Degradation of bacterial lipopolysaccharide by the slime mould Physarum polycephalum.

A strain of the acellular slime mould Physarum polycephalum degraded lipopolysaccharides (LPS) from a variety of bacteria. The anticomplementary (AC) activity of LPS was greatly reduced, as was the content of lauric, myristic, and palmitic acids, and the ability to sensitize erythrocytes to agglutination by antibody. These results indicate that Physarum has enzymes which reduce the lipid A moiety of LPS. In contrast, 2-keto-3-deoxy-D-manno-actanoic acid (KDO), immunodominant sugars, and beta-hydroxymyristic acid were scarcely affected. Both supernates and plasmodial extracts of Physarum had LPS-degradative activity and were able to attack both purified LPS and LPS in killed bacteria.     

Defined and semi-defined media for the growth of amoebae of Physarum polycephalum.

Amoebae of the true slime mould Physarum polycephalum were cultured in two fully-defined liquid media containing amino acids, glucose, three vitamins and a buffered salts solution. Absolute requirements were demonstrated for methionine, haematin, thiamine and biotin, all of which were known to be specific requirements of the plasmodial stage. Methods are described for large-scale culture in three semi-defined media.     

Effects of some protein kinases, cyclic nucleotides and specific inhibitors of phosphorylation on the mitotic cycle of Physarum polycephalum

The true slime mould Physarum polycephalum was treated with various agents by spraying them upon the cell surface 4 hrs before the second synchronous mitosis. The onset of mitosis was considerably approximated after the plasmodium treatment with protein kinases from rat hepatoma or Ph. polycephalum at the late G2 phase. The catalytic and regulatory subunits of cAMP-dependent pig brain protein kinase caused retardation of mitosis, while the holoenzyme, casein kinase and alkaline phosphatase did not affect the timing of mitosis. The cyclic nucleotides and inhibitors of their metabolic enzymes were used to investigate the role of phosphorylation processes in the mitotic cycle.     

Coordinate replication of dispersed repetitive sequences in Physarum polycephalum

The synchronous macroplasmodial growth phase of the slime mould Physarum polycephalum was used to study the in vivo replication of large chromosomal DNA segments. Newly replicated DNA was isolated at various points in S-phase by its preferential association with the nuclear matrix. This DNA was then used to probe cosmid clones of the Physarum genome. The results indicate that certain dispersed repetitive sequences in the genome are coordinately replicated. The observed pattern of replication may be due either to the presence of a replication origin within each repetitive sequence or to the systematic arrangement of these sequences around a replication origin. The latter appears more likely since the repetitive sequences are probably not randomly scattered within the genome.     

Cytoplasmic actin patterns in Physarum as revealed by NBD-phallacidin staining

Fluorescently labeled phallacidin, a F-actin specific drug, was used to demonstrate the morphological variety in the cytoskeletal actin pattern of thin-spread plasmodia of the acellular slime mould Physarum polycephalum. The patterns observed in phallacidin-stained specimens consisted of a polygonal network in the anterior region, and of longitudinal as well as helically twisted fibrils in plasmodial strands of the posterior region. These observations are in complete accordance with our recent results obtained on comparable plasmodia by immunofluorescence microscopy using specific antibodies against actin.     

Physarum vitronectin-like protein: an Arg-Gly-Asp-dependent cell-spreading protein with a distinct NH2-terminal sequence.

A 70-kDa protein cross-reacted with anti-bovine vitronectin was isolated from slime mold Physarum polycephalum. The NH2-terminal amino acid sequence of the protein, referred to as Physarum vitronectin-like protein, did not share any homology with those of animal vitronectins. It had cell-spreading activity, which was specifically inhibited by an Arg-Gly-Asp (RGD)-containing peptide.     

Robust and emergent Physarum logical-computing

There have been many attempts for realization of emergent computing, but the notion of emergent computing is still ambiguous. In an open system, emergence and an error cannot be specified distinctly, because they are dependent on the dis-equilibration process between local and global behaviors. To manifest such an aspect, we implement a Boolean gate as a biological device made of slime mold Physarum polycephalum. A Physarum (slime mold) Boolean gate could be an internally instable machine, while it has the potential for emergent computing. First, we examined whether Physarum Boolean gate works properly, and then examined its behaviors when the gate is collapsed in terms of hardware. The behavior of Physarum changes and self-repairing computing is achieved as a result. The self-repairing against internal failure is one of attributes of emergent and robust computing.     

