Interaction of Dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor. The importance of oligosaccharides which contain phosphodiesters

Mammalian cell lysosomal enzymes or phosphorylated oligosaccharides derived from them are endocytosed by a phosphomannosyl receptor (PMR) found on the surface of fibroblasts. Various studies suggest that 2 residues of Man-6-P in phosphomonoester linkage but not diester linkage (PDE) are essential for a high rate of uptake. The lysosomal enzymes of the slime mold Dictyostelium discoideum are also recognized by the PMR on these cells; however, none of the oligosaccharides from these enzymes contain 2 phosphomonoesters. Instead, most contain multiple sulfate esters and 2 residues of Man-6-P in an unusual PDE linkage. In this study I have tried to account for the unexpected highly efficient uptake of the slime mold enzymes. The results show that nearly all of the alpha-mannosidase molecules contain the oligosaccharides required for uptake, and that each tetrameric, holoenzyme molecule has sufficient carbohydrate for an average of 10 Man8GlcNAc2 oligosaccharides. None of the oligosaccharides or glycopeptides from the lysosomal enzymes bind to an immobilized PMR, but those with 2 PDE show slight interaction. Competition of 125I-beta-glucosidase uptake by various carbohydrate-containing fractions indicates that the best inhibitors are those with 2 PDE, either with or without sulfate esters. Furthermore, the uptake of a lysosomal enzyme isolated from a mutant strain (modA), which produces oligosaccharides with only 1 but not 2 PDE, is about 10-fold less than the uptake of wild-type enzyme which has predominantly 2 PDE. Complete denaturation of 125I-labeled wild-type beta-glucosidase in sodium dodecyl sulfate/dithiothreitol also reduces its uptake by about 10-fold. Taken together, these results suggest that the interactions of multiple, weakly binding oligosaccharides, especially those with 2 PDE, are important for the high rate of uptake of the slime mold enzymes. The conformation of the protein may be important in orienting the oligosaccharides in a favorable position for binding to the PMR.     

Mitogenic stimulation in isoproterenol treated cells of dictyostelium discoideum.

Amoebae of the cellular slime mould Dictyostelium discoideum showed stimulated mitogenic activity when exposed to 200 microM isoproterenol, an activator of adenyl cyclase, for 30 min. Approximately 40% increase in cell proliferation was found at 48 h after isoproterenol treatment. A faster and larger plaque formation as well as higher uptake of FITC-labelled E. coli indicates greater phagocytotic activity in the treated cells. A concurrent increase in DNA and protein syntheses was also recorded in the treated cells. Administration of 400 microM caffeine or 200 microM (+) propranolol brought down the isoproterenol-induced elevation in the cell division rate to control levels. These results are discussed in relation to a precocious activation of adenyl cyclase in the treated cells leading to a transient but significant increase in cell division in this organism.     

Chemotaxis toward carbohydrates and amino acids in Physarum polycephalum

A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- -glucose and 2-deoxy- -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.     

Inhibition of growth and cell wall morphogenesis of Bacillus subtilis by extracellular slime produced by Physarum flavicomum.

Slime secreted by microplasmodia of the myxomycete Physarum flavicomum inhibited the uptake of glucose and amino acids, as well as growth and cell division of the bacterium Bacillus subtilis. Morphological changes such as production of chains, swollen cells, and/or cell lysis, occurred coincident with these physiological inhibitory events. These phenomena were all dependent on the concentration of slime present in the growth medium. Electron microscopy revealed that the cell walls of slime-inhibited cells were undergoing degradation and the process was most pronounced in the swollen cells. Isolated cell walls of B. subtilis were also found to undergo degradation upon incubation with slime. Boiled slime did not exhibit lytic activity on native cell walls, but boiled cell walls were degraded by native slime. The inhibitory effect of slime seemed to be, at least in part, due to an inherent peptidase (protease) activity. B. subtilis eventually overcomes the inhibition exhibited by slime due to the production of an antagonist of slime.     

