The autowave electromechanical activity of the <Emphasis Type="Italic">Physarum polycephalum</Emphasis> plasmodium

The aim of this work is to clarify the role of the electrical activity of the Physarum polycephalum plasmodium in the control of the contractile activity and self-organization of the directed locomotion. This single-celled organism with a non-excitable membrane is a classic object that is used in studies of amoeboid motility. Its patterns of motor behavior and signal systems are common for many tissue cells. The presence of 50 mM KCl in an agar substrate under half of a separate plasmodial strand strongly inhibits the formation of the frontal zone and leads to sharp morphological polarization of the strand, which suggests the involvement of electrical processes in the autowave self-organization of the plasmodial structure. The gigantic sizes of the plasmodium make it possible to record its electrical activity simultaneously at different parts of the cell. It has been established that potentials and currents at parts of the plasmodium that are distant from each other oscillate synchronously and differ only in the shape of the signals, probably due to differences in the phases or the number of excited harmonics. We recorded currents (~50 pA) of single ion channels of the plasmodial membrane using the classical local voltage-clamp method. It has been found that the oscillation spectrum of the current that is generated by the plasmodium has high-frequency fluctuations, which are probably connected with periodic detachments of the membrane from the cytoskeleton during the formation and growth of the pseudopodia. It has been also shown that neomycin, a substrate inhibitor of phospholipase C, prevents oscillations of both the mechanical and electrical activity of the plasmodium. This is consistent with its well-established ability to inhibit mechanosensitive Ca²⁺ channels, which are apparently present in the plasmodial membrane. These data indicate the presence of a general signal system that is linked with the dynamics of the membrane- cytoskeleton association, which could be involved in the galvano- and chemotaxis of amoeboid cells.     

Alkaline phosphatase of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> is insoluble

The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg²⁺-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.     

The effect of inhibitors of cellular respiration on self-oscillating motility in plasmodium <Emphasis Type="Italic">Physarum polycephalum</Emphasis>

This work presents the results of spectral analysis of the time dependences, V(t), of endoplasmic shuttle motility in an isolated strand of plasmodium Physarum polycephalum that were obtained by laser Doppler microscopy after exposure to inhibitors of cellular respiration, viz., potassium cyanide and salicylhydroxamic acid, which lead to the complete cessation of endoplasmic motion. The results confirm the presence of only two harmonic components of V(t) dependences, with frequencies that differ by a factor of 2, ω₂/ω₁ = 1.972 ± 0.028, in different conditions: under normal conditions, without the addition of inhibitors; in a strand that was partially treated with inhibitors; and in the phase of restoring the oscillatory activity after the complete cessation of endoplasmic motion.     

Involvement of extracellular cAMP-specific phosphodiesterase in the control of motile activity of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> plasmodium

Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20–30 times under the action of 1 mM dithiothreitol. In the presence of 1–5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.     

Disruption of actin filaments induces mitochondrial Ca<Superscript>2+</Superscript> release to the cytoplasm and [Ca<Superscript>2+</Superscript>]<Subscript>c</Subscript> changes in <Emphasis Type="Italic">Arabidopsis</Emphasis>root hairs

Background  

Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca²⁺ storage. Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca²⁺ storage, cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c), and the interaction between mitochondrial Ca²⁺ and cytoplasmic Ca²⁺ in Arabidopsis root hairs.     

Ca<Superscript>2+</Superscript> increases the specific coenzyme Q<Subscript>10</Subscript> content in <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Of various metal ions (Ca²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Zn²⁺) added to the culture medium of Agrobacterium tumefaciens at 1 mM, only Ca²⁺ increased Coenzyme Q10 (CoQ₁₀) content in cells without the inhibition of cell growth. In a pH-stat fed-batch culture, supplementation with 40 mM of CaCO₃ increased the specific CoQ₁₀ content and oxidative stress by 22.4 and 48%, respectively. Also, the effect of Ca²⁺ on the increase of CoQ₁₀ content was successfully verified in a pilot-scale (300 L) fermentor. In this study, the increased oxidative stress in A. tumefaciens culture by the supplementation of Ca²⁺ is hypothesized to stimulate the increase of specific CoQ₁₀ content in order to protect the membrane against lipid peroxidation. Our results improve the understanding of Ca²⁺ effect on CoQ₁₀ biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ₁₀ by biological processes.     

