Gliding motility of algae is unaffected by cytochalasin B

The effect of the antibiotic cytochalasin B on gliding motility of Oscillatoria and Navicula was studied using time lapse microcinematography. Contrary to its effects on many other non-muscle motile systems, cytochalasin B did not significantly inhibit motility.     

Preparation,In Vivo Administration,Dose-Limiting Toxicities,and Antineoplastic Activity of Cytochalasin B

An effective and inexpensive protocol for producing cytochalasins A and B is being disclosed to propose a viable method by which to examine the in vivo antineoplastic activity of these congeners in preclinical tumor-bearing mammalian models. In addition, we determine the maximum tolerated doses of cytochalasin B using multiple routes and formulations, characterize the tissue distribution of intravenous bolus cytochalasin B, and assess the in vivo antineoplastic activity of cytochalasin B in comparison in doxorubicin in Balb/c mice challenged intradermally with M109 murine lung carcinoma. We also examine the effects of cytochalasin B against several other murine neoplastic cell lines (Lewis lung, LA4, B16F10, and M5076). Finally, we examine a potential mechanism of the antimetastatic activity of cytochalasin B by observing the effects of the agent on the secretion of N-acetylglucosaminidase (GlcNACase) by B16BL6 and B16F10 murine melanomas in vitro. The results of the study can be summarized as follows: 1) Cytochalasin B can be safely administered intravenously, intraperitoneally, and subcutaneously in murine models, with the maximum tolerated dose of all routes of administration being increased by liposome encapsulation. 2) Cytochalasin B can significantly inhibit the growth of tumors in mice challenged with M109, Lewis lung, LA4, B16F10, or M5076, producing long-term survival against lung carcinomas and adenocarcinomas (M109, Lewis lung, and LA4) and B16F10 melanoma, but not M5076 sarcoma. These effects were comparable to intraperitoneally administered doxorubicin. 4) Low concentrations of cytochalasin B inhibit the secretion of GlcNACase, indicating that cytochalasin B may inhibit metastatic progression by mechanisms not directly associated with its influence on cell adhesion and motility.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Effect of cytochalasin B on formation and properties of muscle F-actin

Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(α-aminoethyl ether)-N,N′-tetraacetic acid and reduced by MgCl₂ at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B.     

INHIBITION OF PHAGOCYTOSIS AND PLASMA MEMBRANE MOBILITY OF THE CULTIVATED MACROPHAGE BY CYTOCHALASIN B : Role of Subplasmalemmal Microfilaments

Functional and morphologic effects of cytochalasin B on the cultivated macrophage were examined to determine the basis for plasma membrane movements of the type required for endocytosis and/or spreading on a substratum. Inhibition of phagocytosis and changes in cell shape by cytochalasin B exhibited nearly identical dose-response curves requiring 2–5 x 10^-6 M and 1–2 x 10^-5 M cytochalasin B to inhibit these functions by 50% and 100%, respectively. In contrast, hexose transport was ten times more sensitive to the drug requiring 2–3 x 10^-7 M cytochalasin B to achieve 50% inhibition of 2-deoxyglucose uptake. Inhibition of phagocytosis and changes in cell shape could not be explained solely by drug effects on hexose transport. Analysis of serial thin sections showed that cytochalasin B doses inhibitory for hexose transport had no effect on distribution or organization of either of the two subplasmalemmal microfilament types. However, cytochalasin B concentrations (2.0 x 10^-5 M) that inhibited phagocytosis and altered cell shape disorganized and/or disrupted oriented bundles of 40–50-Å subplasmalemmal microfilaments, but had no effect on the microfilamentous network. Comparative dose-response studies showing positive correlations among cytochalasin B effects on phagocytosis, changes in cell shape, and alterations in oriented subplasmalemmal microfilament bundles provide additional support for the hypothesis that microfilamentous structures play a role in translocation of plasma membrane required for endocytosis and cell motility.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture

The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-³H]cytochalasin B and transport of $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-[}{}^{14}\text{C]glucose}$ into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a K_D of 2.86 · 10^?7 M, while the low-affinity site had a K_D of 1.13 · 10^?5M. Sugar transport was monitored by $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-}\text{glucose}$ uptake and it was found that cytochalasin B (10^?5M) drastically inhibited transport. However, D-glucose (10^?5M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.     

