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Cytochalasin B (cytochalasin + b)
Selected AbstractsCalyculin-A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggsCYTOSKELETON, Issue 4 2001Yukako Asano Abstract When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a 32P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis. Cell Motil. Cytoskeleton 48:245,261, 2001. © 2001 Wiley-Liss, Inc. [source] Functional importance of the actin cytoskeleton in contraction of bovine iris sphincter muscleAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2002J. A. C. Filipe Summary 1 The contractile capacity of smooth muscle cells depends on the cytoskeletal framework of the cell. The aim of this study was to determine the functional importance of both the actin and the tubulin components of the cytoskeleton in contractile responses of the bovine isolated iris sphincter muscle. 2 In each preparation, two contractions to the muscarinic agonist carbachol were obtained. The maximum responses of the first contractions were taken as 100%. The second contractions to carbachol were elicited in the presence of either cytochalasin B (50 and 5 ,m), an inhibitor of the actin cytoskeleton, or colchicine (100 ,m), an inhibitor of the tubulin cytoskeleton (30 min incubation). 3 Cytochalasin B, at a concentration of 50 ,m, significantly decreased the contractions induced by carbachol, with the maximum response reduced to 21.8 ± 6.6% (n = 12) of the initial maximum. The maximal contractions to carbachol in the presence of colchicine reached 96.2 ± 7.9% (n = 9) of the initial contraction, which was not significantly different from control second responses to carbachol with neither drug present, which reached 113.3 ± 7.6% (n = 7). 4 The effect of cytochalasin B was dose-dependent, since at a lower concentration of 5 ,m, the drug decreased the maximum contraction to carbachol to 60.3 ± 8.8% (n = 6). The effect of cytochalasin B was at least partially reversible, since after the use of the higher concentration of 50 ,m, contractions to carbachol increased to 62.3 ± 15.5% (n = 4) of the maximal response, after 1 h repeated washing of the preparations. 5 Cytochalasin D, at a concentration of 50 ,m, completely abolished the contractions induced by carbachol (n = 4). 6 These findings suggest that in bovine iris sphincter muscle, contractions to carbachol are highly dependent, from a functional point of view, on actin polymerization, and not, to any important degree, on the polymerization of tubulin. [source] CD203c-based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation,CYTOMETRY, Issue 5 2010Eva M. Sturm Abstract Background: The basophil activation test (BAT) based on CD203c upregulation has been validated as a reliable tool for the diagnosis of IgE-mediated allergies. Nevertheless, CD203c-based BAT is hardly comparable with that of CD63-based tests, as the mechanisms of CD203c versus CD63 induction differ considerably. The aim of the present study was to identify potent influencing factors of the CD203c-based BAT and to emphasize differences between CD63 and CD203c detection. Methods: CD203c-based BAT was determined in 82 healthy controls and in 79 allergic patients. The effects of interleukin (IL)-3 and degranulation enhancing substances were investigated and compared with CD63 upregulation. Furthermore, the influence of different storage conditions and incubation times was evaluated and the impact of antiallergic drugs on the test results was assessed. Results: CD203c and CD63 expression was rapidly upregulated reaching a maximum after 20,30 min. Basophil CD203c upregulation assayed after storage times up to 48 h declined already after 4 h. IL-3 treatment increased CD203c and CD63 baseline levels and decreased basophil CD203c responses in a dose-dependent manner. In contrast, cytochalasin B and latrunculin B did not affect CD203c responses but decreased CD63-based BAT. Finally, therapeutic concentrations of dimetindene and desloratadine did not affect CD203c upregulation. Conclusion: CD203c-based basophil activation test should be performed preferentially within 4 h after taking the blood samples. Priming and degranulation-enhancing factors are not required for CD203c-based BAT. In contrast to skin testing, CD203c-based BAT can be performed in patients undergoing antiallergic treatment. © 2010 International Clinical Cytometry Society [source] Characterization of the myo -inositol transport system in Trypanosoma cruziFEBS JOURNAL, Issue 9 2000Marcelo Einicker-Lamas myo -Inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo -inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo -inositol translocation across the parasite cell membrane. myo -Inositol uptake was concentration-dependent in the concentration range 0.1,10 µm with maximal transport obtained at 8 µm. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo -inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+ -ATPase, inhibited the Na+ -dependent and Na+ -independent myo -inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo -inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p -(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 °C. The addition of glucose (5,50 mm) or mannose (10 mm) did not change the myo -inositol uptake, whereas the addition of 10 mm nonlabeled myo -inositol totally inhibited this transport, indicating that the transporter is specific for myo -inositol. Phloretin (0.3 mm) and phoridzin (5 mm), but not cytochalasin B, were efficient inhibitors of myo -inositol uptake. A portion of the accumulated myo -inositol is converted to inositol phosphates and phosphoinositides. These data show that myo -inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters , one Na+ -dependent and the other Na+ -independent. [source] ASSESSING ABSORBABILITY OF BIOACTIVE COMPONENTS IN ALOE USING IN VITRO DIGESTION MODEL WITH HUMAN INTESTINAL CELLJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2010SOON-MI SHIM ABSTRACT This study used a simulated in vitro digestion model coupled with caco-2 cell to assess the digestive stability and absorption of aloin, aloe-emodin and aloenin A. Aloenin A and aloe-emodin were stable and entirely recovered during simulated digestion, but 50% of aloin was lost. Approximately 53.2, 7.3 and 28.7% of aloe-emodin, aloenin A and aloin, respectively, was transported into both apical and basolateral compartments after 1 h incubation in caco-2 cell. The involvement of several transporter proteins for aloin and aloenin A was examined. An inhibitor of SGLT1 on apical surface (phloridzin) or that of GLUT2 on basolateral membrane (cytochalasin B) reduced the absorption of aloin by 40 or 60%, respectively, indicating that aloin is likely to be a partial substrate of SGLT1. In the presence of an efflux transporter inhibitor (verapamil), the transport of aloenin A through an intentinal apical membrane increased up to 2.1 times compared with the control (without verapamil). PRACTICAL APPLICATIONS Our results on both digestive stability and intestinal absorption characteristics of bioactive components in aloe could be of helpful information for promoting its bioavailability. The in vitro technique described in this study provides a rapid and cost-effective alternative for predicting bioavailability of biomarkers in aloe functional food. [source] The basophil activation test in the diagnosis of allergy: technical issues and critical factorsALLERGY, Issue 9 2009G. J. Sturm Background:, The basophil activation test (BAT) is a widely validated and reliable tool especially for the diagnosis of hymenoptera venom allergy. Nevertheless, several pitfalls have to be considered and outcomes may differ because of diverse in-house protocols and commercially available kits. We aimed to identify the factors that may influence results of the CD63-based BAT. Methods:, Basophil responses to monoclonal anti-IgE (clone E124.2.8) and bee and wasp venom were determined by BAT based on CD63. The effect of stimulating factors such as, IL-3, cytochalasin B and prewarming of the samples was investigated. Furthermore, we compared two different flow cytometer systems and evaluated the influence of storage time, different staining protocols and anti-allergic drugs on the test results. Results:, Interleukin-3 enhanced the reactivity of basophils at 300 pM, but not at 75 and 150 pM. Prewarming of samples and reagents did not affect basophil reactivity. CD63 expression assayed after storage time of up to 48 h showed that basophil reactivity already started to decline after 4 h. Basophils stained with HLA-DR-PC5 and CD123-PE antibodies gated as HLA-DRneg/CD123pos cells showed the highest reactivity. No effect on test outcomes was observed at therapeutic doses of dimetindene and desloratadine. Finally, slight differences in the percentage of activated basophils, depending on the cytometer system used, were found. Conclusion:, Basophil activation test should be performed as early as possible after taking the blood sample, preferably within 4 h. In contrast to the skin test, BAT can be performed in patients undergoing treatment with antihistamines. For reasons of multiple influencing factors, BAT should be performed only at validated laboratories. [source] Effects of Gonadotropins on In Vitro Maturation and of Electrical Stimulation on Parthenogenesis of Canine OocytesREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010BS Kim Contents The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 ,s with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 ,s without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines. [source] Growth and reproductive performance of triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814)AQUACULTURE RESEARCH, Issue 2 2009Wenshan Liu Abstract Growth and reproduction of triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were compared in a 30-month study. Triploidy was induced by inhibition of the second polar body formation using 6-dimethylaminopurine (6-DMAP) or cytochalasin B (CB). There were no significant differences in growth and survivorship between triploid and diploid abalone. However, triploid abalone had a more elongated shell and greater foot muscles than diploid abalone. A slightly curvilinear growth in shell length was conformed to all treatments. While diploid abalone had reached sexual maturity and spawned during the study, gonadal development and gamete maturation were abnormal in triploids. Female triploids lacked an apparent gonad at the macroscopic level but microscopic examination revealed that they had a thin layer of oogonia development. In contrast, male triploids were able to form similar-sized gonads to diploids during most of the reproductive period, but with brown-yellow discolouration and stalled gametogenesis at spermatocyte formation. Sex ratio of triploid abalone did not deviate from 1:1. With the onset of sexual maturation, growth and gonadal maturation occurred concurrently in diploid abalone, and there was no indication that growth of (diploid) abalone was reduced. [source] Functional importance of the actin cytoskeleton in contraction of bovine iris sphincter muscleAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2002J. A. C. Filipe Summary 1 The contractile capacity of smooth muscle cells depends on the cytoskeletal framework of the cell. The aim of this study was to determine the functional importance of both the actin and the tubulin components of the cytoskeleton in contractile responses of the bovine isolated iris sphincter muscle. 2 In each preparation, two contractions to the muscarinic agonist carbachol were obtained. The maximum responses of the first contractions were taken as 100%. The second contractions to carbachol were elicited in the presence of either cytochalasin B (50 and 5 ,m), an inhibitor of the actin cytoskeleton, or colchicine (100 ,m), an inhibitor of the tubulin cytoskeleton (30 min incubation). 3 Cytochalasin B, at a concentration of 50 ,m, significantly decreased the contractions induced by carbachol, with the maximum response reduced to 21.8 ± 6.6% (n = 12) of the initial maximum. The maximal contractions to carbachol in the presence of colchicine reached 96.2 ± 7.9% (n = 9) of the initial contraction, which was not significantly different from control second responses to carbachol with neither drug present, which reached 113.3 ± 7.6% (n = 7). 4 The effect of cytochalasin B was dose-dependent, since at a lower concentration of 5 ,m, the drug decreased the maximum contraction to carbachol to 60.3 ± 8.8% (n = 6). The effect of cytochalasin B was at least partially reversible, since after the use of the higher concentration of 50 ,m, contractions to carbachol increased to 62.3 ± 15.5% (n = 4) of the maximal response, after 1 h repeated washing of the preparations. 5 Cytochalasin D, at a concentration of 50 ,m, completely abolished the contractions induced by carbachol (n = 4). 6 These findings suggest that in bovine iris sphincter muscle, contractions to carbachol are highly dependent, from a functional point of view, on actin polymerization, and not, to any important degree, on the polymerization of tubulin. [source] Pancreatic fate of D -[3H] mannoheptuloseCELL BIOCHEMISTRY AND FUNCTION, Issue 3 2001Willy J. Malaisse Abstract D -Mannoheptulose was recently postulated to be transported into cells by GLUT2. The validity of such an hypothesis was assessed by comparing the uptake of tritiated D -mannoheptulose by pancreatic islets versus pieces of pancreas and, in the latter case, by comparing results obtained in control rats versus animals injected with streptozotocin (STZ). The uptake of D -[3H] mannoheptulose by islets represents a time-related and temperature-sensitive process, inhibited by cytochalasin B and enhanced by D -glucose. The uptake of the tritiated heptose was much lower in pieces of pancreatic tissue and inhibited by D -glucose, at least in the STZ rats. Whether in pieces of pancreas exposed in vitro to D -[3H] mannoheptulose or after intravenous injection of the tritiated heptose, the radioactive content of the pancreatic tissue was lower in STZ rats than in control animals. This contrasted with an unaltered radioactive content of liver and muscle in the STZ rats, at least when treated with insulin. Suitably radiolabelled D -mannoheptulose or an analogue of the heptose could thus conceivably be used for quantification of the endocrine pancreatic mass. Copyright © 2001 John Wiley & Sons, Ltd. [source] | ||||||||||||||||