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The presence of mosaicism in the organization of concanavalin agglutinin (Con A) binding sites on murine egg cells was first reported 30 year ago. This discovery has triggered extensive studies into the roles of glycoproteins in gamete interactions in animals. This report comprises the first account of the existence of the mosaicism in higher plants. The distribution of Con A binding sites on both egg cells and central cells of tobacco (Nicotiana tabacum) was found to be polar and apparently determined by the location of the nucleus of the cell. On central cells, Con A binding sites were distributed on the section of the plasma membrane surface near the nucleus. By contrast, the binding sites on egg cells were concentrated away from the nucleus. Therefore, polarity of the plasma membrane component of female cells was confirmed for the first time. It is proposed that such polarized ConA binding sites could be involved in sperm recognition.

Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II.

Cooperativity of molecular adhesion has been proposed as a mechanism for enhanced binding strength of adhesion molecules on the cell surface. Direct evidence for its mechanism, however, has been lacking until now. Atomic force microscopy (AFM) was used to measure the adhesive strength between concanavalin A (Con A) coupled to an AFM tip and Con A receptors on the surface of NIH3T3 fibroblast cells. Cross-linking of receptors with either glutaraldehyde or 3, 3'-dithio-bis(sulfosuccinimidylproprionate) (DTSSP) led to an increase in adhesion that could be attributed to enhanced cooperativity among adhesion complexes. An increase in loading rate due to greater stiffness of fixed cells also contributed to the twofold increase in binding strength. These results show that receptor cross-linking can greatly contribute to a total increase in cell adhesion by creating a shift toward cooperative binding of receptors.

This study was designed to explore the possibility of using ascitic mouse sarcoma cell line (S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180 cells attachment to Concanavalin A-coated surfaces. Inhibition was dependent on concentration, and the IC50 (the concentration that reduced attachment by 50%), of these 2 chemicals was 1.2 x 10(-3) mol/L and 1.0 mol/L, respectively. Another developmental toxicant, hydrocortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also tested and these did not decrease attachment rates. The main results reported here were generally similar to those obtained with ascitic mouse ovarian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not limit attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an alternative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

A new method has been developed for ex vivo preparation of brain cortical cells of BN/BiRijHsd rats to make them suitable for the measurement of the lateral diffusion coefficient of the membrane components by means of fluorescence recovery after photobleaching (FRAP). The method involves chopping the brain cortex into pieces of less than 1 mm. These parts are stained with a fluorescent label (e.g., concanavalin-A-fluorescein, Con-A-FL conjugate) and then gently pressed onto a microscope slide using the coverslip. In the resulting specimen, the largest cells of the cortex can be recognized in phase-contrast image, sufficiently stained by the label and ready for the FRAP measurement. The lateral diffusion coefficient of Con-A-receptor proteins (Dp) was measured in such brain cell preparations of 15 female rats in four age groups (5.6-31.8 months) and 11 males in three age groups (13.8-31.8 months). Highly significant negative, linear age correlation of Dp (R=-0.9958 in females, and -0.9956 in males) were found, the regression equations being D(p female) =(8.8311-0.1425 X)(-10) and D(p male)=(9.3240-0.1630 X)(-10) cm2/s, respectively, where X is age in months. The data confirm that the lateral mobility of plasma membrane proteins represents an important biomarker of cellular aging in the brain cortical cells of BN/BiRijHsd rats.

The loss of neurons by programmed cell death is a normal feature of the nervous system during development and has recently been implicated as a major mechanism of cell death in neurodegenerative diseases. In some cases, programmed cell death is induced by the activation of membrane receptors and is referred to as activation-induced programmed cell death. Activation-induced programmed cell death has been previously described in cells from the immune system, in which the activation of receptors by receptor clustering leads to programmed cell death. To determine whether activation-induced programmed cell death occurs in neurons, Concanavalin A was used to cross-link membrane receptors on cortical neurons. Concanavalin A-induced neuronal death was dose dependent and effective at concentrations previously shown to induce activation-induced programmed cell death in lymphocytes. Programmed cell death was attenuated when Concanavalin A-specific binding to neurons was blocked with methyl alpha-D-mannopyranoside. Succinyl Concanavalin A, which bound to Concanavalin A receptors but was ineffective at cross-linking them, did not induce programmed cell death. Concanavalin A-induced neuronal death exhibited many of the hallmarks associated with programmed cell death, such as membrane blebbing, nuclear condensation and margination, and internucleosomal DNA cleavage. In addition, neurons exposed to Concanavalin A displayed a rapid, robust, and persistent increase in the immediate early gene protein c-Jun. A similar increase in c-Jun precedes programmed cell death induced by beta-amyloid in neurons, and under some conditions an increase in c-Jun has been shown to be required for programmed cell death to occur in neurons. Increased expression of c-jun and other immediate early genes has also been correlated with activation-induced programmed cell death in lymphocytes. These observations suggest that Concanavalin A induces activation-induced programmed cell death in neurons via signals produced from the cross-linking of receptors on neuronal membranes. These results also raise the possibility that beta-amyloid induces programmed cell death in a similar manner, by causing the cross-linking of receptors on neuronal membranes. This mechanism may be relevant to neuronal programmed cell death that occurs during development and neurodegeneration.

