A mechanistic model of nutritional control of protein synthesis in animal tissues

Regulation of mRNA translation has been held responsible for effects of diet, age, alcohol, hormones, hibernation, disease and hypoxia on protein synthesis in animal tissues. Dietary effects are due to concentrations of amino acids and insulin in circulation that affect activities of two key translational regulators, eukaryotic initiation factor 2 (F2) and eukaryotic initiation factor 4E binding protein 1 (Bp). To construct a platform for prediction of global protein synthesis to nutritional stimuli, a dynamic, mechanistic model of translational control in whole tissues was developed. The model was composed of a set of differential equations which describe the dynamics of 11 state variables: tRNA and acyl-tRNA for leucine (Leu), limiting (Laa) and other amino acids (Oaa), inactivated F2 with GDP (F2d), activated F2 with GTP (F2t), F4e, Bp and its complex with F4e (4eBp), available mRNA start codons (AUG), and active ribosomes (Arib). Material was assumed to flow from one variable to another according to mass-action kinetics or Michaelis-Menten form. Uncharged tRNA inhibit GTP exchange on eIF2, and free amino acids and insulin inhibit reversible sequestration of F4e by Bp. Initial conditions and parameters were set for a skeletal muscle fractional synthesis rate of 10%/d and ribosome transit time of 80 s. Between amino acid concentrations of 500 and 4000×10³ nM, protein synthesis increased from 0.9 to 11.7%/d at 0 μU/mL insulin, and from 5.0 to 12.8%/d at 30 μU/mL insulin. Predicted responses to graded levels of a deficient amino acid were asymptotic. A single parameter accomodated differences between tissues in insulin sensitivity. Seven parameters must be changed to simulate initiation and elongation rates in more active tissues such as liver, or in tissues of older mature animals. An increase in uncharged tRNA during insulin stimulation highlighted the physiological importance of coordinated regulation of amino acid supply by insulin. In conclusion, the regulation of F4e release from Bp by Ins and Leu, and of F2d recycling by uncharged tRNA can be tied together to describe a wide range of FSR values across tissues and physiological states.     

Spatial structuring of an evolving life-history strategy under altered environmental conditions

Human disturbances to ecosystems have created challenges to populations worldwide, forcing them to respond phenotypically in ways that increase their fitness under current conditions. One approach to examining population responses to disturbance in species with complex life histories is to study species that exhibit spatial patterns in their phenotypic response across populations or demes. In this study, we investigate a threatened population of fall chinook salmon (Oncorhynchus tshawytscha) in the Snake River of Idaho, in which a significant fraction of the juvenile population have been shown to exhibit a yearling out-migration strategy which had not previously been thought to exist. It has been suggested that dam-related environmental changes may have altered the selective pressures experienced by out-migrating fall chinook, driving evolution of a later and more selectively advantageous migration strategy. Using isotopic analysis of otoliths from returning adult spawners, we reconstructed the locations of individual fish at three major juvenile life stages to determine if the representation of the yearling life history was geographically structured within the population. We reconstructed juvenile locations for natal, rearing and overwintering life stages in each of the major spawning areas in the basin. Our results indicate that the yearling life-history strategy is predominantly represented within one of the main spawning regions, the Clearwater River, rather than being distributed throughout the basin. Previous studies have shown the Clearwater River to have cooler temperatures, later hatch dates, and later outmigration of juveniles, indicating a link between environment and expression of the yearling life history. Our data suggest that this new yearling life history may be disproportionally represented in returning adult spawners, indicating selection for this life history within the population.     

Sex-specific development of avian flight performance under experimentally altered rearing conditions

Numerous studies have examined predation risk resulting fromthe costs of impaired flight performance associated with manykey life-history stages such as reproduction and migration.Interestingly, although avian nestlings experience multipleresource-based physiological trade-offs and undergo considerablemorphological and physiological changes during postnatal development,there is no data available on how nestlings manage the competingdemands of growth and the development of flight ability at thiscritical life-history stage. We examined numerous morphologicaltraits to determine which are responsible for variation in flightperformance in juvenile European starlings (Sturnus vulgaris),a sexually size-dimorphic passerine. We then manipulated maternalquality during chick rearing (via feather clipping) to examinesex-specific sensitivity of fledgling flight performance tothe quality of the rearing environment. Results suggest thatthe mechanics underlying variation in juvenile flight performanceare relatively simple, being principally determined by the ratioof pectoral muscle mass to body mass (BM) and the surface areaof the wings. Interestingly, although the maternal quality manipulationdecreased BM and structural size in daughters, only the flightperformance of sons was negatively affected. Our results suggestthat a survival-related trait can be significantly affectedin the larger sex when raised under stressful conditions. Furthermore,measuring only BM and structural size may not be sufficientin understanding how the sexes are affected by stressful rearingconditions in sexually size-dimorphic species.     

