Alternative pathway of respiration in Physarum polycephalum plasmodia

The external application of inhibitors of glycolysis in the presence of KCN shows a lethal effect on plasmodia of Physarum polycephalum. However, alpha-ketoglutarate, but not succinate, maintains the contraction-relaxation cycle of plasmodial actomyosin in spite of the fact that glycolysis and cytochrome oxidase are inhibited. The oscillations supported by ketoglutarate disappear in the presence of SHAM, an inhibitor of alternative oxidase. These results imply the existence of KCN-resistant, alternative pathway of electron transport in the mitochondria of Physarum polycephalum.     

Calcium binding sites in plasmodia of Physarum polycephalum as revealed by the pyroantimonate technique

Plasmodia of the acellular slime mold, Physarum polycephalum, were treated with an osmium tetroxide fixative containing potassium pyroantimonate to precipitate calcium and thereby localize calcium binding sites and sites of increased calcium concentration. Dense calcium pyroantimonate precipitates were detected within the nucleoli. The distribution of these precipitates during interphase and mitosis coincides with the distribution of the unique minichromosomes in Physarum, i.e., the numerous short pieces of extrachromosomal nucleolar chromatin containing segments of amplified DNA coding for ribosomal RNA. Calcium pyroantimonate precipitates were present as frequent dense granules in the mitochondrial matrix and as fine precipitates in the mitochondrial nucleoid. Large calcium-containing precipitates were seen within cytoplasmic vacuoles, confirming reports by others. In addition, we have identified calcium binding sites along the cytoplasmic surface of the plasma membrane. The distribution of calcium within the plasmodium is discussed in relation to the assembly of the mitotic spindle and the regulation of cell motility.     

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

Molecular Genetics and Genomics - Mutant (“APT”) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of...     

DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum

Nuclei were isolated from synchronized plasmodia of a true slime mold, Physarum polycephalum, in S-phase, and DNA synthesis in the nuclei was studied in vitro. The nuclei catalyzed DNA synthesis at the rate of 0.7 ng DNA/1.0 X 10(6) nuclei/30 min at 25 degrees C, which was 5 times higher than that catalyzed in G2-phase nuclei. The DNA synthesis required Mg2+, four kinds of deoxyribonucleoside 5'-triphosphates and ATP, suggesting that the mode of synthesis is a replicative-type, but not a repair-one. Sedimentation analysis of the DNA products revealed that the nuclei produced 2-4S DNA fragments mainly during a 30-sec pulse incubation, and 2-4S, 5-12S and longer fragments during a 15-min incubation. The pulse- and chase-labeling experiments showed that the 2-4S fragments shifted discontinuously to longer fragments. These results indicate that the nuclei catalyze the formation of 2-4S Okazaki fragments first and then their subsequent ligation. Eighty % and 96% of the DNA synthesis was inhibited by 200 micrograms/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 microM 2',3'-dideoxythymidine 5'-triphosphate. These results suggest that the DNA synthesis is catalyzed by the alpha-type DNA polymerase of Physarum polycephalum.     

Calcium fluxes and intracellular distribution in clonal osteosarcoma cell lines

Calcium efflux patterns were investigated in two clonal osteosarcoma cell lines, ROS $\text{17}\text{2}$ and ROS $\text{2}\text{3}$. Efflux was measured after equilibrating the cells with ⁴⁵calcium in medium containing 1% or 10% serum. The results of ⁴⁵calcium efflux were analyzed by fitting them to a model of three exponential terms, using a computer program based on the non-linear least square method. The results indicated the presence of three exchangeable calcium pools which differ in their rate constants of calcium efflux: a “very fast turnover” (S₁) a “fast turnover” (S₂) and a “slow turnover” (S₃). Washing the labelled cells with sucrose solutions, at pH 7.8 and pH 4, removed most of the calcium localized in S₁, indicating that this calcium is membrane bound. The parameters of calcium efflux in ROS cells were found to be different from those measured previously in cultured bone cells: (1) There was no difference between efflux patterns in cells incubated in medium containing 1% and 10% serum; (2) Rates of calcium fluxes were much lower in ROS cells than those in bone cells; and (3) The amount of calcium in S₃ was very small.     

