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)

Explanation into culture of dorsal root ganglia (DRG) latently infected with herpes simplex virus type 1 (HSV-1) causes reactivation of the virus. Previous studies have suggested that either latency-associated transcripts (LATs) were removed as an early consequence of reactivation or, alternatively, there was a population of latently infected cells which did not contain LATs. We have now attempted to detect this population of neurons by inserting a reporter gene (Escherichia coli lacZ gene), under the control of promoters other than LAT, into the HSV-1 strain 17 mutant in 1814, which was used in the earlier studies. One of these promoters, the human cytomegalovirus enhancer, resulted in weak expression of beta-galactosidase in DRG neurons for at least 5 months. The pattern of staining was predominantly homogeneous in neurons at 3 or 5 days post-infection or at 3 days post-explanation, and was predominantly speckled in latently infected neurons (1 to 5 months post-infection). About 30% of the beta-galactosidase-positive neurons did not contain LATs by in situ hybridization. However, the detergents used to enable penetration of the substrate for beta-galactosidase had also reduced the levels of the LATs; in neurons which originally had only small numbers of LATs this may have reduced levels to below those detectable by the methods used. There was, therefore, no unequivocal evidence for a population of latently HSV-1-infected cells which did not express LATs.
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PMID:Expression of beta-galactosidase in neurons of dorsal root ganglia which are latently infected with herpes simplex virus type 1. 778 83

Viral vectors are a means by which genes can be delivered to specific sites in the adult central nervous system. Nevertheless, the interaction between the viral vector and cells of the nervous system, which forms the basis for specific gene transfer, is not well understood. In this study a nonreplicating defective herpes simplex virus type 1 vector, expressing the marker gene lacZ, was stereotaxically injected at varying titers into the rat central nervous system. Three sites were targeted: the caudate nucleus, dentate gyrus, and cerebellar cortex, and the resulting patterns of beta-galactosidase activity were examined. Many cells of neuronal and glial morphology, and of differing neuronal subtypes, expressed beta-galactosidase at each of the injection sites. However, beta-galactosidase activity was also detected in distant secondary brain areas, the neurons of which make afferent connections with the primary sites. This strongly suggested that the retrograde transport of defective virus was the basis for the enzyme activity observed at a distance. Moreover, retrograde transport to secondary sites was found to be highly selective and restricted to certain retrograde neuroanatomical pathways in a specific and titer dependent fashion. The pathways observed were predominantly, but not exclusively, monoaminergic in origin. This finding is supported by reports of specific tropism by HSV for monoaminergic circuits in experimental encephalitis and transneuronal tracing studies. Our observations suggest that certain functional neuronal populations, which are permissive for the retrograde transfer of defective HSV-1 vectors, might be specifically targeted for gene transfer using this approach. Conversely, a knowledge of the pathways permissive for viral uptake, retrograde transfer, and subsequent gene expression will be essential in order to predict the consequences of gene transfer using viral vectors.
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PMID:Specific patterns of defective HSV-1 gene transfer in the adult central nervous system: implications for gene targeting. 782 88

The in vivo function of the herpes simplex virus type 1 immediate early gene ICP22 has been investigated in mice and guinea pigs using a deletion mutant (del22Z) of HSV-1(F) that lacks all but 18 nucleotides of the ICP22 coding sequence. This mutant carries the bacterial lacZ gene at the site of the deletion and makes functional beta-galactosidase, but is unable to synthesize any detectable ICP22 messenger RNA or protein in vitro. Del22Z was impaired in its ability to cause death in mice following intracerebral, intraperitoneal, or intravaginal inoculation. The mutant failed to produce lesions or other visible signs of infection after bilateral corneal infection of mice but could be recovered from trigeminal ganglia explanted at day 30 after inoculation. Del22Z replicated poorly after intravaginal inoculation of mice and guinea pigs in comparison to the parental virus, and was not recoverable from the dorsal root ganglia of either species. Nevertheless, del22Z sequences could be detected in the dorsal root ganglia of guinea pigs at day 30 by the polymerase chain reaction. These studies demonstrate that the ICP22 gene product is required for acute infection and virulence in two standard in vivo animal models.
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PMID:A herpes simplex virus type 1 ICP22 deletion mutant is altered for virulence and latency in vivo. 782 4

