SAXS study of the snake toxin α-crotamine

-crotamine is a small toxic protein (42 amino acid residues with three disulphide bridges) present in the venom of Crotallus durissus terrificus. Molecular parameters (R _g=13.7 Å, S=3,000 Ų, V=9,200 ų and D _max=40 Å) were derived from SAXS curves obtained from a solution of this protein at pH=4.5. An excellent agreement between the experimental distance distribution curve and that calculated from a model consisting of two lobes linked by the Cys(18)-Cys(30) disulphide bridge.     

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide–mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1–4), and an aryl dicysteinyl peptide (CP 5) can serve as effective “double anchors” to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg₂(peptide)₂ complex. In the presence of excess peptide, Hg(peptide)₂ complexes are also detected. Notably, increasing the distance between the ligating groups or “anchor points” in CP 1–5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH₁ ∼ −91 kJ mol⁻¹ and ΔH₂ ∼ −66 kJ mol⁻¹) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH₁ ∼ −66 kJ mol⁻¹ and ΔH₂ ∼ 46 kJ mol⁻¹). The corresponding entropy change (ΔS) values (ΔS₁ ∼ −129 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −116 J K⁻¹ mol⁻¹) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS₁ ∼ −48 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −44 J K⁻¹ mol⁻¹). Generally, these associations result in a decrease in entropy, indicating that these peptide–mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.     

Regulation of sulfate uptake and xylem loading of poplar roots (Populus tremula x P. alba)

Sulfate transport processes and its regulation were studied in roots of poplar trees (Populus tremula x P. alba). From the exponential increase in sulfate uptake with temperature an activation energy (E_a) of 9.0±0.8 kJ mol^–1 was calculated. In the concentration range 0.005–10 mM sulfate uptake showed biphasic Michaelis-Menten kinetics with a K_m of 3.2±3.4 M and a V_max of 49±11 nmol SO₄^2– g^–1 FW h^–1 for the high-affinity uptake system (phase 1) and a K_m of 1.33±0.41 mM and a V_max of 255±25 nmol SO₄^2– g^–1 FW h^–1 for the low-affinity system (phase 2). Xylem loading decreased linearly with temperature and remained unchanged within the sulfate concentration range studied. Regulation of sulfate uptake and xylem loading by O-acetyl serine (OAS), Cys, reduced glutathione (GSH), Met and S-methylmethionine (SMM) were tested by perfusion into the xylem sap with the pressure probe and by addition to the incubation medium. When added directly to the transport medium, Cys and GSH repressed, and OAS stimulated sulfate uptake; xylem loading was stimulated by Cys, repressed by GSH and only slightly affected by OAS. When perfused into the xylem, none of the compounds tested affected sulfate uptake of excised roots, but xylem loading was stimulated by SMM and OAS and repressed by Met. Apparently, the site of application strongly determined the effect of regulatory compounds of sulfate transport processes.     

Stomatal,biochemical and morphological factors limiting photosynthetic gas exchange in the mangrove associate Hibiscus tiliaceus under saline and arid environment

The effect of salinity on leaf area and the relative accumulation of Na⁺ and K⁺ in leaves of the mangrove associate Hibiscus tiliaceus were investigated. Photosynthetic gas exchange characteristics were also examined under arid and non-arid leaf conditions at 0, 10, 20 and 30‰ substrate salinity. At salinities ≥ 40‰, plants showed complete defoliation followed by 100% mortality within 1 week. Salinities ≤ 30‰ were negatively correlated with the total leaf area per plant (r² = 0.94). The reduction in the total plant leaf area is attributed to the reduction in the area of individual leaves (r² = 0.94). Selective uptake of K⁺ over Na⁺ declined sharply with increasing salinity, where K⁺/Na⁺ ratio was reduced from 6.37 to 0.69 in plants treated with 0 and 30‰, respectively. Under non-arid leaf condition, increasing salinity from 0 to 30‰ has significantly reduced the values of the intrinsic components of photosynthesis V_c,max (from 50.4 to 18.4 μmol m⁻² s^-1), J_max (from 118.0 to 33.8 μmol photons m⁻² s⁻¹), and V_TPU (from 6.90 to 2.30 μmol m⁻² s⁻¹), while stomatal limitation to gas phase conductance (S_L) increased from 14.6 to 38.4%. Water use efficiency (WUE) has subsequently doubled from 3.20 for the control plants to 8.93 for 30‰ treatment. Under arid leaf conditions, the stomatal factor (S_L) was more limiting to photosynthesis than its biochemical components (73.4 to 26.6%, respectively, at 30‰). It is concluded that salinity causes a drastic decline in photosynthetic gas exchange in H. tiliaceus leaves through its intrinsic and stomatal components, and that the apparent phenotypic plasticity represented by the leaf area modulation is unlikely to be the mechanism by which H. tiliaceus avoids salt stress.     

