Begging for milk: evolution of teat massaging in suckling pigs

We present a model that simulates the evolution of teat massaging (begging) by suckling piglets (Sus scrofa) and milk distribution among teats (provisioning) by their mothers. Contrary to previous begging models, this one incorporates an ontogenetic dimension in that the inherited begging and provisioning rules are repeatedly allowed to interact, and their consequences for milk intake, growth and death probability are assessed, during each nursing. We test the model under three selection regimes differing in the relative importance of the between-litter selection component. We show that the selection regime with the strongest between-litter selection component leads to lowest begging levels and most effective milk utilization, thus supporting the hypothesis that selection based on whole litters may attenuate sibling competition.     

Catalytically active filaments - pyruvate decarboxylase from Neurospora crassa. pH-controlled oligomer structure and catalytic function

Pyruvate decarboxylase is a key enzyme in organisms whose energy metabolism is based on alcoholic fermentation. The enzyme catalyses the nonoxidative decarboxylation of 2-oxo acids in the presence of the cofactors thiamine diphosphate and magnesium ions. Pyruvate decarboxylase species from yeasts and plant seeds studied to date are allosterically activated by their substrate pyruvate. However, detailed kinetic studies on the enzyme from Neurospora crassa demonstrate for the first time the lack of substrate activation for a yeast pyruvate decarboxylase species. The quaternary structure of this enzyme species is also peculiar because it forms filamentous structures. The complex enzyme structure was analysed using a number of methods, including small-angle X-ray solution scattering, transmission electron microscopy, analytical ultracentrifugation and size-exclusion chromatography. These measurements were complemented by detailed kinetic studies in dependence on the pH.     

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.Pyruvate decarboxylases (EC 4.1.1.1) catalyze the non-oxidative decarboxylation of pyruvate, yielding acetaldehyde and carbon dioxide. Together with the enzyme alcohol dehydrogenase (EC 1.1.1.1), which reduces the acetaldehyde to ethanol with the help of the co-substrate NADH, it represents the metabolic pathway of alcoholic fermentation. PDC³ is localized in the cytosol of cells from yeasts, plant seeds, and a few bacteria. The catalytic activity of PDC depends on the presence of the cofactor thiamine diphosphate (ThDP), which is bound mainly via a divalent metal ion (magnesium in most cases) to the protein moiety. Many detailed kinetic studies have been published on yeast PDC wild types (1–9). A number of ScPDC variants were analyzed, too (1–9). Some active site variants (E51A, D28A, E477Q) proved to be almost catalytically inactive. PDCs are multisubunit enzymes. The typical molecular mass of one subunit is 59–61 kDa. The tetramer is the catalytically active state of most PDCs. Higher oligomers (octamers) have been described for PDCs from plant seeds (10, 11) or some fungi (12). However, studies on structure function relationships of yeast PDCs showed that the dimer is the minimum functional unit of the enzyme displaying considerable catalytic activity (13, 14). The two closely related pyruvate decarboxylases from Saccharomyces cerevisiae (ScPDC) and Kluyveromyces lactis (KlPDC) are well characterized ThDP-dependent enzymes, which share 86.3% identical amino acid residues. They have been studied in great detail by means of kinetic investigations and spectroscopic studies. Both enzymes are allosterically regulated as reflected by sigmoid steady state kinetics and lag phases in their progress curves. The substrate PYR activates the initially inactive yeast PDCs in a time-dependent manner. Kinetic studies reveal a slow isomerization as triggered by substrate binding to a separate regulatory site (15). A number of substrate surrogates have been identified, which are able to activate PDC as well. The effects of pyruvamide (PA; for the chemical structure, see Scheme 1) on the activation kinetics have been studied in detail for ScPDC (15) and for KlPDC (16). Phosphonate analogues (among them methyl acetylphosphonate, MAP, Scheme 1) of pyruvate have been applied to elucidate the catalytic cycle (17–21) or to trap reaction intermediates in crystal structures (22–24). Chemical modification of PDCs with group-specific reagents pointed to an important role of cysteine residues (25). Site-directed mutagenesis of cysteine residues to alanine or serine demonstrated that residue Cys-221 might be the decisive one for enzyme activation (1, 4, 26, 27). Consequently, it was postulated that the region around Cys-221 is the regulatory site of PDC, and formation of a thiohemiketal at this side chain was proposed. However, a number of questions remained elusive. (i) How is the activator fixed at the regulatory site? (ii) What are the prime structural properties of the active state as compared with the inactive state? (iii) How is the signal transmitted from the regulatory to the active site? (iv) Which are the decisive features of the active site in the activated state that render efficient catalysis possible? To answer these questions, we present here the crystal structures of KlPDC with the bound substrate surrogate MAP and of the ScPDC variants D28E and E477Q with bound substrate PYR along with kinetic studies on the activating effect of both activators and binding studies using the small angle x-ray solution scattering (SAXS) method.Open in a separate windowSCHEME 1.Chemical structures of the substrate pyruvate, the activators pyruvamide and methyl acetylphosphonate, and the thiohemiketal from pyruvate and cysteine, respectively.     

Pyruvate decarboxylase from Kluyveromyces lactis. An enzyme with an extraordinary substrate activation behaviour.

Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady-state kinetics, stopped-flow measurements, and kinetic dilution experiments. The dependence of the observed activation rate constant on the substrate concentration of pyruvate decarboxylase from K. lactis showed a minimum at a pyruvate concentration of 1.5 mm. According to the mechanism of substrate activation suggested this local minimum occurs due to the big ratio of the dissociation constants for the binding of the first (regulatory) and the second (catalytic) substrate molecule. The microscopic rate constants of the substrate activation could be determined by a refined fit procedure. The influence of the artificial activator pyruvamide on the activation of the enzyme was studied.     

