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Permeation grouting – injections at low-pressure values of either microfine cements or chemical products – is frequently adopted to increase the mechanical/hydraulic properties of soils, and standard design approaches are currently either empirical or based on simplified analytical solutions. In this paper, some fundamental hypotheses of these analytical solutions are discussed by carrying out a campaign of finite-element numerical analyses, in which the injection phase in a water-saturated soil is analysed, a Newtonian rheology for the grout is implemented and the hypothesis of immiscibility for the two liquids is assumed. The effect of injection source geometry is discussed, as well as the role of gravity and capillarity. The authors analyse the conditions, in terms of injection flow rate, grout viscosity, soil intrinsic permeability and retention curve, under which the analytical solutions provide reliable results. The numerical results have been compared with the simplified analytical solution herein derived for a one-dimensional spherical geometry, in terms of the ‘characteristic curves’ for the system: the relationship between injection pressure/grout front advancement and injection time.

This book summarizes physical and biological aspects from Kongsfjorden (78° 59’ N, 11–12° E), Svalbard. The contributions to this volume cover atmospheric conditions above Ny-Alesund, as well as physical conditions in Kongsfjorden. The chapters about oceanographic dynamics and sea ice conditions are based on time-series observations of interannual variability, whereas the chapter about the underwater light regime focuses on seasonal dynamics. The pelagic system is covered by reviews of pelagic production, phytoplankton and zooplankton communities. Benthic flora studies address microphytobenthos and macroalgal biodiversity, as well as the physiology of kelp related to stress perception and responses. Benthic fauna communities are described with associated environmental drivers of change. An outline of an Arctic fjord ecosystem model for Kongsfjorden- Krossfjorden is presented. Data that go into models come from sampling at different stations in the marine environment, with an important contribution from long-term data series at stations. Some of the long-term data are based on recordings from autonomous underwater observatories. Finally, one summary presents Kongsfjorden as harbinger of the future Arctic.

Gutt, Julian ... et al.-- 17 pages, 4 figures, The biodiversity, ecosystem services and climate variability of the Antarctic continent and the Southern Ocean are major components of the whole Earth system. Antarctic ecosystems are driven more strongly by the physical environment than many other marine and terrestrial ecosystems. As a consequence, to understand ecological functioning, cross-disciplinary studies are especially important in Antarctic research. The conceptual study presented here is based on a workshop initiated by the Research Programme Antarctic Thresholds – Ecosystem Resilience and Adaptation of the Scientific Committee on Antarctic Research, which focussed on challenges in identifying and applying cross-disciplinary approaches in the Antarctic. Novel ideas and first steps in their implementation were clustered into eight themes. These ranged from scale problems, through risk maps, and organism/ecosystem responses to multiple environmental changes and evolutionary processes. Scaling models and data across different spatial and temporal scales were identified as an overarching challenge. Approaches to bridge gaps in Antarctic research programmes included multi-disciplinary monitoring, linking biomolecular findings and simulated physical environments, as well as integrative ecological modelling. The results of advanced cross-disciplinary approaches can contribute significantly to our knowledge of Antarctic and global ecosystem functioning, the consequences of climate change, and to global assessments that ultimately benefit humankind, In addition to the employers of the authors this study was funded by the SCAR SRPs AnT-ERA, AntClim21 and AntEco

The large diversity of marine microorganisms harboured by oceans plays an important role in planet sustainability by driving globally important biogeochemical cycles; all primary and most secondary production in the oceans is performed by microorganisms. The largest part of the planet is covered by cold environments; consequently, cold-adapted microorganisms have crucial functional roles in globally important environmental processes and biogeochemical cycles cold-adapted extremophiles are a remarkable model to shed light on the molecular basis of survival at low temperature. The indigenous populations of Antarctic and Arctic microorganisms are endowed with genetic and physiological traits that allow them to live and effectively compete at the temperatures prevailing in polar regions. Some genes, e.g. glycosyltransferases and glycosylsynthetases involved in the architecture of the cell wall, may have been acquired/retained during evolution of polar strains or lost in tropical strains. This present work focusses on temperature and its role in shaping microbial adaptations; however, in assessing the impacts of climate changes on microbial diversity and biogeochemical cycles in polar oceans, it should not be forgotten that physiological studies need to include the interaction of temperature with other abiotic and biotic factors.

