Rice bundle sheath cell shape is regulated by the timing of light exposure during leaf development.

Plant leaves contain multiple cell types which achieve distinct characteristics whilst still coordinating development within the leaf. The bundle sheath possesses larger individual cells and lower chloroplast content than the adjacent mesophyll, but how this morphology is achieved remains unknown. To identify regulatory mechanisms determining bundle sheath cell morphology we tested the effects of perturbing environmental (light) and endogenous signals (hormones) during leaf development of Oryza sativa (rice). Total chloroplast area in bundle sheath cells was found to increase with cell size as in the mesophyll but did not maintain a 'set-point' relationship, with the longest bundle sheath cells demonstrating the lowest chloroplast content. Application of exogenous cytokinin and gibberellin significantly altered the relationship between cell size and chloroplast biosynthesis in the bundle sheath, increasing chloroplast content of the longest cells. Delayed exposure to light reduced the mean length of bundle sheath cells but increased corresponding leaf length, whereas premature light reduced final leaf length but did not affect bundle sheath cells. This suggests that the plant hormones cytokinin and gibberellin are regulators of the bundle sheath cell-chloroplast relationship and that final bundle sheath length may potentially be affected by light-mediated control of exit from the cell cycle.

Effects and Related Mechanisms of the Senolytic Agent ABT-263 on the Survival of Irradiated A549 and Ca9-22 Cancer Cells.

Senolytic agents eliminate senescent cells and are expected to reduce senescent cell-mediated adverse effects in cancer therapy. However, the effects of senolytic agents on the survival of irradiated cancer cells remain unknown. Here, the effects of the senolytic agent ABT-263 on the survival of irradiated A549 and Ca9-22 cancer cells were investigated. ABT-263 was added to the culture medium after irradiation. SA-ß-gal activity and cell size, which are hallmarks of cell senescence, were evaluated using a flow cytometer. The colony-forming assay and annexin V staining were performed to test cell survival. We first confirmed that radiation increased the proportion of cells with high SA-ß-gal activity and that ABT-263 decreased it. Of note, ABT-263 decreased the survival of irradiated cancer cells and increased the proportion of radiation-induced annexin V+ cells. Furthermore, the caspase inhibitor suppressed the ABT-263-induced decrease in the survival of irradiated cells. Intriguingly, ABT-263 decreased the proportion of SA-ß-gal low-activity/large cells in the irradiated A549 cells, which was recovered by the caspase inhibitor. Together, these findings suggest that populations maintaining the ability to proliferate existed among the irradiated cancer cells showing senescence-related features and that ABT-263 eliminated the population, which led to decreased survival of irradiated cancer cells.

Low-level laser therapy as a modifier of erythrocytes morphokinetic parameters in hyperadrenalinemia.

Low-level laser therapy (LLLT) is widely used in clinical practice for treatment of various pathologies. It is assumed that LLLT impact on microcirculation is among the mechanisms underlying its therapeutic effect. The microcirculation disorder is observed in the pathogenesis of any inflammatory process and is significantly influenced by red blood cells (RBCs). On this point, studying the RBCs morphology under the influence of LLLT on alterated organism is of scientific interest and practical importance. The aim of the present study was to analyze the LLLT effect on morphokinetic parameters of RBCs in hyperadrenalinemia. The LLLT effect was analyzed on rats intraperitoneally injected with adrenaline hydrochloride solution (0.1 mg/kg). As the comparison groups, the effects of LLLT, adrenaline, or saline injection as well as the parameters of intact animals were studied. LLLT was applied on the occipital region of rats for 10 min. The light irradiation with pulse frequency 415 Hz at 890 nm wavelength and average power density in the plane of the output window at 193 µW/cm2 was used. The dynamics of morphological characteristics of RBCs was studied by phase interference microscopy; the RBC electrophoretic mobility was tested by microelectrophoresis technique; photometric analyses of the RBCs amount, hemoglobin content, and osmotic fragility were performed. The adrenaline injection resulted in a significant increase in the amount of RBC pathological forms and a decrease in discocytes and normocytes by more than 50%. An increase in the optical density of RBC phase portraits, a decline in osmotic resistance, and electronegativity of RBC membranes and a reduction of their number in peripheral blood were also registered. The revealed effects persisted for 1 week after the adrenaline administration. LLLT did not significantly impact on the RBC parameters 1 h after adrenaline injection. However, a day later, LLLT reduced the severity of the adrenaline effect on RBSs, which was manifested in a decreased amount of the pathological forms of RBCs, restored RBC phase portraits, higher electrophoretic mobility and osmotic resistance, and RBSs amount in peripheral blood restored up to the level of intact animals. We suppose that the mechanism of LLLT action is realized both at cellular level through the laser radiation effect on RBC membranes, and at systemic level through the activation of stress-realizing systems of the organism with subsequent limitation of inflammatory response.

