Disappearance Of Fluorescence Research Articles

Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing.

Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification.

A colorimetric and fluorescent cyanide chemosensor based on dicyanovinyl derivatives: Utilization of the mechanism of intramolecular charge transfer blocking

Chemosensor (CS1) was designed and synthesized by simple green chemistry procedure. CS1 exhibited both colorimetric and fluorescence turn-off responses for cyanide (CN−) ion in aqueous solution. The probe showed an immediate visible color changes from yellow to colorless and green fluorescence disappearance when CN− was added. The mechanism of chemosensor reaction with CN− was studied using 1HH NMR and 13C NMR spectroscopies and mass spectrometry. Moreover, test strips based on the sensor were fabricated, which served as convenient and efficient CN− test kits.

DNA detection assay based on fluorescence quenching of rhodamine B by gold nanoparticles: The optical mechanisms

The different ability of single- and double-stranded oligonucleotides to stabilize gold nanoparticles (GNPs) in solution has recently been used to design several label-free hybridization assays on the basis of optical changes associated with GNP aggregation. DNA hybridization can be detected through changes in dye fluorescence quenching by GNPs. Here we examine the mechanisms behind a fluorescent DNA assay for model systems containing DNA oligonucleotides, 15-nm GNPs, and Rhodamine B (RB). There was a direct correlation between complete disappearance of fluorescence and complete adsorption of all RB molecules on nonaggregated GNPs, as revealed by an analysis of the colloids' supernatant liquids. We show that both the inner filter effect and the quenching of the dye owing to its adsorption on GNPs contribute to the observed changes in fluorescence intensity. Therefore, both factors should be properly adjusted to optimize the assay sensitivity. In particular, the low detection limit of the fluorescent DNA assay lies in the range 30–100pM, which is close to the data reported previously for colorimetric and dynamic light scattering DNA assays.

Kv2.1 Cell Surface Clusters Promote Maturation of Clathrin-Coated Pits

The voltage-gated potassium channel Kv2.1 localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. These surface structures are specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells [Deutsch et al., MBoC 15, pp 2917-29 (2012)]. Internalization of Kv2.1 occurs through clathrin-mediated endocytosis and clathrin-coated pits (CCP) localizes adjacent to these micro-domains. This study examines the relationship between Kv2.1 clusters and CCP maturation.TIRF-microscopy was used to study GFP-tagged-clathrin light chain CCPs in live HEK293 cells. HEK cells do not express endogenous Kv2.1, making them a suitable model system in which to investigate the role of Kv2.1 in clathrin-mediated endocytosis. We tracked individual CCPs and measured their lifetimes. This analysis is obtained from the appearance and disappearance of GFP fluorescence within the evanescent field illumination. We compare the dynamics of CCPs in control cells transfected with GFP-CLC and cells co-transfected with Kv2.1 or a Kv2.1 mutant lacking the last 318 amino acids of the C-terminus (ΔC-Kv2.1) necessary for cluster formation.In control cells, CCPs had a mean lifetime of 12.6±0.3 s (mean±sem). The lifetime of CCPs in cells co-expressing Kv2.1 and GFP-CLC was reduced by 50%. When GFP-CLC was co-transfected with the non-clustering ΔC-Kv2.1, the lifetime of CCPs increased by 17%. ΔC-Kv2.1 also decreased the rate of channel endocytosis by 12%.These data reveal that Kv2.1, specifically the C-terminal tail, has a direct effect on CCP lifetimes and thereby maturation. Cells expressing clustering Kv2.1 exhibit more rapidly maturing CCPs as seen by the rate of Kv2.1 internalization. Non-clustering Kv2.1 increases CCP lifetimes, and complete loss of Kv2.1 results in even longer lifetimes, i.e. slower maturation. These results indicate that Kv2.1 cluster formation remodels clathrin-mediated endocytosis.

