Validation of the doubly labeled water method in growing precocial birds: the importance of assumptions concerning evaporative water loss

The doubly labeled water (DLW) method was validated against respiration gas analysis in growing precocial chicks of the black-tailed godwit (Limosa limosa) and the northern lapwing (Vanellus vanellus). To calculate the rate of CO2 production from DLW measurements, Lifson and McClintock's equations (6) and (35) were employed, as well as Speakman's equation (7.17) (all single-pool models). The average errors obtained with the first two equations (+7.2% and -11.6%, respectively) differed significantly from zero but not the error obtained with Speakman's equation (average: -2.9%). The latter error could be reduced by taking a fractional evaporative water loss of 0.13, instead of the value of 0. 25 recommended by Speakman. Application of different two-pool models resulted in relative errors of the DLW method of -15.9% or more. After employing the single-pool model with a fractional evaporative water loss value of 0.13, it was found that there was no relationship between the relative growth rate of the chick and the relative error of the DLW method. Recalculation of previously published results on Arctic tern (Sterna paradisaea) chicks revealed that the fit of the validation experiment could be considerably improved by employing a single-pool model and assuming a fractional evaporative water loss of 0.20 instead of the value of 0.50 taken originally. After employing the value of 0.20, it was found that there was no relationship between the relative growth rate of the chick and the relative error of the DLW method. This suggests that isotope incorporation into new body substances does not cause a detectable error. Thus, the DLW method seems to be applicable in young birds growing as fast as 20% d-1, after making adjustments for the fractional evaporative water loss. We recommend Speakman's equation (7.17) for general use in growing birds when evaporation is unknown.     

Determination of myoglobin concentration and oxidative capacity in cryostat sections of human and rat skeletal muscle fibres and rat cardiomyocytes

Myoglobin plays various roles in oxygen supply to muscle mitochondria. It is difficult, and in some cases impossible, to study the relationship between the myoglobin concentration and the oxidative capacity of individual muscle cells because myoglobin has to be fixed in situ whereas determination of oxidative capacity, for example, succinate dehydrogenase activity, requires unfixed cryostat sections. We have investigated whether a vapour-fixation technique allows the use of serial sections to study the relationship between myoglobin and succinate dehydrogenase activity. The technique is used to study a rat soleus muscle, two human skeletal muscle biopsies and biopsies of two patients with chronic heart failure, and in a control and hypertrophied rat heart. Staining intensities were quantified by microdensitometry. The absorbance values were calibrated using sections cut from gelatine blocks containing known amounts of myoglobin. The results show that it is possible to use serial sections for the determination of the myoglobin concentration and succinate dehydrogenase activity, and indicate that myoglobin can lead to a substantial reduction (18–60%) of the extracellular oxygen tension required to prevent an anoxic core in muscle cells.     

Interactions between ethylene and gibberellins in phytochrome-mediated shade avoidance responses in tobacco

Plants respond to proximate neighbors with a suite of responses that comprise the shade avoidance syndrome. These phytochrome-mediated responses include hyponasty (i.e. a more vertical orientation of leaves) and enhanced stem and petiole elongation. We showed recently that ethylene-insensitive tobacco (Nicotiana tabacum) plants (Tetr) have reduced responses to neighbors, showing an important role for this gaseous plant hormone in shade avoidance. Here, we investigate interactions between phytochrome signaling and ethylene action in shade avoidance responses. Furthermore, we investigate if ethylene acts in these responses through an interaction with the GA class of hormones. Low red to far-red light ratios (R:FR) enhanced ethylene production in wild-type tobacco, resulting in shade avoidance responses, whereas ethylene-insensitive plants showed reduced shade avoidance responses. Plants with inhibited GA production showed hardly any shade avoidance responses at all to either a low R:FR or increased ethylene concentrations. Furthermore, low R:FR enhanced the responsiveness of hyponasty and stem elongation in both wild-type and Tetr plants to applied GA(3), with the stem elongation process being more responsive to GA(3) in the wild type than in Tetr. We conclude that phytochrome-mediated shade avoidance responses involve ethylene action, at least partly by modulating GA action.     

