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.

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.

Feedback inhibition of key glycolytic enzymes in liver: action of free fatty acids.

Increasing concentrations of sodium octanoate were progressively inhibitory to the activities of glucokinase, hexokinase, phosphofructokinase, and pyruvate kinase. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases were also markedly inhibited. Other enzymes of carbohydrate metabolism such as lactate dehydrogenase, phosphohexose isomerase, and fructose-1,6-diphosphatase were not decreased. Among the key glycolytic enzymes, the inhibition of pyruvate kinase by the fatty acid was most marked. The biological significance of the inhibition of the key glycolytic enzymes is interpreted as a feedback inhibitory mechanism in regulation of fatty acid biosynthesis. The mechanism may function for rapid adaptation by which the organism can use the fatty acid level as a metabolic directional switch in decreasing glycolysis and turning on gluconeogenesis.

Decreased sensitivity to colchicine-mediated inhibition of metabolite uptake in isolated hepatoma cells.

The effects of colchicine on the incorporation of [3H]-leucine and [3H]thymidine were studied in inbred BUF rats bearing Morris hepatomas and in isolated hepatoma cells and hepatocytes. The results confirmed previous work by other investigators that uptake of amino acids in hepatomas can be inhibited by colchicine (250 micrograms/100 body wt) under conditions in which uptake is not impeded in host liver. Uptake and incorporation of [3H]thymidine in hepatomas were also more sensitive to inhibition by colchicine. Evidence was obtained that these inhibitory effects on incorporation were diminished in isolated cells and were similar for isolated hepatoma cells and hepatocytes. The data did not exclude alternative mechanisms, but they were compatible with a previous suggestion that an effect of colchicine on circulation in hepatomas may be a factor in the inhibitory action on metabolite uptake.

Effects of cyanate, thiocyanate, and amygdalin on metabolite uptake in normal and neoplastic tissues of the rat.

Thiocyanate was found to resemble cyanate in its inhibitory effects on [3H]thymidine incorporation and the uptake of [32P]phosphate and [3H]amino acids in transplanted tumors of the BUF rat. The capacity to inhibit metabolite uptake in hepatomas and a colon tumor under conditions in which uptake was unchanged or increased in host liver was concluded to be a common feature of the action of cyanate and thiocyanate. Inhibition of [32P]phosphate uptake and [3H]thymidine incorporation into DNA of tumors was also observed after treatment of rats with amygdalin. With this drug, however, the action on tumors and livers of host rats was similar.

Selective effects of two chloromethyl ketones on amino acid and phosphate uptake in rat liver and tumors.

Injection of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) at a level of 10 mg/100 g body weight inhibited the incorporation of 3H-labeled amino acids into protein in Morris hepatomas 7777and 9618A2. The degree of inhibition was similar in cytoplasmic proteins and in histone and nonhistone nuclear protein fractions. There was no inhibitory effect on 3H-labeled amino acid incorporation in the livers of the tumor-bearing rats. The inhibitory effect of N-tosyl-L-lysine chloromethyl ketone (TLCK) on incorporation of 3H-labeled amino acids was observed in both the slowly growing hepatoma 7787 and the rapidly growing hepatoma 7777. In hepatoma 7777, TLCK (2.5 mg/100 g body wt) exerted a greater inhibitory effect on incorporation when administered 60 minutes before [3H]leucine injection than when injected simultaneously. Studies on tissue uptake of amino acids, thymidine, and phosphate indicated that inhibitory effects of TPCK and TLCK on active transport may be a major factor in the action of these drugs on macromolecular synthesis. The inhibitory effects of TPCK and TLCK seen in transplanted hepatomas and a colon tumor were not generally seen in normal tissues of the tumor-bearing rats.

Effects of nicotinamide, isonicotinamide, and bleomycin on DNA synthesis and repair in rat hepatocytes and hepatoma cells.

