Journal articles: 'Gap-FRAP'

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Abbaci, Muriel, Muriel Barberi-Heyob, Jean-René Stines, et al. "Gap junctional intercellular communication capacity by gap-FRAP technique: A comparative study." Biotechnology Journal 2, no. 1 (2007): 50–61. http://dx.doi.org/10.1002/biot.200600092.

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Li, Longkun, Chonghe Jiang, Ping Hao, Weibing Li, Caiping Song, and Bo Song. "Changes of gap junctional cell-cell communication in overactive detrusor in rats." American Journal of Physiology-Cell Physiology 293, no. 5 (2007): C1627—C1635. http://dx.doi.org/10.1152/ajpcell.00122.2007.

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To evaluate the changes in intercellular communication through gap junctions in detrusor overactivity (DO), we studied 23 adult female Wistar rats with DO after partial outflow obstruction (DO group) and 13 sham-operated rats (control group). The two groups were compared by means of urodynamics, light and electron microscopy, expression of Cx40, Cx43, and Cx45 mRNA genes with RT-PCR, Cx43 protein with Western blot analysis, and functional intercellular communication with scrape loading dye transfer (SLDT) and fluorescence recovery after photobleaching (FRAP). The number of gap junctions and the expression of connexin mRNA and Cx43 protein were increased in DO rats, and intercellular communication through gap junctions increased after 6 wk of partial outflow obstruction as assessed with SLDT and FRAP techniques. The findings provide a theoretical rationale for using Cx43 antagonists and gap junction inhibitors in the treatment of patients with overactive detrusor secondary to partial bladder outflow obstruction.

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Kuzma-Kuzniarska, Maria, Clarence Yapp, Thomas W. Pearson-Jones, Andrew K. Jones, and Philippa A. Hulley. "52 IN VITROANDEX VIVOASSESSMENT OF GAP JUNCTION FUNCTION IN TENDON USING FRAP." British Journal of Sports Medicine 48, Suppl 2 (2014): A34.1—A34. http://dx.doi.org/10.1136/bjsports-2014-094114.52.

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Yi, Chenju, Jérémy Teillon, Annette Koulakoff, Hugues Berry, and Christian Giaume. "Monitoring gap junctional communication in astrocytes from acute adult mouse brain slices using the gap-FRAP technique." Journal of Neuroscience Methods 303 (June 2018): 103–13. http://dx.doi.org/10.1016/j.jneumeth.2018.03.005.

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HAN Su-li, 韩素立, 李. 超. LI Chao, 郭. 峰. GUO Feng, and 邵. 晶. SHAO Jing. "Velocity profile measurement of oil films in a confined gap based on FRAP." Optics and Precision Engineering 25, no. 1 (2017): 141–47. http://dx.doi.org/10.3788/ope.20172501.0141.

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Kamioka, Hiroshi, Yoshihito Ishihara, Hans Ris, et al. "Primary Cultures of Chick Osteocytes Retain Functional Gap Junctions between Osteocytes and between Osteocytes and Osteoblasts." Microscopy and Microanalysis 13, no. 2 (2007): 108–17. http://dx.doi.org/10.1017/s143192760707016x.

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The inaccessibility of osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to osteocyte and between adjacent osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18α-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent osteocytes and between osteoblasts and osteocytes. This role is consistent with the ability of osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.

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Hu, G. L., Y. D. Fu, Q. L. Zeng, Z. P. Xu, and H. Chiang. "STUDY ON GAP JUNCTIONAL INTERCELLULAR COMMUNICATION INHIBITION BY ELF MAGNETIC FIELDS USING FRAP METHOD." Electromagnetic Biology and Medicine 21, no. 2 (2002): 155–60. http://dx.doi.org/10.1081/jbc-120006787.

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Lemcke, Heiko, Janine Peukert, Natalia Voronina, Anna Skorska, Gustav Steinhoff, and Robert David. "Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes." Journal of Molecular and Cellular Cardiology 98 (September 2016): 117–27. http://dx.doi.org/10.1016/j.yjmcc.2016.07.008.

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Anders, Juanita J., Suzanne Niedermair, Elaine Ellis, and Maureen Salopek. "Response of rat cerebral cortical astrocytes to freeze- or cobalt-induced injury: An immunocytochemical and gap-FRAP study." Glia 3, no. 6 (1990): 476–86. http://dx.doi.org/10.1002/glia.440030606.