Dimethyl sulfoxide inhibits capping of surface receptors

The microfilaments of cellular slime mold cells are known to be dislocated from their attachment to the plasma membrane by 10% dimethyl sulfoxide (DMSO). We report here that 10% DMSO inhibited capping of lentil lectin receptors on the surface of vegetative cells of the cellular slime mold, Dictyostelium mucoroides When the DMSO was washed from the cells, capping took place. Capping of surface immunoglobulin of mouse lymphocytes was also reversibly inhibited by 10% DMSO.     

Spore germination and swarm cell morphogenesis in the acellular slime mold Fuligo septica

This report describes conditions under which spores of the acellular slime mold Fuligo septica underwent a very rapid, synchronous and complete (100%) germination followed by morphogenesis of motile, flagellated swarm cells from the released protoplasts. This developmental sequence was initiated immediately upon wetting the spores with a surfactant and was completed within 40–50 min in the absence of any exogenous nutrient other than sodium phosphate buffer. Oxygen was required for germination. The rate and percentage of germination diminished with increasing spore concentration suggesting the existence of an autoinhibitor. The morphological sequence of events in the differentiation process was examined by scanning electron microscopy.     

Cell size in Dictyostelium

Cellular slime mold amoebae have become a model system for the study of cell motility and the cytoskeleton. A basic problem which all cells face that involves the cytoskeleton is how to control their size. The varied ways in which cellular slime mold amoebae change their cell size--by changing the size at which division occurs, by cell fusion, and by control over cytokinesis--are reviewed. A model is presented which attempts to explain how the mechanisms affected in certain cytokinesis mutants in Dictyostelium discoideum known as phg mutants could be involved in control of cell size in the predatory slime mold Dictyostelium caveatum.     

Anti-leukemic activities of Dictyostelium secondary metabolites: a novel aromatic metabolite, 4-methyl-5-n-pentylbenzene-1,3-diol, isolated from Dictyostelium mucoroides suppresses cell growth in human leukemia K562 and HL-60 cells

It has previously been shown that DIF-1, a differentiation-inducing factor of the cellular slime mold Dictyostelium discoideum, possesses antitumor activities in mammalian tumor cells and that neuronal differentiation of PC12 cells can be induced with furanodictines (FDs), aminosugar analogs found in D. discoideum, or dictyoglucosamines (DGs), N-acetyl glucosamine derivatives (DG-A from D. purpureum and DG-B from D. discoideum). Thus, cellular slime molds are attractive natural resources that may provide valuable lead compounds to be utilized in the field of pharmacology and medicine. In this study, we have isolated a novel aromatic compound, 4-methyl-5-n-pentylbenzene-1,3-diol (MPBD), from fruiting bodies of the cellular slime mold D. mucoroides and assessed the in vitro antiproliferative activities of MPBD, FDs, and DGs in human leukemia K562 and HL-60 cells. MPBD at 20-80 microM dose-dependently suppressed cell growth in both K562 and HL-60 cells. While FDs at 10-80 microM did not affect cell growth, DGs at 10-40 microM dose-dependently suppressed cell growth in the cells. Although we failed to find the roles of FDs and DGs in the original organisms, MPBD at 5-20 microM was found to promote stalk cell formation in D. discoideum. The present results indicate that MPBD, DGs or their derivatives may have therapeutic potential in the treatment of cancer and confirm our expectations regarding cellular slime molds as drug resources.     

The function of slime from Physarum flavicomum in the control of cell division.

A haploid cell of the myxomycete Physarum flavicomum undergoes cytokinesis, producing a large population of cells. However, after syngamy, cytokinesis no longer occurs but karyokinesis does and subsequent growth results in the formation of a diploid syncytial plasmodium. Slime, which is produced by the plasmodium but not the haploid cells, was aseptically isolated and purified, and tested for its effect as a cytokinetic regulator. Slime (a viscous, high molecular weight, acidic glycoprotein) affected cytokinesis of the haploid myxamoebae growing in pure culture in soluble media, and the effect was concentration dependent. In simple media, a slime concentration of about 6 10(-5) mug protein per cell suppressed cytokinesis about 50%, unequally inhibited the synthesis of protein, RNA, and DNA, but stimulated respiration. The biological activity of slime was not species specific and it also affected the bacterium Bacillus subtilis by inhibiting cytokinesis, stimulating oxygen uptake, and producing an aberrant cell morphology. Slime was inactivated by heat, fragmentation, and incubation with dithiothreitol, mercaptoethanol, and the proteolytic enzyme papain (EC 3.4.22.2). The inhibitory effect of slime on cell division of haploid cells could not be achieved using mucin or various polyanions. The possible role of slime in the production of the diploid syncytium is discussed.     

