A Mathematical Model on Water Redistribution Mechanism of?the?Seismonastic Movement of Mimosa Pudica

A theoretical model based on the water redistribution mechanism is proposed to predict the volumetric strain of motor cells in Mimosa pudica during the seismonastic movement. The model describes the water and ion movements following the opening of ion channels triggered by stimulation. The cellular strain is related to the angular velocity of the plant movement, and both their predictions are in good agreement with experimental data, thus validating the water redistribution mechanism. The results reveal that an increase in ion diffusivity across the cell membrane of <15-fold is sufficient to produce the observed seismonastic movement.     

Redistribution of potassium,chloride and calcium during the gravitropically induced movement of Mimosa pudica pulvinus

When the leaves of Mimosa pudica are changed from their normal position in the gravitational field, they perform reversible compensatory movements by means of pulvini. These movements are not the result of growth processes but involve reversible turgor variations. These variation are concomitant with ion migrations within pulvini: during the gravitropic movement, K⁺ and Cl^- shift towards the adaxial half of the motor organ whereas Ca²⁺ shifts towards the abaxial half. Compounds known to affect K⁺ transport, tetraethylammonium chloride and valinomycin, do not hinder the gravitropic movement but inhibit strongly the seismonastic reaction. The same general result is obtained with compounds affecting anion transport, disulfonic stilbenes and 9-anthracene carboxylic acid. Calcium chelators inhibit the gravitropic movement more efficiently than the seismonastic reaction and the calcium ionophore A 23 187 increases both movements. The data obtained with these various compounds indicate that ions do not have the same functional importance in the regulation of the two different pulvinar movements.Abbreviations abx abaxial half of the pulvinus - adx adaxial half of the pulvinus - 9-AC 9-anthracene carboxylic acid - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid - TEA tetraethylammonium chloride     

Migration of Calcium and Its Role in the Regulation of Seismonasty in the Motor Cell of Mimosa pudica L

Volume and conformational changes of the contractile tannin vacuoles of the abaxial motor cells of the primary pulvinus of Mimosa pudica L. parallel the seismonastic leaf movement. Since such changes in cells and organelles of animal systems are often regulated by calcium, we studied Ca²⁺ movement in the motor cells and tissue. By fixation with Lillie's neutral buffered formalin, followed by staining with alizarin red sulfate (ARS), calcium was localized in the tannin vacuoles of the motor cells of the primary pulvinus. After treatment with ethylenediaminetetraacetate, 8-hydroxyquinoline, and several other calcium-complexing or extracting agents, the color reaction due to alizarin red sulfonate was no longer present. By using an analytical method, it was shown that the effluent from stimulated pulvini has significantly more Ca²⁺ than that from unstimulated controls. Ten millimolar LaCl₃ inhibits recovery of the tannin vacuole in vivo in 10 mm CaCl₂ or in distilled water. Quantitative data obtained by microspectrophotometry demonstrated calcium migration during the bending movement of the primary pulvinus. In the adaxial motor cells a small amount of calcium migrates from the tannin vacuole, and calcium on the cell wall moves to the central vacuole. In the abaxial half, a large amount of calcium from the tannin vacuole moves to the central vacuole of the motor cell. It is probable that the calcium binds to the microfibrillar contents of the central vacuole. These observations support the contention that Ca²⁺ migrates between the surface of the tannin vacuole and the inside of the central vacuole. The recovery and maintenance of the tannin vacuole in the spherical form may play a role in maintaining turgor in the motor cells of the abaxial half of the primary pulvinus of Mimosa.     

