Oligomeric-State-Dependent Conformational Change of the BLUF Protein TePixD (Tll0078)

The photochemical reaction dynamics of a BLUF (sensors of blue light using FAD) protein, PixD, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) were studied by pulsed laser-induced transient grating method. After the formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as expansion of partial molar volume with a time constant of 40 μs, whereas the other reaction phase represented a change in the diffusion coefficient D [i.e., the diffusion-sensitive conformational change (DSCC)]. D decreased from 4.9 × 10^− 11 to 4.4 × 10^− 11 m² s^− 1 upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 × 10^− 11 m² s^− 1 upon formation of the second intermediate. From a global analysis of signals at various grating wavenumbers, the time constant of D-change was determined to be 4 ms. Although the magnitude and rate constant of the faster volume change were independent of protein concentration, the amplitude of the signal that reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution: a reactive species exhibiting the DSCC, and a second species that is nonreactive. The fraction of these species was found to be dependent on the concentration. The difference in reactivity was attributed to the different oligomeric states of TePixD (i.e., pentamer and decamer). The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in the signal transduction of this photosensory protein.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

Community metabolism and buffering capacity of nitrogen in a ruppia cirrhosa meadow

Fluxes of oxygen, inorganic nitrogen (DIN) and denitrification (isotope pairing) were measured from January 1997 to February 1998 via intact cores incubation in a shallow brackish area within the eutrophic Valli di Comacchio (northern Adriatic coast, Italy). Rates were measured in the light and in the dark in sediments colonized by the rooted macrophyte Ruppia cirrhosa and in adjacent sediments with benthic microalgae. Ruppia biomass (25-414 g DW m^− 2) exhibited a seasonal evolution whilst that of microphytobenthos (12-66 mg chl a m^− 2) was more erratic. Net (NP) and gross (GP) primary productivity was 1.15 and 6.89 mol C m^− 2y^− 1 for bare and 25.4 and 51.7 mol C m^− 2y^− 1 for Ruppia vegetated sediments. Nitrogen pools in Ruppia standing stock varied from 43.6 to 631.4 (annual average 201.2) mmol N m^− 2; the macrophyte N content was correlated with DIN concentration in the water column. Estimated N pool in microphytobenthos was one order of magnitude lower (from 2.4 to 14.5 mmol N m^− 2, annual average 7.2). Theoretical DIN assimilation calculated from NP was 127.8 and 1112.6 mmol N m^− 2y^− 1 whilst that calculated from GP was 765 and 2282 mmol N m^− 2y^− 1 for microphytobenthos and Ruppia respectively. Measured annual fluxes of DIN were 974.6 and − 577 mmol N m^− 2y^− 1 in bare and Ruppia vegetated sediments meaning that the two sites were a source and sink for DIN and that from 25 to 50% of Ruppia annual DIN requirements came from the water column. During the period of this study total denitrification was lower in the macrophyte colonized (92.3 mmol N m^− 2y^− 1) compared to bare sediments (163.3 mmol N m^− 2y^− 1) as a probable consequence of higher competition between denitrifiers and phanerogams. At both sites the ratio between denitrification of water column nitrate (D_W) and denitrification coupled to nitrification (D_N) was >1.6 due to little oxygen penetration in reducing sediments (< 1.2 mm) and scarce nitrification activity. D_W (0-35 µmol N m^− 2h^− 1) was significantly correlated with water column NO₃⁻  (2-16 µM). Theoretical DIN assimilation to denitrification ratio varied from 12.0 to 24.8 for Ruppia vegetated and from 0.8 to 4.7 for unvegetated sediments.At Valle Smarlacca, Ruppia may influence nitrogen cycling by incorporating large DIN pools in biomass which is scattered in surrounding areas and fuels intense bacterial activity. With increasing anthropogenic nutrient input and insignificant organic matter export in the open sea the already severe eutrophic conditions are enhanced and may accelerate the decline of the macrophyte meadow.     