Nitrate and phosphate uptake kinetics of the harmful diatom Coscinodiscus wailesii,a causative organism in the bleaching of aquacultured Porphyra thalli

The large diatom Coscinodiscus wailesii is one of the problematic species which indirectly cause bleaching damage to “Nori” (Porphyra thalli) cultivation through competitive utilization of nutrients during its bloom. In the present study, we experimentally investigated the nitrate (N) and phosphate (P) uptake kinetics of C. wailesii, Harima-Nada strain. Maximum uptake rates (ρ_max), obtained by short-term experiments, were 58.3 and 95.5 pmol cell⁻¹ h⁻¹ for nitrate and 41.9 and 59.1 pmol cell⁻¹ h⁻¹ for phosphate at 9 and 20 °C, respectively. The half saturation constants for uptake (K_s) were 2.91 and 5.08 μM N and 5.62 and 6.67 μM P at 9 and 20 °C, respectively. The ρ_max values of C. wailesii, much higher than those of other marine phytoplankton species, suggest that C. wailesii is able to take up large amounts of nutrients from the water column. On the other hand, V_max/K_s (V_max; V_max = ρ_max/Q₀, Q₀; minimum cell quota) values of C. wailesii, which is a better measure to evaluate the competitive ability for nutrient uptake, were low in dominant diatom species. This parameter indicates that C. wailesii is disadvantaged compared to other diatom species in competing for nutrients, and the decreasing nutrient concentrations from winter to spring is an important factor limiting C. wailesii blooming in early spring.     

In vitro assays on the susceptibility of four species of nematophagous fungi to anthelmintics and chemical fungicides/antifungal drug

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC₅₀) of each tested drug to inhibit the germination of fungal spores. EC₅₀ ranged between 0·7 and 47·2 μg ml⁻¹ for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5–4057·8 μg ml⁻¹). EC₅₀ of tested fungicides was 0·6–2·3 μg ml⁻¹ for carbendazim, 55·9–247·4 μg ml⁻¹ for metalaxyl, 24·4–45·2 μg ml⁻¹ for difenoconazole, and 555·9–1438·3 μg ml⁻¹ for pentachloronitrobenzene (PCNB). EC₅₀ of two antifungal drugs was 0·03–3·4 μg ml⁻¹ for amphotericin B and 0·3–10·9 μg ml⁻¹ for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.     

Activation of Archaeoglobus fulgidus Cu-ATPase CopA by cysteine

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu⁺ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅ 800%) the activity of CopA and other P_IB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅ 1 μM) together with the low Cu⁺-Cys K_D (< 10^− 10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu⁺ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu⁺, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K_m suggesting no changes in the E₁ ↔ E₂ equilibrium. Characterization of Cu⁺ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu⁺ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu⁺-Cys complex.     

Uptake kinetics and storage capacity of dissolved inorganic phosphorus and corresponding dissolved inorganic nitrate uptake in Saccharina latissima and Laminaria digitata (Phaeophyceae)