Human bile contains cholesterol crystallization-stimulating proteins that can be isolated by concanavalin A-Sepharose chromatography. In the past few years an increasing number of different pronucleating proteins have been identified in the concanavalin A-binding fraction. In this study we attempted to estimate the relative contribution of a number of these proteins to total concanavalin A-binding pronucleating activity. For this purpose, concanavalin A-binding glycoproteins were isolated from gallbladder bile samples from 12 patients with gallstones. The role of IgA, IgG and IgM and alpha 1-acid glycoprotein was investigated by means of immunoextraction. No decrease in crystallization-promoting activity was observed after precipitation of more than 98% of the different immunoglobulins. In addition, removal of more than 95% of alpha 1-acid glycoprotein from different concanavalin A-binding fractions had no significant effect on cholesterol crystallization-promoting activity. The influence of fibronectin was estimated by addition of physiological concentrations to a model bile system. At these concentrations fibronectin did not promote crystallization. From these data we conclude that immunoglobulins, alpha 1-acid glycoprotein and probably also fibronectin do not significantly contribute to total concanavalin A-binding activity.

In myelinated axons, voltage-dependent sodium channels are segregated at high density at nodes of Ranvier (Rosenbluth, 1976; Waxman and Quick, 1978; Black et al., 1990; Elmer et al., 1990), a distribution that is critical for the saltatory conduction of action potentials (Huxley and Stampfli, 1949). The factors that specifically control the organization and immobilization of sodium channels at nodes are unknown. Recently we have reported that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to maintain sodium channel distribution during differentiation of myelinated nerve (Joe and Angelides, 1992). The specific recruitment of sodium channels at these early stages of myelination and the conspicuous absence of other axon membrane components suggest that the factors governing sodium channel cluster formation show molecular specificity. However, it is not clear whether these clustered sodium channels originate from a redistribution of preexisting diffusely distributed sodium channels. To determine how Schwann cells might regulate sodium channel distribution during myelination we have examined the lateral mobility of fluorescently labeled sodium channels at defined stages of myelination by fluorescence photobleach recovery using tetramethylrhodamine (TmRhd)-labeled Tityus gamma, a sodium channel-specific fluorescent toxin. First, to test whether Schwann cells, in addition to modulating sodium channel distribution, affect the mobility of sodium channels, we cultured dorsal root ganglion neurons in the presence or absence of Schwann cells and monitored sodium channel mobility on cell bodies, axon hillocks, and axons. Even in the absence of Schwann cells, approximately 80% of the sodium channels were immobile on the time scale of the fluorescence photobleach recovery measurement (DL < or equal to 10(-12) cm2/sec), although the remaining fraction of channels are mobile with diffusion coefficients of 5-13 x 10(-11) cm2/sec. Most importantly, in contrast to the effects of Schwann cells on altering the distribution of sodium channels, we found that Schwann cells did not alter the rate of lateral mobility or the mobile fraction of axonal sodium channels. Therefore, although sodium channel distribution depends on Schwann cell contact, immobilization of sodium channels is independent of Schwann cell contact. These effects appear to be specific for sodium channels because 45% of the succinyl concanavalin-A receptors on the axon are mobile, a fraction that decreases to 25% in the presence of Schwann cells later in development. To determine how sodium channels might be immobilized other than by Schwann cell contact, TmRhd-Tityus gamma-labeled dorsal root neurons were treated with 0.5% Triton X-100.(ABSTRACT TRUNCATED AT 400 WORDS)

The expression of the actin-sequestering peptide, thymosin beta 4, was analyzed in proliferating rat thymocytes, activated by diverse stimuli, during the early G1 phase and the S phase. In the presence of concanavalin A a 6.3-fold increase of thymosin beta 4 occurred already after 1 h of stimulation without elevation of the corresponding mRNA level. In contrast, during the S phase the increase of thymosin beta 4 (2.5-fold) was accompanied by a higher mRNA level, but did not exceed the growth related increase of total protein. Stimulation with a crosslinked antibody against rat T cell antigen receptor or stimulation with phorbol 12-myristate 13-acetate (PMA) and Ca(2+)-ionophore A23187, separately or in combination, did not lead to the marked increase of the thymosin beta 4 concentration in the early G1 phase but resulted in elevated thymosin beta 4 peptide and mRNA levels during the S phase. It therefore appears that protein kinase C activation and a rise in cytoplasmic Ca(2+)-concentration are not exclusively responsible for the stimulation of thymosin beta 4 specific translation in thymocytes. This assumption was reinforced by the observation that inhibition of the protein kinase C activity by 1-(5-isoquinolinylsulfony)-2-methylpiperazine (H-7) did not affect the cellular thymosin beta 4 content 1 h and 48 h after concanavalin A (Con A) stimulation.