Simultaneous study of particle reworking, irrigation transport and reaction rates in sediment bioturbated by the polychaetes Heteromastus and Marenzelleria

The present study employed simultaneously methods to investigate particle and solute transport and reaction rates in sandy sediments inhabited by two worms (2500 m^− 2) with different feeding modes. Heteromastus filiformis is a head-down deposit-feeder and the main activities exerted by this worm are transport of particles as faecal pellets from subsurface to surface sediments and burrow ventilation. Marenzelleria viridis is a surface deposit-feeder that actively searches for food by burrowing near the sediment surface, producing a network of ventilated galleries in this zone. M. viridis exhibited 1.5 to 2.2 times higher particle mixing rates (D_b = 3.3 to 4 × 10^− 3 cm^− 2 d^− 1) compared to H. filiformis. In M. viridis treatments, continuous advection (eddy diffusion) was the major factor influencing solute transport resulting in apparent diffusion rates (D_a = 2.2 cm^− 2 d^− 1), which were 3 times higher than molecular diffusion within the sediment. In H. filiformis inhabited sediments, the transport of solutes was discontinuous and driven by a surprisingly high nonlocal exchange (α = 1.1-1.3 d^− 1), emphasizing its strong irrigation effects. Accordingly, the enhancement of solute fluxes was more pronounced for H. filiformis compared to M. viridis. Depth integrated TCO₂ production derived from diagenetic modelling, which takes into account three reaction zones, is in good agreement with rates obtained from measured fluxes, indicating the applicability of both approaches to get reliable rates. However, the reaction rates showed that the presence of animals had a modest effect on microbial carbon oxidation. The results proved that transport conditions are deeply related to feeding modes. Exchange of solutes was the most important transport process by H. filiformis, while M. viridis affected both mixing and solute transport.     

Identification of a Mechanical Rheostat in the Hydrophobic Core of Protein L

The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilised variants characterised, only one residue (I60V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (ΔF_I60V-WT = − 36 pN at 447 nm s^− 1, Δx_uI60V-WT = 0.2 nm). Φ-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13 ± 11 pN at 447 nm s^− 1. By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72 ± 13 pN at 447 nm s^− 1. These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.     

The Contribution of a Covalently Bound Cofactor to the Folding and Thermodynamic Stability of an Integral Membrane Protein

The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol^− 1 to 23.4 ± 1.5 kcal mol^− 1 at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol^− 1 towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol^− 1 to 10.9 ± 1.0 kcal mol^− 1, indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3-6 kcal mol^− 1. Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k_u^H2O, from ~1 × 10^− 16 s^− 1 to ~1 × 10^− 4 s^− 1 at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol^− 1 in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein-retinal interactions that increase the rigidity and compactness of the polypeptide chain.     

Ni(II) coordination compounds based on mixed phthalate and aromatic amine ligands: synthesis, crystal structures and magnetic properties

Three new coordination compounds, [Ni(Pht)(Py)₂(H₂O)₃] (1), [Ni(Pht)(β- Pic)₂(H₂O)₃] · H₂O (2) and [Ni(Pht)(1-MeIm)₂(H₂O)₃] (3) (where Pht²⁻ = dianion of o-phthalic acid; Py = pyridine, β-Pic = 3-methylpyridine, 1-MeIm = 1-methylimidazole), have been synthesized and characterized by IR spectroscopy and thermogravimetric analysis. Crystallographic studies 1-3 reveal that each Ni(II) center has a distorted octahedral geometry being coordinated by two nitrogen atoms of aromatic amines, one oxygen atom from a carboxylate group of a phthalate ligand and three water molecules. Pht²⁻ anions act as monodentate ligands, while the remaining uncoordinated carboxylate oxygen atoms participate in the formation of hydrogen bonding. The uncoordinated oxygen atoms form hydrogen bonds with the coordinated water molecules from adjacent complexes creating a centrosymmetric dimer unit. Further, these dimer units are connected by O-H?O hydrogen bonds in double-chains. Depending on the nature of aromatic amines, the arrangement of these double-chains differs. The double-chains are held together only by van der Waals interactions in 1. In contrast, in 2 these chains form layers by π-π interactions between antiparallel molecules of β-Pic as well as by π-π interactions between β-Pic and Pht aromatic rings. In complex 3, the double-chains are knitted together via C-H?O hydrogen bonds between the methyl group of 1-MeIm and the coordinated carboxylate oxygen atom of Pht, as well as π-π contacts involving antiparallel 1-MeIm cycles. The thermal dependence of the magnetic susceptibilities for compounds 1 and 2 shows a weak antiferromagnetic interaction between the two Ni²⁺ ions of the hydrogen bonded dimers. For compound 3, a ferromagnetic interaction could be observed. Modeling the experimental data with MAGPACK resulted in: g = 2.22, |D| = 4.11 cm⁻¹ and J = −0.29 cm⁻¹ for compound 1, g = 2.215, |D| = 3.85 cm⁻¹ and J = −0.1 cm⁻¹ for compound 2 and g = 2.23, |D| = 4.6 cm⁻¹ and J = 0.22 cm⁻¹ for compound 3.     