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Alam, Mohammad Khairul, Rumana Tuli, Mohammad Sharif Khan, et al. "Chromatographic Assessment of Polyphenolic Profile and Total Phenolic Content and Antioxidant Activity of Common Leafy Vegetables in Bangladesh." Current Chromatography 7, no. 1 (2020): 40–50. http://dx.doi.org/10.2174/2213240607999200421144940.

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Background: Polyphenolic compounds are known to provide health benefits and protect against degenerative chronic diseases. Utilization and identification of foods with a high content of these compounds are gaining greater attention nowadays. Objective: The present study reports the total phenolic content (TPC), polyphenolic composition and antioxidant activity (DPPH, FRAP and TEAC) of 10 commonly consumed leafy vegetables growing in Bangladesh. Materials and Methods: The samples were collected from different locations of Bangladesh and mixed together to ensure sample representativeness. Folin-Ciocalteu method was used for the analysis of TPC, and quantification of polyphenolic components was done by high-performance liquid chromatography (HPLC- DAD). Additionally, antioxidant activities of the selected vegetables were also analysed by utilizing DPPH, FRAP & TEAC. Results and Discussion: TPC ranged from 23.64 ± 1.20 to 45.59 ± 3.04 mg gallic acid equivalent (GAE)/g freeze-dried sample (fds). The polyphenolic spectrum ranged from 0.30 ± 0.02 to 647.42 ± 147.12 mg/100 g fds; quantity and spectrum of which varied in the vegetables. Among the studied vegetables, Centella asiatica contained the highest amount of TPC (45.59 ± 3.04 mg GAE/g fds) and also exhibited high antioxidant capacities, as documented by DPPH, FRAP and TEAC assays. Moreover, Principal component analysis (PCA) of investigated variables clearly separated Centella asiatica from other samples. Conclusion: Phenolic compounds being strong antioxidants reduce the risk of chronic diseases and the finding of this study would aware the people to take vegetables rich in phenolics. It would also fill up the data gap in the existing food composition table of Bangladesh.

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Stines, J. R., M. Abbaci, W. Blondel, et al. "Optimisation instrumentale de la technique de Gap-FRAP visant à discriminer différents types cellulaires selon l'activité des jonctions communicantes." ITBM-RBM 26, no. 4 (2005): 279–81. http://dx.doi.org/10.1016/j.rbmret.2005.06.015.

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Świsłocka, Renata, Ewa Regulska, Joanna Karpińska, Grzegorz Świderski, and Włodzimierz Lewandowski. "Molecular Structure and Antioxidant Properties of Alkali Metal Salts of Rosmarinic Acid. Experimental and DFT Studies." Molecules 24, no. 14 (2019): 2645. http://dx.doi.org/10.3390/molecules24142645.

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The molecular structure of alkali metal rosmarinates was studied in comparison to rosmarinic acid using FT-IR, FT-Raman, 1H and 13C NMR spectroscopy, as well as density functional theory (DFT) calculations. The B3LYP/6-311+G(d,p) method was used to calculate optimized geometrical structures of studied compounds, atomic charges, dipole moments, energies, as well as the wavenumbers and intensities of the bands in vibrational and NMR spectra. Theoretical parameters were compared to experimental data. Antioxidant activity was determined using two spectrophotometric methods: (i) Assessing the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical and (ii) assay of antioxidant power of ferric ions reducing (FRAP). The linear correlations were found between HOMO–LUMO (highest occupied molecular orbital–lowest unoccupied molecular orbital) energy gap and the reducing power expressed as FRAP (R = 0.77) as well as between IC50 values (the ability of quenching DPPH radicals) and Δνas-s(COO) in IR spectra (differences between asymmetric and symmetric stretching vibrations bands) (R = 0.99). Photochemical properties of studied compounds were also evaluated. The influence of alkali metal on the electronic system of the rosmarinic acid molecule was discussed.

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Quesada, Ivan, Esther Fuentes, Etelvina Andreu, Paolo Meda, Angel Nadal, and Bernat Soria. "On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells." American Journal of Physiology-Endocrinology and Metabolism 284, no. 5 (2003): E980—E987. http://dx.doi.org/10.1152/ajpendo.00473.2002.

