Externalization of transferrin receptor in established human cell lines

The externalization of transferrin receptors was found in established human tumor cell lines at the rate of 10-35 ng/hour/10(6) cells, when they were incubated with transferrin at 37 degrees C. This externalization is inhibited by lowering the incubation temperature to 4 degrees C or eliminating the ligand from the culture medium. Metabolic inhibitors such as sodium azide, colchicine, cytochalasin B and chloroquine also decreased the rate of externalization. Almost 95% of released transferrin receptors were precipitated by centrifugation at 100,000 x g for 30 min, suggesting that transferrin receptor is externalized into the medium as a vesicular form.     

Concanavalin A-induced redistribution of surface receptors in Acanthamoeba castellanii at different growth phases

Concanavalin A (ConA)-induced redistribution of surface receptors has been studied in Acanthamoeba castellanii at different growth phases utilizing double fluorescent techniques and transmission electron microscopy. When the amoebae were incubated with 2 micrograms and 10 micrograms tetramethylrhodamine isothiocyanate (TRITC)-ConA/ml for 4 min and 15 min at 28 degrees C the staining pattern was characterized by various numbers of scattered aggregates of fluorescent ConA. Double labeling of the amoebae showed that the fluorescent aggregates represented internalized label, and the internalization was not preceded by any aggregation of ConA receptors on the cell surface as visualized by incubating with anti-ConA serum followed by fluorescein isothiocyanate-conjugated anti-IgG. Following exposure of the amoebae to 10 micrograms TRITC-ConA/ml for 4 min and 15 min at 28 degrees C intracellular accumulation of some of the fluorescent aggregates in cap-like structures occurred at the logarithmic and postlogarithmic growth phases but not at the early stationary growth phase. Electron microscopic observation of amoebae labeled with ferritin-conjugated ConA at 28 degrees C revealed a uniform surface labeling and an intracellular accumulation of the label in vesicular and tubular structures, and occasionally in cap-like structures. Surface capping of ConA receptors in Acanthamoeba was induced by treating the amoebae with ConA and anti-ConA serum at 0 degrees C followed by incubation at 28 degrees C. The formation of surface caps in Acanthamoeba showed growth-phase dependency, too. The visualization of the surface caps at the electron microscopic level was performed by indirect staining utilizing protein A-colloidal gold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internalization of lectins in neuronal GERL

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin- labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.     

EFFECTS OF COLCHICINE, CYTOCHALASIN B, AND 2-DEOXYGLUCOSE ON THE TOPOGRAPHICAL ORGANIZATION OF SURFACE-BOUND CONCANAVALIN A IN NORMAL AND TRANSFORMED FIBROBLASTS

The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37°C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37°C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37°C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.     

Effects of local anesthetics on membrane properties II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Effects of local anesthetics on membrane properties. II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins.

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Lectin-mediated agglutination of murine lymphoma cells. Cell surface deformability and reversibility of agglutination by saccharides

Agglutination of S49 mouse lymphoma cells by Ricinus communis I agglutinin can be reversed by the competing haptenic saccharide, lactose, soon after agglutination, but after further incubation in the absence of lectin the agglutination reaction could not be reversed by lactose and the cells remained as multicell aggregates. The irreversibility of S49 cell agglutination was time, temperature and lectin concentration dependent and its onset correlated with ultrastructurally observed deformation of adjacent cell surfaces and an increase in the proportion of adjacent cell surface areas in close apposition within multicell aggregates. Pretreatment of S49 cells with cytochalasin B or cytochalasin B plus vinblastine enhanced R. communis I agglutinin-mediated agglutination, while vinblastine alone and fluoride plus azide had essentially no effect. When drug-treated cells were agglutinated and then incubated in lectin-free drug-containing media for various times prior to lactose addition, the drug effects were more pronounced. Cytochalasin B alone or with vinblastine inhibited lactose reversal of S49 cell agglutination compared to the drug-free controls, while fluoride plus azide enhanced hapten reversibility. Electron microscopic analysis revealed that the onset of agglutination irreversibility correlated with cell surface deformation in the drug-treated cells. Cell aggregates that were more readily reversible by lactose (fluoride plus azide) were unchanged or less deformed, while S49 aggregates treated with cytochalasin B plus vinblastine were more deformed compared to controls without drugs. These experiments suggest a role for cell surface deformability as an important secondary effect during lectin-mediated cell agglutination of 849 lymphoma cells.     

Characterization of colchicine-binding activity in Dictyostelium discoideum.

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.     

Lectin-mediated agglutination of murine lymphoma cells. Cell surface deformability and reversibility of agglutination by saccharides.

