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Measuring glucocorticoids such as cortisol is a useful tool for exploring relationships among behavior, physiology, and well‐being in primates. As cortisol circulates in blood, it moves into biological matrices such as hair, urine, feces, and saliva. Saliva sampling is a simple, noninvasive method to measure cortisol that can be easily implemented by training animals to voluntarily provide samples. The temporal lag between elevation of cortisol in the blood and elevation of cortisol in saliva likely varies by species and must be characterized to identify appropriate sampling regimens. In the present study we characterized the time course of cortisol changes in saliva following an acute psychological stressor in captive tufted capuchin monkeys (Sapajus apella). We trained eight free‐moving female tufted capuchin monkeys to voluntarily produce clean saliva samples. We exposed them to the acute stressor of a veterinary catch net and observed behavior pre and post exposure. We collected salivary samples immediately pre exposure (0 min) and 30, 45, 60, 75, 90, and 120 min after exposure. Salivary cortisol was quantified using a Salimetrics kit. Behavioral and cortisol measures were compared within individuals to a control condition in which no stressor was presented. Capuchins showed a clear behavioral response to the stressor by demonstrating increased freezing and pacing, decreased feed foraging, nonsocial play, and scratching, and decreased willingness to provide saliva samples after stressor presentation. After stressor presentation, average salivary cortisol began to increase at 30 min and continued to increase through the 120 min sample period. There was individual variation in absolute cortisol levels, the timing of the cortisol increase, and the timing of the peak. Our results suggest that no single time‐point can be reliably used to evaluate salivary cortisol response to an acute stressor across individuals, and instead we recommend the collection of a prolonged time series. [ABSTRACT FROM AUTHOR]

In birds, relative growth rates of morphological characters change in response to restricted food intake during development. Differential allocation of limited resources is hypothesized to reflect functional priorities for developing chicks. Body mass, wing, and flight feathers have been identified as potential priorities for seabird chicks. We used allometry to examine allocation in captive Common Murre chicks fed within a range of natural provisioning. During days 10-45 post-hatch, chicks were fed one of four diets that varied in biomass, energy content, and composition. Energy intake had a more profound effect on growth and development than diet composition; it significantly reduced absolute growth of body mass, manus, and tarsus. Between day 15 and day 20, allocation changed in all treatments: growth of manus was maintained at the expense of body mass. Chicks in more restricted treatments shifted allocation to manus at a lower body mass than those in less restricted groups, but subsequently allocated similarly. Wing loading was higher for chicks than for adult alcids, but scaled similarly. Growth of primary feathers was the most sensitive to small differences in diet composition. Our data also suggest that some changes in allocation may be ontogenetically determined rather than part of an adaptive response to reduced food intake. Key words: alcid, allometry, Common Murre, development, food intake, Uria aalge, wing loading. Chez les oiseaux, le taux de croissance relatif aux traits morphologiques change en reponse a une consommation de nourriture limitee au cours du developpement. L'hypothese d'une allocation differentielle des ressources, lorsqu'elles sont limitees, est avancee afin de refleter les priorites fonctionnelles des oisillons en developpement. La masse corporelle, les ailes et les plumes de vol ont ete identifiees comme des priorites potentielles chez les oisillons d'oiseaux marins. Nous avons utilise l'allometrie pour examiner l'allocation chez des Uria aalge captifs nourris avec une gamme d'aliments naturels. Au cours des jours 10-45 suite l'eclosion, les oisillons ont ete nourris avec une des quatre dietes qui variaient en termes de biomasse, de contenu energetique et de composition. La consommation energetique a eu un effet plus important sur la croissance et le developpement que la composition dietetique, ce qui a significativement reduit la croissance absolue de la masse corporelle, de l'aile et du tarse. Entre le jour 15 et le jour 20, l'allocation a change dans tousles traitements : la croissance de l'aile a ete maintenue au depend de la masse corporelle. Les oisillons soumis a des traitements plus restrictifs ont modifie l'allocation destinee a l'aile lorsque leur masse corporelle devenait plus faible que celles des oisillons dans les groupes moins restreints. L'allocation devenait similaire par la suite. La charge de l'aile etait plus grande pour les oisillons que pour les adultes, mais dans un meme ordre de grandeur. La croissance des plumes primaires etait la plus sensible aux petites differences dans la composition dietetique. Nos donnees suggerent que certains changements dans l'allocation pourraient etre determines de maniere onthogenetique plutot que d'etre induits par une reponse adaptative a une consommation de nourriture reduite.

