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 herpes simplex thymidine kinase (HStk) gene induces regression of epithelial tumors after ganciclovir (GCV) administration. This observation has been attributed to both gene transfer and metabolic cooperation between cells (the bystander effect). This study evaluates the relative roles of the bystander effect and gene delivery of the HStk gene by the LTKOSN.2 vector. MC38 colon adenocarcinoma cells, syngeneic for C57/B16 mice, were used. Whereas in vitro proliferation assays demonstrated a bystander effect, significantly greater inhibition of proliferation occurred with HStk gene transfer. In mixtures containing 75 per cent MC38 cells with no vector (MC38 NV) and 25 per cent MC38 pretransduced with LTKOSN.2 (MC38 TK), proliferation was inhibited by 62 +/- 5 per cent. In mixtures containing 75 per cent MC38 NV with 25 per cent HStk vector-producing cells (LTKOSN.2 VPC), proliferation was inhibited by 97 +/- 1 per cent. In vivo subcutaneous mixture experiments utilized MC38 NV cells inoculated at a 1:1 ratio with various treatment cell groups followed by administration of GCV. Tumor volumes (mean +/- standard error) at 30 days were: 264 +/- 66 mm3 for MC38 TK, 0 for LTKOSN.2 VPC, 1009 +/- 335 mm3 for lacZ VPC (beta-galactosidase VPC), and 1012 +/- 212 mm3 for NIH3T3 (nontransduced cells). These data suggest that in vivo, the bystander effect alone causes tumor inhibition, but gene transfer is necessary for complete tumor elimination in immunocompetent mice.
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PMID:Herpes simplex thymidine kinase gene transfer is required for complete regression of murine colon adenocarcinoma. 920 36

The suitability of non-replicating thymidine kinase deficient herpes simplex virus type 1 expressing bacterial beta-galactosidase (tk-lacZ HSV-1) as a transfer vehicle into sympathetic preganglionic neurons in vivo was assessed. Many sympathoadrenal preganglionic neurons (451 +/- 105) with normal morphology were identified using beta-galactosidase histochemistry two days after inoculation of tk-lacZ HSV-1 into the adrenal gland of hamsters. Beta-galactosidase activity co-localized with nicotinamide adenine dinucleotide phosphate-diaphorase-positive sympathetic preganglionic neurons in the nucleus intermediolateralus, pars principalis. The maximal number of beta-galactosidase expressing neurons was found two days post-inoculation but this number dropped dramatically after this time. An inflammatory infiltrate was abundant around infected neurons and in the white matter at five days and infected neurons appeared morphologically abnormal. At 26 days, the infiltrate was still present but no infected sympathoadrenal preganglionic neurons were detected. Approximately 25% fewer nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the nucleus intermediolateralis, pars principalis were counted ipsilaterally than contralaterally in animals infected for 14, 21 or 26 days with tk-lacZ HSV-1, compared to the 3% difference in animals mock-infected for 26 days. Approximately 33% of the estimated number of sympathoadrenal preganglionic neurons infected with tk-lacZ HSV-1 at five days were apoptotic or necrotic. About 60% of neurons infected with tk-lacZ HSV-1 at two days no longer expressed nicotinamide adenine dinucleotide phosphate-diaphorase at 14-26 days. In conclusion, the non-replicating thymidine kinase deficient HSV-1 was efficiently retrogradely transported from the adrenal gland to infect sympathoadrenal preganglionic neurons. These gene transfer experiments using tk-lacZ HSV-1 suggest that foreign gene expression in sympathetic preganglionic neurons in vivo may be maximal two days after inoculation when beta-galactosidase was expressed in the greatest number of sympathetic preganglionic neurons. After two days, fewer neurons expressed beta-galactosidase and the presence of tk-lacZ HSV-1 appeared to be altering protein expression in sympathetic preganglionic neurons and/or leading to the demise of the infected neuron.
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PMID:Gene transfer into sympathetic preganglionic neurons in vivo using a non-replicating thymidine kinase-deficient herpes simplex virus type 1. 927 1

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.
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PMID:Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene. 931 59

Transduction of the herpes simplex virus thymidine kinase (HSV-tk) into vascular endothelial cells using a replication-defective adenoviral vector (Ad.CMV-tk) to confer sensitivity to ganciclovir (GCV) was investigated. The cytotoxic sensitivity of bovine aortic endothelial cells (BAEC) to GCV following Ad.CMV-tk transduction at multiplicity of infection of 100 was ten-fold that of 9L glioma cells in vitro. Deoxyribonucleic acid fragmentation was detected in these BAEC. A co-culture experiment using BAEC transduced with Ad.CMV-tk (BAEC-tk) and 9L cells expressing beta-galactosidase (9L-Lac Z) showed about 70% tumoricidal effect under the conditions of one BAEC-tk cell in 10 9L-Lac Z cells. Tumor-bearing Fisher 344 rats, an experimental brain tumor model, received Ad.CMV-tk intratumorally at 7 days after tumor implantation, and were subsequently treated with intraperitoneal GCV (100 mg/kg). Histological examination found the vascular endothelial cells adjacent to 9L glioma tissue revealed apoptosis. These results suggest that vascular endothelial cells are an attractive target for adenoviral-mediated HSV-tk gene therapy.
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PMID:Effect of adenoviral-mediated thymidine kinase transduction and ganciclovir therapy on tumor-associated endothelial cells. 936 32

