Cytotoxicity activities and chemical characteristics of exopolysaccharides and intracellular polysaccharides of Physarum polycephalum microplasmodia.

BACKGROUND: Microbial polysaccharides have been reported to possess remarkable bioactivities. Physarum polycephalum is a species of slime mold for which the microplasmodia are capable of rapid growth and can produce a significant amount of cell wall-less biomass. There has been a limited understanding of the polysaccharides produced by microplasmodia of slime molds, including P. polycephalum. Thus, the primary objectives of this research were first to chemically characterize the exopolysaccharides (EPS) and intracellular polysaccharides (IPS) of P. polycephalum microplasmodia and then to evaluate their cytotoxicity against several cancer cell lines. RESULTS: The yields of the crude EPS (4.43 ± 0.44 g/l) and partially purified (deproteinated) EPS (2.95 ± 0.85 g/l) were comparable (p > 0.05) with the respective crude IPS (3.46 ± 0.36 g/l) and partially purified IPS (2.45 ± 0.36 g/l). The average molecular weight of the EPS and IPS were 14,762 kDa and 1788 kDa. The major monomer of the EPS was galactose (80.22%), while that of the IPS was glucose (84.46%). Both crude and purified IPS samples showed significantly higher cytotoxicity toward Hela cells, especially the purified sample and none of the IPSs inhibited normal cells. Only 38.42 ± 2.84% Hela cells remained viable when treated with the partially purified IPS (1 mg/ml). However, although only 34.76 ± 6.58% MCF-7 cells were viable when exposed to the crude IPS, but the partially purified IPS displayed non-toxicity to MCF-7 cells. This suggested that the cytotoxicity toward MCF-7 would come from some component associated with the crude IPS sample (e.g. proteins, peptides or ion metals) and the purification process would have either completely removed or reduced amount of that component. Cell cycle analysis by flow cytometry suggested that the mechanism of the toxicity of the crude IPS toward MCF-7 and the partially purified IPS toward Hela cells was due to apoptosis. CONCLUSIONS: The EPS and IPS of P. polycephalum microplasmodia had different chemical properties including carbohydrate, protein and total sulfate group contents, monosaccharide composition and molecular weights, which led to different cytotoxicity activities. The crude and partially purified IPSs would be potential materials for further study relating to cancer treatment.

Biological activities and chemical compositions of slime tracks and crude exopolysaccharides isolated from plasmodia of Physarum polycephalum and Physarella oblonga.

BACKGROUND: The myxomycetes derive their common name (slime molds) from the multinucleate trophic stage (plasmodium) in the life cycle, which typically produces a noticeable amount of slimy materials, some of which is normally left behind as a "slime track" as the plasmodium migrates over the surface of a particular substrate. The study reported herein apparently represents the first attempt to investigate the chemical composition and biological activities of slime tracks and the exopolysaccharides (EPS) which cover the surface of the plasmodia of Physarum polycephalum and Physarella oblonga. RESULTS: Chemical analyses indicated that the slime tracks and samples of the EPS consist largely of carbohydrates, proteins and various sulphate groups. Galactose, glucose and rhamnose are the monomers of the cabohydrates present. The slime tracks of both species and the EPS of Phy. oblonga contained rhamnose, but the EPS of Ph. polycephalum had glucose as the major monomer. In term of biological activities, the slime tracks displayed no antimicrobial activity, low anticancer activity and only moderate antioxidant activity. However, EPSs from both species showed remarkable antimicrobial activities, especially toward Candida albicans (zone of inhibition ≥20 mm). Minimum inhibitory concentrations of this fungus were found to be 2560 µg/mL and 1280 µg/mL for EPS from Phy. oblonga and Ph. polycephalum, respectively. These EPS samples also showed moderate antioxidant activities. However, they both displayed cytotoxicity towards MCF-7 and HepG2 cancer cells. Notably, EPS isolated from the plasmodium of Phy. oblonga inhibited the cell growth of MCF-7 and HepG2 at the half inhibitory concentration (IC50) of 1.22 and 1.11 mg/mL, respectively. CONCLUSIONS: EPS from Ph. polycephalum plasmodium could be a potential source of antifungal compounds, and EPS from Phy. oblonga could be a potential source of anticancer compounds.

