Induction of mitochondrial migration in the slime mold Physarum polycephalum

Mitochondrial migration in a microplasmodium of Physarum polycephalumwas studied by litgh and electron microscopy. The mitochondriawere dispersed evenly in the microplasmodium of Physarum polycephalumin shaken cultures but when the microplasmodia were left unshakenin a liquid culture for more than 3 hr, the mitochondria migratedtoward the peripheral area and came into contact with an semi-electrontransparent layer beneath the cell membrane. Once the peripherallocalization of mitochondria was established in unshaken culture,subsequent reversal to the shaken cultures induced a reversion.These results suggest that mitochondrial migration is reversiblyindicated by culture condition. (Received June 19, 1978; )     

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)     

Habitat selection and patchiness in Scapholeberis: horizontal distribution and migration of S.mucronata in a small pond

The horizontal distribution of Scapholeberis mucronata in asmall pond was studied by transect and pattern sampling. Densitiesare high (>1000 animals m^–2) in the littoral zone andlow in the open water (no aquatic vegetation; <<50 animalsm^–2). The preference for the littoral zone is, however,less well expressed during the night, indicative of a diurnalhorizontal migration. Within the littoral zone, the distributionof Scapholeberis is highly aggregated. The relative degree ofpatchiness is inversely correlated with density. The relativeabundance of males and ephippial females was not invariablyhigher in than out of patches. It is hypothesized that the observedhabitat preference, as well as the aggregative behaviour, ofScapholeberis are mechanisms to avoid predation by fishes.     

Phototactic Orientation in Plasmodia of the Acellular Slime Mold, Physarum polycephalum

Phototactic orientation to lateral light was studied in plasmodiaof the yellow-pigmented strain (CL) and a white mutant of theslime mold Physarum polycephalum. The orientation changes wereperformed by either a turn or a reversal of the internal polarityof the plasmodium. Fluence rate-response curves for white andmonochromatic light showed positive phototaxis with low fluencerates and negative phototaxis with higher ones. The wavelengthdependences for both strains were similar and indicated thepredominant role of a UV/blue light-absorbing photoreceptor,which was not the same as one of the yellow pigments found inthe wild-type strains. Wavelengths >500 nm induced only aninsignificant effect. The control of the motor apparatus andthe possible linkage of the photoresponse with the other sensoryprocesses known in Physarum are discussed. (Received June 3, 1983; Accepted October 11, 1983)     

Wild Oat/Barley Interactions: Varietal Differences in Competitiveness in Relation to K+ Supply

Eight cvs of barley (Hordeum vulgare L.) were separately plantedwith Wild Oats (Avena fatua L., genetically pure line CS40)in a sand culture with two external K⁺ concentrations. Substantialdifferences were observed among barley cvs in their abilityto compete with wild oat. The variety Fergus was highly competitiveat both high and low [K⁺]_e, whereas Steptoe was competitiveonly at high [K⁺]_e, and Compana was only weakly competitivewith wild oat. The differences between barley cvs were relatedto their previously reported efficiencies of K⁺ uptake and utilization. Hordeum vulgare L., Avena fatua L., barley, wild oat, competition, K⁺ nutrition, utilization efficiency     

Isolation and purification of the extracellular ribonuclease from Physarum polycephalum

Summary The myxomycete Physarum polycephalum has been reported to produce an extracellular ribonuclease. A quick procedure is described for the isolation and purification of the ribonuclease from the culture supernatant. It involves an initial concentration of the culture supernatant by slow freezing, followed by ammonium sulfate precipitation. The enzyme was adsorbed onto DEAE-cellulose and eluted with a sodium chloride gradient.     

Amino acids stimulate phosphorylation of p70S6k and organization of rat adipocytes into multicellular clusters

In previousstudies we have shown that rat adipocytes suspended in Matrigel andplaced in primary culture migrate through the gel to form multicellularclusters over a 5- to 6-day period. In the present study,phosphorylation of the insulin-regulated 70-kDa ribosomal protein S6kinase (p70^S6k) was observedwithin 30 min of establishment of adipocytes in primary culture. Twoinhibitors of the p70^S6ksignaling pathway, rapamycin and LY-294002, greatly reducedphosphorylation of p70^S6k andorganization of adipocytes into multicellular clusters. Of all thecomponents of the cell culture medium, amino acids, and in particular asubset of neutral amino acids, were found to promote bothphosphorylation of p70^S6k andcluster formation. Lowering the concentrations of amino acids in themedium to levels approximating those in plasma of fasted rats decreasedboth phosphorylation of p70^S6kand cluster formation. Furthermore, stimulation ofp70^S6k phosphorylation by aminoacids was prevented by either rapamycin or LY-294002. These findingsdemonstrate that amino acids stimulate thep70^S6k signaling pathway inadipocytes and imply a role for this pathway in multicellularclustering.

