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)

Application of neurotrophic factors (NFs) to the cut stump of motor nerves of neonatal rats confers neuroprotection from trauma-induced neuronal death. To test whether motoneurons are capable of responding to endogenously produced NFs, facial motoneurons were genetically modified in vivo to express several NFs and then tested for their response to peripheral nerve damage. Replication-defective adenoviral vectors [Adv. Rous sarcoma virus (RSV)-nf] representing three families of NFs were constructed that carried genes for brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. Media from cultured cells transduced with Adv. RSV-nf contained NFs that supported the survival of cultured chick sensory neurons in the same manner as recombinant NF standards. When Adv.RSV-nf or an adenoviral vector containing the beta-galactosidase gene (Adv.RSV-beta-gal) were injected into the facial muscles of neonatal rats the vectors were retrogradely transported to the facial nucleus where the NFs or beta-gal were expressed. A fraction (approximately 10%) of the neurons were transduced as demonstrated by reverse transcriptase-PCR, histochemistry, and immunocytochemistry. In the case of Adv.RSV-BDNF, Adv.RSV-CNTF, and Adv.RSV-GDNF, a significant portion of the facial nucleus neurons was protected, 16.5, 18.2, and 53.3%, respectively, from death after axotomy, showing that neurons are capable of transporting the Adv. RSV-nf, expressing the recombinant NF genes, and responding to the NFs. In the case of Adv.RSV-GDNF, a greater number of facial nucleus motoneurons survived than were transduced, indicating that neighboring untransduced neurons were protected by the GDNF expressed by the transduced neurons by a paracrine mechanism.
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PMID:Targeted transduction of CNS neurons with adenoviral vectors carrying neurotrophic factor genes confers neuroprotection that exceeds the transduced population. 925 62

An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac+ strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays. Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.
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PMID:Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR. 936 4

A system to study the fidelity of internal strand transfer events was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. The homology occurred over a 119-base internal region of the donor which coded for the N-terminal portion of the alpha-lac gene. Polymerase chain reaction (PCR) was used to amplify DNA synthesis products. The PCR products were then digested with PvuII and EcoRI and ligated into a vector which had this same region excised. Transformed Escherichia coli were screened for the ability to produce a functional beta-galactosidase protein by blue-white phenotype analysis with white colonies scored as those with errors in alpha-lac. Products synthesized on the donor were used to assess the error rate of human immunodeficiency virus-RT while products transferring to and subsequently extended on the acceptor (transfer products) were used to monitor transfer fidelity. Human immunodeficiency virus-RT made approximately 1 error per 7500 bases copied in the assay. Nucleocapsid protein (NCp), although stimulating strand transfer 3-fold, had no effect on RT fidelity. Transfer products in the absence of NCp had essentially the same amount of errors as donor-directed products while those produced with NCp showed a slight increase in error frequency. Overall, strand transfer events on this template were highly accurate. Since experiments with other templates have suggested that transfer is error prone, the fidelity of strand transfer may be highly sequence dependent.
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PMID:High fidelity of internal strand transfer catalyzed by human immunodeficiency virus reverse transcriptase. 943 Jun 86

Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in unwanted events, such as the formation of recombinant viruses and the mobilization of therapeutic vectors. Using sensitive reverse transcriptase PCR assays, we investigated human and murine gene therapy packaging cell lines for incorporation of endogenous retrovirus transcripts into murine leukemia virus (MLV) vector particles and, conversely, whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were efficiently packaged in particles produced by the murine AM12 packaging system. For every seven MLV-derived beta-galactosidase (beta-Gal) vector genomes present in the particles, one copy of VL30 was also packaged. Although human FLY packaging cells expressed several classes of HERV transcripts (HERV-K, HuRT, type C, and RTVL-H), none was detectable in the MLV vector particles released from the cells. Nonspecific packaging of the MLV Gag-Pol expression vector transcripts was detected in the FLY virions at a low level (1 in 17,000 sequences). These findings indicate that human packaging cells produce retrovirus particles far less contaminated by endogenous viral sequences than murine packaging cells. Human teratocarcinoma cells (GH cells), which produce HERV-K particles, were transduced with an MLV-derived beta-Gal vector. Although both HERV-K and RTVL-H sequences were found in association with the particles, beta-Gal transcripts were not detected, indicating that HERV Gag proteins do not efficiently package MLV-based vectors.
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PMID:Packaging of endogenous retroviral sequences in retroviral vectors produced by murine and human packaging cells. 952 84

