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Cisco (Coregonus artedi) are threatened by climate change and lake eutrophication, and their oxythermal habitat can be assessed with TDO3, the water temperature at which dissolved oxygen equals 3 mg·L−1. We assessed the influence of TDO3 on cisco habitat use, genetic diversity, diets, and isotopic niche in 32 lakes ranging from oligotrophic to eutrophic. Results showed that as TDO3 increased, cisco were captured higher in the water column, in a narrower band, with higher minimum temperatures and lower minimum dissolved oxygen. TDO3 was also negatively related to cisco allelic richness and expected heterozygosity, likely driven by summer kill events. Moreover, TDO3 influenced the isotopic niche of cisco, as fish captured deeper were more depleted in δ13C and more enriched in δ15N compared to epilimnetic baselines. Lastly, cisco in high TDO3 lakes consumed more Daphnia, had fewer empty stomachs, and achieved larger body size. Our work identifies specific characteristics of cisco populations that respond to climate change and eutrophication effects and provides a framework for understanding responses of other cold-water species at the global scale.

SynopsisMitochondrial function is critical for energy homeostasis and should shape how genetic variation in metabolism is transmitted through levels of biological organization to generate stability in organismal performance. Mitochondrial function is encoded by genes in two distinct and separately inherited genomes—the mitochondrial genome and the nuclear genome—and selection is expected to maintain functional mito-nuclear interactions. The documented high levels of polymorphism in genes involved in these mito-nuclear interactions and wide variation for mitochondrial function demands an explanation for how and why variability in such a fundamental trait is maintained. Potamopyrgus antipodarum is a New Zealand freshwater snail with coexisting sexual and asexual individuals and, accordingly, contrasting systems of separate vs. co-inheritance of nuclear and mitochondrial genomes. As such, this snail provides a powerful means to dissect the evolutionary and functional consequences of mito-nuclear variation. The lakes inhabited by P. antipodarum span wide environmental gradients, with substantial across-lake genetic structure and mito-nuclear discordance. This situation allows us to use comparisons across reproductive modes and lakes to partition variation in cellular respiration across genetic and environmental axes. Here, we integrated cellular, physiological, and behavioral approaches to quantify variation in mitochondrial function across a diverse set of wild P. antipodarum lineages. We found extensive across-lake variation in organismal oxygen consumption and behavioral response to heat stress and differences across sexes in mitochondrial membrane potential but few global effects of reproductive mode. Taken together, our data set the stage for applying this important model system for sexual reproduction and polyploidy to dissecting the complex relationships between mito-nuclear variation, performance, plasticity, and fitness in natural populations.

Introductions of Coregonus artedi from Lake Superior into several inland lakes in Minnesota in the 1920s and 1930s constituted a natural experiment of how phenotypic plasticity and adaptation can shape cisco diversity. Genetic data were consistent with a Lake Superior origin for the introduced populations, which resembled contemporary Lake Superior cisco in possessing large, elongated bodies and short fins, and in maturing at older ages. Native inland cisco had smaller, deeper bodies, longer fins, and matured at younger ages. Introduced populations also retained the Lake Superior characteristic of inhabiting the upper portion of the water column and showed a high degree of planktivory. However, introduced cisco exhibited small but measurable shifts in body shape and fin lengths toward native inland forms. These morphological shifts were possibly associated with greater benthivory after translocation. Assuming that native inland populations were well adapted to inland lakes and that selective pressures on native and introduced populations were similar, these results indicated that substantial further phenotypic, behavioral, and life history change is required before the introduced populations become similarly adapted to their new environments. The findings suggest that anthropogenic environmental alterations such as climate change and eutrophication might occur at a faster pace than adaptive evolutionary responses. In addition, translocated Great Lakes coregonines could possibly maintain traits exhibited in their source lake, and may not fully restore ecological functions of extirpated populations.