H fluxes in excised samanea motor tissue : I. Promotion by light

Previous investigators revealed that white light-promoted leaflet opening in Samanea saman (Jacq) Merrill depends upon K⁺ uptake by extensor cells and efflux from flexor cells of the pulvinus, while dark-promoted closure depends upon K⁺ fluxes in the opposite directions. We now monitored H⁺ fluxes during pulvinar movement to test a model proposing coupled H⁺/K⁺ fluxes. H⁺ fluxes were monitored by measuring changes in the pH of a weakly buffered solution (initial pH = 5.5) bathing excised strips of extensor or flexor tissue. White light at hour 3 of the usual dark period promoted pulvinar opening, H⁺ efflux from extensor cells and uptake by flexor cells, while darkness at hours 2 to 4 of the usual light period promoted pulvinar closure, H⁺ uptake by extensor cells and efflux from flexor cells. The following conditions altered H⁺ fluxes during dark-promoted closure. (a) Light reversed the directions of the fluxes in both extensor and flexor cells. (b) Anoxia increased the rate of H⁺ uptake by extensor cells and promoted H⁺ uptake (rather than efflux) by flexor cells, consistent with an outwardly directed H⁺ pump. KCN showed similar effects initially, but they were transient. (c) Increase in external pH from 5.5 to 6.7 promoted H⁺ efflux (rather than uptake) by extensor cells and increased the rate of H⁺ efflux from flexor cells, presumably by decreasing the rate of inward diffusion. (d) Change in external K⁺ did not alter H⁺ fluxes by extensor cells, but removal of external K⁺ decreased the rate of H⁺ efflux from flexor cells by 70%. These observations support a model for coupled H⁺/K⁺ fluxes in pulvinar cells during light-and dark-promoted leaflet movements.     

STRUCTURAL AND ULTRASTRUCTURAL FEATURES OF CORTICAL CELLS IN MOTOR ORGANS OF SENSITIVE PLANTS

(1) The movements are only expressed in motor cells, regardless of the nature of the stimulation or its point of application. Therefore, these cells have structures capable of traducing the different stimulation-induced messages which are received in parts incapable of movement. (2) K⁺, Cl^- and Ca²⁺ are the major ions. Their fluxes have been followed during nyctinastic movements as well as during stimuli-induced movements. At the moment, the location and the role of these ions are being studied. (3) The movement results from the integrated activity of all (n) motor cells in the pulvini (i.e. n > 350 × 10³ in primary pulvini 3 mm long and 1·9 mm thick). (4) The motor cell is a full-grown cell whose osmotic activity induces turgor variations allowing foliar movements. (5) The motor cell is a highly differentiated cell, which, up to now, has never been able to dedifferentiate in order to produce callus. (6) The motor cell has original features in its apoplastic compartment (large meatuses, wall foldings, large periplasm with membranes) and in its symplastic compartment (double vacuolar apparatus, morphological polarity given by the tannin vacuole location near the nucleus, abundant mitochondria). (7) Its cytoskeleton includes microtubules, cytoplasmic and vacuolar fibrils (in particular in the tannin vacuole), and a wall with special properties. (8) The motor cell is supposed to contain contractile proteins, whose nature and location are being investigated. (9) The shape change of the motor cell is obvious after pulvinar bending. This change is probably associated with a volume change in several intracellular compartments (vacuole, mitochondria, vesicles). (10) At the cellular and subcellular level the same general features are observed in motor cells of non-seismonastic and of seismonastic species. Probably, functional differences depend upon differences occurring at the molecular level. (11) The motor cell is an interesting model for the study of the osmoregulation mechanism in plant cells, to test the effect of toxic products, in particular to find their optimal efficiency in the circadian cycle.     

A circadian rhythm in oxygen uptake by samanea pulvini

The rate of O₂ uptake by excised Samanea pulvini oscillates with a circadian rhythm during 52 hours of darkness. Rates of respiration increase during pulvinar opening and decrease prior to and during closure, consistent with the concept that opening requires a greater expenditure of energy. Externally supplied sucrose, necessary for perpetuation of the leaflet movement rhythm, has a small promotive effect on the rate of respiration.     

