Investigating the binding interactions of galantamine with β-amyloid peptide

The anti-Alzheimer’s agent galantamine is known to possess anti-amyloid properties. However the exact mechanisms are not clear. We studied the binding interactions of galantamine with amyloid peptide dimer (Aβ_1–40) through molecular docking and molecular dynamics simulations. Galantamine’s binding site within the amyloid peptide dimer was identified by docking experiments and the most stable complex was analyzed by molecular dynamics simulation. These studies show that galantamine was interacting with the central region of the amyloid dimer (Lys16–Ala21) and the C-terminal region (Ile31–Val36) with minimum structural drift of Cα atom in those regions. Strikingly, a significant drift was observed at the turn region from Asp23-Gly29 (Cα atom RMSD = 9.2 Å and 11.6 Å at 50 fs and 100 fs respectively). Furthermore, galantamine’s binding mode disrupts the key pi–pi stacking interaction between aromatic rings of Phe19 (chain A) and Phe19 (chain B) and intermolecular hydrogen bonds seen in unbound peptide dimer. Noticeably, the azepine tertiary nitrogen of galantamine was in close proximity to backbone CO of Leu34 (distance <3.5 Å) to stabilize the dimer conformation. In summary, the results indicate that galantamine binding to amyloid peptide dimer leads to a significant conformational change at the turn region (Asp23–Gly29) that disrupts interactions between individual β-strands and promotes a nontoxic conformation of Aβ_1–40 to prevent the formation of neurotoxic oligomers.     

The effect of substitution of Phe181 and Phe182 with Ala on activity,substrate specificity and stabilization of substrate at the active site of Bacillus thermocatenulatus lipase

Steric hindrance leads to limitation in the access of substrate into the enzyme active site. In order to decrease steric hindrance, two conserved residues, Phe¹⁸¹ and Phe¹⁸², in the lid domain of Bacillus thermocatenulatus lipase were substituted with alanine by using site-directed mutagenesis. As a result, three mutant lipases were produced. Circular dichroism (CD) spectroscopy showed that the secondary structure of all lipases is similar to one another. F181A mutation increased the distance between phe¹⁸¹ and catalytic ser¹¹⁴, which is buried in the active site by 3.24 Å. It can be suggested that such an increase in distance may lead to a decrease in steric hindrance. F181A mutation increased overall lipase activity by up to 2.6-fold (4670 U mg⁻¹) toward C8 substrate. It also resulted in optimal lipase activity at 65 °C rather than 55 °C. F182A mutation increased the distance between phe¹⁸² and catalytic ser¹¹⁴ by 1.54 Å but failed to induce any significant effect on lipase activity. However, F181A–F182A mutation significantly decreased the activity due to decreased van der Waals interactions between the phenyl group of phenylalanines and the acyl chain of triacylglycerol. These results indicate that presence of one of the two residues, Phe¹⁸¹ or Phe¹⁸², is important for stabilizing triacylglycerols in active site.     

Rapid measurement of long-range distances in proteins by multidimensional <Superscript>13</Superscript>C–<Superscript>19</Superscript>F REDOR NMR under fast magic-angle spinning

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are ¹³C–¹⁵N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~?5 Å due to the low gyromagnetic ratios of ¹⁵N and ¹³C. Here we present a robust 2D ¹³C–¹⁹F REDOR experiment to measure multiple distances to ~?10 Å. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The ¹³C–¹⁹F REDOR sequence is combined with 2D ¹³C–¹³C correlation to resolve multiple distances in highly ¹³C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved ¹³C spectra, the deleterious effect of the large ¹⁹F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2D ¹³C–¹³C resolved ¹³C–¹⁹F REDOR technique on ¹³C, ¹⁵N-labeled GB1. A 5-¹⁹F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-¹⁹F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical ¹⁹F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza B M2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2D ¹³C–¹⁹F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~?10 Å range.     

Computational identification of a phospholipidosis toxicophore using 13C and 15N NMR-distance based fingerprints

