Isoelectric focusing analysis of detergent extracted renal 11 beta-hydroxysteroid dehydrogenase

11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3-10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosterone in equilibrium 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities. In order to test whether renal 11-HSD represents a uniform oxido-reductase as generally assumed, or a dual enzyme system as has been recently proposed an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (= delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.     

Root,shoot and soil parameters required for process-oriented models of crop growth limited by water or nutrients

A review is given of the prospects for using process-oriented models of water and nutrient uptake in improving integrated agriculture. Government-imposed restrictions on the use of external inputs will increase the likelihood of (temporary) nutrient or water stress in crop production in NW Europe and thus a better understanding is required of shoot-root-soil interactions than presently available. In modelling nutrient and water uptake, three approaches are possible: 1) models-without-roots, based on empirically derived efficiency ratios for uptake of available resources, 2) models evaluating the uptake potential of root systems as actually found in the field and 3) models which also aim at a prediction of root development as influenced by interactions with environmental factors. For the second type of models the major underlying processes are known and research can concentrate on model refinement on the one hand and practical application on the other. The main parameters required for such models are discussed and examples are given of practical applications. For the third type of models quantification of processes known only qualitatively is urgently needed.     

Moulting hormone,juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera)

Summary In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis is cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.     

First genetic evidence that invasive bullhead (Cottus L. 1758) in Scotland is of English origin and the difficulty of resolving the European Cottus species taxonomy

The European bullhead (Cottus gobio) is widely distributed across Europe, and within the UK is native to England and Wales, where it is protected under the Habitats Directive. In Scotland, however, the species is considered invasive and thriving populations are recorded in the Forth and Clyde river catchments, and the Ale Water in the Scottish Borders. The genetic identity of the Scottish populations has not been established. There is also debate about the status of the European bullhead and its validity as single species, a species complex with several unresolved species, or distinct different species in its European distribution range. There is therefore a need to determine the taxonomy and likely source of the novel Scottish populations. Genetic analyses using cytochrome oxidase 1 (COI) mitochondrial DNA sequences were undertaken on specimens from the Forth and Clyde catchments, and combined with the results of morphological characteristics to provide a comprehensive assessment of the taxonomic classification for Scottish bullheads. There was considerable variation in morphological characteristics between populations within Scotland and a wider range of variability than previously recorded for English populations. Genetically the Scottish populations were very closely related to English specimens, supporting the hypothesis of introduction directly from England to Scotland. In terms of broader relationships, Scottish specimens are genetically more closely related to the ostensible species Chabot fluviatile Cottus perifretum, which has been suggested as one of a complex of species across Europe. Morphologically they exhibit characteristics on the spectrum between C. perifretum and C. gobio. There is an urgent need for the clarification of the taxonomy of Cottus sp(p). to avoid confusion in future publications, legislation and management practices relating to bullheads throughout the UK and Europe.     

Selection of a minimum-boundary reserve network using integer programming

In the conservation literature, heuristic procedures have been employed to incorporate spatial considerations in reserve network selection with the presumption that computationally convenient optimization models would be too difficult or impossible to formulate. This paper extends the standard set-covering formulation to incorporate a particular spatial selection criterion, namely reducing the reserve boundary to the extent possible, when selecting a reserve network that represents a set of target species at least once. Applying the model to a dataset on the occurrence of breeding birds in Berkshire, UK, demonstrated that the technique resulted in significant reductions in reserve boundary length relative to solutions produced by the standard set-covering formulation. Computational results showed that moderately large reserve network selection problems could be solved without issue. Alternative solutions may be produced to explore trade-offs between boundary length, number of sites required or alternative criteria.     

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

Genome sequence of Listeria monocytogenes Scott A, a clinical isolate from a food-borne listeriosis outbreak

Listeria monocytogenes is an opportunistic food-borne pathogen and the causative agent of listeriosis in animals and humans. We present the genome sequence of Listeria monocytogenes Scott A, a widely distributed and frequently used serovar 4b clinical isolate from the 1983 listeriosis outbreak in Massachusetts.     