In the present study we have identified a 72-kDa cell surface concanavalin A binding glycoprotein (cbg 72) involved in the chick embryo fibroblast (CEF) adhesion onto laminin (LM) substrate. The cbg 72 was shown to interact specifically with immobilized laminin and to be resistant to Triton X-100 extraction when CEF were plated on laminin substrate but not on fibronectin (FN) substrate. This behavior suggested that cbg 72 could interact with cytoskeletal elements during cell spreading onto LM. This assumption is also in good agreement with the partitioning of cbg 72 in Triton X-114. Isolated cbg 72 specifically inhibited CEF spreading onto LM after their initial attachment, whereas cbg 72 did not impair the spreading of CEF onto FN. These data provide a molecular explanation to the inhibition of CEF spreading onto LM observed in the presence of the lectin concanavalin A (P. Codogno, M.-A. Doyennette-Moyne, J. Botti, and M. Aubery, 1988, J. Cell Physiol. 136, 463-470). Moreover, these results provide evidence for the role of a novel LM binding glycoprotein during the adhesion of mesenchymal derived cells. The relationship between cbg 72 and other known cell surface LM binding sites or receptors is discussed.

Gallbladder bile contains nucleation-promoting activity that binds to concanavalin A. The activity was found in gallbladder bile from cholesterol gallstone patients but also in gallbladder bile from patients without stones and patients with pigment stones. Bile from patients with multiple cholesterol gallstones contained high concanavalin A-binding nucleation-promoting activity. The activity was much lower in bile samples from pigment stone patients, patients without stones and patients with a solitary cholesterol stone. Serum contained very little activity and no concanavalin A-binding nucleation-promoting activity could be demonstrated in gallbladder mucosa. This suggests that concanavalin A-binding nucleation promoter is produced in the liver or bile duct epithelium. The activity was fully resistant to digestion with pronase but was heat labile and could be destroyed by prolonged incubation with a mixed glycosidase preparation indicating that sugar residues are important for this activity. On a Superose 12 gel permeation column, promoting activity eluted in two major peaks at apparent molecular weights of 150 +/- 30 kD (n = 5) and less than 5 kD respectively. The mobility on the column was not influenced by pronase digestion. The factor with the higher molecular weight could be isolated further by polyacrylamide gel electrophoresis under nondenaturing conditions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular weight of the glycoprotein was 130 kD. In conclusion, gallbladder bile contains nucleation-promoting activity that binds to concanavalin A. The activity is increased in bile from patients with multiple cholesterol gallstones and could therefore play an important role in the pathogenesis of gallstone disease.

A study was performed to determine quantitative differences in the total protein concentration of gallbladder bile from gallstone patients and to isolate nucleation-promoting factors from the bile. Total protein concentrations in cholesterol gallstone bile (3.6 +/- 0.6 mg/ml, mean +/- SD, n = 10), calcium bilirubinate gallstone bile (4.2 +/- 1.1 mg/ml, n = 10), black pigment gallstone bile (1.9 +/- 0.6 mg/ml, n = 4) and control gallbladder bile (2.3 +/- 0.5 mg/ml, n = 9) were not significantly different. Also no statistically significant differences in cholesterol saturation index were found among these groups. Gallbladder bile from cholesterol gallstone patients showed significantly faster nucleation than that of controls, calcium bilirubinate gallstone, or black pigment gallstone patients. We partially purified biliary glycoproteins proteins from cholesterol gallstone bile or calcium bilirubinate gallstone bile by chromatography on concanavalin A Sepharose. Nucleation time was measured following the addition of these proteins to control bile in vitro. The glycoproteins obtained from cholesterol gallstone bile had significant nucleation-promoting activity, but nucleation time was not changed following the addition of biliary glycoproteins from calcium bilirubinate gallstone patients. These results suggest that qualitative differences in individual proteins of gallbladder bile are responsible for nucleation-promoting activity in vitro.

