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)

Targeted expression of genetic material within the vascular endothelium is potentially a powerful tool for the investigation of endothelial cell (EC) biology. We developed, optimized, and characterized an efficient somatic transgenic model of EC-specific gene transfer. Rat carotid arteries were infused with adenovirus expressing a beta-galactosidase (beta-gal) gene. The level and cell-type specificity of recombinant gene expression were measured by assaying beta-gal activity in vessel extracts and by counting transduced cells in histological sections. Toxicity was evaluated by counting total ECs (3 days) and by measuring neointimal formation (14 days). Effects of transduction on the proliferation of vascular cells were measured with bromodeoxyuridine and [3H]thymidine. Maximum recombinant gene expression resulted from infusion of 1 x 10(10) to 1 x 10(11) plaque-forming units (pfu) per milliliter; approximately 35% of luminal ECs were transduced. A high degree of EC specificity (90% to 98% of total transduced cells) was maintained over this range of virus concentrations. More highly concentrated virus resulted in loss of beta-gal expression and a large decrease in luminal EC number (97% decrease, P < .001). Gene transfer at 4 x 10(10) pfu/mL was efficient, preserved EC integrity, and caused minimal neointimal formation. After gene transfer, there were early (3-day) increases in both EC and smooth muscle cell proliferation. At 14 days, only EC proliferation remained elevated (18% versus 1.4% in vehicle-infused arteries, P = .005). This animal model permits efficient highly EC-specific gene transfer. Vascular toxicity is minimal, although the EC proliferative index is elevated. This model will be useful in experiments that elucidate the biological role of EC gene products and define pathways of EC gene regulation and signal transduction in vivo.
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PMID:Endothelium-specific in vivo gene transfer. 764 20

Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues. Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells. Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses. Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers. Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery. Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days. As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models.
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PMID:Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats. 764 77

Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.
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PMID:Generation of thymidine kinase-deficient mutants of infectious laryngotracheitis virus. 777 65

Serial passage of nuclear polyhedrosis viruses (NPVs) through cultured cell lines results in the appearance of mutants with a complex phenotype referred to as the 'few polyhedra' (FP) phenotype. The altered plaque morphology and reduced occlusion production associated with the FP phenotype have been observed in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) bearing mutations in the gene encoding the 25 kDa protein (25K gene). In this study, we sequenced the 25K genes of four spontaneously occurring AcMNPV FP mutants. These mutants, together with an artificially generated FP mutant (AcFP beta gal, in which the gene for beta-galactosidase is fused in frame with the 25K ORF), were examined at the ultrastructural level to see if they exhibited the reduced virion occlusion and intranuclear envelopment which is associated with the FP phenotype. Observations on Spodoptera frugiperda Sf9 cells infected with the FP mutants revealed that all five mutants were impaired in virion occlusion and intranuclear nucleocapsid envelopment. The 25K mutants were also found to release two- to fivefold more infectious virus (p.f.u.) into the media of infected Sf9 cells. Marker rescue of AcFP beta gal restored wild-type virion occlusion, intranuclear envelopment and levels of infectious virus production.
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PMID:Mutations in the Autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production. 778 73

Trichoplusia ni larvae have been injected with a mixture of wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and a mutant derivative, AcRP8.UW1.lacZ, which lacks the polyhedrin gene, and has the p10 gene replaced by the Escherichia coli beta-galactosidase gene. Following plaque assay of the haemolymph and subsequent staining for beta-galactosidase activity and scoring for polyhedra, recombinant plaques were identified and the recombination frequency estimated as 6.6%.
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PMID:Recombination between genetically modified and unmodified Autographa californica nuclear polyhedrosis virus in Trichoplusia ni larvae. 779 54