Mitochondrial function is critical for energy homeostasis and should shape how genetic variation in metabolism is transmitted through levels of biological organization to generate stability in organismal performance. Mitochondrial function is encoded by genes in two distinct and separately inherited genomes – the mitochondrial genome and the nuclear genome – and selection is expected to maintain functional mito-nuclear interactions. Nevertheless, high levels of polymorphism in genes involved in these mito-nuclear interactions and variation for mitochondrial function are nevertheless frequently observed, demanding an explanation for how and why variability in such a fundamental trait is maintained.Potamopyrgus antipodarumis a New Zealand freshwater snail with coexisting sexual and asexual individuals and, accordingly, contrasting systems of separate vs. co-inheritance of nuclear and mitochondrial genomes. As such, this snail provides a powerful means to dissect the evolutionary and functional consequences of mito-nuclear variation. The lakes inhabited byP. antipodarumspan wide environmental gradients, with substantial across-lake genetic structure and mito-nuclear discordance. This situation allows us to use comparisons across reproductive modes and lakes to partition variation in cellular respiration across genetic and environmental axes. Here, we integrated cellular, physiological, and behavioral approaches to quantify variation in mitochondrial function across a diverse set of wildP. antipodarumlineages. We found extensive across-lake variation in organismal oxygen consumption, mitochondrial membrane potential, and behavioral response to heat stress, but few global effects of reproductive mode or sex. Taken together, our data set the stage for applying this important model system for sexual reproduction and polyploidy to dissecting the complex relationships between mito-nuclear variation, performance, plasticity, and fitness in natural populations.

The proteins responsible for mitochondrial function are encoded by two different genomes with distinct inheritance regimes, rendering rigorous inference of genotype-phenotype connections intractable for all but a few model systems. Asexual organisms provide a powerful means for addressing these challenges because offspring produced without recombination inherit both nuclear and mitochondrial genomes from a single parent. As such, these offspring inherit mitonuclear genotypes that are identical to the mitonuclear genotypes of their parents and siblings and different from those of other asexual lineages. Here, we compared mitochondrial function across distinct asexual lineages ofPotamopyrgus antipodarum,a New Zealand freshwater snail model for understanding the evolutionary consequences of asexuality. Our analyses revealed substantial phenotypic variation across asexual lineages at three levels of biological organization: mitogenomic, organellar, and organismal. These data demonstrate that different asexual lineages have different mitochondrial function phenotypes and that there exists heritable variation (that is, the raw material for evolution) for mitochondrial function inP. antipodarum.The discovery of this variation combined with the methods developed here sets the stage to useP. antipodarumto study central evolutionary questions involving mitochondrial function, including whether mitochondrial mutation accumulation influences the maintenance of sexual reproduction in natural populations.

The proteins responsible for mitochondrial function are encoded by 2 different genomes with distinct inheritance regimes, rendering rigorous inference of genotype-phenotype connections intractable for all but a few model systems. Asexual organisms provide a powerful means to address these challenges because offspring produced without recombination inherit both nuclear and mitochondrial genomes from a single parent. As such, these offspring inherit mitonuclear genotypes that are identical to the mitonuclear genotypes of their parents and siblings but different from those of other asexual lineages. Here, we compared mitochondrial function across distinct asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail model for understanding the evolutionary consequences of asexuality. Our analyses revealed substantial phenotypic variation across asexual lineages at 3 levels of biological organization: mitogenomic, organellar, and organismal. These data demonstrate that different asexual lineages have different mitochondrial function phenotypes, likely reflecting heritable variation (i.e., the raw material for evolution) for mitochondrial function in P. antipodarum. The discovery of this variation combined with the methods developed here sets the stage to use P. antipodarum to study central evolutionary questions involving mitochondrial function, including whether mitochondrial mutation accumulation influences the maintenance of sexual reproduction in natural populations.

Despite their important roles in cell adhesion, motility, immune function, and other processes, little is known about the expression control of the Ras-family small GTPases RAP1A and 1B, located on...

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.

Hepatocyte growth factor (HGF), a potent mitogen, is released into plasma at increased levels following injury to certain tissues, including the liver. Early increases in plasma HGF are not due to a release from the injured liver, but rather from distal organs, particularly the lung. We have investigated the ability of norepinephrine (NE), which rises rapidly in plasma after liver resection, to trigger elevated production of HGF in MRC-5 human embryonic lung fibroblasts. Levels of HGF released to culture media and of HGF mRNA increased when cultures were exposed to NE, or to other adrenergic agonists. While stimulation of either beta- or alpha 1 -adrenergic receptors increased HGF expression, responses to NE appear to be mediated primarily via beta receptors. Since NE has already been shown to act as a comitogen with HGF, our findings suggest that adrenergic hormones may act both to induce production of HGF at distal sites, and to enhance the response to HGF at target tissues.

