Fertilization in the sea urchin arbacia punctulata inhibited by fluorescein dyes: Evidence for a plasma membrane mechanism

Fertilization and ionophore activation of the sea urchin Arbacia punctulata were inhibited in the presence of six analogs of the dye fluorescein. The concentration of any one dye needed for blockage of sperm or Ca-ionophoremediated activation in 50% of the eggs (I₅₀) was a function of the dye's lipid solubility. Substantially higher concentrations of each dye were required to block activation by Ca-ionophore (A23187) than were needed to inhibit sperm activation. A detailed study of the action of Erythrosin B (tetraiodofluorescein) showed that its effects were readily reversible. The I₅₀ concentration of Erythrosin B increased as temperature increased from 10 to 25°C. The kinetics of blockage indicated that Erythrosin B blocked some early step in the program of fertilization. The results suggest that these anionic dyes may inhibit fertilization by preventing successful sperm-egg fusion.     

Food dye, erythrosin B, inhibits ATP-dependent calcium ion transport by brain microsomes

The widely-used food dye Erythrosin B inhibited ATP-dependent Ca2+ accumulation by rat brain microsomes, half-maximal inhibition requiring 1 microM dye. Addition of 0.5-20 microM dye to microsomes preloaded with Ca2+ did not cause any net Ca2+ release. 10 microM dye produced a constant inhibition of Ca2+ accumulation as the intravesicular free Ca2+ was lowered suggesting that, at low concentrations, it acts on the uptake system only. Ca2+ accumulation was ten-fold more sensitive to the dye than Erythrosin B-induced neurotransmitter release reported by others. Higher dye concentrations (100 microM) caused Ca2+ release.     

Structural and functional degradation of Ca2+:Mg2+-ATPase rich sarcoplasmic reticulum vesicles photosensitized by erythrosin B

Erythrosin B (Red Dye No. 3) and Rose Bengal photosensitize the destruction of the Ca2+:Mg2+-ATPase pump protein in sarcoplasmic reticulum (SR) vesicles with respective quantum efficiencies of (1.53 +/- 0.19) X 10(-3) and (1.25 +/- 0.18) X 10(-3). Damage to vesicle function was assayed by measurements of increases in passive Ca2+ permeability. Rates of passive Ca2+ movement into the SR lumen were increased by dye photosensitization in proportion to radiation absorbed. Active Ca2+ transport into SR vesicles was blocked independent of radiation absorbed by Erythrosin B and Rose Bengal at free concentrations of 0.69 microM and 1.16 microM, respectively. The photochemical lability of the Ca2+ pump protein and alterations in passive and active Ca2+ transport may be dependent on the concentration of the dye in the membrane. The photosensitization results may have implications with respect to the suitability of Erythrosin B usage in vivo, since the brightness of our irradiation source is comparable to that of sunlight at 480 nm.     

Interactions of erythrosin B (U.S. F,D & C Red 3) with rat cortical membranes

Erythrosin B inhibits Na, K-ATPase in rat brain tissue as demonstrated by studying glycoside binding, ATPase activity and ion fluxes. The potency of the noncompetitive inhibition of [³H]-ouabain binding by erythrosin B is influenced by glycoside concentration, monovalent cation concentration, and incubation time. [¹⁴C]- Erythrosin B binds to synaptic membranes prepared from rat cortex. Erythrosin B and some of its structural analogs inhibit both [³H]-ouabain and [¹⁴C]-erythrosin B binding, but ouabain and other glycosides do not inhibit the binding of [¹⁴C]-erythrosin B. Subcellular distributions of [³H]-ouabain and [¹⁴C]- erythrosin B binding in fractionated cortical tissue preparations are equivalent and parallel ATPase activity. The dissimilar response of [³H]-ouabain binding and [¹⁴C]-erythrosin B binding to changes in tissue preparation, incubation temperature, and partial solubilization of binding sites by deoxycholate (DOC) Suggests two separate binding sites for erythrosin and ouabain to rat cortical membranes.     