Agglutination of S49 mouse lymphoma cells by Ricinus communis I agglutinin can be reversed by the competing haptenic saccharide, lactose, soon after agglutination, but after further incubation in the absence of lectin the agglutination reaction could not be reversed by lactose and the cells remained as multicell aggregates. The irreversibility of S49 cell agglutination was time, temperature and lectin concentration dependent and its onset correlated with ultrastructurally observed deformation of adjacent cell surfaces and an increase in the proportion of adjacent cell surface areas in close apposition within multicell aggregates. Pretreatment of S49 cells with cytochalasin B or cytochalasin B plus vinblastine enhanced R. communis I agglutinin-mediated agglutination, while vinblastine alone and fluoride plus azide had essentially no effect. When drug-treated cells were agglutinated and then incubated in lectin-free drug-containing media for various times prior to lactose addition, the drug effects were more pronounced. Cytochalasin B alone or with vinblastine inhibited lactose reversal of S49 cell agglutination compared to the drug-free controls, while fluoride plus azide enhanced hapten reversibility. Electron microscopic analysis revealed that the onset of agglutination irreversibility correlated with cell surface deformation in the drug-treated cells. Cell aggregates that were more readily reversible by lactose (fluoride plus azide) were unchanged or less deformed, while S49 aggregates treated with cytochalasin B plus vinblastine were more deformed compared to controls without drugs. These experiments suggest a role for cell surface deformability as an important secondary effect during lectin-mediated cell agglutination of S49 lymphoma cells.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37 degrees C for 30 min or 18 degrees C for 2 h, washing free of cell surface receptor-bound tracer at 4 degrees C and then reincubating at 37 degrees C. The cells preloaded at 37 degrees C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min-1 (t1/2 = 39 min). When the preloaded cells were incubated in the presence of 100 micrograms/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 microM colchicine, 20 microM cytochalasin B, 20 microM chloroquine, 10 mM NH4Cl, 10 microM monensin or 20 microM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoorosomucoid were observed with cells preloaded at 18 degrees C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

Lymphocyte capping induced by polycationized ferritin

In order to better understand the mechanism of lymphocyte surface receptor redistribution induced by externally added ligands, polycationized ferritin (PCF), a nonconventional ligand, was tested using both fluorescence and electron microscopy for its ability to cause patching and capping of anionic molecules on the surface of both transformed and normal mouse lymphocytes. Binding of PCF at 0 degree C for 1 hour induces the appearance of patches; subsequent incubation at 37 degrees for 30--60 minutes causes the formation of a cap structure with the lymphoid cells tested (T-lymphoma cells and splenic lymphocytes). Using various experimental treatments (e.g., sodium azide, cytochalasin B and D, colchicine, prefixation, and cold temperatures), PCF-induced capping has been found to be temperature sensitive, and to require metabolic energy and an intact cytoskeletal system. In addition, using double immunofluorescence techniques which involve rhodamine-labeled PCF and fluorescein-conjugated heavy meromyosin, it has been observed that the formation of the PCF-induced cap coincides with an accumulation of intracellular actin directly beneath the cap structure. Furthermore, agents such as dibutyryl cyclic AMP and theophylline, which cause an increase in intracellular cyclic AMP, have been shown to stimulate PCF-associated capping. This study suggests that increasing levels of intracellular cyclic AMP may activate, directly or indirectly, membrane-associated contractile elements required for the aggregation of membrane proteins into patches and caps.     

Hormonal and pharmacologic agents affecting choline uptake and incorporation in Schistosoma mansoni adults

Initial uptake of choline by Schistosoma mansoni (2-min uptake) revealed no differences between male, female or paired worms for any of the control or experimental groups. After a 30-min uptake period, however, males showed significantly higher uptake of choline in the presence of mitomycin C, cytochalasin B and calcium ionophore A23187, while paired worms showed significantly reduced uptake in the presence of actinomycin D, puromycin, mitomycin C, cytochalasin B, colchicine, insulin, thyroxine and lysine. Choline uptake by females was elevated, in the presence of cytochalasin B at 30 min, although not significantly. Significantly increased incorporation of choline into phosphatidylcholine was observed following a 30-min incubation with 5-hydroxytryptamine (males), puromycin and thyroxine (females) and calcium ionophore A23187 (males, females and pairs). These effects on phosphatidylcholine synthesis are discussed in relation to the uptake data and to previous work concerning the outer membrane complex of the parasite as an important facet of parasite resistance to the host immune response.     