Some of the most rapidly changing ecosystems on our planet are located in the polar regions (IPCC 2007; Turner et al. 2009; SWIPA 2011). In some areas of the Arctic and Antarctic, atmospheric temperatures are rising at rates more than double the global average. In addition, there are other direct human impacts on polar regions such as pollution, exploitation and development. Polar ecosystems and the biodiversity they support are already responding to this change and it is expected that even more profound impacts will occur this century. Compounding the risk to polar biodiversity is the fact that many polar ecosystems have limited functional redundancy; in the event of the loss of a single keystone species, they may potentially be exposed to cascading effects and complete ecosystem restructuring (Post et al. 2009). Rapid climate change affecting the polar regions will also have profound physical and ecological consequences for the rest of the planet since the ice-covered Arctic Ocean, the Antarctic contine...

In the last decade crystallographic evidence for endogenous coordination at the sixth coordination site of the heme iron has been reported for monomeric haemoglobins (Hbs) in both the ferrous (haemochrome) and ferric (haemichrome) oxidation state. Usually, the sixth ligand is provided by the imidazole side chain of a His, the only putative ligand normally present in the distal site of the heme pocket. More recently, structural and spectroscopic evidence has been reported, which show that the bis-histidyl adduct in the ferric state represents a common accessible ordered state also for several tetrameric Hbs isolated from Antarctic fish, both in the solid and solution state. Bis-histidyl coordination was also discovered in the crystals of horse met-Hb exposed to acidic pH. All these crystal structures are characterised by a different binding state of α and β chains. Tetrameric Hbs with all chains in the bis-histidyl coordination state have not yet been found. Herein we review the structural details of the recent results in this field, together with solution studies on the pathway of haemichrome formation

This laboratory has reported previously that fragmentation of brain mitochondrial fractions resulted in a 7–25 fold increase of the antimycin A-sensitive NADH oxidase, provided that ionic compounds such as inorganic phosphate were present in the incubation media used for enzymatic determination ( Giuditta and Strecker, 1959 ). The oxidation of NADH and of succinate by the Keilin-Hartree heart muscle preparation has been shown also to be stimulated by ionic compounds and by globin ( Keilin and Hartree, 1949 ; Slater, 1949 ; Bonner, 1954 ). Similar observations have been made now with liver and heart mitochondria. Evidence is presented to indicate that the stimulatory effects obtained with added ionic compounds depends at least partially on interaction with a heat stable non-dialyzable factor, which appears to be present exclusively in the mitochondrial fractions of heart, liver or brain homogenates.

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 A˚ resolution. This structure reveals a 5C state of the heme iron for both the a- and b-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs

Many fish hemoglobins (Hbs) are endowed with Root effect, that produces a decrease in oxygen affinity at low pH, associated with complete loss of cooperativity. Over the years, several hypotheses on the structural determinants of the Root effect have been suggested. Up to now, all the structural explanations of the Root effect have been based on the two-state model, in which the Root effect is related to an increase of the allosteric equilibrium between the R and T state at acidic pH [1-3]. Here, we report the crystal structures of the deoxy and carbomonoxy forms of the non-Root-effect major component Hb 1 isolated from the Antarctic fish (Af) Trematomus newnesi (Hb1Tn). Surprisingly, in the deoxy state of the non-Root effect Hb1Tn, the inter-aspartic hydrogen bond between Asp95α and Asp101β at the α1β2 interface, which is believed to be important in Root effect [2], is observed. A combined Resonance Raman / x-ray crystallography of this AfHb has revealed heterogeneity in the deoxy coordination. Novel ligated states are observed in both a T-like state and a R/T intermediate quaternary state. Three of four independent CO coordination states are not assisted by the hydrogen bond of the distal histidyl, that swings out of the heme pocket. These un-assisted CO coordination states (supported by FT-IR spectroscopy solution studies) are associated with unusually small thermal fluctuations which characterise both α and β CD corners. The accessibility of ligated states within three different quaternary structures (T, R and R/T intermediate [4]) suggests a novel structural explanation of protein allostery, based on a three-state Edelstein’s model [5]. Grant Sponsors: PRIN , PNRA. [1] Mazzarella et al. Proteins, 2006, 65(2), 490-498. [2] Mazzarella et al. Proteins, 2006, 62(2), 316-321 [3] Yokoyama et al. J. Biol. Chem. 2004, 279, 28632-28640. [4] Vitagliano et al. J. Am. Chem. Soc., 2008, 130, 10527-10535. [5] Edelstein, J. Mol. Biol., 1996, 256, 737-744.