To assess the potential of an in vivo, adenovirus-mediated gene therapy approach for the treatment of malignant melanoma, the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-Ek) transfer and administration of ganciclovir (GCV) was investigated using a nude mouse model. Initially, B16 murine melanoma cells were efficiently transduced in vitro by a recombinant replication-defective adenovirus containing the HSV-tk gene (ADV/RSVtk), and rendered sensitive to cell killing by 10 micrograms/ml GCV. A significant "bystander effect" was observed at low multiplicity of infection in comparison of cell killing to control B16 transduction by adenovirus containing the beta-galactosidase gene (ADV/RSV-beta-gal). In vivo, melanomas established from subcutaneous injection of 4 x 10(5) B16 cells were injected after 14 d with 1 x 10(10) ADV/RSV-tk viral particles. Subsequent treatment for 6 d with GCV resulted in an inhibition of melanoma growth, with an approximately 40-50% reduction in melanoma volume in comparison to controls in repeated experiments. These data demonstrate that adenovirus-mediated gene transfer can function as an efficient delivery system to reduce established tumor burden in vivo. This result may hold significant promise for the development of effective in situ gene therapy for melanoma in humans.
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PMID:Inhibition of melanoma growth by adenoviral-mediated HSV thymidine kinase gene transfer in vivo. 786 Sep 93

The efficacy of adenovirus (ADV)-mediated gene therapy to treat brain tumors was tested in a syngeneic glioma model. Tumor cells were transduced in situ with a replication-defective ADV carrying the herpes simplex virus thymidine kinase (HSV-tk) gene controlled by the Rous sarcoma virus promoter. Expression of the HSV-tk gene enables the transduced cell to convert the drug ganciclovir to a form that is cytotoxic to dividing cells. Tumors were generated in Fischer 344 rats by stereotaxic implantation of 9L gliosarcoma cells into the caudate nucleus. Eight days later, the tumors were injected either with the ADV carrying the HSV-tk (ADV-tk) gene or a control ADV vector containing the beta-galactosidase (ADV-beta gal) gene and the rats were treated with either ganciclovir or saline. Tumor size was measured 20 days after implantation of 9L cells or at death. Rats treated with ADV-beta gal and ganciclovir or with ADV-tk and saline had large tumors. No tumors were detected in animals treated with ADV-tk and with ganciclovir at doses > or = 80 mg/kg. An infiltrate of macrophages and lymphocytes at the injection site in animals treated with ADV-tk and ganciclovir indicated an active local immune reaction. In survival studies, all animals treated with ADV-tk and ganciclovir have remained alive longer than 80 and up to 120 days after tumor induction whereas all untreated animals died by 22 days. These results demonstrate that ADV-mediated transfer of HSV-tk to glioma cells in vivo confers sensitivity to ganciclovir, and represents a potential method of treatment of brain tumors.
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PMID:Adenovirus-mediated gene therapy of experimental gliomas. 788 26

During herpes simplex virus latency, transcripts accumulate from a single transcription unit of the viral genome. The promoter for these latency-associated transcripts (LAT) has been located, and a number of studies have documented the specific regions of this promoter which are important in transient assays of neuronal cells in culture. To examine the regulation of this promoter from the viral genome, both in vitro and in vivo, a series of seven promoter deletion viruses which drive the expression of the reporter gene beta-galactosidase was constructed. Rabbit skin cells were infected in cell culture with viruses bearing each promoter mutation, and the LAT promoter activity was compared with that obtained by infecting two neuronal cell lines, ND7 cells and C1300 neuroblastoma cells. Mouse dorsal root ganglia were also infected with these recombinant viruses by footpad inoculations, and beta-galactosidase activity was measured. Infected neuronal cells lines and dorsal root ganglia exhibit much more LAT promoter activity than infected rabbit skin cells, suggesting that the region upstream of -250 may contain one or several neuronal specific DNA-binding sites. However, a comparison of LAT promoter activities within the deletion series revealed many differences between neurons of the dorsal root ganglia infected in vivo and the two neuronal cell lines infected in vitro. These results suggest that neurons may vary extensively in the quantity or kind of transcription factors they contain.
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PMID:In vivo deletion analysis of the herpes simplex virus type 1 latency-associated transcript promoter. 788 73

Vectors derived from herpes simplex virus type 1 (HSV-1) have allowed foreign genes to be expressed in differentiated postmitotic neurons, but the usefulness of these infected neurons for electrophysiological studies has not been examined. Using a latent, non-pathogenic recombinant HSV-1 virus, we expressed E. coli beta-galactosidase in identified rat cholinergic and dopaminergic neurons in culture. The electrophysiological properties of the infected cholinergic neurons were examined. The neurons remained electrically excitable and responsive to neurotransmitter.
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PMID:Cultured neurons infected with an HSV-1-derived vector remain electrically excitable and responsive to neurotransmitter. 791 15

