The importance of sample size in marine megafauna tagging studies.

Telemetry is a key, widely used tool to understand marine megafauna distribution, habitat use, behavior, and physiology; however, a critical question remains: "How many animals should be tracked to acquire meaningful data sets?" This question has wide-ranging implications including considerations of statistical power, animal ethics, logistics, and cost. While power analyses can inform sample sizes needed for statistical significance, they require some initial data inputs that are often unavailable. To inform the planning of telemetry and biologging studies of marine megafauna where few or no data are available or where resources are limited, we reviewed the types of information that have been obtained in previously published studies using different sample sizes. We considered sample sizes from one to >100 individuals and synthesized empirical findings, detailing the information that can be gathered with increasing sample sizes. We complement this review with simulations, using real data, to show the impact of sample size when trying to address various research questions in movement ecology of marine megafauna. We also highlight the value of collaborative, synthetic studies to enhance sample sizes and broaden the range, scale, and scope of questions that can be answered.

Convergence of marine megafauna movement patterns in coastal and open oceans.

The extent of increasing anthropogenic impacts on large marine vertebrates partly depends on the animals' movement patterns. Effective conservation requires identification of the key drivers of movement including intrinsic properties and extrinsic constraints associated with the dynamic nature of the environments the animals inhabit. However, the relative importance of intrinsic versus extrinsic factors remains elusive. We analyze a global dataset of ∼2.8 million locations from >2,600 tracked individuals across 50 marine vertebrates evolutionarily separated by millions of years and using different locomotion modes (fly, swim, walk/paddle). Strikingly, movement patterns show a remarkable convergence, being strongly conserved across species and independent of body length and mass, despite these traits ranging over 10 orders of magnitude among the species studied. This represents a fundamental difference between marine and terrestrial vertebrates not previously identified, likely linked to the reduced costs of locomotion in water. Movement patterns were primarily explained by the interaction between species-specific traits and the habitat(s) they move through, resulting in complex movement patterns when moving close to coasts compared with more predictable patterns when moving in open oceans. This distinct difference may be associated with greater complexity within coastal microhabitats, highlighting a critical role of preferred habitat in shaping marine vertebrate global movements. Efforts to develop understanding of the characteristics of vertebrate movement should consider the habitat(s) through which they move to identify how movement patterns will alter with forecasted severe ocean changes, such as reduced Arctic sea ice cover, sea level rise, and declining oxygen content.

Induction of histone acetylation in rat liver and hepatoma by organosulfur compounds including diallyl disulfide.

In previous studies, diallyl disulfide induced histone acetylation and differentiation in DS19 mouse erythroleukemic cells. In the present work the potential induction of histone acetylation in tumor-bearing rats was examined. Increased acetylation of histones in liver and Morris hepatoma 7777 was induced by treatment of rats with diallyl disulfide (200 mg/kg body weight), allyl mercaptan (100 mg/kg body weight) and butanethiol (100 mg/kg body weight). The level of histone acetylation was greater in liver than in the hepatoma and the response to the organosulfur compounds tended to be less in the tumor. The data suggested that compounds in garlic or their metabolites may increase the acetylation of core nucleosomal histones and thereby favor cell differentiation.

Induction of histone acetylation in mouse erythroleukemia cells by some organosulfur compounds including allyl isothiocyanate.

In previous studies we observed that some allyl sulfides can cause increased acetylation of histones and differentiation in DS19 mouse erythroleukemia cells. In the present work we observed increased acetylation of histones with allyl isothiocyanate and butanethiol but not with butyl sulfide or butyl disulfide. Increased acetylation of histones was established by change in electrophoretic mobility, incorporation of [3H]acetate or immunoblotting. Histone deacetylase in nuclei of DS19 cells was inhibited 74% by 0.5 mM allyl mercaptan and 43% by 0.5 mM butanethiol but was not significantly affected by 0.5 mM allyl isothiocyanate. There was some degree of reversibility in the effect of allyl isothiocyanate when the cells were incubated for 15 hr in fresh medium. The data suggested that allyl isothiocyanate may stimulate histone acetylation rather than inhibit histone deacetylation. Addition of allyl isothiocyanate, however, had very little or no additional effect on the induction of histone acetylation caused by trichostatin A. Histone acetyltransferase activity determined in cell homogenates was not increased by preincubation of cells with allyl isothiocyanate or inclusion of allyl isothiocyanate in the assay medium. It was concluded that treatment of mouse erythroleukemia cells with allyl isothiocyanate can cause increased acetylation of histones but the mechanism for this effect requires further elucidation.