We have characterized homologous DNA recombination activity in nuclear protein extracts prepared from quiescent and regenerating rat livers. Activity measured in regenerating liver extracts was elevated approximately 35-fold above control, and its appearance closely mirrored the first wave of DNA synthesis, peaking 24 hours after a regenerative stimulus, and returning fairly rapidly to basal levels. We also identified a strand-transferase protein of approximately 100 kDa whose presence in these extracts correlates with homologous recombination activity. Recent evidence suggests that mammalian somatic cells possess a recombinational DNA repair mechanism analogous to that described in the yeast Saccharomyces cerevisiae. Our results indicate that this recombinational repair process may be regulated in vivo by, or play a role in, progression through the cell division cycle.

The α1 receptor mediates the effects of catecholamines on DNA synthesis, as observed in rat liver following a 2/3 partial hepatectomy and in serum-free primary cultures of adult rat hepatocytes exposed to epidermal growth factor.In vitro norepinephrine action at this receptor heterologously down-regulates epidermal growth factor receptors.In vivo, the α1 receptor's effect on DNA synthesis is restricted to early time points after partial hepatectomy. α1 receptor binding capacity does not vary until 48 hr after liver resection (at which time binding is reduced), but an uncoupling of receptor binding from membrane phosphoinositide turnover occurs between 8 and 16 hr after partial hepatectomy. This change is preceded by a fall in membrane-associatedras p21 detected by radioimmunoassays (46% of control levels by 2 hr after partial hepatectomy). Whether this change represents a loss of p21 protein from membranes or a modification that results in a loss of immunoreactivity is not known.

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.

Rap1 proteins are capable of competing with Ras p21 for binding to effectors, and of antagonizing some Ras-induced effects, but their participation in normal growth regulation has not been established. The level of Rap1 protein and the expression of the rap1A gene were examined by immunoblotting and Northern analysis during the regenerative growth response in rat liver following partial hepatectomy. Protein and mRNA were significantly down-regulated prior to and during the onset of DNA synthesis. The timing of this effect is consistent with a model in which expression of Rap1 is turned off or down to allow the initiation of proliferation.

Studies with regenerating liver and hepatocyte cultures have shown that the alpha-1 adrenergic receptor (A1AR) is involved in the early events which transmit a mitogenic signal to hepatocytes after 2/3 partial hepatectomy. In this study, we investigated the role of A1AR in DNA synthesis associated with the augmentative hyperplasia stimulated by the xenobiotic hepatic tumor promoters phenobarbital (PB) and alpha-hexachlorocyclohexane (alpha-HCH), and the peroxisome proliferator ciprofibrate. Male F344 rats were treated with each of the three xenobiotics to stimulate hepatic DNA synthesis. When either phenobarbital or alpha-HCH administration was preceded and accompanied by the A1AR antagonist prazosin, DNA synthesis was significantly inhibited, as measured by [3H]thymidine incorporation or 5-bromo-2'-deoxyuridine (BrdU) nuclear labeling index. There was no inhibition of DNA synthesis by prazosin in the ciprofibrate treated group. The inhibition of hepatic DNA synthesis by prazosin was accompanied by non-significant changes in the number of alpha-1 binding sites in the PB and alpha-HCH treated groups, but a significantly reduced number of alpha-1 binding sites in the ciprofibrate treated group. These studies suggest that A1AR is involved in generating the mitogenic signal leading to hepatic DNA synthesis induced by xenobiotic hepatic tumor promoters phenobarbital and alpha-HCH. A1AR is not involved in the mitogenic pathway generated by the peroxisome proliferator ciprofibrate.