Because inhibitors of poly (ADP-ribose) synthetase have been found to influence DNA synthesis in some systems, the possibility that nicotinamide or isonicotinamide might potentiate the effect of bleomycin on DNA replication and repair was examined. After a 30-minute incubation with bleomycin (200 micrograms/ml), tritiated thymidine ([3H]dThd) incorporation into DNA was stimulated during a subsequent 30-minute incubation with hepatocytes of inbred BUF rats but was decreased in HTC cells of BUF rats. When unscheduled DNA synthesis was measured in the presence of 10 mM hydroxyurea, bleomycin (200 micrograms/ml) increased [3H]dThd incorporation in both cell types. A dose of 20 mM nicotinamide and isonicotinamide caused an approximately 50% inhibition of total [3H]dThd incorporation in HTC cells. Significant inhibitory effects of 20 mM nicotinamide and isonicotinamide on unscheduled DNA synthesis were observed after preincubation of hepatocytes and HTC cells with bleomycin. When the effects of bleomycin on DNA structure were assessed fluorometrically with ethidium bromide after mild alkaline incubation, nicotinamide and isonicotinamide did not significantly affect the damage revealed with bleomycin alone. When HTC cells were incubated for 48 hours with bleomycin (20 micrograms/ml), the increase in cell numbers was about 50% of that in control cultures. Nicotinamide and isonicotinamide also inhibited the proliferation of HTC cells, but the effects were not additive with the effect of bleomycin.

Uptake of 14C-labeled dicarboxylic amino acids in hepatocytes and hepatoma cells.

In previous studies, we observed decreased uptake of 14C-labeled L-aspartate and L-glutamate in s.c. transplants of several rapidly growing hepatomas relative to that in normal liver. The present report extends these observations to isolated cells and indicates that circulation differences cannot be the major factor. Mean net uptakes for the two dicarboxylic amino acids in cells from the rapidly growing Morris Hepatomas 7288ctc and 7777 were 5 to 26% of corresponding values for normal hepatocytes. Rates for net uptake in Hepatoma 7787 cells were intermediate between those of the rapidly growing hepatomas and hepatocytes, while the rates for Hepatoma 5123C cells and hepatocytes were similar. The contribution of sodium-dependent uptake to the mean total net uptake of [14C]aspartate and [14C]glutamate tended to be higher in hepatoma cells than in hepatocytes. Studies with isolated hepatocytes and Hepatoma 5123C cells showed no significant effect on uptake by 10 mM alpha-(methylamino)isobutyric acid and 10 mM 2-amino-2-carboxybicyclo[2.2.1]heptane. On the other hand, L-cysteic acid, L-alanosine, and N-phosphonacetyl-L-aspartic acid were shown to be effective inhibitors of sodium-dependent uptake in Hepatoma 5123C cells. The data suggest that the A and L systems are not major contributors to the uptake of dicarboxylic amino acids in hepatic cells. It was concluded that decreased uptake of dicarboxylic amino acids in rapidly growing hepatomas may accompany decreased metabolism of these dietary nonessential amino acids.

Spermine-induced variations in the adenosine 5'-diphosphate ribosylation patterns of nuclear proteins from rat liver and hepatoma.

Rat liver and hepatoma nuclei were incubated in vitro with [3H]nicotinamide adenine dinucleotide to allow synthesis of a polymer of adenosine diphosphoribose subunits joined in an 1',2' ribose-ribose linkage. The addition of 1 mM spermine altered the adenosine 5'-diphosphate (ADP) ribosylation patterns of nuclear proteins in hepatoma, host liver, and regenerating liver. Spermine-treated nuclei showed a greater incorporation of ADP-ribose into H1 histones and nonhistone nuclear proteins with isoelectric points between pH 3.0 and 6.0 when separated on polyacrylamide gels. Conversely, a large reduction in ADP ribosylation was seen in core histones (H2A, H2B, and H3) from the same nuclei. The proportion of ADP-ribose incorporated into histones was reduced in the nuclei from proliferating cells relative to their respective control livers. These results imply that polyamines, which are higher in concentration in rapidly dividing cells, may elicit a regulatory function by causing the preferential ADP ribosylation of H1 histones, as well as the more acidic of the nuclear proteins.

Effects of cyanate on the distribution of isotope-labeled H2O and extracellular markers in rat liver and tumors.