Concanavalin A-mediated attachment and ingestion of yeast cells by macrophages

The ConA-mediated interaction of yeast cells with macrophages was brought about in two steps. The first step involved the interaction of either macrophages or yeast cells with ConA, MConA or YConA in brief, respectively. The second step consisted of interacting the ConA-coated cells with their non-coated counterpart, yielding MConA-Y or M-YConA. The extent of yeast cell attachment to macrophages depends on the degree of saturation of ConA binding sites on the cell coated with ConA in the first step and on the temperature at which the two cell types interact. The temperature dependence in the range of 10–25 °C implies that cell-cell attachment is sensitive to the physical state of membrane lipids as reflected in increased lateral mobility of ConA receptors in the membrane plane. The extent of ConA-mediated cell association is not influenced significantly by colchicine, cytochalasin B (CB) or hydrocortisone. A mild treatment of macrophages with glutaraldehyde reduces, however, the association of yeast cells, further indicating a need for lateral mobility of ConA receptors. ConA-mediated yeast cell attachment could be totally reversed by α-methyl mannoside in the case of MConA-Y and only partially in the case of M-YConA. Yeast cell ingestion is highly temperature-dependent; in MConA-Y a 50% interiorization of the associated yeast cells is reached at 32 °C and detectable interiorization starts only above 19 °C, while in M-YConA a 50% value of interiorization is reached at 18 °C and about 15% of yeast cells are interiorized already at 5 °C. Interiorization of attached yeast cells is not affected by colchicine. Cytochalasin B (CB) (10 μg/ml) inhibits 82% of yeast interiorization in MConA-Y and only 12% in M-YConA. Hydrocortisone has a similar differential effect of inhibition of ingestion; at 25 °C inhibition in MConA-Y amounts to 78% and in M-YConA to 22%. Sodium azide inhibits 90% of interiorization of yeast cells in both MConA-Y and M-YConA. The following working hypothesis was proposed to explain both the characteristics of attachment and the remarkable difference in ingestion pattern of yeast cells in MConA-Y and M-YConA. ConA-mediated yeast cell attachment to macrophages involves multipoint interaction between the two cells achieved by a certain clustering of ConA receptors in the membrane plane. To achieve interiorization a higher extent of bridge formation between the cells is required, and a higher number of ConA-membrane receptors have to be recruited to the area of apposition of the two membranes. This requires lateral mobility of either ConA receptors conjugates (in the case of MConA) or of mobile non-crosslinked ConA receptors in macrophages interacting with YConA). Mobility of ConA receptor conjugates is more sensitive to membrane fluidity than that of non-crosslinked receptors and hence the differential temperature-dependence of ingestion. The effect of CB suggests an involvement of the cytoskeleton in the reorganization of ConA receptors at the membrane level.     

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles.     

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

农村生活污水处理厂中阿米巴原虫的定量检测

【目的】自由生活的棘阿米巴属(Acanthamoeba spp.)和哈曼属原虫(Hartmannella vermiformis)普遍存在于自然界的土壤和各种水体中,这两个属中的某些种类被认为对人和动物具有潜在的致病性,应用染料法实时荧光定量PCR技术建立特异性强、灵敏度高及重复性好的快速检测阿米巴虫的方法具有实际意义。【方法】采用非培养方法选择适合低拷贝基因检测的荧光染料BRYT Green? dye用于农村生活污水处理厂不同工艺阶段水样中Acanthamoeba spp.和H. Vermiformis 18S rRNA基因的检测和定量分析。【结果】在整个处理工艺流程中均检测到Acanthamoeba spp.和H. Vermiformis,并呈现出不同的变化趋势,进水中分别达到8.70×105拷贝/L和1.84×106拷贝/L。与进水相比,调节池、好氧池和膜池中阿米巴原虫的数量均降低了1?2个数量级,但是出水中Acanthamoeba spp. 则出现增加趋势。【结论】对阿米巴原虫可能造成的潜在健康危害应引起重视,并有必要作为污水处理达标的补充标准。     

Ligand-dependent redistribution of the IgA receptor on cultured rat hepatocytes and its disturbance by cytochalasin B

The topography and dynamics of IgA-secretory component (SC) complexes on the surface of cultured hepatocytes and its disturbance by cytochalasin B were investigated using the colloidal gold technique in conjunction with surface replication. The distribution of IgA-gold conjugates after incubation at 4 degrees C was similar in normal and cytochalasin B-treated hepatocytes and was characterized by diffusely scattered single and clustered particles, the latter often associated with coated pits. After raising the temperature to 37 degrees C, redistribution of particles and their gradual uptake into coated vesicles was observed in control cultures. This ligand-induced redistribution led to a progressive gathering of single and grouped particles in larger clusters (50-200 particles), which appeared to be the site of the most intensive endocytotic activity. In contrast, huge patches of IgA-gold conjugates were formed at the cell periphery of cytochalasin B-treated hepatocytes within 20-60 min at 37 degrees C, while central areas were cleared. Patch formation was triggered by binding of both unlabeled and labeled IgA, but could not be observed with the unoccupied receptor as demonstrated by gold-labeled antibodies against SC. These results show that the topography of SC is markedly changed by binding of its ligand, IgA, and suggest that the dynamics of the IgA-SC complexes in hepatocyte plasma membrane are affected by microfilaments.     

The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis

From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.