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  • 1
    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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  • 2
    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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  • 3
    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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  • 4
    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
    Type of Medium: Electronic Resource
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  • 5
    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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  • 6
    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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  • 7
    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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  • 8
    ISSN: 1573-093X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract The ‘EPAC’ instrument on Ulysses is sensitive to relativistic cosmic rays when far from Jupiter and in the absence of energetic solar particles. Measurement of the latitude gradient of these particles, after correction for time variations, has been made for the 1993–1994 south polar pass. The average magnitude of the gradient is about the same or smaller than predicted by a model which includes full gradient drift. However, the latitude dependence of the solar plasma output into interplanetary space (including fast-stream and magnetic turbulence effects) seems to be important in determining the magnitude of modulation.
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  • 9
    ISSN: 1573-2703
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mathematics , Technology
    Notes: Abstract The paper discusses the fundamental singularity of Stokes flow (the stokeslet) in the context of applications to locomotion and feeding currents in micro-organisms. The image system for a Stokeslet in a rigid plane boundary may be derived from Lorentz's mirror image technique [1] or by an appropriate limit of Oseen's solution for a sphere near a plane boundary [2]. An alternative derivation using Fourier transform methods [3] leads to an immediate physical interpretation of the image system in terms of a stokeslet and its multipole derivatives. The schematic illustration of a stokeslet and its image system in a plane boundary are exploited to explain the fluid dynamical principles of ciliary propulsion. For a point force oriented normal to the plane boundary, the resulting axisymmetric motion leads to a Stokes stream function representation which illustrates the toroidal eddy structure of the flow field. A similar eddy structure is also obtained for the two-dimensional system, although in this case, the toroidal structure is replaced by two eddies. This closed streamline model is developed to model chaotic filtration through the concept of a ‘blinking stokeslet’, a stokeslet alternating its vertical position according to a specific protocol. The resulting behaviour is illustrated via Poincaré sections, particle dispersion and length of particle path tracings. Sessile micro-organisms may exploit a similar process so they can filter as large a volume of liquid as possible in search of food and nutrients.
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  • 10
    ISSN: 1573-2703
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mathematics , Technology
    Notes: Abstract The boundary integral method is applied to model the initial motion of two-dimensional or cylindrical deformable gas bubbles in an inviscid, incompressible fluid. Following the success of recent boundary integral studies to predict the qualitative behaviour of a single gas bubble, this numerical study is extended to consider the interaction of several bubbles. Surface tension, relative initial position and volume are all found to be important factors affecting the bubble interaction, jet formation, “trapping” of fluid between bubbles and bubble shedding. As well as computing the evolution of the bubble surfaces, consideration of the pressure fields and resulting instantaneous streamlines is given.
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