Accommodation properties of isolated hippocampal neurons under conditions of an experimental model of epilepsy

A pilocarpine/lithium model of status epilepticus is an effective tool allowing one to study the principles of development of temporal epilepsy. It is believed that, in addition to the corresponding modifications of the efficacy of synaptic transmission, changes in the endogenous properties of neuronal activity can promote repetitive epileptiform activity. We measured the accommodation parameters of spike generation by isolated neurons of the CA1 hippocampal area obtained from 14-day-old rats 2 or 24 h after they had been subjected to an epileptization procedure, as well as from control rats of the same age. The spike activity of the neurons was initiated by their depolarization with a long-lasting stimulus in a current-clamp mode under conditions of perforated patch clamp. We found that the initial phase of accommodation manifested as a rapid increase in interspike intervals immediately after application of the depolarizing stimulus became significantly shorter in rats 24 h after epileptization; at the same time, the characteristics of the late phase of accommodation underwent no changes. In addition, the mean number of generated action potentials dropped. Such changes were not found in neurons of rats 2 h after epileptization. It is hypothesized that the above effect is compensatory and not injuring; it can develop because of prolonged abnormal activation of neurons in the course of epileptic attacks. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 211–218, May–June, 2006.     

Application of an Escherichia coli green fluorescent protein-based lysine biosensor under nonsterile conditions and autofluorescence background

AIMS: To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism. METHODS AND RESULTS: Escherichia coli lysine auxotroph GFP based biosensor was used to determine the percentage of bioavailable lysine in two samples of soybean-, cottonseed-, and meat and bone meal under nonsterile conditions. The fluorescence emitted by GFP was successfully measured using a spectrofluorimeter to monitor bacterial growth response to protein-derived lysine and lysine containing small peptides. The autofluorescence of analyzed feed samples at different concentrations could also be estimated. CONCLUSIONS: When feed protein concentrations are decreased, autofluorescence interference can be avoided. SIGNIFICANCE: The E. coli lysine auxotroph GFP-based biosensor can successfully be used for the determination of bioavailable lysine in these selected animal feed proteins under nonsterile conditions. IMPACT OF THE STUDY: E. coli GFP biosensor for lysine has potential for routine application in animal feeds.     

Seasonal regulation of reproduction: altered role of melatonin under naturalistic conditions in hamsters

The seasonal reproductive cycle of photoperiodic rodents is conceptualized as a series of discrete melatonin-dependent neuroendocrine transitions. Least understood is the springtime restoration of responsiveness to winter-like melatonin signals (breaking of refractoriness) that enables animals to once again respond appropriately to winter photoperiods the following year. This has been posited to require many weeks of long days based on studies employing static photoperiods instead of the annual pattern of continually changing photoperiods under which these mechanisms evolved. Maintaining Siberian hamsters under simulated natural photoperiods, we demonstrate that winter refractoriness is broken within six weeks after the spring equinox. We then test whether a history of natural photoperiod exposure can eliminate the requirement for long-day melatonin signalling. Hamsters pinealectomized at the spring equinox and challenged 10 weeks later with winter melatonin infusions exhibited gonadal regression, indicating that refractoriness was broken. A photostimulatory effect on body weight is first observed in the last four weeks of winter. Thus, the seasonal transition to the summer photosensitive phenotype is triggered prior to the equinox without exposure to long days and is thereafter melatonin-independent. Distinctions between photoperiodic and circannual seasonal organization erode with the incorporation in the laboratory of ecologically relevant day length conditions.     

Development of hind limb bones in chicken embryos under altered temperature and humidity conditions

The data on growth and development of hind limb bones in chicken embryos under normal (standard) and altered incubation conditions are presented.     

Genetically altered animal models in the kallikrein-kinin system

Transgenic and gene-targeting technologies allowing the generation of genetically altered animal models have greatly advanced our understanding of the function of specific genes. This is also true for the kallikrein-kinin system (KKS), in which some, but not yet all, components have been functionally characterized using such techniques. The first genetically altered animal model for a KKS component was supplied by nature, the brown Norway rat carrying an inactivating mutation in the kininogen gene. Mice deficient in tissue kallikrein, B1 and B2 receptors, some kinin-degrading enzymes, and factor XII followed, together with transgenic rat and mouse strains overexpressing tissue kallikrein, B1 and B2 receptors, and degrading enzymes. There are still no animal models with genetic alterations in plasma kallikrein, kininases I and some other degrading enzymes. The models have confirmed an important role of the KKS in cardiovascular pathology, inflammation, and pain, and have partially elucidated the distinct function of the two receptors. This created the basis for rational decisions concerning the putative use of kinin receptor agonists and antagonists in therapeutic applications. However, a more thorough analysis of the existing models and the generation of new, more sophisticated transgenic models will be necessary to clarify the still elusive issue as to where and by which mechanisms the kinins exert their actions.     