Gastrulation in the sea urchin Strongylocentrotus purpuratus is blocked by the fluorescein dye erythrosin B

Erythrosin B, a food, drug, and cosmetic dye, arrested gastrulation in embryos of the sea urchin Strongylocentrotus purpuratus. A 30 min pulse of 5 microM erythrosin B added at 18 hr postinsemination blocked gastrulation scored at 50 hr postinsemination when control embryos had completed gastrulation. Dye addition at later times had no detectable effects on development through 50 hr postinsemination. The dye may block primary invagination via its known effects on plasma membrane permeability and fluidity.     

Preliminary histological observations on grapevine affected by esca disease

Esca is a destructive disease of the woody tissues of grapevine (Vitis vinifera L.) and due to the complexity of disease many aspects of host-pathogen interactions are not clearly understood. The histological characteristics of esca symptomatic petioles and internodes, collected from Cabernet Sauvignon and Sangiovese grapevine were studied. The tissues were fixed in FAA, dehydrated and embedded in Histoplast. To identify the lignified cell walls the sections were stained by Crystal violet and Erythrosin B and observed using an optical microscope. The main feature of tissues infected by esca disease was the minor lignification of vascular tissues, which was observed in petiole tissues before appearance of esca symptoms. The opportunity to utilize the histological examination of tissues as a method for the early detection of esca infections is hypothesized for the future application.     

Metabolism ofAedes aegypti cells grown in vitro. II. Determination of cell viability

Summary A dye exclusion test for differentiating between live and dead tissue culture cells ofAedes aegypti is described. Erythrosin B at a final concentration of 20 mg/100 ml cell suspension stained these cells differentially; dead cells were stained red but the live ones did not take up the dye. There was a close correlation between the number of unstained cells and incorporation of¹⁴C-leucine. No significant increase was observed in the number of stained cells over a 1-hr staining period. Keeping the cells at 5°C up to 24 hr prior to addition of the dye affected neither total cell number nor the proportion of stained cells. Contribution 202, based on a paper read at the 21st Annual Meeting of the Tissue Culture Association held at Washington, D. C., June 15–18, 1970.     

Determination of human serum albumin using an intramolecular charge transfer fluorescence probe: 4'-dimethylamino-2,5-dihydroxychalcone

A new intramolecular charge transfer fluorescence probe, namely, 4'-dimethylamino-2,5-dihydroxychalcone (DMADHC), exhibited dramatic enhancement of fluorescence intensity with an accompanying blue shift of the emission maximum when the concentration of human serum albumin (HSA) was increased. Binding to HSA also caused a progressive shift in the absorption spectrum of DMADHC, and a clear isosbestic point appeared. The binding site number and binding constant were calculated. Thermodynamic parameters were given and possible binding site was speculated. The optimum conditions for the determination of HSA were also investigated. A new, fast, and simple spectrofluorimetric method for the determination of HSA was developed. In the detection of HSA in samples of human plasma, this method gave values close to that of the Erythrosin B method.     

Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis

Synexin was isolated from bovine liver and found to aggregate adrenal chromaffin granules in the same Ca2+-dependent manner as previously described for adrenal synexin. The chromaffin granule aggregating activity of liver synexin was blocked in vitro by the addition of an antibody prepared to the 47,000 molecular weight band extracted from an SDS gel of an adrenal medullary synexin preparation. Chromaffin granules aggregated by synexin fused when exposed to cis-unsaturated fatty acids at concentrations comparable to those released from phospholipids by stimulated secretory cells. The synexin-induced aggregation reaction was blocked by Erythrosin B, a common food coloring, and by the phenothiazine antipsychotic trifluoperazine and promethazine. The aggregation and fusion of chromaffin granules thus appears to be a useful model system for studying synexin from diverse tissues and for testing pharmacologically or toxicologically active substances for effects on secretory systems.     

Erythrosin B (USFD&C RED 3) inhibits calcium transport and atpase activity of muscle sarcoplasmic reticulum

Erythrosin B (USFD&;C RED 3) inhibits the transport of calcium ions into isolated rabbit muscle sarcoplasmic reticulum vesicles with an IC₅₀ of ～ 0.5 μM and inhibits the Ca²⁺Mg²⁺ ATPase activity with an IC₅₀ of ～ 1 μM. The dye also binds to this tissue with an apparent K_d of ～ 300 nM. Other iodinated and brominated fluorescein analogs and blue dextran also inhibit ATPase activity and displace bound dye, suggesting that erythrosin may bind to a site near to but not identical with the nucleotide site. The dye should prove to be a useful probe for transport and ATPase activity.     

