Influence of season, age, sex, and time of day on the endocrine profile of the common bottlenose dolphin (Tursiops truncatus).

Understanding baseline hormone levels, the magnitude of intra-individual variability, and their variation as a function of life history is difficult in toothed whales (e.g. dolphins and porpoises) because of the effects of capture stress. To determine the endocrine profile of the common bottlenose dolphin (Tursiops truncatus) as a function of season, time of day (TOD), age, sex, and reproductive status, blood corticosteroids, thyroid hormones, and catecholamines were repeatedly measured in a managed-care population exposed to ambient light and water temperatures of San Diego Bay. Additionally, fecal hormone metabolites were assessed for cortisol, aldosterone, and triiodothyronine. Samples were collected at two to four-week intervals over a period of two years, and multiple times within a day at monthly intervals over a year. Samples were collected through the voluntary participation of the dolphins in the blood draws and fecal collections in order to avoid the effects of handling stress. All serum hormones except aldosterone significantly varied with season and all serum hormones except total thyroxine significantly varied as a function of TOD. Fecal glucocorticoid metabolites significantly correlated with circulating cortisol levels, and there was a significant seasonal effect on triiodothyronine fecal metabolites. Strong seasonal effects demonstrated complex interactions with age and sex suggesting that contextual information is critical to interpreting differences in endocrine profiles. Strong circadian patterns further suggest that sampling design is important to the interpretation of blood or fecal collections, particularly since diurnal changes in some serum hormone levels are similar to the magnitude of seasonal differences. Despite potential impacts of feeding schedules on diurnal patterns, managed care populations can provide important insights into seasonal and age-related endocrine changes in toothed whales.

Variation in adrenal and thyroid hormones with life-history stage in juvenile northern elephant seals (Mirounga angustirostris).

The classical approach to quantifying the impact of stressors on wildlife is through characterization of hormones associated with the generalized stress response. However, interpretation of hormone data can be difficult due to the range of natural variation within a species and potential confounds of individual and life-history variables. Blood adrenal and thyroid hormones were measured in 144 chemically immobilized yearling northern elephant seals (Mirounga angustirostris) to characterize variation between sexes and across semiannual haul-outs. There was no relationship between hormone concentrations and time needed for collecting blood nor evidence of diel patterns, suggesting that collection of samples for baseline values can be accomplished without bias due to handling artifacts or time of day. Serum cortisol concentrations did not vary with gender or across haul-out fasts but increased dramatically during molting. Cortisol was correlated with aldosterone across all measured life-history stages. Thyroid hormone levels were lower in females and decreased with fasting in both sexes during the fall haul-out. Cortisol concentrations were inversely associated with total triiodothyronine (T3) and positively associated with reverse T3 concentrations across all measured life-history stages suggesting an important impact of cortisol on deiodinase enzymes and thyroid function. Epinephrine concentrations increased across fasts and norepinephrine concentrations were higher in males than in females. Significant variation in stress hormone concentrations with gender and life-history stage emphasizes the importance of contextual variables when interpreting serum hormone concentrations.

Deadly diving? Physiological and behavioural management of decompression stress in diving mammals.

Decompression sickness (DCS; 'the bends') is a disease associated with gas uptake at pressure. The basic pathology and cause are relatively well known to human divers. Breath-hold diving marine mammals were thought to be relatively immune to DCS owing to multiple anatomical, physiological and behavioural adaptations that reduce nitrogen gas (N(2)) loading during dives. However, recent observations have shown that gas bubbles may form and tissue injury may occur in marine mammals under certain circumstances. Gas kinetic models based on measured time-depth profiles further suggest the potential occurrence of high blood and tissue N(2) tensions. We review evidence for gas-bubble incidence in marine mammal tissues and discuss the theory behind gas loading and bubble formation. We suggest that diving mammals vary their physiological responses according to multiple stressors, and that the perspective on marine mammal diving physiology should change from simply minimizing N(2) loading to management of the N(2) load. This suggests several avenues for further study, ranging from the effects of gas bubbles at molecular, cellular and organ function levels, to comparative studies relating the presence/absence of gas bubbles to diving behaviour. Technological advances in imaging and remote instrumentation are likely to advance this field in coming years.

Environment and activity affect skin temperature in breeding adult male elephant seals (Mirounga angustirostris).

