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Endothelial Cell Signaling During Wound Healing
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
HUVEC were then examined with antitalin and rhodamine-conjugated phalloidin staining under the same conditions (Figure 6, part B). Normal cells revealed well-formed focal adhesions distributed throughout their ventral surface (part B, panel a). Their tyrphostin-treated counterparts did not have discernible focal adhesions (part B, panel b). A diffuse brightness was again seen. Many cells treated with tyrphostin did not spread as well as untreated cells. HUVEC plated in control conditions demonstrated a dense array of well-developed stress fibers of polymerized actin that were visualized by staining with phalloidin (part B, panel c). In most HUVEC treated with tyrphostin, however, stress fibers were sparse and short (part B, panel d). These small stress fibers seemed to radiate from perinuclear foci. A rim of peripheral brightness was also seen.
Molecular Organization of Entamoeba Histolytica
Published in Roberto R. Kretschmer, Amebiasis: Infection and Disease by Entamoeba histolytica, 2020
Isaura Meza, Haydee K. Torres-Guerrero, Marco A. Meraz
Actively moving amebas do not show anisotropic filaments when studied with polarized microscopy or when stained by specific anti-actin antibodies. Nor is actin seen in filamentous form when phalloidin, a specific compound that binds to structured actin, is used. However, actin is present in the trophozoites, organized in diverse forms and actively participating in motility related processes. Actin was first identified in amebas using specific antibodies55,56 and was later isolated from E. histolytica HM1 cell extracts.57,58 The characterization of this abundant protein showed that it lacks the property of binding to and inhibiting DNAse I, a characteristic of all other eukaryotic actins that have been isolated. Purified amebic actin can be induced to polymerize into 7 nm filaments that are decorated with heavy meromyosin. In vivo, actin appears diffused in the cytoplasm, or in the form of aggregates in the leading pseudopod of actively moving amebas. In cells fixed at 37°C actin can be found organized in endocytic invaginations.36 Bailey et al.59,60 described the induction of actin phagocytic mouths at contact sites by challenging trophozoites with red blood cells, red blood cell ghosts or liposomes. Actin can be induced to form “adhesion plates” by contact of trophozoites with fibronectin or laminin-coated surfaces.36,61
Leukocyte/Endothelial Cell Adhesion and Ischemia/Reperfusion Injury
Published in John H. Barker, Gary L. Anderson, Michael D. Menger, Clinically Applied Microcirculation Research, 2019
Gary D. Dunn, D. Neil Granger, Ronald J. Korthuis
The accumulation of neutrophils in postischemic tissues is well-documented and it is assumed that these extravasated leukocytes are responsible for the production of parenchymal cell injury. Recent studies indicate that alterations in the endothelial cell cytoskeleton play an important role in neutrophil accumulation in inflamed tissues. For example, microfilament-rich endothelial cell lamellopodia extend luminally and envelope leukocytes as they emigrate.170 In addition, stabilization of F-actin in endothelial cells with phalloidin prevents the alterations in cytoskeletal architecture and leukocyte emigration that occurs in response to a variety of proinflammatory agents both in vitro and in vivo.171,172 Recent studies indicate that postischemic microvascular dysfunction and neutrophil sequestration can be attenuated by stabilization of microfilamentous cytoskeletal elements in the endothelium.46 Intravital microscopic studies indicate that phalloidin does not alter the increased leukocyte adherence to venular endothelium induced by I/R or venular exposure to PAF, LTB4, and FMLP.124,173 However, this agent completely prevented leukocyte emigration induced by these interventions.173 These studies suggest that prevention of leukocyte emigration by use of compounds that stabilize the architecture of the endothelial cell cytoskeleton may represent a useful new therapeutic approach to ameliorate neutrophil-dependent I/R injury.
