Activation of Cytoplasmic Streaming as an Adaptive Mechanism of Cultured Muskmelon Cells under Hypoxia Stress

Cytoplasmic granules were activated and moved along the cytoplasmicstrands as the cultured muskmelon (Cucumis melo L.) cells weretransferred from the k-8 agar medium to liquid medium. CytochalasinB reversibly inhibited the streaming, suggesting that the motiveforce for streaming was generated by microfilament. Besides,KCN and 2,4-dinitrophenol lowered the streaming velocity, showingthat the streaming required metabolic energy, ATP. Fluoresceindiacetate (FDA) staining was used to estimate cell viability.Variety reticulatus (CR), which had been regarded as floodinguntolerant (Crawford 1982, Liu and Chen 1987), had more viablecells than the flooding tolerant variety saccharinus (CS) whentheir calluses were transferred to liquid media. Meanwhile,variety CR had more streaming cells in the liquid medium, andwas maintained steadily between 6th and 9th day after transfer.It is considered that cytoplasmic streaming occurring in theCR cells served as an adaptive mechanism under flooding stress. (Received April 11, 1988; Accepted October 20, 1988)     

Substrate flexibility regulates growth and apoptosis of normal but not transformed cells

One of the hallmarks of oncogenictransformation is anchorage-independent growth (27). Herewe demonstrate that responses to substrate rigidity play a major rolein distinguishing the growth behavior of normal cells from that oftransformed cells. We cultured normal or H-ras-transformedNIH 3T3 cells on flexible collagen-coated polyacrylamide substrateswith similar chemical properties but different rigidity. Compared withcells cultured on stiff substrates, nontransformed cells on flexiblesubstrates showed a decrease in the rate of DNA synthesis and anincrease in the rate of apoptosis. These responses on flexiblesubstrates are coupled to decreases in cell spreading area and tractionforces. In contrast, transformed cells maintained their growth andapoptotic characteristics regardless of substrate flexibility. Theresponses in cell spreading area and traction forces to substrateflexibility were similarly diminished. Our results suggest that normalcells are capable of probing substrate rigidity and that propermechanical feedback is required for regulating cell shape, cell growth,and survival. The loss of this response can explain the unregulated growth of transformed cells.

     

Effects of n-Alkylamines on the Motility and Viability of Heterosigma akashiwo Cells

The cytotoxic effects of positively charged liposomes and theircomponents on Heterosigma akashiwo cells were investigated.Positively charged liposomes reduced cell motility and eventuallycaused cytolysis. Negatively charged and non-charged liposomeshad little effect on cell motility and/or cell viability. Damagewas also induced by a single application of some n-alkyl-amines.Among n-alkylamines tested, laurylamine (C₁₂) was most effectivein reducing motility and causing cytolysis of cells. The extentof the deleterious effects increased with increasing concentrationsof laurylamine and with the duration of treatment. The extentof damage to cells by laurylamine changed periodically duringthe cell cycle. The effects of laurylamine began to increaseat the forth hour of the light period and began to decreaseat the first hour of the dark period, under condition of 12hours of light and 12 hours of darkness. Laurie and myristicacids, which each have a carboxyl group in place of the aminogroup of the corresponding alkylamines, laurylamine and myristylamine,had little effect on the cells. (Received December 21, 1988; Accepted March 29, 1989)     

Effect of Ferricyanide on Potassium Uptake by Intact Epidermal Tissue and Guard Cell Protoplasts

The effect of ferricyanide on K^$ fluxes in epidermis and inguard cells of Commelina communis L. were studied. Ferricyanideenhanced guard cell protoplasts swelling, which results fromenhanced K^$ uptake. In intact epidermis ferricyanide inhibitedK^$ uptake and consequently, stomatal opening. This was foundin floated and submerged epidermal tissues, indicating thatthe degree of contact with the solution does not affect theresponse to ferricyanide. Investigation of the rate of plasmolysisand de-plasmolysis of guard cells in epidermal tissue revealedthat ferricyanide enhances deplasmolysis, caused by K^$ uptake,only in completely plasmolysed cells, which resemble protoplastsin situ. (Received January 21, 1988; Accepted March 24, 1988)     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