Probing Single-Molecule Enzymatic Dynamics of B-Glucosidase using Zero-Mode Waveguides

β-glucosidases (BGLs, EC 3.2.1.21) are exo-type enzymes that hydrolyze β-glucosidic bonds from the non-reducing end of their substrates. BGLs are present in all living organisms (bacteria, archaea, and eukarya), and perform a range of functions. In bacteria and fungi, BGLs play an important role in cellulose saccharification, which catalyzes the hydrolysis of cellobiose and short cellooligosaccharide to glucose. Due to their potential biotechnological importance, a large number of BGLs from bacteria and fungi have been cloned and characterized. Interestingly, these enzymes often exhibit the enzymatic properties such as substrate and product inhibition. Thus, the dynamic molecular properties are too complicated to be fully understood by the use of conventional ensemble techniques. We then employed a single-molecule assay to probe the enzymatic dynamics of BGL.BGL1B from the wood-rotting basidiomycete Phanerochaete chrysosporium was used in this study. As the enzyme has a relatively low affinity for cellobiose (Km = ∼200 μM), µM concentration of fluorescent cellobiose is required to monitor the enzymatic reaction. Therefore, we employed a single-molecule assay using zero-mode waveguides (ZMWs). ZMWs comprise nanoscale holes in an aluminum film deposited on a fused silica coverslip, and can reduce the observation volume by more than three orders of magnitude relative to conventional microscopic techniques, allowing single-molecule observations at µM concentrations of fluorescent molecules in solution. Biotinylated BGL1B was immobilized in ZMWs through a biotin-streptavidin linkage. The BGL1B was immersed in a solution containing 1 μM tetramethlyrhodamine-conjugated cellobiose (TMR-cellobiose). The repeated appearance and disappearance of TMR fluorescence were often observed, indicating that immobilized BGL1B hydrolyzes TMR-cellobiose in ZMWs. Surprisingly, we have found that the enzymatic reaction is inhibited by glucose non-competitively at the lower concentration and competitively at the higher concentration.

Multifunctional silica nanoparticles for optical and magnetic resonance imaging

The surface of spherical, nonporous silica nanoparticles (SiO2-NPs) was modified with gadolinium (Gd) complexes, fluorophores, and cell-penetrating peptides to achieve multifunctionality on a single particle. The Gd surface concentrations were 9-16 μmol/g resulting in nanomaterials with high local longitudinal and transversal relaxivities (~1×10(5) and ~5×10(5) /mm/s/NP, respectively). Rapid cellular uptake was observed in vitro; however, larger extracellular agglomerates were also formed. In vivo administration revealed a fast distribution throughout the body followed by a nearly complete disappearance of fluorescence in all organs except the lungs, liver, and spleen after 24 h. Such NPs have the potential to serve as efficient multimodal probes in molecular imaging.

Efficiency of Endoscopic Imaging in Repetitive Probing following Unsuccessful Probing

To show the efficiency of repetitive probing performed with intranasal endoscopic imaging in cases who underwent unsuccessful probing and the accompanying nasal pathologies. Eighty-four eyes of the 64 patients in whom primary probing was found to be unsuccessful were included in the study. Cases were divided into 3 groups, depending on the number of unsuccessful attempts (1 [group 1], 2 [group 2], and 3 [group 3] unsuccessful probings). Diagnosis of congenital nasolacrimal duct obstruction was confirmed by history taking, clinical examination, and fluorescence disappearance test. All cases underwent nasal endoscopic-assisted probing and were followed up on the first week, first month, and third month following the procedure. There were 50 eyes in group 1, 23 in group 2, and 11 in group 3. The causes of failure that were determined with endoscopy were as follows: functional insufficiency 8.3%, lower nasal concha tension 4.7%, thin membrane in Hasner valve 3.6%, thick membrane and fibrosis in the distal side of the duct 21.4%, probe progressed submucosally on the nasal wall 33.3%, different exit location in the nose 20.2%, nasal mucosa ballooning in the distal side of the duct 4.7%, mucocele 2.4%, and nasal polyposis 1.2%. The success rate in the groups was estimated as 86% in the first group, 69.5% in the second group, and 54.5% in the third group. Performing the repetitive probings via nasal endoscopy provided information about the location and the shape of the obstruction and the opportunity to approach, according to the cause of the failure.

