A lectin-mediated resistance of higher fungi against predators and parasites.

Fruiting body lectins are ubiquitous in higher fungi and characterized by being synthesized in the cytoplasm and up-regulated during sexual development. The function of these lectins is unclear. A lack of phenotype in sexual development upon inactivation of the respective genes argues against a function in this process. We tested a series of characterized fruiting body lectins from different fungi for toxicity towards the nematode Caenorhabditis elegans, the mosquito Aedes aegypti and the amoeba Acanthamoeba castellanii. Most of the fungal lectins were found to be toxic towards at least one of the three target organisms. By altering either the fungal lectin or the glycans of the target organisms, or by including soluble carbohydrate ligands as competitors, we demonstrate that the observed toxicity is dependent on the interaction between the fungal lectins and specific glycans in the target organisms. The toxicity was found to be dose-dependent such that low levels of lectin were no longer toxic but still led to food avoidance by C. elegans. Finally, we show, in an ecologically more relevant scenario, that challenging the vegetative mycelium of Coprinopsis cinerea with the fungal-feeding nematode Aphelenchus avenae induces the expression of the nematotoxic fruiting body lectins CGL1 and CGL2. Based on these findings, we propose that filamentous fungi possess an inducible resistance against predators and parasites mediated by lectins that are specific for glycans of these antagonists.

A real-time PCR method to quantify spores carrying the Bacillus thuringiensis var. israelensis cry4Aa and cry4Ba genes in soil.

AIM: To develop a rapid real-time PCR method for the specific detection and quantification of Bacillus thuringiensis var. israelensis (Bti) spores present in the environment. METHODS AND RESULTS: Seven soil samples as well as one sediment sample obtained from various regions of Switzerland and characterized by different granulometry, pH values, organic matter and carbonate content were artificially inoculated with known amounts of Bti spores. After DNA extraction, DNA templates were amplified using TaqMan real-time PCR targeting the cry4Aa and cry4Ba plasmid genes encoding two insecticidal toxins (δ-endotoxins), and quantitative standard curves were created for each sample. Physicochemical characteristics of the samples tested did not influence DNA extraction efficiency. Real-time PCR inhibition because of the presence of co-extracted humic substances from the soil was observed only for undiluted DNA extracts from samples with very high organic matter content (68%). The developed real-time PCR system proved to be sensitive, detecting down to 1 × 10(3) Bti spores per g soil. One-way analysis of variance confirmed the accuracy of the method. CONCLUSIONS: Direct extraction of DNA from environmental samples without culturing, followed by a specific real-time PCR allowed for a fast and reliable identification and quantification of Bti spores in soil and sediment. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed real-time PCR system can be used as a tool for ecological surveys of areas where treatments with Bti are carried out.

Molecular identification of Bacillus thuringiensis var. israelensis to trace its fate after application as a biological insecticide in wetland ecosystems.

AIMS: To determine the fate of viable Bacillus thuringiensis var. israelensis (Bti) spores dispersed in the environment, using a universally applicable molecular detection methodology. METHODS AND RESULTS: Soil samples were spread on growth medium, after a temperature selection of the spores. A PCR amplification of the cry4Aa and cry4Ba insecticidal genes was applied on the colonies. Ribotyping was performed subsequently. This combined molecular method proved to be very specific for Bti, which was easily differentiated from the other B. thuringiensis serovars. A site regularly treated with Vectobac-G was chosen within the 'Bolle di Magadino' natural reserve, and monitored throughout 1 year for the detection of Bti spores. The results showed that the numbers were relatively high after insecticidal applications (1.4 x 10(5) CFU g(-1)), and decreased approx. 10-fold after 220 days. A successive treatment induced a new increase. CONCLUSIONS: The results show that yearly repeated use of Vectobac-G does not seem to have a major ecological impact on the 'Bolle di Magadino' natural reserve. Bti spores followed a trend leading to their eventual disappearance from the ecosystem, despite the seasonal application of this biological insecticide for more than a decade. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular identification of Bti cells through the PCR analysis of the delta-endotoxins genes coupled to ribotyping, is an innovative method, that has enabled the identification of this organism into wetland environments.

[Role of transesophageal echocardiography in the diagnosis of cardiac disorders]. / Apport de l'échocardiographie transoesophagienne dans le diagnostic des affections cardiaques.

Transesophageal echocardiography provides a new window for ultrasound examination of the heart. The approach allows ultrasonic access to the heart in patients with transthoracic images of inadequate quality. Furthermore, transesophageal examination may provide information additional to that from the conventional transthoracic approach in the search for lesions including complications of endocarditis, mitral prosthesis dysfunction, aortic dissection, embolism of cardiac origin and selected cardiac malformations.

