Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at hippocampal synapses studied with novel quenching methods.
(9/29)
The kinetics of exo-endocytotic recycling could restrict information transfer at central synapses if neurotransmission were entirely reliant on classical full-collapse fusion. Nonclassical fusion retrieval by kiss-and-run would be kinetically advantageous but remains controversial. We used a hydrophilic quencher, bromophenol blue (BPB), to help detect nonclassical events. Upon stimulation, extracellular BPB entered synaptic vesicles and quenched FM1-43 fluorescence, indicating retention of FM dye beyond first fusion. BPB also quenched fluorescence of VAMP (synaptobrevin-2)-EGFP, thus indicating the timing of first fusion of vesicles in the total recycling pool. Comparison with FM dye destaining revealed that kiss-and-run strongly prevailed over full-collapse fusion at low frequency, giving way to a near-even balance at high frequency. Quickening of kiss-and-run vesicle reuse was also observed at higher frequency in the average single vesicle fluorescence response. Kiss-and-run and reuse could enable hippocampal nerve terminals to conserve scarce vesicular resources when responding to widely varying input patterns. (+info)
Administration of novel dyes for intraocular surgery: an in vivo toxicity animal study.
(10/29)
PURPOSE: To investigate the effect of intravitreal injections of new vital dyes on the retina, the retinal pigment epithelium (RPE) and the choroid in an in vivo rat model. METHODS: Rats were injected intravitreally with four dyes: light-green SF yellowish (LGSF), copper(II)phthalocyanine-tetrasulfonic acid (E68), bromphenol blue (BPB), and Chicago blue (CB) dissolved in physiologic saline solution (PSS) at concentrations of 0.5% and 0.02%. PSS served as the control. Additional animals were treated with single injections of 0.5%, 0.02%, 0.002%, and 0.0002% ICG or 0.002% E68 into one eye. Adverse effects on anterior and posterior segments were evaluated by slit lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by histology and retinal ganglion cell (RGC) quantification 7 days after dye administration. RESULTS: Eyes treated with 0.5% E68, 0.5% ICG, or 0.5% CB showed discrete staining of both cornea and lens not seen at lower concentrations or with other dyes. Histology revealed dose-dependent reactions after E68 administration. ICG 0.5% induced significant thinning of inner retinal layers compared with PSS. ICG 0.02% caused focal degenerative changes of the outer retina in three of seven eyes, whereas 0.002% and 0.0002% ICG did not. CB led to heterogeneous morphologic alterations. BPB- or LGSF-treated eyes showed normal retinal morphology. ICG at all tested concentrations induced significant RGC loss, as did E68 at 0.5% but not at lower concentrations. CONCLUSIONS: BPB or LGSF produced no significantly detectable toxic effects on the retina in vivo. The safety of these new dyes must be established in other models and/or in preclinical studies before the clinical use of any of these dyes. (+info)
Vitreoretinal surgery using bromphenol blue as a vital stain: evaluation of staining characteristics in humans.
(11/29)
OBJECTIVE: To evaluate the staining characteristics of bromphenol blue used during vitreoretinal surgery in humans. PATIENTS AND METHODS: 13 patients with epiretinal membranes were included. Before and after surgery a complete clinical examination including best corrected visual acuity, funduscopy, fluorescein angiography, OCT (Stratus), Goldmann perimetry and multifocal ERG as well as photography of the macular area was performed. Bromphenol blue was used in concentrations of 0.2% in most patients. Removed epiretinal tissue was evaluated using electron microscopy. RESULTS: Using dye concentrations of 0.2% a good demarcation of epiretinal membranes was seen in 11/13 patients. Staining of vitreous remnants at the vitreous base was seen in all patients. No dye-related adverse events were seen during follow-up in the functional tests (VA, ERG, perimetry) performed. Histological evaluation of epiretinal membranes showed unremarkable aspects of epiretinal cellular layers and unremarkable retinal surface of the internal limiting membrane (ILM). CONCLUSION: Bromphenol blue appears to be a very helpful and safe tool in posterior segment surgery. The staining characteristics need to be further evaluated in prospective study settings and larger numbers of patients. (+info)
Protein error of pH indicators in the presence of detergents.