Imaging Single Endocytic Events Reveals Diversity in Clathrin, Dynamin and Vesicle Dynamics

The dynamics of clathrin-mediated endocytosis can be assayed using fluorescently tagged proteins and total internal reflection fluorescence microscopy. Many of these proteins, including clathrin and dynamin, are soluble and changes in fluorescence intensity can be attributed either to membrane/vesicle movement or to changes in the numbers of individual molecules. It is important for assays to discriminate between physical membrane events and the dynamics of molecules. Two physical events in endocytosis were investigated: vesicle scission from the plasma membrane and vesicle internalization. Single vesicle analysis allowed the characterization of dynamin and clathrin dynamics relative to scission and internalization. We show that vesicles remain proximal to the plasma membrane for variable amounts of time following scission, and that uncoating of clathrin can occur before or after vesicle internalization. The dynamics of dynamin also vary with respect to scission. Results from assays based on physical events suggest that disappearance of fluorescence from the evanescent field should be re-evaluated as an assay for endocytosis. These results illustrate the heterogeneity of behaviors of endocytic vesicles and the importance of establishing suitable evaluation criteria for biophysical processes.

Ab Initio Based Surface-Hopping Dynamics Study on Ultrafast Internal Conversion in Cyclopropanone

Cyclopropanone exhibits an intriguing phenomenon that the fluorescence from the S(1) state disappears below 365 nm. This is ascribed to the ultrafast S(1) → S(0) internal conversion process via conical intersection, which deprives opportunity of the fluorescence emission. In this work, we have used ab initio based surface hopping dynamics method to study vibrational-mode-dependent S(1) → S(0) internal conversion of cyclopropanone. A new conical intersection between the S(1) and S(0) states is determined by the state-averaged CASSCF/cc-pVDZ calculations, which is confirmed to play a critical role in the ultrafast S(1) → S(0) internal conversion by the nonadiabatic dynamics simulations. It is found that the internal conversion occurs more efficiently when the initial kinetic energies are distributed in the four vibrational modes related to the C═O group, especially in the C-O stretching and the O-C-C-C out-of-plane torsional modes. Meanwhile, the S(1) lifetime and the time scale of the S(1) → S(0) internal conversion are estimated by the ab initio based dynamics simulations, which is consistent with the ultrafast S(1) → S(0) internal conversion and provides further evidence that the ultrafast internal conversion is responsible for the fluorescence disappearance of cyclopropanone.

Single-molecule Study on the Decay Process of the Football-shaped GroEL-GroES Complex Using Zero-mode Waveguides

It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shaped complex, is unclear. We investigated the decay process of the football-shaped complex at a single-molecule level. Because submicromolar concentrations of fluorescent GroES are required in solution to form saturated amounts of the football-shaped complex, single-molecule fluorescence imaging was carried out using zero-mode waveguides. The single-molecule study revealed two insights into the GroEL-GroES reaction. First, the first GroES to interact with GroEL does not always dissociate from the football-shaped complex prior to the dissociation of a second GroES. Second, there are two cycles, the "football cycle " and the "bullet cycle," in the chaperonin reaction, and the lifetimes of the football-shaped and the bullet-shaped complexes were determined to be 3-5 s and about 6 s, respectively. These findings shed new light on the molecular mechanism of protein folding mediated by the GroEL-GroES chaperonin system.

Conical Intersection Is Responsible for the Fluorescence Disappearance below 365 nm in Cyclopropanone

The photodissociation dynamics of cyclopropanone was explored with the complete active space self-consistent field (CASSCF) calculations and ab initio nonadiabatic molecular dynamics simulations. The related minima, transition state (TS) and minimum-energy conical intersections (MECIs) were obtained as well as energetics. In the static CASSCF calculations, one MECI was found to be responsible for the fluorescence disappearance below 365 nm because the ultrafast internal conversion (IC) via this MECI deprived the opportunity of the fluorescence emission. Further evidence of this ultrafast IC event came from the subsequent ab initio nonadiabatic molecular dynamics simulations.

Visualization of radiation-induced cell cycle-associated events in tumor cells expressing the fusion protein of Azami Green and the destruction box of human Geminin

Ionizing radiation (IR) influences cell cycle-associated events in tumor cells. We expressed the fusion protein of Azami Green (AG) and the destruction box plus nuclear localization signal of human Geminin, an inhibitor of DNA replication licensing factor, in oral tumor cells. This approach allowed us to visualize G2 arrest in living cells following irradiation. The combination of time-lapse imaging analysis allowed us to observe the nuclear envelope break down (NEBD) at early M phase, and disappearance of fluorescence (DF) at the end of M phase. The duration from NEBD to DF was not much affected in irradiated cells; however, most of daughter cells harbored double-strand breaks. Complete DF was also observed in cells exhibiting abnormal mitosis or cytokinesis. We conclude that the fluorescent Geminin probe could function as a stable cell cycle indicator irrespective of genome integrity.