The ability to direct foreign gene expression from the herpes simplex virus type 1 (HSV-1) genome during an acute or latent infection is a subject of increasing importance in the utilization of HSV vectors for gene therapy. Little is known about the types of transcription factors present in neurons or about whether different neuronal populations within a ganglion vary in their complement of these factors. With respect to HSV-1 latency, it is not known how or why the latency-associated transcript (LAT) promoter is able to function continually during latency while all other viral promoters are inactive. To further studies of these two phenomena, we constructed seven recombinant viruses with various promoter constructs driving expression of the lacZ reporter gene. Each construct was inserted into HSV-1 at the glycoprotein C locus, and recombinant viruses were evaluated for the ability to express beta-galactosidase during acute and latent viral infections in murine dorsal root ganglia. During acute infection of murine dorsal root ganglia, the activities of the promoters varied over a wide range. Constructs containing the murine metallothionein promoter (MT1), the phosphoglycerate kinase promoter, the Moloney murine leukemia virus long terminal repeat (LTR), or the region upstream of and including the HSV LAT core promoter (LAT) were active during the acute but not the latent phase of infection. The addition of transcription factor binding sites present in the upstream LAT region to the MT1 and LTR promoters (LAT-MT1 and LAT-LTR, respectively) significantly increased acute-phase expression. Despite these high initial rates of transcription, of all the promoter constructs only LAT-LTR was able to remain transcriptionally active after the establishment of a latent state. Thus, the Moloney murine leukemia virus LTR provides a DNA element which functions to prevent promoter inactivation during latency. An analogous HSV long-term-expression element is evidently not present in the upstream LAT promoter, indicating that the HSV long-term-expression function is provided by a region outside of that which gives high-level neuronal expression during the acute phase of infection.
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PMID:Long-term promoter activity during herpes simplex virus latency. 793 97

We constructed three recombinant vectors derived from the herpes simplex virus type 1 mutant tsK, each of which contained a different transgene under the control of the herpes simplex virus type 1 immediate early 3 promoter inserted into the thymidine kinase locus: the prokaryotic enzymes beta-galactosidase and chloramphenicol acetyl transferase, and a fusion gene consisting of human tissue inhibitor of metalloproteinases linked to the last exon of Thy-1, which encodes for a glycosyl-phosphatidyl-inositol membrane anchor. Infection of postmitotic neocortical and hippocampal neurons in low-density primary cultures with these vectors, achieved reliable expression of all three foreign gene products in various neocortical cell types, e.g. pyramidal neurons, non-pyramidal neurons, and glial cells. The percentage of neurons expressing transgenes ranged from 1 to 46% depending on the multiplicity of infection (highest assayed = 5); the percentage of glial cells expressing transgenes ranged from 0.5 to 98% (highest multiplicity assayed = 3.4). Expression of transgenes could be detected for up to three days in approximately 20% of neurons infected at a multiplicity of infection of 1. Infection of neurons with tk K-derived recombinant vectors inhibited their protein synthesis by 40-50% at a multiplicity of infection of 10, but no effect was observed at a multiplicity of infection of 1. Infection of glial cells with the same vectors at a multiplicity of infection of 1 inhibited protein synthesis by more than 90%. Analysis of neuronal viability at different times post-infection indicated that more than 98% of neurons expressing transgenes 48 h post-infection were viable. Thus, low-density neuronal cultures can be used to assess the efficiency of herpes simplex virus type 1-derived gene transfer vectors and transgene expression in developing cortical postmitotic cells, before and after they establish polarity. In addition, we show that two cytoplasmic enzymes, beta-galactosidase and chloramphenicol acetyl transferase, are able to diffuse freely in the cytoplasm reaching even growth cones in young neurons, while the chimeric protein tissue inhibitor of metalloproteinases/Thy-1 is correctly targeted to the plasma membrane via a glycosyl-phosphatidylinositol anchor. This model system should be useful for investigation of cellular and molecular aspects of the development and establishment of neuronal polarity, as well as for analysis of signals involved in protein targeting in postmitotic neurons.
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PMID:Use of recombinant vectors derived from herpes simplex virus 1 mutant tsK for short-term expression of transgenes encoding cytoplasmic and membrane anchored proteins in postmitotic polarized cortical neurons and glial cells in vitro. 793 6

Promoters whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in vivo. We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice. In this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1; see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence. Transgenic mice were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer. Whereas little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA in double-transgenic mice induced expression of the reporter genes up to several thousand-fold. This induction was abrogated to basal levels upon administration of tetracycline. These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control of transgene expression is required.
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PMID:Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. 793 60


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