Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation.

The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.     

Endocytosis of α1-acid glycoprotein variants by human monocytic lineage cells

Summary— Human α₁-acid glycoprotein (AGP or orosomucoid) is a major glycoprotein of plasma. AGP can be separated on immobilized concanavalin A into three variants bearing none (AGP A), one (AGP B) or two (AGP C) biantennary glycans. In this paper, we show, using flow cytometry and confocal microscopy, that AGP C which is eluted from concanavalin A with mannose, binds to human monocytes, monocyte-derived macrophages as well as human promonocytic cell lines such as THP1 or U937. Conversely HL60, a promyelocytic cell line, does not express the surface AGP C binding protein. AGP C is internalized and degraded with an efficiency depending on the state of differentiation of these cells. In contrast, AGP A which is not recognized by concanavalin A, does not bind to any of these cells.     

Pathways of Differentiation in Chick Embryo Neuroretinal Cultures

Markers of neuronal cell differentiation (GABA accumulation, choline acetyltransferase activity) are shown to increase initially and then decline sharply in monolayer cultures of 9 day embryo neuroretinal (NR) cells. A glial marker (glutamine synthetase, GSase) is precociously inducible by hydrocortisone (HC) in dense'monolayer' NR cultures (containing aggregates of neuronal cells overlying the glial sheet) as well as in chick embryo retinal explants. The induced level of GSase activity is not maintained in the continued presence of HC, but rather declines by 20 days in vitro. Choline acetyltransferase (CAT) activity is higher in HC-treated cultures than in controls only during the period when induced GSase activity is detectable. Furthermore, the subsequent transdifferentiation of lens cells (monitored as δ crystallin content) in these cultures is delayed by 10 days and much reduced in extent when HC is present throughout the culture period.
We suggest a simple model to account for these results, on the basis of recent evidence that lens cells are derived mainly from the retinal epithelial cells (immature Müller glia) of 9-day embryonic NR, and that transdifferentiation results from a change in cell determination during the early stages of'monolayer' culture. In outline, our model proposes that early dedetermination of the retinal glia is associated with a decline of neuronal cell markers (dedifferentiation) followed eventually by loss of the neuronal cells. Hydrocortisone, by inducing transient glial cell differentiation (GSase activity), both prolongs the expression of a neuronal marker (CAT) and also reduces later transdifferentiation into lens.     

Pathways of differentiation in chick embryo neuroretinal cultures

Markers of neuronal cell differentiation (GABA accumulation, choline acetyltransferase activity) are shown to increase initially and then decline sharply in monolayer cultures of 9 day embryo neuroretinal (NR) cells. A glial marker (glutamine synthetase, GSase) is precociously inducible by hydrocortisone (HC) in dens "monolayer' NR cultures (containing aggregates of neuronal cells overlying the glian sheet) as well as in chick embryo retinal explants. The induced level of GSase activity is not maintained in the continued presence of HC, but rather declines by 20 days in vitro. Choline acetyltransferase (CAT) activity is higher in HC-treated cultures than in controls only during the period when induced GSase activity is detectable. Furthermore, the subsequent transdifferentiation of lens cells (monitored as delta crystalline content) in these cultures is delayed by 10 days and much reduced in extent when HC is present throughout the culture period. We suggest a simple model to account for these results, on the basis of recent evidence that lens cells are derived mainly from the retinal epithelial cells (immature Müller glia) of 9-day embryonic NR, and that transdifferentiation results from a change in cell determination during the early stages of "monolayers' culture. In outline, our model proposes that early determination of the retinal glia is associated with a decline of neuronal cell markers (dedifferentiation) followed eventually by loss of the neuronal cells. Hydrocortisone, by inducing transient glial cell differentiation (GSase activity), both prolongs the expression of a neuronal marker (CAT) and also reduces later transdifferentiation into lens.     

Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.     