Disorganized distribution of homogalacturonan epitopes in cell walls as one possible mechanism for aluminium-induced root growth inhibition in maize

Aluminium (Al) toxicity is one of the most severe limitations to crop production in acid soils. Inhibition of root elongation is the primary symptom of Al toxicity. However, the underlying basis of the process is unclear. Considering the multiple physiological and biochemical functions of pectin in plants, possible involvement of homogalacturonan (HG), one of the pectic polysaccharide domains, was examined in connection with root growth inhibition induced by Al. An immunolabelling technique with antibodies specific to HG epitopes (JIM5, unesterified residues flanked by methylesterifed residues; JIM7, methyl-esterified residues flanked by unesterified residues) was used to visualize the distribution of different types of HG in cell walls of root apices of two maize cultivars differing in Al resistance. In the absence of Al, the JIM5 epitope was present around the cell wall with higher fluorescence intensity at cell corners lining the intercellular spaces, and the JIM7 epitope was present throughout the cell wall. However, treatment with 50 microm Al for 3 h produced 10 % root growth inhibition in both cultivars and caused the disappearance of fluorescence in the middle lamella of both epitopes. Prolonged Al treatment (24 h) with 50 % root growth inhibition in 'B73', an Al-sensitive cultivar, resulted in faint and irregular distribution of both epitopes. In 'Nongda3138', an Al-resistant cultivar, the distribution of HG epitopes was also restricted to the lining of intercellular spaces when a 50 % inhibition to root growth was induced by Al (100 microm Al, 9 h). Altered distribution of both epitopes was also observed when of roots were exposed to 50 microm LaCl(3) for 24 h, resulting in 40 % inhibition of root growth. Changes in HG distribution and root growth inhibition were highly correlated, indicating that Al-induced perturbed distribution of HG epitopes is possibly involved in Al-induced inhibition of root growth in maize.

Manufacturable plastic microfluidic valves using thermal actuation

A low-cost, manufacturable, thermally actuated, plastic microfluidic valve has been developed. The valve contains an encapsulated, temperature-sensitive fluid, which expands, deflecting a thin elastomeric film into a fluidic channel to control fluid flow. The power input for thermal expansion of each microfluidic valve can be controlled using a printed circuit board (PCB)-based controller, which is suitable for mass production and large-scale integration. A plastic microfluidic device with such valves was fabricated using compression molding and thermal lamination. The operation of the valves was investigated by measuring a change in the microchannel's ionic conduction current mediated by the resistance variation corresponding to the deflection of the microvalve. Valve closing was also confirmed by the disappearance of fluorescence when a fluorescent solution was displaced in the valve region. Valve operation was characterized for heater power ranging from 36 mW to 80 mW. When the valve was actuating, the local channel temperature was 10 to 19 degrees C above the ambient temperature depending on the heater power used. Repetitive valve operations (up to 50 times) have been demonstrated with a flow resulting from a hydrostatic head. Valve operation was tested for a flow rate of 0.33-4.7 microL/min.

A New Transepithelial Phototherapeutic Keratectomy Mode Using the NIDEK CXIII Excimer Laser

To evaluate epithelial healing, postoperative pain, and best spectacle-corrected visual acuity (BSCVA) after transepithelial photorefractive keratectomy (PRK) performed with a new phototherapeutic keratectomy (PTK) mode using the NIDEK CXIII excimer laser. Fifteen eyes from 10 patients with myopia underwent transepithelial PRK using a multistage program to perform PTK followed by PRK. The PTK incorporated Flex Scan, which accounts for the loss of radial ablation efficiency on the peripheral cornea. The epithelium was removed with the excimer laser by monitoring the disappearance of blue fluorescence during the ablation. Epithelial healing was evaluated by taking slit-lamp photographs every 24 hours until complete reepithelialization. Postoperative pain was measured according to the Faces Pain Rating Scale. All outcomes are reported for 3 months postoperatively. Haze was graded by two ophthalmologists, each masked to the other's result. Mean reepithelialization took 3.50+/-0.85 days, mean pain score was 3.00+/-1.20, and BSCVA was 20/20 for 9 eyes, 20/30 for 3 eyes, and 20/40 for 3 eyes. All patients had haze below grade 2. The outcomes of the preliminary study show that the incorporation of the Flex Scan algorithm in the PTK mode is as safe and effective as conventional PTK algorithms. The primary advantage of this new PTK mode may be more consistent epithelial removal. Additional studies are needed to determine long-term outcomes.

