Prespore and prestalk differentiation of Dictyostelium discoideum as examined by changes of wheat germ agglutinin binding proteins

1987 ◽  
Vol 65 (1) ◽  
pp. 68-75 ◽  
Author(s):  
Akiko Kumagai ◽  
Hideo Mori ◽  
Shinoi Osuka ◽  
Koji Okamoto
Keyword(s):  
Dictyostelium Discoideum ◽  
Wheat Germ Agglutinin ◽  
Culture System ◽  
Wheat Germ ◽  
Binding Proteins ◽  
Normal Development ◽  
Maximum Level ◽  
Cell Types ◽  
The Other ◽  
Two Dimensional

Prespore- and prestalk-specific, wheat germ agglutinin (WGA) binding proteins in Dictyostelium discoideum were identified on two-dimensional gels by the use of a peroxidase–antiperoxidase method. Using these proteins as markers, differentiation of the two presumptive cell types was examined during the development. In normal development, two groups of prespore-specific WGA-binding proteins were found: one was detectable when cells formed discrete aggregates without tips (10.5 h) and reached a maximum level at 12 h, while the other appeared at the time of slug formation (17 h). On the other hand, a prestalk-specific WGA-binding protein, having an unusually high pI value (pI ca. 9.5), began to accumulate just before slug formation (13.5 h). The changes of WGA-binding proteins in a shake culture system were similarly analysed and compared with those in normal development.

scholarly journals Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides.

1986 ◽  
Vol 103 (4) ◽  
pp. 1369-1382 ◽  
Author(s):  
L M Greenberger ◽  
K H Pfenninger
Keyword(s):  
Sialic Acid ◽  
Growth Cone ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Growth Regulation ◽  
Binding Proteins ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Adult Brain ◽  
Two Dimensional

A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.

Effects of cyclic AMP on contact formation and differentiation in Dictyostelium discoideum

1982 ◽  
Vol 56 (1) ◽  
pp. 223-232
Author(s):  
M. Oyama ◽  
K. Okamoto ◽  
I. Takeuchi
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Wheat Germ Agglutinin ◽  
Culture System ◽  
Wheat Germ ◽  
Cell Contact ◽  
Contact Formation

It has been shown previously that Dictyostelium discoideum NC4 cells dissociated at the early aggregation stage form cell clumps and differentiate into prespore cells in a shaking culture containing glucose, albumin, EDTA and cyclic AMP. In this culture system, we found that the cells neither differentiate nor form cell clumps in the absence of cyclic AMP. Wheat-germ agglutinin (WGA) completely inhibited the cyclic AMP-induced formation of cell contact and the inhibition was nullified by the addition of N-acetyl-D-glucosamine. When the cells were prevented from forming contact by either rapid shaking or the addition of WGA, they were unable to differentiate even in the presence of cyclic AMP, indicating that contact formation is a prerequisite for prespore differentiation. Cells dissociated from migrating slugs formed cell clumps in shaking culture, with or without cyclic AMP, and the cell contact was sensitive to WGA. In the absence of cyclic AMP, prespore cells lost their differentiated state, even though the cells were in contact. This indicates that cyclic AMP has a second effect, that of pomoting differentiation, in addition to the effect of inducing contact formation. Both effects were required for prespore differentiation of strain NC4 cells.

Chemotaxis-associated properties of separated prestalk and prespore cells of Dictyostelium discoideum

1986 ◽  
Vol 64 (8) ◽  
pp. 722-732 ◽  
Author(s):  
J. D. Mee ◽  
D. M. Tortolo ◽  
M. B. Coukell
Keyword(s):  
Light Scattering ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Binding Sites ◽  
Large Fraction ◽  
Cell Types ◽  
The Other ◽  
Deficient Mutant ◽  
Camp Phosphodiesterase ◽  
Intracellular Cgmp

During development, prestalk and prespore cells of Dictyostelium discoideum become organized in multicellular structures. This physical association makes it difficult to characterize the two cell types biochemically and physiologically. In the present study, we have separated prestalk and prespore cells from 16-h slugs by the method of Tsang and Bradbury and have examined a number of chemotaxis-associated properties of these cells. When assayed on phosphate-buffered agar under both gradient and nongradient conditions, isolated prestalk cells responded chemotactically to cAMP and, unexpectedly, to folate and certain folate derivatives. In contrast, separated prespore cells failed to respond appreciably to any of these compounds. Neither prestalk nor prespore cells of strain HC91 exhibited a cAMP-induced increase in intracellular cGMP. However, a cGMP response was observed in both prestalk and prespore cells of strain NP368, a cGMP phosphodiesterase deficient mutant. Both cell types exhibited comparable cAMP-mediated light-scattering changes and possessed similar levels of surface cAMP- and folate-binding sites. On the other hand, prestalk cells had at least fourfold higher cAMP phosphodiesterase and folate deaminase activities than prespore cells, and a large fraction of both activities was on the cell surface. Therefore, the greater chemotactic response of prestalk cells to cAMP and folate on agar might be due, in part, to their increased capacity to generate a chemoattractant gradient. Results obtained in this study demonstrate that prestalk and prespore cells separated by this procedure can be used in certain physiological as well as biochemical experiments.

scholarly journals Compartmentalization of intracellular membrane glycocomponents is revealed by fracture-label.

