Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant, Petasites japonicus, via the Induction of Murine Dendritic Cell Maturation.

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8+ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.

A coarse-grained multiscale model to simulate morphological changes of food-plant tissues undergoing drying.

Numerical modelling has emerged as a powerful and effective tool to study various dynamic behaviours of biological matter. Such numerical modelling tools have contributed to the optimisations of food drying parameters leading to higher quality end-products in the field of food engineering. In this context, one of the most recent developments is the meshfree-based numerical models, which have demonstrated enhanced capabilities to model cellular deformations during drying, providing many benefits compared to conventional grid-based modelling approaches. However, the potential extension of this method for simulating bulk level tissues has been a challenge due to the increased requirement for higher computational time and resources. As a solution for this, by incorporating meshfree features, a novel coarse-grained multiscale numerical model is proposed in this work to predict bulk level (macroscale) deformations of food-plant tissues during drying. Accordingly, realistic simulation of morphological changes of apple tissues can now be performed with just 2% of the previous computational time in microscale and macroscale simulations can also be conducted. Compared to contemporary multiscale models, this modelling approach provides more convenient computational implementation as well. Thus, this novel approach can be recommended for efficiently and accurately simulating morphological changes of cellular materials undergoing drying processes, while confirming its potential future expansion to efficiently and accurately predict morphological changes of heterogeneous plant tissues in different spatial scales.

Application of a coupled smoothed particle hydrodynamics (SPH) and coarse-grained (CG) numerical modelling approach to study three-dimensional (3-D) deformations of single cells of different food-plant materials during drying.

Numerical modelling has gained popularity in many science and engineering streams due to the economic feasibility and advanced analytical features compared to conventional experimental and theoretical models. Food drying is one of the areas where numerical modelling is increasingly applied to improve drying process performance and product quality. This investigation applies a three dimensional (3-D) Smoothed Particle Hydrodynamics (SPH) and Coarse-Grained (CG) numerical approach to predict the morphological changes of different categories of food-plant cells such as apple, grape, potato and carrot during drying. To validate the model predictions, experimental findings from in-house experimental procedures (for apple) and sources of literature (for grape, potato and carrot) have been utilised. The subsequent comaprison indicate that the model predictions demonstrate a reasonable agreement with the experimental findings, both qualitatively and quantitatively. In this numerical model, a higher computational accuracy has been maintained by limiting the consistency error below 1% for all four cell types. The proposed meshfree-based approach is well-equipped to predict the morphological changes of plant cellular structure over a wide range of moisture contents (10% to 100% dry basis). Compared to the previous 2-D meshfree-based models developed for plant cell drying, the proposed model can draw more useful insights on the morphological behaviour due to the 3-D nature of the model. In addition, the proposed computational modelling approach has a high potential to be used as a comprehensive tool in many other tissue morphology related investigations.

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes.

Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H+ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes: the 14-3-3 content in the membranes increased 2- to 3-fold, while the H+-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H+-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H+ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins.

Analysis of pigment accumulation heterogeneity in plant cell population by image-processing system.

Plant-cultured cells are often highly heterogeneous in secondary metabolite productivity. The industrial application for large-scale metabolite production requires establishment of a stable high-producing cell line. In this study, image analysis of the individual cell is investigated as a method for evaluation of a heterogeneous cell population, and compared with the conventional method of estimation, which is based on average-cell productivity. Among strawberry cells producing anthocyanins, cells with a wide-range of pigment concentration were observed and maximum anthocyanin content was 10 times higher than the average value. In addition, a change of the frequency distribution was revealed in batch cultivation.

Enhanced post-thaw viability of cryopreserved cells by oxygenated perfluorocarbon or Pluronic F-68.

