Alkaloids of callus tissues and redifferentiated plantlets in the papaveraceae

The callus tissues from 11 representative species of the Papaveraceae and the redifferentiated plantlets from four species were successfully derived and maintained. The alkaloids in the callus tissues and redifferentiated plantlets were examined in comparison with those of the original plants. All the callus tissues are similar in their alkaloid chemistry and contain benzophenanthridine, protopine and aporphine type alkaloids. By contrast, the plantlets have a more specific alkaloid pattern, being similar in content to the original plants.     

Involvement of Proteasome-Ubiquitin System in Wound-Signaling in Tobacco Plants

Treatment of wounded tobacco leaves with a proteasome-specificinhibitor lactacystin resulted in; (1) enhanced expression ofproteasome subunit genes, (2) suppressed expression of proteasomeactivity, (3) induced accumulation of Ub-conjugated proteins,and (4) suppressed expression of wound-inducible genes, indicatingthat the Ub-proteasome system is involved in wound-signalingin plants. ³ Present address: Department of Plant Breeding, National AgricultureResearch Center, 3-1-1 Kannondai, Tsukuba, Ibaraki, 305-8666Japan.     

Viability and Virulence of Experimentally Stressed Nonculturable Salmonella typhimurium

Maintenance of pathogenicity of viable but nonculturable Salmonella typhimurium cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities.     

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.Like several readily culturable pathogenic bacterial species, Salmonella enterica has been shown to enter into a viable-but-nonculturable (VBNC) state in response to environmental stresses (25, 33). In this state, cells display integrity and activities but escape detection by conventional culture-based monitoring (24). The physiological significance of this phenotype is unclear: some authors have proposed that it is part of an adaptive response aimed at long-term survival under adverse conditions (22, 32); others argue that it is a consequence of stochastic cellular deterioration and that VBNC cells are on their way to death (4, 10, 12, 23). In any case, the existence of VBNC pathogens is a public health concern since they may constitute unrecognized sources of infection if they retain their pathogenicity.To date, many studies have addressed the ability of VBNC pathogens to remain infectious, but the conclusions of some investigators are conflicting (15, 36). In vitro experiments have shown that VBNC cells of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Oranienburg can recover their culturability (13, 27, 30, 31). This phenomenon, called resuscitation, confirms that at least some VBNC cells ultimately remain able to multiply and are therefore potentially infectious. On the other hand, most in vivo studies ruled out the ability of S. Typhimurium VBNC cells to initiate infection in mice and chicken or to resuscitate during their passage in the animal gut (6, 17, 34, 35). However, one study reported evidence of the maintenance of pathogenicity by VBNC cells of S. Oranienburg in a model of morphine-immunosuppressed mice (1). An assumption could explain these apparently opposite results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state (8, 19, 26). Experiments intended for testing the ability of VBNC cells to retain their pathogenicity cannot be fully conclusive if the inocula still contain culturable cells. Therefore, all previously published animal experiments with S. Typhimurium were conducted on populations with VBNC-cell/culturable-cell ratios around 10,000:1. Such populations were obtained after strong exposure to stress, either under intense stressing factors for a short period (e.g., germicidal UV-C for 2 min [6]) or under mild stressing factors for a long period (e.g., starvation for a minimum of 1 week [35]). In such populations, most VBNC cells could exceed the stage where they are still infectious, and the negative outcomes of infection studies could actually reflect their inability to specifically address the fraction of recent VBNC cells.A Radioselectan density gradient centrifugation technique was shown to fractionate stationary-phase populations of Escherichia coli into two subpopulations (10, 12, 18). Interestingly, the VBNC cells formed during a 48-h E. coli culture were specifically recovered in the high-density (HD) subpopulation (12). This technique thus gives the opportunity to increase the VBNC-cell/culturable-cell ratio without increasing exposure to stress and, consequently, to work with a larger proportion of cells having recently entered the VBNC state.Here, this technique was used to discriminate different stationary-phase S. Typhimurium subpopulations. We further investigated the invasiveness of these cell subpopulations by using both gene expression assays of invasion genes and in vitro invasion tests. Thus, the aim of this study was to assess the invasiveness of the cell subpopulations in accordance with their cellular states.     

Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis,Lucinidae) and Its Symbiont Population

