Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with ¹²⁵I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.     

The integration of the corneal and limbal fibrils in the human eye.

The precise orientation of the collagen fibrils in human cornea and sclera and the method by which these two areas fuse together at the limbus have never been determined, despite the importance of this information. From a consideration of the mechanics of the system, fibril orientation in the tissue has the potential to affect the curvature of the cornea so, by inference, refractive problems such as astigmatism involving an incorrect curvature of the cornea may be related to fibril orientation. The high intensity and small beam size of a synchrotron x-ray source has enabled us to study fibril orientation in post-mortem human cornea and sclera. Previously we have revealed two preferred directions of orientation in the cornea (Meek, K. M., T. Blamires, G. F. Elliot, T. Y. Gyi, and C. J. Nave. 1987. Curr. Eye Res. 6:841-846) and a circumcorneal annulus in the limbus (Newton, R. H., and K. M. Meek. 1998. Invest. Ophthalmol. & Visual Sci. 39: 1125-1134). Here we present the results of our investigation into the relationship between these two features. Our measurements indicate that the corneal fibrils oriented in the two preferred directions bend at the limbus to run circumferentially. On the basis of these results we propose a model as to how the human cornea and sclera fuse together.     

The dynamics of parental care in Choughs(Pyrrhocorax pyrrhocorax)

Summary The dynamics of parental investment throughout the nestling stage and the factors affecting it were studied in the Chough(Pyrrhocorax pyrrhocorax), a species whose patterns of apportioning parental care are largely unknown. The occurrence of important trade-offs between the sexes, among the different activities of parental care and between parents' survival and current offspring survival were estimated. The parental contributions of both sexes were assessed mainly in terms of food provisioning rate and nest attendance time. Only the female brooded young nestlings while the two sexes contributed equally in food deliveries and nest sanitation. Nestling age greatly affected nest attendance time. The female spent a long time brooding in the first 10 days after hatching. Both sexes increased attendance towards the end of the nestling stage. Conversely, feeding rate and feeding rate per nestling remained approximately constant throughout the nestling period. Nestlings in smaller broods received more feeding visits than those in larger broods. The shape of the per-nestling feeding rate curve was concave-up, supporting Nur's (1984) trade-offs model rather than the Lack-Gibb hypothesis. Maintaining a high feeding frequency in broods already above the modal value might be disadvantageous, implying few benefits and large energy costs (i.e. the reduction of the parents' residual reproductive value). Female brooding time in relation to brood size showed the same decreasing concave-up trend line. Short-term trade-offs proved to be important determinants of the dynamics of parental care. Specifically, the distance from the feeding areas greatly affected the delivery rate: pairs spent a disproportionately longer time foraging in more distant patches than in closer ones. Diurnal variations and changes owing to weather conditions were also examined.

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Die Dynamik elterlicher Investition bei der Alpenkrähe(Pyrrhocorax pyrrhocorax)

Zusammenfassung Der elterliche Aufwand und die geschlechtliche Verteilung des Brutaufwandes bei Alpenkrähen ist weitgehend unbekannt. Ziel der Arbeit war es deshalb, die verschiedenen Aktivitäten der elterlichen Brutversorgung und deren Konsequenzen für die Überlebensverhältnisse der Eltern und des Nachwuchses näher zu untersuchen. Die Fütterung der Brut und die Anwesenheit und Betreuung am Nest standen im Mittelpunkt. Während nur das Weibchen brütete, teilten sich die Eltern die Jungenaufzucht und die Pflege des Nestes etwa gleichmäßig, wobei das Nestlingsalter einen erheblichen Einfluß auf die Nestversorgung hatte. In den ersten 10 Tagen huderte das Weibchen intensiv. Beide Eltern steigerten ihre Brutpflege zum Ende der Nestlingszeit. Dagegen blieben die Fütterungsrate und die Anzahl Fütterungen je Nestling über die gesamte Nestlingszeit in etwa konstant. Junge in kleineren Bruten erhielten mehr Fütterungen als solche in großen. Der Verlauf der Abhängigkeit der Fütterungen je Nestling von der Brutgröße stützt mehr die Hypothese von Nur (1984) als die von Lack und Gibb. Die Aufrechterhaltung einer hohen Fütterungsrate auch bei großen Bruten dürfte nachteilig sein, da sie nur wenig Nutzen bei einem hohen Aufwand (Beeinträchtigung der späteren Brutmöglichkeiten) bringt. Der Huderaufwand des Weibchens zeigt in etwa denselben Zusammenhang mit der Brutgröße. Kurzzeitige elterliche Entscheidungen scheinen eine wichtige Rolle in der Regulation der elterlichen Brutpflege zu spielen. Dabei kommt gerade der räumlichen Lage der Nahrungsplätze eine große Bedeutung zu: an weiter entfernt gelegenen Nahrungsplätzen verbrachten die Eltern unverhältnismäßig mehr Zeit als an nahen Futterplätzen. Daneben haben die Tageszeit und das Wetter einen Einfluß auf die elterliche Brutfürsorge der Alpenkrähen.