Effect of abscisic acid on vitellogenesis in Sarcophaga bullata

In the carnivorous dipteran Sarcophaga bullata Parker, vitellogenesis was partially inhibited by injection of two doses of 12 g abscisic acid (ABA). There was no significant difference between injections on day 2 and 4, or on day 4 and 6. Higher and lower doses were less effective. The mixture of isomers inhibited vitellogenesis more than the cis-trans isomer. ABA had no effect on the total lipid concentration of the haemolymph but it inhibited the sharp increase in total protein concentration of the haemolymph and particularly that of vitellogenin, which normally occurred within 24 hr following liver feeding on day 4. Since vitellogenin synthesis is under the control of moulting hormone (MH), the MH activity was measured by radioimmunoassay to find out whether ABA might interact with vitellogenin synthesis via its hormonal inductor. Eight hours after liver feeding, there was a MH peak in the control groups while following ABA treatment this peak occurred after 18 hr. The inhibitory effect of ABA on vitellogenesis could be overruled by feeding sugar impregnated with ecdysterone (5 mg/g), not by topical application of JH. Our results suggest that ABA might interact with a mechanism which phytophagous and non-phytophagous insects share in common. If in phytophagous insects the same amount of ABA per gram weight, as was effective in Sarcophaga (about 200 g/g), is needed to reduce fecundity, it is not probable that this plant hormone plays a role in the seasonal synchronisation of the growth and reproduction of insects with the senescence of their host plants.

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Résumé La vitellogenèse du diptère carnivore, Sarcophaga bullata a été partiellement inhibée par injection de deux doses de 12 g d'acide absisique (ABA). Il n'y avait pas de différence significative entre les injections du deuxième et quatrième jour, ou du quatrième et sixième jour. La mixture d'isomères a plus inhibé la vitellogenèse que le cis-trans isomère.ABA n'a pas eu d'effect sur la concentration totale des lipides de l'hémolymphe, mais il a inhibé l'augmentation brutale de la concentration protéique totale de l'hémolymphe, et particulièrement celle de la vitellogénine qui se produit normalement dans les 24 h après consommation de foie le 4ème jour. La synthèse de la vitellogénine étant sous le contrôle de l'hormone de mue (MH), l'activité MH a été mesurée par radio-immunologie, pour découvrir si l'ABA agissait sur la synthèse de la vitellogénine par intermédiaire de son inducteur hormonal. Huit heures après alimentation sur foie, le taux de MH chez les témoins était élevé tandis qu'après traitement avec ABA ce pic n'apparaissait qu'après 18 h. L'effet inhibiteur de l'ABA sur la vitellogénine a pu être neutralisé en fournissant aux mouches du sucre impregné d'ecdystérone (5mg/g), mais non par application locale d'hormone juvénile. Nos résultats suggèrent qu'ABA agit probablement sur un mécanisme commun aux insectes phytophages et non phytophages.Il est peu probable que l'acide absisique joue un rôle dans la synchronisation saisonnière de la croissance et de la reproduction des insectes avec la sénescence des plantes-hôtes.

     

In vivo uptake and metabolism of [3H]ecdysone in the vitellogenic follicles of Sarcophaga bullata (Diptera) and its localisation by autoradiography

Within 4 h after injection of [³H]ecdysone, almost all tritiated material has disappeared from the haemolymph, indicating that the uptake by the tissues is very fast. After only 15 min, 19% of the label was found in the ecdysterone fraction and 4% in the highly polar products (HPP) fraction. The uptake of [³H]ecdysone by the ovary (mid-vitellogenic) is almost complete within 1 h after injection. The pattern of [³H]ecdysteroids in the ovaries follows a well ordered sequence: firstly, [³H]ecdysone is the major component of the [³ H]ecdysteroids but it disappears within 2 h, next a peak value of [³H]ecdysterone was found at 1 h, whereafter this also disappeared, and from 2 h on, there was a considerable increase in HPP. The HPP consisted of 3 fractions (A, B and C). Glusulase treatment revealed that apparently only fraction B consisted of glucuronide and/or sulphate-conjugates of ecdysteroids. Autoradiographic experiments confirmed that the uptake of [³H]ecdysone was a very rapid process. In ovaries fixed 1 h after injection, the silver grains were abundant in the ooplasm but were also found in the follicle cell cytoplasm and in trophocytes. In follicles examined 16 h after injection, only a few silver grains were observed in the trophocytes and follicle cells. However, the cytoplasm of the oocyte was labelled. The border cells also accumulated label.

The major results indicate that all cell types of the follicle seem to be able to absorb ecdysone from the haemolymph and that there seems to be a rather selective uptake of ecdysone. In the ooplasm, ecdysone is converted to highly polar conjugates.