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Pancreatic β-cells constitute a well-communicating multicellular network that permits a coordinated and synchronized signal transmission within the islet of Langerhans that is necessary for proper insulin release. Gap junctions are the molecular keys that mediate functional cellular connections, which are responsible for electrical and metabolic coupling in the majority of cell types. Although the role of gap junctions in β-cell electrical coupling is well documented, metabolic communication is still a matter of discussion. Here, we have addressed this issue by use of a fluorescence recovery after photobleaching (FRAP) approach. This technique has been validated as a reliable and noninvasive approach to monitor functional gap junctions in real time. We show that control pancreatic islet cells did not exchange a gap junction-permeant molecule in either clustered cells or intact islets of Langerhans under conditions that allowed cell-to-cell exchange of current-carrying ions. Conversely, we have detected that the same probe was extensively transferred between islet cells of transgenic mice expressing connexin 32 (Cx32) that have enhanced junctional coupling properties. The results indicate that the electrical coupling of native islet cells is more extensive than dye communication. Dye-coupling domains in islet cells appear more restricted than previously inferred with other methods.

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Iranidokht, Vahid, Anestis Kalfas, Reza Abhari, Shigeki Senoo, and Kazuhiro Momma. "Sensitivity analysis on the impact of geometrical and operational variations on turbine hub cavity modes and practical methods to control them." Journal of the Global Power and Propulsion Society 5 (April 19, 2021): 66–78. http://dx.doi.org/10.33737/jgpps/133736.

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This paper presents an experimental investigation on the impact of different design and operational variations on the instabilities induced at the hub cavity outlet of a turbine. The experiments were conducted at the “LISA” test facility at ETH Zurich. The axial gap at the 2nd stage hub cavity exit was varied, and also three different flow deflectors were implemented at the cavity exit to control the cavity modes (CMs). Furthermore, the turbine pressure ratio was altered to mimic the off-design condition and study the sensitivity of the CMs to this parameter. Measurements were performed using pneumatic, and Fast Response Aerodynamic Probes (FRAP) at stator and rotor exit. In addition, unsteady pressure transducers were installed at the cavity exit wall to measure the characteristic parameters of the CMs. For the small axial gap, distinct and strong CMs were generated, which actively interacted with stator and rotor hub flow structures. Increasing the gap damped the fluctuations; however, a broader range of frequencies was amplified. The flow deflectors successfully suppressed the CMs by manipulating the shear layer velocity profile and blocking the growing instabilities. Eventually, the increase in the turbine pressure ratio strengthened the CMs and vice versa.

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Clair, Caroline, Cécile Chalumeau, Thierry Tordjmann, et al. "Investigation of the roles of Ca2+ and InsP3 diffusion in the coordination of Ca2+ signals between connected hepatocytes." Journal of Cell Science 114, no. 11 (2001): 1999–2007. http://dx.doi.org/10.1242/jcs.114.11.1999.

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Glycogenolytic agonists induce coordinated Ca2+ oscillations in multicellular rat hepatocyte systems as well as in the intact liver. The coordination of intercellular Ca2+ signals requires functional gap-junction coupling. The mechanisms ensuring this coordination are not precisely known. We investigated possible roles of Ca2+ or inositol 1,4,5-trisphosphate (InsP3) as a coordinating messengers for Ca2+ spiking among connected hepatocytes. Application of ionomycin or of supra-maximal concentrations of agonists show that Ca2+ does not significantly diffuse between connected hepatocytes, although gap junctions ensure the passage of small signaling molecules, as demonstrated by FRAP experiments. By contrast, coordination of Ca2+ spiking among connected hepatocytes can be favored by a rise in the level of InsP3, via the increase of agonist concentrations, or by a shift in the affinity of InsP3 receptor for InsP3. In the same line, coordination cannot be achieved if the InsP3 is rapidly metabolized by InsP3-phosphatase in one cell of the multiplet. These results demonstrate that even if small amounts of Ca2+ diffuse across gap junctions, they most probably do not play a significant role in inducing a coordinated Ca2+ signal among connected hepatocytes. By contrast, coordination of Ca2+ oscillations is fully dependent on the diffusion of InsP3 between neighboring cells.

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Liu, Wei, Rainer Duden, Robert D. Phair, and Jennifer Lippincott-Schwartz. "ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells." Journal of Cell Biology 168, no. 7 (2005): 1053–63. http://dx.doi.org/10.1083/jcb.200410142.

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Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with ∼40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly–disassembly cycle of the COPI coat in vivo.