Modified 3D-SDAR fingerprints combining ¹³C and ¹⁵N NMR chemical shifts augmented with inter-atomic distances were used to model the potential of chemicals to induce phospholipidosis (PLD). A curated dataset of 328 compounds (some of which were cationic amphiphilic drugs) was used to generate 3D-QSDAR models based on tessellations of the 3D-SDAR space with grids of different density. Composite PLS models averaging the aggregated predictions from 100 fully randomized individual models were generated. On each of the 100 runs, the activities of an external blind test set comprised of 294 proprietary chemicals were predicted and averaged to provide composite estimates of their PLD-inducing potentials (PLD+ if PLD is observed, otherwise PLD−). The best performing 3D-QSDAR model utilized a grid with a density of 8 ppm × 8 ppm in the C–C region, 8 ppm × 20 ppm in the C–N region and 20 ppm × 20 ppm in the N–N region. The classification predictive performance parameters of this model evaluated on the basis of the external test set were as follows: accuracy = 0.70, sensitivity = 0.73 and specificity = 0.66. A projection of the most frequently occurring bins on the standard coordinate space suggested a toxicophore composed of an aromatic ring with a centroid 3.5–7.5 Å distant from an amino-group. The presence of a second aromatic ring separated by a 4–5 Å spacer from the first ring and at a distance of between 5.5 Å and 7 Å from the amino-group was also associated with a PLD+ effect. These models provide comparable predictive performance to previously reported models for PLD with the added benefit of being based entirely on non-confidential, publicly available training data and with good predictive performance when tested in a rigorous, external validation exercise.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Isolation,spectroscopic characterization,X-ray,theoretical studies as well as in vitro cytotoxicity of Samarcandin

Samarcandin 1, a natural sesquiterpene-coumarin, was isolated as well as elucidated from F. assa-foetida which has significant effect in Iranian traditional medicine because of its medicinal attitudes. The crystal structure of samarcandin was determined by single-crystal X-ray structure analysis. It is orthorhombic, with unit cell parameters a = 10.8204 (5) Å, b = 12.9894 (7) Å, c = 15.2467 (9) Å, V = 2142.9 (2) Å³, space group P2₁2₁2₁ and four symmetry equivalent molecules in the unit cell. Samarcandin was isolated in order to study for its theoretical studies as well as its cellular toxicity as anti-cancer drug against two cancerous cells. In comparison with controls, our microscopic and MTT assay data showed that samarcandin suppresses cancer cell proliferation in a dose-dependent manner with IC₅₀ = 11 μM and 13 for AGS and WEHI-164 cell lines, respectively. Density functional theory (DFT) and time-dependent density functional theory (TD-DFT) of the structure was computed by three functional methods and 6-311++G⁷ standard basis set. The optimized molecular geometry and theoretical analysis agree closely to that obtained from the single crystal X-ray crystallography. To sum up, the good correlations between experimental and theoretical studies by UV, NMR, and IR spectra were found.     

Small-angle neutron scattering study of the ultrastructure of chloroplast thylakoid membranes — Periodicity and structural flexibility of the stroma lamellae

The multilamellar organization of freshly isolated spinach and pea chloroplast thylakoid membranes was studied using small-angle neutron scattering. A broad peak at ~ 0.02 Å^? 1 is ascribed to diffraction from domains of ordered, unappressed stroma lamellae, revealing a repeat distance of 294 Å ± 7 Å in spinach and 345 Å ± 11 Å in pea. The peak position and hence the repeat distance of stroma lamellae is strongly dependent on the osmolarity and the ionic strength of the suspension medium, as demonstrated by varying the sorbitol and the Mg⁺⁺-concentration in the sample. For pea thylakoid membranes, we show that the repeat distance decreases when illuminating the sample with white light, in accordance with our earlier results on spinach, also regarding the observation that addition of an uncoupler prohibits the light-induced structural changes, a strong indication that these changes are driven by the transmembrane proton gradient. We show that the magnitude of the shrinkage is strongly dependent on light intensity and that the repeat distance characteristic of the dark state after illumination is different from the initial dark state. Prolonged strong illumination leads to irreversible changes and swelling as reflected in increased repeat distances. The observed reorganizations are discussed within the frames of the current structural models of the granum-stroma thylakoid membrane assembly and the regulatory mechanisms in response to variations in the environmental conditions in vivo. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol^− 1 and 8.93 kcal mol^− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol^− 1 and 9.09 kcal mol^− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.     

The crystallographic structure of Panicum Mosaic Virus (PMV)

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9 Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2₁ with a = 411.7 Å, b = 403.9 Å and c = 412.5 Å, with β = 89.7°. The asymmetric unit was two entire T = 3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ～20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca⁺⁺ ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1–53, 1–49 and 1–21 of the A, B and C subunits, respectively, and the four C-terminal residues (239–242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent “arm” that intertwines with symmetry equivalent “arms” at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A–B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.     