Assessment of myocardial metabolism with iodine-123 heptadecanoic acid: effect of decreased fatty acid oxidation on deiodination.

Terminally radioiodinated fatty acid analogs are of potential use for the noninvasive delineation of regional alterations of fatty acid metabolism by gamma imaging. Since radioactivity from extracted iodine-123 heptadecanoic acid [( 123I]HDA) is released from the myocardium in form of free radioiodide (123I-) the present study was performed to determine whether deiodination of [123I]HDA is related to free fatty acid metabolism. Myocardial production of free radioiodide was measured in rat hearts in vitro and in vivo both under control conditions and after inhibition of fatty acid oxidation. In isolated rat hearts perfused at constant flow with a medium containing [123I]HDA, release of 123I- was markedly reduced during cardioplegia and pharmacologic inhibition of mitochondrial fatty acid transfer with POCA by 67% (p less than 0.005) and 72% (p less than 0.005), respectively. In fasted rats in vivo, 1 min after i.v. injection of [123I]HDA, 51 +/- 5% of myocardial radioactivity was recovered in the aqueous phase, containing free iodide, of myocardial lipid extracts. Aqueous activity was significantly decreased in fed (20 +/- 2%; p less than 0.002) and POCA pretreated (30 +/- 3.7%; p less than 0.05) animals exhibiting reduced oxidation of [14C]palmitate. Thus, deiodination of [123I]HDA was consistently reduced during inhibition of fatty acid oxidation in vitro and in vivo. The results apply to the interpretation of myocardial clearance curves of terminally radioiodinated fatty acid analogs.

Binding of the delta endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae).

The insecticidal delta endotoxin of Bacillus thuringiensis was labeled with iodine-125. Brush-border membrane vesicles, prepared from the midgut epithelium of Pieris brassicae larvae, known to be highly susceptible to the toxin, and from a non-target tissue: the small intestine of rat, were examined for binding of 125I-toxin. The toxin was bound specifically only to insect vesicles. Its binding to the insect membrane system was competitively inhibited by 127I-toxin and non-iodinated toxin, whereas the binding of the 125I-toxin to the mammalian membrane system was not affected by unlabeled toxin. Vesicles of P. brassicae possess two individual binding-site populations for iodinated toxin with dissociation constants of 46 nM and 490 nM. The Hill coefficients of both sites were approximately 1 and the binding capacities were 0.2 pmol and 30 pmol/mg vesicle protein for the high and the low-affinity sites respectively. The estimation of the dissociation constant for non-iodinated toxin, using a competition experiment, revealed only one binding-site population which possessed a dissociation constant of 235 nM. It is concluded that this is the binding site for the native toxin. This site was sensitive towards treatment with proteases or mixed glycosidases. It is suggested that it is a protein or a glycoprotein.

Specificity of Bacillus thuringiensis Delta-Endotoxin.

The insecticidal activity of the delta-endotoxins of 14 Bacillus thuringiensis strains belonging to 12 subspecies was determined against Pieris brassicae, Heliothis virescens, and Spodoptera littoralis. Larvae of P. brassicae were highly susceptible to purified crystals of strains of B. thuringiensis subsp. thuringiensis and B. thuringiensis subsp. morrisoni, whereas H. virescens responded best to B. thuringiensis subsp. kenyae and B. thuringiensis subsp. kurstaki. The crystals of the B. thuringiensis subsp. entomocidus strain were the most potent against S. littoralis. It was shown that the solubility of the crystals within the gut of the three insect species is a first important step in the mode of action. Predissolution of the crystals especially enhanced the insecticidal activity against H. virescens. When in vitro-activated toxins were applied, the relative potency range varied greatly from one insect species to another. It can be concluded that at least three factors influence the potency of B. thuringiensis delta-endotoxins: the strain-related origin of the toxin, the degree of solubility of the crystals in the gut juice, and the intrinsic susceptibility of the insect to the toxin.

Free fatty acid metabolism in "stunned" myocardium.