Heat Shock Induced Change in Protein Ubiquitination in Chlamydomonas

Ubiquitin was purified from pea (Pisum sativum L.) and its antibodywas produced. Western blot analysis showed that the antibodycross-reacted with ubiquitins from a green alga Chlamydomonasreinhardtii, a brown alga Laminaria angustata and a red algaPorphyridium cruentum but not with ubiquitin from a blue-greenalga Synechococcus sp. In Chlamydomonas, the antibody also reactedwith some ubiquitinated proteins including 28- and 31-kDa polypeptides.The isoelectric points of Chlamydomonas ubiquitin and the 28-and 31-kDa ubiquitinated proteins were 8.0, 8.9 and 10.3, respectively.The ubiquitinated proteins, including the 28- and 31-kDa polypeptideswere detected after in vitro ATP-dependent ubiquitination ofChlamydomonas cell extract with ^l25I-labeled bovine ubiquitin.Heat treatment of Chlamydomonas cells (>40°C) causeddrastic increase of ubiquitinated proteins with high mol wt(>60kDa), and coordinated redistribution or decrease of otherubiquitinated proteins and free ubiquitin. Quantitative analysisrevealed that the 28- and 31-kDa ubiquitinated proteins showeddifferent responses against heat stress, i.e. the former beingmore sensitive than the latter. (Received July 10, 1988; Accepted October 4, 1988)     

Orientation of Wall Microfibrils in Avena Coleoptiles and Mesocotyls and in Pisum Epicotyls

Microfibrils (MFs) on the inner surface of the walls of Avenacoleoptile and mesocotyl cells and of Pisum epicotyl cells wereexamined by a replica method. In the elongating epidermis ofthese three organs, cells having MFs that were transverse, obliqueor longitudinal to the elongation axis were intermingled. Inthe elongating parenchymal tissues, all cells deposited MFstransversely. In non-elongating cells of Avena coleoptiles andPisum epicotyls, the orientation of MFs on the inner wall surfaceof both epidermal and parenchymal cells was more longitudinalthan in elongating cells. These observations on the orientationsof MFs are compatible with those our previously reported observationson the orientations of microtubules (MT) (Iwata and Hogetsu1988). Disruption of MTs of Avena coleoptiles by treatment withamiprophosmethyl caused changes in the orientation of depositionof MFs. These results support the idea that MFs are usuallyco-aligned with MTs in organ cells and that the orientationof MFs is controlled by MTs. The averaged direction of MFs, visualized under polarized light,showed a clear difference between the epidermal and inner-tissuecell walls in the elongating regions of the three organs. Inalmost all elongating and non-elongating epidermal cells, theaveraged direction of MFs was longitudinal, while it was transversein all inner-tissue cells. (Received December 16, 1988; Accepted April 28, 1989)     

Effect of Ca2+ on Tonoplast Potential of Permeabilized Characeae Cells

Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca²⁺ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca²⁺, the tonoplast potential(E_M became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl^–-channelinhibitor, E_m became more negative and the response of E_m toCa²⁺ was significantly suppressed. It is suggested that Ca²⁺activates Cl^–-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl^–-channelin N. axilliformis. ¹Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988)     

Orientation of Microtubules during Regeneration of Cell Wall in Selected Giant Marine Algae

The microtubules in highly synchronized aplanospores of twogiant marine algae, Boergesenia forbesii and Valonia ventricosa,were examined by immunofluorescence microscopy throughout theregeneration of the cell wall. Microtubule orientation was alwaysrandom up to 20 h after wounding, although the orientation ofcellulose microfibrils changed from random to parallel withinthat time period. When the rhizoid cells were in the stage ofelongation at 7 to 10 days after wounding, highly ordered microtubuleswere always observed along the longitudinal cell axis exceptat the very tip of the cells where random ones were found. Incontrast, the microfibrils in the innermost lamellae of newlysynthesized cell walls showed three different orientations,that is, transverse, longitudinal and oblique to the longitudinalcell axis. These observations suggest that microtubules maycontrol cell shape, but not the orientation of microfibrils.The mechanism of cell wall construction in these algae is discussedin relation to the self-assembly mechanism thought to operatein the construction of helicoidal cell walls. ³ Present address: Polymer Research Laboratory, Mitsui ToatsuChemicals, Inc., Yokohama, Kanagawa 244, Japan. (Received November 18, 1987; Accepted April 11, 1988)     

Hormonal Induction of Vascular Differentiation in Cultured Zinnia Leaf Disks

Mesophyll cells differentiated into tracheary elements whenZinnia leaf disks were cultured in media containing sufficientauxin and cytokinin. Moderate increases in the levels of phytohormonesinduced the recruitment of adjacent cells into the preexistingvasculature, resulting in the "expansion" of leaf veins. Higherhormonal concentrations induced unpatterned differentiationinto tracheary elements throughout the mesophyll and epidermis.This novel system should facilitate the study of organized vasculardifferentiation. (Received July 30, 1988; Accepted October 31, 1988)     