Cytochalasin B. VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells

Cytochalasin B competitively inhibits the transport of uridine and thymidine by Novikoff rat hepatoma cells growing in suspension culture with apparent K_i''s of 2 and 6 µM, respectively, but has no effect on the intracellular phosphorylation of the nucleosides. Choline transport is not affected by cytochalasin B. Results from pulse-chase experiments indicate that cytochalasin B has no direct effect on the synthesis of RNA, DNA, or uridine diphosphate-sugars. The inhibition of uridine and thymidine incorporation into nucleic acids by cytochalasin B is solely the consequence of the inhibition of nucleoside transport.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A,B, C,D and E: Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of β-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 2 μM cytochalasin A, C >μM cytochalasin B ? 4–5 μM cytochalasin D, E. While maximal rates of O₂^? release and extents of exocytosis require extracellular calcium (1–2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na⁺, K⁺ or choline inhibited either cytochalasin B- or E-stimulated O₂^? production with IC₅₀ values of 5–10 mM and inhibition occurs whether Cl^?, NO₃^? or SCN^? is the anion added with Na⁺ or K⁺. Release of β-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl (IC₅₀ ≈ 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K⁺ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of β-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O₂^? or β-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

The effect of cytochalasin B,colchicine and vinblastine on the adhesion of Trichomonas vaginalis to glass surfaces

The attachment of Trichomonas vaginalis to glass surfaces was studied in the presence of cytochalasin B, colchicine and vinblastine. Marked inhibition of adhesion was noted with high concentrations of cytochalasin B. Colchicine and vinblastine were without effect. These findings suggest that Trichomonas vaginalis adhesion is at least partially mediated by the extension of cellular probes, due to the action of cytochalasin-sensitive microfilaments.     

Effect of cytochalasins on surfactant release from alveolar type II cells

Cytochalasins enhanced surfactant secretion from primary cultures of [³H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[³H]choline ([³H]PC) in a dose-dependent manner with EC₅₀ values of 1, 2, 0.5, 0.1 and 1 μM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1–1.0 μM for cytochalasins C and D between 1 and 10 μM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [³H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the β-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.     

GLIDING MOTILITY OF OSCILLATORIA: ULTRASTRUCTURAL AND CHEMICAL CHARACTERIZATION OF THE FIBRILLAR LAYER1,2

Ultrastructural examination of platinum-carbon replicated trichomes or wall fragments of 3 Oscilatoria species readily displayed the fibrillar components of the presumed motility system. These fibrils had a lateral organization as well as longitudinal continuity and wavelike distortions, which are consistent with the hypothesized movement mechanism. The fibrils were sensitive to proteolytic enzymes but resistant to hydrolytic agents, suggesting a protein composition. The fibrils and motility were unaffected by cytochalasin B.     

Effect of inhibitors on glucose transport in malaria (Plasmodium berghei) infected erythrocytes

Effect of inhibitors on glucose transport in malaria (Plasmodium berghei) infected erythrocytes. International Journal for Parasitology16: 441–446. The effect of cytochalasin B and phloretin on transport of d-glucose and 2-deoxy-d-glucose into Plasmodium berghei-infected mouse erythrocytes was studied. Both the inhibitor-sensitive and insensitive fractions of transport in the infected erythrocytes were increased compared with normal erythrocytes. The i₅₀ values (concentrations of inhibitor producing 50% inhibition) were similar for both infected and normal erythrocytes, indicating that the binding affinities of the carrier were not substantially changed, but the turnover number, availability, or the number of the carrier may have increased in infection. There was a large increase in the transport of l-glucose into infected erythrocytes. Neither inhibitor showed any effect on transport of l-glucose into infected or normal erythrocytes. d-Galactose and d-fructose also showed a large transport increase mostly insensitive to cytochalasin B. The specificity of the transport increase raises the possibility of the presence of a new pathway other than simple diffusion, or the carrier-mediated pathway revealed by cytochalasin B or phloretin inhibition.     