Impact of X-irradiation on microglia.

Irradiation is widely used to treat brain tumors, and also to create bone marrow (BM) chimeras. BM chimeras are widely used to dissect functions and origin of microglia and blood-derived mononuclear cells under homeostatic or pathological conditions. This is facilitated by the fact that microglia survive irradiation and are thus regarded radio-resistant. In this study, we tested whether microglia are indeed radio-resistant and looked for potential mechanisms that might explain this phenomenon. We analyzed the radio-resistance of microglia independently of their physiological brain environment compared to other mononuclear cells from spleen and brain after X-irradiation with 7 Gy or 30 Gy. Furthermore, we investigated long-term effects of X-irradiation on microglia using organotypic hippocampal slice cultures (OHSCs). We found a significant higher survival rate of isolated microglia 4 hr after X-irradiation with 30 Gy accompanied by a decreased proliferation rate. Investigations of apoptosis-related genes revealed no regulation of a specific antiapoptotic pathway but ataxia telangiectasia mutated (ATM), a DNA-repair-related gene, was significantly upregulated in isolated microglia 4 hr after 30 Gy. Irradiation of OHSCs with 7 and 30 Gy revealed a highly and significantly decreased cell number, morphological changes and an increase in migration velocity of microglia. Furthermore, cell loss, increased soma size and process length of microglia was also found in BM chimeras irradiated with 9.5 Gy 5 weeks after irradiation. Here, we present new evidence implying that microglia are not a homogeneous population of radio-resistant cells and report on long-term alterations of microglia that survived irradiation.

Reactivation of Latent Epstein-Barr Virus: A Comparison after Exposure to Gamma, Proton, Carbon, and Iron Radiation.

Among the many stressors astronauts are exposed to during spaceflight, cosmic radiation may lead to various serious health effects. Specifically, space radiation may contribute to decreased immunity, which has been documented in astronauts during short- and long-duration missions, as evidenced by several changes in cellular immunity and plasma cytokine levels. Reactivation of latent herpes viruses, either directly from radiation of latently infected cells and/or from perturbation of the immune system, may result in disease in astronauts. Epstein‒Barr virus (EBV) is one of the eight human herpes viruses known to infect more than 90% of human adults and persists for the life of the host without normally causing adverse effects. Reactivation of several latent viruses in astronauts is well documented, although the mechanism of reactivation is not well understood. We studied the effect of four different types of radiation, (1) 137Cs gamma rays, (2) 150-MeV protons, (3) 600 MeV/n carbon ions, and (4) 600 MeV/n iron ions on the activation of lytic gene transcription and of reactivation of EBV in a latently infected cell line (Akata) at doses of 0.1, 0.5, 1.0, and 2.0 Gy. The data showed that for all doses used in this study, lytic gene transcription was induced and median viral loads were significantly higher for all types of radiation than in corresponding control samples, with the increases detected as early as four days post-exposure and generally tapering off at later time points. The viability and size of EBV-infected Akata cells were highly variable and exhibited approximately the same trend in time for all radiation types at 0.1, 0.5, 1.0, and 2.0 Gy. This work shows that reactivation of viruses can occur due to the effect of different types of radiation on latently infected cells in the absence of changes or cytokines produced in the immune system. In general, gamma rays are more effective than protons, carbon ions, and iron ions in inducing latent virus reactivation, though these high-energy particles did induce more sustained and later reactivation of EBV lytic gene transcription. These findings also challenge the common relative biological effectiveness concept that is often used in radiobiology for other end points.

