Time Learning and Anticipatory Activity in Groups of Arctic Charr

Time learning and anticipatory activity were studied in five groups of 16–17 Arctic charr (Salvelinus alpinus) using self‐feeding devices with individual recognition (PIT (Passive Integrated Transponders)‐tags). The fish were kept under a LD 12:12 h cycle and periods of free access to the food were alternated with periods of time‐limited (2 h) access to food. Self‐feeding activity was significantly related to the light period in unrestricted conditions while related to the feeding periods during time‐limited access to food. The fish learned to concentrate their feeding activity to the restricted mealtime (>50% of the daily self‐feeding activity) within 10 d. The food anticipatory activity, measured as an increased self‐feeding activity before the feeding time and as aggressive interactions close to the trigger, was significant in both cases. The dominant individuals increased the trigger activity in advance of the time‐restricted reward period and subdominant individuals approached the trigger also in advance, inducing aggressive interactions. Thus, anticipating and learning a temporally predictable food source was pronounced in groups of self‐feeding Arctic charr.     

A Randomised Controlled Trial of Consent Procedures for the Use of Residual Tissues for Medical Research: Preferences of and Implications for Patients,Research and Clinical Practice

Background

Despite much debate, there is little evidence on consequences of consent procedures for residual tissue use. Here, we investigated these consequences for the availability of residual tissue for medical research, clinical practice, and patient informedness.

Methods

We conducted a randomised clinical trial with three arms in six hospitals. Participants, patients from whom tissue had been removed for diagnosis or treatment, were randomised to one of three arms: informed consent, an opt-out procedure with active information provision (opt-out plus), and an opt-out procedure without active information provision. Participants received a questionnaire six weeks post-intervention; a subsample of respondents was interviewed. Health care providers completed a pre- and post-intervention questionnaire. We assessed percentage of residual tissue samples available for medical research, and patient and health care provider satisfaction and preference. Health care providers and outcome assessors could not be blinded.

Results

We randomised 1,319 patients, 440 in the informed consent, 434 in the opt-out plus, and 445 in the opt-out arm; respectively 60.7%, 100%, and 99.8% of patients’ tissue samples could be used for medical research. Of the questionnaire respondents (N = 224, 207, and 214 in the informed consent, opt-out plus, and opt-out arms), 71%, 69%, and 31%, respectively, indicated being (very) well informed. By questionnaire, the majority (53%) indicated a preference for informed consent, whereas by interview, most indicated a preference for opt-out plus (37%). Health care providers (N = 35) were more likely to be (very) satisfied with opt-out plus than with informed consent (p = 0.002) or opt-out (p = 0.039); the majority (66%) preferred opt-out plus.

Conclusion

We conclude that opt-out with information (opt-out plus) is the best choice to balance the consequences for medical research, patients, and clinical practice, and is therefore the most optimal consent procedure for residual tissue use in Dutch hospitals.

Trial Registration

Dutch Trial Register NTR2982     

Structural differences between insulin receptors in the brain and peripheral target tissues

Insulin receptors in various brain regions (olfactory tubercle, hippocampus, and hypothalamus) were photoaffinity labeled using the photoreactive analogue of insulin B2(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). A protein with an apparent Mr of 400,000 was specifically labeled with 125I-NAPA-DP-insulin in all three brain regions. When radiolabeled proteins were reduced with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a protein with an apparent Mr of 115,000 and to a much lesser extent in a protein with an apparent Mr of 83,000. The size of these receptor proteins, based on their electrophoretic mobilities, was consistently smaller than insulin receptor proteins in adipocytes. The covalent labeling of insulin receptors in brain by 125I-NAPA-DP-insulin was not blocked by anti-insulin receptor antiserum. Additionally, in contrast to effects observed in peripheral target tissues, this antisera did not inhibit the binding of 125I-insulin to brain membranes. Neuraminidase treatment resulted in an increase in the electrophoretic mobilities of insulin receptor subunits in adipocytes, but, had no effect on receptor subunits in brain. Solubilized insulin receptors from adipocytes were retained by wheat germ agglutinin columns and specifically eluted with N-acetylglucosamine. In contrast, solubilized insulin receptors from brain did not bind to these columns. The results from this study indicate that structural differences, including molecular weight, antigenicity, and carbohydrate composition exist between insulin receptors in brain and peripheral target tissues.     