Ca<Superscript>2+</Superscript> regulation in the absence of the <Emphasis Type="Italic">iplA</Emphasis> gene product in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). The [Ca²⁺]_i-change is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-entry. The significance of the [Ca²⁺]_i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca²⁺-buffers in the cytosol indicates that a [Ca²⁺]_i-increase is required for chemotaxis. Yet, the iplA ^- mutant disrupted in a gene bearing similarity to IP₃-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca²⁺]_i-transient which favours the view that [Ca²⁺]_i-changes are insignificant for chemotaxis.     

Ca<Superscript>2+</Superscript> reduces the effect of hypoxia in mosses <Emphasis Type="Italic">Mnium undulatum</Emphasis> and <Emphasis Type="Italic">Polytrichum commune</Emphasis>

Gametophores of mosses Mnium undulatum and Polytrichum commune were submerged in distilled water or in calcium chloride solution (0.9 mM Ca²⁺) to induce hypoxia. The net photosynthetic (P_N) and dark respiration rate (R_D) were measured in the air containing 300–400 μmol(CO₂)·mol⁻¹(air) and 0.21 mol(O₂)·mol⁻¹(air). P_N of M. undulatum gametophores decreased to 58 % of the control after 1-h submersion in water, whereas to 80 % of the control in P. commune gametophores. A smaller decrease in P_N was observed when the gametophores were immersed in CaCl₂ solution. In hypoxia, R_D in the tested mosses species was a little higher than in the control.     

cAMP controls cytosolic Ca<Superscript>2+</Superscript> levels in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) that is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca²⁺]_i.     

Membrane Stretching Triggers Mechanosensitive Ca<Superscript>2+</Superscript> Channel Activation in <Emphasis Type="Italic">Chara</Emphasis>

In order to confirm that mechanosensitive Ca²⁺ channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca²⁺ (Δ[Ca²⁺]_c). However, the observed Δ[Ca²⁺]_c decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (ΔE _m) and stretching or compression of the plasma membrane. Significant ΔE _m values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). ΔE _m appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large ΔE _m values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara.     

The synthesis of <Emphasis Type="Italic">N</Emphasis><Superscript>2</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>6</Superscript>-substituted diaminopurine ribosides

A series of new 2,6-substituted diaminopurine riboside derivatives were synthesized by activation of protected xantosine with sulfonyl chlorides followed by treatment with various amines. The relationship between the reactivity of intermediates and the nature of the activating agents was studied.     

Antagonistic effect of divalent cations Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> on the morphological development of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> var. <Emphasis Type="Italic">geldanus</Emphasis>

The automated docking program DOCK 5.3.0 was applied to screening for quorum sensing inhibitors (QSIs) of Peudomonus aeruginosa from a database containing 51 active components of Traditional Chinese Medicines with antibacterial activity. Five potential QSIs were revealed by the computer-based virtual screening. The compounds 3, 4, 5, 6, 7 inhibit biofilm formation of P. aeruginosa at a concentration of 200 μM. Compound 4 (baicalein) does not inhibit the growth of P. aeruginosa; however, it significantly inhibits biofilm formation of the bacteria at a lower concentration of 20 μM and promoted proteolysis of the signal receptor TraR protein in Escherichia coli at 4–40 mM. Baicalein and ampicillin showed synergistic activity against P. aeruginosa. These results suggested that baicalein can interfere with quorum sensing system of P. aeruginosa and will be developed as antibacterial agent with novel target.     

The effects of inhibitors and magnesium ions on the activity of the thermostable extracellular cAMP-Specific phosphodiesterase of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> plasmodium

Plasmodium of myxomycete Physarum polycephalum produces cyclic nucleotide phosphodiesterase (PDE). The extracellular PDE is cAMP-specific and highly thermostable. This study demonstrates that the extracellular PDE of Ph. polycephalum is weakly inhibited by caffeine, isobutylmethylxantine and theophiline (type I mammalian PDE nonspecific inhibitors), dipyridamole (mammalian PDE5, PDE6, PDE8 and PDE10 inhibitors), and erythro-9-[3-(2-hydroxynonyl)]-adenine (mammalian PDE2 inhibitor). The enzyme does not require Mg²⁺ for the activity. The results show that the Ph. polycephalum extracellular PDE differs from class I PDEs, represented by mammalian PDE1-PDE11, and, most likely, belongs to a poorly investigated class II PDEs.     