P-bRS: a physarum-based routing scheme for wireless sensor networks.

Routing in wireless sensor networks (WSNs) is an extremely challenging issue due to the features of WSNs. Inspired by the large and single-celled amoeboid organism, slime mold Physarum polycephalum, we establish a novel selecting next hop model (SNH). Based on this model, we present a novel Physarum-based routing scheme (P-bRS) for WSNs to balance routing efficiency and energy equilibrium. In P-bRS, a sensor node can choose the proper next hop by using SNH which comprehensively considers the distance, energy residue, and location of the next hop. The simulation results show how P-bRS can achieve the effective trade-off between routing efficiency and energy equilibrium compared to two famous algorithms.

Effects of medium composition on the growth and lipid production of microplasmodia of Physarum polycephalum.

Physarum polycephalum is a plasmodial slime mold. One of the trophic stages in the life cycle of this organism is a plasmodium. In submerged culture, plasmodia are fragmented into microplasmodia. The latter both lack cell walls and are capable of rapid growth. There has been limited information on the effects of medium composition on the growth and lipid accumulation of microplasmodia. In this study, optimization of medium components by response surface methodology showed that tryptone and yeast extract concentrations had the most significant effects on lipid and biomass production; significant synergistic interactions between glucose and tryptone concentration on these responses were also recorded. The optimal medium was composed of 20 g/L of glucose, 6.59 g/L of tryptone, and 3.0 g/L of yeast extract. This medium yielded 13.86 g/L of dry biomass and 1.97 g/L of lipids. These amounts are threefold higher than those of the American Type Culture Collection (ATCC) medium. In addition, biomass and lipid production reached maximal values between only 4 and 5 days. Fatty acid compositions analysis by gas chromatography-mass spectrometer (GC-MS) revealed that P. polycephalum lipids consisted mainly of oleic acid (40.5%), linoleic acid (10%), and octadecynoic (15.8%). This is the first report on the fatty acid composition of P. polycephalum microplasmodia. These results suggest that the biomass of microplasmodia could be used as a source of material for direct conversion into biodiesel because of the absence of cell walls or it could also be used as a supplemental source of beneficial fatty acids for humans, albeit with some further evaluation needed.

Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy.

Brain glioma treatment with checkpoint inhibitor antibodies to cytotoxic T-lymphocyte-associated antigen 4 (a-CTLA-4) and programmed cell death-1 (a-PD-1) was largely unsuccessful due to their inability to cross blood-brain barrier (BBB). Here we describe targeted nanoscale immunoconjugates (NICs) on natural biopolymer scaffold, poly(ß-L-malic acid), with covalently attached a-CTLA-4 or a-PD-1 for systemic delivery across the BBB and activation of local brain anti-tumor immune response. NIC treatment of mice bearing intracranial GL261 glioblastoma (GBM) results in an increase of CD8+ T cells, NK cells and macrophages with a decrease of regulatory T cells (Tregs) in the brain tumor area. Survival of GBM-bearing mice treated with NIC combination is significantly longer compared to animals treated with single checkpoint inhibitor-bearing NICs or free a-CTLA-4 and a-PD-1. Our study demonstrates trans-BBB delivery of tumor-targeted polymer-conjugated checkpoint inhibitors as an effective GBM treatment via activation of both systemic and local privileged brain tumor immune response.

Mitochondrial numbers increase during glucose deprivation in the slime mold Physarum polycephalum.

Glucose deprivation in the slime mold Physarum polycephalum leads to a specific morphotype, a highly motile mesoplasmodium. We investigated the ultrastructure of both mesoplasmodia and non-starved plasmodia and found significantly increased numbers of mitochondria in glucose-deprived mesoplasmodia. The volume of individual mitochondria was the same in both growth forms. We conjecture that the number of mitochondria correlates with the metabolic state of the cell: When glucose is absent, the slime mold is forced to switch to different metabolic pathways, which occur inside mitochondria. Furthermore, a catabolic cue (such as AMP-activated protein kinase (AMPK)) could stimulate mitochondrial biogenesis.

Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens betagamma-crystallin domain fold.

BACKGROUND: The betagamma-crystallins belong to a superfamily of two-domain proteins found in vertebrate eye lenses, with distant relatives occurring in microorganisms. It has been considered that an eukaryotic stress protein, spherulin 3a, from the slime mold Physarum polycephalum shares a common one-domain ancestor with crystallins, similar to the one-domain 3-D structure determined by NMR. RESULTS: The X-ray structure of spherulin 3a shows it to be a tight homodimer, which is consistent with ultracentrifugation studies. The (two-motif) domain fold contains a pair of calcium binding sites very similar to those found in a two-domain prokaryotic betagamma-crystallin fold family member, Protein S. Domain pairing in the spherulin 3a dimer is two-fold symmetric, but quite different in character from the pseudo-two-fold pairing of domains in betagamma-crystallins. There is no evidence that the spherulin 3a single domain can fold independently of its partner domain, a feature that may be related to the absence of a tyrosine corner. CONCLUSION: Although it is accepted that the vertebrate two-domain betagamma-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage. The spherulin 3a protomer stability appears to be dependent on domain pairing. Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.

Kinetic and thermodynamic stabilization of the betagamma-crystallin homolog spherulin 3a from Physarum polycephalum by calcium binding.

Globular proteins may be stabilized, either intrinsically, at the various levels of the structural hierarchy, or extrinsically, by ligand binding. In the case of the dormant all-beta protein spherulin 3a (S3a) from the slime mold Physarum polycephalum, binding of calcium ions causes extreme kinetic and thermodynamic stabilization. S3a is the only known single-domain member of the two Greek key superfamily of betagamma-crystallins sharing the extreme long-term stability of its homologs in vertebrate eye lens. Spectral analysis allows two Ca2+-binding sites with KD=9 microM and 200 microM to be distinguished. Unfolding in the absence and in the presence of Ca2+gives evidence for extreme kinetic stabilization of the protein: In the absence of Ca2+, the half-time of unfolding in 2. 5 M guanidinium chloride (GdmCl) equals 8.3 minutes, whereas in the presence of Ca2+, even in 7.5 M GdmCl, it exceeds nine hours. To reach the equilibrium of unfolding in the absence and in the presence of Ca2+takes one day and eight weeks, respectively. The corresponding Gibbs free energies (based on the two-state model) are 77 and 135 kJ/mol. Saturation of S3a with Ca2+leads to an upward shift of the temperature-induced equilibrium transition by ca 20 deg. C. The in situ Ca2+concentration in the spherules is sufficient for the complete complexation of S3a in vivo.

Ca2+-loaded spherulin 3a from Physarum polycephalum adopts the prototype gamma-crystallin fold in aqueous solution.

Spherulin 3a is the most abundantly expressed cytosolic protein in spherulating plasmodia of the slime mold Physarum polycephalum. High yields of unlabeled, uniformly 15N and uniformly 13C/15N-labeled recombinant spherulin 3a from Escherichia coli could be produced by a simple protocol described here. The three-dimensional solution structure of Ca2+-loaded spherulin 3a was determined by homo- and heteronuclear NMR spectroscopy. The structure of monomeric spherulin 3a consists of two pleated beta-sheets plus a short alpha-helix arranged into the gamma-crystallin fold. The beta-sheets comprise two intertwined Greek-key motifs. An additional N-terminal beta-strand is unique to spherulin 3a. Complexation of calcium ions greatly enhances overall conformational stability of the protein. The average atomic root-mean-square deviations (r.m.s.d.) for heavy atoms in beta-strands were 0.34(+/-0.16) A for the backbone atoms and 0.73(+/-0.40) A for all atoms. The corresponding r.m.s.d. values for heavy atoms in the whole protein were 0.62(+/-0.42) A for the backbone atoms and 0.99(+/-0.65) A for all atoms. We show the structural relationship between spherulin 3a, a myxomycete dormancy protein, and crystallins from the vertebrate eye lens. Since spherulin 3a has a structure corresponding to one domain of bovine gammaB(II)-crystallin, it represents a hypothetical ancestral gamma-crystallin precursor structure.