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Li, Zhen, Xian-Ming Lin, Pei-Li Gong, Fan-Dian Zeng, and Guan-Hua Du. "Effects of Gingko biloba Extract on Gap Junction Changes Induced by Reperfusion/Reoxygenation After Ischemia/Hypoxia in Rat Brain." American Journal of Chinese Medicine 33, no. 06 (2005): 923–34. http://dx.doi.org/10.1142/s0192415x05003430.

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Gap junction communication between astrocytes plays an important role in the brain. The purpose of this study was to investigate the effects of Gingko biloba extract (GBE) on the changes of connexin 43 (Cx43) mRNA and protein expression levels of rat cortex and hippocampus induced by ischemia-reperfusion and astrocyte gap junction intercellular communication (GJIC) induced by hypoxia-reoxygenation. After 2 hours of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion, there was obvious neurological deficit in rats. Cx43 mRNA and protein expression levels of rat cortex and hippocampus in the ischemia hemisphere were decreased significantly. When GBE at doses of 50 and 100 mg/kg body weight was administrated by p.o. daily for 7 days, the neurological deficit was improved, and lower Cx43 mRNA and protein expression levels induced by ischemia-reperfusion were recovered to normal. The i.p. injection of nimodipine (0.7 mg/kg weight body) also showed improvement on neurological deficit and Cx43 expression levels. Astrocyte GJIC was measured by the fluorescence recovery after photobleaching (FRAP). Hypoxia-reoxygenation induced a significant decrease in GJIC. Pretreatment with GBE (100 mg/l) and nimodipine (1.6 mg/l) significantly prevented the hypoxia-reoxygenation inhibition of GJIC. These results suggest that GBE could exert its neuroprotective effects by improvement of Cx43 expression and GJIC induced by hypoxia/ischemia-reoxygenation/ reperfusion injury.

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Rhett, J. Matthew, Bennett W. Calder, Stephen A. Fann, Heather Bainbridge, Robert G. Gourdie, and Michael J. Yost. "Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2." American Journal of Physiology-Cell Physiology 313, no. 3 (2017): C314—C326. http://dx.doi.org/10.1152/ajpcell.00229.2016.

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Connexin-based therapeutics have shown the potential for therapeutic efficacy in improving wound healing. Our previous work demonstrated that the connexin43 (Cx43) mimetic peptide juxtamembrane 2 (JM2) reduced the acute inflammatory response to a submuscular implant model by inhibiting purinergic signaling. Given the prospective application in improving tissue-engineered construct tolerance that these results indicated, we sought to determine the mechanism of action for JM2 in the present study. Using confocal microscopy, a gap-FRAP cell communication assay, and an ethidium bromide uptake assay of hemichannel function we found that the peptide reduced cell surface Cx43 levels, Cx43 gap junction (GJ) size, GJ communication, and hemichannel activity. JM2 is based on the sequence of the Cx43 microtubule binding domain, and microtubules have a confirmed role in intracellular trafficking of Cx43 vesicles. Therefore, we tested the effect of JM2 on Cx43-microtubule interaction and microtubule polymerization. We found that JM2 enhanced Cx43-microtubule interaction and that microtubule polymerization was significantly enhanced. Taken together, these data suggest that JM2 inhibits trafficking of Cx43 to the cell surface by promoting irrelevant microtubule polymerization and thereby reduces the number of hemichannels in the plasma membrane available to participate in proinflammatory purinergic signaling. Importantly, this work indicates that JM2 may have therapeutic value in the treatment of proliferative diseases such as cancer. We conclude that the targeted action of JM2 on Cx43 channels may improve the tolerance of implanted tissue-engineered constructs against the innate inflammatory response.

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Khan, Shakeel Ahmad, Sammia Shahid, Waqas Bashir, Sadia Kanwal, and Ahsan Iqbal. "Synthesis, characterization and evaluation of biological activities of manganese-doped zinc oxide nanoparticles." Tropical Journal of Pharmaceutical Research 16, no. 10 (2017): 2331–39. http://dx.doi.org/10.4314/tjpr.v16i10.4.

