Molecular Organization of Entamoeba Histolytica
Roberto R. Kretschmer in Amebiasis: Infection and Disease by Entamoeba histolytica, 2020
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
John H. Barker, Gary L. Anderson, Michael D. Menger in Clinically Applied Microcirculation Research, 2019
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.
Engineering Escherichia coli to Combat Cancer
Ananda M. Chakrabarty, Arsénio M. Fialho in Microbial Infections and Cancer Therapy, 2019
Next, we demonstrated that SIEC was able to inject a biologically active effector protein into tumor HeLa cells. During infection EPEC bacteria translocate their own specific receptor Tir (translocated intimin receptor) to the infected cells. The translocated Tir gets inserted in the host plasma membrane, and its extracellular domain is recognized by the OM protein Int exposed on the bacterial surface [98, 103]. The Int-Tir interaction leads to the intimate attachment of the bacteria to the infected host cell and triggers a localized F-actin polymerization underneath the attached bacteria that produces actin-rich pedestal-like protrusions of the host plasma membrane at the sites of bacterial intimate adhesion (Fig. 7.6A). These actin-rich accumulations are visible by fluorescence microscopy after staining of infected cells with a fluorophore phalloidin conjugate [97]. To test whether SIEC could inject Tir effector, we integrated an additional eLEE operon (called eLEE5) encoding Tir, Int, and the Tir-specific chaperone CesT in the flu locus (Antigen 43) of SIEC and SIECΔp1. Following integration, we obtained strains SIEC-eLEE5 and SIECΔp1-eLEE5, in which we confirmed the expression of Int and Tir.
Lepiota cristata does not contain amatoxins or phallotoxins
Published in Toxin Reviews, 2018
Ismail Yilmaz, Ilgaz Akata, Sinan Bakirci, Ertugrul Kaya
Amatoxins are a group of bicyclic octapeptides that occur in some Amanita, Galerina and Lepiota species. Alpha amanitin (AA), beta amanitin (BA), gamma amanitin (GA), epsilon amanitin, amaninamide, amanin and amanullin are some of the members of the amatoxin group. The phallotoxins include phalloidin (PHN), phallacidin (PCN), phallolisin, phallin and phallisasin (Enjalbert et al., 1999; Kaya et al., 2014a; Kaya et al., 2015). Intoxications by amatoxin-containing mushrooms account for only a small part of all poisonings (3%), which does not reduce their importance because almost all lethal mushroom poisonings are caused by amatoxins. It is assumed that AA is the most toxic agent in mushrooms. Amatoxins are more toxic than phallotoxins. While the toxic effect of phallotoxins is mild and causes alterations of the cellular membrane of enterocytes, amatoxins are the agents mainly responsible for fatal clinical poisonings (Bakirci et al., 2015; Escudié et al., 2007; Kaya et al., 2014b; Yilmaz et al., 2014). Edible and toxic mushrooms are often misidentified by mushroom collectors. One mushroom that is frequently confused in this respect is Lepiota cristata; its consumption can create a hazardous situation.
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).
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