The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the "crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.     

Parallel Evolution,not Always so Parallel: Comparison of Small Benthic Charr, <Emphasis Type="Italic">Salvelinus alpinus</Emphasis>, from Grímsnes and Thingvallavatn,Iceland

Synopsis Iceland is unique in terms of geologically young freshwater systems and rapid adaptations of fresh water fishes to diverse habitats, e.g. lava with ground water flow. Iceland has six species of freshwater fishes, including Arctic charr, Salvelinus alpinus. Previous research has shown great diversity within this species. Four different morphs of Arctic charr are found in one lake, Thingvallavatn, including a small benthivorous charr. Similar populations of small benthic charr are known from several other Icelandic freshwater locations, including Nautavakir in Grímsnes. Our comparison of the small benthic charr morphs in Thingvallavatn and in Grímsnes showed that they are similar in morphology but distinguishable in several characteristics. Small benthic charr in Grímsnes and Thingvallavatn demonstrate similar adaptations and are an example of parallel evolution. However, subtle morphological differences between them indicate further specialized adaptations at each location.     

Endothelial induced EMT in breast epithelial cells with stem cell properties

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.     

The Rejection of a Theoretical Beauty: The Foot of the Continental Slope in Maritime Boundary Delimitations Beyond 200 Nautical Miles

This article addresses maritime boundary delimitation concerning the continental shelf beyond 200 nautical miles. The focal point is how the foot of the continental slope can be used as the point of departure in drawing the provisional equidistance line in outer continental shelf boundary delimitations between neighboring states. The article examines the strength and weaknesses of this approach and asks whether the International Tribunal for the Law of the Sea indirectly rejected this approach in the 2012 Bangladesh v. Myanmar Case.     

The Importance of Egg Size and Social Effects for Behaviour of Arctic Charr Juveniles

Early behaviour can determine food intake and growth rate with important consequences for life history and survival in fishes. Egg size is known to affect growth rate of young Arctic charr but its influence on the development of behaviour is poorly documented. It is believed that egg size influence on growth and potentially on the behaviour of young fish decreases over time, minimized by the effects of social factors. Shortly after first feeding, we examined differences in mobility and foraging of Arctic charr in relation to egg size and social environment. The behaviour of juveniles from small and large eggs was compared five times over the course of development and in three different experimental settings: long‐term isolation (isolation before hatching), short‐term isolation vs. group rearing and mixed size group vs. homogeneous size groups. Egg size affected foraging behaviour and mobility of fish: fish coming from large eggs were more mobile and foraged more than fish coming from small eggs. Social environment affected foraging behaviour, mobility and space use: fish in a group were more mobile, foraged more and responded faster to food delivery than isolated fish. The interaction of egg size and social effects was seen primarily in foraging activities but did not affect mobility or space use. Large fish in groups foraged more than the three other groups: large fish in isolation, small fish in groups and small fish in isolation. Agonistic behaviour was rarely observed and there was no significant effect of group composition on agonistic behaviour. We discuss the importance of egg size and social effects at early stages of development with a focus on the evolutionary ecology of Arctic charr.     

Drift versus selection as drivers of phenotypic divergence at small spatial scales: The case of Belgjarskógur threespine stickleback

Divergence in phenotypic traits is facilitated by a combination of natural selection, phenotypic plasticity, gene flow, and genetic drift, whereby the role of drift is expected to be particularly important in small and isolated populations. Separating the components of phenotypic divergence is notoriously difficult, particularly for multivariate phenotypes. Here, we assessed phenotypic divergence of threespine stickleback (Gasterosteus aculeatus) across 19 semi‐interconnected ponds within a small geographic region (~7.5 km²) using comparisons of multivariate phenotypic divergence (PST), neutral genetic (FST), and environmental (EST) variation. We found phenotypic divergence across the ponds in a suite of functionally relevant phenotypic traits, including feeding, defense, and swimming traits, and body shape (geometric morphometric). Comparisons of PSTs with FSTs suggest that phenotypic divergence is predominantly driven by neutral processes or stabilizing selection, whereas phenotypic divergence in defensive traits is in accordance with divergent selection. Comparisons of population pairwise PSTs with ESTs suggest that phenotypic divergence in swimming traits is correlated with prey availability, whereas there were no clear associations between phenotypic divergence and environmental difference in the other phenotypic groups. Overall, our results suggest that phenotypic divergence of these small populations at small geographic scales is largely driven by neutral processes (gene flow, drift), although environmental determinants (natural selection or phenotypic plasticity) may play a role.     