Combined effects of electromagnetic field and low-level laser increase proliferation and alter the morphology of human adipose tissue-derived mesenchymal stem cells.

In recent years, electromagnetic field (EMF) and low-level laser (LLL) have been found to affect various biological processes, the growth and proliferation of cells, and especially that of stem cells. The aim of this study was to investigate the effects of EMF and LLL on proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and thus to examine the impact of these therapeutic physical modalities on stem cell engraftment. hAT-MSCs were isolated from subcutaneous adipose tissue of six persons ranging in age from 21 to 56 years. EMF was applied for a period of 7 days, once a day for 30 min, via a magnetic cushion surface at a frequency of 50 Hz and an intensity of 3 mT. LLL was applied also for 7 days, once a day for 5 min, at radiation energies of 3 J/cm2, with a wavelength of 808 nm, power output of 200 mW, and power density of 0.2 W/cm2. Nonexposed cells (control) were cultivated under the same culture conditions. Seven days after treatment, the cells were examined for cell viability, proliferation, and morphology. We found that after 7 days, the number of EMF-treated hAT-MSCs was significantly higher than the number of the untreated cells, LLL-treated hAT-MSCs were more numerous than EMF-treated cells, and hAT-MSCs that were treated with the combination of EMF and LLL were the most numerous. EMF and/or LLL treatment did not significantly affect hAT-MSC viability by itself. Changes in cell morphology were also observed, in terms of an increase in cell surface area and fractal dimension in hAT-MSCs treated with EMF and the combination of EMF and LLL. In conclusion, EMF and/or LLL treatment accelerated the proliferation of hAT-MSCs without compromising their viability, and therefore, they may be used in stem cell tissue engineering.

Non-Ionotropic NMDA Receptor Signaling Drives Activity-Induced Dendritic Spine Shrinkage.

The elimination of dendritic spine synapses is a critical step in the refinement of neuronal circuits during development of the cerebral cortex. Several studies have shown that activity-induced shrinkage and retraction of dendritic spines depend on activation of the NMDA-type glutamate receptor (NMDAR), which leads to influx of extracellular calcium ions and activation of calcium-dependent phosphatases that modify regulators of the spine cytoskeleton, suggesting that influx of extracellular calcium ions drives spine shrinkage. Intriguingly, a recent report revealed a novel non-ionotropic function of the NMDAR in the regulation of synaptic strength, which relies on glutamate binding but is independent of ion flux through the receptor (Nabavi et al., 2013). Here, we tested whether non-ionotropic NMDAR signaling could also play a role in driving structural plasticity of dendritic spines. Using two-photon glutamate uncaging and time-lapse imaging of rat hippocampal CA1 neurons, we show that low-frequency glutamatergic stimulation results in shrinkage of dendritic spines even in the presence of the NMDAR d-serine/glycine binding site antagonist 7-chlorokynurenic acid (7CK), which fully blocks NMDAR-mediated currents and Ca(2+) transients. Notably, application of 7CK or MK-801 also converts spine enlargement resulting from a high-frequency uncaging stimulus into spine shrinkage, demonstrating that strong Ca(2+) influx through the NMDAR normally overcomes a non-ionotropic shrinkage signal to drive spine growth. Our results support a model in which NMDAR signaling, independent of ion flux, drives structural shrinkage at spiny synapses. SIGNIFICANCE STATEMENT: Dendritic spine elimination is vital for the refinement of neural circuits during development and has been linked to improvements in behavioral performance in the adult. Spine shrinkage and elimination have been widely accepted to depend on Ca(2+) influx through NMDA-type glutamate receptors (NMDARs) in conjunction with long-term depression (LTD) of synaptic strength. Here, we use two-photon glutamate uncaging and time-lapse imaging to show that non-ionotropic NMDAR signaling can drive shrinkage of dendritic spines, independent of NMDAR-mediated Ca(2+) influx. Signaling through p38 MAPK was required for this activity-dependent spine shrinkage. Our results provide fundamental new insights into the signaling mechanisms that support experience-dependent changes in brain structure.