Calcium uptake and release by skeletal-muscle mitochondria

THE EGTA — ruthenium red quench technique was used to obtain initial-velocity plots of Ca²⁺ uptake by skeletal-muscle mitochondria. The K_m was 5 μM and the Hill coefficient 1.9 at both 0° and 10°C. Inorganic phosphate stimulated and Mg²⁺ inhibited initial rates of transport. In experiments on Ca²⁺ release, the $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{2+}}$ exchange was demonstrated. Factors influencing Ca²⁺ release during anaerobiosis include phosphate concentration and extent of Ca²⁺ loading. The results are discussed in relation to the possible participation of mitochondria in the calcium-ion regulation of muscle.     

Cytoplasmic droplets produced by the effect of adenine on Physarum plasmodia: Comparison with caffeine droplets and effect of calcium

Three independently established Drosophila cell lines, Schneider's line 3 (S3), Dübendorfer's line 1 (D1) and MDR3, an adenine salvage deficient clone of the Kc line, all cease to proliferate in the presence of ecdysterone. This is also observed with hybrids between S3 and MDR3 and between D1 and MDR3. It is shown that cells derived directly from wild-type Drosophila embryos can be hybridized with MDR3. Of nine such hybrids all proved to be able to proliferate in the presence of ecdysterone.     

The blue-light reaction in plasmodia of Physarum polycephalum is coupled to respiration

The influence of inhibitors of energy metabolism (2-deoxy-D-glucose, monoiodoacetate, KCN) as well as various substrates for respiration (sodium acetate, glycine, glutamine, α-ketoglutarate, pyruvate) were investigated with respect to the effect of blue light (450 nm) on contractile behaviour of plasmodial strands of Physarum polycephalum. When the energy metabolism is not experimentally modified, blue light induces a prolongation of the period of the contraction-relaxation cycle. This effect appears within 2–3 min and seems to represent the primary reaction of this organism to blue light. Inhibition of respiration by KCN completely abolished this response to blue-light irradiation. In contrast, an impediment of glycolysis enhanced the effect. This indicates that the reaction to blue light is related to respiration, i.e., to the function of mitochondria. Among different substrates for respiration only α-ketoglutarate combined with pyruvate and applied in the presence of inhibitors of glycolysis showed an enhancement of the photoresponse, i.e., a prolongation of the period and an increase of the amplitude of the force oscillations. This indicates that the pyruvate and α-ketoglutarate-dehydrogenase complexes functioning in mitochondrial respiration are involved in the primary blue-light reaction of plasmodia of Physarum polycephalum.     

Specific nuclear elimination in polyploid plasmodia of the slime mold Physarum polycephalum

In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.     

Calcium binding capacity and calcium sensitive protein content in denervated frog muscles

Total and ionic calcium content, calcium binding capacity of sarcoplasmic proteins and calcium insensitive proteins were examined in atrophying leg muscles of frog after 1-5 months period of denervation. Different muscles showed different levels of atrophy and the total calcium content varied with reference to the type of muscle. Ionic calcium levels doubled in the gastrocnemius muscle after three months denervation. Calcium binding capacity of proteins and calcium insensitive proteins decreased rapidly up to four months after denervation in the gastrocnemius muscle. However no significant changes in the levels of calcium binding capacity and calcium insensitive proteins were found with reference to the type of muscle. Since total calcium content remains constant and wet muscle mass (expressed as atrophy) decreased markedly, an apparent increase in calcium concentration occurs in each muscle on denervation.     

