Occupational asthma to the slime mold Dictyostelium discoideum

Dictyostelium discoideum is a slime mold that exists in a unicellular amoeboid form under certain nutritional conditions. In this form, it produces unique lysosomal enzymes that are valuable in studying cell-to-cell signaling systems. We report on a research microbiologist who developed rhinoconjunctivitis and asthma after release of D. discoideum from a pressurized canister. Immediate skin test reactivity was demonstrated to whole and lysed organisms. Enzyme-linked immunosorbent assay results revealed IgE antibody against D. discoideum whole organism, lysed organism, and lysosomal enzymes with the strongest response being directed toward lysosomal enzymes. Pulmonary function testing showed a decline in forced expiratory volume in 1 second and forced expiratory flow after modified laboratory exposure to D. discoideum. This case represents the first report of occupational rhinoconjunctivitis and asthma from slime mild.     

A new collection of thermosensitive endocytosis mutants in the cellular slime mold Dictyostelium discoideum

We used a photoactivatable fluid-phase marker to isolate a new collection of thermosensitive endocytosis mutants in the cellular slime mold Dictyostelium discoideum. All the strains were thermosensitive for growth on bacteria or axenic medium at 27 degrees C. Initial rates of endocytosis rapidly decreased upon incubation at the restrictive temperature, but surprisingly most of the strains showed a transient recovery of activity with prolonged exposure to 27 degrees C. Endocytosis and exocytosis activities were uncoupled for some of the cell lines at 27 degrees C whereas the others had to be shifted to 29 degrees C. Further molecular analysis of these mutants could lead to the discovery of new proteins involved in endocytosis and its regulation.     

Structural organization and processing of the genetic transcript in the cellular slime mold Dictyostelium discoideum

30 patients suffering from generalized lichen planus (= L.p.) have been examined by the following methods in order to more precise information regarding their immune-status: 1. Quantitative determination of immuno-globulins including complement fraction (beta 1C/A) 2. Intracutaneous tests with recall-antigens 3. Investigation of cutaneous sensitivity to DNCB 4. Lymphocytemigrationinhibition- and Lymphocytetransformationstests. The results obtained were not statistically significant when comparing the results of this investigation in patients suffering from L.p. with the control group. Therefore, the theory that immunological processes are the basis of these occurences is still under discussion. Further studies applying more specific methods should be conducted in order to clarify this problem.     

Green fluorescent protein production in the cellular slime molds Polysphondylium pallidum and Dictyostelium discoideum

The green fluorescent protein-encoding gene from Aequorea victoria has been cloned into several different transforming vectors and expressed in the cellular slime molds, Polysphondylium pallidum and Dictyostelium discoideum. We find that the protein is stable and non-toxic in both species, can be easily visualized in living and fixed specimens, and can be used to purify rare cells by fluorescence-activated cell sorting (FACS).     

A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis

A series of aggregation-deficient (aggregateless) mutants were isolated in genetically marked haploid strains of the cellular slime mould Dictyostelium discoideum. Diploids were produced from pairs of such haploid mutants by a fusion system based on this organism's parasexual cycle. The diploids were isolated from the haploids by using complementation of non-allelic growth-temperature-sensitive mutations and selection at the restrictive temperature. Complementation between the aggregateless mutations uas then assessed in 419 diploids so formed. The non-complementing aggregateless mutations fell into five complementation groups (ago A, B, C, D, and E) and a dominant aggregation class that allowed little or no aggregation when present in a diploid with any of the other mutations tested or the parental wild type. Complicating factors, including partial dominance, multiple mutations, and possible interallelic conplementation, are discussed. Data on the linkage of the aggregateless mutations was obtained by using recessive drug resistance mutations on three linkage groups to segregate haploids from the diploids. Calculations from our results suggest a genetic complexity of about 50 genes that are specific and essential for aggregation.     

UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from Dictyostelium discoideum recognizes serine-containing peptides and eukaryotic cysteine proteinases

Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins. Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs. We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues. Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them. Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency. Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase. CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM. SDS denaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all. Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc-1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, Km = 5.6 microM. Thus, Ser:GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs. This may be physiologically important for establishing or maintaining non-overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium proteins that reside in functionally distinct endo-lysosomal vesicles.     