A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of its ability to suppress mucoidy in Escherichia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by beta-galactosidase expression in lon- cps-lac fusion strains of E. coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rnc- E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E. coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid-borne C. burnetii era. Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35, 27 and 25 kDa. Antibodies against purified E. coli Era protein cross-reacted with the 35 kDa protein of C. burnetii on Western blots.
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PMID:Analysis of the rnc locus of Coxiella burnetii. 783 May 73

Three independently selected spontaneous thymidine kinase-negative mutants (TK-phenotype) and a recombinant with Escherichia coli beta-galactosidase gene (LacZ+ phenotype) inserted in the viral thymidine kinase gene (tk) were derived from a plaque-cloned isolate of K-1 ectromelia virus strain (TK+ phenotype). Dramatically decreased virulence of TK- variants was observed for all routes of mouse inoculation. The kinetics of TK+ and TK- variants in various target organs indicated a significant decrease of production and dissemination of TK- mutants and recombinant in the organs of mice. In the spleen and liver of intranasally or intracerebrally infected mice TK- virus was not detected during the entire period of observation. Analysis of organs homogenates of mice intranasally infected by a mixture of recombinant with TK-LacZ+ phenotype and parental isolate with TK+LacZ- phenotype on the monolayers of TK- cells indicated that only white plaques (LacZ-) with the TK+ phenotype appeared from liver and spleen homogenates. Thus, the mouse acts as a live filter much more efficiently than any other selective systems. Ultrastructural studies showed that viral damage in animals infected by TK- variants was far less than that observed in mice, infected with wild type of ectromelia virus and pathological lessions were slight and reversible. Replication of ectromelia virus TK- variants was blocked at the viroplasma stage in cells with a high level of differentiation in contrast to TK+ variants. Most likely, such restriction of target cells assortment is the general reason of reduced virulence in the case of tk-gene inactivation.
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PMID:Fine mechanisms of ectromelia virus thymidine kinase-negative mutants avirulence. 783 64

The dismal results of conventional therapy for primary malignant brain tumors has justified exploring gene therapy approaches for this disease. Transduction of animal brain tumor models in vivo has been reported previously with retroviruses and herpes viruses. Because adenoviruses have the advantage of transducing quiescent and actively dividing tumor cells, they may prove to be more effective in such therapy. We used a replication-deficient recombinant adenovirus bearing the Escherichia coli beta-galactosidase gene in a rat C6 glioma tumor model. Transduced cells were detected by X-5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to reveal beta-galactosidase activity. Initial experiments in vitro showed 50% and 90% transduction at vector titers of approximately 10(7) and 10(8) plaque-forming units/ml, respectively. Although no cytopathic effects were seen at 10(7) plaque-forming units/ml, more than 50% reduction in tumor cell growth was noted at 10(8) plaque-forming units/ml both in vitro and in vivo. Stereotactic delivery of the recombinant adenovirus into the frontal lobe of normal rat brains resulted in intense staining of all cell types, that is, neurons, astrocytes, and ependymal cells. Stereotactic injection into C6 glioma brain tumors in rats stained 25 to 30% of the tumor cells. We conclude that adenovirus vectors can be used to transfer genes to central nervous system tumors in vivo. Using stereotactic delivery, adenovirus vectors can transfer genes into the central nervous system intended for tumor therapy.
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PMID:Stereotactic delivery of a recombinant adenovirus into a C6 glioma cell line in a rat brain tumor model. 783 41

A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection. The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques remain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility. In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.
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PMID:Intralaboratory optimization and standardization of mutant screening conditions used for a lambda/lacI transgenic mouse mutagenesis assay (I). 787 99

Four insect cell lines were used to isolate two recombinant baculoviruses which had the beta-galactosidase (beta-gal) gene for colorimetric assay purposes. Plaque assays were performed using two Trichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and two Spodptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blue beta-gal+ recombinants) formed in the Trichoplusia cells was higher than in the Spodoptera cells. The appearance of Autographa californica NPV polyhedra was also faster in the T. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.
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PMID:Cell lines used for the selection of recombinant baculovirus. 806 52


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