Chara buckellii, a Euryhaline Charophyte from an Unusual Saline Environment : III. Time Course of Turgor Regulation

The time course of turgor regulation of the euryhaline giant-celled alga, Chara buckellii, is presented. Isolated intermodal cells were challenged by increasing or decreasing the external osmotic pressure by 150 milliosmoles per kilogram with all ions in the media or by dilution, respectively. Regulation following hypotonic stress was complete within 48 hours whereas regulation following hypertonic stress required between 96 and 144 hours. The change in internal osmotic pressure could be entirely accounted for by changes in vacuolar KCl in response to hypotonic stress, but this ion pair only accounted for 45% of the change in response to hypertonic stress. The membrane potential of C. buckellii is normally hyperpolarized with respect to the equilibrium potential for K⁺ (E_K). The membrane depolarized to a level close to E_K in response to hypotonic treatment and this was accompanied by a transient increase in membrane conductance. In response to hypertonic stress, the membrane hyperpolarized transiently, then repolarized to a level close to the control. This was accompanied by a temporary decrease in membrane conductance. The data are discussed with respect to the ecological significance of the time course and ion transport mechanisms during turgor regulation.     

渗透压对血管内皮细胞中NO合成酶活性的影响及其机制研究

目的：研究非等渗压浓度对血管内皮细胞NO合成酶活性的影响,并探索其发生机制。方法：使血管内皮细胞暴露于低渗（205mOsm)或高渗透压（410mOsm)培养液,用Griess法测定NO合成酶（NOS）活性,以Northern blot ting观测细胞iNOS和eNOS基因表达的变化。结果： 非等渗压浓度可使血管内皮细胞中NOS活性显著升高。细胞NOS活性变化具有明显的时间效应规律,低渗透压浓度效应产生的效应早于高渗透压浓度,且低渗透压浓度的影响较高渗透压浓度更为明显。Dexamethasone对这种非等渗透压诱导的NOS活性没有明显作用,给予cycloheximide,不影响非等渗压诱导的这种差异。Nothern blot分析表明：非等渗压浓度不诱导iNOS基因表达,而使eNOSmRNA表达增加。结论：非等渗透压浓度诱导血管内皮细胞NOS活性升高,eNOS基因表达增强是其主要机制之一。     

Persistent Symmetry Frustration in Pollen Tubes

Pollen tubes are extremely rapidly growing plant cells whose morphogenesis is determined by spatial gradients in the biochemical composition of the cell wall. We investigate the hypothesis (MP) that the distribution of the local mechanical properties of the wall, corresponding to the change of the radial symmetry along the axial direction, may lead to growth oscillations in pollen tubes. We claim that the experimentally observed oscillations originate from the symmetry change at the transition zone, where both intervening symmetries (cylindrical and spherical) meet. The characteristic oscillations between resonating symmetries at a given (constant) turgor pressure and a gradient of wall material constants may be identified with the observed growth-cycles in pollen tubes.     

渗透压对痢疾志贺菌水通道蛋白glpF基因表达的影响

目的探讨细菌水-甘油通道蛋白（GlpF）的生理功能及其对生长繁殖的影响。方法将用简并PCR发现表达水通道蛋白glpF基因的痢疾志贺菌接种于不同渗透压的液体培养基和添加了GlpF功能抑制剂的相应培养基中,培养不同时间后取培养液检测其生长繁殖量,RT-PCR分析其GlpF的表达。结果痢疾志贺菌GlpF的表达随培养基渗透压的改变而变化,在低渗培养基中的表达低于在等渗环境中;在高渗透压的培养基中,其表达显著高于在等渗培养基中。在加入Hg2＋抑制剂抑制GlpF的表达后,在低渗培养基中,未明显影响细菌的生长繁殖,但在较高渗透压的培养基中,细菌的繁殖量显著少于在未加Hg2＋抑制剂的同样渗透压培养基中。结论在非等渗环境中,细菌GlpF的表达对细菌细胞内外水分的调节,维持胞内环境稳定起到重要作用,尤其在高渗透压环境中更为明显。     