Enhanced generation of Alzheimer’s amyloid‐β following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C

Alzheimer's amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCalpha and PKCepsilon isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid-beta (Abeta) fragments of APP, although identification of the PKC substrate phospho-state-sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum-free medium is associated with several phenomena, namely: (i) PKCalpha down-regulation; (ii) PKCepsilon up-regulation; (iii) accumulation of APP and/or APP carboxyl-terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Abeta. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Abeta generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Abeta generation. These data have implications for the design of amyloid-lowering strategies based on modulating PKC activity.

Identification of an Autoinhibitory Mechanism That Restricts C1 Domain-mediated Activation of the Rac-GAP α2-Chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.

Difference in Tear Clearance Times between Standard Fluorescein Solution and a Novel Muco-Adhesive Fluorescein Solution

RationaleA non-irritative methylcellulose solution with muco-adhesive properties (MucoAd™) prolongs the effect of the decongestant, xylomethazoline, on nasal mucosa and has no irritative effects on rabbit conjunctiva. This double-blind study provides information on how the polymer solution behaves on the ocular surface. Delayed tear clearance would be beneficial in dry eye syndrome (DES) and would improve drug delivery to the conjunctival sack as in allergic conjunctivitis.MethodsTear clearance was assessed in 10 healthy volunteers by disappearance of fluorescein film from the eye. Following informed consent, medical history, general physical and eye examinations, standard fluorescein solution (control) and polymer solution-bound fluorescein (test) were dropped in each eye in random sequence. Fluorescence was observed by ocular biomicroscopy, and the time between solution administration and disappearance of fluorescence from the corneal surface was recorded. Upon conclusion of the measurement in each eye, the subject rated unpleasant sensations on a Visual Analogue Scale (VAS) from 0 (worst) to 100 mm (none). Wilcoxon Signed Ranks test was used for the analysis.ResultsCompared to the control solution, the muco-adhesive solution demonstrated longer clearance time (in minutes): 8,13 [median] (6,89 - 13,52) [25th - 75th interquartile range] vs. 5,93 (4,62 - 10,37), exact significance p = 0.002, and improved tolerability: VAS scores for the muco-adhesive solution was 100 (93 - 100) vs. 94 (55-100) for the control solution, exact significance p = 0.031.ConclusionA non-irritative methylcellulose solution with increased muco-adhesiveness delayed tear clearance assessed by fluorescein compared to standard, diagnostic fluorescein solution. The muco-adhesive solution was better tolerated. RationaleA non-irritative methylcellulose solution with muco-adhesive properties (MucoAd™) prolongs the effect of the decongestant, xylomethazoline, on nasal mucosa and has no irritative effects on rabbit conjunctiva. This double-blind study provides information on how the polymer solution behaves on the ocular surface. Delayed tear clearance would be beneficial in dry eye syndrome (DES) and would improve drug delivery to the conjunctival sack as in allergic conjunctivitis. A non-irritative methylcellulose solution with muco-adhesive properties (MucoAd™) prolongs the effect of the decongestant, xylomethazoline, on nasal mucosa and has no irritative effects on rabbit conjunctiva. This double-blind study provides information on how the polymer solution behaves on the ocular surface. Delayed tear clearance would be beneficial in dry eye syndrome (DES) and would improve drug delivery to the conjunctival sack as in allergic conjunctivitis. MethodsTear clearance was assessed in 10 healthy volunteers by disappearance of fluorescein film from the eye. Following informed consent, medical history, general physical and eye examinations, standard fluorescein solution (control) and polymer solution-bound fluorescein (test) were dropped in each eye in random sequence. Fluorescence was observed by ocular biomicroscopy, and the time between solution administration and disappearance of fluorescence from the corneal surface was recorded. Upon conclusion of the measurement in each eye, the subject rated unpleasant sensations on a Visual Analogue Scale (VAS) from 0 (worst) to 100 mm (none). Wilcoxon Signed Ranks test was used for the analysis. Tear clearance was assessed in 10 healthy volunteers by disappearance of fluorescein film from the eye. Following informed consent, medical history, general physical and eye examinations, standard fluorescein solution (control) and polymer solution-bound fluorescein (test) were dropped in each eye in random sequence. Fluorescence was observed by ocular biomicroscopy, and the time between solution administration and disappearance of fluorescence from the corneal surface was recorded. Upon conclusion of the measurement in each eye, the subject rated unpleasant sensations on a Visual Analogue Scale (VAS) from 0 (worst) to 100 mm (none). Wilcoxon Signed Ranks test was used for the analysis. ResultsCompared to the control solution, the muco-adhesive solution demonstrated longer clearance time (in minutes): 8,13 [median] (6,89 - 13,52) [25th - 75th interquartile range] vs. 5,93 (4,62 - 10,37), exact significance p = 0.002, and improved tolerability: VAS scores for the muco-adhesive solution was 100 (93 - 100) vs. 94 (55-100) for the control solution, exact significance p = 0.031. Compared to the control solution, the muco-adhesive solution demonstrated longer clearance time (in minutes): 8,13 [median] (6,89 - 13,52) [25th - 75th interquartile range] vs. 5,93 (4,62 - 10,37), exact significance p = 0.002, and improved tolerability: VAS scores for the muco-adhesive solution was 100 (93 - 100) vs. 94 (55-100) for the control solution, exact significance p = 0.031. ConclusionA non-irritative methylcellulose solution with increased muco-adhesiveness delayed tear clearance assessed by fluorescein compared to standard, diagnostic fluorescein solution. The muco-adhesive solution was better tolerated. A non-irritative methylcellulose solution with increased muco-adhesiveness delayed tear clearance assessed by fluorescein compared to standard, diagnostic fluorescein solution. The muco-adhesive solution was better tolerated.