Differentiation for aggregation in the cellular slime moulds: the emergence of autonomously signalling cells in Dictyostelium discoideum

The results of experiments on small populations of Dictyostelium discoideum, directed towards the measurement of the development in time of the competence of the cells to signal autonomously, are reported. This competence is quantified by X3, the intrinsic probability that a given cell may turn autonomous. The data show an early exponential growth in time of X3, followed by saturation. The saturation value depends on the population size suggesting that the differentiation is a co-operative phenomenon. The differentiation of autonomous cells starts roughly 7 h after the removal of food and saturates within 21 h.     

Sexual hormone in the cellular slime mould Dictyostelium purpureum

The existence of sexual hormone in Dictyostelium purpureum was revealed when the extracelluar medium from certain strains (Dp6 or Dp7) induced macrocyst formation when added to cells of the opposite mating type (Dp2). Our results suggest that mating in cellular slime moulds may involve a secreter-responder system whereby one mating-type strain (Dp6 or Dp7) releases sexual hormone while the opposite strain (Dp2) responds. However, the existence of a hormone released by the responding strain has not been completely ruled out by our experiments. The sexual hormones of cellular slime moulds appear to be species-specific since hormone from D. purpureum cannot induce macrocyst formation in responder strains of D. discoideum and hormone from D. discoideum has no effect on D. purpureum cells.     

Signal relay by single cells during wave propagation in cellular slime mold

The autolysin of Streptococcus cremoris had the specificity of an endo-N-acetylmuramidase as it hydrolysed the linkage between N-acetylmuramic acid and N-acetylglucosamine. The enzyme had no amidase or endopeptidase action. It reached highest activity in the exponential phase of growth and in the electron microscope seemed to fragment the coccal wall at the equatorial ring.     

DNA polymerase of Dictyostelium discoideum

Only one molecular weight species of DNA polymerase was found in different developmental stages of the eukaryotic microorganism Dictyostelium discoideum. The molecular weight of this DNA polymerase is estimated to be about 127 000 by sucrose gradient centrifugation. The enzyme is present in all stages of growth and development, including dormant spores. All DNA polymerase activity is lost upon incubation of the crude extract with N-ethylmaleimide. The reaction properties, molecular weight and N-ethylmaleimide sensitivity of the D. discoideum DNA polymerase are similar to those of the DNA polymerase-alpha from mammalian sources.     

Inhibition of growth of Dictyostelium discoideum amoebae by bisphosphonate drugs is dependent on cellular uptake

PURPOSE: The aim of the study was to determine whether bisphosphonates are internalised by Dictyostelium amoebae and whether cellular uptake is required for their growth-inhibitory effects. Bisphosphonates inhibit growth of amoebae of the slime mould Dictyostelium discoideum, by mechanisms that appear to be similar to those that cause inhibition of osteoclastic bone resorption. METHODS: Cell-free extracts prepared from amoebae that had been incubated with bisphosphonates were analysed by 31P-n.m.r, spectroscopy or ion-exchange f.p.l.c., to identify the presence of bisphosphonates or bisphosphonate metabolites respectively. The growth-inhibitory effect of bisphosphonates towards Dictyostelium amoebae was also examined under conditions in which pinocytosis was inhibited. RESULTS: All of the bisphosphonates studied were internalised by Dictyostelium amoebae, probably by fluid-phase pinocytosis, and could be detected in cell-free extracts. Amoebae that were prevented from internalising bisphosphonates by pinocytosis were markedly resistant to the growth-inhibitory effects of these compounds. In addition, bisphosphonates encapsulated within liposomes were more potent growth inhibitors of Dictyostelium owing to enhanced intracellular delivery of bisphosphonates. CONCLUSIONS: All bisphosphonates inhibit Dictyostelium growth by intracellular mechanisms following internalisation of bisphosphonates by fluid-phase pinocytosis. It is therefore likely that bisphosphonates also affect osteoclasts by interacting with intracellular, rather than extracellular, processes.     