The decline of honeybee colonies and their eventual collapse is a widespread phenomenon in the Northern hemisphere of the globe, which severely limits the beekeeping industry. This dramatic event is associated with an enhanced impact of parasites and pathogens on honeybees, which is indicative of reduced immunocompetence. The parasitic mite Varroa destructor and the vectored viral pathogens appear to play a key-role in the induction of this complex syndrome. In particular, the Deformed Wing Virus (DWV) is widespread and is now considered, along with Varroa, one of the major causes of bee colony losses. Several lines of evidence indicate that this mite/DWV association severely affects the immune system of honeybees and makes them more sensitive to the action of other stress factors. The molecular mechanisms underpinning these complex interactions are currently being investigated and the emerging information has allowed the development of a new functional model, describing how different stress factors may synergistically concur in the induction of bee immune alteration and health decline. This provides a new logical framework in which to interpret the proposed multifactorial origin of bee colony losses and sets the stage for a more comprehensive and integrated analysis of the effect that multiple stress agents may have on honeybees.

This review highlights some aspects of the biochemistry of cold-adapted hemoproteins in fish and bacteria, without claiming to be exhaustive. Heme hexacoordination where the sixth ligand is provided by an internal amino-acid residue, in cold-adapted hemoproteins will be discussed.

Fishes thriving in polar habitats offer many opportunities for comparative approaches to understanding protein adaptations to temperature. Notothenioidei, the dominant suborder in the Antarctic ocean, have evolved reduction of hemoglobin concentration and multiplicity, perhaps as a consequence of temperature stability and other environmental parameters. In the icefish family, the blood pigment is absent. In contrast, similar to other acanthomorph teleosts, Arctic fish, thriving in a more complex oceanographic system, have maintained higher hemoglobin multiplicity and a highly diversified globin system in response to environmental variability and/or variations in metabolic demands. This review summarises the current knowledge on the structure, function and phylogeny of hemoglobins of fish living in polar habitats. In particular, on the basis of crystallographic analysis, a novel guideline to the interpretation of the Root effect in terms of a three-state model is suggested, implying the accessibility of an R/T intermediate quaternary structure, frequently observed in Antarctic fish hemoglobins. The occurrence of bis-histidyl and penta-coordinate states in ferric forms of polar fish hemoglobins suggests additional redox properties.

Crystallographic and spectroscopic evidences on Antarctic fish hemoglobins (AFHbs) have revealed that their ferric tetramers at physiological pH are in a mixed |α(aquo-met)/β(bis-histidyl) coordination state and show a quaternary structure intermediate between the classical R and T states (H state). Ferric bis-histidyl adducts (hemichromes) have been also observed in some mammalian Hbs. In order to clarify whether hemichrome in AFHbs can be converted into a ferrous bis-histidyl adduct (hemochrome), at least in the crystal phase, chemical reduction of ferric hemoglobins from Trematomus bernacchii (HbTb) single crystals has been followed via Raman microscopy. The results of this analysis reveal that in HbTb, upon reduction, the bis-histidyl coordination is disrupted in favor of a penta-coordinated ferrous deoxy state, with no evidence of hemochrome. These data are in agreement with UV/Vis absorption spectra in solution. Furthermore, our data are also indirectly supported by the observation that upon reduction with dithionite, the ferric HbTb crystals crack and lose their diffraction power: in the crystalline state, the quaternary structure transition from the H to the T state is not compatible with the crystal packing. Altogether these data indicate that if bis-histidyl adducts have a functional significance in AFHbs, this function refers to a stable ferric state, or to a transient, though never detected, ferrous species.

Pagothenia borchgrevinki has a higher haemoglobin concentration than other Antarctic notothenioids and the high oxygen capacity may correlate with the relatively active mode of life of this fish. The fish has five haemoglobins (Hb C, Hb 0, Hb 1, Hb 2 and Hb 3) with Hb 1 accounting for 70-80% of the total, and Hb C being present in trace amounts. Hb 1 and Hb 2 are functionally similar in terms of Bohr and Root effects. Hb 3 has a weaker Bohr effect than Hb I and Hb 2, and the Root effect is similar to that of Hb 1. Hb 0 has a strong Bohr effect and the Root effect is enhanced to a larger extent by the physiological effectors chlorides and phosphates than that of the other components with the exception of Hb C. The heats of oxygenation are lower than those of temperate fish haemoglobins. Temperature variations may have a different effect on the functional properties of each haemoglobin, and chloride and phosphates may play an important role in the conformational change between the oxy and deoxy structures. The complete amino acid sequences of Hb 1 and Hb 0, as well as partial N-terminal or internal sequences of the other haemoglobins, have been established. The high multiplicity of functionally distinct haemoglobins indicates that P. borchgrevinki has a specialized haemoglobin system.