Concanavalin A (Con A), a tetravalent lectin, was shown to impair 8 chick embryo fibroblast (8 d CEF) spreading on a laminin (LM) substrate but not on a fibronectin substrate (FN), suggesting that cell surface Con A binding proteins could be involved in 8 d CEF spreading on a LM substrate. The interaction of Con A-binding proteins with Con A is dependent upon the carbohydrate moieties of the isolated glycoproteins; since they interact strongly with Con A-Sepharose and are eluted with 0.3 Mol/l alpha-methylmannopyranoside, the isolated Con A binding-proteins inhibit 8 d CEF adhesion to a Con A substrate to the same extent as alpha-methylmannopyranoside. Furthermore, the isolated Con A binding proteins specifically inhibit in a dose-dependent manner 8 d CEF spreading on LM but not on FN.

The effect of delta HOP on the formation of concanavalin A (Con A) caps and steady state anisotropy (rs) obtained in 1,6-diphenyl-1,3,5-hexatriene (DPH) labeled thymocyte plasma membranes was compared with the effect of glucocorticoids on these experiments. Results demonstrate that delta HOP decreased cap formation "in vivo" as well as "in vitro" and that it did not change fluorescence anisotropy "in vitro". Glucocorticoids had an opposite effect, increasing fluidity apparently through a glucocorticoid specific mechanism not exerted by delta HOP.

The redistribution of surface membrane immunoglobulin (SmIg) and concanavalin A (Con-A) was studied in peripheral mononuclear cells of three patients who had hairy cell leukemia. Fluorescein-labeled polyvalent goat antihuman immunoglobulin and fluoresceinated Con-A were used as ligands. The capping of ligand-receptor complexes was temperature-dependent and was more prominent at 37 C. A significant drop in the percentages of SmIg and Con-A capping was observed when cytochalasin B was added. In contrast, no decline in the percentages of SmIg and Con-A capping was seen in the presence of colchicine. The inhibitory action of cytochalasin B on the capping of SmIg and Con-A receptors in hairy cells suggests that these receptors are integral macromolecules of the plasma membrane and their redistribution is regulated by cytoskeletal structures.

Fluorescence redistribution after photobleaching (FRAP) was utilized to select a "fast" lateral mobility clone from Kirsten murine sarcoma virus-transformed 3T3 (KMSV-3T3) fibroblasts. The clone, E7G1, demonstrated a lateral mobility for membrane wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors of (2.1 +/- 1.6) X 10(-9) cm2/s and (2.7 +/- 2.3) X 10(-9) cm2/s, respectively. These mobilities were approximately equivalent to phospholipid mobility (2.8 +/- 1.9 X 10(-9) cm2/s). The fast mobility phenotype is observed when the cells are unattached and spherical. Upon attachment, the mobility decreases to (0.19 +/- 0.19) X 10(-9) cm2/s. In addition, the ability of Con A to initiate global modulation was completely lost in spread as well as spherical cells in the E7G1 fast mobility clone. A comparison of F-actin patterns between untransformed Balb/c fibroblasts and the E7G1-transformed line suggests a correlation between well-developed stress fiber assemblies and the ability to induce global modulation. The fast mobility clone was stable for at least 23 passages.

Ligand binding to cell surface receptors induces rapid internalization of ligand-receptor complexes by receptor mediated endocytosis. We have examined the intracellular movement of endocytic vesicles, induced by the lectin concanavalin A (Con A), in cultured rat ovarian granulosa cells using fluorescence and electron microscopy. Within 20 minutes of ligand treatment at 37 degrees C, numerous Con A-containing endocytic vesicles form, which migrate to the cell center by 60 minutes. Double label fluorescence microscopy, using fluorescein-Con-A and rhodamine immunofluorescent staining of tubulin or vimentin, indicates that during vesicle migration microtubules and 10-nm filaments are altered in their organization. By 30 minutes, vesicles are associated with microtubule bundles, which subsequently collapse around the nucleus. Similarly, 10-nm filaments accumulate around the nucleus in conjunction with the perinuclear aggregation of endocytic vesicles. Electron microscopy of Con A-horseradish peroxidase-labeled cells demonstrates that endocytic vesicles fuse to form large receptosome-like structures during intracellular migration and these structures are associated with cytoplasmic microtubules and 10-nm filaments. Taxol, a drug that stabilizes microtubules, prevents endocytic vesicle translocation to the Golgi region. Nocodazole, which causes microtubule disassembly, results in the collapse of 10-nm filaments and the central aggregation of endocytic vesicles. The data indicate that the cytoskeleton participates in the directed intracellular movement of endocytic vesicles; the possible subcellular basis for this movement is discussed.

In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, two states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coefficient of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion constant of mobile receptors was partially reversed by treatment with microtubule-disrupting agents such as Colcemid and Vinca alkaloids. High doses of soluble Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was observed. These experiments provide additional support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the molecular level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.