Mammalian play: training for the unexpected.

In this review, we present a new conceptual framework for the study of play behavior, a hitherto puzzling array of seemingly purposeless and unrelated behavioral elements that are recognizable as play throughout the mammalian lineage. Our major new functional hypothesis is that play enables animals to develop flexible kinematic and emotional responses to unexpected events in which they experience a sudden loss of control. Specifically, we propose that play functions to increase the versatility of movements used to recover from sudden shocks such as loss of balance and falling over, and to enhance the ability of animals to cope emotionally with unexpected stressful situations. To obtain this "training for the unexpected," we suggest that animals actively seek and create unexpected situations in play through self-handicapping; that is, deliberately relaxing control over their movements or actively putting themselves into disadvantageous positions and situations. Thus, play is comprised of sequences in which the players switch rapidly between well-controlled movements similar to those used in "serious" behavior and self-handicapping movements that result in temporary loss of control. We propose that this playful switching between in-control and out-of-control elements is cognitively demanding, setting phylogenetic and ontogenetic constraints on play, and is underlain by neuroendocrinological responses that produce a complex emotional state known as "having fun." Furthermore, we propose that play is often prompted by relatively novel or unpredictable stimuli, and is thus related to, although distinct from, exploration. We present 24 predictions that arise from our new theoretical framework, examining the extent to which they are supported by the existing empirical evidence and contrasting them with the predictions of four major alternative hypotheses about play. We argue that our "training for the unexpected" hypothesis can account for some previously puzzling kinematic, structural, motivational, emotional, cognitive, social, ontogenetic, and phylogenetic aspects of play. It may also account for a diversity of individual methods for coping with unexpected misfortunes.     

Dimensions of maternal behaviour characteristics in domestic and wildxdomestic crossbred sows

We examined the maternal behaviour of seven domestic and seven wildxdomestic primiparous sows during 10 days post partum to investigate two questions: (1) Did maternal behaviour change during domestication? (2) Can the interindividual variability of maternal behaviour be subsumed into a few dimensions of maternal temperament? We recorded: (a) willingness to leave the nest for food on Day 2; (b) reaction to a playback of squeezed piglet distress vocalisation on Day 2; (c) spontaneous nursing behaviour and spontaneous lying-down behaviour on Day 5 (from an overnight video recording); (d) reactions to playbacks of various piglet distress vocalisations on Day 6 and (e) reactions to a human in the 'nest' with piglets on Day 9. Moreover, data on baseline cortisol saliva concentration and its increase during a brief transportation period and novel environment challenge at the age of 5 months were available. Crossbred sows did not differ from domestic ones in any aspect of maternal behaviour except for a higher tendency to terminate final massage during nursings and a higher frequency of changing posture from lying to standing and back during the night. Factor analysis (based on correlation matrix of 11 behaviour and cortisol variables calculated for all 14 sows after removing the effect of breed) indicated that 82% of the variability in the data could be explained by three factors: first, 'calmness' on which low night time frequency of major posture changes, carefulness of lying-down behaviour and high propensity to remain in nursing position after milk ejection loaded positively while cortisol concentrations during challenge loaded negatively; second, 'protectiveness' with high loadings of the reaction scores to the playbacks of piglet distress calls and the human presence near the piglets; and third, 'nursing activity' which was strongly positively associated with nursing frequency, and negatively with the proportion of nutritive nursings and baseline cortisol values. The results indicate that most aspects of pig maternal behaviour have not been significantly changed by domestication and that substantial variability in maternal behaviour exists between sows, perhaps in the form of several behaviour characteristics which encompass both behaviour and endocrine profiles of the sows.     

The Influence of Protein Concentration on Oligomer Structure and Catalytic Function of Two Pyruvate Decarboxylases

As a general rule protein concentration typical for structural studies differs considerably from that chosen for kinetic investigations. Consequently, structure-function relationships are often postulated without appropriate knowledge, whether the functional behaviour of the enzyme is the same in both protein concentration ranges. To deal with this question, substrate activation kinetics of two well-characterised yeast pyruvate decarboxylases, from Saccharomyces cerevisiae and from Kluyveromyces lactis, were analysed over the broad protein concentration range 2-2,000 μg/mL. Analytical ultracentrifugation and small-angle X-ray scattering were used to analyse the enzymes’ oligomer structure in aqueous solution. For the upper part of the concentration range the determined parameters, like catalytic activity, observed substrate activation rates, sedimentation coefficients and scattering parameters are independent on enzyme concentration changes. No indication of protein aggregation is detectable. However, significant changes occur at low enzyme concentration. The catalytically active tetramer dissociates progressively into dimers with comparable catalytic activity, but with significantly accelerated substrate activation.     

Phosphorylation of serine 264 impedes active site accessibility in the E1 component of the human pyruvate dehydrogenase multienzyme complex

At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residues in the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHc correlates mostly with phosphorylation of serine 264, which is located at the entrance of the substrate channel leading to the active site of E1. Despite intense investigations, the molecular mechanism of this inactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in human wild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation of Ser264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvate decarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active site via the substrate channel and the subsequent reductive acetylation of the E2 component are severely slowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Glu determined by X-ray crystallography reveals no differences in the three-dimensional architecture of the phosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However, the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant. By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glu suggest that phosphorylation blocks access to the active site by imposing a steric and electrostatic barrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant, which carries no charge at position 264, is also selectively deficient in pyruvate binding and reductive acetylation of E2, we conclude that mostly steric effects account for inhibition of PDHc by phosphorylation.