Effects of Colchicine, Vinblastine, Cytochalasin B and Phalloidin on the Seismonastic Movement of Mimosa pudica Leaf and on Motor Cell Ultrastructure

Fleurat-Lessard, P., Roblin, G., Bonmort, J. and Besse, C. 1988.Effects of colchicine, vinblastine, cytochalasin B and phalloidinon the seismonastic movement of Mimosa pudica leaf and on motorcell ultrastructure.—J. exp. Bot. 39: 209–221. Colchicine at 1 x 10^–3 mol dm^–3 does not affectthe seismonastic movement of Mimosa pudica leaves but disruptsmicrotubules in motor cells. Vinblastine at 5 x 10^–3 moldm^–3 does not affect this movement and partly disruptsmicrotubules. Vinblastine at 1 x 10^–4 mol dm^–3 alwaysdisrupts microtubules, even after a 12 h reversibility whenthe movement is restored. These drugs, applied at the same respectiveconcentrations, do not alter cytoplasmic and vacuolar fibrils.Cytochalasin B and phalloidin alter the seismonastic movementof Mimosa leaves when applied at concentrations of 1.25 x 10^–3and 2.4 x 10^–4 mol dm^–3 respectively. These drugs,used at the same respective concentrations, also affect themotor cell structure and, in particular, modify the arrangementand the structure of the fibrils but they do not destroy themicrotubules. These data suggest that microtubules are not directly involvedin the seismonastic reaction whereas fibrils, formed by thin(3.0 nm wide) filaments, may be implicated in this reaction. Key words: Colchicine, cytochalasin B, phalloidin, Mimosa pudica, motor cells, vinblastine     

A Hydrazine Coupled Cycling Assay Validates the Decrease in Redox Ratio under Starvation in Drosophila

A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD⁺/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP⁺ and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD⁺ and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation.     

EVIDENCE FOR CELL-SURFACE GLYCOSYLTRANSFERASES : Their Potential Role in Cellular Recognition

Intact chicken embryo neural retina cells have been shown to catalyze the transfer of galactose-¹⁴C from uridine diphosphate galactose (UDP-galactose) to endogenous acceptors of high molecular weight as well as to exogenous acceptors. Four lines of evidence indicate that the galactosyltransferases catalyzing these reactions are at least partly located on the outside surface of the plasma membrane: (a) there is no evidence for appreciable uptake of sugar-nucleotides by vertebrate cells nor did unlabeled galactose, galactose 1-phosphate, or UDP-glucose interfere with the radioactivity incorporated during the reaction; (b) the cells remained essentially intact during the course of the reaction; (c) there was insufficient galactosyltransferase activity in the cell supernatants to account for the incorporation of galactose-¹⁴C into cell pellets; and (d) the intact cells could transfer galactose to acceptors of 10⁶ daltons, and the product of this reaction was in the extracellular fluid. Appropriate galactosyl acceptors interfered with the adhesive specificity of neural retina cells; other compounds, which were not acceptors, had no effect. These results suggested that the transferase-acceptor complex may play a role in cellular recognition.     

Transport of the Auxin 2,4-Dichlorophenoxyacetic Acid Through Absiccion Zones, Pulvini, and Petioles of Phaseolus vulgaris

Measurements were made of the transport of 2,4-dichlorophenoxyacetic acid-¹⁴C (2,4-D) through segments cut from the region of the distal abscission zone in young and old primary leaves of Phaseolus vulgaris L. When old leaves were used basipetal transport of 2,4-D in segments including pulvinar tissue, abscission zone, and petiolar tissue was much less than in wholly petiolar segments. In both young and old plants, segments consisting entirely of pulvinar tissue transported 2,4-D basipetally at a velocity about half that in petiolar tissue. At both ages the flux of 2,4-D through pulvinar tissue was less than that through petiolar tissue. In segments from old leaves the flux through pulvinar tissue was much less than in young plants; the flux through petiolar tissue changed little with age. There was no change with age in the velocity of basipetal transport. The distribution of ¹⁴C along segments including the abscission zone showed no marked discontinuity. It was concluded that the pulvinus limited the basipetal movement of 2,4-D through segments from old leaves which included both pulvinar and petiolar tissue, but there was no evidence that the abscission zone itself was a barrier to auxin transport.     