We have investigated the role of cytoskeletal contraction in the capping of surface proteins crosslinked by concanavalin A on mutant Dictyostelium cells lacking conventional myosin. Measurements of cellular deformability to indicate the development of cortical tension show that cells of the wild-type parental strain, AX4, stiffen early during capping and relax back towards the softer resting state as the process is completed. Mutant cells lacking myosin (mhcA-) have a lower resting-state stiffness, and fail to stiffen and to cap crosslinked proteins on binding concanavalin A. Hence conventional myosin is essential both for capping and for the concomitant increase in cell stiffness. Furthermore, depletion of cellular ATP by azide causes a 'rigor' contraction in AX4 cells which makes them stiffen and become spherical. By contrast, the mhcA- cells fail to respond in these ways. These measurements of cortical tension in non-muscle cells can thus be directly correlated with the presence of conventional myosin, demonstrating that contractile tension generated by myosin can drive both a change of cell shape and the capping of crosslinked surface receptors.

Untreated mouse peritoneal macrophages as well as macrophages treated with concanavalin A (ConA) were incubated in the presence of untreated or ConA-treated epimastigotes and trypomastigotes of Trypanosoma cruzi. Treatment of epimastigotes or trypomastigotes with ConA increased or decreased their uptake by macrophages, respectively. Treatment of their macrophages with ConA reduced by 70% and increased by five times the ingestion of epimastigotes and trypomastigotes, respectively. These results are discussed in relation to previous studies on the mobility of ConA receptors in the membrane of the parasite. Using fluorescein- or ferritin-labelled ConA we observed that ConA binding sites located on the plasma membrane of macrophages are internalized during endocytosis of T. cruzi, and observed in association with the membrane of the endocytic vacuole. Vacuoles without parasites showed a uniform distribution of ConA binding sites, while these sites were distributed in patches in vacuoles containing parasites. These results, in association with others previously reported, suggest the involvement of glycoproteins and/or glycolipids localized on the cell surface of T. cruzi and macrophages during the T. cruzi-macrophage interaction.

Aequorin luminescence has been utilized to determine the spatial and temporal fluctuations of the free calcium ion concentration [Ca++] in Chaos carolinensis during ameboid movement, pinocytosis, and capping. The [Ca++] increases above approximately 10(-7) M during normal ameboid movement. Three types of luminescent signals are detected in cells: continuous luminescence, spontaneous pulses, and stimulated pulses. Continuous luminescence is localized in the tails of actively motile cells, and spontaneous pulses occur primarily over the anterior regions of cells. We are sometimes able to correlate the spontaneous pulses with extending pseudopods, whereas stimulated pulses are induced by mechanical damage, electrical stimulation, concanavalin A-induced capping, and pinocytosis. The localization of both distinct actin structures and sites where [Ca++] increases suggests cellular sites of contractile activity. The independent evidence from localizing actin structures and the distribution of [Ca++] can also be viewed in relation to the solation-contraction coupling hypothesis defined in vitro.

The lateral diffusion coefficient, D, of concanavalin A receptors and receptor complexes on the surface of lymphocytes and RDM4 lymphomas is enhanced by several orders of magnitude to D greater than 5 X 10(-9)cm2/sec by induction of swelling of the cells to bulbous form. Treatments with concanavalin A or 7-nitrobenz-2-oxa-1,3-diazole-phallacidin induce blebs and the bulbous form. The resulting separation of the plasma membrane from most of the F-actin cytoskeleton is accompanied by release of constraints on lateral diffusion of the cell surface receptors, which allows the diffusivity of these glycoproteins to increase nearly to the limit allowed by membrane viscosity.

Using double immuno-fluorescence techniques on frozen-thick sections, we have examined the fate of intermediate filaments during Con A receptor capping in lymphoid cells. Our results indicate that during capping intermediate filaments are preferentially aggregated between the surface receptor cap structure and the cell nucleus. It is possible, therefore, that intermediate filaments are directly involved in lymphocyte capping.

A uniform electric field of 10 V/cm applied across the surface of embryonic toad Xenopus muscle cells results in the asymmetric accumulation of concanavalin A (Con A) receptors toward one side of the cells within 10 min, as visualized by postfield fluorescent Con A labeling. This field produces an extracellular voltage difference of 20 mV across these 20-microns wide cells. The effect is reversible in two respects: (a) Additional exposure of the cell to the same field of opposite polarity for 10 min completely reverses the asymmetric accumulation to the other side of the cell. (b) Relaxation occurs after the removal of the field and results in complete recovery of the uniform distribution in 30 min. Both the accumulation and the recovery movements are independent of cell metabolism, and appear to be electrophoretic and diffusional in nature. The threshold field required to induce a detectable accumulation by the present method is between 1.0 and 1.5 V/cm (corresponding to a voltage difference of 2-3 mV across a 20-microns wide cell). The electrophoretic mobility of the most mobile population of nonliganded Con A receptors is estimated to be about 2 x 10(-3) microns/s per V/cm, while their diffusion coefficient is in the range of 4-7 x 10(-10) cm2/s. Extensive accumulation of the Con A receptors by an electric field results in the formation of immobile aggregates. The Con A receptors appear to consist of a heterogeneous population of membrane components different in their charge properties, mobility, and capability in forming aggregates.