Uptake rates of dissolved inorganic phosphorus and dissolved inorganic nitrogen under unsaturated and saturated conditions were studied in young sporophytes of the seaweeds Saccharina latissima and Laminaria digitata (Phaeophyceae) using a “pulse‐and‐chase” assay under fully controlled laboratory conditions. In a subsequent second “pulse‐and‐chase” assay, internal storage capacity (ISC) was calculated based on V_M and the parameter for photosynthetic efficiency F_v/F_m. Sporophytes of S. latissima showed a V_S of 0.80 ± 0.03 μmol · cm^?2 · d^?1 and a V_M of 0.30 ± 0.09 μmol · cm^?2 · d^?1 for dissolved inorganic phosphate (DIP), whereas V_S for DIN was 11.26 ± 0.56 μmol · cm^?2 · d^?1 and V_M was 3.94 ± 0.67 μmol · cm^?2 · d^?1. In L. digitata, uptake kinetics for DIP and DIN were substantially lower: V_S for DIP did not exceed 0.38 ± 0.03 μmol · cm^?2 · d^?1 while V_M for DIP was 0.22 ± 0.01 μmol · cm^?2 · d^?1. V_S for DIN was 3.92 ± 0.08 μmol · cm^?2 · d^?1 and the V_M for DIN was 1.81 ± 0.38 μmol · cm^?2 · d^?1. Accordingly, S. latissima exhibited a larger ISC for DIP (27 μmol · cm^?2) than L. digitata (10 μmol · cm^?2), and was able to maintain high growth rates for a longer period under limiting DIP conditions. Our standardized data add to the physiological understanding of S. latissima and L. digitata, thus helping to identify potential locations for their cultivation. This could further contribute to the development and modification of applications in a bio‐based economy, for example, in evaluating the potential for bioremediation in integrated multitrophic aquacultures that produce biomass simultaneously for use in the food, feed, and energy industries.     

Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l⁻¹ of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (μ) and the 1,3-PDO yield from glucose (Y_P/S) in the disruptant were higher than those in the non-disruptant (ΔglpF, μ = 0·08 ± 0·00 h⁻¹, Y_P/S = 0·06 mol mol-glucose⁻¹; BW25113, μ = 0·06 ± 0·00 h⁻¹, Y_P/S = 0·02 mol mol-glucose⁻¹). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.     

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution

Chloride (Cl⁻) is essential for O₂ evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P⁺Q_A⁻), rates of water oxidation steps (S-states), rate of proton evolution, and flash O₂ yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O₂ evolution rate (−20 %) and proton release (−36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S₂ → S₃ → S₀’) slow with increasing light intensity and during O₂/water exchange (S₀’ → S₀ → S₁), while the non-protonogenic S₁ → S₂ transition is kinetically unaffected. As flash rate increases in Cl⁻ cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br⁻. The Cl⁻ advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H₃O⁺Cl⁻ in dry water channels vs. separated Br⁻ and H⁺ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br⁻ cultures for both proton evolution and less PSII charge recombination. In Br⁻ cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br⁻ with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.     

Implication of storage conditions on luminescence response from Photobacterium phosphoreum in free and immobilized form

Bioluminescent bacteria in the form of a cell suspension for on-site hazard analysis are not suitable as in vivo luminescence in free cells fluctuates and may lead to erroneous results. Furthermore, the culture broth cannot be stored for long durations to continue sensing analytes as the luminescence ceases over time. Factors that affect luminescence response include growth dynamism, and ambient environmental conditions. The present study investigated the effect of storage conditions such as temperature (25 ± 2°C, room temperature; 4°C; and −20°C) and ambient aqueous environment (M1: sucrose, 1.02 M; M2, bioluminescent media [tryptone, 10 g L⁻¹; NaCl, 28.5 g L⁻¹; MgCl₂.7H₂O, 4.5 g L⁻¹; CaCl₂, 0.5 g L⁻¹; KCl 0.5 g L⁻¹; yeast extract, 1 g L⁻¹; H₂O, 1 L]; M3, bioluminescent media and 95% glycerol, 1:1 ratio) on the luminescence emission from the calcium alginate-immobilized Photobacterium phosphoreum (S_b) against the cells in free suspension for an extended period. The results indicated that both the parameters that were undertaken markedly affected the luminescence. In the study, S_b showed an enhanced luminescence emission than the control up to 18.5-fold and for a prolonged period which can be efficiently utilized for rapid biosensing of hazardous materials.     