Enhanced intracellular Ca<Superscript>2+</Superscript> concentrations in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> after addition of oligosaccharide elicitors

Elicitation can lead to overproduction of secondary metabolites in plants and microbes. Potential changes in cytosolic Ca²⁺ levels in bacteria were studied in response to elicitation. We report, for the first time, the effect of oligosaccharide elicitors on intracellular Ca²⁺ levels. The apoaequorin gene was cloned into Escherichia coli DH5α and Bacillus subtilis 1604 cultures. Addition of elicitors, oligoguluronate and mannan oligosaccharides, to the cultures caused up to 11-fold increase in cytosolic Ca²⁺ in E. coli and tenfold increase in B. subtilis. These increases in Ca²⁺ levels could therefore contribute to the enhancement of secondary metabolite levels.     

Correlation between Unrepaired Radiation-induced DNA Strand Breaks and Mitotic Delay in <Emphasis Type="Italic">Physarum polycephalum</Emphasis>

THE DNA of cells exposed to ionizing radiation incurs strand breaks and certain other types of damage (for review see ref. 1). Single-strand breaks are repaired both in prokaryotes^2,3 and in eukaryotes^4–6. But although double-strand break repair has been reported for phage DNA in lambda phage-infected bacteria⁷, for the radioresistant bacterium Micrococcus radiodurans⁸ and for the Chinese hamster ovary cell⁹, this type of repair has not been demonstrated in other bacterial species³ and mammalian cell lines^5,6,10, suggesting that double-strand, rather than single-strand breaks are the lesions primarily responsible for the lethal effects of ionizing radiation^3,6,11.     

Enhancing yield of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by controlling NH<Subscript>4</Subscript><Superscript>+</Superscript> concentration

Yield of S-adenosylmethionine was improved significantly in recombinant Pichia pastoris by controlling NH₄ ⁺ concentration. The highest production rate was 0.248 g/L h when NH₄ ⁺ concentration was 450 mmol/L and no repression of cell growth was observed. Within very short induction time (47 h), 11.63 g/L SAM was obtained in a 3.7 L bioreactor.     

Ca<Superscript>2+ </Superscript>and Cu<Superscript>2+ </Superscript>supplementation increases mannitol production by <Emphasis Type="Italic">Candida magnoliae</Emphasis>

Supplementation with CaCl₂·2H₂O (50 mg l⁻¹) or CuSO₄·5H₂O (10 mg l⁻¹) improved mannitol production by Candida magnoliae by 14.5 and 18.6% (25 and 32 g/L), respectively. When used in combination, they acted synergistically: Ca²⁺ decreased the intracellular concentration of mannitol 30%, whereas Cu²⁺ increased the intracellular activity of mannitol dehydrogenase 1.6-times more than control. Ca²⁺ probably works by altering the permeability of cells to mannitol, whereas, Cu²⁺ increases the activity of an enzyme responsible for mannitol biosynthesis.     

Coupling of phospholipase C and PI3K/PTEN signaling pathways in <Emphasis Type="Italic">Physarum polycephalum</Emphasis>: The action of U73122 on motile and autooscillatory activity of plasmodium

The possibility of tight coupling of phospholipase C with the signal pathway PI3K/ PTEN, a ubiquitous mechanism for the control of chemotaxis and cell shape in free-living amoebae and mammalian tissue cells, has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that on the maintenance of contractile autooscillations and protoplasmic shuttle streaming, U73122, an inhibitor of the signal transduction to phospholipase C, induces degradation of the plasmodium frontal zone, decreases efficiency of locomotion and suppresses the chemotaxis toward glucose as well as the response of oscillator to this attractant. The identity of the effects of U73122 with those shown for wortmannin and LY294002, widely used PI3K inhibitors (Matveeva et al. 2008. Biophysics. 53, 533–538), suggests a tight coupling of the signal pathways of phospholipase C and PI3K/PTEN. U73122 increases the period of contractile oscillations and abolishes its cyclic changes attributed for the plasmodium migration. The results indicate that motile behavior of the plasmodium is under the receptor-mediated control.     

Purification and characterization of cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript> from <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Cytochrome c ₆ , (cyt c ₆) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E _m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.