Periodic organisation of foldback sequences in Physarum polycephalum nuclear DNA.

Nuclear DNA from the slime mould Physarum polycephalum is shown to contain interspersed inverted repeat sequences, such that denatured fragments of DNA containing pairs of these sequences form intra-chain duplexes under appropriate conditions. The organisation and distribution of the nucleotide sequences responsible for the formation of foldback structures in Physarum DNA have been investigated using the electron microscope. The majority of foldback duplexes have sizes ranging up to 800 base pairs, and about 60-80% of DNA molecules 2.2 X 10(4) bases in length contain interspersed foldback elements. The size of individual foldback duplexes, and also the length of the intervening sequences which separate them, are non-random. The results can best be explained by a model in which separate foldback foci in Physarum DNA are spaced periodically at regular intervals. The regions containing foldback foci are thought to contain smaller, tandemly-arranged sequences of discrete sizes, in some cases related to other nucleotide sequences of a similar nature in the same locality in Physarum DNA.     

Structure of a beta-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated beta-D-galactan ([alpha]D+8degrees) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 leads to 4)-, but also of (1 leads to 3)-, and (1 leads to 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extracellular polysaccharide produced by P. polycephalum.     

Amino-acid sequence data of beta-tubulin from Physarum polycephalum myxamoebae

Starting with 7.7 mg of a beta-tubulin isolated from myxamoebae of the slime mould Physarum polycephalum, 90% of the sequence has been determined by the Edman degradation of peptides generated by cyanogen bromide, trypsin and Staphylococcus aureus protease. Differences to other beta-tubulins are mainly conservative and spread evenly throughout the chain except for a high concentration at the C-terminus. The Physarum beta-tubulin shows most homology to Chlamydomonas beta-tubulin (90.5%) and least homology to yeast beta-tubulin (S. cerevisiae, 73.4%). Two tryptic peptides were isolated in approximately equal quantities which were identical except in one position (S/ALTVPELTQRMFDA) showing that at least two beta-tubulins are present in myxamoebae. However, since this was the only heterogeneity found, these beta-tubulins are probably very similar.     

The electron-transport system of mitochondria from the slime mould Physarum polycephalum

A method is described for the preparation of mitochondria from the slime mould Physarum polycephalum; the mitochondria were not coupled. P. polycephalum mitochondria oxidized added NADH via a rotenone-insensitive pathway, but the oxidation of malate plus glutamate was rotenone sensitive; both of these substrates reduced much less cytochrome b than did succinate, in both aerobic and anaerobic steady states. Spectroscopy at 77 degrees K separated three absorption maxima in the alpha-band region, at 560nm, 553nm and one at 547nm due to cytochrome c. The absorption at 553nm was increased in the aerobic steady state by the addition of 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that it was due to a b-type cytochrome. All three absorption maxima appeared in the aerobic steady state after the addition of a range of substrates. The respiratory activity with different substrates and the response to inhibitors of respiration were similar to those previously described for fungus mitochondria (Weiss et al., 1970; Erickson & Ashworth, 1969). When grown under conditions of haem limitation the mitochondria contained a lower concentration of cytochromes than normal.     

The linear extrachromosomal DNA of Physarum polycephalum replicates and is maintained under non-selective conditions in two different lower eukaryotes.

The slime mould Physarum polycephalum contains 100 to 200 molecules of extrachromosomal linear DNA (PeDNA). Two sets of the 19S and 26S ribosomal genes are located on each molecule of PeDNA. In the nonmitotic phase of the cell cycle PeDNA is localised in the nucleolus. The molecules are maintained throughout vegetative growth. In order to study the signals responsible for its maintenance, PeDNA was purified and introduced into the two distantly related yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Surprisingly, intact PeDNA transforms both yeasts with high frequency and PeDNA sequences are maintained in the absence of selective pressure.     

