Dietary restriction causes chronic elevation of corticosterone and enhances stress response in red-legged kittiwake chicks

Release of corticosterone in hungry kittiwake chicks facilitates begging and allows them to restore depleted energy reserves by increasing parental food provisioning. However, in order to avoid detrimental effects of chronic elevation of corticosterone, chicks might suppress adrenocortical activity in response to prolonged food shortages. In this study we examined temporal dynamics of corticosterone release in red-legged kittiwake (Rissa brevirostris) chicks exposed to prolonged restrictions in energy content and/or nutritional quality (low versus high lipid content) of their food. Starting at the age of 15 days, chicks were fed either high- or low-lipid fish at 40%, 65%, and 100% of ad libitum energy intake. Body mass measurements and baseline plasma samples were taken on a weekly basis after beginning of the treatment. After 3 weeks of treatment, chicks were exposed to a standardized acute handling and restraint stress protocol, where in addition to a baseline sample, three plasma samples were taken at intervals up to 50 min. We found that food-restricted chicks had lower body mass, chronically (during 2-3 weeks) elevated baseline and higher acute stress-induced levels of corticosterone compared to chicks fed ad libitum. Low lipid content of food further exacerbated these effects. An increase in baseline levels of corticosterone was observed within a week after energy requirements of food-restricted chicks exceeded their daily energy intake. A tendency for suppression of adrenocortical activity was observed in treatments fed low-lipid diets only at the end of the experiment. We suggest that nest-bound chicks, if food-stressed, might suffer deleterious effects of chronic elevation of corticosterone.     

Primers for the amplification of sequence‐characterized loci in Cryphonectria cubensis populations

We describe the development of DNA markers for the fungal pathogen of Eucalyptus, Cryphonectria cubensis. These markers originated from cloned intershort sequence repeat polymerase chain reactions, which enrich for medium to highly repetitive DNA sequences. In total, 10 markers were isolated, eight of which were polymorphic, and these can subsequently be applied to study populations of C. cubensis.     

Plasmids in Leuconostoc oenos

A new procedure was used to isolate 11 plasmids from eight Leuconostoc oenos strains. Plasmid DNA was not detected in 34 other strains of this species. Plasmid sizes ranged from 2.47 to 4.61 kilobase pairs. This is the first report of extrachromosomal elements in L. oenos.     

N-terminal-methionylated interleukin-1 beta has reduced receptor-binding affinity

The receptor-binding affinity of recombinant-derived interleukin-1 beta containing unprocessed N-terminal methionine (MAPV-) was 10-fold lower than protein containing the authentic N-terminal sequence (APV-). Structural analysis of the methionylated and non-methionylated proteins by NMR spectroscopy detected no (or minor) conformational differences. The differences in binding affinity, therefore, suggest that the additional N-terminal methionine causes a small, direct or indirect, perturbation of the receptor-binding region.     

A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin

Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.     

Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.     

Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing Escherichia coli by Real-Time PCR

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.     

Determination of the positions of bound water molecules in the solution structure of reduced human thioredoxin by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy.

The presence of bound water molecules in the solution structure of reduced human thioredoxin has been investigated using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. It is demonstrated that the backbone amide protons of Lys21, Lys39, Lys82, Gly83 and Asn102, as well as the side-chain amide group of Asn102, are in close proximity to bound water molecules. Examination of the high-resolution solution structure of reduced human thioredoxin reveals that these results are best accounted for by four bound water molecules. Subsequent simulated annealing calculations carried out on the basis of interproton distance and hydrogen bonding restraints to the bound water molecules, supplemented by the original set of experimental restraints used in the calculation of the three-dimensional structure of human thioredoxin, permit a more precise localization of the bound water positions. Potential hydrogen bonds to these water molecules are described and a comparison is made to corresponding bound water molecules in the crystal structure of oxidized Escherichia coli thioredoxin.     

Enzymology of ubiquinone-utilizing electron transfer complexes in nonionic detergent.

The enzymology of isolated succinate: ubiquinone reductase and ubiquinone: cytochrome c reductase in nonionic detergents (alkyl polyoxyethylene derivatives) was studied. In the membrane the two multiprotein complexes and their hydrophobic substrates ubiquinone and dihydroubiquinone, are embedded in a common lipid bilayer. In detergent solutions the complexes are each inserted into micelles. Detergent micelles also serve as a solvent for the complexes hydrophobic substrates. As a consequence the isolated complexes are in a discontinuous phase with respect to their hydrophobic substrates and with respect to each other. Three types of assays were used. Firstly, single enzyme assays in which the hydrophobic substrates had to transfer from free micelles to the complex-bound micelles in order for enzyme reactions to occur. Secondly, assays in which the enzymic reactions were coupled to auxiliary nonenzymic reactions which rapidly converted the hydrophobic products back into substrates within the complex-bound micelle. Dichloroindophenol was used for the oxidation of dihydroubiquinone and dihydroduroquinone for the reduction of ubiquinone. Thirdly, assays in which the succinate: ubiquinone reductase reaction was coupled with the ubiquinone: cytochrome c reductase reaction. With the first type of assay, the kinetics of the substrate transfer reaction was dependent upon the type of detergent. In detergents with small polyoxyethylene head groups the transfer reactions were rate-limiting, and in detergents with large polyoxyethylene head groups the transfer reactions were fast and the enzymic reactions were rate-limiting...