nitrate, and lipase hydrolysis of the diacylglycerol derived from the lipid. The structure of the thionsulfolipid was identified as 6-sulfo-alpha-D-quinovopyranosyl(1-->1')-2'-O-acyl-3'-O-thioacy l -2-glycerol. When cells of HL 60, as a human lymphoma, were cultured with thionsulfolipid, 61% of the cell growth was inhibited at the concentration of 200 micrograms/ml. The lipid was toxic against minnows (Tanichtys albonubes). The LD50 was 20 ppm. Thioic O-acid ester-containing lipid (thionsulfolipid) has not been found in any other photosynthetic organisms.
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PMID:Thioic O-acid ester in sulfolipid isolated from freshwater picoplankton cyanobacterium, Synechococcus sp. 833 48

Deficiency in p53-mediated cell death is common in human cancer, contributing to both tumorigenesis and chemoresistance. In an attempt to restore p53, we evaluated in vitro infectivity and cytotoxicity of a wild type (w.t.) p53-expressing adenovirus (Ad-p53) toward a panel of human cancer cell lines (n = 19). At a multiplicity of infection of 30, both Ad-p53 and adenovirus expressing beta-galactosidase (Ad-LacZ) infected greater than 99% of cells derived from brain, lung, breast, ovarian, colon, and prostate cancer, but failed to infect leukemia or lymphoma cells. Ad-p53, but not Ad-LacZ, infection of cancer cells was followed by nuclear accumulation of the CDK inhibitor p21WAFI/CIPI, cell cycle arrest and loss of viability. Ad-p53 induced apoptotic death in cancer cells that express mutant p53, including multi-drug resistant cells, but fewer deaths were observed in some w.t. p53 expressing cells. Ad-p53-infected SKBr3 breast cancer cells were more sensitive to cytotoxicity of the DNA damaging drugs mitomycin C or Adriamycin, but not the M-phase specific drug vincristine. Our results suggest that Ad-p53 is capable of infecting and killing cancer cells of diverse tissue origins (including multi-drug resistant cancer cells), that p21WAFI/CIPI may be a useful marker of p53 infectivity and that there may be synergy between Ad-p53 and either mitomycin C or Adriamycin induced cell death in tumors with p53 mutations.
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PMID:In vitro evaluation of a p53-expressing adenovirus as an anti-cancer drug. 870 13

Cationic liposomes are considered to be safe vectors for gene transfer, but they are less efficient at delivering DNA to cells when compared with retroviral vectors. Cationic liposomes complexed with DNA were targeted to specific cells in vitro by means of monoclonal antibodies (mAbs) or ligands associated with the liposomes. Significant increases in expression of a beta-galactosidase reporter gene were observed in vitro in mAb-targeted liposomes, compared with non-targeted liposomes, in both an adherent tumor cell line (human adenocarcinoma) and a suspension cell line (human T-lymphoma). Also, use of asialofetuin as a targeting ligand significantly increased expression of the reporter gene in human hepatoma cells. Our results suggest that site-specific targeting of cationic liposomes is a good strategy for increasing both the selectivity and the efficiency of DNA delivery to cells and with further development may lead to targeted DNA delivery in vivo.
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PMID:Use of targeted cationic liposomes in enhanced DNA delivery to cancer cells. 885 50

The hamster polyomavirus (HaPV) is associated with spontaneously appearing skin epithelioma of the Syrian hamster Z3 strain. Virus particles prepared from the skin epithelioma cause lymphoma and leukemia when injected into newborn hamsters from a distinct Syrian hamster colony (HaP); in contrast to the skin epithelioma the hemopoietic tumors are virus free but accumulate viral DNA. To study the humoral immune response of HaPV-infected Z3 hamsters we produced recombinant HaPV proteins in Escherichia coli as beta-galactosidase-, TrpE- and dihydrofolate reductase-fusion proteins or as non-fused proteins. Recombinant plasmids carried segments of all putative early and late HaPV proteins. The recombinant proteins were detected in stained SDS polyacrylamide gels and in Western blots using monoclonal anti-TrpE and anti-beta-galactosidase antibodies and sera of HaPV-infected hamsters. Sera from HaPV-infected Z3 hamsters and crude lysates of all clones were applied to Western blots to characterize the humoral immune response in the animals. HaPV-specific antibodies were found to be directed against early protein segments translated from the first common exon and from the second unique exon of LT and MT, resp., as well as against the late proteins VP1 and VP2/3. The almost complete VP2 was recognized by all sera whereas VP1 was detected only by a half of the sera. Our data suggest the presence of at least 2 immunodominant regions in VP2, one in the C-terminal VP1 and at least 4 in early proteins.
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PMID:Hamster polyomavirus-encoded proteins: gene cloning, heterologous expression and immunoreactivity. 888 64