The large body size and high rates of metabolic heat production associated with male mating success in polygynous systems creates potential thermoregulatory challenges for species breeding in warm climates. This is especially true for marine predators carrying large blubber reserves intended for thermoregulation in cold water and fuel provision during extended fasts. Thermographic images were used to measure changes in skin temperature (T(S)) in adult male northern elephant seals (Mirounga angustirostris) over the breeding season. Environmental variables, primarily ambient temperature and solar radiation, were the principal determinants of mean and maximum T(S). When controlled for environmental variables, dominance rank significantly impacted mean T(S), being highest in alpha males. Behavioral activity significantly influenced T(S) but in a counter-intuitive way, with inactive males exhibiting the highest T(S). This was likely due to strong impacts of environmental variables on the kinds of behavior exhibited, with males being less active on warm, humid days at peak solar radiation. We classified thermal windows as areas in which T(S) was one standard deviation greater than mean T(S) for the individual seal within a thermograph. Thermal features suggest active physiological thermoregulation during and after combat and significant circulatory adaptations for heat dumping, as evidenced by recurring locations of thermal windows representing widely varying T(S) values. Frequent observations of localized T(S) above 37°C, particularly after combat, suggest the production of thermoregulatory stress during breeding behavior. Our findings demonstrate the importance of environmental drivers in shaping activity patterns during breeding and provide evidence for thermoregulatory costs of successful breeding in large polygynous males.

Investigation of the potential for vascular bubble formation in a repetitively diving dolphin.

The production of venous gas emboli (VGE) resulting from altered dive behavior is postulated as contributing to the stranding of beaked whales exposed to mid-frequency active sonar. To test whether nitrogen gas uptake during repetitive breath-hold diving is sufficient for asymptomatic VGE formation in odontocetes, a bottlenose dolphin (Tursiops truncatus Montagu) was trained to perform 10-12 serial dives with 60 s surface intervals to depths of 30, 50, 70 or 100 m. The dolphin remained at the bottom depth for 90 s on each dive. Doppler and/or two-dimensional imaging ultrasound did not detect VGE in the portal and brachiocephalic veins following a dive series. Van Slyke analyses of serial, post-dive blood samples drawn from the fluke yielded blood nitrogen partial pressure (P(N(2))) values that were negligibly different from control samples. Mean heart rate (HR; +/-1 s.d.) recorded during diving was 50+/-3 beats min(-1) and was not significantly different between the 50, 70 and 100 m dive sessions. The absence of VGE and elevated blood P(N(2)) during post-dive periods do not support the hypothesis that N(2) supersaturation during repetitive dives contributes to VGE formation in the dolphin. The diving HR pattern and the presumed rapid N(2) washout during the surface-interval tachycardia probably minimized N(2) accumulation in the blood during dive sessions.

Effects of brief yoga exercises and motivational preparatory interventions in distance runners: results of a controlled trial.

OBJECTIVE: To examine the efficacy of two preparatory interventions on one mile run performance in 90 high school long distance runners. METHOD: After participants had completed a one mile baseline run, they were randomly assigned to participate in either one of two interventions (brief yoga exercises, motivational shouting exercises) or a no intervention control condition. Experimental conditions were implemented one week after the baseline run about 20 minutes before a second one mile trial. RESULTS: Participants assigned to the motivational intervention improved their running performance significantly more than those assigned to the other two conditions. Although the magnitude of the effect was small, participants assigned to yoga exercises showed significant improvements in running performance relative to control condition participants. Consumer satisfaction ratings indicated that participants who were assigned to the motivational and yoga exercise groups liked their interventions more than those assigned to the control group. CONCLUSION: Motivational and yoga interventions designed to improve long distance running performance were equally acceptable to the participants, but the former had a greater effect.

A functional imaging study of cooperation in two-person reciprocal exchange.

Cooperation between individuals requires the ability to infer each other's mental states to form shared expectations over mutual gains and make cooperative choices that realize these gains. From evidence that the ability for mental state attribution involves the use of prefrontal cortex, we hypothesize that this area is involved in integrating theory-of-mind processing with cooperative actions. We report data from a functional MRI experiment designed to test this hypothesis. Subjects in a scanner played standard two-person "trust and reciprocity" games with both human and computer counterparts for cash rewards. Behavioral data shows that seven subjects consistently attempted cooperation with their human counterpart. Within this group prefrontal regions are more active when subjects are playing a human than when they are playing a computer following a fixed (and known) probabilistic strategy. Within the group of five noncooperators, there are no significant differences in prefrontal activation between computer and human conditions.

Renal function in suckling and fasting pups of the northern elephant seal.