Role of Calcium in Phototaxis of Physarum polycephalum

The calcium transport blockers, ruthenium red and lanthanumions, inhibit negative phototactic orientation in the acellularslime mold, Physarum polycephalum. Likewise, agents known toenhance calcium fluxes (caffeine and phosphatidic acid) affectphoto-orientation as well as trimethyl-phenyl-phosphonium^$ whichimpairs electrochemical gradients across membranes. The resultssuggest that calcium fluxes are involved in the sensory transductionof phototactic orientation in this organism. The cation transportthrough the channels occurs passively along a previously establishedgradient built by energy dependent calcium pumps. Inhibitionof these pumps by poly-L-lysine also impairs phototactic orientation.The location of the calcium transport phenomena is discussed. (Received April 17, 1985; Accepted August 12, 1985)     

In situ accumulation of calcium by organelles of squid axoplasm

Existing morphological and physiological evidence indicates that axoplasm of squid axons sequesters calcium by both mitochondrial and non-mitochondrial buffers. The present work demonstrates that essentially all of the non-mitochondrial component is located in organelles. Extruded axoplasm was loaded with varying amounts of calcium by mixing with small volumes of solutions containing pH buffered ⁴⁵Ca. Ethyleneglycol-bis(β-amino-ethyl ether)N,N′-tetraacetic acid (EGTA) or diethylenetriamine pentaacetic acid (DTPA) was used to stabilize the free calcium. The axoplasm was then sucked up in a polyethylene tube and centrifuged at 100,000 g for 2–3 hours to produce a loose pellet comprising 10–20% of the axoplasm volume. After centrifugation, the tube was frozen, sliced into segments, and counted by liquid scintillation. No significant pellet accumulation of exogenous calcium occurred at physiological concentrations of free calcium (ca. 50 nM); however, a threshold for accumulation existed at 150–200 nM. Essentially complete pellet sequestration of the exogenous load occurred at a free calcium concentration above 1 μM. About half of the pellet buffering capacity was sensitive to carbonyl cyanide, p-trifluoromethoxy phenylhydrazone (FCCP). Variation of exogenous load between 0.1 – 3 mmole/kg axoplasm did not affect the buffering capacity of either the FCCP sensitive or insensitive components when the free calcium concentration was above threshold.     

Chemotaxis: not a prerequisite for plasmodia coalescence in Physarum polycephalum.

The sea mussel, Mytilus edulis, forms an adhesive substance which is an extremely stable, alkali-soluble protein complex. Hydrolysates of the adhesive were processed using ion-exchange chromatography and fluorescent fractions compared to authenic dityrosine. UV spectra in acid solutions, fluorescent spectra, and migration on thin layer chromatography indicated that the fluorescent fraction was identical to authenic dityrosine. The tyrosine complexes function to link peptide chains into a stable threedimensional network with unique chemical properties.     

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).     

Isolation and partial characterization of haemagglutinins from plasmodia of Physarum polycephalum.

The soluble haemagglutinins produced by plasmodia of Physarum polycephalum were purified by chromatographic methods and resolved into haemagglutinins I and II. On SDS-PAGE, purified haemagglutinins I and II each gave a single band with an apparent molecular mass of 6 and 11 kDa, respectively. The results of gel-filtration chromatography suggested that both haemagglutinins were dimers of the respective subunits under non-denaturing conditions. Rabbit erythrocytes were preferentially agglutinated by both haemagglutinins. The human type A, B and O erythrocytes were agglutinated by haemagglutinin II to an equal degree but were not agglutinated by haemagglutinin I. Simple sugars failed to inhibit the activities of both haemagglutinins. The activities, however, were effectively inhibited by the addition of thyroglobulin. Other glycoproteins such as fetuin, orsomucoid and transferrin inhibited the activity of haemagglutinin I but not that of haemagglutinin II. These haemagglutinins were detected in a slime fraction obtained from the culture media of starved plasmodia, suggesting that they are released to the outside of the plasmalemma to become associated with the slime layer on the plasmodial surface.