Metabolic limits on dive duration and swimming speed in the southern elephant seal Mirounga leonina.

The ability of air-breathing marine predators to forage successfully depends on their ability to remain submerged. This is in turn related to their total O(2) stores and the rate at which these stores are used up while submerged. Body size was positively related to dive duration in a sample of 34 adult female southern elephant seals from Macquarie Island. However, there was no relationship between body size and dive depth. This indicates that smaller seals, with smaller total O(2) stores, make shorter dives than larger individuals but operate at similar depths, resulting in less time being spent at depth. Nine adult female elephant seals were also equipped with velocity time depth recorders. In eight of these seals, a plot of swimming speed against dive duration revealed a cloud of points with a clear upper boundary. This boundary could be described using regression analysis and gave a significant negative relationship in most cases. These results indicate that metabolic rate varies with activity levels, as indicated by swimming speed, and that there are quantifiable limits to the distance that a seal can travel on a dive of a given swimming speed. However, the seals rarely dive to these physiological limits, and the majority of their dives are well within their aerobic capacity. Elephant seals therefore appear to dive in a way that ensures that they have a reserve of O(2) available.

Induction of histone acetylation and growth regulation in eryrthroleukemia cells by 4-phenylbutyrate and structural analogs.

The objective of this investigation was to study the relationship between histone acetylation and growth inhibition by 4-phenylbutyrate and structural analogs. Inhibition of growth of DS19 mouse erythroleukemia cells and K562 human leukemic cells by 4-phenylbutyrate did not appear to be mediated by glutamine depletion. Vanadate blocked differentiation of DS19 cells but did not affect the hyperacetylation of histones. 2-phenylbutyrate was a more effective inhibitor of cell proliferation than 3-phenylbutyrate but was less effective as an inducer of histone acetylation. 4-Phenylbutyrate was a more effective inhibitor of histone deacetylase and inducer of histone acetylation than the structural analogs examined including 2- and 3-phenylbutyrate, cinnamate, methoxycinnamate, 2-phenoxybutyrate and phenoxyacetate.

Increased acetylation of histones induced by diallyl disulfide and structurally related molecules.

In previous studies, diallyl disulfide induced differentiation in DS19 mouse erythroleukemic cells. A mechanism mediated by increased histone acetylation was investigated. Diallyl disulfide caused increased acetylation of H4 and H3 histones in DS19 cells and K562 human leukemic cells. Diallyl disulfide was more effective than diallyl monosulfide and diallyl sulfone. Acetylation was also induced in rat hepatoma and human breast cancer cells by diallyl disulfide or its metabolite, allyl mercaptan. Allyl mercaptan was a more potent inhibitor of histone deacetylase than diallyl disulfide. Differentiation in erythroleukemic cells by diallyl disulfide and allyl mercaptan may be mediated through induction of histone acetylation.

Induction of reporter gene expression by inhibitors of histone deacetylase.

The relationship between histone acetylation and induction of gene expression was studied in Ros 17/2.8 rat osteosarcoma cells transfected with the pCH110 plasmid. This plasmid is commonly used in cotransfections as a measure of transfection efficiency. Cells were incubated for 48 hours with sodium butyrate, phenylbutyrate, 3-bromopropionate or trichostatin A. There was an approximate relationship between the extent of beta-galactosidase induction and the degree of histone hyperacetylation. Trichostatin A was the most effective agent followed by sodium butyrate and then phenylbutyrate. The toxicity of 3-bromopropionate made it difficult to compare its action with the other agents. Phenylbutyrate was less effective than sodium butyrate in causing induction of gene expression and histone hyperacetylation but this action may be a factor in the growth-inhibitory and differentiating activity of phenylbutyrate which has also been attributed to glutamine depletion.

Differential expression of cytochrome P4502E1 (CYP2E1) in Morris hepatomas and livers of tumor bearing rats.

The objective of this investigation was to determine if the decrease of cytochrome P4502E1 (CYP2E1) in hepatomas is related to the tumor growth rate. There was a significant correlation between N-nitrosodimethylamine (NDMA) demethylase activities and CYP2E1 protein levels in rat liver and hepatomas. The levels of NDMA demethylase activities and CYP2E1 protein content were lower in hepatomas than in the corresponding host livers. NDMA demethylase activities and CYP2E1 protein levels were greater in hepatomas of slow and intermediate growth rate than in fast growing hepatomas. A similar trend was also observed with CYP2E1 mRNA levels. The results demonstrated an inverse relationship between growth rate of rat hepatomas and the expression of CYP2E1.