Induced movements of giant vesicles by millimeter wave radiation.

Our previous study of interaction between low intensity radiation at 53.37GHz and cell-size system - such as giant vesicles - indicated that a vectorial movement of vesicles was induced. This effect among others, i.e. elongation, induced diffusion of fluorescent dye di-8-ANEPPS, and increased attractions between vesicles was attributed to the action of the field on charged and dipolar residues located at the membrane-water interface. In an attempt to improve the understanding on how millimeter wave radiation (MMW) can induce this movement we report here a real time evaluation of changes induced on the movement of giant vesicles. Direct optical observations of vesicles subjected to irradiation enabled the monitoring in real time of the response of vesicles. Changes of the direction of vesicle movement are demonstrated, which occur only during irradiation with a "switch on" of the effect. This MMW-induced effect was observed at a larger extent on giant vesicles prepared with negatively charged phospholipids. The monitoring of induced-by-irradiation temperature variation and numerical dosimetry indicate that the observed effects in vesicle movement cannot be attributed to local heating.

Functional diversity of photobiological traits within the genus Symbiodinium appears to be governed by the interaction of cell size with cladal designation.

Dinoflagellates of the genus Symbiodinium express broad diversity in both genetic identity (phylogeny) and photosynthetic function to presumably optimize ecological success across extreme light environments; however, whether differences in the primary photobiological characteristics that govern photosynthetic optimization are ultimately a function of phylogeny is entirely unresolved. We applied a novel fast repetition rate fluorometry approach to screen genetically distinct Symbiodinium types (n = 18) spanning five clades (A-D, F) for potential phylogenetic trends in factors modulating light absorption (effective cross-section, reaction center content) and utilization (photochemical vs dynamic nonphotochemical quenching; [1 - C] vs [1 - Q]) by photosystem II (PSII). The variability of PSII light absorption was independent of phylogenetic designation, but closely correlated with cell size across types, whereas PSII light utilization intriguingly followed one of three characteristic patterns: (1) similar reliance on [1 - C] and [1 - Q] or (2) preferential reliance on [1 - C] (mostly A, B types) vs (3) preferential reliance on [1 - Q] (mostly C, D, F types), and thus generally consistent with cladal designation. Our functional trait-based approach shows, for the first time, how Symbiodinium photosynthetic function is governed by the interplay between phylogenetically dependent and independent traits, and is potentially a means to reconcile complex biogeographic patterns of Symbiodinium phylogenetic diversity in nature.

Meniscal allograft sterilisation: effect on biomechanical and histological properties.

Sterilisation of allografts are a crucial step in ensuring safety and viability. Current sterilisation standards such as 25 kGy gamma irradiation (γ) can have adverse effects on the ultrastructure and biomechanical properties of allograft tissue. Supercritical CO2 (SCCO2) technology, represents an improved sterilisation process that potentially preserves tissue properties. This study aimed to test the effect of SCCO2 sterilisation on the biomechanical and histological properties of the meniscus and compare this to the current standard of γ. Thirty-two 18-month old ovine menisci were randomly assigned into three groups for sterilisation (SCCO2, γ and control). After treatment, biomechanical indentation testing (stiffness and stress relaxation) or histological analysis [percentage of void, cells and extracellular matrix (ECM) per slide] was undertaken. Both SCCO2 and gamma groups displayed an increase in stiffness and stress relaxation as compared to control, however, this difference was lesser in samples treated with SCCO2. No significant histological quantitative differences were detected between SCCO2 and control specimens. Gamma-treated samples demonstrated a significant increase in void and decrease in ECM. Interestingly, both treatment groups demonstrated a decreasing mean void and increasing ECM percentage when analysed from outer to inner zones. No significant differences were detected in all-endpoints when analysed by section. SCCO2 sterilisation represents a potential feasible alternative to existing sterilization techniques such as γ.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.

