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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian cells transfected with the bacterial neomycin gene. We found that COS-1 cells stably transfected with the neomycin resistance gene had a greater than 50% reduction in cell-associated glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP). A similarly reduced amount of AP was also observed in wild-type COS-1 cells incubated in the presence of G418 or other aminoglycoside antibiotics. The AP was released from cells into the culture supernatant in its GPI-anchored form. Our data suggest that the G418-induced reduction of AP involves a vesiculation process of COS-1 cells.
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PMID:Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins. 922 84

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.
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PMID:Helper-free foamy virus vectors. 985 18

The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a useful screen for putative bone differentiating factors.
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PMID:Establishing an immortalized human osteoprecursor cell line: OPC1. 1049 Dec 20

Human growth hormone (hGH) is frequently used clinically for growth abnormalities in children and also in adults with growth hormone deficiency. The hormone is usually administered to the individuals by frequent injections. In the present study we investigated the potential of bone marrow stromal cells as vehicles to deliver the GH in vivo by infusion of cells transduced with hGH cDNA into mice femurs. The effect of the hormone on the transduced cells in vitro was also assessed. Bone marrow stromal cells established from a mouse model of human osteogenesis imperfecta mice (oim) were transduced with a retrovirus containing hGH and neomycin resistance genes. The hGH-expressing cells were selected in a medium containing G418 and were then assessed for the hGH expression in vitro. The selected cells synthesized 15 ng/10(6) cells of hGH per 24 h in vitro and exhibited alkaline phosphatase activity when they were treated with the human recombinant bone morphogenetic protein 2 (rhBMP-2). The transduced cells also proliferated faster than the LacZ transduced cells but they did not exhibit a higher rate of matrix synthesis. When 2 x 10(6) hGH+ cells were injected into the femurs of mice, hGH was detected in the serum of the recipient mice up to 10 days after injection. The highest level of growth hormone expression, 750 pg/ml, was detected in the serum of the recipient mice I day after injection of the transduced cells. hGH was also detected in the medium conditioned by cells that were flushed from the femurs of the recipient mice at 1, 3, and 6 days after cell injection. These data indicate that bone marrow stromal cells could potentially be used therapeutically for the delivery of GH or any other therapeutic proteins targeted for bone. The data also suggest that GH may exert its effects on bone marrow stromal cells by increasing their rate of proliferation.
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PMID:In vivo expression of human growth hormone by genetically modified murine bone marrow stromal cells and its effect on the cells in vitro. 1097 31

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.
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PMID:Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells. 1114 68

The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9P35AcV5-1 and Sf9P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9P35AcV5-1 and Sf9P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.
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PMID:Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion. 1151 84

Bone morphogenetic proteins (BMP) play a pivotal role in growth and differentiation of osteoblastic lineage cells. BMPs are potent stimulators of bone formation in various animal models. To understand the mechanism of BMP action in bone cells, we have investigated the effects of overexpression of the BMP-2 gene on proliferation and differentiation of UMR-106 rat osteosarcoma cells. A stable UMR-106 cell line overexpressing the BMP-2 gene was established by transfection of cells using a mammalian expression vector harboring human BMP-2 cDNA followed by G418 selection. After introduction of the BMP-2 gene, UMR-106 cells appeared more spindle-shaped in morphology compared to the predominantly cuboidal appearance of the parental cells. Overexpression of BMP-2 markedly inhibited proliferation as measured by cell counting and [3H]thymidine incorporation assays. Extracellular matrix (ECM) derived from cells overexpressing BMP-2 exhibited a less supportive effect on proliferation of UMR cells than did ECM derived from parental cells. Furthermore, cell-cell communication through gap junctions was reduced more than 50% as determined by nondisruptive fluorescent dye transfer assays. Overexpression of BMP-2 significantly stimulated expression of osteocalcin and alkaline phosphatase genes, indicating its role in osteoblastic differentiation. There was little effect on osteopontin gene expression.
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PMID:Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells. 1190 Apr 83

