Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Fluorescent analysis of alpha-keto acids in serum and urine by high-performance liquid chromatography

A selective procedure using synthetic substrates for determination of exo-1,4,-beta-glucanases in a mixture of exoglucanases , endoglucanases , and beta-glucosidases is formulated. The heterobiosides , p- nithrophenyl -beta-D- cellobioside ( pNPC ) or p-nitrophenyl-beta-D-lactoside ( pNPL ), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases , which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of beta-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of D-glucono-1,5-delta-lactone, a specific inhibitor of beta-glucosidases. The interference of endoglucanases , which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Selective labeling of α-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions

The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C. S., Manshouri, T., and Atassi, M. Z. (1987)J. Prot. Chem. 6, 455–461; Garcia-Borron, J. C., Bieber, A. L., and Martinez-Carrion, M. (1987)Biochemistry 26, 4295–4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR-mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC-Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule.     

Effect of cholera toxin on rat intestinal permeability assessed with fluorescent dextran 3000

Abstract The effect of culture filtrate containing cholera toxin (CT) on rat intestinal permeability was studied using fluorescein isothiocyanate-labelled dextran 3000 (FITC-D3, M _r, 3000) as probe molecule. CT was given either perorally, via a gastric tube 90 min before, or locally in conjunction with the permeability measurement in the distal ileum. Compaired to the control animals, either mode of administration resulted in increased permeation of FITC-D3 from the intestine to portal blood. The effect of the local treatment was apparent after 5–10 min and prevailed during the 60-min measurement period. The results indicate that CT not only affects net water transport at the intestinal mucosa but also the passage of larger molecules across the intestinal wall.     

Size Effects on the Uptake of Particles by Populations of Tetrahymena pyriformis Cells

Flow cytometry has been used to make direct measurements of rates of uptake of latex microspheres from dilute, monodisperse suspensions by Tetrahymena pyriformis. Measurements were made for five different sizes of microspheres, ranging from 1.09 to 6.17 μm diameter. Fractions of cells in the population that did not ingest the microspheres offered were also determined. In addition, the size distributions, as indicated by the forward angle light scattering intensity which is measured by the instrument, were determined for the whole population and for the subpopulations of cells that did and did not ingest the particles, for each particle size used. It was found that the fraction of cells that did not ingest the particles was small and independent of particle size when this was less than about 2.7 μm, but increased with particle size when particle size was increased above this value. The so-called maximum clearance rate, which can be calculated from the data, was found to increase monotonically with particle size if it were based only on those cells which actually ingested the particles offered. However, a plot of maximum clearance rate vs. particle size exhibited a maximum if the clearance rate were based on all cells present in the population.     

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI propidium iodide - SLO streptolysin O     

Relations between grapevine replant disease and root colonization of grapevine (Vitis sp.) by fluorescent pseudomonads and endomycorrhizal fungi

In pot experiments cuttings of grapevine rootstock cultivar 5C were grown on a soil from a grapevine nursery affected with replant disease (replant soil) and on a similar soil that had not been planted with grapevines before (non-replant soil). Plants were also inoculated with the vesicular-arbuscular (VA) mycorrhizal fungus,Glomus mosseae, or left without mycorrhizal fungus inoculation. Shoot and root growth, mycorrhization of roots and numbers of total aerobic bacteria and fluorescent pseudomonads on the rhizoplane of grapevines were determined at several sampling dates. On replant soil, numbers of fluorescent pseudomonads on the rhizoplane were higher compared to non-replant soil, before differences in shoot and root weight between replant and non-replant soil occurred. Without inoculation withG. mosseae, the mycorrhization of roots was much lower on replant soil (13%) than on non-replant soil (51%). On replant soil, inoculation withG. mosseae increased mycorrhization to 39% and increased shoot length, leaf area and shoot weight. The beneficial effect of VA-fungus inoculation on replant soil was not due to increased nutrient concentrations in leaves. On replant soil, the inoculation withG. mosseae reduced the number of fluorescent pseudomonads on rhizoplane of grapevine, while the numbers of total aerobic bacteria were not influenced by inoculation withG. mosseae. These results suggest a direct or indirect role of fluorescent pseudomonads in replant disease of grapevine.