Structures,magnetic properties,and XPS of cyanide-bridged NdIII/SmIII/GdIII–CrIII complexes

Synthesis, structural characterization, and spectroscopic and magnetic properties of three new cyano-bridged 3d–4f bimetallic complexes, Ln^III(DMF)₄(H₂O)₃Cr^III (CN)₆ · nH₂O (Ln = Nd, Sm, Gd), have been described. The Nd–Cr complex crystallizes in the monoclinic P2₁/n space group with the following unit cell parameters: a = 20.063(7) Å, b = 8.967(4) Å, c = 18.023(6) Å, b = 96.12(3)°, V = 3224(2) Å³, and Z = 4. The neodymium (III) ion, which adopts anti-prism eight-coordination environment, is linked to the [Cr^III(CN)₆]³⁻ moiety through a bridging cyanide ligand with Nd–N = 2.550(4) Å and Nd–N–C = 164.4(4)°. The variable-temperature (0.5 T at 2–300 K) and variable-field (0–5 T at 2 and 5 K) magnetic measurements reveal that the weak interaction of Gd–Cr complexes differs from that of Nd–Cr and Sm–Cr ones mainly because of the lack of orbital angular momentum. The XPS and diffuse reflectance electronic spectra were also measured to discuss charge transfer transitions concerning π-backdonation from the viewpoint of magneto-optical functions.     

Structure and water permeability of fully hydrated diphytanoylPC

Diphytanoylphosphatidylcholine (DPhyPC) is a branched chain lipid often used for model membrane studies, including peptide/lipid interactions, ion channels and lipid rafts. This work reports results of volume measurements, water permeability measurements P_f, X-ray scattering from oriented samples, and X-ray and neutron scattering from unilamellar vesicles at T = 30 °C. We measured the volume/lipid V_L = 1426 ± 1 Å³. The area/lipid was found to be 80.5 ± 1.5 Å² when both X-ray and neutron data were combined with the SDP model analysis (Ku?erka, N., Nagle, J.F., Sachs, J.N., Feller, S.E., Pencer, J., Jackson, A., Katsaras, J., 2008. Lipid bilayer structure determined by the simultaneous analysis of neutron and X-ray scattering data. Biophys. J. 95, 2356–2367); this is substantially larger than the area of DOPC which has the largest area of the common linear chain lipids. P_f was measured to be (7.0 ± 1.0) × 10^?3 cm/s; this is considerably smaller than predicted by the recently proposed 3-slab model (Nagle, J.F., Mathai, J.C., Zeidel, M.L., Tristram-Nagle, S., 2008. Theory of passive permeability through lipid bilayers. J. Gen. Physiol. 131, 77–85). This disagreement can be understood if there is a diminished diffusion coefficient in the hydrocarbon core of DPhyPC and that is supported by previous molecular dynamics simulations (Shinoda, W., Mikami, M., Baba, T., Hato, M., 2004. Molecular dynamics study on the effects of chain branching on the physical properties of lipid bilayers. 2. Permeability. J. Phys. Chem. B 108, 9346–9356). While the DPhyPC head–head thickness (D_HH = 36.4 Å), and Hamaker parameter (H = 4.5 × 10^?21 J) were similar to the linear chain lipid DOPC, the bending modulus (K_C = 5.2 ± 0.5 × 10^?21 J) was 30% smaller. Our results suggest that, from the biophysical perspective, DPhyPC belongs to a different family of lipids than phosphatidylcholines that have linear chain hydrocarbon chains.     

Discriminating the endogenous and exogenous urinary estrogens in human by isotopic ratio mass spectrometry and its potential clinical value

Estrogens were prohibited in the food producing animals by European Union (96/22/EC directive) and added to the Report on Carcinogens in United States since 2002. Due to very low concentration in serum or urine (～pg/mL), the method of control its abuse had not been fully developed.The endogenous estrogens were separated from urines of 18 adult men and women. The exogenous estrogens were chemical reference standards and over the counter preparations. Two patients of dysfunctional uterine bleeding (DUB) administered exogenous estradiol and the urines were collected for 72 h. The urinary estrogens were separated by high-performance liquid chromatography (HPLC) and confirmed. The exogenous and exogenous estrogens were analyzed by gas chromatography combustion isotope ratio mass spectrometry (GC–C–IRMS) to determine the ¹³C/¹²C ratio (δ¹³C‰).The δ¹³C‰ values of reference standard of E1, E2, and E3 were ?29.36 ± 0.72, ?27.98 ± 0.35, ?27.62 ± 0.51, respectively. The δ¹³C‰ values of the endogenous E1, E2, and E3 were ?21.62 ± 1.07, ?22.14 ± 0.98, and ?21.88 ± 1.16, with P < 0.01 (t-test). Two DUB patients’ urinary estradiol δ¹³C‰ values was depleted to ?28.02 ± 0.33 after the administration. The progesterone, 17α-hydroxyprogesterone, pregnanediol, as well as desogestrel and ethinylestradiol from contraceptives were also determined.Stable carbon isotope analysis can distinguish the endogenous and exogenous urinary estrogen in human.     