Fine-scale parallel patterns in diversity of small benthic Arctic charr (Salvelinus alpinus) in relation to the ecology of lava/groundwater habitats

It is critical to study factors that are important for origin and maintenance of biological diversity. A comparative approach involving a large number of populations is particularly useful. We use this approach to study the relationship between ecological factors and phenotypic diversity in Icelandic Arctic charr (Salvelinus alpinus). Numerous populations of small benthic charr have evolved in lava springs in Iceland. These charr appear morphologically similar, but differ in important morphological features related to feeding. We found a clear relationship between diversity in morphology, diet, and ecological factors among populations. In particular, there were clear differences in morphology and diet between fish coming from habitats where the lava spring flowed on as a stream compared to habitats where the lava spring flowed into a pond. Our study shows that ecological factors are important for the origin and maintenance of biological diversity. The relationship between phenotype and ecological factors are observed on a fine scale, when comparing numerous populations that are phenotypically similar. This strongly suggests that for understanding, managing, and conserving biological diversity important ecological variables have to be taken into the account.     

Extreme Sexual Brain Size Dimorphism in Sticklebacks: A Consequence of the Cognitive Challenges of Sex and Parenting?

Selection pressures that act differently on males and females produce numerous differences between the sexes in morphology and behaviour. However, apart from the controversial report that males have slightly heavier brains than females in humans, evidence for substantial sexual dimorphism in brain size is scarce. This apparent sexual uniformity is surprising given that sexually distinct selection pressures are ubiquitous and that brains are one of the most plastic vertebrate organs. Here we demonstrate the highest level of sexual brain size dimorphism ever reported in any vertebrate: male three-spined stickleback of two morphs in an Icelandic lake have 23% heavier brains than females. We suggest that this dramatic sexual size dimorphism is generated by the many cognitively demanding challenges that males are faced in this species, such as an elaborate courtship display, the construction of an ornate nest and a male-only parental care system. However, we consider also alternative explanations for smaller brains in females, such as life-history trade-offs. Our demonstration of unprecedented levels of sexual dimorphism in brain size in the three-spined stickleback implies that behavioural and life-history differences among the sexes can have strong effects also on neural development and proposes new fields of research for understanding brain evolution.     

Inshore migration and otolith microstructure/microchemistry of anguillid glass eels recruited to Iceland

The timing of catches of anguillid glass eels and their otolith microstructure and microchemistry were studied in southwest Iceland, where the European eel, Anguilla anguilla and American eel, A. rostrata have been thought to live sympatrically, to learn about their early life history and the possible mechanism of the separation between these two species ranges. Catches at the site studied suggest that glass eels may have started upstream migration as the river temperature warmed in late June and early July. The glass eels were mitochondrially identified into two species, A. anguilla and A. rostrata, although the latter were likely hybrids between the two species based on a different study. Otolith analyses showed no sharp increases in otolith increment width or sharp decrease of otolith Sr:Ca ratio in either species, which are the characteristic changes corresponding to the onset of metamorphosis in many anguillid species including A. rostrata collected in North America and A. anguilla in Europe. The mean age at recruitment determined for the glass eels in Iceland were similar between the two species (336.6 ± 41.7 and 319.3 ± 36.0 days for A. anguilla and A. rostrata, respectively), as were their total lengths (range 58.0–78.5 mm and 58.5–73.0 mm). In addition, mean age at metamorphosis (278.0 ± 36.8 and 254.0 ± 47.7 days) and total age (372.3 ± 50.8 and 352.9 ± 42.6 days) were also similar between the two species. However, these ages of A. rostrata in Iceland were older than those in North America, and those of A. anguilla collected in Iceland were roughly intermediate between the rest of Europe and North Africa. These findings support the hypothesis that the timing of metamorphosis is a key factor for determining the place of recruitment of glass eels and maintaining the geographic separation between the two species.     

Effects of replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli

Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold-adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal-binding sites in the active site of APs, and all three metal ions participate in catalysis. The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs. In contrast, two of the residues that bind the third metal ion (numbered 153 and 328 in Escherichia coli AP) are different in various APs. This may explain their different catalytic efficiencies, as the Mg2+ most often present there is important for both structural stability and the reaction mechanism. We have mutated these key residues to the corresponding residues in E. coli AP to obtain the double mutant Asp116/Lys274, and both single mutants. All these mutants displayed reduced substrate affinity and lower overall reaction rates. The Lys274 and Asp116/Lys274 mutants also displayed an increase in global heat stability, which may be due to the formation of a stabilizing salt bridge. Overall, the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold-active Vibrio AP both locally and globally, and this influences kinetic properties.