Pure populations of myogenic cells were obtained by cloning satellite cells from human skeletal muscle biopsies. Cell-surface glycoproteins at various stages of myogenesis were analysed by one- and two-dimensional gel electrophoresis. A total of 14 distinct proteins were detectable at the cell surface, on the basis of their susceptibility to desialation by exogenous neuraminidase or their iodination by exogenous lactoperoxidase. Reproducible changes in lectin binding or iodination of eight of these proteins occurred during myogenesis. Only two of the developmentally regulated proteins were components of the detergent-insoluble extracellular matrix fraction. Developmental regulation of these two proteins was unaffected by growth of cultures in 5-bromo-2'-deoxyuridine to inhibit myogenesis. In contrast, developmental regulation of the other cell-surface proteins was inhibited by growth in 5-bromo-2'-deoxyuridine, suggesting that changes in these proteins are tightly coupled to satellite cell differentiation. These studies represent the first systematic analysis of the surface proteins of pure, clonally derived, primary cultures of normal myogenic cells.

The lectin concanavalin A (Con A) was used as a model probe to study the behavior of molecules bound to the surface of recently transformed schistosomula of Schistosoma mansoni. Con A binding was saturable (150-180 pg/organism) and specifically competed by alpha-methyl mannoside. Both FITC-Con A and 125-I-Con A were lost from the surface of schistosomula with a halftime of 8-10 h in culture in defined medium. A comparable decrease in the binding of Con A to schistosomula cultured and then labeled with the lectin indicated that the labeling procedure itself was not inducing the observed change. Internalization of Con A was not seen by either fluorescence microscopy or electron microscope radioautography. In addition, 70-80% of the radioactivity lost from the parasite was recoverable by TCA precipitation from the culture medium as intact Con A (27,000 mol wt on SDS PAGE). Thus, the mechanism of clearance of bound Con A from the surface of cultured schistosomula is apparently by sloughing of Con A molecules intact into the culture media and not by endocytosis and degradation. Con A binding sites, visualized with hemocyanin by scanning electron microscopy, appeared homogeneously distributed over the surface of schistosomula when organisms were labeled at 4 degree C or after fixation with glutaraldehyde. However, Con A and hemocyanin formed aggregates on the surface of schistosomula when labeling was performed at 37 degrees C, which suggests that lectin binding sites have lateral mobility within the plane of the membrane. These aggregates are likely independent of metabolism by the parasite because aggregation also occurs on the surface of organisms killed with azide.

Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We have used the technique of fluorescence photobleaching recovery to study the lateral mobility of cell-surface constituents during the differentiation of neuroblastoma clone N1E-115 cells. The concanavalin A (Con A) binding sites appear as discrete patches distributed over the entire cell surface and exhibit lateral mobility in undifferentiated cells comparable with that of surface glycoproteins of other cells. After induction of differentiation, however, the vast majority of Con A binding sites become immobilized, and we present data which suggest that the mechanism of this immobilization may involve linkage to the internal actin network.

Concanavalin A (Con A), insulin and epidermal growth factor (EGF) surface membrane receptors on cultured bovine odontogenic epithelial cells, were demonstrated by immunofluorescent reactivity with fluorescein-isothiocyanate (FITC) conjugated Con A, FITC-insulin, or EGF and anti-EGF, traced with FITC goat anti-rabbit globulin. Con A, insulin and EGF receptors co-capped with each other and with cytoplasmic actin; the latter was demonstrated by anti-actin antibody (AAA) traced with fluorescein or rhodamine-labelled goat anti-human globulin. Capping of receptors was promoted by prior treatment of cells with cytochalasin B or colchicine, but inhibited by the presence of the drugs throughout the experimental procedure. Binding of a ligand to its receptor promoted capping of an unrelated ligand-bound receptor, a phenomenon that was inhibited by cytochalasin B or colchicine. These results suggest that surface receptors for insulin, EGF and Con A interact with each other and with cytoplasmic actin.

