The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

Sites of Tubulin Polymerization in PC 12 Cells

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.     

Cell migration within the embryonic limb primordium of Drosophila as revealed by a novel fluorescence method to visualize mRNA and protein

 We report a new technique using fluorescent probes to detect a mRNA and a protein simultaneously in the Drosophila embryo. For in situ hybridization, 3-hydroxy-N-2′-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR was used as a fluorescent substrate for alkaline phosphatase. It was possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. We applied this technique to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. We stained embryos bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA was detectable in the ventral region of the limb primordium, and β-galactosidase protein in the dorsal region. In the middle, both mRNA and protein were detectable. These results suggest that the Dll enhancer is activated in the ventral region of the limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium. Received: 7 April 1997 / Accepted: 15 May 1997     

Dye-flow apparatus to measure the variation in axial xylem permeability over a stem cross-section

Abstract Equipment and methodology are described that allows the radial variation in axial xylem permeability (hydraulic conductivity) over a tree cross-section to be measured and the flow paths to be identified by the strictly controlled flow of dye through a specimen. The apparatus can be calibrated so that the point-to-point variation of absolute permeability over a xylem cross-section can be calculated from the dye-flow patterns, which otherwise show only relative variations in permeability. The effect of using different dyes and dye concentrations on the penetration time and the shape of the dye patterns was investigated. The penetration time through the wood of identical end-matched specimens is appreciably longer for fixing dyes than for non-fixing dyes, and for the fixing dyes it depends strongly on the dye concentration. However, the dye patterns of the end-matched specimens were indistinguishable with fixing and non-fixing dyes, and independent of dye concentration. The fixing dye toluidine blue at 0.25% to 0.5% (w/w) was found most suitable as it yields a clear permanent pattern.     

Antigenic differences between Anabaena azollae fresh from the Azolla fern leaf cavity and free-living cyanobacteria

Fluorescent antibody staining indicated differences in surface antigenicity in Anabaena azollae cells fresh from the leaf cavities of the fern, Azolla caroliniana, and algae which were isolated and subcultured from this fern. Such results suggest that either changes in antigenicity occur in this phycobiont during culturing or that isolation selects for an antigenically different mutant strain capable of in vitro growth.Non-Standard Abbreviations FA fluorescent antibody staining - PBS phosphate buffered saline - W microwatt - Anti-F antiserum prepared against fresh cells - Anti-N antiserum prepared against Newton's culture - FTTC fluorescein isothiocyanate To whom offprint requests should be sent     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

Clostridium thermoaceticum forms methanol from carbon monoxide in the presence of viologen dyes

Whole cells of Clostridium thermoaceticum, crude extracts of such cells as well as the supernatant of 100 000 × g centrifugations catalyse the reduction of carbon monoxide to methanol in the presence of viologens or cobalt sepulchrate. Without such a mediator methanol could not be detected. The reaction shows a marked optimum at pH 5. The incubation of [5-¹⁴C]methyltetrahydrofolate led only to the formation of ¹⁴C-labeled ethanol; the radioactivity in methanol was negligible. The reaction seems to be catalysed by carbon monoxide dehydrogenase.     

Calix[4]arene based molecular sensors with pyrene as fluoregenic unit: Effect of solvent in ion selectivity and colorimetric detection of fluoride

Fluorescent molecular sensors having excimer emission property have been designed and synthesised incorporating calix[4]arene derivatives in cone and 1,3-alternate conformation as ionophore and two pyrene moieties at close proximity as fluorophore. They exhibit strong excimer emission around 515 nm, which is used to monitor interaction of metal ions with the ionophores. Ion-binding study of these fluoroionophore has been investigated in acetonitrile-chloroform and THF-H₂O with a wide range of cations and anions and the recognition process is monitored by luminescence, UV-Vis and ¹H NMR (for F⁻) spectral changes. The present study demonstrated profound influence of solvent in ion selectivity, in acetonitrile-chloroform they formed complexes with Hg²⁺, Pb²⁺, Cu²⁺ and Ni²⁺, whereas in THF-H₂O they exhibit selectivity only for Cu²⁺. In the case of anions, selectivity for only F⁻ with color change is observed. Composition of the complexes formed was determined from mass spectrometry and the binding constants were determined from fluorescence titration data. The reasons for formation of excimer emission, quenching of it in presence of certain metal ions, role of solvent in selectivity and energy/electron transfer process involved in the ion-recognition event have been discussed on the basis of experimental data.     

Comparative study of different fluorescent dyes for the detection of proteins on membranes using the peroxyoxalate chemiluminescent reaction

We have previously shown that the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2) chemiluminescent reaction in acetone can be used for the detection of proteins labeled with the fluorescent reagent 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. To improve this method, in this work we have designed and constructed a cell that allows us to perform this chemiluminescent reaction on PVDF membranes with a homogeneous distribution of the reagents. Using this cell we have examined the analytical properties of several recently developed fluorescent protein dyes chemically different from MDPF. We have found that the metal chelate dye SYPRO Ruby can also be excited by the high-energy intermediate produced in the TCPO-H(2)O(2) reaction.     

Morphology of the first-instar nymph and adult female of Kermes echinatus Balachowsky,with a comparison to K. vermilio Planchon (Hemiptera,?Coccoidea,?Kermesidae)

Thefirst-instar nymph and the adult female of Kermes echinatus Balachowsky (Hemiptera, Coccoidea, Kermesidae) are described and illustrated. This species is compared with Kermes vermilio Planchon, a morphologically similar species known in the Palaeractic region.