Toxicity of Xanthene Dyes to Entomopathogenic Fungi

Effects of xanthene dyes on mycelial growth and conidial germination in three species of entomopathogenic fungi, Beauveria bassiana, Metarhizium anispoliae and Paecilomyces fumosoroseus, were evaluated in a variety of assay systems. In a disk-diffusion assay, erythrosin B and phloxine B (but not eosin B) produced zones of inhibition in colonies of all three species under continuous exposure to light at disk-loadings of 100mug. None of the dyes produced zones of inhibition in the absence of light at disk loadings of 100mug. Both erythrosin B and phloxine B inhibited mycelial growth of all three species in the light in a dose-dependent manner. Weaker dose-responses for inhibition of growth in the dark were observed for some fungus/dye combinations. Erythrosin B, tested singly, completely inhibited conidial germination in the light in all eight fungal strains tested at 100mug ml-1 medium, but failed to inhibit conidial germination in any of these strains in the dark at the same concentration of dye. For single strains of each of the three fungi, erythrosin B and phloxine B inhibited conidial germination in a dose-dependent manner in the light with IC50s < 6.2mug dye ml-1 medium for all fungus/dye combinations. Phloxine B was a more potent inhibitor of germination than erythrosin B for all three fungal species. At fixed dosages of erythrosin B and phloxine B, inhibition of conidial germination in all three species increased with time of exposure to light. These results constitute the first quantitative demonstration of photodynamic inhibition of conidial germination in fungi by xanthene dyes.     

Erythrosin and pH gradient induced photo-voltages in bilayer membranes

Summary Erythrosin and light flashes induce voltage transients across bilayer membranes in the presence of transmembrane pH gradients. Fast voltage transients, which rise in <50 nsec and fall in 500 nsec, are attributed to photo-deprotonation of dye sorbed in the glycerol region of phospholipid membranes. Six other halogenated xanthene dyes induce similar effects, which apparently resulted from triplet states of monoanionic dye. No photo-effects were observed with fluorescein.     

Fluorescein Dyes as Bacterial Stains with Special Reference to their Use for Soil Preparations

Erythrosin and rose bengal have been suggested in the past as bacterial stains, particularly in the case of the direct study of microorganisms in soil. The writers have made a further investigation of these two dyes and others of the same group (thirteen in all) to determine their relative merits for this purpose. It is found that practically all the deeper colored dyes of the group stain bacteria in pure culture satisfactorily; but that in the case of soil infusions some of the dyes have such an affinity for the dead organic matter as to obscure the bacteria present. Best results in the present work were obtained with rose bengal B; but it is pointed out that good staining effects may be obtained with the phloxines and erythrosins. The behavior of any one of these dyes when applied to a soil preparation varies according to the reaction of the material stained; and this is mentioned as a possible explanation as to why others have found better results with erythrosin than with rose bengal.     

Application of a non-hazardous vital dye for cell counting with automated cell counters

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.     

Determining binding sites of polycyclic aromatic small molecule-based amyloid-beta peptide aggregation modulators using sequence-specific antibodies

Numerous aromatic small molecule modulators of amyloid-beta peptide (Aβ) monomer aggregation and neurotoxicity have been identified with the ultimate goal of Alzheimer’s disease (AD) treatment. Determining binding sites of these modulators on Aβ monomer is an important topic in the mechanistic understanding of AD pathology and drug development. However, Aβ monomer binding sites have been reported for only a very limited number of Aβ modulators. In this article, we present a convenient method for determining aggregation-modulating polycyclic aromatic small molecule ligand binding sites on Aβ monomer using immunostaining with a panel of Aβ sequence-specific antibodies. To validate our technique, we first examined one modulating aromatic ligand, Congo Red, with known binding sites, which yielded consistent results with previous findings. Then, using the same technique, binding sites on Aβ of four known Aβ monomer aggregation modulators, Erythrosin B, Eosin Y, Phloxine B, and Rose Bengal, were determined. The identified ligand binding sites were also confirmed by a separate fluorescence quenching-based assay using a panel of overlapping Aβ sub-fragments. The technique described here greatly increases researchers’ ability to determine the Aβ monomer binding site(s) of aggregation-modulating aromatic small molecule ligands and to screen for new ligands that bind specific regions on Aβ.     