Stability of a homo-dimeric Ca(2+)-binding member of the beta gamma-crystallin superfamily: DSC measurements on spherulin 3a from Physarum polycephalum.

Spherulin 3a (S3a) from Physarum polycephalum represents the only known single-domain member of the superfamily of beta gamma eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization and Ca(2+)-binding. The temperature and denaturant-induced unfolding of S3a in the absence and in the presence of Ca2+ were investigated by differential scanning calorimetry and fluorescence spectroscopy. To accomplish reversibility without chemical modification of the protein during thermal denaturation, the only cysteine residue (Cys4) was substituted by serine; apart from that, the protein was destabilized by adding 0.5-1.8 M guanidinium chloride (GdmCl). The Cys4Ser mutant was found to be indistinguishable from natural S3a. The equilibrium unfolding transitions obey the two-state model according to N2-->2 U, allowing thermodynamic parameters to be determined by linear extrapolation to zero GdmCl concentration. The corresponding transition temperatures TM for the Ca(2+)-free and Ca(2+)-loaded protein were found to be 65 and 85 degrees C, the enthalpy changes delta Hcal, 800 and 1280 kJ/mol(dimer), respectively. The strong dependencies of TM and delta Hcal on the GdmCl concentration allow the molar heat capacity change delta Cp to be determined. As a result, delta Cp = 18 kJ/(K mol(dimer)) was calculated independent of Ca2+. No significant differences were obtained between the free energy delta G degree calculated from delta Hcal and TM, and extrapolated from the stability curves in the presence of different amounts of denaturant. The free energy derived from thermal unfolding was confirmed by the spectral results obtained from GdmCl-induced equilibrium transitions at different temperatures for the Ca(2+)-free or the Ca(2+)-loaded protein, respectively. Within the limits of error, the delta G degree values extrapolated from the transitions of chemical denaturation to zero denaturant concentration are identical with the calorimetric results.

A unique U5-->A substitution in the Physarum polycephalum U1 snRNA: evidence at the RNA and gene levels.

The 5' terminal sequence of U1 snRNA that base-pairs with the intron 5' splice site in the course of spliceosome assembly was considered to be universally conserved. A study of the P polycephalum U1 snRNA at both RNA and gene levels shows that there are exceptions to this rule: the P polycephalum U1 snRNA has a U to A substitution at position 5, that is partially compensated by a high frequency of T residue at position +4 of introns. In contrast to the yeast genome, the P polycephalum genome contains several U1 snRNA coding sequences (about 20). They either encode the U1A snRNA expressed in microplasmodia or correspond to the previously cloned U1B coding sequence. Both coding sequences show the U5A substitution. The ratio of U1A versus U1B coding sequences is of about 3. A U1A gene was cloned. The 60 nt region upstream of the coding sequence has the same sequence as in the U1B gene. The U1B gene is probably expressed at another stage of the P polycephalum life cycle.

Tropomyosin is localized in the nuclear matrix and chromosome scaffold of Physarum polycephalum.

The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum. The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl. SDS-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight. Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence, suggesting the presence of the antigen in them. Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold. Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.

Crystallization and preliminary X-ray diffraction studies of a 40 kDa calcium binding protein specifically expressed in plasmodia of Physarum polycephalum.

A calcium binding protein with a molecular mass of 40 kDa (CBP40), the gene product of plasmodial-specific LAV1-2 of Physarum polycephalum, was crystallized in the presence of EDTA. The crystals diffracted X-rays up to a resolution of 3.0 A. They belonged to the trigonal space group, P3221 (or P3121), with unit cell dimensions of a = b = 64.4 A and c = 207.2 A. Ca2+-bound crystals were obtained by soaking in a CaCl2 solution, which gave diffraction data of similar quality. The Ca2+-soaked crystals belonged to the same space group as those crystallized in the presence of EDTA with unit cell dimensions of a = b = 64.4 A and c = 209.4 A.