Magneto-structural relationships for a mononuclear Co(II) complex with large zero-field splitting

A mononuclear cobalt(II) complex, [Co(ac)₂(H₂O)₂(MeIm)₂], with heteroleptic coordination sphere possessing the {CoO₂O′₂N₂} chromophore has been prepared and structurally characterized. The magnetic data down to 2 K show an enhanced magnetic anisotropy manifesting itself in a large zero-field splitting (ZFS) parameter. As a consequence, the magnetization deviates substantially from the Brillouin-function behavior. A fit to the zero-field splitting model gave the following set of magnetic parameters: D/hc = +95 cm⁻¹, g_x = 2.530, zj/hc = −0.078, χ_TIP = 16.7 × 10⁻⁹ m³ mol⁻¹, (g_z = 2.0). The Griffith-Figgis model and the Generalized Crystal-Field model lie beyond the spin-Hamiltonian formalism; they gave analogous, although not identical ZFS parameters: D/hc = 109 cm⁻¹, and D/hc = 77 cm⁻¹, respectively. The absorption spectrum taken in the FAR-IR region exhibits manifold absorption peaks referring to the transitions among the crystal-field multiplets of the parent ⁴A_2g + ⁴E_g terms (D_4h), originating in a crystal-field splitting of the octahedral ⁴T_1g ground term.     

Characterisation of the electron transfer and complex formation between Flavodoxin from D. vulgaris and the haem domain of Cytochrome P450 BM3 from B. megaterium

Investigation of the complex formation and electron transfer kinetics between P450 BMP and flavodoxin was carried out following the suggested involvement of flavodoxin in modulating the electron transfer to BMP in artificial redox chains bound to an electrode surface. While electron transfer measurements show the formation of a tightly bound complex, the NMR data indicate the formation of shortly lived complexes. The measured k_obs ranged from 24.2 s^− 1 to 44.1 s^− 1 with k_on ranging from 0.07 × 10⁶ to 1.1 × 10⁶ s^− 1M^− 1 and K_d ranging from 300 μM to 24 μM in buffers of different ionic strength. This apparent contradiction is due to the existence of two events in the complex formation prior to electron transfer. A stable complex is initially formed. Within such tightly bound complex, flavodoxin rocks rapidly between different positions. The rocking of the bound flavodoxin between several different orientations gives rise to the transient complexes in fast exchange as observed in the NMR experiments. Docking simulations with two different approaches support the theory that there is no highly specific orientation in the complex, but instead one side of the flavodoxin binds the P450 with high overall affinity but with a number of different orientations. The level of functionality of each orientation is dependent on the distance between cofactors, which can vary between 8 and 25 Å, with some of the transient complexes showing distances compatible with the measured electron transfer rate constants.     