Previous work has shown that administration of sodium cyanate inhibits the uptake of several metabolites in tumors under conditions in which there is generally no inhibition in normal tissues of the rat. In the present work, it was found that cyanate treatment inhibits the distribution of 3H2O, [3H]methoxyinulin, and [14C]sucrose in rats with greater effects in the tumors than the normal tissues examined. Tumor-bearing rats received i.p. injections of sodium cyanate (250 mg/kg body weight). After 60 min, the rats received s.c. injections of 3H2O. Treatment with cyanate decreased the radioactivity in blood and liver, but greater effects were seen in five transplanted tumors (LK1 colon tumor and Morris hepatomas 5123C, 7288CTC, 7777, and 9618A2). At 10 min after injection of 3H2O, the mean radioactivities in tumors of cyanate-treated rats were 11 to 23% of control values and in some tumors were still less than in controls at 60 min after isotope injection. Evidence was obtained that the action of cyanate was not due to osmotic effects or loss of water from the tissues. The distribution of the extracellular markers [3H]methoxyinulin and [14C]sucrose was also decreased in hepatomas in cyanate-treated rats. The data do not exclude effects on membrane permeability but suggested that cyanate decreased circulation in the tumors.

Binding of metabolically activated benzo(a)pyrene to nuclear macromolecules.

The binding of metabolically activated [3H]benzo(a)pyrene ([3H]BP) to the DNA, RNA, histones, and nonhistones of isolated rat liver and lung nuclei was studied. Conditions for optimal binding to the nuclear components were determined. Upon incubation with isolated liver nuclei and reduced nicotinamide adenine dinucleotide phosphate, [3H]BP was able to bind to nuclear components. The binding appeared to be covalent in nature. Treatment of the rats with 3-methylcholanthrene induced the nuclear aryl hydrocarbon hydroxylase (AHH) activity and also increased the level of carcinogen binding. The addition of rat liver microsomes to the incubation systems greatly enhanced the level of [3H]BP binding to the macromolecules in the nuclei from both the control and 3-methylcholanthrene-treated rats, and the maximal levels of binding obtained with these two types of nuclei were similar. The binding was inhibited by 7,8-benzoflavone or glutathione. Lung nuclei from control rats had very low AHH activity and did not exhibit appreciable carcinogen binding, whereas those from 3-methylcholanthrene-pretreated animals had slightly higher AHH activity and caused low levels of binding. The binding of [3H]BP to lung nuclei was greatly enhanced by liver microsomes but only slightly by lung microsomes, which had rather low AHH activity. Several lines of evidence indicate that, in the control experiments (no reduced nicotinamide adenine dinucleotide phosphate added), the radioactivity associated with the macromolecule fractions is probably a background value rather than due to the binding caused by a specific interaction between benzo(a)pyrene and cytochrome P-450. The present study clearly demonstrates that a carcinogen activated at the microsomes can enter into the nucleus and react with its macromolecules; the carcinogen can also be activated by the monoxygenase system of the nuclear envelope. It appears that both the endoplasmic reticulum and the nuclear envelope are potentially important sites of carcinogen activation.

Nuclear protein changes in rat hepatomas correlating with growth rate.

The nature of nuclear proteins that are soluble in 8 M urea-50 mM phosphate, pH 7.6, was compared in rat liver and Morris hepatomas, Isoelectric focusing, using carrier ampholytes for a pH gradient of 3.5 to 10, indicated that with increasing growth rate of the hepatomas there was a progressive tendency for a decrease in nonhistone nuclear proteins with isoelectric points in the range 7.5 to 8.9 and an increase in the range 5.1 to 6.7. Studies on the influence of time on the pH gradient revealed that a nonuniform drift provided a better resolution of the pH range 7.5 to 8.9 at 7 hr than at 24 hr, while the latter time for electrofocusing gave an improved resolution of the pH range 5.1 to 6.7 Polyarcylamide gel electrophoresis in a urea-acetic acid system showed that 8 M urea-50 mM phosphate; pH 7.6 extracted a small part of the histones from nuclei of both liver and hepatomas. There was less extraction of histones from the hepatoma nuclei, especially in two rapidly growing hepatomas with the most notable difference being seen in the lysine-rich H1 histone. The results suggested that in addition to qualitative or quantitative changes in nonhistone nuclear proteins in liver cancer there are alterations in the binding of histones to chromatin.

