Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.     

CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.

An algorithm is presented for the calculation of metal ion concentrations from given total metal concentrations (and vice versa) in physiological media containing metal-chelating compounds. In such media, conditions differ from those used for stability constant determination of metal-chelator equilibria; therefore calculated metal ion concentrations are incorrect. We recompute stability constants to reflect the effects of ionic strength and temperature of physiological solutions. Twelve different equilibria can be considered per metal-chelator pair. The computer program also calculates the contribution of ionized species of metals, chelator, complexes and pH buffers to ionic strength. Measurements with a Ca-selective electrode and with fura-2 show that calculated ionic Ca2+ concentrations are correct from 10 nM up to the millimolar range. The importance of the correct calculation of metal ion concentrations in physiological experiments is demonstrated by data, and derived kinetic parameters, on Na+/Ca2+ exchange and the ATP-dependent Ca2+ pump of enterocyte plasma membrane vesicles. The program is written in Turbo Pascal and will run on IBM-compatible computers. It is menu-driven and supports the use of a Microsoft mouse.     

Study on the enzymatic 5′-deiodination of 3′,5′-diiodothyronine using a radioimmunoassay for 3′-iodothyronine

A radioimmunoassay for 3′-iodothyronine has been developed. All iodothyronine analogues (except 3,3′-diiodothyronine) showed very little (0.02% at most) cross-reactivity, and the assay was sensitive to 1 pg 3′-iodothyronine/ tube. We have studied the 5′-deiodination of 3′,5′-diiodothyronine by rat liver microsomal fraction in the presence of dithiothreitol. Production of 3′-iodothyronine at 37°C was found to be linear with time of incubation up to 30 min and with concentration of microsomal protein up to 100 μg/ml. The reaction rate reached a limit on increasing 3′,5′-diiodothyronine concentration to 10 μM. The effect of pH on 3′-iodothyronine production was found to depend on 3′,5′-diiodothyronine concentration. Increasing 3′,5′-diiodothyronine concentration from 0.1 to 10 μM resulted in a shift of the pH optimum from 6–6.5 to 7.5. Similar effects on the 5′-deiodination of 3,3′,5′-triiodothyronine were observed, supporting the hypothesis that these reactions are catalysed by a single enzyme (iodothyronine 5′-deiodinase).     

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors'' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants'' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.     

The costs of egg production and incubation in great tits (Parus major).

The costs of egg production and incubation may have a crucial effect on avian reproductive decisions, such as clutch size and the timing of reproduction. We carried out a brood-size enlargement experiment on the great tit (Parus major), in which the birds had to lay and incubate extra eggs (full costs), only incubate extra eggs (free eggs) or did not pay any extra cost (free chicks) in obtaining a larger brood. We used female fitness (half the recruits produced plus female survival) as a fitness measure because it is the female which pays the costs of egg production and incubation, and because clutch size is under female control. Female fitness decreased with increasing costs (fitness of free chicks females is higher than that of free eggs females which is higher than that of full costs females). These fitness differences were due to differences in female survival rather than in the number of recruits produced. This is the first time that the costs of egg production and incubation have been estimated using such a complete fitness measure, including, as our measure does, the local survival to the following year of both the female and her offspring. Our results emphasize that reproductive decisions cannot be understood without taking egg production and incubation costs into account.     

Localization of99mTc in bone by means of autoradiography

The uptake of two different preparations of^99mTechnetium-methylene diphosphonate in fetal rat calvaria is compared. The localization of^99mTc after administration of^99mTc(Sn)-MDP and^99mTc-MDP showed equal distribution in autoradiography.     

Ruddy turnstones Arenaria interpres rapidly build pectoral muscle after raptor scares

To cope with changes in the environment, organisms not only show behavioural but also phenotypic adjustments. This is well established for the digestive tract. Here we present a first case of birds adjusting their flight machinery in response to predation risk. In an indoor experiment, ruddy turnstones Arenaria interpres were subjected to an unpredictable daily appearance of either a raptor or a small gull (as a control). Ruddy turnstones experiencing threat induced by a flying raptor model, longer than after similar passage by the gull model, refrained from feeding after this disturbance. Pectoral muscle mass, but not lean mass, responded in a course of a few days to changes in the perceived threat of predation. Pectoral muscle mass increased after raptor scares. Taking the small increases in body mass into account, pectoral muscle mass was 3.6% higher than aerodynamically predicted for constant flight performance. This demonstrates that perceived risk factors may directly affect organ size.