Elephant seals fast for prolonged periods without access to water. This is made possible, in part, by reductions in urine production. However, the mechanisms involved in reducing urine production are not understood. In this study, glomerular filtration rate (GFR) was measured in five northern elephant seal pups (Mirounga angustirostris) via the inulin clearance technique. Measurements were made during day 9 and day 18-22 of nursing and the second and eighth week of the postweaning fast. Plasma aldosterone and cortisol concentrations, quantified by radioimmunoassay, were measured in eight other weanlings during the second and eighth week of the fast. Mean GFR was 79.3+/-29.3 ml/min during the early suckling period and 78.2+/-17.1, 89.8+/-52.7, and 80.4+/-12.2 ml/min during the late suckling, early fasting and late fasting periods, respectively. Differences between nursing and fasting were insignificant, possibly because reduced protein oxidation during suckling and rapid recruitment of protein for tissue synthesis obviated the need for postprandial hyperfiltration. Alternatively, maintenance of GFR during fasting may facilitate urea concentration by compensating for reductions in the fractional excretion of urea. It is further hypothesized that aldosterone is primarily responsible for mediating renal water reabsorption in this system.

Protein catabolism in suckling and fasting northern elephant seal pups (Mirounga anglstirostris).

Nursing elephant seal pups are hypothesized to be preadapted to the postweaning fast, yet no comparison of lipid or protein use for meeting metabolic costs has been made between these contrasting nutritional periods. To address this, protein catabolism was estimated in five elephant seal pups from measurements of urea turnover made twice during nursing and twice during the postweaning fast. Changes in body composition were measured in ten separate weaned pups via tritiated water dilution and matched to fasting urea turnover measurements in order to assess errors in protein catabolism derived from urea turnover rates. Estimates of lean mass loss based upon urea turnover and tritiated water dilution were in general agreement, supporting estimates of protein catabolism derived from urea turnover measurements. Protein catabolism was estimated to contribute less than 4% to the average metabolic rate of suckling and fasting pups implying strict protein conservation during both periods and supporting the shypothesis that suckling pups are pre-adapted to fasting. It is proposed that strict protein conservation across suckling and fasting compensates for relative reductions in maternal investment associated with the abbreviated lactation period of the elephant seal.

Can diving-induced tissue nitrogen supersaturation increase the chance of acoustically driven bubble growth in marine mammals?

The potential for acoustically mediated causes of stranding in cetaceans (whales and dolphins) is of increasing concern given recent stranding events associated with anthropogenic acoustic activity. We examine a potentially debilitating non-auditory mechanism called rectified diffusion. Rectified diffusion causes gas bubble growth, which in an insonified animal may produce emboli, tissue separation and high, localized pressure in nervous tissue. Using the results of a dolphin dive study and a model of rectified diffusion for low-frequency exposure, we demonstrate that the diving behavior of cetaceans prior to an intense acoustic exposure may increase the chance of rectified diffusion. Specifically, deep diving and slow ascent/descent speed contributes to increased gas-tissue saturation, a condition that amplifies the likelihood of rectified diffusion. The depth of lung collapse limits nitrogen uptake per dive and the surface interval duration influences the amount of nitrogen washout from tissues between dives. Model results suggest that low-frequency rectified diffusion models need to be advanced, that the diving behavior of marine mammals of concern needs to be investigated to identify at-risk animals, and that more intensive studies of gas dynamics within diving marine mammals should be undertaken.

Classification of dolphin echolocation clicks by energy and frequency distributions.

Dolphins demonstrate an adaptive control over echolocation click production, but little is known of the manner or degree with which control is exercised. Echolocation clicks (N approximately 30,000) were collected from an Atlantic bottlenose dolphin (Tursiops truncatus) performing object discrimination tasks in order to investigate differential click production. Seven categories of clicks were identified using the spectral conformation and relative position of -3 and -10 dB peaks. A counterpropagation network utilizing 16 inputs, 50 hidden units, and 8 output units was trained to classify clicks using the same spectral variables. The network classified novel clicks with 92% success. Additional echolocation clicks (N > 24,000) from two other dolphins were submitted to the network for classification. Classified echolocation clicks were analyzed for animal specific differences, changes in predominant click type within click trains, and task-related specificity. Differences in animal and task performance may influence click type and click train length.

Enhancement of cardiac function by cyclocreatine in models of cardiopulmonary bypass.