Kinetic analysis of biochemical differentiation in Dictyostelium discoideum

The metabolic network leading to accumulation of cellulose, trehalose, and mucopolysaccharide during development of Dictyostelium discoideum was simulated on a computer. The program consists of a metabolic map, the measured specific activity of the enzymes involved at each stage in development, and the substrate and inhibitor affinities. The Km values of four enzymes, amylase, UDP-galactose polysaccharide transferase, UDP-galactose epimerase, and cellulose synthetase, were determined for this study. At each iteration (1 min) during the period simulated (1500 min), the in vivo activity was calculated for each enzyme using Michaelis-Menten equations and new values for metabolites and end products were generated. The computed values for the concentration of both metabolites and polysaccharides were in close agreement with the measured values at all stages of development. We conclude that the in vitro measured values correlate well with the measured in vivo rates when treated in this manner. The program was modified to simulate the alterations in carbohydrate metabolism which might be expected in mutant strains with reduced activity of various enzymes. Trehalose was found to overaccumulate when either the peak value of the developmentally controlled increase in the specific activity of UDPGlc pyrophosphorylase was reduced. Trehalose accumulation was decreased in simulations of mutants lacking glycogen phosphorylase or glycogen synthetase. The interaction of these metabolic pathways is discussed.     

Structural roles of the spore coat proteins in Dictyostelium discoideum

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wild-type strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricin A, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.     

Structural and functional analysis of gp64, a membrane protein of the cellular slime mold Polysphondylium pallidum

We examined 38 patients with an arthroscopic bioabsorbable tack repair for anterior shoulder instability in a prospective evaluation. The mean follow-up was 22 months (range 12 to 33). The average age was 28.4 years (range 15 to 57), the operation was performed at average of 50 months (3 to 244 months) after injury. Assessment using the Rowe score revealed excellent results in 33 and good results in 3 patients. 1 patient had a fair result and 1 had a poor result. 26 should obtained full range of motion, 11 had minor (< 10 degrees) loss of external rotation, 1 experienced greater (< 20 degrees) loss of external rotation. 3 of the 38 patients (8%) had recurrent instability, 1 patient with 2 preceding operations and atraumatic and voluntary dislocation, respectively. The recurrence rate of arthroscopic Bankart repair with bioabsorbable tacks are comparable to open Bankart procedures. Success of the procedure depends on appropriate surgical technique and suitable selection of patients with unidirectional, posttraumatic, anterior instability who are found to have well-developed ligamentous tissue.     

Isolation and characterization of glycogen synthase in Dictyostelium discoideum

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K(m) values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS.     

Production and secretion of recombinant proteins in Dictyostelium discoideum

We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection.     

ATPase kinetics of the Dictyostelium discoideum myosin II motor domain

Structural characterization of the mode of interaction of nucleotides bound to myosin has relied upon the crystal structure of the Dictyostelium discoideum myosin II motor domain. This fragment, denoted S1dC, lacks the regulatory domain and light chain subunits and may therefore fail to display the normal ATPase activity of the intact myosin molecule. Here we show that the elementary steps of the S1dC ATPase pathway and the effects of actin are similar to those of the complete myosin head fragment. This indicates that truncation at residue E759, with the removal of the light chain binding sites, is not crucial to catalytic activity. In particular, S1dC does not show the anomolous tight binding of ADP displayed by slightly shorter M754 construct reported elsewhere. We also show that the fluorescent analogue Cy3-EDA-ATP is a good substrate for S1dC and demonstrate the use of fluorescence correlation spectroscopy to determine the affinity of Cy3-EDA-ADP using microgram quantities of proteins.     

Glycoproteins of the plasma membrane of Dictyostelium discoideum during development

Polysomal RNA from cultured sublines of baby hamster kidney (BHK) cells directed protein synthesis in an in vitro system derived from wheat germ extract. One product of the in vitro synthesis was dihydrofolate reductase (DHFR), as confirmed by methotrexate-substituted Sepharose affinity chromatography followed by SDS-polyacrylamide slab gel electrophoresis and autoradiography of the proteins labeled with 35S-methionine. The DHFR synthesized in vitro comigrates in the gel with authentic BHK DHFR, indicating that the molecular weights and structures of the in vivo and in vitro enzymes are probably the same. Polysomal RNA obtained from the methotrexate-resistant BHK subline (A5), which possesses some 140 times higher DHFR levels than the methotrexate-sensitive parents subline (B1), directed the synthesis of approximately 70 times more DHFR per unit of total in vitro synthesized protein than did B1 polysomal RNA. Assuming then that the rates of translation of A5 and B1 DHFR mRNAs in the wheat germ cell-free system are the same, our results show that a major part of the high DHFR levels observed in A5 cells is due to the presence of elevated quantities of DHFR mRNA.