Visualization of Rab3A dissociation during exocytosis: A study by total internal reflection microscopy

Rab3A is a small G protein in the Rab3 subfamily, and is thought to act at late stage of exocytosis. However, the detailed mechanism of its action is not completely understood. To study the role of Rab3A in exocytosis, we used a total internal reflection fluorescence microscope to examine the fluorescence changes of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles in PC12 cells upon stimulation. The fluorescence of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles decreased while showing different patterns. The NPY-EGFP-labeled vesicles that exocytosed showed a transient fluorescence increase before NPY-EGFP fluorescence disappearance, which represents fusion and NPY release. This transient increase was diminished in cells that co-expressed the GDP-bound Rab3A mutant. The fluorescence of EGFP-Rab3A-labeled vesicles dispersed before disappearance, which represents the dissociation of Rab3A from the vesicles. The dispersion was not found in GTP-bound Rab3A mutant-labeled vesicles. Interestingly, EGFP-Rab3A F59S, a mutant unable to bind rabphilin, dissociates slower from the vesicles than wild type Rab3A and caused a slower release of NPY-EGFP. The results provide direct evidence to support the hypothesis that GTP hydrolysis and rabphilin are involved in Rab3A dissociation from the vesicles and the occurrence of exocytosis.

Cargo Regulates Clathrin-Coated Pit Dynamics

Clathrin-coated pits (CCPs) are generally considered a uniform population of endocytic machines containing mixed constitutive and regulated membrane cargo. Contrary to this view, we show that regulated endocytosis of G protein-coupled receptors (GPCRs) occurs preferentially through a subset of CCPs. Significantly, GPCR-containing CCPs are also functionally distinct, as their surface residence time is regulated locally by GPCR cargo via PDZ-dependent linkage to the actin cytoskeleton. Such cargo-regulated CCPs show delayed recruitment of dynamin and can undergo an abortive event in which clathrin coats separate from the plasma membrane without concomitant receptor endocytosis. Segregation of cargo into CCP subsets, combined with cargo-dependent control of CCP dynamics, suggests a simple kinetic mechanism to generate functional specialization early in the endocytic pathway and reduce competition between diverse endocytic cargo.