Concanavalin A-mediated agglutination and distribution of concanavalin A-binding sites in Acanthamoeba following treatment with colchicine and cytochalasin B

Incubation of Acanthamoeba castellanii (Neff strain) with FITC-ConA (15 micrograms/ml) resulted in the appearance of patches of fluorescence on the amoebae within 2 min of incubation. These patches disappeared following treatment of the amoebae with alpha-MeMan. Pretreatment of the amoebae with colchicine or cytochalasin B or with colchicine and cytochalasin B in combination did not significantly alter the distribution pattern of fluorescence in the amoebae. 2,4-Dinitrophenol and incubation at 4 degrees C on the other hand decreased the degree of patching of the amoebae. Pretreatment with 2,4-dinitrophenol and incubation at 4 degrees C also decreased the ConA-mediated agglutination of the amoebae. No effect on the ConA-mediated agglutination was, however, observed following pretreatment of the amoebae with colchicine and cytochalasin B neither alone nor in combination. Our results indicate that ConA-mediated agglutination and long-range ConA-receptor mobility in the Acanthamoeba are not under the control of structures sensitive to cytochalasin B or colchicine.     

Response of the resident macrophage to concanavalin A : Alterations of surface morphology and anionic site distribution

We have characterized certain resident guinea-pig peritoneal macrophage surface alterations following treatment with concanavalin A (ConA) and succinyl-ConA (S-ConA). Studies employing scanning electron microscopy (SEM) have demonstrated that ConA, a tetrameric lectin, decreases dramatically the number of macrophage surface folds and ruffles although S-ConA, a dimeric derivative, is apparently not active. However, incubation of S-ConA-treated cells with rabbit anti-ConA (anti-ConA) restored this effect. The decrease in surface folds could not be observed in the presence of α-methyl- -mannoside (αMM), a hapten sugar of ConA. We have performed several receptor-labeling transmission electron microscopy (TEM) studies employing ferritin-conjugated ConA (FT-ConA) and cationized ferritin (CF). These experiments indicate that the ConA receptor-FT-ConA complexes form (1) clusters on the plasmalemma and (2) adsorptive pinocytic vesicles lined with the ferritin label. At the same time scale, we have observed a redistribution of macrophage surface anionic sites following treatment with ConA or S-ConA plus anti-ConA but not S-ConA alone. The redistribution of anionic sites is abolished in the presence of αMM. The specificity of the CF label was checked by pre-incubating cells with poly- -lysine (PLL) or neuraminidase and by employing normal ferritin. These studies provide evidence which support the concept of a directed movement of surface charges during adsorptive pinocytosis. We discuss the possible relevance of this concept with regard to regional alterations of pH at the plasmalemma and the contribution of these anions to the trans-pinosomal pH gradient through the Donnan potential.     

The role of microfilaments in the capping of epidermal growth factor receptor in A431 cells

Capping of the EGF receptor (EGF-R) on the surface of suspended and adherent epidermoid carcinoma cells, A431, is studied. It was induced at 20 degrees C after treating cells with monoclonal antibody to the EGF receptor followed by the second antibody conjugated with FITC. Accumulation of cortical actin under the caps was detected by rhodamine-phalloidin. Destruction of the actin stress-fiber-like bundles was observed during incubation of cells with the ligands at 0 degrees C. Two processes appear to take place at 20 degrees C: redistribution of the EGF-R with cortical actin into the caps within 15-30 min and reconstruction of cytoplasmic actin bundles over 45-60 min. Dihydrocytochalasin B prevented cap formation in adherent cells, but small patches of EGF-R colocalized with actin aggregates under plasma membrane were observed. The function of different actin-containing cytoskeleton structures in the process of capping is discussed.     