(12/29)
The characteristics of color development due to a protein error in the dye-binding method in the presence of a non-ionic detergent has been investigated by the calculations based on the chemical equilibrium of a protein error. The calculation results were compared with those obtained using three pH indicators (Bromophenol Blue, Bromocresol Green and Bromocresol Purple) and three non-ionic detergents in the pH region from 1 to 13. In the experiments, the color development increased with the lower concentrations of the detergents, but decreased at higher concentrations. The pH where the color development reached a maximum value shifted to a higher pH as the detergent was added. These experimental results were reproduced by the calculation when the molar absorptivity of the dye-protein complex was assumed to increase due to adding the detergent. Such agreement between the experimental and the calculated results indicates that the characteristics of the color development in the dye-binding method in the presence of a non-ionic detergent can be analyzed by calculations based on the chemical equilibrium of a protein error. (+info)
Rapid recovery of releasable vesicles and formation of nonreleasable endosomes follow intense exocytosis in chromaffin cells.
(13/29)
Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of < or =20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K(+) and 2 mM Ca(2+)) provoking intense exocytosis (51 +/- 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 +/- 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in <2 min. The remaining nonreleasable fraction was distributed mainly as fluorescent spots ( approximately 0.7 microm) selectively labeled by 40- to 70-kDa dextrans and was suppressed by a phosphatidylinositol-3-phosphate kinase inhibitor, suggesting that it had been formed by a bulk retrieval mechanism. We concluded that chromaffin cells can rapidly recycle significant fractions of their total vesicle population, and that this pathway prevails when cholinergic agonists are used as secretagogues. When exocytosis exceeded approximately 20% of plasma membrane, an additional mechanism was activated, which was unable to produce secretory vesicles in our experimental time frame but appeared crucial to maintaining membrane surface homeostasis under extreme conditions. (+info)
Determination of oseltamivir quality by colorimetric and liquid chromatographic methods.
(14/29)
Influence of pH for the determination of serum albumin by a dye-binding method in the presence of a detergent.
(15/29)
In the dye-binding method, the absorbance increase caused by a protein error of a pH indicator is observed only in a restricted pH range. However, this pH range in the presence of a detergent has not yet been examined. Thus, the author investigated the pH (pH(UL)) where the absorbance increase becomes zero by a calculation based on the chemical equilibrium of a protein error of a pH indicator, and by experiments using four sulfonephthalein dyes. The pH(UL) value changed only with the detergent concentration, but did not change at all due to the dye, buffer solution or protein concentrations. Although the pH(UL) value was different according to the kind of dye used, it correlated well with the pK(D) values (dissociation constant) of BPB, BCG, BCP and BTB. The characteristics of pH(UL) in the reactions of the four dyes indicated good agreement with that obtained by a calculation. (+info)
Bioprocess of triphenylmethane dyes decolorization by Pleurotus ostreatus BP under solid-state cultivation.
(16/29)
With an aim to evaluate dye decolorization by white rot fungus on natural living conditions, reproducing by solidstate fermentation, the process of triphenylmethane dyes decolorization using the white rot fungus P. ostreatus BP, cultivated on rice straw solid-state medium, has been demonstrated. Three typical dyes, including malachite green, bromophenol blue, and crystal violet, were almost completely decolorized by the fungus after 9 days of incubation. During the process of dye decolorization, the activities of enzyme secreted by the fungus, and the contents of soluble components, such as phenolic compounds, protein, and sugar, changed regularly. The fungus could produce ligninolytic, cellulolytic, and hemicellulolytic enzymes and laccase was the most dominant enzyme in solid-state medium. Laccase, laccase isoenzyme, and the laccase mediator could explain the decolorization of malachite green, bromophenol blue, and crystal violet by the fungus in solid medium, respectively. It is worth noting that the presence of the water-soluble phenolic compounds could stimulate the growth of fungus, enhance the production of laccase, and accelerate dye decolorization. (+info)