Recovery of Aging-Related Size Increase of Skin Epithelial Cells: In vivo Mouse and In vitro Human Study

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20–40% for cells of older passage (6–8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.     

Curvularia lunata, a New Source of Cytochalasin B

A biologically active metabolite was found in crude extracts of Curvularia lunata (Wakker) Boedijn (ATCC 34690) isolated from decayed tissues of litchi fruit (Litchi chinensis Sonn.). The fungus was grown on a shredded wheat-yeast extract-sucrose medium, and cultures were extracted with chloroform after 3 weeks of growth at 21°C. Chloroform extracts were toxic to day-old cockerels and caused abnormal distortion of wheat coleoptile segments. The major toxin had a 50% lethal dose of 700 mg/kg (oral dose) and was a colorless crystalline material with a melting point of 218°C. Elemental and high-resolution mass spectral analyses indicated a formula of C₂₉H₃₇NO₅ and a molecular weight of 479.62. The crystalline preparation was identified as 97% cytochalasin B and 3% cytochalasin A. The yield of cytochalasin B from C. lunata cultures grown on 4.5 kg of shredded wheat and 9 liters of yeast extract-sucrose medium was 6.24 g of purified material.     

Effect of Colchicine,Vinblastine, and Cytochalasin B on Cell Surface Anionic Sites of Tritrichomonas foetus1

ABSTRACT. Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic sitecontaining macromolecules located on the cell surface of T. foetus.     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Effect of cytochalasin B on the evagination in vitro of leg imaginal discs

Cytochalasin B (1 μg/ml) completely inhibited the evagination of isolated leg imaginal discs cultured in vitro in a synthetic medium (ME) containing α-ecdysone (3 μg/ml). In discs precultured for 6 hr in medium ME without the drug, then transferred to cytochalasin B-containing medium, continuation of evagination was stopped immediately. The inhibition of evagination was completely reversible, provided pretreatment with cytochalasin B did not exceed 8 hr. Results are discussed in view of what is known on the effect of cytochalasin B on other developmental systems. Findings are compatible with the primary action of the drug being an alteration of cell surface properties, thus bringing to light the importance of these properties in the course of normal imaginal disc evagination.     

Modification of Salmonella Typhimurium motility by the probiotic yeast strain Saccharomyces boulardii

Background

Motility is an important component of Salmonella enterica serovar Typhimurium (ST) pathogenesis allowing the bacteria to move into appropriate niches, across the mucus layer and invade the intestinal epithelium. In vitro, flagellum-associated motility is closely related to the invasive properties of ST. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is widely prescribed for the prophylaxis and treatment of diarrheal diseases caused by bacteria or antibiotics. In case of Salmonella infection, S.b-B has been shown to decrease ST invasion of T84 colon cell line. The present study was designed to investigate the impact of S.b-B on ST motility.

Methodology/Principal Findings

Experiments were performed on human colonic T84 cells infected by the Salmonella strain 1344 alone or in the presence of S.b-B. The motility of Salmonella was recorded by time-lapse video microscopy. Next, a manual tracking was performed to analyze bacteria dynamics (MTrackJ plugin, NIH image J software). This revealed that the speed of bacterial movement was modified in the presence of S.b-B. The median curvilinear velocity (CLV) of Salmonella incubated alone with T84 decreased from 43.3 µm/sec to 31.2 µm/sec in the presence of S.b-B. Measurement of track linearity (TL) showed similar trends: S.b-B decreased by 15% the number of bacteria with linear tract (LT) and increased by 22% the number of bacteria with rotator tract (RT). Correlation between ST motility and invasion was further established by studying a non-motile flagella-deficient ST strain. Indeed this strain that moved with a CLV of 0.5 µm/sec, presented a majority of RT and a significant decrease in invasion properties. Importantly, we show that S.b-B modified the motility of the pathogenic strain SL1344 and significantly decreased invasion of T84 cells by this strain.

Conclusions

This study reveals that S.b-B modifies Salmonella''s motility and trajectory which may account for the modification of Salmonella''s invasion.