Viability and growth following cryopreservation have been examined following (i) culture of Oryza sativa cv. Taipei 309 (Japonica rice) in medium overlaying oxygenated perfluorocarbon (PFC) liquid (Flutec PP6), and (ii) culture of two cvs. of Japonica rice (Taipei 309, Tarom), and Lolium multiflorum in media with Pluronic F-68 (0.01-0.2% w/v). The mean viability of cv. Taipei 309 rice cells following 4 days in medium overlaying oxygenated Flutec (0.45 +/- 0.07; n = 20) was significantly (P < 0.05) greater than control (0.35 +/- 0.08). Cell viability of cv. Tarom was increased by 400% (P < 0.001) with 0.1% (w/v) Pluronic F-68 in the recovery medium. Mean cell viability was also elevated 3-fold (P < 0.001) and 2-fold (P < 0.05) with 0.2% (w/v) and 0.01% (w/v) Pluronic, respectively. A 2-fold increase (P < 0.001) in viability occurred for Taipei 309 cells exposed to 0.01% (w/v) Pluronic F-68; that of Lolium cells was increased by 31% (P < 0.01) under similar conditions. Pluronic F-68 (0.01% w/v) also stimulated biomass, by 29-32%, in both cvs. Taipei 309 and Lolium at 30 days post-thawing.

Studies on selection and characterization of a stress-tolerant sugarcane cell line.

A sugarcane (Saccharum sinensis Roxb.) cell line R932 resistant to growth inhibition by the proline analogue hydroxyproline was selected. R932 showed greater tolerance to PEG and low temperature stress than the donor. The line R932 showed larger accumulation of proline (x3.2) than the sensitive donor. In vitro enzymic analysis of gamma-glutamyl kinase involved in the proline biosynthesis showed that the enzyme in the R932 was less sensitive to inhibition by 50-mM exogenous proline than that in the donor. The results suggested that the change in gamma-glutamyl kinase properties might lead to excessive accumulation of proline, and the elevated proline contents might in turn lead to an increase in tolerance to environmental stress.

Cloning of the BamHI repeat from Amaranthus and study of its methylation in genomic DNA during dedifferentiation.

A 670 bp BamHI repeat was isolated from the genomic DNA of Amaranthus paniculatus and was cloned in pUC19 vector (pABH3). The level of methylation of total genomic DNA and the BamHI repeat therein was studied by employing methylation sensitive enzymes to digest genomic DNA isolated from whole seedlings, hypocotyl, callus, and suspension culture of Amaranthus. The restriction pattern indicated double methylation of the target site CCGG. Methylation pattern of both the total genomic DNA and the cloned repeat was found to be similar during dedifferentiation indicating that there may not be a general relationship between hypomethylation and dedifferentiation.

Spatial and temporal gene expression patterns occur during corm development.

We investigated gene expression patterns that occur during taro corm development. Two-dimensional gel electrophoresis identified several different prevalent proteins that accumulate during corm development. Microsequencing studies indicated that some of these proteins are related to taste-modifying proteins, such as curculin and miraculin, and proteins found in other storage organs, such as sporamin and the Kunitz trypsin inhibitor. A curculin-encoding cDNA clone, designated as TC1, was identified that corresponds to a highly prevalent 1-kb corm mRNA. The TC1 mRNA accumulates during corm development, is more prevalent in corm apical than basal regions, and is either absent, or present at low concentrations, in other vegetative organs such as the leaf and root. In situ hybridization experiments showed that the TC1 mRNA is highly concentrated in corm storage parenchyma cells and is absent, or present in reduced concentrations, in other corm cells and tissues. Our results show that corm development is associated with the differentiation of specialized cells and tissues, and that these differentiation events are coupled with the temporal and spatial expression of corm-specific genes.

[Effect of cultured Polyscias cells on the activity of components of the protein-synthesizing system of rabbit liver]. / Vliianie kul'tiviruemykh kletok polistsiasa na aktivnost' komponentov beloksinteziruiushchei sistemy pecheni krolikov.

The authors studied teh effect of cultured Polyscias filcifolia Bailey cells on protein biosynthesis in an acellular system obtained from the liver of rabbits in experimental myocardial ischemia. It was found that the preparation normalizes the values of protein biosynthesis, the duration of the average polypeptide chain synthesis, and the activity of aminoacyl-tRNA synthetases.

Rapid control of purity for the cytoplasm of male-sterile seed stocks by means of a dot hybridization assay.