     

Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their ⁵⁹Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (K_d ≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K₃MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (K_d ≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10⁹ cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.     

Morphology and development of the haptors among the Monocotylidae (Monogenea)

Monocotylid monogeneans inhabit a wide diversity of sites on their chondrichthyan hosts including the skin, gills, nasal fossae, urogenital system and coelom. The large variation in the morphology of the haptor appears to reflect this diversity in attachment sites. We demonstrate that the complexity of the haptor can be related to the habitat of the parasite. Generally, those parasites which live in habitats subject to strong water currents such as the gills and dorsal skin surface have more complex haptors than those in environments exposed to weaker or no water currents including the nasal fossae, urogenital system and body cavity. However, there can be considerable variation in haptoral components, even among congeners, living on the ‘gills’ of their hosts. The microhabitat was determined for Monocotyle helicophallus and M. spiremae, both from the gills of the pink whipray, Himantura fai, and M. corali from the gills of the cowtail ray, Pastinachus sephen. We demonstrate that differences in the morphology of the hamuli and the number and morphology of septal sclerites and marginal papillae among these species of Monocotyle can be related directly to their microhabitat. It also appears that different haptoral structures are important for attachment to the host at different stages in the development of the parasite, based on studies on the development and distribution of Neoheterocotyle rhinobatidis from the gills of the common shovelnose ray Rhinobatos typus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Does a Low Nitrogen Supply Necessarily Lead to Acclimation of Photosynthesis to Elevated CO2?

Long-term exposure of plants to elevated partial pressures of CO₂ (pCO₂) often depresses photosynthetic capacity. The mechanistic basis for this photosynthetic acclimation may involve accumulation of carbohydrate and may be promoted by nutrient limitation. However, our current knowledge is inadequate for making reliable predictions concerning the onset and extent of acclimation. Many studies have sought to investigate the effects of N supply but the methodologies used generally do not allow separation of the direct effects of limited N availability from those caused by a N dilution effect due to accelerated growth at elevated pCO₂. To dissociate these interactions, wheat (Triticum aestivum L.) was grown hydroponically and N was added in direct proportion to plant growth. Photosynthesis did not acclimate to elevated pCO₂ even when growth was restricted by a low-N relative addition rate. Ribulose-1, 5-bisphosphate carboxylase/oxygenase activity and quantity were maintained, there was no evidence for triose phosphate limitation of photosynthesis, and tissue N content remained within the range recorded for healthy wheat plants. In contrast, wheat grown in sand culture with N supplied at a fixed concentration suffered photosynthetic acclimation at elevated pCO₂ in a low-N treatment. This was accompanied by a significant reduction in the quantity of active ribulose-1, 5-bisphosphate carboxylase/oxygenase and leaf N content.     

Adeno-Associated Virus Vector-Mediated Transgene Integration into Neurons and Other Nondividing Cell Targets

The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.     

Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to an hrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s. pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn²⁺/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H₂O₂ in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.     

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by ¹H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg⁻¹. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.