Transport Systems of Ventricaria ventricosa: Asymmetry of the Hyper- and Hypotonic Regulation Mechanisms

Hyper- and hypotonic stresses elicit apparently symmetrical responses in the alga Ventricaria. With hypertonic stress, membrane potential difference (PD) between the vacuole and the external medium becomes more positive, conductance at positive PDs (Gmpos) increases and KCl is actively taken up to increase turgor. With hypotonic stress, the membrane PD becomes more negative, conductance at negative PDs (Gmneg) increases and KCl is lost to decrease turgor. We used inhibitors that affect active transport to determine whether agents that inhibit the K(+) pump and hypertonic regulation also inhibit hypotonic regulatory responses. Cells whose turgor pressure was held low by the pressure probe (turgor-clamped) exhibited the same response as cells challenged by hyperosmotic medium, although the response was maintained longer than in osmotically challenged cells, which regulate turgor. The role of active K(+) transport was confirmed by the effects of decreased light, dichlorophenyldimethyl urea and diethylstilbestrol, which induced a uniformly low conductance (quiet state). Cells clamped to high turgor exhibited the same response as cells challenged by hypo-osmotic medium, but the response was similarly transient, making effects of inhibitors hard to determine. Unlike clamped cells, cells challenged by hypo-osmotic medium responded to inhibitors with rapid, transient, negative-going PDs, with decreased Gmneg and increased Gmpos (linearized I-V), achieving the quiet state as PD recovered. These changes are different from those exerted on the pump state, indicating that different transport systems are responsible for turgor regulation in the two cases.     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.     

Optimization of osmotic shock process variables for enhancement of the release of periplasmic interferon-α2b from Escherichia coli using response surface method

The osmotic shock process for the release of periplasmic recombinant human interferon-α2b from Escherichia coli was optimized using response surface method (RSM). The process parameters such as pH, buffer concentration and sucrose concentration in hypertonic solution, cell concentration to hypertonic solution, contact time of cells with hypertonic solution, temperature of hypertonic solution, cell concentration to hypotonic solution, contact time of cells with hypotonic solution and temperature of hypotonic solution were initially screened using Plackett Burman design. Further optimization was carried out using central composite design (one of the design in RSM) for sucrose concentration in hypertonic solution as well as cell concentration to hypertonic and hypotonic solutions. The optimal cell concentration was 0.05 g/mL in hypertonic solution and 0.2 g/mL in hypotonic solution. The use of hypertonic solution containing 18% sucrose with a combination of 100 mM Tris and 2.5 mM EDTA buffer (pH 8.0 and 25 °C) and cold water (4 °C) as a hypotonic solution gave the optimum release of interferon-α2b. Increased product concentration in the final solution resulted from the optimized process would reduce the downstream steps during purification. The concept of reuse of hypertonic solution was also demonstrated.     

Pressure–Induced Cell Wall Instability and Growth Oscillations in Pollen Tubes

In the seed plants, the pollen tube is a cellular extension that serves as a conduit through which male gametes are transported to complete fertilization of the egg cell. It consists of a single elongated cell which exhibits characteristic oscillations in growth rate until it finally bursts, completing its function. The mechanism behind the periodic character of the growth has not been fully understood. In this paper we show that the mechanism of pressure – induced symmetry frustration occurring in the wall at the transition-perimeter between the cylindrical and approximately hemispherical parts of the growing pollen tube, together with the addition of cell wall material, is sufficient to release and sustain mechanical self-oscillations and cell extension. At the transition zone, where symmetry frustration occurs and one cannot distinguish either of the involved symmetries, a kind of ‘superposition state’ appears where either single or both symmetry(ies) can be realized by the system. We anticipate that testifiable predictions made by the model () may deliver, after calibration, a new tool to estimate turgor pressure from oscillation frequency of the periodically growing cell. Since the mechanical principles apply to all turgor regulated walled cells including those of plant, fungal and bacterial origin, the relevance of this work is not limited to the case of the pollen tube.     