Tat is a virally expressed regulatory protein involved in the replication of HIV-1, the etiological agent of AIDS. To investigate the effect of tat inhibition on HIV replication, we constructed a retroviral vector to express an anti-tat hammerhead ribozyme as part of the 3' untranslated region of beta-galactosidase transcripts. Initial testing of this vector in tat-expressing COS-7 cells reduced tat activity by 85-95% as measured by tat-dependent CAT assays. Amphotropic and HIV-pseudotyped retroviral particles generated with this vector were used in HIV challenge experiments to determine the ability of this reagent to control HIV replication. CD4(+) peripheral blood lymphocytes (PBLs) stably transduced with this vector were subsequently challenged with HIV. These cells were able to resist HIV infection for up to 20 days as measured by cell death and reverse transcriptase activity. These data yield proof of principle that a pseudotyped retroviral vector can target and deliver a protective ribozyme to CD4(+) cells.
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PMID:Inhibition of HIV-1 replication by an anti-tat hammerhead ribozyme. 953 87

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
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PMID:Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. 958 6

Estrogens may influence the expression of various cytokines, adhesion molecules, von Willebrand factor and prostacyclin produced by endothelial cells. However, reports concerning expression of the estrogen receptor in endothelial cells are controversial. Primary human umbilical vein endothelial cells (HUV-EC), the non continuous human umbilical vein endothelial cell line HUV-EC-C (ATCC CRL 1730) and endothelial cells from 10 frozen umbilical cords were analyzed for the expression of the estrogen receptor. Immunological studies using estrogen receptor specific antibodies failed to detect the expression of the receptor in all human umbilical vein endothelial cells tested. No estrogen receptor transcripts were found in primary HUV-EC or HUV-EC-C by reverse transcriptase-polymerase chain reaction. Weak hybridization signals were detected when the PCR amplicons were hybridized with estrogen receptor cDNA sequences as a probe. In vitro protein-DNA interaction studies revealed no complexes between a fully consensus estrogen response element and HUV-EC-C extracts. Finally, transient transfection studies in HUV-EC-C could not demonstrate 17beta-estradiol-induced transcription of the beta-galactosidase reporter gene linked to a consensus estrogen response element. These observations suggest that human umbilical vein endothelial cells lack the estrogen receptor.
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PMID:Human umbilical vein endothelial cells lack expression of the estrogen receptor. 983 29