Salinity induced changes in haemolymph osmolality and total metabolic rate of the mud crab Rhithropanopeus harrisii Gould, 1841 from Baltic coastal waters

The specific metabolic rate (SMR) and haemolymph osmolality (HO) of the mud crab Rhithropanopeus harrisii Gould, 1841 from Baltic brackish waters were measured at a habitat salinity of 7 psu (T = 15 °C, full air saturation) and after step-wise acclimation to a salinity range of 3-27 psu. Values of SMR at 7 psu varied between 0.40 and 3.89 J g^− 1 WW h^− 1 (n = 25, wet weight range 0.051-1.142 g) and were significantly (p < 0.05) related to the specimen's wet weight (WW) according to the power regression SMR = 0.94 WW^− 0.41 (R² = 0.68). The SMR of females did not differ significantly (p > 0.05) from those of males. When exposed to higher salinities, the SMR of R. harrisii decreased significantly (p < 0.05) and reached a minimum value at 23 psu (0.55 ± 0.05 J g^− 1 WW h^− 1, n = 6). Mean haemolymph osmolality at 7 psu amounted to 581 ± 26 mOsm kg^− 1 (n = 5) and was 2.9 times higher than that of the external medium. R. harrisii hyperosmoregulated its body fluids up to 24 psu (727 mOsm kg^− 1) at which salinity the isosmotic point was reached.     

Laboratory experiments on the effects of variable suspended sediment concentrations on the ecophysiology of the porcelain crab Petrolisthes elongatus (Milne Edwards, 1837)

The porcelain crab Petrolisthes elongatus is a particulate suspension feeding species common to coastal areas of New Zealand (NZ). Consistent with the responses of other suspension feeding species, it is likely to be negatively influenced by elevated suspended sediment concentrations. Laboratory experiments were conducted to quantify the effect of temperature (12 °C, 15 °C and 18 °C) and suspended sediment concentration (total particulate matter (TPM): low < 100 mg L^− 1; medium 100-1000 mg L^− 1; high > 1000 mg L^− 1) on the clearance rate (CR in L h^− 1), oxygen uptake rate (VO₂ in mL h⁻¹), net absorption efficiency (AE), and net energy budget (NEB in J h^− 1) of P. elongatus across a range of sizes. Variation in CR and AE was independent of temperature and of body size, but were significantly different (P < 0.05) at low and medium suspended sediment concentrations compared with high suspended sediment concentrations. CR responded in a non-linear manner to changes in TPM, increasing with TPM up to a maximum value at medium-low concentrations (approximately 250 mg L^− 1) and then decreasing thereafter. CR had almost completely shut down at TPM concentrations of > 1000 mg L^− 1 and at particulate organic matter (POM) concentrations of > 250 mg L^− 1. AE was zero at approximate TPM and POM values of 1200 mg L^− 1 and 300 mg L^− 1, respectively. VO₂ was positively correlated with body size and with temperature, but was independent of TPM. NEB values for P. elongatus were low (approx 110 J g^− 1 h^− 1) at low sediment concentrations, were high (approx 320 J g^− 1 h^− 1) at medium sediment concentrations, and were negative (approx − 114 J g^− 1 h^− 1) at high sediment concentrations. These findings indicate that P. elongatus is likely to be food-limited at sediment concentrations of < 100 mg L^− 1, and severely negatively affected at sediment concentrations of > 1000 mg L^− 1, at least for the duration of such events which may persist for 2-3 days in coastal environments where this crab occurs.     

Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid

Crude glycerol is a major byproduct of the biodiesel industry; previous research has proved the feasibility of producing docosahexaenoic acid (DHA, 22:6 n − 3) through fermentation of the algae Schizochytrium limacinum on crude glycerol. The objective of this work is to investigate the cell growth kinetics, substrate utilization efficiency, and DHA production of the algae through a continuous culture. Steady-state biomass yield, biomass productivity, growth yield on glycerol, specific glycerol consumption rate, and fatty acid composition were investigated within the range of dilution rate (D) from 0.2 to 0.6 day⁻¹, and the range of feed crude glycerol concentration (S₀) from 15 to 120 g/L. The maximum specific growth rate was determined as 0.692 day⁻¹. The cells had a true growth yield of 0.283 g/g but with a relatively high maintenance coefficient (0.2216 day⁻¹). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L due to the higher DHA content at S₀ = 90 g/L. The biomass and DHA productivity of the continuous culture was comparable to those of batch culture, while lower than the fed-batch culture, mainly because of the lower DHA content obtained by the continuous culture. Overall, the results show that continuous culture is a powerful tool to investigate the cell growth kinetics and physiological behaviors of the algae growing on biodiesel-derived crude glycerol.     