Effects of low-intensity ultrasound on gramicidin D-induced erythrocyte edema.

OBJECTIVES: To determine whether low-intensity ultrasound (US) can reduce red blood cell (RBC) edema and, if so, whether the US activity is associated with aquaporin 1 (AQP-1), a water channel in the cell membrane. METHODS: Red blood cell edema was induced by gramicidin D treatment at 40 ng/mL for 20 minutes and evaluated by a hematocrit assay. Low-intensity continuous wave US at 1 MHz was applied to RBCs for the last 10 minutes of gramicidin D treatment. To determine whether US activity was associated with AQP-1, RBCs were treated with 40 µM mercuric chloride (HgCl(2)), an AQP-1 inhibitor, for 20 minutes at the time of gramicidin D treatment. Posttreatment morphologic changes in RBCs were observed by actin staining with phalloidin. RESULTS: Red blood cell edema increased significantly with gramicidin D at 20 (1.8%), 40 (6.7%), 60 (16.7%), and 80 (11.3%) ng/mL, reaching a peak at 60 ng/mL, compared to the control group (20 ng/mL, P = .019; 40, 60, and 80 ng/mL, P < .001). No significant RBC hemolysis was observed in any group. Edema induced by gramicidin D at 40 ng/mL was significantly reduced by US at 30 (3.4%; P = .003), 70 (4.4%; P = .001), and 100 (2.9%; P = .001) mW/cm(2). Subsequent experiments showed that edema reduction by US ranged from 7% to 10%. Cotreatment with HgCl(2) partially reversed the US effect and showed a significantly different level of edema compared to gramicidin D-alone and US-cotreated groups (P = .001). These results were confirmed by microscopic observation of RBC morphologic changes. CONCLUSIONS: Low-intensity US could reduce gramicidin D-induced RBC edema, and its effect appeared to at least partly involve regulation of AQP-1 activity. These results suggest that low-intensity US can be used as an alternative treatment to control edema and related disorders.

[The epididymal adipose tissue of mice after nanosecond pulse-periodic microwave irradiation].

The effect of pulse-periodic microwave radiation (PPMR) of the pulse repetition frequency of 8-25 pulseper second, the peak power density of 1500 W/cm2 on the epididymal adipose tissue of micewas investigated. The effect was assessed by the changes in the fat mass and size of the irradiated adipocytes. It was found that the fat mass and size distribution of adipocytes are affected by irradiation. The effects depend on the pulse repetition frequency and intensity of exposure.

Short-term hyperosmolality pretreatment on cells can reduce the radiosensitivity via RVI and Akt1 activation.

BACKGROUND/AIMS: The ionizing radiation (IR) has been applied in clinical treatment for many years and the radiosensitivity is crucial to the treatment. Radiosensitivity of cells is subjected to many environmental factors, such as hypoxia and temperature. Hyperosmolality as a common environmental factor has been demonstrated to be associated with survival and apoptosis of cells in many studies. Thus we investigated the influence of hyperosmolality on cells radiosensitivity. METHODS: We examined the viability and surviving fraction of L-O2 cells of irradiated L-O2 cells, and detected the effect on AHH-1 cells by flow cytometry, in order to investigate the effect of short-term hyperosmolality pretreatment on cells radiosensitivity. Comet assay was used to assess the DNA strand breaks. Then the detection of Akt1 by western blot and the process of regulatory volume increase by CYSY-TT were involved in the mechanism. RESULT: We demonstrated that a short-term hyperosmolality pretreatment on cells could reduce their radiosensitivity. Further research indicated that the short-term hypertonic condition could induce regulatory volume increase (RVI), which activated Akt1 and degenerated the IκB-α. This process was associated with reduced cells radiosensitivity. Finally, we used the flufenamic acid (FFA), a blocker to cation channels (HICCs) to inhabit RVI and consequently inhabit the protective effect of hyperosmolality on irradiated cells. CONCLUSION: a short-term hyperosmolality pretreatment could reduce the cells radiosensitivity by RVI and following activation of Akt1.