This study takes a look at the effects of supplementary plasma testosterone (T) on paternal care in the chestnut-collared longspur. Study results indicate that the pattern of male longspurs' lessened responsiveness to T during the nestling phase may be reflective of the relative importance of male care to nest success in the species.

In birds with facultative brood reduction, survival of the junior chick is thought to be regulated primarily by food availability. In black-legged kittiwakes (Rissa tridactyla) where parents and chicks are provided with unlimited access to supplemental food during the breeding season, brood reduction still occurs and varies interannually. Survival of the junior chick is therefore affected by factors in addition to the amount of food directly available to them. Maternally deposited yolk androgens affect competitive dynamics within a brood, and may be one of the mechanisms by which mothers mediate brood reduction in response to a suite of environmental and physiological cues. The goal of this study was to determine whether food supplementation during the pre-lay period affected patterns of yolk androgen deposition in free-living kittiwakes in two years (2003 and 2004) that varied in natural food availability. Chick survival was measured concurrently in other nests where eggs were not collected. In both years, supplemental feeding increased female investment in eggs by increasing egg mass. First-laid (“A”) eggs were heavier but contained less testosterone and androstenedione than second-laid (“B”) eggs across years and treatments. Yolk testosterone was higher in 2003 (the year with higher B chick survival) across treatments. The difference in yolk testosterone levels between eggs within a clutch varied among years and treatments such that it was relatively small when B chick experienced the lowest and the highest survival probabilities, and increased with intermediate B chick survival probabilities. The magnitude of testosterone asymmetry in a clutch may allow females to optimize fitness by either predisposing a brood for reduction or facilitating survival of younger chicks. [ABSTRACT FROM AUTHOR]

Abstract: In order to understand the physiological role of serotonin in regulating aggressive behaviour it is important to understand how this neuromodulator acts within the context of a naturally fluctuating social and physical environment. To accomplish this, we examined the effect of the selective serotonin reuptake inhibitor fluoxetine during the breeding season in free-living male American tree sparrows (Spizella arborea) in Northern Alaska. During this time period males are maximally aggressive towards territorial intruders. Male, territorial sparrows were injected with either vehicle or a 10mg/kg dose of fluoxetine. One hour later, aggression was measured using a simulated territorial intrusion. Depending upon when birds were sampled, the aggression scores for vehicle and fluoxetine treatments were grouped according to the number of days after territorial behaviour was initiated. The three groups were: early, days 1–5; middle, days 6–10; and late, days 11–15. There was a significant overall difference between groups (F(5,36) = 5.18, P < 0.0015). Post hoc analysis demonstrated that the level of aggression did not differ between the three groups of saline injected birds. However, fluoxetine injected birds showed a time dependent decrease in aggression. When compared to control birds the middle and late fluoxetine groups had significantly less aggression. Furthermore, the late group of fluoxetine treated birds were significantly less aggressive than the early group of fluoxetine birds. This demonstrates that free-living male ATSPs show a rapid change in their behavioural response to fluoxetine across the first 2 weeks of the breeding season. The rapidity of the change in responsiveness suggests a dynamic sensitivity of the serotonergic system. [Copyright &y& Elsevier]