Locomotion of Fundulus Deep Cells during Gastrulation

SYNOPSIS. Mechanism of locomotion of deep cells of Fundulusheteroclitus was studied in vivo during gastrulation with theaid of time lapse cinemicrography (Nomarski differential interferencecontrast optics), scanning electron microscopy of cells knownto be moving at the time of fixation, and cell culture. Theseare our findings. 1) Deep cells usually move rapidly, at about10–15 µ/min, regardless of whether they move byblebbing or spreading. Evidence suggests that this high speedis associated with weak adhesion of the trailing edge: it remainsrounded, without large retraction fibers, and it advances continuouslywith advance of the leading edge, not sporadically, as it wouldif it adhered strongly. 2) In contrast, when stationary cellsin close contact separate, they remain connected by retractionfibers, suggesting strong punctate adhesions. 3) Locomotionby shortening of a long lobopodium is really a form of spreadingmovement; the tip of a lobopodium always spreads. Also, sincespeed of shortening decreases with continuance, it may dependprimarily on elastic recoil rather than active contraction.4) Fundulus deep cells appear to move in two ways: a) protrusionof blebs, followed by much cytoplasmic flow; b) protrusion oflamellipodia, accompanied by filopodia and frequent cell shortening.5) Filopodia were not found except at the leading edge of aspreading lamellipodium and often spread themselves; perhapsfilopodia and lamellipodia are interconvertible. 6) A lamellipodialmargin may form undulations in vivo that move backward likeruffles in vitro. 7) At all times, whether stationary or moving,the surface of deep cells is smooth, raising unanswered questionsconcerning the source of surface for their rapid protrusiveactivity.     

Changes in Light-Induced Electron Transfer Through Cytochrome b-563 Depending on the Oxidation-Reduction Conditions in Intact Cells of the Cyanobacterium Synechocystis PCC 6714

Flash-induced redox changes of cytochrome b-563 were studiedin intact cells of the cyanobacterium Synechocystis PCC 6714.The redox reactions of this cytochrome depended on the redoxstate of the electron transport system, as determined by a balancebetween the influx of electron from substrates and the effluxto molecular oxygen. Under aerobic conditions, flash-inducedchanges in cytbchrome b-563 were almost insignificant in thephotoheterotrophic cells grown in the presence of DCMU (0.1mM) and glucose (60 mM), whereas a rapid reduction of the cytochrome,followed by a re-oxidation, was clearly observed when the respiratoryoxidase was inhibited by KCN (1 mM). Under anaerobic conditions,when cytochrome b-563 was fully reduced in the dark, a prominentoxidation of the cytochrome was observed after a flash; thesubsequent re-reduction was very slow. The oxidation of cytochromeb-563 was inhibited by the addition of 2-heptyl-4-hydroxyquinolineN-oxide (HQNO). These results suggest that electron transferin the cytochrome bf complex occurs by a modified Q-cycle mechanism,the reaction sequence of which depends on the redox states ofcytochrome b-563 and plastoquinone. Flash-induced oxidationand reduction of cytochrome b-563 in cells grown under photoautotrophicconditions were less prominent, but the patterns of the reactionswere consistent with the suggested interpretation. (Received May 9, 1988; Accepted August 15, 1988)     

Armoured dinoflagellates in the NE Atlantic during the BOFS cruises, 1988-90

The armoured dinoulagellates present in 90 plankton samplescollected by the use of an Apatein closing net were enumerated.The samples were collected from various stations around the20W meridian and between 44 and 60N during the 1988, 1989and 1990 Biogeochemical Ocean Flux Study (BOFS) cruises. Atmost stations, samples were obtained from various depths, althoughin 1990 only the surface zone was sampled. A total of 126 specieswere identified, of which 49 have chloroplasts and are thoughtto be autotrophic, 47 are assumed to be heterotrophic and thenutritional type of the remainder is unknown. The samples collectedduring July 1988 were dominated by large numbers of the twophotosynthetic species Gonyaulax polygramma and Protoceratiunreticulatum (=Gonyaulax grindleyi). The much more intensivesampling of 1989 revealed several Ceratiun species, C.fusus,C.furca and C.lineazum, together with Gonyaulax polygramma,as the most common dinoflagellates. In 1990 the samples, whichwere taken during a Lagrangian survey in May-June, were alsodominated by Ceratium species. This time C.azoricwn was a majorcomponent and Protoceratium reticulatum was again present inhigh numbers as in 1988. A number of analyses were carried outon the data collected. It was found that the majority of themore frequent species were autotrophs and most were membersof the genus Ceraziwn. The effects of depth were shown to resultin reduced numbers of cells and species, but no clear associationwas found between species and depth. After the application ofDetrended Correspondence Analysis (DECORANA) to all surfacesamples, there was found to be a clear association between thespecies composition and both time of year and latitude. Watercolumn stability is also probably an important factor in speciescomposition and cell numbers. Seasonal changes in the frequencyof the main species were also noted with some, such as C.lineatum,being more important early in the summer and others, such asProtoceratium reticulatum and particularly G.polygramma, becomingdominant later. The use of Two-way Indicator Analysis (TWINSPAN)revealed some potential species associations.     