L929, 3T12-3, B16, 3LL, and YAC1 cells with cytostatic factor (CF)-inducing activity from Lactobacillus casei-elicited murine peritoneal macrophages (LCEPM) were susceptible to the cytostatic activity of LCEPM and to LCEPM-produced CF, but L1210, P388D1, and Colon 26 cells, which have no CF-inducing activity, were resistant to that of LCEPM and and to the CF. The resistance of P815 cells to that of LCEPM was stronger than that of 3T12-3 cells, but the CF-inducing activity of P815 cells was about 50% weaker than that of 3T12-3 cells. Release of CF from LCEPM was also caused by heat-killed (100 degrees C, 10 min) 3T12-3 or P815 cells, and this release was inhibited by D-mannose. The CF-inducing activity of heat-killed 3T12-3 or P815 cells was reduced by mild trypsin digestion (37 degrees C for 10 min). A D-mannose-containing glycopeptide or glycoprotein (GP) was separated from 3T12-3 or P815 cells by concanavalin A (Con A) or wheat germ agglutinin (WGA) affinity chromatography. The CF were released from LCEPM by stimulation with the Con A-binding GP of the tumour cells, but the WGA-binding GP had little activity. It is suggested that tumour cells with CF-inducing activity may be susceptible to the cytostatic activity of LCEPM, and those without CF-inducing activity may be resistant to the cytostatic activity of LCEPM and the release of CF from activated macrophages may be caused by the Con A-binding GP of the tumour cell surface.

Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation.

A concanavalin A (Con A)-mediated polyclonal helper assay system was established by using the thymus cells or splenic T cells as a source of helper T cells. When splenic B cells were cocultured with thymus cells or splenic Lyt-2- T cells in the presence of an optimal dose of Con A, B Cells were polyclonally activated and differentiated into immunoglobulin-secreting cells. This Con A-mediated helper activity was completely inhibited by the addition of alpha-methyl-D-mannoside and could not be substituted by culture supernatant of Con A-stimulated thymocytes or splenic T cells. Almost all the activity of the thymus cells was carried by peanut agglutinin low binding population. Genetic restriction between T and B cells was not observed in this helper function. In ontogeny, Con A-mediated helper activity in the thymus was first detected at a few days after birth and reached the adult level at about 1 week of age. The polyclonal helper assay system developed in the present study provides a sensitive system to analyse the helper function of thymus cells and also to delineate the early phase of the differentiation of helper T cell population.

CM factor (CVF) - treated normal mouse serum produced a profound inhibition of the antibvody responses, affecting IgG responses more than IgM, and being T-dependent more than T-independent, but no part of the response being unaffected...

Comparative studies on the cellular morphology of culturd mouse T-lymphoma cells (with particular emphasis on organelles and membrane-associated materials) were conducted using both frozen thin sections and epon thin sections. Due to the fact that the frozen thin sectioning technique allows antigenicity to be retained and also permits good accessibility of the external macromolecular reagents to the interior of the cell, we have been able to explore the intracellular localization of some membrane glycoproteins such as Con A-binding sites and viral membrane glycoprotein, gp 69/71. Our data indicate that most of the membranous cellular structures (e.g., rough endoplasmic reticulum, vesicles, Golgi and nuclear envelope) contain the Con A-specific sugars, mannose, and glucose. In addition, we have found that intracellular gp 69/71 molecules exist in an aggregated form at the terminal region of cisternae of rough endoplasmic reticulum and in vesicles of two size ranges (0.1 to 0.15 microns and 0.3 to 0.4 microns) as well as in the cytoplasm close to the plasma membrane. These findings have not only confirmed some of the previous biochemical data but have also provided new information concerning the biochemical nature of intracellular membrane components and the possible biosynthetic fate of membrane precursor molecules.

The distribution and lateral mobility of Con A receptors in the course of the cell cycle has been studied on human gastric low-differentiated mucous adenocarcinoma cells (MGC 80-3). Synchronized MGC 80-3 cells in monolayer were labeled with F-ConA in the different phases of cell cycle. The discontinuous pattern of surface labelling was present during G1, S and G2, while there were marked differences among them. The cells in mitosis displayed uniform distribution of Con A receptors complex with strong fluorescence. The fluorescence photobleaching recovery method has been used to determine the lateral mobilities of Con A receptors complex. There was marked difference among various phases for mobility pattern and diffusion coefficient. The pattern of mobility of Con A receptor complex was appeared diffusion mobility in M and G1 phases, while flow pattern occurred in S and G2 phases. The diffusion coefficient (D) G1 greater than M, the rate of flow S greater than G2, but mobile fraction in G2 phase is higher than other phases. These results showed that dynamic properties of Con A receptor are apparently different which were regulated by the cell cycle.

Concanavalin A (con A) receptors on the surface of cultured Xenopus myoblasts redistributed in response to monopolar, pulsed electric fields. The prefield uniform distribution of the receptors became asymmetrical, and was polarized toward the cathodal pole, in the same way as in DC fields. The extent of asymmetry depended on the duration of field exposure, pulse width (or alternatively, interpulse interval), frequency, and intensity. This relationship was most conveniently expressed by using duty cycle, a quantity determined by both pulse width and frequency. Pulses of average intensity 1.5 V/cm induced detectable asymmetry within 5 min. At the lowest average field intensity used, 0.8 V/cm, significant asymmetry was detected at 150 min. For pulses of high duty cycle (greater than 25%), steady state was reached after 30 min exposure and the steady state asymmetry was dependent on average field intensity. For low duty cycle fields, the time required to reach steady state was prolonged (greater than 50 min). Before reaching a steady state, effectiveness of the pulses, as compared with DC fields of equivalent intensity, was a function of duty cycle. A low duty cycle field (fixed number of pulses at low frequency or long interpulse interval) was less effective than high duty cycle fields or DC.