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Purpose: To synthesize, characterize and investigate the antimicrobial properties of pure and manganese-doped zinc oxide nanoparticles.Method: Un-doped and manganese-doped zinc oxide (Mn-doped ZnO) nanoparticles were prepared using co-precipitation method. The synthesized Mn-doped ZnO nanoparticles were characterized using energy-dispersive x-ray spectroscopy (EDX), scanning electron microscopy (SEM), and x-ray diffraction (XRD) spectroscopic techniques. Their band gap energies were measured with ultraviolet-visible (UVVis) spectroscopy, while their antioxidant properties were evaluated by ferric reducing antioxidant power (FRAP), DPPH radical-scavenging, ferric thiocyanate (FTC) and total phenolic content (TPC) assays. The antimicrobial activities of the nanoparticles against different bacterial strains were determined using agar diffusion method.Result: Results from XRD, SEM, EDX and UV-Vis analyses demonstrated successful synthesis of undoped and Mn-doped ZnO nanoparticles as seen in their hexagonal, wurtzite structures. The un-doped and Mn-doped ZnO nanoparticles had average grain sizes of 16.72 nm and 17.5 nm, and band gap energies of 3.585 eV and 2.737 eV, respectively. Significant antibacterial activity was manifested by Mndoped ZnO against E. coli, S. aureus, Klebsiella and B. subtilis, with zones of inhibition (ZOIs) of 13 ± 0.09 mm, 14 ± 0.01 mm, 18 ± 0.07 mm and 20 ± 0.10 mm, respectively. The Mn-doped ZnO nanoparticles also exhibited effective and significant antioxidant potential relative to butylated hydroxytoluene (BHT) and un-doped ZnO nanoparticles.Conclusion: Mn-doped ZnO nanoparticles demonstrate significant antimicrobial and antioxidant activities. Thus, the preparation is a good candidate for further development into therapeutic formulations.Keywords: Mn-doped ZnO, Nanoparticles, Properties, Antioxidant, Antibacterial

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Pidoux, Guillaume, Pascale Gerbaud, Sédami Gnidehou, et al. "ZO-1 is involved in trophoblastic cell differentiation in human placenta." American Journal of Physiology-Cell Physiology 298, no. 6 (2010): C1517—C1526. http://dx.doi.org/10.1152/ajpcell.00484.2008.

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Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.

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Pfau, A., J. Schlienger, D. Rusch, A. I. Kalfas, and R. S. Abhari. "Unsteady Flow Interactions Within the Inlet Cavity of a Turbine Rotor Tip Labyrinth Seal." Journal of Turbomachinery 127, no. 4 (2003): 679–88. http://dx.doi.org/10.1115/1.2008973.

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This paper focuses on the flow within the inlet cavity of a turbine rotor tip labyrinth seal of a two stage axial research turbine. Highly resolved, steady and unsteady three-dimensional flow data are presented. The probes used here are a miniature five-hole probe of 0.9 mm head diameter and the novel virtual four sensor fast response aerodynamic probe (FRAP) with a head diameter of 0.84mm. The cavity flow itself is not only a loss producing area due to mixing and vortex stretching, it also adversely affects the following rotor passage through the fluid that is spilled into the main flow. The associated fluctuating mass flow has a relatively low total pressure and results in a negative incidence to the rotor tip blade profile section. The dominating kinematic flow feature in the region between cavity and main flow is a toroidal vortex, which is swirling at high circumferential velocity. It is fed by strong shear and end wall fluid from the pressure side of the stator passage. The static pressure field interaction between the moving rotor leading edges and the stator trailing edges is one driving force of the cavity flow. It forces the toroidal vortex to be stretched in space and time. A comprehensive flow model including the drivers of this toroidal vortex is proposed. This labyrinth seal configuration results in about 1.6% turbine efficiency reduction. This is the first in a series of papers focusing on turbine loss mechanisms in shrouded axial turbines. Additional measurements have been made with variations in seal clearance gap. Initial indications show that variation in the gap has a major effect on flow structures and turbine loss.

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Caviston, Juliane P., Mark Longtine, John R. Pringle, and Erfei Bi. "The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast." Molecular Biology of the Cell 14, no. 10 (2003): 4051–66. http://dx.doi.org/10.1091/mbc.e03-04-0247.

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The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36Gallele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36Gor GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.

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Santiquet, Nicolas W., Yann Develle, Anthony Laroche, Claude Robert, and François J. Richard. "Regulation of Gap-Junctional Communication Between Cumulus Cells During In Vitro Maturation in Swine, a Gap-FRAP Study1." Biology of Reproduction 87, no. 2 (2012). http://dx.doi.org/10.1095/biolreprod.112.099754.

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Lemcke, Heiko, Natalia Voronina, Gustav Steinhoff, and Robert David. "Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy." Journal of Visualized Experiments, no. 124 (June 19, 2017). http://dx.doi.org/10.3791/55870.