Regulation of Carbohydrate Breakdown of Chlorella Mutant No. 20, Studied by 31P NMR Spectroscopy and Enzymatic Analysis 2. Regulation in the Presence of Exogenous Glucose

In the preceeding paper (Ruyters 1988) the regulation of bluelight-stimulated carbohydrate degradation and respiration wasinvestigated in cells of the chlorophyll-free Chlorella mutantno. 20. Since some results are not in agreement with the commonconception of control, for comparison the regulation of carbohydratebreakdown, especially that of its glycolytic pathway, was studiedafter addition of exogenous glucose to Chlorella mutant cells. Evidence is presented that principle regulatory mechanisms remainthe same in blue light- as in glucose-stimulated metabolism.Pyruvate kinase is proven to be the control enzyme of the glycolyticpathway in Chlorella mutant no. 20, whereas there is no suchindication for phosphofructokinase. The possible cause and consequenceof the opposite changes in the ATP/ADP ratio for the regulationof carbohydrate breakdown are discussed, leading to the viewthat PEP plays a major regulatory role in Chlorella mutant no.20, while the adenine nucleotides seem to be of minor importance. (Received October 5, 1987; Accepted January 7, 1988)     

A TYROSINASE-LIKE ENZYME IN CEPAEA NEMORALIS AND ITS POSSIBLE RELATION WITH SHELL BANDING POLYMORPHISM

Shell banding polymorphism in Cepaea nemoralis may be relatedto tyrosinase activity in this gastropod, as the hyalozonatephenotype shows bands with no pigmentation. We aimed to establishif there is tyrosinase activity in Cepaea, and this activitywas analyzed for different organs on electrophoresis gel slabsand oxygen electrode. We have found two isocnzymatic forms,one of them polymorphic, with high activity in the foot andmantle, organs both involved with the pigmentation of body andshell. We propose that several isoenzymatic forms are functional inthe melanosome. These may be activated during the cell lysis,as hyalozonate samples display tyrosinase activity. (Received 22 June 1988; accepted 5 September 1988)     

Regulation of Tubulin Degradation in Isolated Zinnia Mesophyll Cells in Culture

Tubulin degradation in isolated Zinnia mesophyll cells in culturewas investigated by pulse-chase labeling with [³⁵S]-methionineand two-dimensional electrophoresis. Tubulin degradation changesdynamically during culture. Almost no tubulin degradation occursin the cells on the first day in culture. Treatment of thesecells with colchicine activates the degradation of tubulin,but not of proteins other than tubulin. In the presence of colchicine,the and ß-subunits of tubulin are degraded togetherand the half life of each subunit is approximately 6 h. After2 d in culture, there is active degradation of tubulin evenin the absence of colchicine. Colchicine did not inhibit new synthesis of tubulin in Zinniacells. This is very different from the results reported in culturedmammalian cells, whereby unpolymerized tubulin elevated by colchicine-treatmentdepresses its own synthesis. These and previous results dealing with changes in the leveland synthesis of tubulin in cultured Zinnia cells (Fukuda 1987),are discussed in relation to the regulation of tubulin metabolismin cultured Zinnia cells. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Aoba-yama, Sendai, 980 Japan. (Received September 5, 1988; Accepted December 20, 1988)     

An immunofluorescent survey of the brown tide chrysophyte Aureococcus anophagefferens along the northeast coast of the United States