Parallelized small-scale production of uniformly C-labeled cell extract for quantitative metabolome analysis

The need for quantitative intracellular metabolome information is central to modern applied biotechnology and systems biology. In most cases, sample preparation and metabolite analysis result in degradation of metabolites and signal suppression due to metabolite instability and matrix effects during LC–MS analysis. Therefore the application of uniformly (U) ¹³C-labeled cell extract as an internal standard has gained interest in recent years. In this study a multiple-step protocol has been developed for efficient preparation of U-¹³C-labeled Escherichia coli cell extracts in stirred-tank bioreactors on a milliliter scale with a minimal supply of costly ¹³C-labeled substrate. Significant reduction of fermentation medium salt concentration in the U-¹³C-labeled cell extract was achieved to reduce ion-suppression effects during mass-spectrometric analysis. Additionally, variation of reaction conditions in parallel-operated stirred-tank bioreactors on a milliliter scale enables the simultaneous preparation of U-¹³C-labeled cell extracts with varying metabolite concentrations, which is shown by an example of the labeled phosphoenolpyruvate level in E. coli.     

Protective immunity against lethal anaphylactic reaction in Toxoplasma gondii-infected mice by DNA vaccination with T. gondii-derived heat shock protein 70 gene

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce lethal anaphylactic reaction in T. gondii-infected mice through platelet-activating factor (PAF)-mediated, but not classical IgE-dependent, pathway via TLR4/MyD88 signal pathway. The effector cells generating PAF and causing T.g.HSP70-induced anaphylactic reaction were CD11b⁺ and CD11c⁺ cells, although the reaction was enhanced by marked IFN-γ production by CD11b⁺, CD11c⁺, CD4⁺ and CD8⁺ splenocytes. In the present study, the effects of T.g.HSP70 gene vaccine targeting peripheral dendritic cells were evaluated against T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice. C57BL/6 mice receiving T.g.HSP70 gene vaccine showed prolonged survival. Platelets of peripheral blood, which completely disappeared during the T.g.HSP70-induced anaphylactic reaction, were partially restored with the T.g.HSP70 gene vaccination. The T.g.HSP70-induced marked production of PAF and IFN-γ from splenocytes of infected mice during the T.g.HSP70-induced anaphylactic reaction was shown to decrease after the T.g.HSP70 gene vaccination. Thus, T.g.HSP70 gene vaccine induced protective immunity against T.g.HSP70-induced PAF-mediated lethal anaphylactic reaction in T. gondii-infected mice.     

The dynamic structure of the Escherichia coli cell envelope as probed by 15N nuclear magnetic resonance spectroscopy

Proton decoupled ¹⁵N NMR spectroscopy is shown to be a useful tool for probing the dynamic structure of the bacterial cell envelope. The proton decoupled ¹⁵N NMR spectra of Escherichia coli whole cells, cell envelopes and outer membranes were obtained and displayed resonances originating from protein side-chain groups, phosphatidylethanolamine, and peptidoglycan. Removal of phospholipids from the cell envelope resulted in a decrease in the motional freedom of peptidoglycan and cell envelope proteins. The mobility of the protein Arg side-chain groups is incresed in the absence of peptidoglycan. These data provide insights into the effect of supramolecular organization on the dynamic structure of the E. coli cell envelope.     

The Role of Na+/K+-ATPase during Chick Skeletal Myogenesis

The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na⁺/K⁺-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na⁺/K⁺-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM) and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na⁺/K⁺-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na⁺/K⁺-ATPase.     

The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi: Activation of a Plasmalemma K/H Exchange Mechanism

Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K⁺ which was accompanied by an equimolar net influx of H⁺. These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K⁺ during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K⁺ and H⁺ fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K⁺ concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K⁺ efflux and H⁺ influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K⁺/H⁺ exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death.     