Visualization of binding sites for bovine parathyroid hormone (PTH 1-84) on cultured kidney cells with a biotinyl-b-PTH (1-84) antagonist

Parathyroid hormone (PTH) receptors have been found in a subpopulation of kidney cells. In this report, we investigated the feasibility of techniques that apply a partial antagonist of PTH conjugated to biotin to localize receptors cytochemically on bovine kidney cortical cells in monolayer culture at the light microscopic level. Biotinylated bovine PTH (1-84) (biotinyl-PTH) was bound to the cultured cells for 1-30 min at 37 degrees C in the amounts of 10(-5) -10(-10) M. In a different set of experiments, the cells were also exposed to a solution containing 10(-6) M biotinylated PTH and an excess of unlabeled PTH, insulin, adrenocorticotropin, or calcitonin for 10 and 30 min at 37 degrees C to test the specificity of the binding. The cells were then fixed in 2.5% glutaraldehyde and stained with the avidin-biotin peroxidase complex (ABC) technique. Diffuse labeling was evident on 30% of the cells in 10 min with concentrations of biotinyl-PTH as low as 10(-8) M. The stain was diffuse, but more intense after 1-10 min in higher concentrations (10(-6) M). If a 15-1500-fold excess of unlabeled PTH was added to the biotinyl-PTH, no staining was observed. The other peptides (insulin, ACTH or calcitonin) had no effect on binding. Longer times in biotinyl-PTH (10(-6) M for 10-30 min) resulted in intense patches of label on the cells resembling caps (in addition to the pale diffuse label). The percentage of labeled cells in the monolayer (30%) did not change with time. These studies show that a partial antagonist of PTH can be used as a cytochemical probe for specific PTH receptors in a subpopulation of cultured cortical kidney cells.     

Ultra-structure and localisation of formazan formed by human neutrophils and amoebae phagocytosing virulent and avirulent Legionella pneumophila

Legionella pneumophila (LP) strains of differing virulence were incubated with a solution of nitroblue-tetrazolium (NBT) at a concentration of 1 mg.ml-1 in the presence of Acanthamoeba polyphaga or human polymorphonuclear neutrophils (PMN). Reduction of NBT to formazan occurred at a faster rate in the presence of virulent strains. Reduction appeared to be temperature dependent; at 37 degrees C the reaction rate was higher than at 20 degrees C. On microscopic examination, deposits of formazan around Legionella cells were observed inside amoebae similar to those deposited in human neutrophils. Electron microscopy revealed electron-dense particles surrounding virulent legionellae, which appeared to be associated with formazan formation. Formazan formation inside amoebae may suggest the presence of a respiratory burst against LP, which is more intense with virulent strains.     

Functional integrity of cytokineplasts: specific chemotactic and capping responses

Cytokineplasts (CKP) are motile, membrane-bound, anucleate, granule-poor cytoplasmic fragments that are induced from human blood polymorphonuclear leukocytes (PMN) by the brief application of heat. We examined CKP with respect to specific chemotactic and capping responses, the presence of the N-formyl-peptide chemotactide receptor, and evidence of respiratory burst activity and compared them with CB-cytoplasts, which are fragments created by the centrifugation of cytochalasin B (CB)-treated PMN at high speeds. Under agarose, CKP responded chemotactically to both N-formyl-methionyl-leucyl-phenylalanine (fmlp) and zymosan-activated serum; CB-cytoplasts responded to neither chemoattractant. Despite the functional differences, both fragments retained N-formyl-peptide receptors as measured by affinity labeling with N-formyl-norleu-leu-phe-norleu-125I-tyr-lys and autoradiography of dried SDS-PAGE gels. For studies of capping we used a murine monoclonal antibody, PMN7C3, which binds a specific, widely distributed membrane component of intact PMN, and on warming, promptly induces capping of ligand-receptor complexes. Rhodamine-conjugated PMN7C3 at 4 degrees C labeled the surface of CKP homogeneously. As the CKP warmed to 37 degrees C, label became concentrated in small fluorescent caps at the rear of migrating fragments. Although CB-cytoplasts also bound the fluorochromed antibody homogeneously in the cold, on warming they were unable to concentrate the label normally. With respect to respiratory burst activity, the situation in the two fragments was reversed: CKP did not generate superoxide anion when stimulated either with phorbol myristate acetate or with fmlp after pretreatment with CB; CB-cytoplasts, as noted earlier by other investigators, did. These two types of cytoplasts with markedly different capabilities have complementary roles in the analysis of PMN function.     