Growth, secondary production and gonad development of two co-existing amphioxus species (Branchiostoma belcheri and B. malayanum) in subtropical Hong Kong

The present study investigated the growth, secondary production and gonad development of two co-existing amphioxus species Branchiostoma belcheri and B. malayanum in subtropical Hong Kong from June 2005 to June 2006. Based on the modal progression analysis, amphioxus populations were decomposed into separate cohorts. The von Bertalanffy growth models were also estimated according to the size incremental data. From the growth models, the size ranges of one, two and three-year-old B. belcheri were estimated to be 5-28 mm, 28-38 mm and 38-45 mm BL, respectively; while the one and two-year-old B. malayanum were estimated to be 7-30 mm and 30-35 mm BL, respectively. The secondary production was calculated at 1.15 g m^− 2 yr^− 1 DW or 0.63 g m^− 2 yr^− 1 AFDW for B. belcheri with density 424 ind m^− 2, and 0.51 g m^− 2 yr^− 1 DW or 0.40 g m^− 2 yr^− 1 AFDW for B. malayanum with density 121 ind m^− 2. The production to biomass ratio (P/B) was 1.13 for B. belcheri and 0.98 for B. malayanum. Changes in the gonad length index indicated that B. belcheri spawned mainly in June and July, while B. malayanum mainly in April and August. As compared with B. belcheri, B. malayanum was characterized by rapid growth, shorter life span, early maturity and lower population density. Such differences in population dynamics may allow both species to share a similar habitat and co-exist in subtropical waters of Hong Kong.     

The effects of algae species and densities on the population growth of the marine rotifer, Colurella dicentra

Colurella dicentra clones isolated from bay water in the Mississippi Gulf Coast were cultured with artificial seawater. Experiments were conducted to determine the effects of six algae species (Nannochloropsis oculata, Tetraselmis chuii, Chaetoceros gracilis, Rhodomonas salina, Isochrysis galbana, and Prorocentrum micans), six C. gracilis densities, and six N. oculata densities (25,000, 50,000, 100,000, 250,000, 500,000, and 1,000,000 cells ml^− 1) on C. dicentra population growth. Algae type influenced rotifer production (p < 0.0001). C. gracilis treatment (9120 ± 3351SD) produced the highest number of rotifers followed by N. oculata (5760 ±2232SD). P. micans had the lowest number of rotifers, although not significantly different from numbers in T. chuii, R. salina, and I. galbana treatments (p > 0.05).The population growth rate (r) varied with algae species treatment. The highest values were recorded for C. gracilis treatment (0.22 to 0.26 d^− 1), followed by N. oculata (0.21 to 0.24 d^− 1), and the lowest for P. micans (− 0.19 to 0.14 d^− 1). C. gracilis and N. oculata densities had significant effects (p < 0.0001) on C. dicentra population growth. The highest rotifer production was recorded at a C. gracilis density of 100,000 cells ml^− 1, followed by 250,000 cells ml^− 1 and 50,000 cells ml^− 1. Algae densities of 500,000 cells ml^− 1 and above produced the lowest rotifer numbers. Population growth rate (r) varied with C. gracilis densities. The highest values were observed for C. gracilis concentrations of 100,000 cells ml^− 1 (0.17 to 0.19 d^− 1), and the lowest for concentrations of 500,000 cells ml^− 1 and above (− 0.19 to 0.09 d^− 1). The 100,000 cells ml^− 1N. oculata density gave the highest rotifer production followed by 50,000, 250,000, 25,000, and 500,000 cells ml^− 1. Algae densities of 1,000,000 cells ml^− 1 produced the lowest rotifer numbers. Population growth rate (r) varied with N. oculata densities, with the highest values obtained for algae densities of 100,000 cells ml^− 1 (0.35 to 0.40 d^− 1), and the lowest for concentrations of 1,000,000 cells ml^− 1 (0.05 to 0.012 d^− 1). This is the first report of C. dicentra in Mississippi Coastal waters, and perhaps the smallest marine rotifer species (93 by 49 μm) ever cultured successfully.     