In rat liver parenchyma, two subpopulations of hepatocytes can be distinguished by the absence or presence of the marker enzyme, glutamine synthetase (GS). Hepatocytes in the perivenous zone immediately adjacent to the hepatic venules in the liver acinus are positive for GS. Using autoradiography in combination with immunocytochemistry, the response of these two hepatocyte populations (GS positive and GS negative) to a variety of growth factors (defined compounds or complex stimuli) was investigated in vitro. Irrespective of the individual growth-promoting activity (which varied considerably), all stimuli led to much higher labeling indices in GS-negative cells as compared to GS-positive cells. In GS-negative cells, the strongest effect was exerted by serum obtained from partially hepatectomized rats (labeling index, 67%) and the conditioned media of JM1 and JM2 hepatoma cells (63%-82%), followed by a combination of insulin and either norepinephrine (46%) or epidermal growth factor (EGF; 42%). In contrast, serum had the weakest influence on GS-positive cells (0.3%), while the other potent stimuli enhanced the labeling index of these cells by between 6% and 15% within 48 h. The percentage of labeled nuclei was higher in mononucleated than in binucleated GS-positive hepatocytes. The time course of thymidine incorporation was also different for the two subpopulations. Under all growth-promoting conditions, the stimulation of GS-negative cells peaked between 72 and 96 h, while it increased continuously in GS-positive cells for at least 120 h, particularly in the case of serum. In proliferating cultures, both the absolute and the relative number of GS-positive hepatocytes decreased, while no such effect was found in various nonproliferating control cultures maintained at low and high cell density. Similar results were found for GS activity. In contrast, the hormonal induction of tyrosine aminotransferase (TAT) was not affected. It is suggested that these differences in the growth response of GS-positive and -negative cells contribute to the acinar gradient in hepatocyte proliferation that occurs during liver regeneration. Furthermore, the striking phenotypic instability of GS-positive cells that have undergone DNA synthesis and mitosis supports the hypothesis that cellular reprogramming depends on passage through the cell cycle.

Norepinephrine (NE) produced a dose-dependent inhibition of 125I-epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an alpha 1-specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity.

The role of adrenergic agents in DNA synthesis was investigated in two models of stimulated hepatocyte growth: in vitro primary serum-free cultures of adult parenchymal hepatocytes, and in vivo liver regeneration after two-thirds partial hepatectomy. In both systems the alpha 1-adrenergic receptor appeared to be involved in mediating stimulatory effects. In primary hepatocyte cultures norepinephrine acted via this receptor to enhance the DNA synthesis stimulated by epidermal growth factor (EGF), and heterologously downregulated EGF receptors. In liver regeneration the administration of an alpha 1 blocking agent interfered with the first wave of regenerative DNA synthesis, and this effect was preceded by an elevation in EGF receptor number. Measurements of plasma catcholamines demonstrated that elevated levels of norepinephrine and epinephrine were in circulation within 2 h after partial hepatectomy. Surgical hepatic sympathectomy also interfered with early liver regeneration, suggesting that locally delivered adrenergic agents are important to initiation of DNA synthesis. These data suggest that stimulation at the alpha 1-adrenergic receptor is among the early signals for liver regeneration and that heterologous regulation of EGF receptors, similar to that observed in vitro, may be a part of the regenerative response.

Stimulation of DNA synthesis by peroxisonte proliferators, including DEHP, should be viewed differently from the stimulation of DNA synthesis by xenobiotic chemicals which stimulate restorative hyperplasia after hepatic necrosis induced by the toxicity of the chemical. The emerging picture of the control mechanisms for hepatocyte proliferation suggests that rather few and distinct factors are involved. The stimulation of DNA synthesis by peroxisome proliferators should be examined in the context of the effects of these factors. Comparisons with other xenobiotics show that induction of DNA synthesis at rates comparable to those of peroxisome proliferators is not sufficient to explain the rates of carcinogenicity associated with peroxisonie proliferators. These considerations lead to the conclusion that although DNA synthesis enhances the incidence of neoplasia, it should not be viewed as a complete carcinogen, nor should it be considered as resulting in initiation at rates that can explain the carcinogenic potency of compounds such as peroxisome proliferators.

Primary cultures of adult rat hepatocytes are stimulated to enter DNA synthesis by norepinephrine (NE). This stimulation is maximal if the hepatocytes are incubated with NE for more than 12 hr, beginning no later than 2-4 hr after the cells are first plated. After 24 hr in culture, hepatocytes are unresponsive to NE stimulation. A strong synerergistic interaction between NE and epidermal growth factor (EGF) may be observed in cultures incubated with both EGF and NE, or pretreated with NE, then exposed to EGF. This interaction may be related to the finding that NE, in similarity with other factors that enhance EGF stimulation, reduces binding at the EGF receptor during the first 24 hr in culture.