     

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

Cytochrome systems in cells of a denitrifying bacterium, Pseudomonasstutzeri (VAN NIEL strain), grown under different atmosphericconditions were compared with reference to the effects of nitrateand nitrite on cytochrome synthesis. When a culture was sufficiently aerated (aerobic conditions),synthesis of all cytochrome components was repressed, regardlessof the presence or absence of nitrate and nitrite. When aeratedmoderately (semi-aerobic conditions), both soluble and paniculatecytochromes c-552 and cytochrome b-558 contents markedly increasedeven in the absence of nitrate and nitrite. Under anaerobic or semi-aerobic conditions, nitrite inducedcytochrome a₂–c synthesis. This inductive effect of nitritewas counteracted by nitrate. Nitrate also repressed particulatecytochrome c-552 synthesis to some extent but nitrite did not. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan (Received June 24, 1969; )     

The Photometabolism of Acetate by Chlorella pyrenoidosa

Chlorella pyrenoidosa can utilize sodium acetate as a carbonsource for growth in the light. Growth proceeds under aerobicconditions both in the presence and in the absence of carbondioxide, but under anaerobic conditions only in its presence.The assimilation of acetate does not result from oxidation tocarbon dioxide followed by photosynthetic fixation because theproducts of ¹⁴C-acetate assimilation are different from theproducts of ¹⁴CO₂ fixation in the presence of unlabelled acetate. In aerobic conditions 10-⁶ M DCMU induces a pattern of acetateassimilation in the light similar to that in the dark. Thus,in the presence of DCMU in the light, less acetate carbon isincorporated into cells, particularly into lipids, polysaccharide,and protein, and more is released as carbon dioxide than inits absence. The effect of 4 x 10^-3 M MFA on acetate assimilationin the presence of 10^-6 M DCMU is the same in light and dark.Acetate assimilation is unaffected by desaspidine and sodiumbisulphite. The mean generation time of C. pyrenoidosa growing on acetatein the light under aerobic conditions is 20 hours. When 10^-5M DCMU is added the mean generation time is 60 hours, the sameas that for Chlorella growing on acetate in the dark. The activityof the enzymes of the glyoxylate cycle, isocitrate lyase (E.C.4.1.3.1.)and malate synthetase (E.C.4.1.3.2.) is repressed in the light,but activity of both enzymes increases markedly when DCMU isadded.     

Mass Culture of a Slime Mold, Physarum polycephalum

The slime mold, Physarum polycephalum, was cultivated in a soluble natural medium in shake flasks and in 30-liter and 50-gal conventional baffled fermentors. Yields of 6 to 10 g (dry weight) per liter were obtained in the large-scale fermentations. Because of the slow growth of the myxomycete, particular attention had to be paid to aseptic technique. The inability of this organism to withstand the normal degree of agitation employed with most aerobic fermentations made it difficult to obtain adequate aeration. Conditions for growth of the organism on a pilot-plant scale are presented.     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL     

Production and degradation of β-Poly-L-malate in cultures of Physarum polycephalum

β-Poly-L-malate (PMA) is synthesized by plasmodia of Physarum polycephalum during growth and secreted into the culture medium. There it is degraded to L-malate after growth has ceased. Its concentration is highest in cell nuclei, where it probably performs a plasmodium-specific function.     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

Modification in Cell Shape Unrelated to Cellulose Microfibril Orientation in Growing Thallus Cells of Chaetomorpha moniligera

Cellulose microfibril orientation patterns in thallus cellsof Chaetomorpha moniligera were studied, and the relationshipbetween the microfibril and the peripheral microtubule arrangementsduring cell-shape modification by colchicine was examined. Inthe cuttings from growing thalli, linearly arranged cylindricalcells developed into cask-shaped cells during 4–6 daysof culture at 27?C. In the cylindrical cells, microfibrils formingthe innermost portion of the wall were arranged alternatelyin longitudinal and transverse directions, but peripheral microtubuleswere always arranged only in a longitudinal direction. Thesefeatures were also noted in the cask-shaped cells. Colchicineat 10^–3M and 3?10^–3M accelerated both cell expansionand wall thickening with matrix deposition, but the directionsin which both microfibrils and microtubules were arranged werethe same as those of the cylindrical cells. These results indicatethat (1) the microfibril and microtubule arrangements of Chaetomorphaare not necessarily correlated, (2) changes in cell shape ofChaetomorpha are not necessarily accompanied by changes in thearrangement of cell-wall microfibrils, and (3) colchicine playsa role in the loosening and thickening of cell walls by enhancingmatrix deposition. (Received June 2, 1986; Accepted February 13, 1987)     

Growth and differentiation of wild type amoebae of Physarum polycephalum in liquid culture

A method for growing wild type amoebal strains of Physarum polycephalum in two-membered liquid culture is presented. The medium is a simple buffered salts solution. We found that a minimal level of divalent cation was required for growth. All amoebal strains tried to date have grown under our conditions in stationary culture. Growth under gyratory conditions was only successful at 60 rpm or less and consistent growth required a period of adaptation over several transfers. Differentiation of two apogamic strains, CL and CH1, were compared. Contrary to the results seen on agar plates, the time of onset for the first committed amoebae was identical for both strains in liquid culture. Attempts to demonstrate mating between two genetically compatible amoebal strains grown together in liquid culture were not successful.     