Denitrification, nitrate turnover, and aerobic respiration by benthic foraminiferans in the oxygen minimum zone off Chile

Population density, nitrate turnover, and oxygen respiration of benthic foraminiferans were investigated in the oxygen minimum zone (OMZ) off the Chilean coast. Live foraminiferans were found predominantly in the upper 3 mm of the sediment, and the nitrate accumulating species Nonionella cf. stella and Stainforthia sp. dominated with a combined standing stock of 2.0 × 10⁶ Rose Bengal stained specimens m^− 2. The rate of denitrification in cells of N. cf. stella analyzed with nitrous oxide microsensors during acetylene inhibition was 84 ± 33 pmol C individual^− 1 d^− 1. Multiplied with the standing stock of N. cf. stella and Stainforthia sp. this yielded a minimum benthic denitrification rate of 173 µmol N m^− 2 d^− 1 by foraminiferans. Foraminiferal denitrification, which seemed to account for almost all benthic denitrification at the investigated site will be overlooked by most conventional methods measuring benthic denitrification. Compared to the denitrification rates, the potential rates of nitrate accumulation and oxygen respiration by N. cf. stella were an order of magnitude higher (864 pmol N individual^− 1 d^− 1 and 760 ± 87 pmol C individual^− 1 d^− 1, respectively), which seems an adaptation to the infrequent availability of nitrate and oxygen in the sediment surface.     

Particle reworking and solute transport by the sediment-living polychaetes Marenzelleria neglecta and Hediste diversicolor

This experimental study quantified and compared particle-mixing and solute transport by the polychaetes Marenzelleria neglecta (2 g ww, 3200 ind. m^− 2) and Hediste diversicolor (2 g ww, 800 ind. m^− 2) in Baltic Sea sediments. Particle tracers (luminophores) were added to the sediment surface and their vertical distribution in the sediment was measured after 10 d. The rate of particle mixing was quantified using a gallery-diffusion model calculating the biodiffusion coefficient D_b and the non-local transport parameter r. Bioirrigation was measured by adding an inert solute tracer (bromide) to the overlying water 1, 1.5 and 2 d before the end of the experiment, and quantified by calculating the net bromide flux and fitting the bromide profiles to a 1D diffusion model providing an apparent biodiffusion coefficient D_a. The two polychaete worms displayed similar particle-mixing and solute transport efficiencies (based on total biomass) despite different modes of bioturbation. However, H. diversicolor was a more efficient particle-reworker and M. neglecta a more efficient bioirrigator, on an individual level. H. diversicolor buried a higher percentage (13%) of luminophores below the top 0.5 cm surface layer than M. neglecta (6%). D_b did not differ between the two species (2.4 × 10^− 3 cm² d^− 1) indicating a similar rate of diffusive mixing of the top sediment, however, the non-local transport parameter r was 2.5 y^− 1 for H. diversicolor and zero for M. neglecta, suggesting no significant particle-transport below the biodiffusive layer by M. neglecta. The average individual net bromide fluxes obtained were ca. 0.01 mL min^− 1 for H. diversicolor and 0.003 mL min^− 1 for M. neglecta, corresponding to an area-specific rate of ca. 12 L m^− 2 d^− 1 at the used densities. D_a did not differ between the two polychaetes, suggesting a higher individual solute exchange efficiency of M. neglecta considering the much higher ventilation rates reported for H. diversicolor than for Marenzelleria sp. The ongoing colonization of Baltic Sea sediments by M. neglecta at high densities may thus lead to an enhanced soluble release of both nutrients and contaminants. These results add information to the understanding of the potential effects of the invasion of M. neglecta on sediment biogeochemistry when competing with and/or replacing native species.     

