kDa FITC-dextran Research Articles

Electric field mediated loading of macromolecules in intact yeast cells is critically controlled at the wall level

The mechanism of electric field mediated macromolecule transfer inside an intact yeast cell was investigated by observing, under a microscope, the fluorescence associated to cells after pulsation in a buffer containing two different hydrophilic fluorescent dyes. In the case of a small probe such as propidium iodide, a long lived permeabilized state was induced by the field as classically observed on wall free systems. Penetration of a 70 kDa FITC dextran was obtained only by using drastic conditions and only a very limited number of yeast cells which took up macromolecules remained viable. Most dextrans were trapped in the wall. A dramatic improvement in transfer of dextrans was observed when the cells were treated by dithiothreitol before pulsation. A cytoplasmic protein leakage was detected after the electric treatment suggesting that an irreversible damage took place in the walls of many pulsed cells. Electroloading of macromolecules in intact yeast cells appears to be controlled by a field induced short lived alteration of the envelope organization.

Introduction of foreign DNA into Chlorella saccharophila by electroporation

Plasmid, pBI221, was introduced into protoplasts of Chlorella saccharophila c-211-1a prepared from the cells in the stationary phase by electroporation. Transient expression of the introduced plasmid was observed under a field strength of between 600 and 900 V/cm, and a pulse duration of around 400 ms, where high membrane permeability to 70-kDa FITC-dextran was ascertained.

In Vitro Development of the Sea Urchin Male Pronucleus

We have developed a cell-free extract from fertilized or unfertilized sea urchin eggs which promotes formation of male pronuclei from exogenously added permeabilized sperm nuclei. Using a buffer to simulate egg cytoplasmic conditions, three states of nuclear condensation can be distinguished: condensed (conical), partially decondensed (conical or ovoid), and decondensed (spherical). The in vitro system meets several in vivo criteria established by microinjection experiments. Decondensation is promoted at elevated pH and in activated egg cytoplasm, but does not require Ca2+. Pronuclear development is supported to >100 male nuclei per egg-equivalents as in vivo. Pronuclear development requires addition of an ATP-generating system and is blocked by two kinase inhibitors (6-DMAP and staurosporine) at the same concentrations effective in vivo. Decondensed nuclei form by 40 min of incubation and acquire a putative nuclear envelope shown by exclusion of 150 kDa FITC-dextran by 1-2 hr. The rates of decondensation and nuclear envelope formation are accelerated by addition of GTP. Protease inhibition experiments suggest a role for nonhistone protein degradation in pronuclear progression. This system should prove useful for investigating mechanisms of the postmeiotic sea urchin male chromatin remodeling which follows fertilization, previously accessible only in vivo.

The synthesis, characterization and biological testing of a novel class of mucosal permeation enhancers

Abstract A series of chemical derivatives of 24,25-dihydrofusidic acid have been prepared and tested as nasal permeation enhancers in an in vivo rat model. The derivatives were synthesized by coupling basic, acidic, and neutral functionalities to the C21 carbon of 24,25-dihydrofusidic acid. The pH-solubility of the derivatives was a function of the ionization state of the molecule. The CMC values, determined by the wavelength shift in the maximum absorbance, were remarkably similar (0.5–2 mM) for all the derivatives which formed micelles. The absorption enhancement potential of each derivative was evaluated by co-administration with insulin, hGH, and a 20 kDa FITC-labeled dextran. With insulin and hGH, the acidic derivatives showed a similar enhancement effect which was greater than that observed with the basic derivatives. The absolute bioavailabilities were approximately 38% for insulin and 8% for hGH with the acidic derivatives. In the case of the 20 kDa FITC-dextran compound, both the acidic and the basic derivatives showed similar enhancement effects. The data suggest a specific interaction of the basic compounds with hGH and insulin resulting in diminished enhancement. The enhancement potentials of the chemical derivatives synthesized in this study do not appear to be due to the chemical properties of the side chain, or a specific side chain binding to protein or a receptor interaction with the side chain, but instead they are likely due to the physical and chemical properties of the 24,25-dihydrofusidate steroidal nucleus.

FITC-dextran as a probe for endosome function and localization in kidney

Fluorescein isothiocyanate (FITC)-labeled endosomes were localized in kidney epithelial cells after tissue fixation and sectioning, and specific membrane transport properties of isolated endocytic vesicles were measured using the same probe. Rats were infused intravenously with 10 kDa FITC-dextran, and kidneys were fixed with paraformaldehyde lysine periodate. FITC-labeled vesicles were visualized in semithin (1 micron) frozen sections of excised tissue by epifluorescent microscopy and by electron microscopy after a photoconversion reaction. Most FITC-labeled endosomes were apically located in epithelial cells lining the urinary tubules. By immunocytochemistry the anti-lysosomal glycoprotein LGP 120 was absent from most of the FITC-labeled vesicles, although some colocalization was noted. The limiting membrane of FITC-labeled endosomes contained a vacuolar proton pump (pHmin = 6.23 +/- 0.033) and a water channel (osmotic water permeability coefficient, Pf = 0.052 +/- 0.005 cm/s) and was highly permeable to ethylene glycol and urea but relatively impermeable to glucose. Methods allowing the attribution of specific membrane functions to vesicles that can be visualized in the apical endocytic pathway of epithelial cells should be of general use for the study of endocytic pathways in a variety of systems.

The number of molecules taken up by electroporated cells: quantitative determination

Fluorescent and fluorescent-labeled molecules were used with calibrated flow cytometric fluorescence measurements of electrically pulsed cells (intact yeast: Saccharomyces cerevisiae) to demonstrate a method for determining the net number of molecules transported into electroporated cells. For the conditions used, a single pulse of width 50 μs and magnitude 8.0 ± 0.5 kV cm resulted in an average net molecular uptake which is large, n̄ = 1.4 × 10 5 molecules of 70 kDa FITC-dextran (supplied extracellular concentration of 500 μM), and − n = 1.0 × 10 8 molecules of 660 Da propidium iodide (PI; 80 μM). Both molecules were present in pulsed cells at less than equilibrium values, consistent with a transient uptake mechanism. Intracellular FITC-dextran is present in soluble form, while PI is predominantly bound to nucleic acids. A broad, statistically significant distribution of molecular uptake was also observed. Such quantitative determinations should be important for guiding applications of electroporation, and for testing models of electroporation mechanisms. Further, the use of PI, which is well established as a membrane exclusion dye, provides additional support for the interpretation that both PI and FITC-dextran were internalized as a result of an electrical pulse. Electroporation; Molecular transport; Number of molecules; Flow cytometry; Yeast