The blood of Channichthyidae, the most phyletically derived notothenioid family, is devoid of haemoglobin (Hb). Three of the 15 channichthyid species retain inactive genomic remnants of the major adult notothenioid a-globin gene, but have lost the gene encoding adult β-globin. In an attempt to understand the mutational events leading to loss of globin expression in channichthyds, the organisation of globin genes in red-blooded notothenioid species was examined. Globin genes in fish are often organised as alβ pairs linked in 5' to 5' orientation, and the major adult α- and β-globins of the Antarctic yellowbelly rockcod Notothenia coriiceps have this organisation. A second globin gene cluster from N. coriiceps contains two sets of linked α- and β-globin genes but the deduced amino acid sequences of the globins differ from those of the adult globins. To test the hypothesis that the globins may be expressed at different stages of ontogenesis. total RNA from head kidney and erythrocytes of adult and juvenile N. coriiceps were probed for transcripts corresponding to the four globin genes. The data suggest that the globin genes of this cluster are transcribed maximally in the early phase of development. Southern analysis of the genomes of red- and white-blooded notothenioids revealed that the genes of this complex are absent in Hb-less icefish.

The importance of the Arctic, in contributing to the knowledge of the overall ensemble of adaptive processes influencing the evolution of marine organisms, calls for investigations on molecular adaptations in Arctic fish. Unlike the vast majority of Antarctic Notothenioidei, several Arctic species display high hemoglobin multiplicity. The blood of four species, the spotted wolffish of the family Anarhichadidae and three Gadidae, contains three functionally distinct major components. Similar to many Antarctic notothenioids, Arctic Liparis tunicatus (suborder Cottoidei, family Liparidae) has one major hemoglobin (Hb 1) accompanied by a minor component (Hb 2). This paper reports the structural and functional characterisation of Hb 1 of L. tunicatus. This hemoglobin shows low oxygen affinity, and pronounced Bohr and Root effects. The amino-acid sequence of the β chain displays an unusual substitution in NA2 (β2) at the phosphate-binding site, and the replacement of Val E11 (β67) with Ile. Similar to some Antarctic fish Hbs, electron paramagnetic resonance spectra reveal the formation of a ferric penta-coordinated species even at physiological pH. The amino-acid sequences have also been used to gain insight into the evolutionary history of globins of polar fish. L. tunicatus globins appear close to the notothenioid clades as predicted by teleostean phylogenies. Close phylogenetic relationships between Cottoidei and Notothenioidei, together with their life style, seem to be the main factor driving the globin-sequence evolution.

The influence of hydrostatic pressure on the stability and activity of glutamate dehydrogenase (GDH) from the liver of the Antarctic fish Chaenocephalus aceratus (Notothenioidei: Channichthyidae) has been investigated using the homologous bovine enzyme for comparison. At gradually increasing pressure, the fish GDH retains all activity up to 140 MPa, whereas in the same pressure range the bovine enzyme is slightly inactivated. The kinetics of pressure-induced inactivation of Antarctic GDH displays an exponentially decreasing residual activity as a function of the incubation time, whereas the bovine enzyme shows a complex pattern of behaviour in the first 20 min of incubation, attributable to the transient formation of aggregates. The coenzyme NAD increases the stability of both enzymes. The activation volume calculated for the reaction catalysed under pressure by fish GDH is higher than that of the bovine enzyme. These results indicate that the molecular properties of fish and bovine GDH are affected differently by hydrostatic pressure. A common mode of inactivation under extreme conditions of pressure and temperature of the cold-adapted Antarctic fish and mesophilic bovine GDH is deduced and discussed.

Antarctic fish hemoglobins (AF-Hbs) exhibit a peculiar oxidation process. Our previous crystallographic and spectroscopic investigations have demonstrated that, upon oxidation, these proteins show a remarkable propensity to evolve toward the formation of lowspin hexa-coordinated species. The crystal structures of the fully oxidized forms of AF-Hbs, isolated from Trematomus newnesi and Trematomus bernacchii, have also shown that and chains follow different oxidation pathways. Interestingly, the quaternary structures of these forms are intermediate between the physiological R and T hemoglobin states. In order to obtain additional information on the structural features of the intermediate species along the oxidation pathway, we are currently characterizing AF-Hbs exposed to air for different time periods. Preliminary data reveal the presence of novel forms with unexpected structural properties. In particular, we detected (1)the presence of partially liganded forms with structures that are intermediate between the R and the T state, (2) the existence of hybrid alpha(aquomet)-beta(penta-coordinated Fe3+) forms, and (3) the occurrence of novel subunit-subunit interactions at the alpha 1beta2 interface.