Lactose Binding to Galectin-1 Modulates Structural Dynamics, Increases Conformational Entropy, and Occurs with Apparent Negative Cooperativity

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K₁ = 21 ± 6 × 10³ M^− 1) than the second (K₂ = 4 ± 2 × 10³ M^− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K₁ = 20 ± 10 × 10³ M^− 1 and K₂ = 1.67 ± 0.07 × 10³ M^− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general.     

A cyano-bridged FeRe(CN)2 cluster incorporating two high-magnetic anisotropy building units

The pentagonal bipyramidal high-spin iron(II) complex, [(TPA^2C(O)NHtBu)Fe(CF₃SO₃)]⁺, is shown to exhibit a high-anisotropy ground state, with fits to dc magnetization data providing an axial zero-field splitting parameter of D = − 7.9 cm⁻¹. The utility of this compound as a building unit is demonstrated, as its reaction with [ReCl₄(CN)₂]²⁻ affords the cyano-bridged dinuclear cluster (TPA^2C(O)NHtBu)FeReCl₄(CN)₂. dc magnetic susceptibility measurements reveal intracluster ferromagnetic exchange interactions between Fe^II and Re^IV centers, with J = +3.0 cm⁻¹, giving rise to a spin ground state of S = 7/2. Moreover, fits to dc magnetization data obtained for the FeRe cluster show the presence of strong axial anisotropy, with D = −2.3 cm⁻¹. Finally, variable-frequency ac susceptibility measurements reveal the onset of slow magnetic relaxation at low temperature, suggesting that the FeRe cluster is a single-molecule magnet.     

Aminocarboxylate complexes of vanadium(III): Electronic structure investigation by high-frequency and -field electron paramagnetic resonance spectroscopy

Aminocarboxylate complexes of vanadium(III) are of interest as models for biologically and medicinally relevant forms of this interesting and somewhat neglected ion. The V(III) ion is paramagnetic, but not readily suited to conventional EPR, due to its integer-spin ground state (S = 1) and associated large zero-field splitting (zfs). High-frequency and -field EPR (HFEPR), however, has the ability to study such systems effectively. Three complexes, all previously structurally characterized: Na[V(trdta)] · 3H₂O, Na[V(edta)(H₂O)] · 3H₂O, and [V(nta)(H₂O)₃] · 4H₂O (where trdta stands for trimethylenediamine-N,N,N′,N′-tetraacetate and nta stands for nitrilotriacetate) were studied by HFEPR. All the investigated complexes produced HFEPR responses both in the solid state, and in aqueous solution, but those of [V(nta)(H₂O)₃] · 4H₂O were poorly interpretable. Analysis of multi-frequency HFEPR spectra yielded a set of spin Hamiltonian parameters (including axial and rhombic zfs parameters: D and E, respectively) for these first two complexes as solids: Na[V(trdta)] · 3H₂O: D = 5.60 cm⁻¹, E = 0.85 cm⁻¹, g = 1.95; Na[V(edta)(H₂O)] · 3H₂O: D = 1.4 cm⁻¹, E = 0.14 cm⁻¹, g = 1.97. Spectra in frozen solution yielded similar parameters and showed multiple species in the case of the trdta complex, which are the consequence of the flexibility of this ligand. The EPR spectra obtained in frozen aqueous solution are the first, to our knowledge, of V(III) in solution in general and show the applicability of HFEPR to these systems. In combination with very insightful previous studies of the electronic absorption of these complexes which provided ligand-field parameters, it has been possible to describe the electronic structure of V(III) in [V(trdta)]⁻ and [V(edta)(H₂O)]⁻; the quality of data for [V(nta)(H₂O)₃] does not permit analysis. Qualitatively, six-coordinate V(III) complexes with O,N donor atoms show no electronic absorption band in the NIR region, and exhibit relatively large magnitude zfs (D ? 5 cm⁻¹), while analogous seven-coordinate complexes do have a NIR absorption band and show relatively small magnitude zfs (D < 2 cm⁻¹).