1984 ◽  
Vol 98 (1) ◽  
pp. 29-34 ◽  
Author(s):  
M R Torrisi ◽  
P Pinto da Silva
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Secretory Granules ◽  
The Other ◽  
Rat Tissues ◽  
Intracellular Membrane ◽  
Definition Of

We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes. In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments: one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues.

scholarly journals Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 753-763
Author(s):  
P Van Vlasselaer ◽  
N Falla ◽  
H Snoeck ◽  
E Mathieu
Keyword(s):  
Bone Marrow ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Three Dimensional ◽  
The Other ◽  
Light Scatter ◽  
Murine Bone Marrow ◽  
Osteogenic Cells ◽  
Mineralized Matrix ◽  
Binding Intensity

Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)- treated mice based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA). Four sort gates were established using forward (FSC) and perpendicular (SSC) light scatter and were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the other gates, synthesized alkaline phosphatase, collagen, and osteocalcin and formed a mineralized matrix in culture. The number of osteoprogenitor cells was significantly enriched after depleting the 5- FU bone marrow from cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural killer cells, granulocytes, macrophages, and erythrocytes. Approximately 95% of the FSChigh SSChigh cell population of this “lineage-negative” (Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three additional sort windows were established based on WGA binding intensity and were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright. Cells from the Sca-1+ WGAbright gate, but not from the other gates, synthesized bone proteins and formed a mineralized matrix. However, they lost this capacity upon subcultivation. Further immunophenotypic characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that stain with the von Kossa technique and contain osteoblast and osteocyte-like cells.

scholarly journals Analysis of cholecystokinin-binding proteins using endo-beta-N-acetylglucosaminidase F.

1984 ◽  
Vol 99 (3) ◽  
pp. 1110-1116 ◽  
Author(s):  
S A Rosenzweig ◽  
L D Madison ◽  
J D Jamieson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Neuraminidase Treatment ◽  
Similar Treatment ◽  
Definitive Evidence ◽  
Electrophoretic Mobilities ◽  
Significant Enrichment ◽  
Polypeptide Backbone

We have previously shown that the cholecystokinin (CCK)-binding proteins in rat pancreatic plasma membranes consist of a major Mr 85,000 and minor Mr 55,000 and Mr 130,000 species as revealed by affinity labeling with 125I-CCK-33 using the cross-linker, disuccinimidyl suberate. The glycoprotein nature of these species was investigated using endoglycosidase F (endo F) and neuraminidase treatment and wheat germ agglutinin-agarose chromatography. Treatment of affinity-labeled membranes with endo F resulted in increased electrophoretic mobilities of all three binding proteins, indicating removal of N-linked oligosaccharide side chains. Endo F treatment of each protein in gel slices indicated the following cleavage relationships: Mr 85,000----65,000; Mr 55,000----45,000; Mr 130,000----110,000. Using limiting enzyme conditions to digest each protein contained in excised SDS gel slices, three and four products, respectively, were identified for the Mr 85,000 and 55,000 proteins. Similar treatment of the Mr 130,000 protein revealed only the Mr 110,000 product. These results indicated that the Mr 85,000 protein has at least three, the Mr 55,000 protein has at least four, and the Mr 130,000 protein has at least one, N-linked oligosaccharide side chain(s) on their polypeptide backbone. Neuraminidase treatment of affinity-labeled membranes caused slight increases in the electrophoretic mobilities of all three proteins, indicating the presence of sialic acid residues. Solubilization of affinity-labeled membranes in Nonidet P-40 followed by affinity chromatography on wheat germ agglutinin-agarose revealed that all three CCK-binding proteins specifically interact with this lectin and can be eluted with N-acetyl-D-glucosamine. Analysis of the proteins present in the eluted fractions by silver staining indicated a significant enrichment for proteins having molecular weights corresponding to the major CCK-binding proteins in comparison to the pattern of native membranes. Taken together, these studies provide definitive evidence that the CCK-binding proteins in rat pancreas are (sialo)glycoproteins.

Localization and regulation of acid-base secretory currents from individual epithelial cells

1991 ◽  
Vol 261 (6) ◽  
pp. F963-F974 ◽  
Author(s):  
C. Scheffey ◽  
A. M. Shipley ◽  
J. H. Durham
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Outward Current ◽  
Cell Types ◽  
The Other ◽  
Two Dimensional ◽  
Acid Base ◽  
Electrical Currents ◽  
Inward Current ◽  
Inward Currents

The turtle urinary bladder is composed of different epithelial cell types that are suspected to separately produce electrogenic acid and alkali excretion. We measured the electrical currents produced by individual cells, scanning a two-dimensional vibrating probe over the luminal surface of the bladder. Acidification (outward current) was produced by the type of epithelial cell rich in carbonic anhydrase (CA cells). The measured currents of these cells quantitatively accounted for the total epithelial acidification current. When alkali secretion was induced by adenosine 3',5'-cyclic monophosphate and acidification was inhibited (by luminal pH 4), we measured inward currents localized to a small number of epithelial cells in four bladders but found no localization in the other seven treated bladders. When alkali secretion was localized and induced without inhibiting acidification, we found both cells producing inward current and cells producing outward current, which demonstrated that the two transport functions can occur simultaneously. We conclude that net acid-base secretion can be determined by regulating the transport rates of separate cells.

A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk

1990 ◽  
Vol 68 (4) ◽  
pp. 699-704 ◽  
Author(s):  
Yuzuru Kubohara ◽  
Koji Okamoto
Keyword(s):  
Dictyostelium Discoideum ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Stalk Cell ◽  
Affinity Column ◽  
Western Blots

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5–6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.Key words: Dictyostelium discoideum, Dictyostelium mucoroides, wheat germ agglutinin.

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