Pre-inoculation of Ri T-DNA-transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: an ultrastructural and cytochemical study

The influence exerted by Pseudomonas fluorescens, strain 63-28R, in stimulating plant defense reactions was investigated using an in-vitro system in which Ri T-DNA-transformed pea (Pisum sativum L.) roots were subsequently infected with Pythium ultimum. Cytological investigations of samples from P. fluorescens-inoculated roots revealed that the bacteria multiplied abundantly at the root surface and colonized a small number of epidermal and cortical cells. Penetration of the epidermis occurred through the openings made by the disruption of the fibrillar network at the junction of adjacent epidermal cell walls. Direct cell wall penetration was never observed and bacterial ingress into the root tissues proceeded via an intercellular route. Striking differences in the extent of fungal colonization were observed between bacterized and non-bacterized pea roots following inoculation with P. ultimum. In non-bacterized roots, the pathogen multiplied abundantly through most of the tissues while in bacterized roots, pathogen growth was restricted to the epidermis and the outer cortex. At the root surface, the bacteria interacted with the pathogen, in a way similar to that observed in dual culture tests. Most Pythium cells were severely damaged but fungal penetration by the bacteria was never observed. Droplets of the amorphous material formed upon interaction between the bacteria and the host root were frequently found at the fungal cell surface. Incubation of sections with a -1,4-exoglucanase-gold complex revealed that the cell wall of markedly altered Pythium hyphae was structurally preserved. Successful penetration of the root epidermis was achieved by the few hyphae of P. ultimum that could escape the first defensive line in the rhizosphere. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. The unusual occurrence of polymorphic wall appositions along the host epidermal cells was an indication that the host plant was signalled to defend itself through the elaboration of physical barriers.Abbreviations AGL Aplysia gonad lectin - PGPR plant growth-promoting rhizobacteria The authors wish to thank Sylvain Noël for excellent technical assistance. This study was supported by grants from the Fonds Québécois pour la formation de chercheurs et l'Aide à la Recherche (FCAR), the Natural Sciences and Engineering Council of Canada (NSERC) and the Ministère de l'Industrie, du Commerce, de la Science et de la Technologie (SYNERGIE).     

Two-bond C-C spin-coupling constants in carbohydrates: effect of structure on coupling magnitude and sign

An empirical projection method is described to predict the magnitudes and signs of two-bond ¹³C-¹³C spin-coupling constants (²J_CC) in aldopyranosyl rings. The method has been applied primarily to the interpretation of ²J_CCC values, although the behavior of ²J_COC has also been examined in light of the new approach, producing results which may prove useful in the conformational analysis of O-glycosidic linkages in oligosaccharides. High-level ab initio calculations of ²J_CC values in model compounds were found to be in agreement with the predictions.     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

A general assay for restriction endonucleases and other DNA-modifying enzymes with plasmid substrates

A procedure for measuring the activities of enzymes that alter the covalent structure of DNA is described. The assay utilizes covalently closed circles of DNA as the substrate and yields quantitative data on the fraction of this DNA converted to both open-circle and linear forms.     

Effects of fire on abundance of Eragrostis intermedia in a semi-arid grassland in southeastern Arizona

Abstract. Eragrostis intermedia (Plains lovegrass) is a midheight perennial bunchgrass native to semi-arid grasslands of the southwestern USA, that becomes an abundant and dominant component of these grasslands in areas long protected from livestock grazing. Substantial mortality of plains lovegrass occurred on a large livestock exclosure in southeastern Arizona, after a period of declining precipitation, but only in areas that had not burned in the previous three years. Lovegrass abundance subsequently increased on both undisturbed and burned sites, but remained substantially higher on the burned area. Long-term abundance of plains lovegrass may depend on episodic fire, particularly during periods of reduced precipitation.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Quantitative floral development in Pseudolysimachion (Scrophulariaceae): intraspecific variation and comparison with Veronica and Veronicastrum

Intraspecific variation in floral development was studied in four morphologically distinct strains of Pseudolysimachion longifolium and two of P. spicatum. Size increase of whole buds from living plants of P. longifolium was followed in absolute time. Growth rates were homogeneous within inflorescences and within individual plants, and significantly different between individual plants. Organ growth trajectories (plotted as organ size over gynoecium size) differed little among individuals of Pseudolysimachion belonging to morphologically distinct strains; variation was most pronounced in the calyx. Corolla tube as well as calyx and corolla lobe measurements varied more during the early stages of development than near maturity, suggesting constraints on mature flower form. In addition to intraspecific variation, floral development in Pseudolysimachion was compared with that in related Veronica chamaedrys and Veronicastrum virginicum, using data from a previous study. Mature flowers of Pseudolysimachion have long corolla tubes similar to those of Veronicastrum and long corolla lobes similar to those of Veronica. The trajectories of most organs of Pseudolysimachion were intermediate between those of Veronica and Veronicastrum in position as well as in type of curve, i.e., different trajectories lead to similar end products; thus, the shapes of the mature organs are not strictly homologous and not indicative of close relationship.     

SPERM DISPLACEMENT WITHOUT SPERM TRANSFER IN DROSOPHILA MELANOGASTER

In this paper we show that when Drosophila melanogaster females are mated twice, the semen of the second male causes a reduction of the effective number of resident sperm from the previous mating. This is demonstrated by two different kinds of experiments. In one set of experiments, mated females were remated to two different kinds of sterile males, one with normal semen and the other with deficient semen. The effect on the resident sperm was determined from the number of remaining progeny after mating to the sterile male, with the result that the normal semen reduced the amount of resident sperm in comparison with matings to the males with deficient semen. The second set of experiments employed interrupted matings. These experiments were based on the observation that semen is delivered before sperm during the first 5 min of copulation. The second matings were interrupted instantly, 2 min, and 4 min after the initiation of copulation. Compared to the instant interruptions, the two later interruptions had the effect of reducing the amount of resident sperm. The results of these two experiments clearly indicate that a sperm-incapacitation process plays a role in the well-documented phenomenon of sperm displacement (last-male advantage) in this species. Such a process could play a role in sperm displacement in the many cases where the mechanism is unknown.     

Uneven distribution of protein kinase C-α and -β isozymes in human sarcomas and carcinomas

Protein kinase C (PKC) represents a family of structurally related Ser/Tre kinases which are involved in mitogenic signalling and may contribute to human neoplasia. To address this issue, the messenger RNA and protein levels of PKC isoenzymes α and β were analyzed in several human sarcoma- and carcinoma-derived cell lines. Carcinomas contained low or undetectable levels of either PKC-α or PKC-β. Sarcomas exhibited similar or increased PKC expression compared to human diploid fibroblasts. Moreover, sarcoma cell lines expressing one PKC isoform did not contain detectable levels of the other. When PKC was depleted from the tumor cells, we observed that the PKC overexpressing sarcomas had reduced their malignant properties as determined by their ability to grow in semisolid medium. In addition, epidermal growth factor-stimulated and erbB2-transformed fibroblasts exhibited enhanced cell growth in the absence of PKC. We propose a model for the effect of PKC as a negative regulator of proliferation in epithelial cells and a growth promoter in fibroblasts. © 1994 wiley-Liss, Inc.