A novel thermophilic Gram-positive bacillus, “Bacillus thermoantarcticus”, isolated from geothermal soil near the crater of Mount Melbourne, is described. The organism grows at an optimal temperature of 63°C at pH 6.0, is oxidase-positive, catalase-negative and produces an exopolysaccharide, an exocellular xylanase, an intracellular alcohol dehydrogenase and exo- and endocellularα-glucosidase(s). The sequence of 16S rDNA is very similar to that of “Bacillus thermoglucosidasius”; however, the guanine-plus-cytosine (G + C) content is 8 mol% higher. The type strain is “Bacillus thermoantarcticus” (DSM 9572).

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade.

Fish hemoglobins have been extensively studied, not only for their structural and functional properties, but also because they offer the possibility of investigating functional differentiation and molecular adaptations in marine and freshwater species living in a large variety of environmental conditions. The number of available amino-acid sequences of fish hemoglobins yields enough material to study evolutionary, structural, and functional aspects. Unlike most mammals, including humans, fish often exhibit hemoglobin multiplicity, due to gene-related heterogeneity and gene-duplication events. Here, we review and discuss some new insights about hemoglobin functional differentiation in fish. In this context, the contributions of structural biology extend well beyond the elucidation of mechanisms and hemoglobin-structure/function relationships.

This article summarizes the current knowledge of hematopoietic stem-cell development in fish and suggests new study areas. The interpretation of the complex transcriptional changes that occur during hematopoiesis poses many challenges because the relationship between the molecular mechanisms regulating lineage-specific differentiation and other levels of biological organization is poorly understood.

Since haemoglobins of all animal species have the same haem group, differences in their properties, including oxygen affinity, electrophoretic mobility and pH sensitivity, must result from the interaction of the prosthetic group with specific amino-acid residues in the primary structure. For this reason, fish globins have been the subject of extensive studies in recent years, not only for their structural characteristics, but also because they offer the possibility to investigate the evolutionary history of these ancient molecules in marine and freshwater species living in a great variety of environmental conditions. This review summarizes the current knowledge on the structure, function and phylogeny of haemoglobins of notothenioid fishes. On the basis of crystallographic analysis, the evolution of the Root effect is analysed. Adaptation of the oxygen transport system in notothenioids seems to be based on evolutionary changes, involving levels of biological organization higher than the structure of haemoglobin. These include changes in the rate of haemoglobin synthesis or in regulation by allosteric effectors, which affect the amount of oxygen transported in blood. These factors are thought to be more important for short-term response to environmental challenges than previously believed.

1. Bathydraco marri Norman is a cold-adapted Antarctic teleost (Family: Bathydraconidae), living preferably at depths between 400 and 1200 m. 2. The blood of this species contains a single hemoglobin, in which oxygen binding is pH-regulated (Bohr and Root effects). 3. The complete amino acid sequence of the alpha and beta chains of the hemoglobin of B. marri has been elucidated.

Five isolates of thermophilic bacteria, capable of growing at 65°C, were obtained from samples of soil collected from the Cryptogam Ridge (Mount Melbourne) and in the area north of Edmonson Point, Northern Victoria Land, Antarctica. Electron microscopy, morphological and physiological properties, lipid analyses and GC content of the isolates are described in this paper. On the basis of the results presented the organisms belong to the eubacterial domain.

Crystallographic and spectroscopic evidences on Antarctic fish hemoglobins (AFHbs) have revealed that their ferric tetramers at physiological pH are in a mixed ±(aquo-met)/²(bis-histidyl) coordination state and show a quaternary structure intermediate between the classical R and T states (H state). Ferric bis-histidyl adducts (hemichromes) have been also observed in some mammalian Hbs. In order to clarify whether hemichrome in AFHbs can be converted into a ferrous bis-histidyl adduct (hemochrome), at least in the crystal phase, chemical reduction of ferric hemoglobins from Trematomus bernacchii (HbTb) single crystals has been followed via Raman microscopy. The results of this analysis reveal that in HbTb, upon reduction, the bis-histidyl coordination is disrupted in favor of a penta-coordinated ferrous deoxy state, with no evidence of hemochrome. These data are in agreement with UV/Vis absorption spectra in solution. Furthermore, our data are also indirectly supported by the observation that upon reduction with dithionite, the ferric HbTb crystals crack and lose their diffraction power: in the crystalline state, the quaternary structure transition from the H to the T state is not compatible with the crystal packing. Altogether these data indicate that if bis-histidyl adducts have a functional significance in AFHbs, this function refers to a stable ferric state, or to a transient, though never detected, ferrous species.