Nitrogen uptake kinetics of Prymnesium parvum (Haptophyte)

The uptake rates of different nitrogen (N) forms (NO₃⁻, urea, and the amino acids glycine and glutamic acid) by N-deficient, laboratory-grown cells of the mixotrophic haptophyte, Prymnesium parvum, were measured and the preference by the cells for the different forms determined. Cellular N uptake rates (ρ_cell, fmol N cell⁻¹ h⁻¹) were measured using ¹⁵N-labeled N substrates. P. parvum showed high preference for the tested amino acids, in particular glutamic acid, over urea and NO₃⁻ under the culture nutrient conditions. However, extrapolating these rates to Baltic Seawater summer conditions, P. parvum would be expected to show higher uptake rates of NO₃⁻ and the amino acids relative to urea because of the difference in average concentrations of these substrates. A high uptake rate of glutamic acid at low substrate concentrations suggests that this substrate is likely used through extracellular enzymes. Nitrate, urea and glycine, on the other hand, showed a non-saturating uptake over the tested substrate concentration (1–40 μM-N for NO₃⁻ and urea, 0.5–10 μM-N for glycine), indicating slower membrane-transport rates for these substrates.     

Influence of linear alkylbenzene sulfonate and ethanol on the degradation kinetics of domestic sewage in co-digestion with commercial laundry wastewater

The influence of ethanol on the degradation kinetics of linear alkyl benzene sulfonate (LAS) and organic matter was investigated using batch experiments with different initial LAS concentrations (8.3 mg L⁻¹ to 66.9 mg L⁻¹) and biomass immobilized on sand. Data were fitted with a substrate inhibition model. Concentrations of 2.4 mg LAS L⁻¹ and 18.9 mg LAS L⁻¹ (without and with ethanol) provided the maximum LAS utilization rate by the biomass (S_bm). For LAS degradation, ethanol addition favored a lower decrease in the specific substrate utilization rate (r_obs), even at the LAS concentration usually reported as inhibitory (> 14.4 mg L⁻¹). For organic matter degradation, r_obs was higher with ethanol. Higher biomass differentiation was observed at higher LAS concentrations. With ethanol, microbial selection occurred at LAS concentrations near S_bm. At higher LAS concentrations, the dominance and diversity values did not change significantly with ethanol, whereas without ethanol, their behaviors were irregular.

     

Stomatal,biochemical and morphological factors limiting photosynthetic gas exchange in the mangrove associate Hibiscus tiliaceus under saline and arid environment

The effect of salinity on leaf area and the relative accumulation of Na⁺ and K⁺ in leaves of the mangrove associate Hibiscus tiliaceus were investigated. Photosynthetic gas exchange characteristics were also examined under arid and non-arid leaf conditions at 0, 10, 20 and 30‰ substrate salinity. At salinities ≥ 40‰, plants showed complete defoliation followed by 100% mortality within 1 week. Salinities ≤ 30‰ were negatively correlated with the total leaf area per plant (r² = 0.94). The reduction in the total plant leaf area is attributed to the reduction in the area of individual leaves (r² = 0.94). Selective uptake of K⁺ over Na⁺ declined sharply with increasing salinity, where K⁺/Na⁺ ratio was reduced from 6.37 to 0.69 in plants treated with 0 and 30‰, respectively. Under non-arid leaf condition, increasing salinity from 0 to 30‰ has significantly reduced the values of the intrinsic components of photosynthesis V_c,max (from 50.4 to 18.4 μmol m⁻² s^-1), J_max (from 118.0 to 33.8 μmol photons m⁻² s⁻¹), and V_TPU (from 6.90 to 2.30 μmol m⁻² s⁻¹), while stomatal limitation to gas phase conductance (S_L) increased from 14.6 to 38.4%. Water use efficiency (WUE) has subsequently doubled from 3.20 for the control plants to 8.93 for 30‰ treatment. Under arid leaf conditions, the stomatal factor (S_L) was more limiting to photosynthesis than its biochemical components (73.4 to 26.6%, respectively, at 30‰). It is concluded that salinity causes a drastic decline in photosynthetic gas exchange in H. tiliaceus leaves through its intrinsic and stomatal components, and that the apparent phenotypic plasticity represented by the leaf area modulation is unlikely to be the mechanism by which H. tiliaceus avoids salt stress.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Distribution of Sprattus sprattus phalericus (Risso, 1827) and zooplankton near the Black Sea redoxcline