In this study we describe a new retroviral vector utilizing an internal ribosome entry site (IRES) from encephalomyocarditis virus to co-express two genes. One is the herpes simplex virus type 1 thymidine kinase gene (HSV-TK) which induces sensitivity to ganciclovir, and the second is the bacterial beta-galactosidase gene (LacZ) which was revealed by an histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). We engineered the U937 human cell line to co-express both genes and monitored transduced cells using X-Gal staining. Several transduced clones were selected. The clones exhibiting X-Gal positive cells were sensitive to ganciclovir treatment (1 microgram/ml) while X-Gal negative clones were not. Monoclonal cell lines showed a single copy of the provirus integrated in their genome with the TK-IRES-LacZ sequence stably inserted in all clones. The band distribution pattern of the proviral DNA differed only at the long terminal repeat (LTR) level. Northern blot analysis of an X-Gal positive/ganciclovir sensitive clone showed an mRNA band of 6 kb with both LacZ and TK probes. An X-Gal negative/ganciclovir resistant clone was negative with both probes. This report shows: (1) a therapeutic gene can be linked to a marker gene by an IRES element achieving equivalent expression of both proteins; (2) the co-expression of a marker gene makes fluorescein-di-beta-D-galactopyranoside staining possible, and consequently separation of cells expressing the LacZ gene by fluorescence activated cell sorting. Thus the cells expressing the HSV-Tk gene are enriched; (3) the use of a marker gene such as LacZ could open up interesting perspectives in gene therapy protocols because of the opportunity to monitor the transduced cells using a simple cytochemical stain.
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PMID:Retroviral transfer of herpes simplex virus-thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector. 940 6

The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.
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PMID:Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. 944 13

We generated neurotropic herpes simplex type 1 viruses expressing human placental alkaline phosphatase and studied the utility of this enzyme as a marker of infected neurons. The neurotropism of these viruses was assessed by their ability to infect sympathetic preganglionic neurons after adrenal injection in hamsters. The transneuronal transfer of these viruses was examined by their ability to cross the peripheral synapse from the kidney to renal preganglionic neurons or to cross the central synapse from the adrenal gland to the medulla oblongata. Finally, we injected an alkaline phosphatase-expressing herpes simplex virus into the adrenal gland and a beta-galactosidase-expressing herpes simplex virus (US5gal) into the muscular wall of the small intestine to label two neural circuits in one animal and to assess the feasibility of a dual-virus labelling system. The alkaline phosphatase gene was inserted into the glycoprotein J locus or the virus-induced host shut-off locus in the herpes simplex genome to create viruses which replicate (gJHAP HSV or vhsHAP HSV) or into the thymidine kinase locus to generate a virus that does not replicate in neurons in vivo (TK- HAP HSV). Each of the three viruses was retrogradely transported from the adrenal gland of hamsters to sympathetic preganglionic neurons, suggesting that the neurotropism of these viruses was maintained. gJHAP HSV travelled transneuronally from the kidney to sympathorenal preganglionic neurons and from the adrenal gland to neurons in the rostral ventrolateral medulla. Neuronal infection with alkaline phosphatase-expressing virus could be identified using histochemistry but detailed morphology of these neurons was not revealed. However, staining by anti-herpes simplex virus immunoperoxidase demonstrated that they had normal morphology. Identification of two distinct neural circuits in one animal was achieved with our dual-virus labelling system. The nonreplicating TK- HAP HSV was used in combination with US5gal to identify intestinal and adrenal sympathetic preganglionic neurons. The beta-galactosidase-expressing intestinal neurons were labelled bilaterally in the nucleus intermediolateralis, pars principalis, and alkaline phosphatase-expressing adrenal neurons were found ipsilaterally. Some clusters of sympathetic preganglionic neurons in the nucleus intermediolateralis, pars principalis contained mostly intestinal sympathetic preganglionic neurons and a few adrenal sympathetic preganglionic neurons. In other areas, the opposite pattern occurred. About 3-7% of the labelled sympathetic preganglionic neurons were double-labelled by both markers. The distinct and crisp morphology and dendritic processes of neurons stained by beta-galactosidase histochemistry contrasted with the partial staining of neurons by alkaline phosphatase, revealing beta-galactosidase as a better marker of infected neurons. In conclusion, alkaline phosphatase-expressing herpes simplex viruses are yet neurotropic after insertion of this marker enzyme into any of three different loci of the herpes simplex genome. One replicating alkaline phosphatase-expressing virus travelled transneuronally. These alkaline phosphatase-expressing herpes simplex virus can be used together with beta-galactosidase-expressing herpes simplex viruses to determine the target specificity of sympathetic preganglionic neurons controlling visceral organs or can be used to express two different recombinant genes in two targeted neuronal populations. This study suggests that sympathetic preganglionic neurons controlling the intestine and adrenal gland are almost completely distinct.
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PMID:Simultaneous identification of two populations of sympathetic preganglionic neurons using recombinant herpes simplex virus type 1 expressing different reporter genes. 946 44