We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells (lymphocytes, macrophages, endothelial cells) from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity (adhesion, costimulatory and cytotoxic molecules, cytokines, etc.) at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy.
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PMID:Dissection of tumour and host cells from target organs of metastasis for testing gene expression directly ex vivo. 888 7

The targeted introduction of therapeutic genes into malignant cells based on receptor mediated endocytosis of ligand-DNA conjugates recently was established as a transfection system and provides a promising strategy for cancer therapy. Antiidiotype antibodies could be of particular interest for this approach because their immunoglobulin receptor idiotypes represent highly specific tumor markers. Their safe and specific applicability in vivo, alone or as immunotoxins, has been proven in clinical trials for passive immunotherapy and vaccination strategies. For these reasons we have explored the utility of antiidiotype antibodies for gene delivery systems using the reporter genes beta-galactosidase and luciferase. Two monoclonal antibodies, SIC5 and 5D10, specific for B-lymphoma cell lines, which represent models for murine plasmacytoma (38C13) and human non-Hodgkin's lymphoma (SU-DHL-4) have been covalently linked to polylysine via the heterobifunctional cross-linker SPDP. Highly efficient uptake and internalization of the immunoconjugates have been shown by fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis. Successful transfections have been shown at the RNA and the reporter gene level (beta-galactosidase, luciferase) using different promoter/enhancer systems. Beta-galactosidase activity was detected by flow cytometry (FACS-gal) analysis for both cell lines, and SU-DHL-4 cells showed significant luciferase activity.
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PMID:Antibody-mediated gene delivery for B-cell lymphoma in vitro. 898 39

With the aim of generating a virus-cell system to introduce alterations in proteins of interest--which may be of use in studies of their biological functions--we established a persistent infection on a B-lymphoma cell line (A20.2J) with vaccinia virus (VV) recombinants. As a model, we used a vaccinia virus recombinant expressing the human immunodeficiency virus HIV-1 env gene. In this unique virus-cell system, we found that it is possible to introduce several structural and functional alterations in the env protein with passage numbers. From passage 10-20, two new env products emerged: an uncleaved gp160 and a glycoprotein fragment of 110 kDa. The uncleaved gp160 exhibit interesting properties as an immunogen. This protein forms stable oligomers, is not released from the cells, cannot fuse CD4+ presenting HeLa cells and activates a stronger cellular immune response than the parental cleaved env. In contrast, the 110 kDa product is a poor immunogen, since it lacks the gp41 domain, cannot form oligomers, accumulates intracellularly and cannot fuse CD4+ cells. In the persistently infected cells we have also found alterations in another heterologous protein-beta-galactosidase-a gene inserted in the same locus of VV as the env gene. This alteration resulted in a truncation of the (beta-galactosidase protein from 125 kDa to about 70 kDa. A similar size truncation of env and of beta-galactosidase was observed in many of the isolated VV recombinants.
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PMID:Use of persistent infections with vaccinia virus recombinants to introduce alterations in foreign proteins: an application to HIV-1 env protein. 902 76

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

We have hypothesized that adenoviral vectors might mediate gene transfer into cell lines derived from human lymphocytic malignancies, such as lymphoma, lymphocytic leukemia, and myeloma. A panel of 33 cell lines was studied for their ability to be transduced by an adenoviral (AD) vector encoding the Escherichia coli beta-galactosidase gene (AD-betagal). A cytochemical assay and a flow cytometry assay both demonstrated that a subset of lymphocytic cell lines can be efficiently transduced by adenoviral vectors. In particular, three of three anaplastic large cell lymphoma lines, two of two Hodgkin's disease cell lines, two of seven Burkitt's lymphoma cell lines, and three of five myeloma cell lines exhibited efficient gene transfer. The ability of an AD vector expressing the thymidine kinase (tk) gene from herpes simplex virus-1 (AD-tk) followed by ganciclovir (GCV) to kill 11 of these lymphocytic cell lines was studied. In eight of the cell lines tested, more than 68% of the cells were killed by AD-tk/GCV. Similar results were obtained using an adenoviral vector expressing the wild-type p53 tumor suppressor gene (AD-p53). Thus, AD-tk/GCV and AD-p53 both demonstrated efficient killing of these cell lines. These data document that adenoviral vectors are valuable reagents for the introduction of genes into selected lymphocytic cell lines. These data also suggest that adenoviral vectors might be useful for gene therapy of subsets of lymphocytic malignancy.
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PMID:Adenoviral vectors efficiently target cell lines derived from selected lymphocytic malignancies, including anaplastic large cell lymphoma and Hodgkin's disease. 981 92


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