Toxic and nontoxic microcystis colonies in natural populations can be differentiated on the basis of rRNA gene internal transcribed spacer diversity

Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.     

How do aldehyde side products occur during alkene epoxidation by cytochrome P450? Theory reveals a state-specific multi-state scenario where the high-spin component leads to all side products

A theoretical study of alkene epoxidation, by the high-valent iron-oxo species (Compound I) of cytochrome P450, reveals a multi-state scenario in which the different products are generated in a state specific manner. All the low-spin doublet state processes are effectively concerted epoxide producing pathways. By contrast, all the high-spin quartet processes are stepwise and either lead to epoxide that does not conserve the isomeric identity of the alkene (cis or trans), or/and to by-products such as suicidal complexes and aldehydes. The product/state inventory is the following: (a) The epoxide with conserved alkene stereochemistry is generated from the low-spin doublet states of Compound I in a nonsynchronous but effectively concerted pathways that involve carbon radical (with Fe(III) and Fe(IV)) and cationic intermediates. (b) The epoxide with scrambled alkene stereochemistry is obtained from the quartet high-spin radical intermediate (with Fe(IV)). (c) The suicidal complex, with a C-N bond between the alkene and the porphyrin, is obtained from the high-spin cationic state that possesses one electron in the sigma xy* orbital (the antibonding Fe-N orbital made from dxy and nitrogen sigma-hybrids). (d) The aldehyde by-product is obtained from the high-spin cationic state that possesses one electron in the sigma xy* orbital (the antibonding O-Fe-S orbital made from dz2 and the oxo and sulfur sigma-hybrids). Factors controlling the competition between these processes are discussed.     

Central assumptions of predator-prey models fail in a semi-natural experimental system

The relationship between the encounter rate of predators with prey and the density of this prey is fundamental to models of predator-prey interactions. The relationship determines, among other variables, the rate at which prey patches are depleted, and hence the impact of predator populations on their prey, and the optimal spatial distribution of foraging effort. Two central assumptions that are made in many models are that encounter rate is directly proportional to prey density and that it is independent of the proportion of prey already removed, other than via the decreased density. We show here, using captive great tits searching for winter moth caterpillars in their natural hiding positions, that neither of these assumptions hold. Encounter rate increased less than directly in proportion to prey density, and it depended not only on the current density of prey, but also on the proportion of prey already removed by previous foragers. Both of these effects are likely to have major consequences for the outcome of predator-prey interactions.     

Oncostatin M-stimulated apical plasma membrane biogenesis requires p27(Kip1)-regulated cell cycle dynamics

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.     

Density dependence,territoriality, and divisibility of resources: from optimality models to population processes

Species differ enormously in their territorial systems. Some species defend only small areas surrounded by undefended space, while others defend large contiguous territories. Using an optimization approach, we show that this variation can be explained from the density of two types of resources: divisible and nondivisible. We assume that benefits of territories are monotonously related to the defended amount of divisible resources (hereafter called food). In contrast, no benefits are obtained without a nondivisible resource (hereafter called nest site) in the territory, while more than one nest site does not further increase the benefits. The optimal territory size depends on the relative abundance of these resources. With a low density of nest sites, the optimal territory size is small and includes only the nest site. If the density of nest sites is relatively large, the optimal territory size is high, and territories are contiguous. Competition for these different resources yields contrasting patterns of how populations are regulated. If there is mainly competition for nest sites, we expect density-dependent exclusion through territoriality and no density-dependent reproduction. When competition is mainly for food, we expect density-dependent reproduction because optimal territory size will be compressed at higher densities, resulting in lower reproductive success. These predicted patterns indeed are observed in some well-studied passerine species for which both the territorial system and the occurrence of density dependence is known.