Individual maize chromosomes in the C(3) plant oat can increase bundle sheath cell size and vein density.

C(4) photosynthesis has evolved in at least 66 lineages within the angiosperms and involves alterations to the biochemistry, cell biology, and development of leaves. The characteristic "Kranz" anatomy of most C(4) leaves was discovered in the 1890s, but the genetic basis of these traits remains poorly defined. Oat × maize addition lines allow the effects of individual maize (Zea mays; C(4)) chromosomes to be investigated in an oat (Avena sativa; C(3)) genetic background. Here, we have determined the extent to which maize chromosomes can introduce C(4) characteristics into oat and have associated any C(4)-like changes with specific maize chromosomes. While there is no indication of a simultaneous change to C(4) biochemistry, leaf anatomy, and ultrastructure in any of the oat × maize addition lines, the C(3) oat leaf can be modified at multiple levels. Maize genes encoding phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the 2'-oxoglutarate/malate transporter are expressed in oat and generate transcripts of the correct size. Three maize chromosomes independently cause increases in vein density, and maize chromosome 3 results in larger bundle sheath cells with increased cell wall lipid deposition in oat leaves. These data provide proof of principle that aspects of C(4) biology could be integrated into leaves of C(3) crops.

Manipulation of cell volume and membrane pore comparison following single cell permeabilization with 60- and 600-ns electric pulses.

Intense nanosecond-duration electric pulses (nsEP) open stable nanopores in the cell membrane, followed by cell volume changes due to water uptake or expulsion, as regulated by the osmolality balance of pore-impermeable solutes inside and outside the cell. The size of pores opened by either fifty 60-ns EP (~13 kV/cm) or five, 600-ns EP (~6 kV/cm) in GH3 cells was estimated by isoosmotic replacement of bath NaCl with polyethylene glycols and sugars. Such replacement reduced cell swelling or resulted in transient or sustained cell shrinking in response to EP. depending on the availability of pores permeable to the test solute. Unexpectedly, solute substitutions showed that for the same integral area of pores opened by 60- and 600-ns treatments (as estimated by cell volume changes), the pore sizes were similar. However, the 600-ns exposure triggered significantly higher cell uptake of propidium. We concluded that 600-ns EP opened a greater number of larger (propidium-permeable pores), but the fraction of the larger pores in the entire pore population was insufficient to contribute to cell volume changes. For both the 60- and 600-ns exposures, cell volume changes were determined by pores smaller than 0.9 nm in diameter; however, the diameter increased with increasing the nsEP intensity.

Structural assessment of the impact of environmental constraints on Arabidopsis thaliana leaf growth: a 3D approach.

Light and soil water content affect leaf surface area expansion through modifications in epidermal cell numbers and area, while effects on leaf thickness and mesophyll cell volumes are far less documented. Here, three-dimensional imaging was applied in a study of Arabidopsis thaliana leaf growth to determine leaf thickness and the cellular organization of mesophyll tissues under moderate soil water deficit and two cumulative light conditions. In contrast to surface area, thickness was highly conserved in response to water deficit under both low and high cumulative light regimes. Unlike epidermal and palisade mesophyll tissues, no reductions in cell number were observed in the spongy mesophyll; cells had rather changed in volume and shape. Furthermore, leaf features of a selection of genotypes affected in leaf functioning were analysed. The low-starch mutant pgm had very thick leaves because of unusually large palisade mesophyll cells, together with high levels of photosynthesis and stomatal conductance. By means of an open stomata mutant and a 9-cis-epoxycarotenoid dioxygenase overexpressor, it was shown that stomatal conductance does not necessarily have a major impact on leaf dimensions and cellular organization, pointing to additional mechanisms for the control of CO(2) diffusion under high and low stomatal conductance, respectively.