Relationship of IgE receptor topography to secretion in RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking IgE-receptor complexes with anti-IgE antibody leads to degranulation. Receptor cross-linking also stimulates the redistribution of receptors on the cell surface, a process observed here by labeling the anti-IgE with 15 nm protein A-gold particles that are visible by back-scattered electron imaging in the scanning electron microscope. We report that anti-IgE binding stimulates the redistribution of IgE-receptor complexes at 37 degrees C from a dispersed topography to distributions dominated sequentially by short chains, small clusters, and large aggregates of cross-linked receptors. Cells incubated with 1 microgram/ml anti-IgE, a concentration that stimulates maximum net secretion, redistribute receptors into chains and small clusters during a 15 min incubation period. At 3 and 10 micrograms/ml anti-IgE, net secretion is reduced and the majority of receptors redistribute rapidly into clusters and large aggregates. The addition of Fab fragments with the high anti-IgE concentrations, to reduce cross-linking, delays receptor aggregation and enhances secretion. The progression of receptors from small clusters to large aggregates is prevented in cells treated with dihydrocytochalasin B to prevent F-actin assembly. These results establish that characteristic patterns of receptor topography are correlated with receptor activity. In particular, they link the formation of large receptor aggregates to reduced signalling activity. Cytoskeleton-membrane interaction is implicated in the formation or stabilization of the large receptor clusters.     

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.     

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.     

Biological factors affecting enflagellation of Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate that can be evoked to transform from amoebae to flagellates by subculture to nonnutrient buffer. More than half of the amoebae of strains KUL, nN68, and Lovell became enflagellated 300 min after subculture to amoeba-saline, whereas no amoebae of strains NF66, NF69, and HB4 did. N. fowleri nN68 enflagellated best when grown at 32 or 37 degrees C and subcultured to amoeba-saline at 37 or 42 degrees C. Amoebae from the stationary phase of growth enflagellated more readily than did actively growing amoebae. Incubation in expended culture medium from stationary-phase cultures enhanced the capability of growing amoebae to enflagellate after subculture to amoebasaline. Enflagellation was more extensive when the population density in amoebasaline did not exceed 2 x 10(5) amoebae per ml. Cycloheximide at 1 microgram/ml and actinomycin D at 25 micrograms/ml inhibited growth of N. fowleri nN68. Cycloheximide at 0.5 microgram/ml and actinomycin D at 25 micrograms/ml completely prevented enflagellation when added at time zero. Cycloheximide at 0.5 microgram/ml, added 120 to 300 min after initiation of enflagellation, prevented further differentiation and caused existing flagellates to revert to amoeboid cells. Similarly, actinomycin D at 25 micrograms/ml, added 90 to 300 min after initiation of enflagellation, retarded differentiation and caused flagellates to revert. Radiolabeled precursors were incorporated into macromolecules during differentiation in nonnutrient buffer. Enflagellation of N. fowleri is a suitable model for studying regulation of a eucaryotic protist.     

The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 mumol inorganic phosphate/g muscle in 20 min at 20 degrees C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 degrees C for 18 h caused ectoenzyme inactivation which plateaued at 35-40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 micrograms ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by alpha-methyl D-mannopyranoside. As the temperature increased from 10 to 40 degrees C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 degrees C, with a "break point" and a clear change in slope at 35 degrees C. When treated with ConA the activity increased linearly from 10 to 40 degrees C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 degrees C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.     

