The drug probenecid inhibits the vacuolar accumulation of fluorescent anions in onion epidermal cells

1991 ◽  
Vol 99 (3) ◽  
pp. 557-563
Author(s):  
K. J. OPARKA ◽  
E. A. MURANT ◽  
K. M. WEIGHT ◽  
D. A. M. PRIOR ◽  
N. HARRIS
Keyword(s):  
Structural Changes ◽  
Lucifer Yellow ◽  
Plant Cells ◽  
Epidermal Cells ◽  
Drug Uptake ◽  
Central Vacuole ◽  
Endosomal Membrane ◽  
Lucifer Yellow Ch ◽  
Net Uptake ◽  
Onion Epidermal Cells

The drug probenecid has been shown to inhibit the vacuolar accumulation of a range of fluorescent anions with differing pKa values (Lucifer Yellow CH, Cascade Blue hydrazide, sulphorhodamine G, carboxyfluorescein, FITC) in onion epidermal cells. In the absence of the drug, uptake occurred into the vacuole and was sensitive to extracellular pH. In the presence of the drug, the dyes accumulated exclusively in the cytoplasm and nucleus. Probenecid also induced vesiculation of the tonoplast and caused the cytoplasm to become polar, both of these structural changes being reversible by removal of the drug. In the case of the permeant FITC molecule, probenecid inhibited net uptake into the epidermal cells, and addition of the drug to cells that had already accumulated dye in the central vacuole resulted in rapid dye leakage across the tonoplast. However, the other more impermeant probes failed to leak to the cytoplasm once they had accumulated in the vacuole, even after prolonged exposures to probenecid. The data are discussed in the light of recent evidence for probenecid-sensitive carriers capable of transporting fluorescent anions across the endosomal membrane of macrophages and in relation to the concept of a detoxification system for the sequestration of xenobiotic anions by plant cells

Lucifer Yellow and Fluorescein Isothiocyanate uptake by Cells of Morinda Citrifolia in Suspension Cultures is not Confined to the Endocytotic Pathway

1991 ◽  
Vol 100 (1) ◽  
pp. 237-241
Author(s):  
D. O'DRISCOLL ◽  
G. WILSON ◽  
M. W. STEER
Keyword(s):  
Lucifer Yellow ◽  
Fluorescein Isothiocyanate ◽  
Anion Transport ◽  
Suspension Cultures ◽  
Morinda Citrifolia ◽  
Central Vacuole ◽  
Cell Plasma Membrane ◽  
Transport Inhibitor ◽  
Lucifer Yellow Ch ◽  
Cell Plasma

On the basis of previous reports that the uptake of Lucifer Yellow-CH (LYCH) is restricted to the endocytotic pathway in plant cells, it was anticipated that the probe would provide an estimate of endocytotic activity in suspension cultures of Morinda citrifolia. The uptake of LYCH and fluorescein isothiocyanate (FITC) have been examined in whole cell and protoplast forms of M. citrifolia. Rapid staining of the general cytoplasm and nucleus was observed with both fluorochrome treatments, while both markers were excluded from the central vacuole. In addition, treatment of cells with Probenecid, an anion transport inhibitor, prevented LYCH from entering cells. It is suggested that LYCH utilizes an anion transport mechanism to cross the plant cell plasma membrane. Furthermore, LYCH and FITC are excluded from the central vacuole due to the absence of such a mechanism on the tonoplast.

An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Yu-Miao Zhang ◽  
Jun Wang ◽  
Tao Wu
Keyword(s):  
Subcellular Localization ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Epidermal Cells ◽  
Cyclin Dependent Kinase ◽  
Protein Subcellular Localization ◽  
Protein Protein Interactions ◽  
Efficient System ◽  
Protein Protein Interaction ◽  
Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

scholarly journals Immunocytochemical identification of dye-injected cells.

1982 ◽  
Vol 30 (2) ◽  
pp. 189-191 ◽  
Author(s):  
R L Michaels
Keyword(s):  
Pancreatic Islet ◽  
Islet Cells ◽  
Lucifer Yellow ◽  
Immunofluorescent Staining ◽  
Pancreatic Islet Cells ◽  
Lucifer Yellow Ch ◽  
Before And After ◽  
Coupled Cells ◽  
Indirect Immunofluorescent

Lucifer Yellow CH may be injected into pancreatic islet cells and visualized in Epon sections of the embedded tissue both before and after plastic removal and immunocyto-chemical staining. The dye retains its fluorescence, clearly marking the injected cell and adjacent dye-coupled cells, but does not interfere with the indirect immunofluorescent staining patterns that are characteristic of the islet cells

scholarly journals Gap junction structures after experimental alteration of junctional channel conductance.