The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals

Electron beam damage is the fundamental limit to resolution in electron cryomicroscopy (cryo-EM) of frozen, hydrated specimens. Radiation damage increases with the number of electrons used to obtain an image and affects information at higher spatial frequencies before low-resolution information. For the experimentalist, a balance exists between electron exposures sufficient to obtain a useful signal-to-noise ratio (SNR) in images and exposures that limit the damage to structural features. In single particle cryo-EM this balance is particularly delicate: low-resolution features must be imaged with a sufficient SNR to allow image alignment so that high-resolution features recorded below the noise level can be recovered by averaging independent images. By measuring the fading of Fourier components from images obtained at 200 kV of thin crystals of catalase embedded in ice, we have determined the electron exposures that will maximize the SNR at resolutions between 86 and 2.9 Å. These data allow for a rational choice of exposure for single particle cryo-EM. For example, for 20 Å resolution, the SNR is maximized at ～20 e^?/Ų, whereas for 3 Å resolution, it is maximized at ～10 e^?/Ų. We illustrate the effects of exposure in single particle cryo-EM with data collected at ～12–15 and ～24–30 e^?/Ų.     

Hydrothermal synthesis and structural characterization of an organic–inorganic hybrid material: (H2tptz)2[δ-Mo8O26]·2H2O (tptz=2,4,6-tripyridyltriazine)

The reaction of MoO₃ and 2,4,6-tripyridyltriazine (tptz) in water at 180°C for 48 h and pH 5.5 produces (H₂tptz)₂[Mo₈O₂₆]·2H₂O in 70% yield. The structure is constructed from δ-Mo₈O₂₆ ⁴⁻ clusters, H₂tptz²⁺ and H₃O⁺ cations linked through hydrogen bonding into a network. Crystal data: C₁₈H₁₆Mo₄N₆O₁₄; monoclinic P2₁/n; a=10.2225(5) Å, b=14.0072(6) Å, c=18.1154(8) Å, β=93.896(1)°, V=2587.9(2) Å³, Z=4, D_calc=2.372 g cm⁻³; R₁=0.0271 based on 3212 reflections.     

Effect of ligand congeniality on energy transfer reaction between photo-excited tris(bipyridine)ruthenium(II) and chromate(III) complexes in aqueous solutions

Energy-transfer rate-constants from photo-excited [Ru(N–N)₃]²⁺ (N–N = 2,2′-bipyridine (bpy), 4,4′-dimethyl-2,2′-bipyridine (4dmb), 5,5′-dimethyl-2,2′-bipyridine (5dmb)) to [Cr(O–O)₃]³⁻ (O–O²⁻ = ox²⁻ ((COO⁻)₂), mal²⁻ (CH₂(COO⁻)₂)) and [Cr(CN)₆]³⁻ in encounter complexes were evaluated in aqueous solutions containing alkali metal ion. The rate constant depends on the molecular size of the ruthenium(II) complex: 1.8 × 10⁸ s⁻¹ for [Ru(bpy)₃]²⁺ (molecular radius, r = 5.8 Å), 1.4 × 10⁸ s⁻¹ for [Ru(5dmb)₃]²⁺ (r = 6.1 Å) and 0.96 × 10⁸ s⁻¹ for [Ru(4dmb)₃]²⁺ (r = 6.7 Å) in the system of [Ru(N–N)₃]²⁺–[Cr(ox)₃]³⁻ in aqueous solution. However, the rate constant is much more sensitive to the chromate(III) complex than to ruthenium(II) complex; 1.8 × 10⁸ s⁻¹ and 0.43 × 10⁸ s⁻¹ for [Cr(ox)₃]³⁻ (r = 4.0 Å) and [Cr(mal)₃]³⁻ (r = 4.2 Å) in the [Ru(bpy)₃]²⁺–[Cr(O–O)₃]³⁻ systems, respectively. We conclude that the congeniality between the donor’s and acceptor’s ligands in encounter complex plays an important role in energy transfer in aqueous solution.     

Head-to-tail interaction between amphotericin B and ergosterol occurs in hydrated phospholipid membrane

Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.     