In birds, patterns of development of the adrenocortical response to stressors vary among individuals, types of stressors, and species. Since there are benefits and costs of exposure to elevated glucocorticoids, this variation is presumably a product of selection such that animals modulate glucocorticoid secretion in contexts where doing so increases their fitness. In this study, we evaluated hypothalamo-pituitary-adrenal (HPA) activity in first-hatched free-living seabird nestlings that engage in intense sibling competition and facultative siblicide (black-legged kittiwakes, Rissa tridactyla). We sampled 5 day old chicks (of the ∼45 day nestling period), a critical early age when food availability drives establishment of important parent-offspring and intra-brood dynamics. We experimentally supplemented parents with food ("supplemented") and measured chick baseline corticosterone secretion and capacity to rapidly increase corticosterone in response to an acute challenge (handling and 15 min of restraint in a bag). We also used topical administration of corticosterone to evaluate the ability of chicks to downregulate physiologically relevant corticosterone levels on a short time scale (minutes). We found that 5 day old chicks are not hypo-responsive but release corticosterone in proportion to the magnitude of the challenge, showing differences in baseline between parental feeding treatments (supplemented vs non-supplemented), moderate increases in response to handling, and a larger response to restraint (comparable to adults) that also differed between chicks from supplemented and control nests. Topical application of exogenous corticosterone increased circulating levels nearly to restraint-induced levels and induced downregulation of HPA responsiveness to the acute challenge of handling. Parental supplemental feeding did not affect absorbance/clearance or negative feedback. Thus, while endogenous secretion of corticosterone in young chicks is sensitive to environmental context, other aspects of the HPA function, such as rapid negative feedback and/or the ability to clear acute elevations in corticosterone, are not. We conclude that 5 day old kittiwake chicks are capable of robust adrenocortical responses to novel challenges, and are sensitive to parental food availability, which may be transduced behaviorally, nutritionally, or via maternal effects. Questions remain about the function of such rapid, large acute stress-induced increases in corticosterone in very young chicks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Determinants of individual variation in reallocation of limited resources towards self-maintenance versus reproduction are not well known. We tested the hypothesis that individual heterogeneity in long-term 'somatic state' (i) explains variation in endocrine and behavioural responses to environmental challenges, and (ii) is associated with variation in strategies for allocating to self-maintenance versus reproduction. We used relative telomere length as an indicator of somatic state and experimentally generated an abrupt short-term reduction of food availability (withdrawal of food supplementation) for free-living seabirds (black-legged kittiwakes, Rissa tridactyla ). Incubating male kittiwakes responded to withdrawal by increasing circulating corticosterone and losing more weight compared to continuously supplemented controls. Males with longer telomeres increased time in directed travel regardless of treatment, while experiencing smaller increases in corticosterone. Males with longer telomeres fledged more chicks in the control group and tended to be more likely to return regardless of treatment. This study supports the hypothesis that somatic state can explain variation in short-term physiological and behavioural responses to challenges, and longer-term consequences for fitness. Male kittiwakes with longer telomeres appear to have prioritized investment in self over investment in offspring under challenging conditions.

Current food supply is a major driver of timing of breeding in income-breeding animals, likely because increased net energy balance directly increases reproductive hormones and advances breeding. In capital breeders, increased net energy balance increases energy reserves, which eventually leads to improved reproductive readiness and earlier breeding. To test the hypothesis that phenology of income-breeding birds is independent of energy reserves, we conducted an experiment on food-supplemented ("fed") and control female black-legged kittiwakes (Rissa tridactyla). We temporarily increased energy costs (via weight handicap) in a 2 × 2 design (fed/unfed; handicapped/unhandicapped) during the pre-laying period and observed movement via GPS-accelerometry. We measured body mass, baseline hormones (corticosterone; luteinising hormone) before and after handicap manipulation, and conducted a gonadotropin-releasing hormone challenge. Females from all treatment groups foraged in similar areas, implying that individuals could adjust time spent foraging, but had low flexibility to adjust foraging distance. Consistent with the idea that income breeders do not accumulate reserves in response to increased food supply, fed birds remained within an energy ceiling by reducing time foraging instead of increasing energy reserves. Moreover, body mass remained constant until the onset of follicle development 20 days prior to laying regardless of feeding or handicap, implying that females were using a 'lean and fit' approach to body mass rather than accumulating lipid reserves for breeding. Increased food supply advanced endocrine and laying phenology and altered interactions between the hypothalamic-pituitary-adrenal axis and the hypothalamic-pituitary-gonadal axis, but higher energy costs (handicap) had little effect. Consistent with our hypothesis, increased food supply (but not net energy balance) advanced endocrine and laying phenology in income-breeding birds without any impact on energy reserves., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In birds, exposure to exogenous testosterone during embryonic development can suppress measures of immune function; however, it is unclear whether these effects are due to direct or indirect action via aromatization. Estradiol (E 2 ) is synthesized from testosterone by the enzyme aromatase, and this conversion is a necessary step in many signaling pathways that are ostensibly testosterone-dependent. Many lines of evidence in mammals indicate that E 2 can affect immune function. We tested the hypothesis that some of the immunomodulatory effects observed in response to in ovo testosterone exposure in birds are mediated by conversion to E 2 by aromatase, by using fadrozole to inhibit aromatization of endogenous testosterone during a crucial period of embryonic immune system development in domestic chickens (Gallus gallus). We then measured total IgY antibody count, response to PHA challenge, mass of thymus and bursa of Fabricius, and plasma testosterone post-hatch on days 3 and 18. Because testosterone has a reputation for immunosuppression, we predicted that if modulation of an immune measure by testosterone is dependent on aromatization, then inhibition of estrogen production by fadrozole treatment would lead to elevated measures of that parameter. Conversely, if testosterone inhibits an immune measure directly, then fadrozole treatment would likely not alter that parameter. Fadrozole treatment reduced circulating E 2 in female embryos, but had no effect on males or on testosterone in either sex. Fadrozole-treated chicks had decreased day 3 plasma IgY antibody titers and a strong trend towards increased day 18 thymic mass. Furthermore, fadrozole treatment generated a positive relationship between testosterone and thymic mass in males, and tended to increase day 18 IgY levels for a given bursal mass in females. There was no effect on PHA response, bursal mass, or plasma testosterone at either age post-hatch. The alteration of several indicators of immune function in fadrozole-treated chicks implicates aromatization as a relevant pathway through which developmental exposure to testosterone can affect immunity in birds., (Copyright © 2020. Published by Elsevier Inc.)