1985 ◽  
Vol 101 (5) ◽  
pp. 1741-1748 ◽  
Author(s):  
T M Miller ◽  
D A Goodenough
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Electron Micrographs ◽  
Time Course ◽  
Structural Changes ◽  
Lucifer Yellow ◽  
Freeze Fracture ◽  
Channel Conductance ◽  
Experimental Alteration ◽  
Quick Freezing

Gap junctions are known to present a variety of different morphologies in electron micrographs and x-ray diffraction patterns. This variation in structure is not only seen between gap junctions in different tissues and organisms, but also within a given tissue. In an attempt to understand the physiological meaning of some aspects of this variability, gap junction structure was studied following experimental manipulation of junctional channel conductance. Both physiological and morphological experiments were performed on gap junctions joining stage 20-23 chick embryo lens epithelial cells. Channel conductance was experimentally altered by using five different experimental manipulations, and assayed for conductance changes by observing the intercellular diffusion of Lucifer Yellow CH. All structural measurements were made on electron micrographs of freeze-fracture replicas after quick-freezing of specimens from the living state; for comparison, aldehyde-fixed specimens were measured as well. Analysis of the data generated as a result of this study revealed no common statistically significant changes in the intrajunctional packing of connexons in the membrane plane as a result of experimental alteration of junctional channel conductance, although some of the experimental manipulations used to alter junctional conductance did produce significant structural changes. Aldehyde fixation caused a dramatic condensation of connexon packing, a result not observed with any of the five experimental uncoupling conditions over the 40-min time course of the experiments.

scholarly journals Calicivirus VP2 forms a portal to mediate endosome escape

2018 ◽  
Author(s):  
Michaela Conley ◽  
Marion McElwee ◽  
Liyana Azmi ◽  
Mads Gabrielsen ◽  
Olwyn Byron ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structural Changes ◽  
Host Cells ◽  
Major Step ◽  
Endosomal Membrane ◽  
Endosome Escape ◽  
Animal Pathogens ◽  
Capsid Shell ◽  
Capsid Protein Vp1

AbstractTo initiate the infectious process, many viruses enter their host cells by triggering endocytosis following receptor engagement. The mechanism by which non-enveloped viruses, such as the caliciviruses, escape the endosome is however poorly understood. TheCaliciviridaeinclude many important human and animal pathogens, most notably norovirus, the cause of winter vomiting disease. Here we show that VP2, a minor capsid protein encoded by all caliciviruses, forms a large portal assembly at a unique three-fold symmetry axis following receptor engagement. This feature surrounds an open pore in the capsid shell. We hypothesise that the VP2 portal complex is the means by which the virus escapes the endosome, pene-trating the endosomal membrane to release the viral genome into the cytoplasm. Cryogenic electron microscopy (cryoEM) and asymmetric reconstruction were used to investigate structural changes in the capsid of feline calicivirus (FCV) that occur when the virus binds to its cellular receptor junctional adhesion molecule-A (fJAM-A). Near atomic-resolution structures were calculated for the native virion alone and decorated with soluble receptor fragments. We present atomic models of the major capsid protein VP1 in the presence and absence of fJAM-A, revealing the contact interface and conformational changes brought about by the interaction. Furthermore, we have calculated an atomic model of the portal protein VP2 and revealed the structural changes in VP1 that lead to pore formation. While VP2 was known to be critical for the production of infectious virus, its function has been hitherto undetermined. Our finding that VP2 assembles a portal that is likely responsible for endosome escape represents a major step forward in our understanding of both theCaliciviridaeand icosahedral RNA containing viruses in general.

scholarly journals Ice crystal growth of living onion epidermal cells as affected by freezing rates

2018 ◽  
Vol 21 (1) ◽  
pp. 606-617 ◽  
Author(s):  
Duohua Xu ◽  
Huaiwen Wang ◽  
Yanan Wang ◽  
Zhe Zhang
Keyword(s):  
Crystal Growth ◽  
Epidermal Cells ◽  
Ice Crystal ◽  
Onion Epidermal Cells

scholarly journals Ganglionic and non-ganglionic neurons in frog heart: do they differ?

2017 ◽  
Vol 26 (2) ◽  
pp. 9
Author(s):  
Darius Batulevičius ◽  
Gertrūda Skripkienė ◽  
Greta Graužinytė ◽  
Augustina Grigaitė ◽  
Valdas Skripka
Keyword(s):  
Rana Temporaria ◽  
Lucifer Yellow ◽  
Frog Heart ◽  
Total Length ◽  
Fluorescent Markers ◽  
Lucifer Yellow Ch ◽  
Frog Rana Temporaria ◽  
The Mean ◽  
Morphological Differences ◽  
Intracardiac Neurons

This study was designed to compare the morphology of neurons in relation to their distance from the major nerve trunks in the heart of the frog Rana temporaria. Seventy-nine intracardiac neurons were labelled intracellularly with fluorescent markers Lucifer Yellow CH and Alexa Fluor 568. The neurons located on the extensions of the vagus nerve were considered as ganglionic, while neurons spread loosely at further distance from these extensions were considered as non-ganglionic. The mean area of the soma in ganglionic neurons was about 25% larger than in non-ganglionic neurons. Ganglionic neurons had a higher soma area/nucleus area ratio than non-ganglionic neurons. Although both the total number and the total length of dendrite-like processes was similar between the two groups, ganglionic neurons had significantly fewer dendrite-like processes from the soma (1.5±0.3 vs. 3.9±1.0; P<0.05) and shorter total length of these processes from the soma (63±18 μm vs. 178±51 μm; P<0.05). In conclusion, ganglionic and non-ganglionic frog intracardiac neurons exhibit substantial morphological differences. We hypothesize that these differences may indicate different projections or variations in the number of their preganglionic inputs.

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