The classification of various organisms according to the free amino acid composition change as the result of biological evolution

Summary. The free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflect biological evolution. Cell homogenates were treated with 80–90% ethanol to separate cellular proteins and free amino acids contained in the cells. Different patterns of the free amino acid compositions were observed in the various organisms. Characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. The patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaeobacteria. Rat hepatoma cells (R-Y121B) were cultured in Eagle's minimum essential medium (MEM) containing 5% serum or in a modified MEM lacking arginine, tyrosine and glutamine. No significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. It is suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. Received July 5, 2000 Accepted July 31, 2001     

黄柄钙皮菌无菌培养条件的优化筛选

黄柄钙皮菌是黏菌中的一种重要模式生物,本文筛选了其无菌培养条件,比较了生活史不同阶段的发生时间,同时比较了生活史中孢子萌发、原质团生长及子实体形成3个阶段的培养条件。结果表明,黄柄钙皮菌适宜的培养条件为：在20%水琼脂上于28℃进行孢子萌发;在24–26℃下20%琼脂+40%燕麦培养基上进行原生质团的培养;在24–26℃下1 000Lx光照10–14h时形成子实体。     

A computer simulation of chemical signaling during the aggregation phase of Dictyostelium discoideum

A model for chemical signaling among cells during the aggregation phase of the cellular slime mold Dictyostelium discoideum is simulated using a computer. The resulting chemical concentration profiles are considered in terms of experimental observations. Considerable emphasis is placed on examination of the chemical environment at the center of an aggregation. Its unique nature suggests a mechanism for initially establishing the location of center. Effects of the dimensionality of the simulation universe are also discussed.     

Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage

Transglutaminases are Ca(2+)-dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca(2+)-binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury.     

Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 micrograms/ml (26 microM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of beta-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.     

Effect of monoterpenes on growth of cellular slime mold,Dictyostelium discoideum Ax-2

We examined 12 important monoterpenes found on the forest floor underPinus thunbergii, and monitored their effect on the growth of a slime mold,Dictyostelium discoideum Ax-2. Four concentrations were tested for each compound (3.3, 0.33, 0.033, and 0.0033 μ/mL). Relative growth rates were determined by comparing the cell counts of treated organisms with those from the controls. At a concentration of 3.3 μlml, (1S)-(-)-α-pinene, (-)-menthone, (-)-camphene, (S)-(+)-carvone, and (1 R)-(-)-fenchone strongly inhibited the development of this slime mold. In contrast, (+)-sabinene, (R)-(+)-limonene, and myrcene showed no inhibitory effects, even at the highest concentration tested. By comparing individual growth rates with the control during the incubation period, we could classify these monoterpenes into three groups: I., compounds that were able to inhibit Ax-2 growth at all concentrations; II., compounds that showed a strong inhibitory effect at treatments between 3.3 and 0.033 μl/mL, and mild anti-microbial activity at the lowest concentration; and III., compounds that inhibited growth at higher concentrations (3.3 and 0.33 μl/mL), but enhanced it at lower levels (0.033 and 0.0033 μ/ml.). Based on these results, we suggest that the inhibitory and enhancing effects of selected monoterpenes depend upon the concentration of the individual compound.     

Chemical Characterization of Polysaccharide from the Slime Layer of the Cyanobacterium Microcystis flos-aquae C3-40