Plasmolysis and cell wall deposition in wheat root hairs under osmotic stress

We analysed cell wall formation in rapidly growing root hairs of Triticum aestivum under reduced turgor pressure by application of iso- and hypertonic mannitol solutions. Our experimental series revealed an osmotic value of wheat root hairs of 150 mOsm. In higher concentrations (200–650 mOsm), exocytosis of wall material and its deposition, as well as callose synthesis, still occurred, but the elongation of root hairs was stopped. Even after strong plasmolysis when the protoplast retreated from the cell wall, deposits of wall components were observed. Labelling with DiOC₆(3) and FM1-43 revealed numerous Hechtian strands that spanned the plasmolytic space. Interestingly, the Hechtian strands also led towards the very tip of the root hair suggesting strong anchoring sites that are readily incorporated into the new cell wall. Long-term treatments of over 24 h in mannitol solutions (150–450 mOsm) resulted in reduced growth and concentration-dependent shortening of root hairs. However, the formation of new root hairs does occur in all concentrations used. This reflects the extraordinary potential of wheat root cells to adapt to environmental stress situations.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

An aeropalynological survey in the city of Van,a high altitudinal region,East Anatolia-Turkey

Pollen concentrations in the atmosphere of Van city has been monitored for two consecutive years (2010–2011). This was the first detailed aeropalynological study for the elevated East Anatolia Region of Turkey. The sampling was performed by Hirst-type volumetric sampler, and pollen grains of 35 taxa were identified. The main pollen producers of the pollen flora were recorded as: Poaceae (20.94 %), Cupressaceae (10.53 %), Fraxinus (8.56 %), Chenopodiaceae/Amaranthaceae (7.77 %), Populus (7.75 %), Quercus (6.70 %), Platanus (6.68 %), Morus (5.57 %), Plantago (3.03 %). The pollen spectrum reflected the floristic diversity of the region, and the highest pollen concentration was recorded in April. There were a great percentage of allergenic taxa found in the city atmosphere, otherwise many of them scored under threshold values for risk of pollinosis. Statistical analyses were performed for correlating daily pollen concentrations of dominated pollen types concurrent with the data of meteorological parameters in MPS periods and number of significant correlations found. In addition, comparing 2-year data in terms of pollen concentrations and meteorological factors in MPS durations, many variables were found explanatory and concordant with the data. MPS starting dates of many plant taxa were found nearly a month later compared with western sites and lower altitudes of the country as well as Mediterranean countries; this case is mostly thought the ecological factors of the study area which directly affects the plant growth about the timing.     

Sorption of phthalocyanine dye by erythrocyte membranes in buffered solutions of different tonicities

The sorption of nonpenetrating vital phthalocyanine dye--Heliogen Blue (HB) into the cells was studied on the human red cells incubated in hypertonic, isotonic and hypotonic solutions. The alteration in the outer membrane surface was detected using the monomer-dimer ratio (M/D) deduced from the absorbtion spectra of the sorbed dye, and the concentration (C) of the dye in membrane. The decrease in M/D and C was found in hypotonic and hypertonic conditions, resp. The increase in the number of binding sites caused by conformational changes in membrane is discussed.     

Applied osmotic loading for promoting development of engineered cartilage

This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300 mOsM) or isotonic (330 mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400 mOsM media from day 0 to day 49 yielded a Young's modulus of 513±89 kPa and GAG content of 7.39±0.52%ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young's modulus, reaching 487±187 kPa and 6.77±0.54%ww (GAG) for the 400 mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle–van't Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.     

Glycine modulates membrane potential,cell volume,and phagocytosis in murine microglia

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1–4). In BV-2 cells, glycine (5 mM) led to a rapid Na⁺-dependent depolarization of membrane potential (V _mem). Furthermore, glycine increased CV by about 9 %. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13 %. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na⁺ with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.     

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.     

Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from ≈80% “stage 1” to ≈55% “stage 2”. When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K_m ≈ 9 moles per cubic meter; V_max ≈ 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k_i ≈ 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V_max from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.     