This study tests the hypothesis that the administration of cyclocreatine prior to global ischemia enhances recovery of cardiac function during reperfusion. Two models were used. First, in a Langendorff-working heart model of normothermic cardioplegic arrest, rats (n = 6 per group) were injected intravenously with saline or cyclocreatine (600, 300, or 150 mg/kg). After 30 min or 2 h, hearts were excised and perfused in the Langendorff mode for 5 min and then in the working heart mode for 20 min. Normothermic arrest was induced by infusing warm St. Thomas solution once; then hearts were kept at 37 degrees C for 40 min. Following arrest, hearts were reperfused in the Langendorff mode for 15 min and then in the working mode for 30 min. Cyclocreatine consistently produced significantly better recovery of aortic flow and cardiac output compared to that of saline hearts. Second, in an intact canine model of cold cardioplegic arrest, adult mongrel dogs (n = 3 to 6 per group) underwent aortic cross-clamping for 1 h, followed by reperfusion on bypass for 45 min and off bypass for 4 h. Dogs were injected intravenously with saline or cyclocreatine (500 mg/kg) for 1 h before experiment. Post-bypass segmental contractility and cardiac output were significantly better in cyclocreatine hearts compared to that of controls. In a limited study, after a 3 h aortic cross-clamp time, cyclocreatine hearts achieved 91% baseline function while control hearts failed after 2 h. Results of this study suggest that cyclocreatine, without inotropic or chronotropic effect, protects the heart from global ischemic injury.

Studies of copper transport in cultured bovine chondrocytes.

Copper serves as the cofactor for a number of important enzymes in cartilage, as well as in other tissues, including lysyl oxidase, superoxide dismutase, and cytochrome oxidase. Ceruloplasmin is responsible for the transport of approx. 95% of the copper in serum, but the mechanisms for intracellular copper transport are unknown. We have demonstrated recently that a high-molecular-weight cartilage glycoprotein, referred to as CMGP, has regions of sequence homology with ceruloplasmin. CMGP also binds copper and has at least some oxidase activity similar to that of ceruloplasmin. Other tissues synthesize intracellular ceruloplasmin-like proteins. The present report represents part of an effort to examine the hypothesis that CMGP is a copper transport protein in chondrocytes and to characterize the enzymatic activities of CMGP. These studies demonstrate that CMGP is the principal chondrocyte protein labeled by 67Cu in vitro and that the label is localized to the mitochondria, cytosol, and membrane fractions of sucrose gradients, suggesting copper transport through the cell. In parallel experiments, [3H]leucine was incorporated into proteins corresponding to the subunits and fragments of CMGP, as described previously, and in a similar distribution among the subcellular fractions as labeled copper. Additionally, CMGP has oxidase and ferroxidase activities similar to those of ceruloplasmin.

Evaluation of 3-[18F]fluoro-alpha-fluoromethyl-p-tyrosine as a tracer for striatal tyrosine hydroxylase activity.

3-[18F]Fluoro-alpha-fluoromethyl-p-tyrosine (3-F-FMPT) was evaluated as a tracer for CNS tyrosine hydroxylase (TH) activity in rodents and in a rhesus monkey. Results of in vitro experiments using rat striatal homogenates showed that the introduction of fluorine into the 3-phenyl position did not significantly alter the ability of FMPT to act as a TH-activated L-aromatic amino acid decarboxylase (L-AAAD) inhibitor. These studies further showed that 3-F-FMPT-induced L-AAAD inhibition was dose-dependent. Furthermore, striatal homogenates prepared from rats pretreated with the potent TH inhibitor alpha-methyl-p-tyrosine was found to have diminished 3-F-FMPT-induced L-AAAD inhibition. However, despite these promising in vitro results, the biodistribution of this compound in mice showed low brain uptake and fast clearance through the kidneys. A PET study using a Rhesus monkey injected with 3-[18F]F-FMPT confirmed the results obtained in mice, i.e. negligible brain uptake but high localization in the bladder. We conclude that 3-[18F]F-FMPT would not be useful as a tracer for cerebral TH activity.

The critical phase inspection process: a benchmarking study in search of industries' best practices.

Benchmarking is the orderly process of measuring one's own products, services, and practices against those of companies recognized as leaders. Eli Lilly and Company's Quality Assurance Department formed the Critical Phase Inspection Team to benchmark the processes for selecting and conducting critical phase inspections and reporting inspection findings. The team developed a telephone survey that was conducted with 33 other quality assurance units across the country. Analysis of the phone survey responses resulted in the identification of 5 quality assurance units that we felt could provide valuable information to us on these activities. Site visits to these companies were arranged and information was shared. We present here the analysis and results of our benchmarking endeavor. Through the information sharing involved in the benchmarking process, namely, the telephone surveys and the site visits, fresh ideas emerged and new acquaintances were made. Comparisons and adaptations of our methods with others in the quality assurance business will lead us to breakthrough improvements that will allow us to improve our current processes.

Evidence that a 550,000-dalton cartilage matrix glycoprotein is a chondrocyte membrane-associated protein closely related to ceruloplasmin.

