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)

Recombinant retroviruses encoding the histochemically detectable enzyme beta-galactosidase have been used to investigate lineage in the vertebrate nervous system. Identification of the descendants of individual progenitors is straightforward when progeny cells are arranged in a reproducible, clustered pattern, but difficulties in interpretation arise when progeny migrate extensively and/or in an irregular pattern. To better resolve clonal boundaries, additional histochemical marker viruses that engender distinctive reaction products can be used in combination with lacZ-bearing viruses. To this end, we have created a retrovirus vector, DAP, encoding an easily assayable enzyme, human placental alkaline phosphatase. DAP was found to be at least as useful as a lacZ-encoding retrovirus (e.g., BAG) with respect to high viral titer, stability of expression, and in identification of infected cells in vivo. Moreover, it was found to be neutral with respect to postnatal rodent retinal development and offered superior staining characteristics relative to lacZ. Coinfection of rodent retina with DAP and BAG allowed an examination of the clonal nature of radial arrays of labeled retinal cells that previously had been described as products of a single infected progenitor. Of 1100 radial arrays examined for the presence of both DAP- and BAG-infected cells, only 1.2% were the result of infection with more than one virus.
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PMID:A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. 173 42

Development of the chicken retina was investigated through clonal analysis using retroviral vectors. Replication-incompetent retroviral vectors encoding either human placental alkaline phosphatase, beta-galactosidase, or a viral core protein were used in paired combinations to infect retinal progenitor cells from Embryonic Days 2.5-7.0. Labeled clones were analyzed late in embryonic development after retinal histogenesis was complete. The early accessibility of the chicken retina, combined with its large final size, resulted in the labeling of much larger clones than had been reported previously in other species. The clones were composed of many cell types, supporting previous conclusions from other vertebrates that the progenitor cells of developing retina are multipotent. The majority of clones derived from early infections consisted of multiple tightly clustered arrays of cells accompanied by dispersed individual cells. Clonal complexity and tangential dispersion were greater in peripheral than central retina and decreased considerably with increasing age of infection. These observations suggest that early during retinal development, shortly after optic cup formation, there is considerable mixing of progenitor cells.
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PMID:Clonal analysis in the chicken retina reveals tangential dispersion of clonally related cells. 781 85

Factors that influence early events of primary tumor development have been cumbersome to evaluate because of the need to either wait for tumor palpability after experimental manipulation or use of radiolabel to evaluate cell clearance. To facilitate these and similar analyses of cells in vivo, new methods are described that utilize histochemical marker genes to quantitate tumor cell number in a target tissue through the use of luminescent, enzymatic assays for these gene products. 3T3 Cells transfected with either human placental alkaline phosphatase or bacterial lacZ genes were injected subcutaneously into athymic nude mice. Using luminescent substrates designed for marker gene enzymes, extracts from homogenized tumor cell-bearing skins were assayed for the corresponding marker enzyme activities, which were optimized for recovery from skin extracts and correlated to cell number. The homogenization buffer used for these assays was designed to accommodate the optimal and simultaneous recovery of cytosolic beta-galactosidase and membrane-linked alkaline phosphatase from the skin, as well as from cultured cells. These assays provide an inexpensive, sensitive method for quantitatively monitoring the fate of cells genetically tagged with marker genes in various in vivo environments.
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PMID:Quantitation of two histochemical markers in the same extract using chemiluminescent substrates. 781 4

Sequences 5' of the human blue visual pigment gene have been assayed in transgenic mice for their ability to direct cell-type-specific expression of linked beta-galactosidase (lacZ) and placental alkaline phosphatase (P ALP1) reporters. Constructs containing either 5.4 kilobases (kb) or 0.47 kb of 5' flanking DNA direct expression exclusively to the retina. Within the retina, transgene expression is confined to blue cones and cone bipolar cells, as determined, respectively, by double labeling with anti-cone pigment antibodies and by morphologic analyses. These results imply that blue cones and cone bipolar cells have partially overlapping transcriptional specificities.
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PMID:Blue cones and cone bipolar cells share transcriptional specificity as determined by expression of human blue visual pigment-derived transgenes. 820 64

Two histochemical marker genes, Drosophila alcohol dehydrogenase (ADH) and human placental alkaline phosphatase (ALP), were cloned into the recombinant retroviral vectors pLJ and pgag beta-actin. The resulting vectors were transfected into retroviral producer cell lines, psi CRE and psi CRIP, and stable recombinant retrovirus producers were isolated. Recombinant virus was harvested and used to transduce genes into several cell lines, singly or in conjunction with lacZ (Escherichia coli beta-galactosidase)-containing retrovirus. Cell lines were then stained using standard histochemical methods for recombinant gene expression. We found that multiple gene products could be identified in the same cell populations and in the case of ALP and beta-galactosidase, in the same cells. The resulting reagents should be useful for a variety of cell-marking studies including those involving multiple clonal analysis and developmental studies for gene therapy.
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PMID:Recombinant retroviruses containing novel reporter genes. 851 8