Investigations into the polymorphism of lipid A from lipopolysaccharides of Escherichia coli and Salmonella minnesota by Fourier-transform infrared spectroscopy

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5 degrees and 58 degrees C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41 degrees, 46 degrees, 44 degrees and 47 degrees C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4'-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52 degrees C for E. coli lipid A). The analysis of greater than CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22 degrees, 30 degrees, 27 degrees and 25.5 degrees C (first transition) and at about 34 degrees, 42 degrees, 38.5 degrees and 40.5 degrees C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: Up to about 30 degrees C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of greater than CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Shortened insulin with enhanced in vitro potency

After it has been shown that removal of residues B26-B30 leaves insulin with full biological activity, provided the new C-terminus is amidated (Fischer et al. (1985) Biol. Chem. Hoppe-Seyler 366, 521-525), it is demonstrated here that it does not even preclude enhancement of potency. 7 analogues of des-(B26-B30)-insulin-B25-amide were prepared by trypsin-mediated semisynthesis, the replacements being D-PheB24; HisB25, D-PheB25, TrpB25, TyrB25; D-PheB24,B25 and D-PheB24, TyrB25. Mere conversion of the configuration of B25-phenylalanine reduces in vitro potency to 0.5%. If B25-phenylalanine is, however, substituted by histidine or tyrosine activity is increased to 310 or 230, respectively. According to the features common to these two side chains, the favourable effect should be due to their ring structure with balanced aromatic and polar or H-bonding properties, respectively. The results indicate that in the complete insulin molecule the C-terminal pentapeptide modulates the subtle role that residues B24 and/or B25 play in receptor binding and activity; its presence may have a positive or negative effect. The drastic differences in activity between the shortened analogues are in no ways reflected in the CD spectra which are very similar, though clearly different from that of native insulin.     

Design and biological activity of a new generation of synthetic C3a analogues by combination of peptidic and non-peptidic elements.

Based on published X-ray crystallographic data of the anaphylatoxic complement peptide C3a, we have synthesized a series of peptides with appropriate amino acid exchanges and a maximal length of 13 amino acids. N-terminal acylation of these optimized structures with epsilon-aminohexanoic acid and complex aromatic structures like fluorenylmethoxycarbonyl, 2-nitro-4-azidophenyl, fluoresceinyl and rhodaminyl leads to a dramatic increase in biological activity. The culmination of our synthetic efforts is a C3a analogue with 13 amino acid residues and a biological activity six times that of native C3a.     

Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form.     

Reconstitution of the lipid matrix of the outer membrane of Gram-negative bacteria as asymmetric planar bilayer

Summary This paper is a report on the reconstitution of the lipid matrix of the outer membrane of Gram-negative bacteria as an asymmetric planar bilayer. This is the first time that a planar membrane is described, which consists on one side of a phospholipid (PL) mixture and on the other side of lipopolysaccharide (LPS). Therefore, strong emphasis is placed on a physical characterization of this membrane via its electrical properties. The membranes were prepared from spread monolayers or from vesicle-derived monolayers. Contrary to observations for symmetric phospholipid membranes, specific capacitances of (0.67±0.02) F·cm^–2, breakdown voltages between 200 and 400 mV and specific conductances between 10^–8 and 2×10^–7S·cm^–2 were obtained independent of the preparation method. The LPS-containing membranes were stable up to 3 hr if they were formed and kept at temperatures above the hydrocarbon chain melting temperature of the LPS. For the specific capacitance, a dependence on the aperture radius was observed. This is explained by assuming a toroidal transition zone at the rim of the aperture.First results on the action of the pore-forming -toxin fromStaphylococcus aureus on bilayers of different composition demonstrate particular characteristics of this asymmetric bilayer system. The pore-formation rate is highest in symmetric phospholipid bilayers, considerably lower in asymmetric PL/LPS systems and fully inhibited in LPS/LPS systems.     

Structure-function relationships of shortened [LeuB25]insulins, semisynthetic analogues of a mutant human insulin

Replacement of B25-phenylalanine by leucine in the insulin sequence causes marked inactivation. The effect of this sequence variation was studied here in des-(B26-30)-insulin. [LeuB25]des-(B26-30)-insulin and its B25-amide were prepared by trypsin-mediated semisynthesis from N-terminally protected des-(B23-30)-insulin and synthetic tripeptides. The relative lipogenic potency in isolated rat adipocytes was 8.0% for the truncated analogue with a free B25-carboxyl function, and 18.1% for the amidated analogue. Binding to cultured human IM-9 lymphocytes was 4% and 9%, respectively. Thus, both shortened insulins are markedly more active than [LeuB25]insulin. The PheB25----LeuB25 substitution in both the shortened and the full sequence has a moderate effect on the CD spectrum, indicating that the gross main chain conformation is largely retained in both molecules. Independent of the substitution an absolute increase of the circular dichroism is observed upon amidation of the B25-carboxyl group.