Tumor-selective inhibition of the incorporation of 3H-labeled amino acids into protein by cyanate.

Sodium cyanate at a dose level of 125 or 250 mg/kg i.p. caused an inhibition of incorporation of 3H-labeled amino acids into cytoplasmic and nuclear proteins of the rapidly growing hepatoma 7777 and the slowly growing hepatoma 9618A. There was no inhibitory effect on 3H-labeled amino acid incorporation into protein in the livers of rats bearing these tumors. Studies on the effects of sodium cyanate on incorporation of 3H-labeled amino acids into total acid-insoluble material indicated that a greater than 85% inhibition could be achieved in hepatoma 5123C, hepatoma 9618A2, and the MK3 kidney tumor with either little or no effect in host liver, kidneys, brain, skeletal muscle, intestinal mucosa, and regenerating liver after partial hepatectomy.

Decreased uptake of 14C-labeled dicarboxylic amino acids in rapidly growing hepatomas.

In contrast to the increased uptake of amino acids which has been found in many neoplastic cells, we have observed a decrease in the net uptake of [14C]aspartate and [14C]glutamate in rapidly growing hepatomas relative to rat host liver. When measured 10 min after s.c. injection, the radioactivity from 14C-labeled dicarboxylic amino acids was greater in liver than in all other tissues examined (blood, skeletal, muscle, heart, spleen, lung, and brain) except kidney, where there was an approximately 2-fold greater uptake of aspartate and 10-fold greater uptake of glutamate. Mean uptakes in the rapidly growing Morris hepatomas 7288CTC and 7777 were 19 to 26% of corresponding values for the host livers. Comparison with uptake of 3H2O indicated that these low values were not solely due to differences in circulation. Decreased uptake was not accompanied by equivalent decreases in the concentration of aspartate and glutamate in the tumors. There were small changes in the net uptake of these amino acids in the slowly growing hepatoma 7787 and no significant differences in regenerating liver and hepatoma 5123C, a tumor of intermediate growth rate. The net uptake of [14C]arginine and [14C]lysine in the hepatomas was similar to that in host livers, except for a 250% increase in uptake of [14C]lysine in hepatoma 5123C. A decreased uptake of the magnitude seen with dicarboxylic amino acids in rapidly growing hepatomas has not been observed with other amino acids.

Fractionation of nuclei and analysis of nuclear proteins of rat liver and Morris hepatoma 7777.

The contributions of nuclear populations to the total profile of nuclear proteins in a tissue were examined in normal rat liver and Morris hepatoma 7777. Comparison by sodium dodecyl sulfate polyacrylamide gel electrophoresis of phenol-soluble nuclear proteins from tumor and control liver revealed additional proteins of molecular weight 60,000, 100,00, and 135,000 and the loss of proteins of about 45,000 and 55,000 in the tumor. Subfractionation of liver nuclei on a 30 to 50% sucrose gradient yielded three nuclear classes with nearly identical complements of the phenol-soluble proteins. Similar fractionation performed on the hepatoma nuclei also produced three nuclear populations. In the hepatoma nuclei, several differences in the phenol-soluble proteins were found between the minor, slowly sedimenting nuclear fraction, and the two major fractions, while the two latter fractions were very similar in their protein composition. Histones derived from both tissues were also compared electrophoretically, indicating a decrease in the concentration of histone H1(0)in all nuclear classes derived from the tumor.

Soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA of Novikoff hepatoma cells.

The nature of soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA was studied in Novikoff hepatoma cells. The decreased activity in hepatoma preparations was due to loss of a high-molecular-weight heat-labile factor. Although this factor cochromatographed with arginase activity on Sephadex G-150, it does not appear to result from this activity as judged by the failure of arginine to prevent the inhibitory effect on [3H]thymidine incorporation. Both liver and hepatomas contained a heat-stable factor with inhibitory activity. Studies with ethanol-soluble material suggested that the action was not solely attributable to the presence of unlabeled thymidine, since the apparent molecular weight was too high and since the factor(s) inhibited [3H]leucine incorporation into protein in addition to inhibiting [3H]thymidine incorporation in DNA.