Cell expansion and microtubule behavior in ray floret petals of Gerbera hybrida: responses to light and gibberellic acid.

It is still unclear how light and gibberellins are integrated to regulate petal size. Here, we report that light improves both the length and the width of the ray floret petals in G. hybrid, but GA(3) promotes only the petal length. It is also revealed that the control of the petal size by light and GA(3) depends on modulating the cell size, which is governed by the behavior of cortical microtubule.Light and gibberellins are important regulators of plant organ growth. However, little is known about their roles in petal size determination. Here, we report how light and gibberellic acid (GA(3)) signals are integrated to regulate the ray floret (Rf) size in Gerbera hybrida. The inflorescences of G. hybrida at stages 1.5 were cultivated in vitro for 9 d followed by the determination of the Rf petal size. Results demonstrated that the light signal significantly enhanced both the length and the width of Rf petals, but GA(3) promoted only the petal length. Moreover, GA(3) displayed a synergistic positive effect on the length but an antagonistic effect on the width with the light signal. Measurements of the petal cells revealed that the cell size, not the cell number, exhibited a dominant contribution to the petal size in response to light and GA(3) signals. Furthermore, light and GA(3) signals not only induced an obvious reorientation of cortical microtubules (MTs) into transverse arrays but also promoted the recovery of the MT lengths in petal cells following oryzalin (an MT depolymerizing agent) treatment. Importantly, disruption of the MT lengths and arrays by oryzalin could inhibit the cell expansion and the petal enlargement induced by light or/and GA(3) signals. Taken together, it is concluded that the control of the petal size by light and GA(3) signals mainly depends on modulating the cell size and, moreover, the organization of the cortical MTs plays a crucial role in the control of the cell size and hence the Rf petal growth.

Electroporation-induced changes in normal immature rat myoblasts (H9C2).

Application of a high electric field causes an electric shock to the heart. This is utilized in defibrillation to reestablish normal contraction rhythms during dangerous arrhythmias or in cardiac arrest. If shock-induced transmembrane potentials are large enough, they can cause tissue destruction due to irreversible electroporation (EP). Also electrochemotherapy of nearby tissues may have an adverse effect on the heart. Herein, we present experimental data on effects of electroporation in culture of cardiac cells (H9C2). The electric field was applied in short pulses of 25-3250 V/cm, 50 µs each. The viability of cells was tested by MTT assay after 24 hours. For detection of DNA fragmentation, associated with apoptosis, alkaline and neutral comet assays were performed after EP. Additionally phase contrast images of cells obtained directly after EP were analyzed. Although cell images indicated disruption of cell membranes after EP with high intensities, only a few percent of apoptotic cells and no necrotic effects in the cell nucleus could be observed in comet assay tests performed 2 hours post EP. MTT viability test showed that pulse intensities above 375 V/cm are destructive for myocytes viability.

Cell bathing medium as a target for non thermal effect of millimeter waves.

Non thermal (NT) effect of direct radiation 4 Hz-modulated 90-160 GHz of Millimeter Waves (MMW) and preliminary MMW-treated physiological solution (PS) influence were studied on snail isolated neuron, rat's brain tissue hydration and skin penetration. It was shown that the 4 Hz-modulated low intensity 90-160 GHz MMW direct radiation and MMW-treated PS leads to on single neuron shrinkage, skin and brain tissue dehydration. On the basis of obtained data it was suggested that the cell bathing aqua medium serve as a target through which the NT effect of MMW on cell hydration is realized. The MMW-induced brain tissue dehydration can considering as consequence of MMW-induced skin water structural changes leading to unknown messenger formation able to modulate the brain cell hydration. The extrasensitivity of cell hydration to low intensity of MMW radiation allow to recommend cell hydration as a cellular marker for estimation of the NT biological effect of MMW on cells and organisms.