Surveys were conducted along the northeast coast of the USA.between Portsmouth, NH, and the Chesapeake Bay in 1988 and 1990,to determine the population distribution of Aureococcus anophagefferens,the chrysophyte responsible for massive and destructive ‘browntides’ in Long Island and Narragansett Bay beginning in1985. A species-specific immunofluorescent technique was usedto screen water samples, with positive identification possibleat cell concentrations as low as 10–20 cells ml^–1.Both years.A.anophagefferens was detected at numerous stationsin and around Long Island and Barnegat Bay, NJ, typically athigh cell concentrations. To the north and south of thus ‘center’,nearly half of the remaining stations were positive for A.anophagefferens,but the cells were always at very low cell concentrations. Manyof the positive identifications in areas distant from Long Islandwere in waters with no known history of harmful brown tides.The species was present in both open coastal and estuanne locations,in salinities between 18 and 32 practical salinity units (PSU).The observed population distributions apparently still reflectthe massive 1985 outbreak when this species first bloomed, giventhe number of positive locations and high abundance of A.anophagefferensin the immediate vicinity of Long Island. However, the frequentoccurrence of this species in waters far from this population‘center’ is disturbing. Aureococcus anophagefferensis more widely distributed than was previously thought. Numerousareas thus have the potential for destructive brown tides suchas those associated with the sudden appearance of the speciesin 1985.     

Physical Analysis of Tissue Mechanics in Amphibian Gastrulation

During morphogenesis, intercellular attachments constrain cellmobility so that embryonic tissues may (i) deform as solid sheetsof tightly bound cells, (ii) disperse to migrate as separatecells or (iii) flow as multicellular liquids (in which cellsremain aggregated yet can still slide past one another). Bymodelling deep germ layers as multicellular liquids in amphibiangastrulation, Davis and I have predicted, and then confirmedexperimentally, (i) the area-invariance of deep-germ-layer surfacetensions in vitro, (ii) spontaneous cell slippage in deformeddeep—germ—layer cell aggregates and (iii) correlationsof tissue surface tensions with tissue positioning in deep-germ-layercell-sorting and aggregate—fusion experiments. Liquid—tissueflow involves intercalations of interior cells into expandingtissue interfaces (or withdrawal of surface cells from shrinkingtissue interfaces). Tissue surface tensions are macroscopicreflections of the microscopic, tissue—specific adhesivedifferentials which direct these cell translocations. Such adhesivedifferentials may act independently of, or together with, activecell—shape changes, chemotaxis, contact guidance and/orhaptotaxis in controlling embryonic tissue rearrangements. Deepcell intercalations in vivo occur throughout amphibian gastrulation:during ectodermal epiboly; during marginal—zone extensionand convergence (and therefore blastopore closure); during mesodermalinvolution; and probably during the anterior spreading and axialextension (and therefore dorsal convergence) of involuted mesoderm.Tissue—surface—tension measurements may help determine(i) which of these cell—intercalation processes are activeand which are passive, (ii) the specific contributions of variousmicroscopic cell properties to the regulation of liquid—likegerm—layer assembly and (iii) similarities and differencesbetween in vitro and in vivo control mechanisms governing amphibiangastrulation.     

Blue Light Pulse-Induced Transient Changes of Electric Potential and Turgor Pressure in the Motor Cells of Phaseolus vulgaris L.

Blue light induces both depolarization of membrane potentialin the motor cell and turgor movement in the laminar pulvinusof bean plant. This paper describes the changes of electricpotential and turgor pressure induced in Phaseolus vulgarisL. by blue light pulses. A transient depolarization of membranepotential as large as 40 mV was induced by a short pulse of15 s blue light in motor cells of the laminar pulvinus. Thischange was not an action potential because of the absence ofa refractory period and threshold. The magnitudes of the responsewere dependent on the fluence of light. The response was long-lived,indicating that continuous input of light energy is not requiredfor a sustained response. The potential change was always followedby a transient turgor movement of the pulvinus. A molecular mechanism similar to a model postulated for theblue light response of stomata may operate in the motor cell.However, the direction of the electrical response to blue light(depolarization) in the motor cell was the opposite of thatin the guard cell (hyperpolarization). Turgor change of themotor cell by blue light was also opposite in direction (decrease). (Received February 19, 1988; Accepted June 28, 1988)     

Uptake and Utilization of Sugars in Cultured Rice Cells

Suspension cultured cells of rice (Oryza sativa) were grownin a medium containing sucrose. Sucrose was rapidly hydrolyzedextracellularly in the early stage of subculture with a concomitantdecrease in the medium pH. The hydrolysis may be due to cellwall associated acid invertase and may be promoted by acidificationof the medium. The resulting glucose and fructose seemed tobe utilized equally. The cells grown on either sucrose, glucoseor fructose contained each of these sugars and possessed cellwall associated invertase activity. Protoplasts prepared bycell wall degrading enzymes utilized preferentially glucoseor fructose rather than sucrose. These results suggest thatexogenous sucrose is hydrolyzed by the cell wall associatedinvertase to hexoses, which are then taken up and metabolized. (Received November 25, 1987; Accepted February 8, 1988)