Cartilage matrix glycoprotein (CMGP) is a disulfide-bonded 550,000-dalton protein that is synthesized by chondrocytes and ciliary epithelial cells. We have purified the protein from bovine and porcine articular cartilage and have sequenced two peptides, which both have significant homology with human ceruloplasmin, a copper-binding oxidase. Immunolocation analysis indicates that a commercial polyclonal antiserum to human ceruloplasmin reacts with bovine cartilage CMGP. Chelating columns made with copper bind CMGP from bovine cartilage extracts. CMGP is present in bovine chondrocyte membrane preparations purified from sucrose density gradients. Oligonucleotide probes have been synthesized based on the published sequence of the 3'-untranslated region and a portion of the C terminus of human ceruloplasmin and have been used to amplify a cDNA fragment from bovine cartilage and human liver libraries. CMGP demonstrates oxidase activity towards p-phenylenediamine similar to that of ceruloplasmin. These studies suggest that CMGP is closely related to, if not identical with, ceruloplasmin. It is possible that CMGP may be involved in metal transport into and/or within the chondrocyte.

2-(Aminomethyl)chromans that inhibit iron-dependent lipid peroxidation and protect against central nervous system trauma and ischemia.

A series of 2-(aminomethyl)chromans was developed as potent inhibitors of iron-dependent lipid peroxidation. Compounds within this class are extremely effective at inhibiting lipid peroxidation with IC50's as low as 0.2 microM. Selected members were found to enhance early neurological recovery and survival in a mouse head injury model. In this assay, improvement in the 1-h post-head-injury neurological status (grip test score) by as much as 230% of control was observed. One of the most efficacious compounds (35) was evaluated in two models of cerebral ischemia where significant neuroprotection was observed. These results provide further support for the importance of cerebroprotective antioxidants for the treatment of traumatic and ischemic injury as well as additional evidence for the role of oxygen radicals in postischemic brain damage.

Visualization of dopamine nerve terminals by positron tomography using [18F]fluoro-beta-fluoromethylene-m-tyrosine.

[18F]-6-Fluoro-beta-fluoromethylene-m-tyrosine ([18F]FFMMT) was evaluated as a potential imaging agent for dopamine nerve terminals using positron emission tomography (PET). Biodistribution and time course of this tracer in mice after i.p. injection was consistent with the distribution of dopamine. PET imaging studies involving rhesus macaques showed specific uptake in the dopamine-rich caudate-putamen region. This specific localization was blocked by inhibiting the enzyme L-aromatic amino acid decarboxylase and the transport of the tracer into brain was shown to be stereospecific. These results show the promise of L-[18F]FFMMT as a PET tracer in monitoring degeneration of the CNS dopamine system.

A rhesus monkey model for sexual transmission of a papillomavirus isolated from a squamous cell carcinoma.

Recently we molecularly cloned and characterized a papillomavirus from a lymph node metastasis of a primary penile carcinoma found in a rhesus monkey; this virus species, rhesus papillomavirus type 1 (RhPV-1), is similar to oncogenic human papillomaviruses (HPVs), such as HPV-16 or HPV-18, in that the RhPV-1 DNA was found to be integrated in the tumor cell DNA. To compare the sexual transmission and oncogenic nature of RhPV-1 with these HPVs, we undertook an extensive retrospective study of a group of rhesus monkeys whose sexual mating and offspring histories were known. These animals had mated directly with the index male mentioned above or were secondarily exposed to this virus through intermediate sexual partners. This study combines cytological, histopathological, and several complementary hybridization and DNA amplification techniques on multiple tissue samples to demonstrate the sexually transmitted nature of RhPV-1. The oncogenic potential of RhPV-1 is suggested in several of the infected animals by the presence of various degrees of neoplasia including squamous cell cancer of the cervix.

Novel 21-aminosteroids that inhibit iron-dependent lipid peroxidation and protect against central nervous system trauma.

A novel class of 21-aminosteroids has been developed. Compounds within this series are potent inhibitors of iron-dependent lipid peroxidation in rat brain homogenates with IC50's as low as 3 microM. Furthermore, selected members enhance early neurological recovery and survival in a mouse head injury model. Significant improvement in the 1 h post-head-injury neurological status (grip test score) by as much as 168.6% of the control has been observed. The most efficacious compound in this assay (30) showed an increase in the 1-week survival of 78.6% as compared to 27.3% for the vehicle-treated mice in the head-injury model. Based on its biological profile, 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16 alpha- methylpregna-1,4,9(11)-triene-3,20-dione monomethanesulfonate (30) was selected for further evaluation and is currently entering phase I clinical trials for the treatment of head and spinal trauma.