G-proteins cycle between an inactive GDP-bound state and an active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage display to identify a series of peptides that bind G alpha subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP x AlF4(-)- and GTPgammaS-bound states of G alpha(i) subunits. KB-1753 blocks interaction of G alpha(transducin) with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated G alpha in vitro. The crystal structure of KB-1753 bound to G alpha(i1) x GDP x AlF4(-) reveals binding to a conserved hydrophobic groove between switch II and alpha3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for G alpha(i) subunits.

Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Galpha subunits normally bound to GDP and Gbetagamma. Activated Galpha . GTP then regulates effectors such as adenylyl cyclase. Except for Gbetagamma, no known regulators bind the adenylyl cyclase-stimulatory subunit Galphas in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Galpha(i). Herein, we report that KB-752 binds Galpha(s) . GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPgammaS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Galpha(i) and Galpha(s).

Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.

Background: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites., Results: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range., Conclusions: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

Random mutant libraries with substitutions at the interface between the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c were screened. All residue combinations that have been identified in naturally occurring cytochrome c sequences are found in the libraries. Mutants with these combinations are biologically functional. Enthalpies, heat capacities, and midpoint temperatures of denaturation are used to determine the entropy and Gibbs free energy of denaturation (delta GD) for the ferri form of the wild-type protein and 13 interface variants. Changes in delta GD cannot be allocated solely to enthalpic or entropic effects, but there is no evidence of enthalpy-entropy compensation. The lack of additivity of delta GD values for single versus multiple amino acid substitutions indicates that the helices interact thermodynamically. Changes in delta GD are not in accord with helix propensities, indicating that interactions between the helices and the rest of the protein outweigh helix propensity. Comparison of delta GD values for the interface variants and nearly 90 non-cytochrome c variants to side-chain model data leads to several conclusions. First, hydrocarbon side chains react to burial-like transfer from water to cyclohexane, but even weakly polar side chains respond differently. Second, despite octanol being a poor model for protein interiors, octanol-to-water transfer free energies are useful stability predictors for changing large hydrocarbon side chains to smaller ones. Third, unlike cyclohexane and octanol, the Dayhoff mutation matrix predicts stability changes for a variety of substitutions, even at interacting sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Buried within cytochrome c lies a highly-conserved helix-helix interface formed by the perpendicular packing of the C-terminal helix against the N-terminal helix. This interface involves a peg-in-hole interaction between Gly-6 and Leu-94 and an aromatic-aromatic interaction between Phe-10 and Tyr-97. To gain insight into protein design, we investigated the relationship between the sequence of the interface and the physiological function of yeast iso-1-cytochrome c. A library of mutants at positions 94 and 97 of the C-terminal helix was created to examine the effect of novel amino acid combinations. We isolated 45 of the 400 possible amino acid combinations, 32 of which result in a functional cytochrome c. Contrary to evolutionary conservation of the peg-in-hole and aromatic-aromatic interactions, we find that side-chain volume and conservation of aromatic residues do not play an essential role in determining function. Additionally, we find negatively-charged residues within the interface that result in a functional cytochrome c. Examination of the 45 missense mutants indicates that approximately 120 unique combinations are compatible with function. These results show that the interface is flexible. However, truncation of the C-terminal helix at position 94 abolishes function, suggesting that the interface is essential. The correlation observed between our library of mutants and the mutation matrix compiled by Gonnet et al. [Gonnet, G. H., Cohen, M. A., & Benner, S. A. (1992) Science 256, 1443-1445] demonstrates the potential use of the matrix to predict the effect of sequence changes on natural proteins and to optimize the design of novel proteins.