Amino acid sequence data of alpha-tubulin from myxamoebae of Physarum polycephalum

About 96% of the amino acid sequence of an alpha-tubulin from the slime mould Physarum polycephalum has been determined. Of 430 sequenced amino acids, 30 differ from the deduced amino acid sequence of a recently published alpha-tubulin complementary DNA from the plasmodial form of P. polycephalum. The myxamoebal alpha-tubulin differs from all other known alpha-tubulins in one of the last three C-terminal amino acids that are Gly-Glu-Tyr instead of the usual Glu-Glu-Tyr. These last three amino acids are preceded by 11 residues that appear to be particularly susceptible to mutation. No heterogeneity was found whilst sequencing the myxamoebal alpha-tubulin, indicating that only one type of alpha-tubulin is present in myxamoebae. This alpha-tubulin appears to be less conserved than the previously described plasmodial alpha-tubulin, supporting the hypothesis that the structural constraints on tubulin in axonemes have a significant effect on its rate of mutation.     

Chemically induced changes in the morphology, dynamic activity and cytoskeletal organization of Physarum cell fragments

Spherical cell fragments derived from Physarum polycephalum by caffeine-treatment were used as an experimental system to investigate the influence of 15 externally applied substances on the general morphology, motile behavior and cytoskeletal organization of the acellular slime mold. In comparison to controls, the most obvious changes observed after chemical stimulation proved to be cytokinetic activities, ameboid-like movement phenomena, intense cell surface dynamics and formation of cytoplasmic actin fibrils. The results demonstrate the high adaptability of the microfilament system in Physarum even when subjected to extreme conditions in the external environment.     

Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum

The effects of experimentally-altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts-only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21-fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

The inhibitory Ca2+-regulation of the actin-activated Mg-ATPase activity of myosin from Physarum polycephalum plasmodia

Myosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (greater than 95%), in a high yield (about 10 mg/100 g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500,000 Mr myosin). Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when Ca2+ concentration was increased to 50 microM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3 microM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+. The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2 microM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin. The involvement of this myosin-linked regulatory system in the Ca2+ -control of cytoplasmic streaming is discussed.     

Sequence organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences.

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum

Abstract. The polygonal arrangement of actomyosin fibrils in different stages of the acellular slime mold Physarum polycephalum is correlated with morphogenetic processes at the cell surface. Light and electron microscopic investigations on both endoplasmic drops and thin-spread small plasmodia demonstrate that the differentiation of a polygonal pattern depends on a transient deficiency of plasma membrane invaginations.
Glycerol-extracted specimens show condensation and drastic spatial changes in the organization of the polygonal net after addition of ATP, thus indicating contractile properties of this system. Observations with the polarizing microscope reveal rhythmic changes in fibrillar birefringence intensity corresponding to the protoplasmic streaming activity, i.e., birefringence increases during contraction and decreases during relaxation. Cell fusion experiments, local irradiation with blue light (450 nm), and chemical treatment by impeding the mitochondria1 function with DNP (2,4-di-nitrophenol) demonstrate morphological as well as physiological interdependences of the actomyosin system, the motive force generation, and the expression of a locomotor polarity in plasmodia of Physarum polycephalum.     

Fluctuations in proline and other free amino acids during the mitotic cycle of the myxomycete Physarum polycephalum.

1. During synchronous growth of the acellular slime mould Physarum polycephalum the free amino pool had two maxima, one of 650 units [nmol/plasmodium dry weight (mg)] at metaphase and the other of 780 units in mid G2 with minima of 550 units before and after mitosis. 2. Proline formed 20--25% of the total pool with aspartic acid, glutamic acid, threonine, valine, leucine, lysine and arginine making up 55% of the pool. 3. The fluctuation of proline during the mitotic cycle was quite different from that of the other amino acids and was transiently very low during telophase.     

Rebuilding Iberian motorways with slime mould

Plasmodium of a cellular slime mould Physarum polycephalum is a unique living substrate proved to be efficient in solving many computational problems with natural spatial parallelism. The plasmodium solves a problem represented by a configuration of source of nutrients by building an efficient foraging and intra-cellular transportation network. The transportation networks developed by the plasmodium are similar to transport networks built by social insects and simulated trails in multi-agent societies. In the paper we are attempting to answer the question "How close plasmodium of P. polycephalum approximates man-made motorway networks in Spain and Portugal, and what are the differences between existing motorway structure and plasmodium network of protoplasmic tubes?". We cut agar plates in a shape of Iberian peninsula, place oat flakes at the sites of major urban areas and analyse the foraging network developed. We compare the plasmodium network with principle motorways and also analyse man-made and plasmodium networks in a framework of planar proximity graphs.