Defective herpes simplex virus (HSV) vectors are an efficient means to deliver genes to cells of the central nervous system (CNS). Such vectors containing two independent transcription units would be extremely valuable for many studies, including the comparative analysis of promoter function and expression of multiple gene products in the CNS. We have constructed a 'double-cassette' vector expressing two easily detectable marker enzymes, beta-galactosidase (beta-gal) and human placental alkaline phosphatase (AP). Cells infected in vitro, including neurons and glia, and in vivo expressed both gene products.
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PMID:Co-expression of two gene products in the CNS using double-cassette defective herpes simplex virus vectors. 873 67

A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for beta-galactosidase, beta-glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for beta-galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g., the detection limit for beta-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of beta-galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.
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PMID:Chemiluminescence: sensitive detection technology for reporter gene assays. 878 27

Gene therapy of the lung requires the introduction and expression of a therapeutic gene in airway cells. Although retroviral vectors may be useful in this context, the ability of retroviruses to infect specific cell types in the airway is not known. In this study, we examined the ability of amphotropic recombinant retroviral vectors to transduce primary cultures of rabbit airway epithelial cell populations purified for basal or secretory cells. Transduction efficiencies in basal and secretory cell populations were found to be similar; about 27% after a single exposure to vector, and up to 77% after multiple exposures. The fate of genetically modified cells from the different populations was followed through terminal differentiation using organotypic cultures. The epithelium of the organotypic cultures generated from each population exhibited both pseudostratified and stratified morphology, produced mucin, and stained positively with antibodies specific for basal and ciliated cells. The mucociliary epithelium also showed co-localization of these phenotypic markers with the expression of the vector-encoded beta-galactosidase gene. We conclude that retroviruses can efficiently transduce primary cultures of basal and secretory cells, and that both of these cell types can be progenitor cells of the airway epithelium. In vivo delivery of a retroviral vector containing a human placental alkaline phosphatase gene resulted in expression of the heterologous gene in rabbit tracheal epithelial cells. However, transduction efficiency was low and occurred only in the wounded trachea.
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PMID:Retroviral vectors efficiently transduce basal and secretory airway epithelial cells in vitro resulting in persistent gene expression in organotypic culture. 889 79

It is reported that ethanol enhances DNA synthesis in E. coli cells [Basu, T and Poddar, R. K. (1994), Folia. Microbiol. 39, 3-6]. This communication reports that during growth of E. coli in the presence of 5% v/v ethanol, the derepressed expression of the cytoplasmic enzymes beta-galactosidase and D-serine deaminase per cell increased approximately three fold, while that of the periplasmic enzyme alkaline phosphatase decreased approximately 40% compared to control cell levels. However, in cells transformed with the plasmid pSM 456, bearing phoA-lacZ fusion, the level of induced synthesis of the hybrid protein PhoA-LacZ, controlled by the phoA promoter, was elevated by 25% in the presence of 5% v/v ethanol. This result suggests that the induction of the alkaline phosphatase precursor has also been enhanced by the ethanol treatment, but the inhibition in the export of the precursor across the cytoplasmic membrane, by the influence of ethanol, may represent the reason for the deficient expression of active alkaline phosphatase. It is proposed that there is an ethanol-mediated increase in DNA synthesis, resulting in gene amplification, which may enhance the synthesis of inducible proteins in ethanol-treated cells.
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PMID:Over expression of inducible proteins in Escherichia coli by treatment with ethanol. 916 3

The ability of recombinant adeno-associated virus (AAV) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. In this study, we assessed the ability of several AAV vectors to transduce airway cells in rabbits by measuring marker gene expression. AAV vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (AP) gene were delivered to one lobe of the rabbit lung by use of a balloon catheter placed under fluoroscopic guidance. We observed vector-encoded beta-gal or AP staining almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement. The overall efficiency of transduction in the balloon-treated bronchial epithelium was low but reached 20% in some areas. The majority of the staining was in ciliated cells but was also observed in basal cells and airway smooth muscle cells. We observed an 80-fold decrease in marker-positive epithelial cells during the 60-day period after vector infusion, whereas the number of marker-positive smooth muscle cells stayed constant. Although treatment with the topoisomerase inhibitor etoposide dramatically enhanced AAV transduction in primary airway epithelial cells in culture, treatment of rabbits did not improve transduction rates in the airway. Vector readministration failed to produce additional transduction events, which correlated with the appearance of neutralizing antibodies. These results indicate that both readministration and immune modulation will be required in the use of AAV vectors for gene therapy to the airway epithelium.
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PMID:Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration. 922 83


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