Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.     

Synthesis of multiple N-acylhomoserine lactones is wide-spread among the members of the Burkholderia cepacia complex

Seventy strains of the Burkholderia cepacia complex, which currently comprises six genomic species, were tested for their ability to produce N-acylhomoserine lactone (AHL) signal molecules. Using thin layer chromatography in conjunction with a range of AHL biosensors, we show that most strains primarily produce two AHLs, namely N-octanoylhomoserine lactone (C8-HSL) and N-hexanoylhomoserine lactone (C6-HSL). Furthermore, some strains belonging to B. vietnamiensis (genomovar V) produce additional long chain AHL molecules with acyl chains ranging from C10 to C14. For B. vietnamiensis R-921 the structure of the most abundant long chain AHL was confirmed as N-decanoylhomoserine lactone (C10-HSL) by liquid chromatography-mass spectrometry (LC-MS) in combination with total chemical synthesis. Interestingly, a number of strains, most notably all representatives of B. multivorans (genomovar II), did not produce AHLs at least under the growth conditions used in this study. All strains were also screened for the production of extracellular lipase, chitinase, protease, and siderophores. However, no correlation between the AHL production and the synthesis of these exoproducts was apparent. Southern blot analysis showed that all the B. cepacia complex strains investigated, including the AHL-negative strains, possess genes homologous to the C8-HSL synthase cepI and to cepR, which encodes the cognate receptor protein. The nucleotide sequence of the cepI and cepR genes from one representative strain from each of the six genomovars was determined. Furthermore, the cepI genes from the different genomovars were expressed in Escherichia coli and it is demonstrated that all genes encode functional proteins that direct the synthesis of C8-HSL and C6-HSL. Given that cepI from the B. multivorans strain encodes a functional AHL synthase, yet detectable levels of AHLs were not produced by the wild-type, this suggests that additional regulatory functions may be present in members of this genomovar that negatively affect expression of cepI.     

Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.      

Elektronenmikroskopische Untersuchungen der Phagozytose und Wirkung pyrogener Reizstoffe im Knochenmark

Zusammenfassung Die feinstrukturellen Vorgänge bei Phagozytose intravenös injizierter Tusche im Knochenark der Ratte werden beschrieben.5–15 min nach Injektion werden einzelne Kohlepartikel mittels pinozytotischer Membraneinstülpungen oder fingerförmiger Ausstülpungen von den Sinusendothelzellen aufgenommen und in kleinen, membranumgebenen Vesikeln abgelagert, die sodann zu größeren Zytosomen konfluieren. Die Zytosomen enthalten — neben den phagozytierten Kohleteilchen — mehr oder weniger eines feingranulären Materials. Von den Sinusendothelzellen gelangt die aufgenommene Tusche in die charakteristischen phagozytierenden Retikulumzellen, wo sie zusammen mit großen Mengen feingranulären Materials in Zytosomen abgelagert wird. Freie, extrazelluläre Tusche wird im Markparenchym nur ganz vereinzelt und nur während der ersten 5–15 min p. i. gefunden. Es wird diskutiert, daß die frei im Parenchym angetroffenen Partikel artefiziell dorthin verschleppt wurden. Mittels Auszählung von Tuschepartikeln wurden die zeitlichen Abläufe festgehalten: 5–15 min nach Injektion befinden sich einzelne Tuschepartikel im Lumen der Gefäße und in den Sinusendothelzellen; viel Tusche ist aber auch schon in Form von Zytosomen in den Sinusendothelien abgelagert. 1 Std bis 14 Tage nach Injektion ist Tusche nur noch innerhalb von Zytosomen abgelagert; zum großen Teil in den Sinusendothelzellen, zum kleineren Teil in phagozytierenden Retikulumzellen.4–8 Wochen nach Injektion sind kohlehaltige Zytosomen in Endothel- und phagozytierenden Retikulumzellen gleich häufig. — Injektion von Pyrexal verursachte eine periphere Leukozytose, rief aber im Knochenmark keine deutlichen Veränderungen in Struktur oder Tuscheverteilung hervor.Die Problematik von Tracer-Untersuchungen im feinstrukturellen Bereich und ihrer quantitativen Auswertung wird diskutiert; die Folgerungen, die sich aus den Ergebnissen für das Verständnis von Feinstruktur und Funktion des Knochenmarks als eines Bestandteils des RHS ziehen lassen, werden besprochen.