Aims: The aim of this study was to investigate the lipolytic activity of cold-adapted Antarctic marine bacteria and, furthermore, the combined effect of some environmental factors on this enzymatic process. Methods and Results: Strains were assayed for lipolytic activity on a basal medium amended with seven individual fatty acid esters. A significant activity was observed for 148 isolates (95·5% of the total screened). The interactive effect of pH, temperature and NaCl concentration on the substrates was tested for six representative isolates, identified as Pseudoalteromonas, Psychrobacter and Vibrio. Differences between strains according to NaCl and pH tolerances were observed. Only one strain degraded the substrate more efficiently at 4°C than at 15°C. Conclusions: Our findings demonstrate that the lipolytic activity of Antarctic marine bacteria is rather variable, depending on culture conditions, and occurs in a wide range of salt concentration and pH. Significance and Impact of the Study: Isolation and characterization of bacteria that are able to efficiently remove lipids at low temperatures will provide insight into the possibility to use cold-adapted bacteria as a source of exploitable enzymes. Moreover, research on the interactive effects of salt concentration, pH and temperature will be useful to understand the true enzyme potentialities for industrial applications.

In the framework of the I.C.E.FISH (International Collaborative Effort on Antarctic Fish Adaptive Evolution) project, during the 1998–1999 season at the Italian shore-based Terra Nova Bay Station, the broad fields of adaptation and evolution of the fish suborder Notothenioidei were tackled through the integration of many disciplines. As a representative contribution of I.C.E.FISH, a cytogenetic study of the bathydraconid fish Gymnodraco acuticeps is reported. The Bathydraconidae is a heterogeneous notothenioid taxon whose intra-family relationships are very uncertain. The conventional karyotype and the location of nuclear ribosomal genes, by means of fluorescence in situ hybridisation, contributed to species-specific characterisation and provided the basis for comparative analyses. The karyotype of G. acuticeps consists of 48 chromosomes (2 metacentric, 2 submetacentric and 44 acrocentric); major ribosomal cistrons correspond to a large region in a single chromosome pair. Mapping of the available karyotypic data on a molecular phylogenetic tree provided information on chromosomal diversification during the cladogenesis of the bathydraconids.

Liganded and unliganded forms of two Antarctic fish haemoglobins, from Trematomus newnesi and T. bernacchii, have been crystallized in low-salt media using polyethylene glycol as precipitant. In particular, crystals of air-exposed T. newnesi carbomonoxy haemoglobin were found to be isomorphous to the crystals grown in high-salt media. Preliminary X-ray analysis of the diffraction data revealed that the beta-haem iron of this haemoglobin is in the haemichrome state, with both the proximal and distal histidyl residues linked to the iron. This is the first crystallization of a haemichrome intermediate of a vertebrate haemoglobin.

The Root effect is a widespread property in fish hemoglobins (Hbs) that produces a drastic reduction of cooperativity and oxygen-binding ability at acidic pH. Up to now, the structural explanation of the Root effect has been based on the two-state model, and is related to an over-stabilization of the T quaternary structure. Here, we report the crystal structure of the deoxy and carbomonoxy form of the non-Root effect major component Hb isolated from the Antarctic fish Trematomus newnesi (Hb1Tn). In the deoxy state, the inter-aspartic hydrogen bond at the α1β2 interface between Asp95α and Asp101β is observed. In the carbomonoxy Hb1Tn crystals, both a T-like state and a R/T intermediate quaternary structure are observed. In these crystals, three of four independent CO coordination states are not assisted by the hydrogen bond with the distal histidine, that goes out of the heme pocket. This un-assisted CO coordination states are associated with unusually small thermal fluctuations which characterise both α and β CD corners. The accessibility of ligated states within three different quaternary structure (T, R and R/T intermediate) suggests a novel structural explanation of protein allostery based on a three state Edelstein’s model.