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Inter- and Intracellular Signaling in Plant Cells with Participation of Neurotransmitters (Biomediators)
Published in Akula Ramakrishna, Victoria V. Roshchina, Neurotransmitters in Plants, 2018
Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using anticontractile agents (Figure 9.5) such as (1) binding with actin of microfilaments—cytochalasin В (an inhibitor of actin polymerization in microfilaments) and phalloidin (binds with actin of microfilaments and hinders its depolymerization) and (2) binding with tubulin-containing microtubules—alkaloids colchicine and vinblastine (inhibitors of tubulin polymerization in microtubules) or taxol (stabilizer of microtubules depolymerization by suppressing that disrupts the normal process of dynamic reorganization of the network of microtubules, which is important for cellular functions at the stage of mitosis and cell cycle interphases). Besides above-mentioned reagents, latrunculins A and B are used as inhibitors of G-actin-polymerization, toxins from marine sponges (for example from genus Latrunculia) or phalloidin, bicyclic heptapeptide from fungi Amanita phalloides, that binds with F-actin and stops is depolymerization (Figure 9.5).
Endolysosomal Patch Clamping
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Cheng-Chang Chen, Christian Grimm, Christian Wahl-Schott, Martin Biel
To functionally characterize ion channels in early endosomal membranes with the patch clamp technique, it is essential to selectively enlarge early endosomes. Inhibition of Rab5 activity stimulates membrane fusion between early endosomes, which can be achieved by transfection with Rab5-Q79L, a dominant-negative Rab5 mutant. This manipulation can selectively enlarge early endosomes to up to 5 µm in diameter, allowing ion channels on the early endosomes to be examined by patch clamping (Cang et al., 2015). To transfect Rab5-Q79L into HEK293 cells or COS-1 cells, PolyJet (SignaGen Laboratories) or Turbofect (Thermo Fisher Scientific) can be used; Lipofectamine LTX (Invitrogen) or Lipofectamine 3000 (Invitrogen) should be used for neurons, glial cells or RAW264.7 macrophages (Cang et al., 2015). However, gene transfection in native cells, such as primary macrophages, is very difficult. Therefore, pharmacological tools have also been sought after. Vicenistatin, a natural compound that enhances homotypic fusion between early endosomes, has recently been reported as a possible small molecule tool for early endosome (Rab5-positive) enlargement, and the vacuoles produced by a treatment with 300 nM vicenistatin for 2 hours are suitable for patch clamping (Wang et al., 2017). Alternatively, a combined treatment with two small molecules, wortmannin and latrunculin B, also selectively enlarges Rab5-positive early endosomes but not Rab7-, LAMP1- or Rab11-positive vesicles (Chen et al., 2017b). Typically, cells are treated with 200 nM wortmannin in combination with 10 nM latrunculin B (e.g., 10–15 min for HEK293 cells; 20 min for peritoneal macrophages; 20 min for lung-tissue macrophages) prior to experimentation. The compounds are washed out before the patch clamp experiment. Wortmannin and latrunculin B can be purchased from Sigma-Aldrich (Cat. No. W1628 and L5288, respectively) and stored at −20°C for 2–4 months. Only freshly diluted compounds should be used.
Flow cytometry identifies an early stage of platelet apoptosis produced by agonists of the P2X1 and P2X7 receptors
Published in Platelets, 2022
Joelyn Wong, Ben J. Gu, Harry Teoh, Malgorzata Krupa, Mastura Monif, Mark Slee, James S. Wiley
2ʹ(3ʹ)-O-(4-benzoylbenzoyl)-ATP (BzATP), ATP, αβ-meATP, ADP, bovine thrombin, methyl-beta-cyclodextrin, ethidium bromide, calcium chloride, bovine serum albumin, glucose, sodium chloride, potassium chloride, latrunculin A and apyrase were bought from Sigma Aldrich (St. Louis, USA). Latrunculin A was kept as a frozen stock solution at 500 μM in DMSO. Cyclodextrin was kept as a frozen stock solution at 200 mM in buffered saline. MRS2365 was from Tocris Bioscience and used at a final concentration of 1μM. MitoTracker Green (MTG) and MitoTracker DeepRed (MTdR) were obtained from Molecular Probes (Oregon, USA). Annexin V antibody was bought from eBioscience. Monoclonal antibody CD41 was from Becton Dickenson and CD61 was from Dako. Mouse anti-human P2X7 receptor monoclonal antibody (clone L4) was conjugated with either FITC or Alexa-647 using protein labeling kits as per manufacturer’s instructions [26]. The isotype control was a monoclonal IgG2b (clone WMD7) conjugated with Alexa-647. Sheep anti-P2X7 polyclonal antibody against a nonhomologous epitope of human P2X7 has been previously described [27]. HEPES-buffered saline contained NaCl 140 mM, HEPES 10 mM, MgCl2 0.1 mM, and pH 7.1.
Biochemical and immunocytochemical characterization of coronins in platelets
Published in Platelets, 2020
David R. J. Riley, Jawad S. Khalil, Khalid M. Naseem, Francisco Rivero
Washed platelet suspensions (5 × 108 platelets/ml), either untreated or treated with various substances for the appropriate time, were mixed with an equal volume of fractionation buffer (320 mM sucrose, 4 mM HEPES, 0.5 mM Na3VO4, pH 7.4) supplemented with phosphatase and protease inhibitor cocktail. Latrunculin B (LatB) was used at 20 µM for 20 min to depolymerize F-actin prior to lysis. Samples were subjected to five freeze-thaw cycles in liquid nitrogen. Intact platelets were removed by centrifugation at 1,000 × g for 5 min at 4°C and fractionation was done by centrifugation at 100,000 × g for 60 min at 4°C. The fractions were normalized by volume and analyzed by Western blot.
Structure–activity relationship of a peptide permeation enhancer
Published in Tissue Barriers, 2023
L-R5 is more successful in increasing the permeability of FD-4 compared to the PEs bilobalide and latrunculin A (Figure 2A). The mechanisms of action of these two molecules are similar, as they both influence the cytoskeleton of the cell. By interacting with the adenosine A1 receptor, bilobalide causes contraction of the actin filament, which transiently opens intercellular junctions.41 Latrunculin A enters the cell and disrupts the cytoskeletal filaments, distorting the cell and allowing larger molecules to pass through cellular junctions.42 L-R5 has been designed to inhibit the activity of PKC ζ in phosphorylating TJs proteins. Opening of TJs may be also due to influence of L-R5 cytoskeleton, as the enzyme was shown to be involved in cytoskeletal organization.31,45 The opening induced by L-R5 appears to lead to a higher permeability with a faster onset, however, it should be noted that the applied concentrations of the other two molecules necessary to open TJs are much lower. An equivalent concentration of the peptide may give the same or even better results. The problem is that since latrunculin A is derived from a sponge toxin,46 an increase in concentration may lead to safety issues. By contrast, bilobalide showed no cytotoxicity at a concentration of 625 µM and may be more effective at higher concentrations if no increase in toxicity is observed.41 The fast increase in the first 15 minutes of each condition may be due to a hormetic effect,47 even if this difference is not significant. More specifically, the modification of the ambient environment will inevitably force the cell to adapt. A greater disruption of the cell membrane by FAs is also a potential reason for this increase, which would lead to a short increase in paracellular permeability. Another explanation of this short greater permeation enhancement the formation of micelles between FD-4 and FAs.