Heart rate, swimming speed, and estimated oxygen consumption of a free-ranging southern elephant seal.

Heart rate, swimming speed, and diving behaviour were recorded simultaneously for an adult female southern elephant seal during her postbreeding period at sea with a Wildlife Computers heart-rate time depth recorder and a velocity time depth recorder. The errors associated with data storage versus real-time data collection of these data were analysed and indicated that for events of short duration (i.e., less than 10 min or 20 sampling intervals) serious biases occur. A simple model for estimating oxygen consumption based on the estimated oxygen stores of the seal and the assumption that most, if not all, dives were aerobic produced a mean diving metabolic rate of 3.64 mL O2 kg-1, which is only 47% of the field metabolic rate estimated from allometric models. Mechanisms for reducing oxygen consumption while diving include cardiac adjustments, indicated by reductions in heart rate on all dives, and the maintenance of swimming speed at near the minimum cost of transport for most of the submerged time. Heart rate during diving was below the resting heart rate while ashore in all dives, and there was a negative relationship between the duration of a dive and the mean heart rate during that dive for dives longer than 13 min. Mean heart rates declined from 40 beats min-1 for dives of 13 min to 14 beats min-1 for dives of 37 min. Mean swimming speed per dive was 2.1 m s-1, but this also varied with dive duration. There were slight but significant increases in mean swimming speeds with increasing dive depth and duration. Both ascent and descent speeds were also higher on longer dives.

Organosulfur compounds and cancer.

There is evidence that organosulfur compounds can inhibit the induction and growth of cancer. Several organosulfur compounds are dietary constituents and Allium species are a rich source of such molecules. Some but not all epidemiological studies have suggested that consumption of garlic can decrease cancer incidence. There is substantial evidence that constituents of garlic including diallyl sulfides can inhibit the induction of cancer in experimental animals. Effects on both tumor initiation and promotion have been documented. Some effects may be mediated by modulation of carcinogen metabolism involving altered ratios of phase I and phase II drug metabolizing enzymes. 59,60 Inhibitory actions on the growth of tumor cells have been documented and, for some tumor cells, differentiating effects of diallyl sulfides can occur. A definitive mechanism of action has not been established and evidence exists for effects at several sites in carcinogen metabolism and regulation of tumor growth. It is not always clear that laboratory studies can be extrapolated to reasonable levels of consumption by humans of garlic or other Allium species.

Effects of C18 fatty acid isomers on DNA synthesis in hepatoma and breast cancer cells.

The influence of geometrical isomerism on the growth regulatory effects of 18 carbon unsaturated fatty acids on the incorporation of [3H]thymidine into DNA was studied in 7800NJ rat hepatoma and T47D human breast cancer cells. 9 cis, 12 cis linoleic acid was more inhibitory than the trans 9, trans 12 isomer (linolelaidic acid). The monounsaturated cis isomer, oleic acid, was also more inhibitory than the trans isomer. In contrast to published studies on the proliferation of breast cancer cells, we observed conditions in which linoleic acid was more inhibitory than conjugated linoleic acid for thymidine incorporation into DNA. Increasing the concentration of bovine serum albumin from 1 mg/ml to a physiological concentration of 38 mg/ml greatly diminished inhibitory effects and favored stimulatory effects on hepatoma and breast cancer cells. The results suggested that the growth inhibitory and stimulatory effects of C18 unsaturated fatty acids on cancer cells are influenced by geometrical isomerism and the ratio of the albumin to fatty acid concentrations.

Discordant effects of butyrate analogues on erythroleukemia cell proliferation, differentiation and histone deacetylase.

Actions of butyrate and structural analogues on histone deacetylase activity were compared with effects on proliferation and differentiation of erythroleukemia cells. Proliferation was inhibited by 5 mM tert- butylacetate, phenylacetate, phenylbutyrate, 3-bromopropionate and ethyl butyrate without induction of hemoglobin synthesis. n - Butyramide was a stronger inhibitor of cell proliferation and a more effective inducer of hemoglobin synthesis than isobutyramide. The data from combination studies were compatible with butyramide and isobutyramide being weaker agonists that competed for a common site with butyrate. Butyramide and isobutyramide were weaker inhibitors of histone deacetylase than 4-phenylbutyrate and phenylacetate, which in turn were less effective than 3-bromopropionate and butyrate. Butyrate and analogues had similar inhibitory effects on histone deacetylase activity in nuclei from mouse DS19 cells and human K562 cells. Effects on histone deacetylase did not show a consistent correlation with inhibition of cell proliferation or induction of hemoglobin synthesis.