Trypsinized cells from embryonic chick neural retina redistributed concanavalin A receptors to patches and caps. Between 12 and 16 days of development, the ability to redistribute concanavalin A receptors declined. This restriction in mobility of the receptors was accompanied by changes in susceptibility to the capping-inhibitory drugs colchicine and cytochalasin B.

A mannoside-directed lectin has been isolated and purified from the seeds of Dolichos lablab L. by affinity chromatography. We have established that this glycoprotein, which displays high erythroagglutinating activity without blood group specificity, highly activates murine T lymphocytes, and we have described for the first time its mitogenic properties. Although its main properties are close to those of concanavalin A (Con A), the well-known mannoside-directed mitogen devoid of sugar moiety, several differences were found in some of the early events triggered by the two lectins during lymphocyte mitogenic stimulation: higher level of interleukin-2 (IL-2) synthesis, optimal dose for IL-2 synthesis at suboptimal mitogenic concentration, lack of ecto-5' nucleotidase inhibition, and lack of mitogenic inhibition at high lectin concentration. Because the two lectins did not act on the cell surface in exactly the same way, we have compared their receptors involved in mitogenesis on the plasma membrane of murine lymphocytes. We had previously established that the polyclonal activation of these cells probably occurred through high-molecular-weight receptors (200-230 kDa). Since the mitogenic stimulation of lymphocyte by galactose oxidase (GO), like that of Con A, was inhibited by DLA, we analyzed the cell surface receptors that were common to these three polyclonal mitogens. After labeling the neuraminidase/GO-treated cell surface glycoproteins with NaB3H4, we immunoprecipitated the Con A and DLA receptors which are the target of GO mitogenic action. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the precipitates demonstrated that there exist on the lymphocyte cell surface receptors common to the polyclonal mitogens DLA, Con A, and GO. Because Con A and DLA sterically inhibit GO mitogenic stimulation, the common glycoproteins which represent the necessary sites of oxidative mitogenic action are probably those which are involved in DLA and Con A-triggered mitogenesis, despite the different properties of the two lectins. These differences could be explained by the lower molecular weight receptors of the two lectins which are not identical.

Concanavalin A (Con A) has been shown to induce delays in oral regeneration in the ciliate Stentor coeruleus. Associated with the delayed oral regeneration is a shedding of the cell's extracellular pellicle with the loss of some pigment granules. It is shown that the delays in oral regeneration are not the result of the pigment shedding. The delays are localized in the earliest stages of oral regeneration prior to stage 4. The delays caused by Con A are completely reversible by the addition of 2 mg/ml alpha-D-methyl mannoside either at the time of Con A exposure or 5 min later. Con A clearly binds to the cell surface as shown by the binding of FITC-Con A and its reversal by alpha-methyl mannoside. Crosslinking of Con A receptor molecules may be responsible for the effects of Con A since succinyl Con A, which does not crosslink these receptors, has no effect on oral regeneration even at double the Con A concentration. Calcium ions are also implicated in the action of Con A because an excess of extracellular calcium (10 mM) completely eliminates the Con A delays when added simultaneously with Con A. Examination of the minimum extracellular calcium concentration required for this effect showed that 2 mM calcium can reverse most of the delays but that 5 mM is necessary to completely reverse the delays caused by Con A. If the addition of calcium is delayed for various times after Con A addition, the extracellular calcium is progressively less effective in reversing the Con A delays.

Interaction of multivalent ligands and cell surface receptors can induce redistribution of these receptors to form patches and caps. In this study, we have investigated the role of nucleus-membrane interaction in the capping of membrane components. Mouse L cells and leukemia EL4 cells were enucleated with the aid of cytochalasin B, yielding cytoplasts and karyoplasts. Capping of surface receptors was induced by allo- and hetero-immune sera followed by fluorescein-conjugated antiglobulin serum, or by the plant lectin concanavalin A. Capping could easily be induced in intact cells, but virtually no capping was detected in the nucleus-free cytoplasts. Interestingly, karyoplasts, which posses cell-membrane components but very little cytoplasm, could be easily induced to cap their surface antigens. Hence, cap formation of membrane components seems not to be an autonomous membrane process. The data suggest that interaction of surface membranes and inner cell components associated with the nucleus is involved in the movement of surface membrane receptors.