Investigation of staining,polarization and fluorescence microscopic properties of myoepithelial cells

Summary During studies of early arteriosclerotic lesions fibers with the staining properties of myosins were observed in epithelial cells of various organs. To obtain a basis for further studies, staining, oolarization and fluorescence microscopic properties of classical myoepithelial cells and tonofibrils were investigated. The tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN stain for myosins and related proteins was applied to sections of tongue and skin. In other series various milling dyes, xanthene dyes and Thiazine Red R were substituted for Levanol Fast Cyanine 5RN.Myoepithelial cells of lingual and eccrine sweat glands showed the microscopic properties of smooth muscle cells; tonofibrils had little or no affinity for the dyes tested. The terminal bar-terminal web system of glandular epithelium and the fibrous layer in ducts of eccrine sweat glands resembled myosins and differed significantly from proteins of the epiderminkeratin group, e.g. tonofibrils. In preliminary studies the iodinated xanthene dyes Rose Bengal G, Erythrosin B and Y were found suitable for light, fluorescence and electron microscopic studies.     

Fluorescein Dyes as Bacterial Stains with Special Reference to their Use for Soil Preparations

Erythrosin and rose bengal have been suggested in the past as bacterial stains, particularly in the case of the direct study of microorganisms in soil. The writers have made a further investigation of these two dyes and others of the same group (thirteen in all) to determine their relative merits for this purpose. It is found that practically all the deeper colored dyes of the group stain bacteria in pure culture satisfactorily; but that in the case of soil infusions some of the dyes have such an affinity for the dead organic matter as to obscure the bacteria present. Best results in the present work were obtained with rose bengal B; but it is pointed out that good staining effects may be obtained with the phloxines and erythrosins. The behavior of any one of these dyes when applied to a soil preparation varies according to the reaction of the material stained; and this is mentioned as a possible explanation as to why others have found better results with erythrosin than with rose bengal.     

Activation of phospholipase D by calmodulin antagonists and mastoparan in carnation petals

An in vivo assay for phospholipase D (PLD; EC 3,1,4,4) activity,based on its transphosphatidylation property, is described indetail and was used to study putative post-translational regulationmechanisms of PLD activity in carnation (Dianthus caryophyllusL.) petals. A variety of agents was applied to petal discs.The calmodulin (CaM) antagonists propranolol, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide(W7) and N-(6-aminohexyl)-1-naphthalenesulphonamide (W5), stimulatedPLD activity in a dose-dependent manner. EGTA partially inhibitedthe stimulation by the CaM antagonists. Erythrosin B, an inhibitorof CaM-dependent P-type Ca^z+-ATPases, slightly stimulated PLDactivity. The results suggest that part of the stimulation ofPLD activity by CaM antagonists is due to an increased intracellularCa²⁺-concentration, PLD activity was stimulated by mastoparanin a dose- and time-dependent manner. The signal-like activationkinetics suggests that mastoparan activates PLD (in)directlyvia a G protein. Key words: Phospholipase D, CaM antagonists, mastoparan, Dianthus, calcium     

Plasma membrane ATPase activity during pepino (Solanum muricatum) ripening

Plasma membrane-enriched samples were extracted from pepino fruit (cv. El Camino) by phase partitioning. H⁺-ATPase (EC 3.6.1.35) activity in these samples increased during late fruit development (immediately before the onset of ripening) and western blotting confirmed there was an increase in enzyme abundance at this time. H⁺-ATPase activity decreased during early ripening and then increased again in the final phase of ripening. Immunolocalisation showed the plasma membrane H⁺-ATPase was most abundant in the outer cell layers of the fruit, which are considered to have a major role in determining fruit texture. Fruit softening was not accelerated by harvest and there was no stimulation of H⁺-ATPase activity by harvest. An in vitro tensile test using fruit rings showed tissue softening proceeded faster at low apoplastic pH (4.5) than at pH 6.5; and tissue buffered at pH 6.5 softened less than unbuffered rings. Erythrosin B, an inhibitor of the plasma membrane H⁺-ATPase, also retarded softening in vitro. These data suggest that plasma membrane H⁺-ATPase activity may contribute to the onset of pepino softening through a reduction in apoplastic pH.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.