Cancer-specific antigens are promising targets for the specific delivery of certain drugs or genes to cancer cells in cancer therapy. Carcinoembryonic antigen (CEA) is one of the cancer-associated antigens predominantly detected in the gastrointestinal cancer of the colon and stomach. Targeting strategies for CEA-producing cancer cells have been thoroughly developed mainly by the production of monoclonal antibodies to CEA and further single-chain variable fragment (scFv) antibodies. Here, we have generated Moloney murine leukemia virus-derived retroviral vectors co-displaying an anti-CEA scFv-envelope chimeric protein and an unmodified envelope protein to deliver a gene for herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase. The harvested viruses successfully incorporated the chimeric envelope protein as well as the unmodified envelope into the viral particles, and specifically bound to and infected human CEA-producing cancer cells via recognition of CEA, depending on the CEA-producing phenotype of the target cells. These results may have significant implications for the use of scFv directed against tumor-specific antigens for targeting specific antigen-producing cancer cells, a potential step toward in vivo cancer therapy.
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PMID:Targeting strategy for gene delivery to carcinoembryonic antigen-producing cancer cells by retrovirus displaying a single-chain variable fragment antibody. 947 83

The African swine fever virus (ASFV) strain Malawi LIL20/1 open reading frame (ORF) j13L was expressed in vaccinia virus (VV) from a strong synthetic late promoter as either a complete ORF (vSJ1) or lacking codons 1-31 (vSJ2). Each recombinant VV produced a small plaque which rapidly reverted to a normal size upon passage. The yield of infectious virus from a single cycle infection with vSJ1 or vSJ2 was reduced 50- to 100-fold compared to wild-type (wt) and a revertant virus (vSJ5) in which the j13L ORF was removed and the VV thymidine kinase gene restored. PCR analysis of nine spontaneous large plaque revertant viruses, recovered after passage of vSJ1 in BSC-40 cells, showed that six had lost the j13L ORF and the co-inserted beta-galactosidase gene. Three viruses retained the j13L and beta-galactosidase genes, but in each case the j13L protein was not expressed due to a different single base deletion near the 5' end of the j13L coding region which introduced a stop codon a short distance downstream. The formation of intracellular mature virus (IMV) and extracellular enveloped virus was reduced 50- to 75-fold in cells infected with vSJ1 compared to wt VV and revertant vSJ5. Electron microscopy showed aberrant IMV precursor structures in vSJ1-infected cells, and immunoelectron microscopy demonstrated that these structures contained j13L protein. These results indicate that expression of the j13L protein is toxic for VV replication due to interference with VV morphogenesis prior to IMV formation.
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PMID:Expression of African swine fever virus envelope protein j13L inhibits vaccinia virus morphogenesis. 960 32

The efficacy of adenovirus-mediated gene therapy for treatment of metastatic B16 melanomas, established in syngeneic C57BL/6 mice, was assessed via an ex vivo cytokine vaccine approach or via an in vivo strategy utilizing combination cytokine/herpes simplex virus-thymidine kinase (HSV-tk) suicide gene delivery and treatment with ganciclovir (GCV). In the ex vivo tumor vaccine approach, B16 melanoma cells, transduced in vitro by adenovirus containing either interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha cytokine genes and gamma irradiated, were subcutaneously injected into the flank and a distant subcutaneous challenge injection of unmodified B16 melanoma cells was performed 15 d later. Significant reductions in challenge tumor volume were observed in the IL-2 group (75% reduction; p = 0.02) and in the GM-CSF group (88% reduction; p = 0.0006), whereas the effect for tumor necrosis factor-alpha was not statistically significant. In the in vivo treatment of established melanomas, this cytokine approach was combined with a suicide gene therapy and subcutaneous B16 melanomas were directly injected with (i) IL-2/recombinant, replication-deficient adenovirus (adv) and thymidine kinase (tk)/adv, (ii) GM-CSF/adv, IL-2/adv, and tk/adv, or (iii) control beta-galactosidase (beta-gal)/adv and tk/adv. After intraperitoneal application of GCV (10 mg per kg) for 6 d, the residual tumor masses were excised and the animals challenged with unmodified B16 cells. Challenge tumor growth was reduced by 56% for the IL-2/tk/adv/GCV treatment (p = 0.041) and by 77% for the GM-CSF/IL-2/tk/adv/GCV treatment p (p = 0.037), in comparison with the beta-gal/tk/GCV control group. These data may hold significant promise for the development of effective ex vivo and in vivo gene therapy modalities to counter the highly metastatic nature of human melanoma.
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PMID:Ex vivo and in vivo adenovirus-mediated gene therapy strategies induce a systemic anti-tumor immune defence in the B16 melanoma model. 962 Feb 91


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