Potential sites for the perception of gravity in the acellular slime mold Physarum polycephalum.

Recently a gravisensitivity of the acellular slime mold Physarum polycephalum, which possesses no specialized gravireceptor, could be established by conducting experiments under simulated and under real near weightlessness. In these experiments macroplasmodia showed a modulation of their contraction rhythm followed by regulation phenomena. Until now the perception mechanism for the gravistimulus is unknown, but several findings indicate the involvement of mitochondria: A) During the impediment of respiration the 0g-reaction is inhibited and the regulation is reduced. B) The response to a light stimulus and the following regulation phenomena strongly resemble the behavior during exposure to 0g, the only difference is that the two reactions are directed into opposite directions. In the blue-light reaction a flavin of the mitochondrial matrix seems to be involved in the light perception. C) The contraction rhythm as well as its modulations are coupled to rhythmic changes in the levels of ATP and calcium ions, involving the mitochondria as sites of energy production and of Ca(++)-storage. So the mitochondria could be the site of the regulation and they possibly are the receptor sites for the light and gravity stimuli. Also the observation of a morphologic polarity of the slime mold's plasmodial strands has to be considered: Cross-sections reveal that the ectoplasmic wall surrounding the streaming endoplasm is much thinner on the physically lower side than on the upper side of the strand--this applies to strands lying on or hanging on a horizontal surface. So, in addition to the mitochondria, also the morphologic polarity may be involved in the perception mechanism of the observed gravisensitivity and of the recently established geotaxis. The potential role of the nuclei and of the contractile elements in the perception of gravity is also discussed.

[The characteristics of PSCL32.5 protein from Physarum polycephalum and the variation of the content through cell cycle].

Crude fraction of Arg/Ser-rich proteins (SR proteins) were isolated from plasmodia of Physarum polycephalum and immunoassayed by western blot with a monoclonal antibody against SC35 protein from HeLa cell. Two polypeptides were detected by the antibody, suggesting that they were SCL(SC35-like) proteins. The SCL proteins have their mass weight of 32.5 kD and 82.5 kD, respectively, and so were termed PSCL32.5 and PSCL82.5 in this paper. The PI of PSCI32.5 was ascertained as 6.19 by IEF after a further purification of the protein with SDS-PAGE. The densitometric scanning of the western blot bands of PSCI32.5 isolated at different phases of cell cycle of P. polycephalum demonstrated that the relative content of the protein varied through the cell cycle: it appeared as the lowest at early S phase, showed increases from S phase to G2 phase, and peaked at late G2 phase.

[A SC35-like protein is localized in the nucleus of Physarum polycephalum].

After being labeled with an anti-SC35 antibody, the specimens of Physarum polycephalum at S, G2, prophase, metaphase and ana-telophase were observed with an Hitachi electron microscope and gold particles marking the location of the SC35-like protein were mainly found in the nucleus,indicating the existence of a SC35-like protein in it. Judging from the densities of the gold particles in the individual domains of the nucleus, the SC35-like protein was principally located in the nucleolar domain and interchromatin domain during G2 and prophase, and the protein was distributed in the interchromosome domain at metaphase and ana-telophase when the nucleus was disintegrated, suggesting that the nucleolus and interchromatin (interchromosome) domain are the two main locations of the SC35-like protein in the nucleus. Further observations upon the nucleolus revealed that the density of the gold particles in the dense fibrillar component (DFC) of the nucleolus was much higher than that of the fibrillar center (FC), demonstrating that the protein was largely situated in the DFC rather than FC.

[A cyclin A-like protein is localized in the cells of Physarum polycephalum and functions in the cell cycle].