Altered surface distribution of both C3b receptors and Fc receptors on neutrophils induced by anti-C3b receptor or aggregated IgG

Human neutrophils to which monospecific Fab' or F(ab')2 anti-C3b receptor had been bound at 0 degrees C were incubated for timed intervals at temperatures ranging from 0 degrees C to 37 degrees C, after which the cells were labeled with TRITC -conjugated second antibody. Neutrophils bearing Fab' anti-C3b receptor and incubated for up to 30 min at 37 degrees C, and cells bearing F(ab')2 anti-C3b receptor and incubated at 0 degrees C, exhibited diffusely distributed punctate clusters of receptors. Neutrophils bearing the bivalent anti-receptor and incubated at 30 degrees C or 37 degrees C for 5 min had redistributed C3b receptors into caps and patches that were associated with subplasmalemmal accumulations of myosin. The redistribution of cross-linked C3b receptors was inhibited by pretreatment of the neutrophils with either cytochalasin D or chlorpromazine. On approximately one-half of the cells demonstrating capped C3b receptors there was a corresponding redistribution of Fc receptors, as demonstrated by subsequent binding of FITC-aggregated IgG (FITC agg-IgG). In contrast, capping of C3b receptors did not alter the diffuse distribution of HLA-A on these cells. Cross-linking of Fc receptors on neutrophils by FITC agg-IgG also induced temperature-dependent capping of these receptors that was inhibited by cytochalasin D and chlorpromazine. In approximately one-half of the cells demonstrating capped Fc receptors, subsequent labeling of C3b receptors revealed a similar redistribution of these receptors. Thus, the neutrophil responds to cross-linking of either C3b receptors or Fc receptors by a cytoskeletal-dependent rearrangement of both receptors that causes their overlapping topographic distribution, demonstrating a form of cooperative interaction between these two types of receptors that are involved in the phagocytic reactions of these cells.     

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.     

Alpha-spectrin immunoanalog in Acanthamoeba cells

A monospecific, affinity purified antibody was prepared against chicken erythrocyte alpha-spectrin. The antibody cross-reacted with only one high molecular weight polypeptide (235 kDa) from whole Acanthamoeba cells. The localization of alpha-spectrin-related antigen in Acanthamoeba cells was examined using immunofluorescence and postembedding cytochemical techniques. Three patterns of distribution of alpha-spectrin immunoanalog were distinguished: as submembranous layer, cytoplasmic aggregates and uniform dispersion through the cytoplasm. Immunoelectron microscopic studies showed that the colloidal gold label was located in the cytoplasm in the vicinity of the plasma membrane. The gold particles were also aggregated around unidentified cytoplasmic filamentous structures. The presence of spectrin-related protein in protozoan cells of Acanthamoeba is in accordance with previous assumptions of the widespread occurrence of spectrin-related proteins. The heterogenous distribution of the immunoanalog of alpha-spectrin protein in Acanthamoeba cells is discussed.     

Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.

125I-labeled and ferritin-labeled low density lipoprotein (LDL) were used as visual probes to study the surface distribution of LDL receptors and to examine the mechanism of the endocytosis of this lipoprotein in cultured human fibrobasts. Light microscopic autoradiograms of whole cells incubated with 125I-LDL at 4 degrees C showed that LDL receptors were widely but unevenly distributed over the cell surface. With the electron microscope, we determined that 60-70% of the ferritin-labeled LDL that bound to cells at 4 degrees C was localized over short coated segments of the plasma membrane that accounted for no more than 2% of the total surface area. To study the internalization process, cells were first allowed to bind ferritin-labeled LDL at 4 degrees C and were then warmed to 37 degrees C. Within 10 min, nearly all the surface-bound LDL-ferritin was incorporated into coated endocytic vesicles that were formed by the invagination and pinching-off of the coated membrane regions that contained the receptor-bound LDL. With increasing time at 37 degrees C, these coated vesicles were observed sequentially to migrate through the cytoplasm (1 min), to lose their cytoplasmic coat (2 min), and to fuse with either primary or secondary lysosomes (6 min). The current data indicate that the coated regions of plasma membrane are specialized structures of rapid turnover that function to carry receptor-bound LDL, and perhaps other receptor-bound molecules, into the cell.     