Sex-specific development of cell-mediated immunity under experimentally altered rearing conditions in blue tit nestlings

In birds, poor rearing conditions usually have negative effects on T-cell-mediated immune response. However, earlier studies demonstrate that fitness-related traits such as body mass may show sex-specific patterns when subject to alteration of rearing conditions. Therefore, to investigate whether deterioration of rearing conditions influences the development of immune function differently in male and female nestlings, we performed brood size manipulation experiments on blue tit (Parus caeruleus) nestlings. To alter rearing conditions, some broods were increased by three nestlings soon after hatching, while other broods were left non-manipulated. Immune response was assessed as a hypersensitivity reaction to phytohaemagglutinin in 11-day-old nestlings. Additionally, we studied the consequences of brood size manipulation for fledgling body mass and tarsus length. The enlargement of brood size had different effects on the cellular immune responses of male and female nestlings, with males being more negatively affected than their female nest-mates. Sex-specific effects of poor rearing conditions were also recorded for tarsus length, such that tarsus growth was more retarded in female than in male nestlings. We discuss the effects of deterioration of rearing conditions on sex-specific development of cell-mediated immunity with respect to sexual dimorphism of size and developmental strategies in male and female nestlings.     

Development of hind limb bones in chicken embryos under altered temperature and humidity conditions

The data on growth and development of hind limb bones in chicken embryos under normal (standard) and altered incubation conditions are presented.     

Ethanol inhibition of NMDA receptors under conditions of altered protein kinase A activity

N-methyl-D-aspartate receptors (NMDA) are glutamate-activated ligand-gated ion channels that participate in diverse forms of synaptic plasticity as well as glutamate-dependent excitotoxicity. Inhibition of the NMDA receptor function may underlie some of the behavioral actions associated with acute exposure to ethanol. The sensitivity of NMDA receptors to ethanol is influenced by the subunit composition of the receptor and, by association, with certain cytoskeletal proteins. Previous studies have also suggested that phosphorylation may regulate the sensitivity of NMDA receptors to ethanol. In this study, the ethanol inhibition of recombinant NMDA receptor currents was determined under conditions designed to enhance or inhibit the activity of protein kinase A (PKA). Human embryonic kidney 293 (HEK293) cells were transfected with cDNAs encoding NMDA subunits and channel activity was monitored with whole-cell patch-clamp electrophysiology. Under control recording conditions, ethanol (100 mM) inhibited NR1/2A and NR1/2B receptor currents by approximately 25-30%. The degree of ethanol inhibition was not affected or was slightly enhanced under conditions designed to enhance PKA activity. This included treatment of cells with cAMP analogs, inclusion of phosphatase inhibitors or purified PKA in the pipette filling solution, co-expression of catalytically active PKA, expression of the NR1 PKA-site phosphorylation site mimic (S897D) or by co-expression of the PKA scaffolding protein yotiao or the dopamine D(1) receptor. Ethanol inhibition of NR1/2A and NR1/2B receptors was not altered when PKA effects were suppressed, either by co-expression of a PKI inhibitory peptide or the phosphorylation-deficient NR1 mutants (S897A, S896A, S896A/S897A). In addition, ethanol inhibition of NMDA-induced currents in cultured cortical or hippocampal neurons was not affected by modulators of PKA. These results suggest that PKA does not appear to play a major role in determining the acute ethanol sensitivity of NMDA receptors.     

Evaluation of cell culture flasks designed for experiment under altered gravity-vector conditions.

Cell culture flasks applicable for altered gravity conditions, such as centrifugation, clino-rotation or microgravity in space, were manufactured for trial. The flask has flat polystyrene surface for monolayer culture and gas-permeable film window on the opposite face. The space in-between consists the culture chamber to be filled with liquid medium. To reduce the water loss and bubble formation in the culture fluid, another gas permeable window was placed on top to form a space where distilled water may be filled. The double-decker culture flask can be used for both space and ground-based experiments in common.     

Hydrophilic-hydrophobic properties of microorganisms under various culturing conditions

The index of hydrophobicity and the hydrophilic-hydrophobic properties of seven microbial cultures were determined by studying their adhesion to n-hexadecane. The index of hydrophobicity was shown to be a stable characteristic of a culture grown in media with different carbon sources. As was found for Escherichia coli K-12 and Acinetobacter calcoaceticus K-9 whose hydrophobicity indicates were quite different, the character of cell hydration was virtually independent of the growth phase and did not change upon either submerged or superficial cultivation.