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Sahli, Khaled, Gagan D. Flora, Parvathy Sasikumar, et al. "Structural, Functional and Mechanistic Insights Uncover the Fundamental Role of Orphan Connexin62 in Platelets." Blood, August 21, 2020. http://dx.doi.org/10.1182/blood.2019004575.

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Connexins (Cxs) oligomerise to form hexameric hemichannels in the plasma membrane that can further dock together on adjacent cells to form gap junctions and facilitate intercellular-trafficking of molecules. In this study, we report the expression and function of an 'orphan' connexin, Cx62, in human and mouse (Cx57, mouse homologue) platelets. A novel mimetic peptide (62Gap27) was developed to target the second extracellular loop of Cx62 and 3D structural models predicted its interference with gap junction and hemichannel function. The ability of 62Gap27 to regulate both gap junction and hemichannel-mediated intercellular communication was observed using FRAP analysis and flow cytometry. Cx62 inhibition by 62Gap27 suppressed a range of agonist-stimulated platelet functions and impaired thrombosis and haemostasis. This was associated with elevated PKA-dependent signalling in a cyclic adenosine monophosphate-independent manner, and was not observed in Cx57 deficient mouse platelets (in which the selectivity of 62Gap27 for this connexin was also confirmed). Notably, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Together, our data reveal a fundamental role for a hitherto uncharacterised connexin in the regulation of the function of circulating cells.

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Mida, Kabran Guy Roger, Yeboue Koffi Apollinaire, Adopo Sopie Sheilla Flavie, Mamyrbékova-Békro Janat Akhanovna, and Békro Yves-Alain. "Chemical Characterization and in Vitro Antioxidant Activity of Honey from Different Localities of Côte D’ivoire." Chemical Science International Journal, July 14, 2021, 22–34. http://dx.doi.org/10.9734/csji/2021/v30i630236.

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Aims: Although the honey produced in Côte d'Ivoire is widely used in households, there is no technical sheet mentioning its chemical composition. Thus, the present study aimed to fill this gap while studying the chemical composition and antioxidant power of eleven honey samples from nine localities in Côte d'Ivoire. Methodology: The identification of secondary metabolites in the different honey samples was carried out by TLC and by color reaction tests. The quantification of these was performed by spectrophotometry. The antioxidant power of the honey samples was studied by DPPH and FRAP methods. Results: All honey samples contains secondary metabolites from various families, with varying contents of total phenolic compounds and flavonoids. Overall, the honey samples showed a notable antioxidant profile, making them good free radical scavengers. The relationship established between the different variables analyzed showed in some cases positive and significant correlations. Conclusion: The results of this study show that the honey produced in Côte d'Ivoire is of good quality. Therefore, its recurrent consumption could contribute to fighting certain pathologies caused by oxidative stress.

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Nieves-Morión, Mercedes, Conrad W. Mullineaux, and Enrique Flores. "Molecular Diffusion through Cyanobacterial Septal Junctions." mBio 8, no. 1 (2017). http://dx.doi.org/10.1128/mbio.01756-16.

