A mutational analysis of the globotriaosylceramide-binding sites of verotoxin VT1.

Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb(3) receptors which present the P(k) trisaccharide Galalpha(1-4)Galbeta(1-4)Glcbeta. Crystallographic studies have identified three P(k) trisaccharide (P(k)-glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of P(k)-glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to P(k)-glycoside and pentavalent P(k) trisaccharide (pentaSTARFISH) in solution and Gb(3) presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb(3) involved all three sites. Binding of all single site mutants to liposomal Gb(3) was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb(3) in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb(3,) even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membrane-localized Gb(3). Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb(3) trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction.     

Assessment of tight junctions between pulmonary epithelial and endothelial cells

This study is intended to determine whether qualitative assessment of tight junction integrity from freeze-fracture data is reliable. We used lung parenchyma from a control mongrel dog's cardiac lung lobe, from a mongrel dog subjected to vascular high-pressure pulmonary edema (HPPE), and from a dog subjected to oleic acid-induced low-pressure pulmonary edema (LPPE) (6). Quantitative assessment was done on 115 freeze-fracture micrographs of epithelial tight junctions and on another 158 freeze-fracture micrographs of endothelial junctions from the 3 dogs. Quantitative assessment showed differences between the dogs in junction depth, fibril numbers, density, and complexity. for qualitative assessment, these same 273 micrographs were assessed in a single-blind fashion by having six investigators sort first the epithelial and then the endothelial junctions into normal or damaged categories. Qualitative assessment did not agree with quantitative data, suggesting that it is unreliable.     

Production of15N-labelled hen egg white lysozyme usingAspergillus niger

Summary Aspergillus niger has been used as a host organism for the production of¹⁵N-labelled hen egg white lysozyme (HEWL). In order to achieve maximum incorporation of label, strains expressing the HEWL gene were grown in medium containing ammonium¹⁵N chloride as sole nitrogen source. Yields of HEWL protein were reduced relative to those obtained on more complex media. Gains in yield using complex media were offset by reduction in¹⁵N incorporation. No differences in either yield or kinetics of production were observed when ammonium¹⁵N chloride was replaced by unlabelled ammonium chloride as sole nitrogen source. Yields of¹⁵N-HEWL produced in this way are adequate for, and offer considerable advantages to, NMR studies of structure and folding of mutant and wild-type lysozymes.     

applications of electroporation of adherent cellsIn Situ,on a partly conductive slide

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowT _m of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.     

The migration game in habitat network: the case of tuna

Long-distance migration is a widespread process evolved independently in several animal groups in terrestrial and marine ecosystems. Many factors contribute to the migration process and of primary importance are intra-specific competition and seasonality in the resource distribution. Adaptive migration in direction of increasing fitness should lead to the ideal free distribution (IFD) which is the evolutionary stable strategy of the habitat selection game. We introduce a migration game which focuses on migrating dynamics leading to the IFD for age-structured populations and in time varying habitats, where dispersal is costly. The model predicts migration dynamics between these habitats and the corresponding population distribution. When applied to Atlantic bluefin tunas, it predicts their migration routes and their seasonal distribution. The largest biomass is located in the spawning areas which have also the largest diversity in the age-structure. Distant feeding areas are occupied on a seasonal base and often by larger individuals, in agreement with empirical observations. Moreover, we show that only a selected number of migratory routes emerge as those effectively used by tunas.     

Neodymium yttrium aluminium garnet laser photocoagulation for major haemorrhage from peptic ulcers and single vessels: a single blind controlled study.

A prospective single blind controlled trial was performed to assess the efficacy of the neodymium yttrium aluminium garnet laser in the management of patients bleeding from peptic ulcers and single vessels. Over 20 months 184 patients were found at endoscopy to be bleeding from peptic ulcers and single vessels; 130 of these failed to meet the criteria for entry into the study, but their bleeding stopped with conservative management. Forty-five patients entered the study: in all 25 who were bleeding from ulcers with spots the bleeding stopped irrespective of the treatment allocated. Twenty patients were bleeding from arteries. Eight were allocated to placebo treatment, and all later underwent emergency surgery for further haemorrhage. Twelve were allocated to receive laser treatment, and only one of eight who actually received it required surgery (p less than 0.002). It is concluded that laser treatment reduces the incidence of further haemorrhage and the need for emergency surgery, but the technique is difficult and not always applicable.     

Fermentation of xylose and hemicellulose hydrolysates by an ethanol-adapted culture ofBacteroides polypragmatus

Bacteroides polypragmatus type strain GP4 was adapted to grow in the presence of 3.5% (w/v) ethanol by successive transfers into 1% (w/v)d-xylose media supplemented with increasing concentrations of ethanol. The maximum specific growth rate of the ethanol-adapted culture (=0.30 h^-1) was not affected by up to 2% (w/v) ethanol but that of the non-adapted strain declined by about 50%. The growth rate of both cultures was limited by nutrient(s) contained in yeast extract. The ethanol yield of the adapted culture (1.01 mol/mol xylose) was higher than that (0.80 mol/mol xylose) of the non-adapted strain. The adapted culture retained the ability to simultaneously ferment pentose and hexose sugars, and moreover it was not inhibited by xylose concentrations of 7–9% (w/v). This culture also readily fermented hemicellulose hydrolysates obtained by mild acid hydrolysis of either hydrogen fluoride treated or steam exploded Aspen wood. The ethanol yield from the fermentation of the hydrolysates was comparable to that obtained from xylose.This paper is issued as NRCC No. 26338