Adaptation and constraint in a stickleback radiation.

The evolution of threespine sticklebacks in freshwater lakes constitutes a well-studied example of a phenotypic radiation that has produced numerous instances of parallel evolution, but the exact selective agents that drive these changes are not yet fully understood. We present a comparative study across 74 freshwater populations of threespine stickleback in Norway to test whether evolutionary changes in stickleback morphology are consistent with adaptations to physical parameters such as lake depth, lake area, lake perimeter and shoreline complexity, variables thought to reflect different habitats and feeding niches. Only weak indications of adaptation were found. Instead, populations seem to have diversified in phenotypic directions consistent with allometric scaling relationships. This indicates that evolutionary constraints may have played a role in structuring phenotypic variation across freshwater populations of stickleback. We also tested whether the number of lateral plates evolved in response to lake calcium levels, but found no evidence for this hypothesis.

Quantification of lumen stenoses with known dimensions by conventional angiography and computed tomography: implications of using conventional angiography as gold standard.

BACKGROUND: Quantitative coronary angiography (QCA) has inherent limitations for displaying complex vascular anatomy, yet it remains the gold standard for stenosis quantification. OBJECTIVE: To investigate the accuracy of stenosis assessment by multi-detector computed tomography (MDCT) and QCA compared to known dimensions. METHODS: Nineteen acrylic coronary vessel phantoms with precisely drilled stenoses of mild (25%), moderate (50%) and severe (75%) grade were studied with 64-slice MDCT and digital flat panel angiography. Fifty-seven stenoses of circular and non-circular shape were imaged with simulated cardiac motion (60 bpm). Image acquisition was optimised for both imaging modalities, and stenoses were quantified by blinded expert readers using electronic callipers (for MDCT) or lumen contour detection software (for QCA). RESULTS: Average difference between true and measured per cent diameter stenosis for QCA was similar compared to MDCT: 7 (+/-6)% vs 7 (+/-5)% (p=0.78). While QCA performed better than MDCT in stenoses with circular lumen (mean error 4 (+/-3)% vs 7 (+/-6)%, p<0.01), MDCT was superior to QCA for evaluating stenoses with non-circular geometry (mean error 10 (+/-7)% vs 7 (+/-5)%, p<0.05). In such lesions, QCA underestimated the true diameter stenosis by >20% in 9 of 27 (33%) vs 1 of 29 (3%) in lumen with circular geometry. CONCLUSIONS: QCA often underestimates diameter stenoses in lumen with non-circular geometry. Compared to QCA, MDCT yields mildly greater measurement errors in perfectly circular lumen but performs better in non-circular lesions. These findings have implications for using QCA as the gold standard for stenosis quantification by MDCT.

Parallel evolution of ecomorphological traits in the European whitefish Coregonus lavaretus (L.) species complex during postglacial times.

The extensive phenotypic polymorphism in the European whitefish has triggered evolutionary research in order to disentangle mechanisms underlying diversification. To illuminate the ecological distinctiveness in polymorphic whitefish, and evaluate taxonomic designations, we studied nine Norwegian lakes in three watercourses, which each harboured pairs of divergent whitefish morphs. We compared the morphology and life history of these morphs, documented the extent of genetic differentiation between them, and contrasted the niche use of sympatric morphs along both the habitat and resource axes. In all cases, sympatric morphs differed in the number of gill rakers, a highly heritable trait related to trophic utilization. Individual growth rate, age and size at maturity, diet and habitat use also differed between morphs within lakes, but were remarkably similar across lakes within the same morph. Microsatellite analyses confirmed for all but one pair that sympatric morphs were significantly genetically different, and that similar morphs from different lakes likely have a polyphyletic origin. These results are most compatible with the process of parallel evolution through recurrent postglacial divergence into pelagic and benthic niches in each of these lakes. We propose that sparsely and densely rakered whitefish sympatric pairs may be a likely case of ecological speciation, mediated in oligotrophic lakes with few trophic competitors.

Evolutionary history of the European whitefish Coregonus lavaretus (L.) species complex as inferred from mtDNA phylogeography and gill-raker numbers.