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ABSTRACT Heterocyst-forming cyanobacteria grow as filaments in which intercellular molecular exchange takes place. During the differentiation of N2-fixing heterocysts, regulators are transferred between cells. In the diazotrophic filament, vegetative cells that fix CO2 through oxygenic photosynthesis provide the heterocysts with reduced carbon and heterocysts provide the vegetative cells with fixed nitrogen. Intercellular molecular transfer has been traced with fluorescent markers, including calcein, 5-carboxyfluorescein, and the sucrose analogue esculin, which are observed to move down their concentration gradient. In this work, we used fluorescence recovery after photobleaching (FRAP) assays in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 to measure the temperature dependence of intercellular transfer of fluorescent markers. We find that the transfer rate constants are directly proportional to the absolute temperature. This indicates that the “septal junctions” (formerly known as “microplasmodesmata”) linking the cells in the filament allow molecular exchange by simple diffusion, without any activated intermediate state. This constitutes a novel mechanism for molecular transfer across the bacterial cytoplasmic membrane, in addition to previously characterized mechanisms for active transport and facilitated diffusion. Cyanobacterial septal junctions are functionally analogous to the gap junctions of metazoans. IMPORTANCE Although bacteria are frequently considered just as unicellular organisms, there are bacteria that behave as true multicellular organisms. The heterocyst-forming cyanobacteria grow as filaments in which cells communicate. Intercellular molecular exchange is thought to be mediated by septal junctions. Here, we show that intercellular transfer of fluorescent markers in the cyanobacterial filament has the physical properties of simple diffusion. Thus, cyanobacterial septal junctions are functionally analogous to metazoan gap junctions, although their molecular components appear unrelated. Like metazoan gap junctions, the septal junctions of cyanobacteria allow the rapid intercellular exchange of small molecules, without stringent selectivity. Our finding expands the repertoire of mechanisms for molecular transfer across the plasma membrane in prokaryotes. IMPORTANCE Although bacteria are frequently considered just as unicellular organisms, there are bacteria that behave as true multicellular organisms. The heterocyst-forming cyanobacteria grow as filaments in which cells communicate. Intercellular molecular exchange is thought to be mediated by septal junctions. Here, we show that intercellular transfer of fluorescent markers in the cyanobacterial filament has the physical properties of simple diffusion. Thus, cyanobacterial septal junctions are functionally analogous to metazoan gap junctions, although their molecular components appear unrelated. Like metazoan gap junctions, the septal junctions of cyanobacteria allow the rapid intercellular exchange of small molecules, without stringent selectivity. Our finding expands the repertoire of mechanisms for molecular transfer across the plasma membrane in prokaryotes.

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Ram, Rashmi, Andrew P. Wescott, Tamlyn Thomas, Robert T. Dirksen, and Burns C. Blaxall. "Abstract 070: Interaction between Mena, Connexin43, and Rac1 at the Intercalated Disc." Circulation Research 113, suppl_1 (2013). http://dx.doi.org/10.1161/res.113.suppl_1.a070.

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Background: We previously reported that Mena, a member of Ena/VASP family of actin regulatory protein, is associated with heart failure (HF). Mena is localized to the intercalated disc (ICD) and interacts with ICD proteins implicated in HF. The ICD mediates force transmission and electrical coupling between cardiomyocytes. Slowed conduction in HF is associated with Cx43 remodeling, the predominant connexin isoform in the heart. Mena-/- mice develop cardiac dysfunction, conduction abnormalities, ICD disorganization, and Cx43 lateralization. We hypothesized that: 1) Mena’s interaction with the cytoskeleton is critical for ICD stabilization and maintenance of electrical function; 2) Mena may modulate signal(s) from the cardiac sarcolemma to the ICD for control of expression and localization of Cx43 through the regulation of the small GTPase Rac1. Methods and Results: Interaction of Mena with Cx43 and Rac1 was evaluated in neonatal rat ventricular myocytes (NRVM), HeLa cells, and in transgenic mice overexpressing constitutively active V12Rac1 (RacET). Pull down assay using GST-PAK1 demonstrated a direct association of Mena with active Rac1-GTP in vitro. In NRVMs, siRNA knockdown of Mena significantly increased activity of Rac1-GTPase by 2-fold (p=0.05, n=5). Western blot analysis revealed increased total Cx43 expression in Mena siRNA (1.11±0.2) compared to scramble (0.33±0.1, p<0.01, n=5). Enhanced Cx43 expression was associated with increased rate of recovery after photobleaching between cardiomyocytes by gap-FRAP analysis (Tau 153.0±4.8 vs. 73.0±1.8 sec, p<0.0001). Ongoing experiments with optical mapping of NRVM monolayers will determine whether conduction velocity may be enhanced after Mena knockdown. Confocal imaging revealed altered Cx43 trafficking and localization with Mena knockdown. Additionally in ventricles of RacET hearts, Mena expression was increased by 59% (p=0.01, n=4) compared to control, along with lateral redistribution of Cx43. Conclusions: Our results suggest that Mena is a critical regulator of the ICD at the intersection of Cx43 and Rac1. Increased Rac1 activation is associated with lateralized Cx43, and enhanced cellular coupling. Mena may modulate Cx43 localization through the regulation of Rac1 activity.

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Körner, Arlene, Matias Mosqueira, Markus Hecker, and Nina D. Ullrich. "Substrate Stiffness Influences Structural and Functional Remodeling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes." Frontiers in Physiology 12 (August 19, 2021). http://dx.doi.org/10.3389/fphys.2021.710619.

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Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte’s physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca2+ handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.

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Journal articles on the topic 'Gap-FRAP'

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