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Summary The fine structure of the bone marrow of rats after i. v. injection of carbon particles is described. 5–15 min after injection, sinusendothelial cells ingest carbon particles by means of pinocytotic invaginations or finger like processes of the cell membrane. The ingested carbon particles are deposited in small vesicles which afterwards form larger cytosomes. Besides the phagocytized carbon particles, the cytosomes contain a finely granular material. Later the ingested material is found also in the characteristic phagocytic reticular cells where it is deposited in cytosomes together with large amounts of finely granular material. Free extracellular carbon particles are found in the parenchyma only sparsely and only during the first 5–15 min p. i. It is discussed that the carbon particles found extracellularly in the parenchyma are artefacts. To document the sequence of events the number of particles was determined: 5–15 min p. i. carbon particles are found in the lumen of the vessels and in the sinusendothelial cells; however, some particles are already deposited as cytosomes in the sinusendothelial cells. 1 hour to 14 days after injection, the carbon particles are only deposited within cytosomes; the larger amount of the cytosomes is found in the sinusendothelial cells, the smaller in phagocytic reticular cells. 4–8 weeks p. i. the amount of carbon containing cytosomes in endothelial cells and phagocytic reticular cells is equal. Injection of Pyrexal caused a peripheral leucocytosis, but caused no distinguishable changes in the structure of the carbon distribution. The problems of tracer studies in electron microscopy and of their quantitative evaluation, as well as the conclusions which can be drawn from our results in understanding the fine structure and function of vascular and reticuloendothelial parts of the bone marrow are discussed.

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Studie im Rahmen der Assoziation Hämatologie EURATOM-GSF.

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Wir danken Frl. G. Wochinger und M. Darsow für ihre wertvolle Mitarbeit.     

Abdominal gland secretion ofBledius rove beetles as an effective defence against predators

Recent investigations indicated thatBacillus thuringiensis delta-endotoxins (DET) possess aphidicidal activity in an artificial diet bioassay. Crystalline preparations of CryIIA, CryIIIA and CryIVD solubilized in a slightly alkaline sucrose/amino acid diet clearly imparted toxicity toward adults of potato aphid,Macrosiphum euphorbiae (Thomas) (Homoptera: Aphididae) after 4–5 days of continuous feeding. No obvious feeding deterrence was noted in these assays, as copious honeydew was produced and aphids often died in a feeding position. CryIIIA which was solubilized in aphid diet, but filtered to remove spores or crystalline toxin lacked aphidicidal activity. Spores from an acrystalliferous strain (EG2205) were not toxic by themselves at 7.75×10⁵ spores/ml aphid diet, but did restore toxicity to the filtered CryIIIA solution. Therefore, low levels of spores may be very effective in concert with DET for aphicidal activity. Results also clearly demonstrated that a suspension of crystalline CryIIIA alone, without spores, exhibited toxicity. Therefore, DET may be more toxic to the aphids when imbibed as a fine suspension, perhaps indicating the need for slow solubilization into the aphid midgut.     

The use of general foraging kairomones in a generalist parasitoid

Almost no comparative studies are available on the use of general and specific infochemical cues by generalist parasitoids with hosts from different families feeding on host plants also from different families. Based on literature, two hypotheses were developed and tested with host recognition cues used by the larval parasitoid Lariophagus distinguendus . This generalist parasitizes beetle species from different families developing in seeds of plant species from the Poaceae and Fabaceae. The first hypothesis predicts that for initial encounters with host species, natural enemies should innately use general cues, which are common to all hosts and their food plants. The second hypothesis predicts that natural enemies should learn specific cues from host plant and host after experience with a host species. The first hypothesis was partly confirmed. L. distinguendus innately reacted to faecal cues from several host species and chemical analyses of faeces from these hosts revealed the common occurrence of chemicals that are used for host recognition by L. distinguendus . In disagreement with the first hypothesis, parasitoids did not innately respond to cues from plant seeds. Preference experiments on the influence of experience demonstrated an increased host recognition response towards a host after experience with it. In support of the second hypothesis, L. distinguendus females learned specific cues from herbivore-damaged wheat, rice and cowpea seeds and from the faeces of the bean weevil Callosobruchus maculatus .