The Association of RGS2 and Slug in the Androgen-induced Acquisition of Mesenchymal Features of Breast MDA-MB-453 Cancer Cells
Published in Endocrine Research, 2022
Dana B. Alsafadi, Mohammad S. Abdullah, Randa Bawadi, Mamoun Ahram
Immunostaining of RGS2, Slug, and AR was performed on cells seeded on type 1 collagen-coated glass coverslips as previously described.12 Briefly, cells were fixed in 4% paraformaldehyde and permeabilized by 0.5% Triton X-100. Mouse anti-RGS2, mouse anti-Slug (clone A-7), and rabbit anti-AR antibodies (clone N20) were incubated with the cells for 3 hrs at room temperature, then secondary antibodies, goat anti-rabbit Alexa Fluor 488 (R37116, Life Technologies) and goat anti-mouse Alexa Fluor 594 (R37121, Life Technologies), were added for 1 hr. Phalloidin conjugated with Alexa Fluor 488 (A12379, Life Technologies) was used for F-actin staining. Immunofluorescence was analyzed using Zeiss Axio Imager fluorescent microscope and images were processed using Zen 2012, blue edition (Zeiss, Germany).
l -Carnitine conjugated chitosan-stearic acid polymeric micelles for improving the oral bioavailability of paclitaxel
Published in Drug Delivery, 2020
Tan Yang, Jianfang Feng, Qian Zhang, Wei Wu, Hailan Mo, Lanzhen Huang, Wei Zhang
Chitosan (molecular weight 3–6 kDa, deacetylation degree 93.1%) was obtained from Yuhuan Marine Biochemistry Co., Ltd. (Yuhuan, China). LC-SA was purchased from Shanghai Luzhen Biotechnology Co., Ltd. (Shanghai, China). N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and LC were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Pyrene was obtained from Aldrich Chemical Co., Ltd. (Milwaukee, WI). TRITC-phalloidin was obtained from the AAT Bioquest Co. Ltd. (Shanghai, China). 4′,6-Diamidino-2-phenylindole (DAPI) and Hank’s balanced salt solution (HBSS) were obtained from the Thermo Fisher Scientific Co. Ltd. (Shanghai, China). All other reagents and solvents were of analytical grade and used without further purification. Twenty female and male Sprague-Dawley (SD) rats weighing 220 ± 20 g (n = 5 for each group) were fasted overnight before oral gavage but had free access to water, and then divided randomly into four groups. SD rats were purchased from the Silaikejingda Corporation (Changsha, China). All animal studies were carried out in accordance with the Guidelines for Animal Experimentation of Guilin Medical University and the protocol approved by the Animal Ethics Committee of the institution, and complied with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. Caco-2 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).
Clathrin-mediated integrin αIIbβ3 trafficking controls platelet spreading
Published in Platelets, 2018
Wen Gao, Panlai Shi, Xue Chen, Lin Zhang, Junling Liu, Xuemei Fan, Xinping Luo
Apyrase, PGE1, Fg, indomethacin, and PF-573228 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagen was purchased from chrono-log (Havertown, PA, USA). α-Thrombin was from Enzyme Research Laboratories (South Bend, IN, USA). AP2-mu, tyrphostin A23, cytochalasin, taxol, clathrin antibodies and control IgG were purchased from Santa Cruz (Dallas, TX, USA). Monoclonal antibody SZ21 [20] against human platelet β3 for immunofluorescence was a gift from Xiaodong Xi (Shanghai Jiao Tong University School of Medicine). FITC-conjugated-CD41 antibody was purchased from Beckman Coulter (Brea, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse antibody and rhodamine affinipure goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Anti-LAMP1 rabbit antibody, anti-EEA1 rabbit antibody, pitstop 2 (ab120687), and its negative control (ab120688) were purchased from Abcam (Cambridge, MA, USA). Rhodamine-conjugated phalloidin was from Life Technologies (Gaithersburg, MD, USA). Super signal chemiluminescent substrate was from Pierce (Rockford, IL, USA). Rabbit anti-integrin β3 polyclonal antibody and mouse anti-GAPDH monoclonal antibody were from Cell Signaling Technology (Danvers, MA, USA). Anti-FAK rabbit polyclonal antibody was purchased from EMD Millipore (Billerica, MA, USA). Piceatannol was purchased from Cayman Chemical (Ann Arbor, MI, USA). U0126 was from Gene Operation (Ann Arbor, MI, USA). PP2 was purchased from Calbiochem (Darmstadt, Germany). Dynasore, U73122, apoptozole and Ro 31-8220 were purchased from Selleck (Houston, TX, USA).