To assess whether myocardial lipid metabolism is altered in the "stunned" myocardium we have studied the metabolism of (1-14C)-palmitate during reperfusion in a modified rat heart preparation. Hearts were perfused retrogradely at a physiological flow rate (2 ml/min) in a non-recirculating system with erythrocyte-enhanced Krebs-Henseleit buffer containing albumin 0.4 mM, glucose 11 mM, palmitate 0.4 mM and trace amounts of (1-14C)-palmitate. Left ventricular pressure was measured by a latex balloon in the left ventricular cavity. Control hearts were perfused at constant flow for 120 min. To achieve reversible ischaemic damage, myocardial perfusion was reduced by 95% for 40 min, followed by reperfusion at the control flow rate for 60 min (reperfusion group). For comparison, irreversible damage was produced by calcium free perfusion (calcium paradox group). In the reperfusion group, the developed pressure was severely depressed 5 min after reperfusion to 23% of the value in the control group (p less than 0.05) but recovered to 84% (NS) at 60 min. In the calcium paradox group, mechanical activity ceased completely without recovery. Myocardial uptake of (1-14C)-palmitate in the reperfusion group was similar to the control experiments for the entire reperfusion period, whereas a marked depression was observed in the calcium paradox group. 14CO2 production was severely depressed at the onset of reperfusion in both the reperfusion and calcium paradox group to 42% (p less than 0.05) and 29% (p less than 0.05) respectively. In contrast to the calcium paradox group, 14CO2 production in the reperfusion group recovered progressively to 70% (NS) of the control value during the 60 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and activity of Bacillus thuringiensis delta-endotoxin to invertebrate cells.

Fluorescein isothiocyanate was used as a label to detect delta-endotoxin of Bacillus thuringiensis subsp. thuringiensis and israelensis in binding studies with different in vitro cell systems. Protoxin of the subspecies thuringiensis could be labelled directly whereas the activated toxin had to be traced indirectly with labelled antibodies. Both protoxin and activated toxin bound to primary midgut cell cultures of Pieris brassicae larvae as well as to cells of an established culture of Drosophila melanogaster. No binding with either toxin form could be observed with hemocytes of P. brassicae. Biological activity as shown by the trypan blue viability assay was obtained only with the activated toxin against the midgut cells. Toxin of the subspecies israelensis reacted very unspecifically. Binding followed by rapid destruction was obtained with all the tested cultures.

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer.

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.

Specificities of monoclonal antibodies against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis.

Eight hybrid cell lines secreting monoclonal antibodies directed against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis were grown in BALB/c mice. Ascites fluids were collected, and the antibodies were purified by antigen-affinity chromatography. The specificity of each monoclonal antibody for the toxin and protoxin was established by the indirect enzyme-linked immunosorbent assay. All the antibodies consisted of gamma 1 heavy and kappa light chains. They were reactive with both the native toxin and the protoxin. In contrast to specific goat antiserum, they failed, however, to bind to heat and sodium dodecyl sulfate denatured antigen. These eight cloned cell lines gave rise to five kinds of antibodies distinguished by isoelectric focusing. Competitive antibody binding studies revealed that these five antibodies recognize at least four distinct antigenic determinants of the native toxin and the protoxin. Two of the epitopes are unrelated, whereas three antibodies compete for binding to their antigenic determinants. In the bioassay with larvae of Pieris brassicae, one antibody was found to block the toxin and protoxin activity completely. A second inhibited it partially, whereas the other three antibodies did not affect it at all.

Bacterial mesosomes: method dependent artifacts.

The occurrence of mesosomes was investigated during septum formation of vegetative and sporulating cells of Bacillus cereus. It has been demonstrated that bacterial mesosomes which are considered by numerous microbiologists as an integrated constituent of Gram positive bacteria, are in reality artifacts arising during the preparation for electron microscopy. The conventional fixation methods allowed enough time for the cytoplasmic membrane to react to the changed conditions and to form the typical pocket-like membrane invaginations. With cryofixation followed by freeze-substitution it was shown in ultrathin sections that mesosomes do not occur. The extremely rapid freezing and the substitution of the ice by an organic solvent containing the fixative prevented the formation of membraneous artifacts.

[Blood picture changes in patients hospitalized in an internal medical department]. / Blutbildveränderungen bei internmedizinisch hospitalisierten Patienten.

One quarter, i.e. 810 of 3621 patients admitted to a medical department showed quantitative abnormalities of the blood picture even when leukocytosis is excluded. In contrast, only in 113 cases were hematological diseases the main diagnoses on the hospital charts. 18% of the patients hospitalized for the first time had anemia. On analysis of blood changes according to their causes, 28% were found to be of either iatrogenic or toxic origin, the two causes exhibiting roughly the same frequency. The incidence of iatrogenic and toxic blood changes was equal to that of blood abnormalities due to malignant disease including hemoblastoses. Alcohol and phenacetin were the most frequent exogenous toxins, while anticoagulant, immunosuppressant and ulcerogenic drugs were the most frequent iatrogenic causes of abnormalities.