Inhibitory effect of arginine restriction on hepatoma growth.

The potential effects of arginine depletion on promotion of hepatocarcinogenesis and the proliferation of hepatoma cells was investigated. A promotional effect of an arginine-free diet on tumor incidence in liver and kidney was not detected in rats and mice treated with N-nitrosodimethylamine. Inhibitory effects of an arginine-deficient diet on the growth of transplanted hepatomas were observed. Relative to the effect on body weight, the inhibition was greater in mice than rats. The inhibitory effects of an arginine-deficient diet were not correlated with the arginase activity in the tumors. Studies with hepatoma cells treated with polyethyleneglycol-modified arginase indicated that the inhibitory effects of arginine-deprivation on DNA synthesis need not be related to depletion of polyamine precursors.

Changes in the glucose-6-phosphatase complex in hepatomas.

Hepatomas tend to have a decreased glucose-6-phosphatase activity. We have observed phenotypic stability for this change in Morris hepatomas transplanted in rats. To determine if this decrease is selective for translocase functions or the hydrolase activity associated with glucose-6-phosphatase, we have compared activities in liver and hepatomas with glucose-6-phosphate or mannose-6-phosphate as substrates and with intact or histone-disrupted microsomes. In five out of seven subcutaneously transplanted rat hepatoma lines, the microsomal mannose-6-phosphatase activity was lower than in preparations from liver of normal or tumor-bearing rats. With liver microsomes and with most hepatoma microsomes, preincubation with calf thymus histones caused a greater increase in mannose-6-phosphatase than in glucose-6-phosphatase activity. In studies with liver and hepatoma microsomes there were similar increases in mannose-6-phosphatase activity with total calf thymus histones and arginine-rich histones. A smaller increase was seen with lysine-rich histones. The effect of polylysine was similar to the action of lysine-rich histones. There was only a small effect with protamine at the same concentration (1 mg/ml). Rat liver or hepatoma H1 histones gave only about half the activation seen with core nucleosomal histones. Our data suggested that microsomes of rat hepatomas tend to have decreased translocase and hydrolase functions of glucose-6-phosphatase relative to activities in untransformed liver.

Inhibitory effects of tea extracts and (-)-epigallocatechin gallate on DNA synthesis and proliferation of hepatoma and erythroleukemia cells.

Polyphenols extracted from green or black tea with ethyl acetate were strongly inhibitory for DNA synthesis in HTC rat hepatoma cells and DS19 mouse erythroleukemia cells at concentrations of 0.1-0.2 mg/ml. There was less inhibition with a subsequent black tea fraction extracted with butanol and with the residual water-soluble fraction. Although cell proliferation was inhibited by (-)-epigallocatechin gallate and the tea extracts, there were only marginal effects on differentiation of DS19 cells as judged by hemoglobin synthesis.

Butyramide and monobutyrin: growth inhibitory and differentiating agents.

Butyramide and monobutyrin were chosen for study as neutral compounds that might exhibit some of the growth inhibitory and differentiating effects of butyrate. Inhibitory effects on DNA synthesis in hepatoma cells and on cellular proliferation in mouse erythroleukemia cells were observed. Induction of differentiation by butyramide and monobutyrin was seen in mouse erythroleukemia cells as monitored by increased synthesis of hemoglobin and H1 zero histone. Butyramide and monobutyrin were less effective inducers of hemoglobin synthesis than butyrate but their neutral character may offer an advantage in the induction of tumor cell differentiation.

Inhibitory action of orotate, 2-thioorotate and isoorotate on nucleotide metabolism and nucleic acid synthesis in hepatoma cells.

1. The specificity of the action of orotate on hepatoma cells was investigated. 2. Orotic acid and its methyl ester had similar inhibitory effects on the incorporation of [3H]thymidine into DNA of hepatoma cells. 3. In contrast to previous studies in vivo, incubation of rat kidney cells with orotate caused an increase in the ratio of UTP/ATP concentrations that was similar to effects on hepatic cells. 4. Inhibitory effects of 2-thioorotate and isoorotate on metabolism were found to be less selective and required higher concentrations than with orotate.