To produce hybrids, one member of the parental line is genetically made male-sterile. This male-sterile trait is encoded by mitochondria so that it is maternally inherited. Consequently, the progeny of a male-sterile plant is fully sterile. Nevertheless, during the handling of cytoplasmic male-sterile seed stocks, some mixture with seeds of the maintainer lines can occur. Up to the present time, the only way to check the homogeneity of the cytoplasmic male-sterile seed stock was to grow the plants until flowering time. We have developed a method which can be used immediately after the harvest, allowing us to check samples from both sunflower and sugar beet. We used the mitochondrial plasmid, present only in the maintainer lines, as a probe for the total nucleic acids prepared from the cytoplasmic male-sterile seed stocks which might be contaminated. The signals compared to those of samples artificially contaminated allow us to measure as few as one male-fertile seed in 1000 seeds in a rapid and accurate manner.

[A cytological study of plants growing under exposure to different radiation levels]. / Tsitologicheskoe issledovanie rastenii, proizrastavshikh pod vozdeistviem raznykh urovnei radiatsii.

Genetically significant consequences of emergency at the Chernobyl Atomic Power Plant have been studied on wheat and rye plants. Plants grown in the 30-km zone of the plant after its emergency are determined to have high frequency of chromosome aberrations reaching 2.666% in rye and 1.075-2.572% in wheat and 2.235-3.187% as dependent on the variety, biotype (awnless, semi-awned and awned) and places of occurrence. These levels of aberrations are 5.34, 2.42-5.78 and 4.07-6.71 times higher than their frequency in control plants (0.499, 0.444 and 0.475%, respectively).

Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. I. Relationship between DNA and total protein content and size of nucleus, nucleolus and cell.

There was a linear relation between an increase in DNA content and size of nuclei, nucleoli and cells in callus and proembryos (Theobroma cacao L.). In callus the increase of DNA content was accompanied by proportional increase in nuclear size whereas in proembryos the increase in nuclear size did not match the increasing amount of DNA. The stimulation of embryogenesis by 10(-2) mg/l 2,4-D was associated with increase in nuclear and nucleolar size and with decrease in cell sizes. Inhibition of embryogenesis by 1.0 mg/l 2,4-D+10% coconut water did not change nuclear size, but increased cell size in relation to the control. The process of embryo formation was accompanied by changes in relationship between nuclear, nucleolar and cell size and the total (DNFB-stained) proteins content. In callus as well as in proembryo the increase in total protein content in nucleus was not equivalent to the increasing sizes of nuclei which leads to the decrease in nuclear protein concentration. Similar situation was observed for nucleoli. Differences were found in the concentration of cytoplasmic proteins between the callus and proembryo cells. The stimulation of embryogenesis by low concentration of 2,4-D resulted in decrease in concentration of total proteins in nuclei and nucleoli and the increase in cytoplasm.

Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. II. Relation between basic protein content and size of nucleus, nucleolus and cell.

Embryo formation from callus of Theobroma cacao L. was associated with the changes in relationship between nuclear, nucleolar and cell sizes and the content of basic proteins (FG-FCF-stained). Together with the increase in nuclear size of callus and proembryo cells the increase in the amount of nuclear basic proteins was found. In the callus cells the increase in nucleolar protein content exceeded that in nucleolus size, which led to the rise in basic protein concentration in the nucleolus. However, in the early stage of embryogenesis the increase in protein content was not so marked as that in callus, which indicated that embryogenesis involved a decrease in concentration of nucleolar basic proteins. Differences between callus and proembryo cells were also observed in the concentration of cytoplasmic proteins. The increase in size of callus cells was the same as the increasing amount of cytoplasmic proteins. In proembryos a significant increase in cell size was accompanied by only slight changes in cytoplasmic proteins. The stimulation of embryogenesis by 2,4-D resulted in an increase of nuclear concentration of basic proteins in proembryos. The intensification of embryogenesis involved the decrease of the concentration of nucleolar proteins together with the increase in concentration of basic cytoplasmic proteins.