Isolation of a Potential Neural Stem Cell Line from the Internal Capsule of an Adult Transgenic Rat Brain

A thermosensitive mutation of simian virus 40 large T antigen (LTA) gene, the tsA58 gene, was cloned downstream of the 6-kbp neurofilament light chain promoter in pPOLYIII and injected into the pronucleus of fertilised oocytes of Sprague-Dawley rats to develop a strain harbouring six copies of the transgene. Immunocytochemical staining of hemizygous adult tissues with antibodies to the C-terminus of LTA showed that the inactive form of LTA was expressed only in the fibres of the internal capsule and in the choroid plexus of the brain. Culturing the former region at 33 degrees C, the permissive temperature for LTA, yielded a cell line, NF2C, which produced active LTA and grew at 33 degrees C but which produced only inactive LTA and eventually died at the non-permissive temperature of 39 degrees C. This clonal cell line was heterogeneous at 33 degrees C, producing the precursor neuronal cell marker nestin and the glial-specific markers glial fibrillary acidic protein, vimentin and S100A1, as well as weakly producing the neuronal cell markers 68-kDa neurofilament protein (NF68) and microtubule-associated protein 2 (MAP2) in different subpopulations of cells. However, at 39 degrees C, the cells produced dendritic, neuronal-like processes and elevated levels of NF68 and MAP2, as well as the neuronal markers synaptophysin, neurone-specific enolase, and low levels of tau, all determined by western blotting and immunofluorescent staining. Basic fibroblast growth factor enhanced the growth of the cells at 33 degrees C but also enhanced the formation of dendritic neuronal-like processes at 39 degrees C. It is suggested that NF2C represents a potential stem cell line from adult brain that expresses precursor and glial cell markers at 33 degrees C but undergoes partial differentiation to a neuronal cell phenotype at 39 degrees C.     

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.     

Changes in intramembranous particle topography and concanavalin A receptor mobility associated with myoblast differentiation.

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37 degrees C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.     

Dynamics and histological characteristics of gonadal sex differentiation in pejerrey (Odontesthes bonariensis) at feminizing and masculinizing temperatures

This study evaluated the effects of different temperatures on the histological process of sex differentiation in the pejerrey Odontesthes bonariensis, a fish with marked temperature-dependent sex determination (TSD), at feminizing, neutral, and masculinizing temperatures. Fish reared at three temperatures (17 degrees C, 24 degrees C, and 29 degrees C) from hatching were sampled weekly until 11 weeks and their gonads were examined by histology. The percentages of females at 17 degrees C, 24 degrees C, and 29 degrees C were 100%, 73%, and 0%, respectively. Sex differentiation occurred earlier and at a smaller body size at higher temperatures in both sexes. The first signs of ovarian differentiation were observed at 4 and 7 weeks at 24 degrees C and 17 degrees C, respectively, and those of testicular differentiation at 4 and 7 weeks at 29 degrees C and 24 degrees C, respectively. Body or gonadal growth rates before sex differentiation were not proportional to temperature and showed no sexual dimorphism at 24 degrees C, where both sexes were present. Thus, differential growth rate is probably not a factor in TSD or histological sex differentiation in pejerrey. Blood vessels were formed before sex differentiation in both sexes and at all temperatures, and may be important for sex differentiation. No signs of intersexuality were found in any of the groups, and this characterizes pejerrey as the differentiated type of gonochorist even at feminizing and masculinizing temperatures. Ovaries were formed by the same histological processes at feminizing (17 degrees C) and neutral (24 degrees C) temperatures and without any pathological features such as germ cell degeneration. The process of testicular formation was generally similar at 24 degrees C and 29 degrees C, but some fish at 29 degrees C had widespread germ cell degeneration before sex differentiation. This suggests that pathological processes leading to germ cell death, such as heat-induced dysfunction of the supporting somatic cells, could be involved in masculinization of the genetic females at high temperatures.     

Binding of concanavalin A to isolated hepatocytes and its effect on uptake and degradation of asialo-fetuin by the cells.