Selective modulation of nucleotide levels in rat liver and hepatomas by high-orotate or arginine-deficient diets and by carbamoylating agents.

Transplanted Morris hepatomas in Buffalo-strain rats were found to be resistant to the changes in ribonucleotide levels in rat liver caused by a high-orotate diet or an arginine-deficient diet. The increase in UTP levels and decrease in ATP levels seen in the livers of rats on a 1%-orotate diet were less marked in the livers of BUB- and DBA-strain mice on this diet. Although the changes were less than in rat liver, there was a 2-3-fold increase in UTP concentration in the livers of mice on the high-orotate diet. However, there was a similar response in nucleotide levels in the two species when the animals were maintained on an arginine-deficient diet, and there was a greater than 10-fold increase in the UTP level in the livers of both rats and mice. These diets had much less effect on the levels of deoxyribonucleotides than of ribonucleotides. In contrast to the insensitivity of hepatomas to dietary modulation of nucleotide levels, treatment of hepatoma-bearing rats with carbamoylating agents (sodium cyanate and 2-chloroethyl isocyanate) caused decreases in the levels of nucleotides in the tumors which were generally greater than in host livers. For example, 2-chloroethyl isocyanate depressed ATP levels in the Morris hepatomas 5123C and 20 under conditions in which there was no significant effect on host liver ATP. The data revealed selective modulation of nucleotide levels in normal and neoplastic liver which may be achieved by either dietary modification or drug treatment.

Divergent effects of cyanate on amino acid and phosphate uptake by liver and hepatoma.

The uptake of alpha-aminoiso[3H]butyric acid and 32Pi was observed to be inhibited by sodium cyanate in transplanted hepatomas but was increased in the livers of the tumor bearing rats. Incorporation of 32Pi into macromolecules in hepatomas was also inhibited by cyanate. Treatment with this drug did not influence circulating concentrations of isotope-labeled materials. There were relatively small effects on uptake of 36Cl- in cyanate-treated rats and the action was not tissue specific. The data were compatible with an inhibitory effect of cyanate on active transport in hepatomas which was not seen under the same conditions in host liver.

Effects of carbamoylating agents on tumor metabolism.

Carbamoylation of macromolecules occurs by the displacement of hydrogen on several groups, but the most stable addition at neutral pH is on amino groups. This reaction occurs predominantly with proteins and results from the administration in vivo of inorganic cyanate or organic isocyanates. The latter act more rapidly, but also are more rapidly hydrolyzed in aqueous solution. This instability has been a factor limiting study of the pharmacological properties of organic isocyanates. However, organic isocyanates are released from some nitrosoureas of value in cancer therapy such as 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The carbamoylating activities of BCNU and CCNU are generally considered less significant than their alkylating activity in the action of these drugs on tumors, but carbamoylation may serve to inhibit DNA repair. There is evidence that carbamoylating agents can exert selective inhibitory effects on metabolite uptake and macromolecular synthesis in neoplastic tissues. Such selectivity is much more notable in vivo than in vitro. In the case of cyanate, the selectivity in vivo has been variously attributed to a requirement for metabolic activation, to selective effects on circulation in solid tumors, and to diminished pH in tumors. It is the distinction between such factors and the identification of critical cellular targets which provide major challenges in present studies on the effects of carbamoylating agents on tumor metabolism.

Colchicine affects the distribution of isotope-labeled H2O and extracellular markers in rat liver and hepatomas.

Treatment of hepatoma-bearing rats with colchicine (175 or 250 micrograms/100 g body wt) greatly diminished the radioactivity in tumors when measured 10 min after subcutaneous injection of 3H2O. There were relatively small effects of colchicine treatment on the level of radioactivity in blood and little change in the ratio of radioactivity in blood and liver. Inhibitory effects of colchicine were also observed for the distribution in hepatomas of the extracellular markers [3H] methoxy-inulin and [14C] sucrose. An effect of colchicine on circulation in hepatomas may be a factor in the previously reported inhibitory action on metabolite uptake.