Effects of 60-Hz electric fields on cellular elongation and radial expansion growth in cucurbit roots.

Serial longitudinal and transverse sections were prepared from roots of Cucumis sativus and Cucurbita maxima that had been exposed/sham-exposed to 60-Hz electric fields for 0-2 days. Field exposures were selected to produce a 10-20% or a 70-80% growth inhibition in whole roots of both species. Cortical cell length and diameter were measured using a microscope and eyepiece micrometer; measurements were conducted "blind." In both species, inhibition of cellular elongation was associated with exposure to electric fields (EF). Cellular radial expansion was apparently unaffected by exposure to electric fields. The diameters of radially unexpanded or fully expanded C. sativus cortical cells were about 25-30% smaller than those of comparable cells in C. maxima roots. Previous studies of the relationship between rates of root growth and applied EF strength showed that the response thresholds of C. sativus and C. maxima differed by a similar relative amount. These results are consistent with the postulate that EF-induced effects in roots are elicited by induced transmembrane potentials.

Cell wall elastic constitutive laws and stress-strain behavior of plant vegetative tissue.

The appropriateness of several elastic constitutive laws for apple and potato cell walls is tested using uniform cell inflation data. Whole-tissue stress-strain behavior under uniaxial loading is predicted from an analysis of the compression of a conglomerate of cells in a simple arrangement.

Relative resistance of and delayed toxicity in undifferentiated plant cells to methyl mercury.

Meristematic cells of carrot (Daucus carota L., Ca68-10) and lettuce (Lactuca sativa L., LE-67) cultured in 71-V medium (with 0.15 microgram/ml 2, 4-D) were given one initial dose (0.0001, 0.001, or 0.01 microgram/ml) of methyl mercury (MeHg) at 0 hr, cultured for 288 hr. and then subcultured for 4 weeks. In another experiment, MeHg was added in four daily doses (0.05, 0.10, 0.50, 1.0, 2.5, or 5.0 micrograms/ml) starting at 72 hr for 120 hr and then subcultured for 168 hr. The resulting 50% growth reduction (Gr50) levels were high--1570 and 540 micrograms Hg/g dry wt for carrot and lettuce, respectively. Methyl mercury was taken up completely by cells but retention decreased at higher MeHg concentrations in the medium. The resistance of undifferentiated cells of both species to MeHg was markedly greater than that observed in multicellular plants. Cells derived from Hg-treated cultures did not recover their growth potential when subcultured in Hg-free medium. The Gr50 levels were lowered during successive subculturing in both carrot (50 micrograms Hg/g) and lettuce (10 micrograms Hg/g), indicating increased sensitivity to residual Hg in cells. This effect depended on the initial Hg concentration and on the number of cell divisions. At low MeHg concentrations, it was observed in cells 12 generations after one initial dose of 0.01 microgram/ml MeHg.

Nuclear and nucleolar protein during the cell cycle in differentiating Pisum sativum vascular tissue.

Squash preparations of Pisum sativum fourth internode tissue were stained with a combined Feulgen and dinitrofluorobenzene (DNFB) procedure. Nuclei from differentiating xylem vessel elements, phloem sieve tube elements and phloem fibres were measured for their DNA and protein contents with a Zeiss scanning cytophotometer linked to an interactive computer system. Nuclei were examined from both slowly growing and more rapidly growing internodes. A computer program was constructed to calculate nuclear protein alone as well as the ratio of DNFB (protein) to Feulgen (DNA) staining in each 0.5 X 0.5 micron measuring point. Nuclei were assigned to each of ten interphase fractions based on DNA content, nuclear area and percent condensed chromatin. There was a slight increase of nuclear protein during G1, a gradual increase in S, and a continued, often sharper, rise as G2 proceeded. In all three cell types, there was, on the average, a higher protein content throughout interphase in nuclei from the more rapidly growing internodes than from the slower growing ones. A population of fibre nuclei designated G0, however, differed from phloem and xylem G0 nuclei in the pattern of protein change. The nucleolar protein/DNA ratios of xylem nuclei increased in G1, showed no significant change in S, but increased thereafter.