Recycling of photoaffinity-labeled insulin receptors in rat adipocytes. Dissociation of insulin-receptor complexes is not required for receptor recycling

We have used an iodinated, photoreactive analog of insulin, 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, to covalently label insulin receptors on the cell surface of isolated rat adipocytes. Following internalization of the labeled insulin-receptor complexes at 37 degrees C, we measured the rate and extent of recycling of these complexes using trypsin to distinguish receptors on the cell surface from those inside the cell. The return of internalized photoaffinity-labeled receptors to the cell surface was very rapid at 37 degrees C proceeding with an apparent t 1/2 of 6 min. About 95% of the labeled receptors present in the cell 20 min after the initiation of endocytosis returned to the cell surface by 40 min. Recycling was slower at 25 and 16 degrees C compared to 37 degrees C and essentially negligible at 12 degrees C or in the presence of energy depleters. Addition of excess unlabeled insulin had no effect on the recycling of photoaffinity-labeled insulin receptor complexes, whereas monensin, chloroquine, and Tris partially inhibited this process. These data indicate that dissociation of insulin from internalized receptors is not necessary for insulin receptor recycling. Furthermore, agents which have been shown to prevent vesicular acidification inhibit the recycling of insulin receptors by a mechanism other than prevention of ligand dissociation.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method. Combined transmission and scanning electron microscopic study of ConA binding sites in mouse macrophages

Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-point-dried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the three-dimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0 degree C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0 degree C were further incubated at 37 degrees C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cell-bound ligands.     

Biophysical insight reveals tannic acid as amyloid inducer and conformation transformer from amorphous to amyloid aggregates in Concanavalin A (ConA)

The aggregation phenomenon (amyloid and amorphous) is associated with several pathological complications in human, such as Alzheimer’s, Parkinson’s, Huntington, Cataract diseases, and Diabetes mellitus type 2. In the present study we are offering evidence and breaking the general belief with regard to the polyphenols action as protein aggregate inhibitors. Herein we confirm that tannic acid (TA) is not only an amyloid inducer, but also it switches one type of conformation, ultimately morphology, into another. We ascertain based on our findings that aggregates are not rigid structures and the stability can be challenged under certain conditions. This study also confirms that unfolded and amorphous aggregates can serve as precursors of amyloids and TA interactions with unordered aggregates (amorphous) bringing orderliness in the conformation via amyloidosis. The shifting of unordered conformation toward orderliness is governed by the modulation in surface hydrophobic patches in Concanavalin A (ConA). Hence, a degree of exposed hydrophobic cluster can be claimed as a strong parameter to detect and distinguish the native, amorphous and both types of amyloids. Turbidity and Rayleigh light scattering measurements followed similar pattern while Thioflavin T and 1-anilino-8-naphthalene sulfonate fluorescence assays of the binding with amorphous and amyloid followed an inverse relation. Electron microscopic studies revealed the morphological variation in the ConA at 65°C as amorphous while the ConA treated with TA followed by heat treatment at 65°C was defined as amyloid in nature. Interestingly for the first time we are reporting the slight agglutination activity by the ConA amyloids.     

Down-regulation of transforming growth factor-beta receptors: cooperativity between the types I, II, and III receptors and modulation at the cell surface.

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-beta (TGF-beta) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-beta1 treatment at 37 degrees C) and showed a dose response, between 10 and 200 pM TGF-beta1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-beta-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-beta receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-beta1 at 4 degrees C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI-GFP) and RII indicated that, after treatment with TGF-beta1 at 4 degrees C, RI-GFP formed aggregates at the cell surface at this temperature. RI-GFP was not detected at the surface of these cells after TGF-beta1 treatment at 37 degrees C. Our results suggest a two phase mechanism for TGF-beta1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.     

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.