The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.The entire marine Lucinidae family, found in a wide range of sulfidic habitats, lives in association with chemoautotrophic sulfide-oxidizing bacterial symbionts, generally hosted in the gills of the bivalve. Lucinids are usually found in shallow water, such as intertidal mud or seagrasses (4, 53), in deeper water, e.g., Bathyaustriella thionipta (30), and in deep oceans at a 2,000-m depth, i.e., Lucinoma kazani (21, 55). The chemoautotrophic endosymbionts involved in such relationships are always localized inside specialized cells called bacteriocytes, and they have been found in several genera of the Lucinidae family, such as Codakia (4, 28), Loripes (39, 43), Lucina, and Lucinoma (17). Sulfur granules inside the symbiont cytoplasm have been demonstrated in most of the investigated species. The intracellular symbionts take energy from the oxidation of reduced sulfur compounds (27, 56, 59) and synthesize organic molecules by CO₂ fixation in a Calvin-Benson cycle, translocated to the host (18). This relationship between the host and its symbionts represents the autotrophic pathway for host nutrition (27). It has also been suggested that in symbiotic bivalves, intracellular digestion of the symbionts may be a nutrient source for the host, based on studies of hydrothermal vent and shallow water bivalves (6, 26, 39).The relative importance of the autotrophic versus heterotrophic nutritional pathway can be estimated by measuring the carbon isotope (δ-¹³C) ratios in the host tissue. Measuring this δ-¹³C ratio on a wide range of invertebrates suggested that bivalves, including members of the Lucinidae, that live in reduced sediment may obtain a significant proportion of their organic carbon from chemoautotrophic endosymbionts (4, 10, 51, 52, 57). This suggestion was in agreement with the reduced functional digestive system previously described for the Lucinidae family (2, 53). Structural and morphological studies of gills of a few lucinids (belonging to the genera Lucina and Lucinoma) strongly suggested that symbionts play an important role in host nutrition since they occupy about 30% of the gill tissue and produce most of the host energy (17). Nevertheless, an alternative pathway for feeding, i.e., heterotrophic particulate feeding, could occur in some of the lucinid bivalves, since diatoms were found in the stomach of some lucinids (57). Duplessis et al. (22) showed that particulate feeding could be an important part of the nutritional strategy in symbiont-bearing Lucinoma, as opposed to the anatomical features that gave the impression that this bivalve relied only on symbiont nutrition.In natural habitats, chemoautotrophic bivalves live at the interface between an anoxic sulfide-generating zone and water column oxygenated sediment. However, even if they are not close to a vent, these symbiotic organisms often have to deal with environments that are periodically depleted of oxygen (5, 12) and with extremely low sulfide concentration (13, 15, 16). These natural environmental variations lead to annual and seasonal changes in the δ-¹³C ratio, as observed for some thyasirid species (13, 14). This δ-¹³C ratio variation may be assumed to correspond to the capability of the host to rely on both autotrophic and heterotrophic pathways, and the preponderance of one pathway versus the other in the mixotrophic diet has been considered to be the way in which these organisms deal with changes in the chemical composition of their environment.Apart from the decrease in symbiont abundance suggested by transmission electron microscopy (TEM) analysis and a decrease in sulfur and protein content in the gill tissue of thyasirids (20, 38, 40), little is known about the physiological status of these symbionts and the changes undergone by the symbiotic population of starved bivalves. A previous study of the population was carried out under natural conditions with Codakia orbicularis, a chemoautotrophic bivalve. This tropical bivalve lives in shallow-water sediment among the roots of seagrasses (Thalassia testudinum) (1). Like all lucinids studied so far, it is associated with sulfur-oxidizing symbionts (4, 27, 28) containing elemental sulfur in their cytoplasm (42). The bacterial symbiont of C. orbicularis, environmentally transmitted to the host (31), belongs to a single taxonomic group (Gammaproteobacteria) (25) and is shared by several other tropical lucinids (24, 25, 34, 35). Only a few data are available on the physiology of this symbiont. It was characterized by the presence of Rubisco and ATP sulfurylase enzymes and a δ-¹³C ratio typical of chemoautotrophic bivalves (4). Unlike other related thyasirids tested for nitrate respiration even under oxygenated conditions (37), the symbionts of C. orbicularis use oxygen as the primary electron acceptor (23). Initial investigations of the population of Codakia orbicularis'' symbiont revealed that the symbiotic population hosted by freshly collected individuals contained a high proportion of large bacterial cells containing multiple copies of their genome, typical of actively growing cells, despite the absence of dividing cells (11). It was assumed that the host could maintain a pure culture of the symbiont inside the bacteriocytes by regulating the entry and growth of newly recruited symbionts from sediment and probably regulating symbiont densities by host digestion (11).This study was undertaken to investigate the dynamics of the symbiotic population hosted by C. orbicularis under experimental conditions based on long-term starvation of bivalves, i.e., incubated without planktonic food. We set out the ultrastructural, structural, and physiological changes that occurred in the symbiont population by examining the host gill sections using TEM and fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH). A purified fraction of gill endosymbionts was analyzed by flow cytometry (FCM) to investigate the nucleic acid content and cell size of symbionts and by using the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) method and epifluorescence microscopy to detect the respiratory activity of symbionts. The modifications induced by host starvation in the symbiotic population are described for the period of long-term starvation.     

Jasmonate-dependent plant defense restricts thrips performance and preference

Background  

The western flower thrips (Frankliniella occidentalis [Pergande]) is one of the most important insect herbivores of cultivated plants. However, no pesticide provides complete control of this species, and insecticide resistance has emerged around the world. We previously reported the important role of jasmonate (JA) in the plant's immediate response to thrips feeding by using an Arabidopsis leaf disc system. In this study, as the first step toward practical use of JA in thrips control, we analyzed the effect of JA-regulated Arabidopsis defense at the whole plant level on thrips behavior and life cycle at the population level over an extended period. We also studied the effectiveness of JA-regulated plant defense on thrips damage in Chinese cabbage (Brassica rapa subsp. pekinensis).     

Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E. coli

S-RNase is a style-specific ribonuclease which is associated with gametophytic self-incompatibility. An expression vector of a fusion protein of Pyrus pyrifolia(Japanese pear) S3-RNase with glutathione-S-transferase (GST) was constructed and transformed into E. coli. Using this system, the fusion protein, GST-S3-RNase, was expressed as an active form and can be used for screening pollen S-gene product(s).     

Temperature and external electric field influence in membrane permeability of Babesia infected erythrocytes

1. Modification of erythrocyte membrane properties infected by Babesia canis was studied using the effect of electric pulses of short duration. 2. This process induces the formation of pores in the membrane and the releasing of hemoglobin and other cytoplasmic proteins into the external medium. 3. The rate of molecular permeation across the electrically perforated membranes depends on several factors: electric-field strength, pulse number, pulse duration, temperature and cellular concentration. 4. Even for low parasitemia, differences in the effect of these parameters were observed between infected and non-infected erythrocytes. 5. Here we describe an influence of electric field intensity and temperatures on the opening pores.