Understanding what environmental drivers influence marine predator–prey relationships can be key to managing and protecting ecosystems, especially in the face of future climate change risks. This is especially important in environments such as the Black Sea, where strong biogeochemical gradients can drive marine habitat partitioning and ecological interactions. We used underwater video recordings in the north-eastern Black Sea in November 2013 to observe the distribution and behaviour of the Black Sea sprat (Sprattus sprattus phalericus, Risso 1827) and its zooplankton prey. Video recordings have shown that the Black Sea sprat S. sprattus phalericus tolerates severely hypoxic waters near the redoxcline. The school was distributed in the 33–96 m layer [oxygen concentration (O₂) 277–84 μmol L⁻¹]. Some individuals were observed to leave the school and descended 20 m deeper for foraging on copepods in the 119–123 m layer (O₂ 12–10 μmol L⁻¹). Zooplankton appeared concentrated on the upper boundary of the suboxic zone (O₂ < 10 μmol L⁻¹). No zooplankton were observed below O₂ 6–7 μmol L⁻¹ (128 m). Understanding the ability of this species to tolerate low oxygen waters is crucial to predicting future responses to natural and anthropogenic changes in hypoxia.     

Spectroscopic and computational investigation of three Cys-to-Ser mutants of nickel superoxide dismutase: insight into the roles played by the Cys2 and Cys6 active-site residues

Nickel-dependent superoxide dismutase (NiSOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O₂ ^·−). A distinctive and fascinating feature of NiSOD is the presence of active-site nickel–thiolate interactions involving the Cys2 and Cys6 residues. Mutation of one or both Cys residues to Ser prevents catalysis of O₂ ^·−, demonstrating that both residues are necessary to support proper enzymatic activity (Ryan et al., J Biol Inorg Chem, 2010). In this study, we have employed a combined spectroscopic and computational approach to characterize three Cys-to-Ser (Cys → Ser) mutants (C2S, C6S, and C2S/C6S NiSOD). Similar electronic absorption and magnetic circular dichroism spectra are observed for these mutants, indicating that they possess nearly identical active-site geometric and electronic structures. These spectroscopic data also reveal that the Ni²⁺ ion in each mutant adopts a high-spin (S = 1) configuration, characteristic of a five- or six-coordinate ligand environment, as opposed to the low-spin (S = 0) configuration observed for the four-coordinate Ni²⁺ center in the native enzyme. An analysis of the electronic absorption and magnetic circular dichroism data within the framework of density functional theory computations performed on a series of five- and six-coordinate C2S/C6S NiSOD models reveals that the active site of each Cys → Ser mutant possesses an essentially six-coordinate Ni²⁺ center with a rather weak axial bonding interaction. Factors contributing to the lack of catalytic activity displayed by the Cys → Ser NiSOD mutants are explored.     

Temporal and spatial variability in nitrogen uptake kinetics during harmful dinoflagellate blooms in the East China Sea

Temporal and spatial variability in the kinetic parameters of uptake of nitrate (NO₃⁻), ammonium (NH₄⁺), urea, and glycine was measured during dinoflagellate blooms in Changjiang River estuary and East China Sea coast, 2005. Karenia mikimotoi was the dominant species in the early stage of the blooms and was succeeded by Prorocentrum donghaiense. The uptake of nitrogen (N) was determined using ¹⁵N tracer techniques. The results of comparison kinetic parameters with ambient nutrients confirmed that different N forms were preferentially taken up during different stages of the bloom. NO₃⁻ (V_max 0.044 h⁻¹; K_s 60.8 μM-N) was an important N source before it was depleted. NH₄⁺ (V_max 0.049 h⁻¹; K_s 2.15 μM-N) was generally the preferred N. Between the 2 organic N sources, urea was more preferred when K. mikimotoi dominated the bloom (V_max 0.020 h⁻¹; K_s 1.35 μM-N) and glycine, considered as a dominant amino acid, was more preferred when P. donghaiense dominated the bloom (V_max 0.025 h⁻¹; K_s 1.76 μM-N). The change of N uptake preference by the bloom-forming algae was also related to the variation in ambient N concentrations.     

The Functions of Crucial Cysteine Residues in the Arsenite Methylation Catalyzed by Recombinant Human Arsenic (III) Methyltransferase

Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs³⁺). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs³⁺ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs³⁺ was methylated to dimethylarsinous acid (DMA³⁺). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs³⁺ methylation catalyzed by hAS3MT.