The subcellular distribution of a Cyclin A-like protein in the cells of Physarum polycephalum and the function of the protein in the cell cycle were studied by immunoelectron microscope and anti-Cyclin A antibody blocking. After labeled with an anti-Cyclin A monoclonal antibody, the density of gold particles in the labeled specimens was much higher than that in the control, indicating that a Cyclin A-like protein existed in Physarum polycephalum. In the labeled specimen, the gold particles density of the nucleus was higher than that of cytoplasm, which was similar to that of the control, demonstrating that the Cyclin A-like protein was a nuclear protein. The gold particles density of the nuclei varied during the cell cycle. The highest appeared in S phase and the lowest came in metaphase and ana-telophase, which was close to that in the control. From S phase to metaphase, the gold particle densities dropped down gradually. The changes in the gold particle density showed the changes in the content of the Cyclin A-like protein. After treated with the anti-Cyclin A antibody in S phase and G2 phase respectively, the nuclei of Physarum polycephalum were arrested in the phases and the morphology of these nuclei became irregular. When treated with the anti-Cyclin A antibody in prophase, the nuclei appeared abnormal. These results suggested that the Cyclin A-like protein is important in cell cycle regulation of Physarum polycephalum, essentially in S/G2 and G2/M changes.

[Immunochemical identification of kinesin in Physarum polycephalum].

Physarum polycephalum, a low eukaryote ameba provides an attractive system for studying contractile proteins. In this work, we have identified a kinesin-like protein in the plasmodium of Physarum polycephalum by western blotting, using monoclonal antibody against kinesin (bovine brain). The molecular weight of the polypeptide which immunologically cross-reacts with kinesin from bovine brain is about 137kd. It suggests that the 137kd polypeptide is the heavy chain of the kinesin in Physarum polycephalum.

[Immunocytochemical identification of tropomyosin in nucleoli and nucleolar matrix of Physarum polycephalum].

Nucleoli were isolated from physarum polycephalum, and nucleolar matrix was prepared by digesting the nucleoli respectively with DNase 1, 0.25 mol/L (NH4)2SO4 and 2 mol/L NaCl to remove DNA and most proteins. SDS-PAGE analysis indicated that there were about 20 polypeptides in nucleolar matrix component, including the 37 kD polypeptide which was similar to tropomyosin in molecular weight. The result of indirect immunofluorescence treated with anti-tropomyosin antibody and sheep anti-rabbit IgG antibody labelled with FITC showed that bright fluorescence was observed in the nucleoli and nucleolar matrix, but no bright fluorescence in the controls. Indirect Immunoblotting detection further verified that tropomyosin existed in nucleolar matrix. Protein A-colloidal gold immunoelectron microscopic study showed that there were many gold particles in the specimens labelled with tropomyosin antibody, and there were few gold particles found in the controls. Tropomyosin distributed dispersedly in nucleoli.

Material approximation of data smoothing and spline curves inspired by slime mould.

The giant single-celled slime mould Physarum polycephalum is known to approximate a number of network problems via growth and adaptation of its protoplasmic transport network and can serve as an inspiration towards unconventional, material-based computation. In Physarum, predictable morphological adaptation is prevented by its adhesion to the underlying substrate. We investigate what possible computations could be achieved if these limitations were removed and the organism was free to completely adapt its morphology in response to changing stimuli. Using a particle model of Physarum displaying emergent morphological adaptation behaviour, we demonstrate how a minimal approach to collective material computation may be used to transform and summarise properties of spatially represented datasets. We find that the virtual material relaxes more strongly to high-frequency changes in data, which can be used for the smoothing (or filtering) of data by approximating moving average and low-pass filters in 1D datasets. The relaxation and minimisation properties of the model enable the spatial computation of B-spline curves (approximating splines) in 2D datasets. Both clamped and unclamped spline curves of open and closed shapes can be represented, and the degree of spline curvature corresponds to the relaxation time of the material. The material computation of spline curves also includes novel quasi-mechanical properties, including unwinding of the shape between control points and a preferential adhesion to longer, straighter paths. Interpolating splines could not directly be approximated due to the formation and evolution of Steiner points at narrow vertices, but were approximated after rectilinear pre-processing of the source data. This pre-processing was further simplified by transforming the original data to contain the material inside the polyline. These exemplary results expand the repertoire of spatially represented unconventional computing devices by demonstrating a simple, collective and distributed approach to data and curve smoothing.