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)

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.
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PMID:Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. 130 4

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.
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PMID:Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene. 210 44

Although a T-cell response in human cytomegalovirus (HCMV)-immune individuals exists against the most abundantly expressed protein pp65 of the virus matrix, less is known about the determinants that evoke this response. The aim of the study was to identify regions within HCMV pp65 (ppUL83) that contain sequences for the cellular immune response by the use of three recombinant overlapping beta-galactosidase pp65 fusion proteins (C74, C35, and C47), covering the C-terminal 265 amino acids of the entire pp65 sequence. Two T-cell epitope determinants were recognized by human lymphocytes of healthy, HCMV-seropositive, human leukocyte antigen (HLA)-typed individuals. One T-cell determinant (amino acids [aa] 303-388) was localized in the mid-region of the entire pp65 sequence and a second T-cell determinant (aa 477-561) within the C-terminal region. By fine mapping with synthetic hexadecamer peptides three T-cell epitopes were identified within these two regions: P10-I (aa 361-376) in the mid-region, P3-II (aa 485-499), and P6-II (aa 509-524) in the C-terminal region. Inhibition studies with monoclonal antibodies to HLA class I or class II revealed a class II restricted response to peptides P10-I or P6-II, respectively. P10-I responders shared the HLA-DR11 allele and P6-II responders the -DR3 allele. Therefore, these T-cell epitopes of HCMV pp65 might be presented in association with particular HLA class II alleles.
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PMID:Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes. 913 60

Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulin-containing conjugates to which variants of the simian-virus-SV40 large-tumor antigen (T-ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e6 to the nucleus. NLSs were included either as peptides coupled co-valently to the carrier bovine serum albumin, or within the coding sequence of beta-galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS-containing T-ag beta-galactosidase fusion protein (P10)-(chlorin e6)-insulin, exhibiting an EC50 more than 2400-fold lower than the value for free chlorin e6, and more than 15-fold lower than that of an NLS-deficient beta-galactosidase-(chlorin e6)-insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e6. Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal-membrane-disrupting activity. In the case of the NLS-containing P10-conjugate, co-incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5-fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell-type-specific PDT.
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PMID:Adenoviruses synergize with nuclear localization signals to enhance nuclear delivery and photodynamic action of internalizable conjugates containing chlorin e6. 1032 26

Naturally occurring mutations of the beta subunit of the cyclic guanosine monophosphate (cGMP) phosphodiesterase (beta-PDE) gene in rod photoreceptors of mice and dogs are similar to one of the inherited retinal degenerations termed retinitis pigmentosa in humans. Defects in the rod beta-PDE gene leading to photoreceptor cell degeneration in retinal degenerative (rd) mice can be corrected by transfer of a wild type beta-PDE gene. However, the rapid photoreceptor degeneration in this mutant makes the study of gene therapy difficult. Since the retinal degeneration is slowed in vitro, we have employed retinal explants from rd mice to study factors influencing viral transduction. Retinal explants provide a rapid, efficient method to compare the transduction efficiency of adenoviral vector-mediated reporter gene delivery at different ages in normal and rd mice. Retinal explants from postnatal day (P)2 to P28 control (C57BL/6J) and P2-P42 rd mice were exposed for 20 hr to 2.5 x 10(8) plaque forming units (pfu) ml(-1) of adenoviral vector with a beta-galactosidase (Lac Z) reporter gene (Ad-CMV-Lac Z). After incubation in vector-free media for an additional 3 days, the explants were fixed and histochemically stained for beta-galactosidase to reveal Lac Z gene expression. The explants were also embedded and sectioned for light microscopic observation. Transduction efficiency was higher in rd mice than in controls on all postnatal days examined. In normal retinal explants, expression of the Lac Z gene increased from P2 to a peak around P7-P8, then decreased at subsequent ages; little transduction could be found after P17. In rd mice transduction efficiency of Ad-CMV-Lac Z increased from P2 to P7, decreased by P10 and increased again after P10. The most dramatic increase in the transduction efficiency occurred in the rd retina between P10 and P15 when Lac Z was intensely expressed throughout the retina. Microscopic examination of retinal sections revealed the types and distribution of Lac Z-positive cells responsible for the deep blue staining in the retinal whole mount. In normal and rd mice, Lac Z-positive cells were located throughout the retina. However, larger numbers of Lac Z-positive cells were present at all ages examined in retinal explants from rd mice compared to normal mice. These data indicate a difference in transduction efficiency between normal and rd mice, especially after P12, and suggest efficient adenovirus-mediated gene transfer is more attainable in developing or degenerating retina. Thus, transduction efficiency in rd mice depends on the relationship between development, maturation and the degenerative state of the photoreceptor cells.
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PMID:Adenoviral-mediated gene transfer to retinal explants during development and degeneration. 1532 66