-The increased delivery of serotonin (5-hydroxytryptamine, 5-HT) to the lung aggravates the development of hypoxia-induced pulmonary hypertension in rats, possibly through stimulation of the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). In cultured rat PA-SMCs, 5-HT (10(-8) to 10(-6) mol/L) induced DNA synthesis and potentiated the mitogenic effect of platelet-derived growth factor-BB (10 ng/mL). This effect was dependent on the 5-HT transporter (5-HTT), since it was prevented by the 5-HTT inhibitors fluoxetine (10(-6) mol/L) and paroxetine (10(-7) mol/L), but it was unaltered by ketanserin (10(-6) mol/L), a 5-HT2A receptor antagonist. In PA-SMCs exposed to hypoxia, the levels of 5-HTT mRNA (measured by competitive reverse transcriptase-polymerase chain reaction) increased by 240% within 2 hours, followed by a 3-fold increase in the uptake of [3H]5-HT at 24 hours. Cotransfection of the cells with a construct of human 5-HTT promoter-luciferase gene reporter and of pCMV-beta-galactosidase gene allowed the demonstration that exposure of cells to hypoxia produced a 5.5-fold increase in luciferase activity, with no change in beta-galactosidase activity. The increased expression of 5-HTT in hypoxic cells was associated with a greater mitogenic response to 5-HT (10(-8) to 10(-6) mol/L) in the absence as well as in the presence of platelet-derived growth factor-BB. 5-HTT expression assessed by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization in the lungs was found to predominate in the media of pulmonary artery, in which a marked increase was noted in rats that had been exposed to hypoxia for 15 days. These data show that in vitro and in vivo exposure to hypoxia induces, via a transcriptional mechanism, 5-HTT expression in PA-SMCs, and that this effect contributes to the stimulatory action of 5-HT on PA-SMC proliferation. In vivo expression of 5-HTT by PA-SMC may play a key role in serotonin-mediated pulmonary vascular remodeling.
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PMID:Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. 1002 7

We used adenoviral-mediated gene transfer of human amyloid precursor proteins (h-APPs) to evaluate the role of various h-APPs in causing neuronal cell death. We were able to infect PC12 cells with very high efficiency because approximately 90% of the cells were cytochemically positive for beta-galactosidase activity when an adenoviral vector containing LacZ cDNA was used to infect cells. Cells infected with adenovirus containing h-APP cDNA showed high-level transcription and expression of h-APP as measured by reverse transcriptase-polymerase chain reaction and Western immunoblot analyses, respectively. Intracellular and extracellular levels of h-APP were elevated approximately 17-and 24-fold in cultures infected with recombinant adenovirus containing wild-type mutant and 13- and 17-fold with V642F mutant. No elevation in h-APP was seen in cultures infected with antisense h-APP or null adenovirus. H-APP levels were maximal 3 days after infection. Overexpression of V642F mutant h-APP in PC12 cells and hippocampal neurons resulted in about a twofold increase in death compared with overexpression of wild-type h-APP. These results demonstrate the usefulness of recombinant adenoviral mediated gene transfer in cell culture studies and suggest that overexpression of a familial Alzheimer's disease mutant APP may be toxic to neuronal cells.
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PMID:Death of PC12 cells and hippocampal neurons induced by adenoviral-mediated FAD human amyloid precursor protein gene expression. 1008 85

Newly developed antiviral compounds consisting of an adamantane derivative chemically linked to a water-soluble polyanionic matrix were shown to inhibit HIV-1 infection in lymphoblastoid cells, HeLa CD4+ beta-galactosidase (MAGI) cells and macrophages. The effect of the compounds was recorded by measuring viral reverse transcriptase activity and p24 by ELISA in culture supernatant and by immunoblotting of cell lysates. In this paper we describe the data obtained with one of the most promising compounds, Amant. Amant was not toxic for the host cells at concentrations as high as 1 mg/ml. The inhibition of HIV-1 replication in MT-4 and MAGI cells was observed when Amant was added either before infection or with the virus (0 h of infection), and was expressed even when the compound added at 0 h was removed 1.5 h after infection. Its inhibitory concentration (IC50) against HIV-1 and HIV-2 replication was 2-6 and 93 microg/ml, respectively. The anti-HIV-1 effect of the compound was gradually decreased when it was added 1 and 2 h post infection, and no inhibition was observed when the compound was added 4 h after infection, suggesting that the compound as a membranotropic drug blocks an early step of replication. It completely prevented the transport of Gag proteins into the nuclei. Pretreatment of the virus with Amant did not reduce its infectious activity. The classical adamantane derivatives amantadine and rimantadine hydrochloride did not inhibit HIV replication.
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PMID:Inhibition of HIV-1 replication by newly developed adamantane-containing polyanionic agents. 1032 46


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