We compared mitochondrial DNA and gill-raker number variation in populations of the European whitefish Coregonus lavaretus (L.) species complex to illuminate their evolutionary history, and discuss mechanisms behind diversification. Using single-strand conformation polymorphism (SSCP) and sequencing 528 bp of combined parts of the cytochrome oxidase b (cyt b) and NADH dehydrogenase subunit 3 (ND3) mithochondrial DNA (mtDNA) regions, we documented phylogeographic relationships among populations and phylogeny of mtDNA haplotypes. Demographic events behind geographical distribution of haplotypes were inferred using nested clade analysis (NCA) and mismatch distribution. Concordance between operational taxonomical groups, based on gill-raker numbers, and mtDNA patterns was tested. Three major mtDNA clades were resolved in Europe: a North European clade from northwest Russia to Denmark, a Siberian clade from the Arctic Sea to southwest Norway, and a South European clade from Denmark to the European Alps, reflecting occupation in different glacial refugia. Demographic events inferred from NCA were isolation by distance, range expansion, and fragmentation. Mismatch analysis suggested that clades which colonized Fennoscandia and the Alps expanded in population size 24 500-5800 years before present, with minute female effective population sizes, implying small founder populations during colonization. Gill-raker counts did not commensurate with hierarchical mtDNA clades, and poorly with haplotypes, suggesting recent origin of gill-raker variation. Whitefish designations based on gill-raker numbers were not associated with ancient clades. Lack of congruence in morphology and evolutionary lineages implies that the taxonomy of this species complex should be reconsidered.

Morphological divergence and origin of sympatric populations of European whitefish (Coregonus lavaretus L.) in Lake Femund, Norway.

Combining morphological and genetic analysis, we compared patterns of diversification within and between morphs among sympatric European whitefish (Coregonus lavaretus L.) populations in Lake Femund, Norway. Seven external populations, from potential colonization routes into Lake Femund were included. We found that deep-, shallow-, river- and bay spawning populations are distinct morphs in Lake Femund. Within morphs, populations range from being similar genetically (Fst=0-0.005) among deep-spawning populations to being highly differentiated (Fst=0.153) between bay-spawning populations. Between morphs, genetic differences ranged from a low (Fst=0.008-0.022) between deep- and shallow-spawning populations to high difference (Fst=0.125-0.143) between shallow- and bay-spawning populations. A higher proportion of molecular variance was seen among (3.9%) than within morphs (2.8%). The adaptive gene combinations behind the four morphs seem to have originated within the lake, although the lake could have been colonized from more than one source population.

Evidence that nitroprusside stimulates glucose uptake in isolated rat cardiomyocytes via mitogen-activated protein kinase.

Sodium nitroprusside (SNP), a nitric oxide (NO.) donor, stimulates glucose uptake in skeletal muscle. We investigated the stimulatory effect of SNP on glucose uptake in cardiomyocytes and the possible role of soluble guanylate cyclase, phosphatidylinositol-3-kinase (PI-3-kinase) and the mitogen-activated protein kinases (MAPKs). Cardiomyocytes were isolated from adult male Wistar rats by trypsin/collagenase perfusion and glucose uptake determined from the accumulation of 3H-2-deoxyglucose. SNP caused a dose-dependent increase in glucose uptake with 200-300% increase at 30 mM. Cytochalasin B completely prevented the SNP-induced increase in glucose uptake. 8-Br-cGMP (100 microM) and the NO. donor spermineNONOate (100 microM) were without effect on basal glucose uptake. SNP-stimulated glucose uptake was not inhibited by the guanylate cyclase inhibitor ODQ (10 microM). Sodium ferrocyanide (Na4Fe(CN)6), a compound structurally related to SNP, but without any NO. group, also stimulated glucose uptake in cardiomyocytes suggesting that the effect of SNP could be unrelated to liberation of NO. Wortmannin, an inhibitor of PI-3-kinase, inhibited insulin-stimulated glucose uptake completely but did not affect SNP-stimulated glucose uptake. SNP-stimulated glucose uptake was inhibited by 50 microM PD 098059 (inhibitor of the MAPK-kinases that activate external regulated kinase [ERK1/2]) and by 50 microM SB203580 (inhibitor of p38MAPK). In conclusion, high SNP concentrations dose-dependently stimulate glucose uptake in cardiomyocytes and our data suggest a role for MAPK signalling, but not PI-3-kinase and soluble guanylate cyclase, in stimulation of glucose uptake.

Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation.

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.

Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes.

The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.

Toxicity of acid aluminium-rich water to seven freshwater fish species: a comparative laboratory study.

The present study focuses on the relative sensitivity among freshwater fish species to aqueous aluminium. Seven common Scandinavian fish species were exposed to acidic Al-rich water, acidic Al-poor water, and approximately neutral water as a control. The relative sensitivity among the species to an acute aluminium challenge was documented, and was in the following order: Atlantic salmon, Salmo salar, as the most sensitive; then roach, Rutilus rutilus; minnow, Phoxinus phoxinus; perch, Perca fluviatilis; grayling, Thymallus thymallus; brown trout, Salmo trutta; and Arctic char, Salvelinus alpinus. Substantial mortality was observed in all species when exposed to the Al-rich medium. Some mortality was also observed in minnow, roach, and brown trout exposed to the acidic Al-poor medium and the control medium. A high resistance to aluminium was observed in Arctic char, while perch was found to be more sensitive to aluminium than expected and, for the first time, a toxic response to aqueous aluminium in grayling was documented. Through controlled experimental studies, the results confirm that aluminium is an important factor in the toxicity of acidified waters to freshwater fish species.

Leukaemia and anaemia in AKR/O mice. II. Role of the spleen as an erythropoietic organ during leukaemia development.

In order to study the role of the spleen in erythropoiesis during AKR/O leukaemogenesis, we have cultured bone marrow and spleen erythroid colony-forming units (CFU-E) and burst-forming units (BFU-E) from AKR/O mice (n = 40) with leukemia of varying severity and type of manifestation. Mice with leukaemia/lymphoma had reduced concentrations of bone marrow CFU-E and BFU-E as compared to healthy, age-matched AKR/O mice. The spleen content of CFU-E was increased, and highest in mice with a spleen size between 500 and 1000 mg. The largest spleens had a somewhat lower CFU-E content. Mice with the highest spleen CFU-E content most often had a normal PCV; however, 4/7 had a normal bone marrow CFU-E concentration. During AKR/O leukaemogenesis with development of spleen enlargement, the spleen may act as an erythropoietic organ, and contribute to maintaining a normal PCV. This may be a temporary ability which is reduced or lost with further progress of the disease.

Leukaemia and anaemia in AKR/O mice I. Leukaemia characteristics, haematological variables and erythropoiesis stimulating factor(s).

AKR/O mice were used as a model for studying the pathogenesis of the anaemia accompanying leukaemia/lymphoma. The leukaemia incidence was 87%. Median age at diagnosis was 11.3 months. At diagnosis most of the mice had normal leukocyte counts. Clinically the mice divided into subgroups depending on the relative organ involvement: 1) thymoma group (n = 98), 2) spleen group (n = 144), 3) combined group (n = 27) and 4) mice with moderate organ changes (n = 216). Mice of group 1 were younger than the others, had a rapidly progressive disease, normal to elevated packed cell volume (PCV), and plasma erythropoietin (Epo) was normal or increased. Mice of group 2 were usually anaemic with high plasma Epo estimates and often elevated reticulocyte counts. Group 4 was the oldest group. Some of these mice were severely affected haematologically. Overall there was an inverse relation between PCV and plasma Epo estimate, indicating a normal Epo response to anaemia. In all groups increasing spleen size was associated with increased severity of anaemia and increased reticulocyte counts. The association between anaemia, elevated reticulocyte counts and spleen enlargement suggests haemolysis as a mechanism for anaemia, and also raises the question of compensatory spleen erythropoiesis.