We have constructed pSG5-RAR gamma-neo plasmid containing mouse retinoic acid receptor gamma (RAR gamma) gene and neo gene, and introduced it into embryonic stem ES-5 cells, by calcium phosphate mediated transfection. Some G418-resistant clones were isolated and from RNA dot blot analysis of these clones, a clone overexpressing RAR gamma gene was established, designated as ES-gamma cell line. Northern blot hydridization and Southern blot hydridization analysis of ES-gamma cells (Fig 3, 4) demonstrated that ES-gamma cells overexpressed exogenous RAR gamma mRNA and the exogenous RAR gamma cDNA integrated into the genome of ES cells. ES-gamma cells retained undifferentiated morphology and positive alkaline phosphatase activity (Plate I, Fig. 1, 2), so it resembled ES-5 cells in terms of stem cell characteristics. When ES-gamma cells were subcutaneously inoculated into nude mouse and differentiated in vivo, tumorous nodules containing various tissue structures were obtained, demonstrating their pluripotent properties just like parent ES-5 cells. Contrasting with ES-5 cells, the histological features of tumors showed no cartilage tissues, but abundant muscle tissues and keratinized cyst like structures constituted by stratified squamous epithelia (Plate I, Fig. 3). Differentiating in vitro by hanging drop culture methods, ES-gamma cells differentiated mostly into fibroblast-like cells, (Plate II, Fig. 1-5). The above results indicated that overexpression of RAR gamma gene changed the cell type of ES cells differentiating in vivo and in vitro. During the differentiation of ES-5 cells induced by RA, a large number of cells rounded up, detached from the dish and tended to die. We suspected that this phenomenon may be apoptosis. The ultrastructure appearance of the dying cells displayed typical apoptotic changes including chromatin condensation and nuclear fragmentation (Plate I, Fig. 4, 5). Detection of DNA fragments using agarose gel electrophoresis showed characteristic laddered patterns of apoptotic DNA fragments (Fig. 5). The above results indicated that RA induced apoptosis of ES-5 cells in the course of differentiation. The percentage of apoptosis of ES-5 cells increased accordingly, with the increase of RA concentration (Fig. 6). With the same concentration of RA 10(-7) mol/L, the percentage of apoptotic of ES-gamma death was roughly one times more than that of ES-5 cells (Fig. 7), a fact indicating that RAR gamma may mediate the apoptotic signal transduction of ES cells by RA.
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PMID:[Expression of retinoic acid receptor gamma gene in ES cells and its effect on their differentiation and apoptosis]. 1201 44

CD20 is a specific antigen expressed on normal and neoplastic B cells exclusively. Recent researches showed that in B cell leukemia, CD20 was over-expressed. Therefore monoclonal antibody (McAb) to CD20 may be of clinical value in diagnosis and treatment of some leukemias and lymphomas. In this study, the full length gene of CD20 cDNA were cloned from total RNA of Raji cells, inserted into an eukaryotic expression vector pcDNA3.1, forming a recombinant plasmid pcDNA3.1/CD20. NIH-3T3 cells were transfected with pcDNA3.1/CD20 and selected with G418 for the transfected cells. Alkaline phosphatase against alkaline phosphatase assay(APAAP) experiments showed that the selected cells could express the human CD20 onto its surface. Balb/c mice were immunized with CD20( ) NIH-3T3 cells once three weeks for 3 shuts. Indirect immunofluorescence experiments were done with the Raji cells and the serum of the immunized mice, and the results showed that the spleen of the immunized mice could be used to prepare the McAb to CD20.
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PMID:Cloning and Expression of Human CD20 Gene on NIH-3T3 Cell Membrane. 1207 39

The molecular mechanisms responsible for random integration and gene targeting by recombinant adeno-associated virus (AAV) vectors are largely unknown, and whether vectors derived from autonomous parvoviruses transduce cells by similar pathways has not been investigated. In this report, we constructed vectors based on the autonomous parvovirus minute virus of mice (MVM) that were designed to introduce a neomycin resistance expression cassette (neo) into the X-linked human hypoxanthine phosphoribosyl transferase (HPRT) locus. High-titer, replication-incompetent MVM vector stocks were generated with a two-plasmid transfection system that preserved the wild-type characteristic of packaging only one DNA strand. Vectors with inserts in the forward or reverse orientations packaged noncoding or coding strands, respectively. In human HT-1080 cells, MVM vector random integration frequencies (neo(+) colonies) were comparable to those obtained with AAV vectors, and no difference was observed for noncoding and coding strands. HPRT gene-targeting frequencies (HPRT mutant colonies) were lower with MVM vectors, and the noncoding strand frequency was threefold greater than that of the coding strand. Random integration and gene-targeting events were confirmed by Southern blot analysis of G418- and 6-thioguanine (6TG)-resistant clones. In separate experiments, correction of an alkaline phosphatase (AP) gene by gene targeting was nine times more effective with a coding strand vector. The data suggest that single-stranded parvoviral vector genomes are substrates for gene targeting and possibly for random integration as well.
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PMID:Chromosomal integration and homologous gene targeting by replication-incompetent vectors based on the autonomous parvovirus minute virus of mice. 1464 70


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