1. The binding of 125I-labelled concanavalin A to isolated rat hepatocytes was studied at temperatures between 4 degrees C and 37 degrees C. At the latter temperature, concentrations of concanavalin A from 0.01 to 0.4 mg/ml were used. In all of these experiments, binding reached a plateau after 40--60 min, when 28--35% of the concanavalin A added was bound to the cells (cell density 8 x 10(6) cells/ml). 2. The rate of uptake of 125I-labelled asialo-fetuin by the hepatocytes was lowered to 30% of control values when the cells were preincubated with 0.1 mg of concanavalin A/ml. This decrease could be accounted for by a decrease in the rate of binding of asialo-fetuin to the beta-galactoside receptor of the cells. The binding capacity of the cells was not influenced by preincubation with concanavalin A. 3. Degradation of asialo-fetuin was decreased only if concanavalin A was present during the uptake of asialo-fetuin by the cells. Subcellular fractionation revealed that concanavalin A lowered the rate of entry of endocytosed asialo-fetuin into the lysosomes. The effect of concanavalin A on degradation is distinct from its effect on the rate of uptake of asialo-fetuin by hepatocytes.     

Concanavalin A can trap insulin and increase insulin internalization into cells cultured in monolayer

When rat hepatoma cells (R-Y121B) were incubated with insulin at 37 degrees C, concanavalin A increased insulin internalization into cells. When R-Y121B cells were first incubated with labeled insulin at 4 degrees C then with concanavalin A at various concentrations at 37 degrees C, the total cellular radioactivity was much higher at high lectin concentrations than at low lectin concentrations. This increase was not only due to an increase in insulin internalization into cells but also to an increase in insulin binding to cell surfaces. Concanavalin A can trap insulin on the insulin receptors - a "trapping" effect. It has been concluded that insulin and concanavalin A binding sites are very close to each other on the insulin receptors.     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

Surface membrane redistribution and stabilization of concanavalin A-specific receptors following Yaba tumor poxvirus infection

Monkey kidney cells productively infected with Yaba tumor poxvirus clearly exhibit plasma membrane alterations when treated with both fluorescein-labeled and unlabeled concanavalin A. The convanavalin A-mediated cytoagglutination reaction for Yaba-infected Jinet and CV-1 cells increased linearly from 12 to 16 h post-infection, reaching a maximum by 24-28 h. Treatment of either Yaba-infected CVC-1 or Jinet cells with methyl-D-glucopyranoside before or after addition of concanavalin A completely blocked or reversed the cytoaglutination response. Trypsin treatment of uninfected CV-1 or Jinet cells enhanced concanavalin A-mediated cytoagglutination properties. Conversely, trypsin treatment of Yaba-infected Jinet cells resulted in a reduced cytoagglutination response. Increasing temperature and lectin concentration enhance concanavalin A-mediated cytoagglutination for uninfected, trypsin-treated and Yaba-infected CV-1 cells. Cytosine arabinoside has little or no effect on the Yaba-induced cell cytoagglutination reaction while cycloheximide blocks the cytoagglutinatin response if added prior to 12 h post-infection. Fluorescein-labeled concanavalin A binding studies have revealed that at 4 degrees C, Yaba-infected CV-1 cells display a predominantly 'patchy' pattern of topological fluorescence, while trypsin-treated and uninfected CV-1 cells at 4 degrees C display a uniform pattern of fluorescence binding. Patchy fluorescence, indicative of concanavalin A-suspeptible, receptor-site clustering on the surface membrane, was reduced 50% if Yaba-infected CV-1 cells were treated with glutaraldehyde (2.5%) before addition of fluorescein-labeled concanavalin A at 4 degrees C. Similar pre-fixatin of trypsin-treated CV-1 cells resulted in uniform, fluorescent labelling patterns at all assay temperatures.     

Membrane-bound neuregulin1 type III actively promotes Schwann cell differentiation of multipotent Progenitor cells

Many steps of peripheral glia development appear to be regulated by neuregulin1 (NRG1) signaling but the exact roles of the different NRG1 isoforms in these processes remain to be determined. While glial growth factor 2 (GGF2), a NRG1 type II isoform, is able to induce a satellite glial fate in neural crest stem cells, targeted mutations in mice have revealed a prominent role of NRG1 type III isoforms in supporting survival of Schwann cells at early developmental stages. Here, we investigated the role of NRG1 isoforms in the differentiation of Schwann cells from neural crest-derived progenitor cells. In multipotent cells isolated from dorsal root ganglia, soluble NRG1 isoforms do not promote Schwann cell features, whereas signaling by membrane-associated NRG1 type III induces the expression of the Schwann cell markers Oct-6/SCIP and S100 in neighboring cells, independent of survival. Thus, axon-bound NRG1 might actively promote both Schwann cell survival and differentiation.     

Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells

NR5A2 is a nuclear receptor which regulates the expression of genes involved in cholesterol metabolism, pluripotency maintenance and cell differentiation. It has been recently shown that DLPC, a NR5A2 ligand, prevents liver steatosis and improves insulin sensitivity in mouse models of insulin resistance, an effect that has been associated with changes in glucose and fatty acids metabolism in liver. Because skeletal muscle is a major tissue in clearing glucose from blood, we studied the effect of the activation of NR5A2 on muscle metabolism by using cultures of C2C12, a mouse-derived cell line widely used as a model of skeletal muscle. Treatment of C2C12 with DLPC resulted in increased levels of expression of GLUT4 and also of several genes related to glycolysis and glycogen metabolism. These changes were accompanied by an increased glucose uptake. In addition, the activation of NR5A2 produced a reduction in the oxidation of fatty acids, an effect which disappeared in low-glucose conditions. Our results suggest that NR5A2, mostly by enhancing glucose uptake, switches muscle cells into a state of glucose preference. The increased use of glucose by muscle might constitute another mechanism by which NR5A2 improves blood glucose levels and restores insulin sensitivity.     

Studies on cell division in mammalian cells. VII. A temperature- sensitive cell line abnormal in centriole separation and chromosome movement

A temperature-sensitive Syrian hamster mutant cell line, ts-745, exhibiting novel mitotic events has been isolated. The cells show normal growth and mitosis at 33 degrees C, the permissive temperature. At the nonpermissive temperature of 39 degrees C, mitotic progression becomes aberrant. Metaphase cells and those cells still able to form a metaphase configuration continue through and complete normal cell division. However, cells exposed to 39 degrees C for longer than 15 min can not form a normal metaphase spindle. Instead, the chromosomes are distributed in a spherical shell, with microtubules (MT) radiating to the chromosomes from four closely associated centrioles near the center of the cell. The cells progress from the spherical monopolar state to other monopolar orientations conical in appearance with four centrioles in the apex region. Organized chromosome movement is present, from the spherical shell state to the asymmetrical orientations. Chromosomes remain in the metaphase configuration without chromatid separation. Prometaphase chromosome congression appears normal, as the chromosomes and MT form a stable monopolar spindle, but bipolar spindle formation is apparently blocked in a premetaphase state. When returned from 39 degrees to 33 degrees C, the defective phenotype is readily reversible. At 39 degrees C, the mitotic abnormality lasts 3-5 h, followed by reformation of a single nucleus and cell flattening in an interphase- like state. Subsequent cell cycle events appear to occur, as the cells duplicate chromosomes and initiate a second round of abnormal mitosis. Cell cycle traversion continues for at least 5 d in some cells despite abnormal mitosis resulting in cells accumulating several hundred chromosomes.     

Concanavalin A promotes the uptake of lysosomal hydrolases by human fibroblasts.

Human placental hexosaminidase B and beta-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4 degrees C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37 degrees C is refractory to haptens treatment. These results are interpreted as an initial binding of enzyme to concanvalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Factors affecting the molecular structure and the agglutinating ability of concanavalin A and other lectins.

Ultracentrifugation analyses were performed on lectins under varying conditions of pH, ionic strength and temperature. It has been demonstrated that the phytohemagglutinin from Phaseolus vulgaris, the wheat germ agglutinin and the soybean agglutinin are stable when these parameters are varied, whereas the concanavalin A molecule exhibits a striking reversible dimer-tetramer transition with variation in pH (from 6.0 to 7.2) and temperature (from 4 degrees up to 37 degrees C). It has also been demonstrated that, in agglutination experiments undertaken at different temperatures, cells do eventually aggregate with the first three lectins provided that incubation time is sufficient, whereas the concanavalin-A-induced agglutination was previously found to be temperature-sensitive. These results strongly suggest that the effect of temperature on agglutination by lectins may essentially be due to a structural transition of the lectin itself and nott only to modification of cell surface properties.     

Fusion of SV40-induced endocytotic vacuoles with the nuclear membrane

The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4 degrees C. Following incubation of these modified cells at 37 degrees C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37 degrees C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37 degrees C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.