Effects of prey motility and concentration on feeding in Acartia tonsa and Temora longicornis: the importance of feeding modes

Feeding experiments were conducted with the ambush-feeding copepodAcartia tonsa and the feeding-current-generating copepod Temoralongicornis. The copepods were offered a mixed diet of the dinoflagellateHeterocapsa triquetra and the ciliate Balanion comatum of similarcell size. The dinoflagellate was offered at a constant concentrationof 10–15 cells mL^–1, whereas the ciliate was offeredat a variety of concentrations, ranging from 7 to 57 cells mL^–1.Copepods with different feeding modes possess different mechanismsfor prey detection, suggesting that the two copepods would responddifferently to the two prey types. Both copepods had significantlyhigher clearance rates on the highly motile ciliate than onthe less motile dinoflagellate. In encounters between A. tonsaand its prey, we argue that this is due to the higher hydromechanicalsignal generated by the ciliate. The advection feeding copepodT. longicornis fed on the two prey according to their relativeconcentrations; in this case, we suggest that although B. comatumis capable of detecting feeding-current-generating predators,the feeding current velocity generated by T. longicornis isgreater than the escape velocity of this ciliate.     

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.     

Growth kinetic parameters of two planktonic desmid species under fluctuating phosphorus conditions in continuous-flow culture

Two planktonic desmid taxa, Staurastrum chaetoceras and Cosmariumabbreviatum var. planctonicum, were examined under pulsed phosphorus(P)-limited conditions in continuous culture. Two pulse regimeswere applied, i.e. 2.5 µM P twice a week and 10 µMP once every 2 weeks. Under both pulse regimes, S.chaetocerasshowed a higher maximum P uptake rate (V^max) and affinity forP uptake (V^max/affinity constant K^m) than C.abbreviatum. Affinityfor P uptake in S.chaetoceras just before pulsing was higherthan shortly after pulsing, whereas C.abbreviatum did not showany significant difference. Cell densities and cellular P quotain S.chaetoceras and C.abbreviatum fluctuated in a comparableway, but fluctuations in C.abbreviatum were less pronouncedthan in S.chaetoceras. The mean cell volume of both specieswas greater under the bimonthly than under the biweekly pulseregime, and the lowest under continuous P-limited conditions.Competition between S.chaetoceras and C.abbreviatum under thebimonthly pulse regime was won by S.chaetoceras, but displacementwas less fast than under more frequent pulsing (equalling thesame total P supply). The above-mentioned results are discussedin relation to possible ecological strategies.     

PRODUCTION OF HYDROGEN SULFIDE FROM SULFITE BY A COPPER-ADAPTED YEAST

Activity of hydrogen sulfide production from sulfite was studiedusing a copper-resistant yeast strain (R), its parent strain(P), and the culture of R in the medium without copper addition(R(0)). More hydrogen sulfide was produced under aerobic conditionthan under anaerobic condition. Sulfide producing activity wasin the order of R(0)>P>R under either condition. Stationaryphase cells produced more sulfide than logarithmic phase cellswhen cultured without copper, while the reverse was the casewith R, cultured in copper medium. Sulfide production was inhibitedby high concentrations of sulfite and by salicylaldoxime. Differencein the pathway from sulfite to sulfide was suggested betweenthe resistant strain (R and R(0)) and P in that the former wasmore sensitive to these inhibitors. ¹ Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

Light- and thiosulfate-dependent reduction of nicotinamide adenine dinucleotides in whole cells of Chromatium vinosum

The light-driven, thiosulfate-dependent reduction of nicotinamideadenine dinucleotides under acrobic conditions in whole cellsof Chromatium vinosum was investigated. The total concentration of pyridine nucleotides in whole cellswas about 50 nmoles per µmole of bacteriochlorophyll.Under dark aerobic conditions, the majority of the nucleotidespresent was NAD⁺ with about 20% as NADP⁺. About 40% of the total NAD was reduced under continuous illumination.Thiosulfate or sulfide was needed for the photoreduction, whileorganic acids such as succinate or malate were not. The initialrate of NAD⁺ photoreduction in the presence of thiosulfate wasapproximately 100 nmoles per µmole of bacteriochlorophyllper min. The NAD⁺ photoreduction was strongly inhibited by uncouplersand electron transfer inhibitors. In contrast, an energy transferinhibitor, N, N'-dicyclohexylcarbodiimide, did not affect NAD⁺photoreduction at a concentration at which the light-inducedATP formation was inhibited. A transmembrane electrochemicalH⁺ gradient generated by cyclic electron transfer may be theenergy source for reduction of NAD⁺ in Chromatium vinosum. (Received April 2, 1980; )