The Mechanics of Injury to Isolated Protoplasts following Osmotic Contraction and Expansion

Micro-osmotic manipulation was used to determine the influence of osmotic contraction on the expansion potential of individual protoplasts isolated from rye (Secale cereale L. cv Puma) leaves. For protoplasts isolated from leaves of nonacclimated plants (NA protoplasts), osmotic contraction in sufficiently hypertonic solutions (>1.53 osmolal) predisposed the protoplasts to lysis during osmotic expansion when they were returned to isotonic conditions (0.53 osmolal). In contrast, for protoplasts isolated from leaves of cold acclimated plants (ACC protoplasts), osmotic contraction in either 2.6 or 4.0 osmolal solutions was readily reversible. Following osmotic contraction, the resting tension (γ_r) of NA protoplasts was similar to that determined for protoplasts in isotonic solutions (i.e. 110 ± 22 micronewtons per meter). In contrast, γ_r of ACC protoplasts decreased from 164 ± 27 micronewtons per meter in isotonic solutions to values close to zero in hypertonic solutions. Following expansion in hypotonic solutions, γ_r's of both NA and ACC protoplasts were similar for area expansions over the range of 1.3 to 1.6. Following osmotic contraction and reexpansion of NA protoplasts, hysteresis was observed in the relationship between γ_r and surface area—with higher values of γ_r at a given surface area. In contrast, no hysteresis was observed in this relationship for ACC protoplasts. Direct measurements of plasma membrane tension (γ) during osmotic expansion of NA protoplasts from hypertonic solutions (1.53 osmolal) revealed that γ increased rapidly after small increments in surface area, and lysis occurred over a range of 1.2 to 8 millinewtons per meter. During osmotic expansion of ACC protoplasts from hypertonic solutions (2.6 osmolal), there was little increase in γ until after the isotonic surface area was exceeded. These results are discussed in relation to the differences in the behavior of the plasma membrane of NA and ACC protoplasts during osmotic contraction (i.e. endocytotic vesiculation versus exocytotic extrusion) and provide a mechanistic interpretation to account for the differential sensitivity of NA and ACC protoplasts to osmotic expansion from hypertonic solutions.     

Maturation timing of stamens and pistils in the dioecious plant Silene latifolia

The dioecious plant Silene latifolia depends on nocturnal insects for pollination. To increase the chance of cross-pollination, pollen grains seem to be released and stigmas seem to be receptive simultaneously at night. We divided the floral development of S. latifolia into 1–20 stages, and determined the timetables of male and female function. The corolla of both male and female flowers opens at sunset (1900 hours) and closes at sunrise (0900 hours). To investigate the period of the reproductive phase of male and female function, we measured the germination rate on a pollen medium and the pollen germination rate on stigma during the period when stamens and stigmas were viable in the timetable. Male flowers had early- and late-maturing stamens that had the highest pollen viability, germination rate and pollen tube growth at midnight (0000 hours) at 1 day after flowering (DAF) and 0000 hours at 2 DAF. In contrast, female flowers maintained a germination rate of nearly 100 % from 1800 hours at 1 DAF to 1200 hours at 3 DAF. These results suggested that S. latifolia transferred the matured pollen grains from male flowers to female flowers only at night.     

Purifying nuclei.

Nuclear isolation methods exist since over 50 years and even today new procedures and amendments of standard methods are published. They can be classified into nonaqueous and aqueous methods. The latter can be subdivided into isotonic, hypertonic and hypotonic systems. In most cases the aqueous isolation renders nuclei closer to their physiological status in the cell. A standard method for the hypotonic isolation of nuclei is presented and the methodology of nuclear isolation is discussed.     

Understanding pollen tube growth: the hydrodynamic model versus the cell wall model

Scientific progress stimulates the evolution of models used to understand and conceptualize biological behaviors. The widely accepted cell wall model of pollen tube growth explains stochastic growth of the apical pectin wall, but fails to explain the mechanism driving oscillations in growth and cell signaling. Recent advances led to the formulation of a new hydrodynamic model that explains the mechanism that drives both stochastic and oscillatory growth, as well as oscillations in cell signaling and ion fluxes. A critical analysis of evidence that has been used to challenge the validity of the hydrodynamic model yields new information on turgor pressure, cell mechanical properties and nonlinear dynamics in pollen tube growth. These results may have broader significance for plant cell growth.