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  • 1
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract More than 50 mutations in the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus have been described, yet only 2 alter the AUG initiation codon. One, variant HPRT1151, results in Lesch-Nyhan syndrome (LNS), and the other, HPRTIllinois, results in partial HPRT deficiency. Although previously undetectable, we used a sensitive gel assay to demonstrate that HPRTIllinois is not only active, but has a native Mr indistinguishable from normal. Confirmatory evidence of activity and native Mr is demonstrated following transfection of HPRT cells with expression plasmids containing cDNA sequences representing HPRTIllinois. These data provide support for the hypothesis that patient RT, or variant HPRTIllinois, is spared manifestations of the LNS as a result of translation at the newly formed GUG initiation codon.
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  • 2
    ISSN: 1573-904X
    Keywords: adenoviral vector ; Caco-2 ; gene transfer ; integrin ; intestinal epithelium ; vitronectin receptor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished. Methods. Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were decteted by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the β-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes. Results. The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors. Conclusions. These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficicient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.
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  • 3
    ISSN: 1573-904X
    Keywords: gene expression ; hPepTl ; Caco-2 cells ; adenovirus ; drug screening
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Our goals are to establish an in vitro screening system and to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligo-peptide transporter (hPepTl). This study characterizes the expression of hPepTl in human intestinal Caco-2 cells, rat intestinal epithelial cells (IEC-18), and human cervix epithelial cells (Hela) after adenoviral transduction. Methods. A recombinant replication-deficient adenovirus carrying the hPepTl gene was made and used as a vector for the expression of hPepTl. The increase in the uptake permeability of cephalexin and Gly-Sar was determined. The effects of time, dose, apical pH, and substrate specificity were evaluated. Results. A significant increase in the uptake permeability of Gly-Sar and cephalexin was found in all three cell lines after viral transduction. The increase of Gly-Sar permeability in Hela, IEC-18, and Caco-2 cells was 85-, 46-, and 15-fold respectively. Immunoblotting using an antibody against hPepTl detected high levels of a 85-98-kDa protein in all three infected cell lines. Substrate permeability was dependent on time of infection, inward pH gradients, and multiplicity of infection (MOI). Decreased infectivity and lower hPepTl expression were observed in differentiated Caco-2 cells. The uptake was inhibited by dipeptides and β-lactam antibiotics but not amino acids. Conclusions. Adenoviral infected Hela cells displayed a pronounced level of hPepTl expression with a low background and high specificity to dipeptides. These features make this system a useful tool for screening of potential substrates. The success of overexpression of hPepTl in Caco-2 and IEC-18 cells may lead to a novel approach in improving oral absorption of peptides and peptidomimetic drugs.
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  • 4
    ISSN: 1573-904X
    Keywords: adenovirus ; intestine ; gene therapy ; cyclodextrins ; jejunum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer. In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum. Methods. Transduction efficiency of replication-deficient adenovirus type 5 vectors encoded with either the E. coli beta-galactosidase or the jellyfish green fluorescent protein gene was assessed by X-gal staining or visualization of fluorescence 48 hours after infection. In vivo experiments were performed using an intestinal loop ligation technique. Results. Several formulations of neutral and positively charged beta cyclodextrins significantly enhanced adenoviral-mediated gene transfer in the selected models. The cyclodextrin formulations studied increased adenoviral transduction in the intestine by enhancing both viral binding and internalization. Viral binding was significantly increased on cell membranes treated with positively charged cyclodextrins, as seen with confocal microscopy and rhodamine-labeled virus. Permeability studies and TEER readings revealed that the most successful formulations gently disrupt cell membranes. This enhances internalization of viral particles and results in increased levels of gene expression. Conclusions. These formulations can be of value in gene transfer to cells and tissues in which adenoviral infection is limited due to a lack of fiber and αv integrin receptors. They are simple to prepare and do not affect the ability of the virus to transduce target cells.
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  • 5
    ISSN: 1546-1718
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Previous methods of in vivo gene transfer to differentiated neurons of the adult mammalian brain have been inefficient and associated with technical problems. We have therefore developed a model system of direct gene transfer using a replication–defective adenoviral vector containing a ...
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford [u.a.] : International Union of Crystallography (IUCr)
    Acta crystallographica 51 (1995), S. 1067-1069 
    ISSN: 1600-5759
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford [u.a.] : International Union of Crystallography (IUCr)
    Acta crystallographica 51 (1995), S. 2565-2567 
    ISSN: 1600-5759
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1520-5118
    Source: ACS Legacy Archives
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1540-8183
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We have investigated the safety and efficacy of sealing the femoral arterial puncture site using purified bovine collagen in the catheter laboratory immediately after coronary stent implantation. Ten consecutive patients, mean age 64.6 years, pretreated with heparin, aspirin, and dextran underwent the insertion of a collagen plug immediately after coronary stent implantation. The mean activated clotting time was 512 seconds (range 320–999). Femoral arterial hemostasis was achieved before leaving the catheterization laboratory in nine of ten patients. Seven of these patients received additional mild groin compression with an air cushion compression device after hemostasis was successful. One of ten patients had persistent bleeding after delivery of the collagen plug and later required blood transfusion. The collagen plug may be a useful adjunct to obtaining hemostasis following coronary stent implantation, but further trials are required to test its reliability in fully anticoagulated patients.
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  • 10
    ISSN: 1573-5044
    Keywords: Annona squamosa ; hypocotyl ; node ; silver thiosulphate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A micropropagation system for Annona squamosa L. (Sugar Apple) using hypocotyls of seedlings and nodal cuttings from 3-year-old plants was developed. Shoot proliferation was achieved with Woody Plant Medium supplemented with BA. Silver thiosulphate was added at 0.5 mg l−1 to control leaf abscission. Rooting was obtained when subcultured shoots were preconditioned for 2 weeks in medium with 10 g l−1 activated charcoal before treatment with 43 µm NAA or 39 µm IBA. Rooting was improved when galactose was used instead of sucrose in the rooting medium. The rooted plantlets were acclimatised successfully.
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