The Effects of Manganese (II) But Not Nickel (II) Ions on Enterococcus hirae Cell Growth, Redox Potential Decrease, and Proton-Coupled Membrane Transport

Enterococcus hirae grow well under anaerobic conditions by fermenting glucose, accompanied with the decrease of oxidation–reduction potential (E _h) from positive values to negative ones. It was shown that heavy metals—copper and iron ions—affect E. hirae growth and alter E _h and proton-potassium ions fluxes through the cell membrane. The aim of this study was to establish the effects of manganese (II) ions on bacterial growth within the concentration range of 0.01–1 mM and compare with nickel (II) ions’ effect. The presence of Mn²⁺ during E. hirae ATCC9790 growth had significant effects: The lag phase duration decreased while the specific growth rate was increased; decrease in E _h was shifted. In contrast, no visible changes in bacterial growth and E _h were observed in the case of Ni²⁺. The effects of these ions on proton-potassium ions fluxes through the cell membrane were estimated in the presence and absence of N,N′-dicyclohexylcarbodiimide (DCCD), inhibitor of the F_oF₁ ATPase. Stronger effect of Mn²⁺ on H⁺–K⁺ exchange was detected in the presence of DCCD that can be explained by a possible complex formation between these substances and its direct influence on membrane transport proteins.     

The Catalytic Activity of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 3 Is Required To Sustain CD4+ CD8+ Thymocyte Survival

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4⁺ CD8⁺ (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3^−/− thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.     

In Vivo Fate Imaging of Intracerebral Stem Cell Grafts in Mouse Brain

We generated transgenic human neural stem cells (hNSCs) stably expressing the reporter genes Luciferase for bioluminescence imaging (BLI) and GFP for fluorescence imaging, for multimodal imaging investigations. These transgenic hNSCs were further labeled with a clinically approved perfluoropolyether to perform parallel ¹⁹F MRI studies. In vitro validation demonstrated normal cell proliferation and differentiation of the transgenic and additionally labeled hNSCs, closely the same as the wild type cell line, making them suitable for in vivo application. Labeled and unlabeled transgenic hNSCs were implanted into the striatum of mouse brain. The time profile of their cell fate after intracerebral grafting was monitored during nine days following implantation with our multimodal imaging approach, assessing both functional and anatomical condition. The ¹⁹F MRI demarcated the graft location and permitted to estimate the cell number in the graft. BLI showed a pronounce cell loss during this monitoring period, indicated by the decrease of the viability signal. The in vivo obtained cell fate results were further validated and confirmed by immunohistochemistry. We could show that the surviving cells of the graft continued to differentiate into early neurons, while the severe cell loss could be explained by an inflammatory reaction to the graft, showing the graft being surrounded by activated microglia and macrophages. These results are different from earlier cell survival studies of our group where we had implanted the identical cells into the same mouse strain but in the cortex and not in the striatum. The cortical transplanted cells did not show any loss in viability but only pronounced and continuous neuronal differentiation.     

Study of the reaction of kidney collecting duct principal cells to hypotonic shock. Experiment and mathematical model

The reaction of rat kidney collecting duct principal cells to hypotonic shock was studied. The changes in cell relative volume were measured using fluorescent dye calcein, and a mathematical model based on our experimental results was developed. It was shown that regulatory volume decrease is mainly provided by significant release of osmolytes from the cell and decrease of the plasma membrane water permeability. Using our model, we calculated the membrane water permeability and found it to decrease from 2 · 10⁻¹ to 2 · 10⁻² cm/s. We conclude that for effective RVD to occur, a dramatic increase in the membrane permeability to K⁺, Cl⁻ and organic anions is necessary.     

Potassium Flux and Leaf Movement in Samanea saman : I. Rhythmic Movement

Samanea leaflets usually open in white light and fold together when darkened, but also open and dose with a circadian rhythm during prolonged darkness. Leaflet movement results from differential changes in the turgor and shape of motor cells on opposite sides of the pulvinus; extensor cells expand during opening and shrink during closure, while flexor cells shrink during opening and expand during closure but change shape more than size. Potassium in both open and closed pulvini is about 0.4 N. Flame photometric and electron microprobe analyses reveal that rhythmic and light-regulated postassium flux is the basis for pulvinar turgor movements. Rhythmic potassium flux during darkness in motor cells in the extensor region involves alternating predominance of inwardly directed ion pumps and leakage outward through diffusion channels, each lasting ca 12 h. White light affects the system by activating outwardly directed K⁺ pumps in motor cells in the flexor region.