The effects of several treatments involving alpha-adrenergic mechanisms upon the early stages of rat liver regeneration were examined. Catecholamine concentrations in rat plasma were measured at various times after hepatectomy and were found to be elevated relative to those in plasma from sham-operated rats. Surgical hepatic denervation or injection of an alpha 1-adrenergic receptor antagonist (prazosin) reduced incorporation of [3H]thymidine into liver DNA during the first 24 hr after partial hepatectomy. Chronic guanethidine injections (3 to 6 weeks) reduced liver catecholamine levels, but did not affect its ability to regenerate. The inhibition of regenerative DNA synthesis by prazosin was preceded by an alteration in the binding of epidermal growth factor to regenerating liver, which was apparently the result of an increased number of epidermal growth factor receptors. Thus, alpha 1-adrenergic blockade, which affects both epidermal growth factor receptor binding and subsequent DNA synthesis in hepatocyte primary cultures, can also modulate these processes during liver regeneration in vivo.

Catecholamines, acting via the alpha-1 adrenergic receptor, have been demonstrated to influence adult rat hepatocyte DNA synthesis in primary culture and in vivo during liver regeneration following partial hepatectomy (PHX). Earlier investigations have suggested that the alpha-1 effect on DNA synthesis is significant only during the first day following PHX. We examined receptor binding at several early and late time points after surgery, and we observed a significant loss of specific [3H]-prazosin binding to cells isolated from rat livers 48 and 72 hr after PHX. In contrast, the ability of norepinephrine to stimulate inositol phosphate production in isolated cells prelabeled with [3H]-myo-inositol was transiently reduced between 8 and 16 hr, when alpha-1 binding capacity was virtually unchanged. This uncoupling of phosphoinositide turnover from binding was preceded by a drop in hepatic membrane ras p21 content, as assayed by liquid competition radioimmunoassay. The loss of immunoreactive p21 from membranes was significant by 2 hr after PHX. These findings suggest a role for alpha-1 receptors and ras protein in the early events of liver regeneration.

TGF-beta is a potent inhibitor of EGF-induced DNA synthesis in primary rat hepatocyte cultures. Norepinephrine (NE) was shown to modulate this inhibition of DNA synthesis. It produced a five-fold increase, from 2.8 pM to 14.4 pM, in the ID50 for TGF-beta. The effect was dose-dependent and was significant at concentrations of 10(-6)M NE and greater. The modulation by NE was mediated by the alpha 1-adrenergic receptor as shown by the ability of the alpha 1 antagonist prazosin to block the activity. This effect might be important during liver regeneration in allowing escape of hepatocytes from negative growth control exerted by TGF-beta.

Addition of norepinephrine to primary cultures of adult rat hepatocytes stimulates the incorporation of [3H]thymidine in a dose-dependent manner. This effect has been observed in serum-free medium containing epidermal growth factor and insulin. Stimulation of DNA synthesis by norepinephrine was strongly antagonized by the alpha 1-adrenergic antagonist prazosin but not by an alpha 2 antagonist or by a beta-adrenergic blocker. The beta agonist isoproterenol did not stimulate significant DNA synthesis. These results indicate that catecholamines interact with the alpha 1 adrenoreceptor to stimulate DNA synthesis in hepatocytes. Since alpha 1 receptors are present in most cells, this receptor may be important in cell growth regulation.

The autonomic nervous system in rats has been assessed by means of indirect immunofluorescence using monospecific antibodies to neuron-specific enolase, neurofilaments, glial fibrillary acidic protein and S-100 protein [10 days after partial (70%) hepatectomy]. Different groups of rats were studied: group A: 70% resection and normal dual blood supply (n = 5); group B: 70% resection with only portal blood to the liver remnant (n = 5); group C: 70% resection with only arterial blood to the liver remnant (n = 5); group D: sham operated controls (n = 5). All rats of groups A and D showed normal liver/body weight ratios after 10 days in contrast to groups B and C where liver weights were 50–60% of the preresection weight. In group A the regeneration process was histologically normal and associated with a remarkable increase of autonomic innervation patterns in the portal triad. In contrast, livers of animals in groups B and C showed under the light microscope features of hepatocyte degeneration associated with a decreased autonomic innervation compared to the controls. The changes are identical in groups B and C, and are therefore irrespective of the type of blood deprivation (arterial or portal). These results support the importance of dual blood supply for an optimal regenerative response in liver remnants after liver resection. We suggest that the autonomic nerve supply of the portal triad plays at least an important permissive role in liver regeneration.