Macromolecular material from the slime layer of the cyanobacterium Microcystis flos-aquae C3-40 was defined as material that adhered to cells during centrifugation in growth medium but was dislodged by washing with deionized water and retained within dialysis tubing with a molecular-weight cutoff of 3,500. At each step of this isolation procedure, the slime was observed microscopically. Cells in the centrifugal pellet were surrounded by large amounts of slime that excluded negative stain, whereas cells that had been washed with water lacked visible slime. Two independently isolated lots of slime contained no detectable protein (<1%, wt/wt) and consisted predominantly of anthrone-reacting polysaccharide. Sugars in a hydrolysate of slime polysaccharide were derivatized with trimethylsilylimidazole and examined by gas chromatography-mass spectrometry. The composition of the slime polysaccharide was 1.5% (wt/wt) galactose, 2.0% glucose, 3.0% xylose, 5.0% mannose, 5.5% rhamnose, and 83% galacturonic acid. This composition resembles that of the plant polysaccharide pectin, which was treated in parallel as a control. Consistent with earlier indications that M. flos-aquae slime preferentially binds certain cations, the ratio of Fe to Na in the dialyzed slime was 10⁴ times that in the growth medium. The composition of the slime is discussed with respect to possible mechanisms of cation binding in comparison with other cyanobacterial exopolysaccharides and pectin.     

Bacterial glycoconjugates are natural ligands for the carbohydrate binding site of discoidin I and influence its cellular compartmentalization

Klebsiella pneumoniae, Escherichia coli, and Bacillus subtilis, bacteria commonly eaten by Dictyostelium discoideum, contain glycoconjugates that bind discoidin I, a lectin synthesized by the slime mold as it differentiates. In cells fed bacteria that contain abundant discoidin I-binding glycoconjugates, these ligands and endogenous discoidin I accumulated in specialized structures called multilamellar bodies. In contrast, in cells fed bacteria that had been treated to thoroughly deplete them of discoidin I-binding glycoconjugates, neither endogenous discoidin I nor complementary glycoconjugates were found in the multilamellar bodies. In such cells discoidin I was located in the cytoplasm, as indicated by both immunohistochemistry with the electron microscope and immunoassay of subcellular fractions. The results indicate that a function of the carbohydrate-binding site of discoidin I is to interact with bacterial glycoconjugates, which the slime mold does not degrade. This interaction directs compartmentalization of the lectin in multilamellar bodies and its externalization from the cell in these structures.     

Discoidin-binding polysaccharide from Dictyostelium discoideum

Extracts of Dictyostelium discoideum grown axenically in a chemically defined medium were evaluated for binding to discoidin I and discoidin II, endogenous lectins of this slime mold. Binding activity was measured by competitive inhibition of 125I-lactosyl-bovine serum albumin binding to the immobilized lectins. With the solubilization procedure used extracts of vegetative cells and of early aggregates had no significant inhibitory activity, but an abundant discoidin-binding substance was detected in late aggregates and fruiting bodies. This material was purified by ethanol and acid precipitation followed by precipitation with discoidin. It is a polysaccharide composed of 77% galactose, 15% N-acetylgalactosamine, 5% glucose, and 3% N-acetylglucosamine and may be a biologically functional ligand for the slime mold lectins, in particular discoidin II. Use of axenic cells was critical in these experiments, since extracts of Escherichia coli and Klebsiella aerogenes, commonly used as food for D. discoideum, were found to contain substances that react with discoidin. This would complicate isolation of endogenous discoidin ligands from cells raised on bacteria.     

Effect of carbamate esters on neurite outgrowth in differentiating human SK-N-SH neuroblastoma cells

Carbamate esters are widely used as pesticides and can cause neurotoxicity in humans and animals; the exact mechanism is still unclear. In the present investigation, the effects of carbamates at sublethal concentration on neurite outgrowth and cytoskeleton as well as activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in differentiating human SK-N-SH neuroblastoma cells were studied. The results showed that 50 microM of either aldicarb or carbaryl significantly decreased neurite length in the retinoic acid-induced differentiation of the neuroblastoma cells, compared to cells treated with vehicle. Western blot analyses revealed that neither carbamate had significant effects on the levels of actin, or total neurofilament high molecular proteins (NF-H). However, increased NF-H phosphorylation was observed following carbamate treatment. These changes may represent a useful in vitro marker of carbamate neurotoxicity within a simple model of neuronal cell differentiation. Furthermore, activity of AChE, but not NTE, was significantly inhibited by aldicarb and carbaryl in differentiating cells, which suggested that cytoskeletal protein changes induced by carbamate esters in differentiating cells was associated with inhibition of AChE but not NTE.     

Concanavalin a and wheat germ agglutinin receptors on dictyostelium: Their visualization by scanning electron microscopy with microspheres

The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.