δ15N and δ13C diet–tissue discrimination factors for large sharks under semi-controlled conditions

Stable isotopes (δ¹⁵N and δ¹³C) are being widely applied in ecological research but there has been a call for ecologists to determine species- and tissue-specific diet discrimination factors (?¹³C and ?¹⁵N) for their study animals. For large sharks stable isotopes may provide an important tool to elucidate aspects of their ecological roles in marine systems, but laboratory based controlled feeding experiments are impractical. By utilizing commercial aquaria, we estimated ?¹⁵N and ?¹³C of muscle, liver, vertebral cartilage and a number of organs of three large sand tiger (Carcharias taurus) and one large lemon shark (Negaprion brevirostris) under a controlled feeding regime. For all sharks mean ± SD for ?¹⁵N and ?¹³C in lipid extracted muscle using lipid extracted prey data were 2.29‰ ± 0.22 and 0.90‰ ± 0.33, respectively. The use of non-lipid extracted muscle and prey resulted in very similar ?¹⁵N and ?¹³C values but mixing of lipid and non-lipid extracted data produced variable estimates. Values of ?¹⁵N and ?¹³C in lipid extracted liver and prey were 1.50‰ ± 0.54 and 0.22‰ ± 1.18, respectively. Non-lipid extracted diet discrimination factors in liver were highly influenced by lipid content and studies that examine stable isotopes in shark liver, and likely any high lipid tissue, should strive to remove lipid effects through standardising C:N ratios, prior to isotope analysis. Mean vertebral cartilage ?¹⁵N and ?¹³C values were 1.45‰ ± 0.61 and 3.75‰ ± 0.44, respectively. Organ ?¹⁵N and ?¹³C values were more variable among individual sharks but heart tissue was consistently enriched by ~ 1–2.5‰. Minimal variability in muscle and liver δ¹⁵N and δ¹³C sampled at different intervals along the length of individual sharks and between liver lobes suggests that stable isotope values are consistent within tissues of individual animals. To our knowledge, these are the first reported diet–tissue discrimination factors for large sharks under semi-controlled conditions, and are lower than those reported for teleost fish.     

δ13C and stomatal number variability in the Cretaceous conifer Frenelopsis

The ¹³C/¹²C ratios of leaves of the conifer morphotype Frenelopsis were measured to decipher the influences of water and salt stress on stomatal density (SD), epidermal cell density (ECD) and stomatal index (SI). Three morphospecies were analyzed: F. ugnaensis from freshwater fluvio-lacustrine deposits (Upper Barremian), F. turolensis and alata from coastal deposits (Lower-Middle Albian and Upper Albian respectively). The cuticle δ¹³C values show a large variation from ? 28‰ to ? 21‰. Comparison with previously published marine carbonate δ¹³C records indicate that the difference in cuticle δ¹³C between the different deposits are mainly due to difference in CO₂-plant isotope fractionation rather than to change in isotopic composition of inorganic carbon in the atmosphere and ocean. The less negative δ¹³C and wide range in δ¹³C of F. turolensis and alata (? 27.5 to ? 21‰), compared to F. ugnaensis, (? 28 to ? 25‰) are interpreted as a result of salt and/or water stress. The data as a whole yield a good relationship between the ¹³C/¹²C ratio and SD (r = 0.67, n = 42, p < 0.001), SI (r = 0.53, n = 41, p < 0.001), hence suggesting that the differences in SD and SI between the three morphospecies are related to freshwater/saline environment. Looking at single morphospecies, the SD of F. ugnaensis decreases with increasing δ¹³C value (r = ? 0.57, n = 15, p = 0.026) as well as a decrease of SI (r = ? 0.62, n = 15, p = 0.013), possibly reflecting warmer and drier conditions. Average SI of F. alata does not significantly change with δ¹³C and inferred soil salinity in contrast to SD (p < 0.01).     

CBOD5 treatment using the marshland upwelling system

Five-day carbonaceous biochemical oxygen demand (CBOD₅) removal efficiency was evaluated for the marshland upwelling system (MUS) under both intermediate and saltwater conditions. The MUS treated decentralized wastewater from two private camps and a public restroom in the Grand Bay National Estuarine Research Reserve, Moss Point, Mississippi, and one private camp in the Barataria Terrebonne National Estuary, along Bayou Segnette, Louisiana. Raw wastewater was injected into the surrounding subsurface at a depth of 3.8 or 4.3 m. Various injection flow rates and frequencies were tested in addition to a synthetic wastewater trial. All trials followed a first-order background corrected removal equation, resulting in removal constants ranging from 0.49 to 3.32 m^?1 and predicted surface concentrations from 5.7 to 33.0 mg L^?1. CBOD₅ (unfiltered) influent concentrations of 282 ± 173 mg L^?1 were reduced to an overall effluent mean of 13 ± 13 mg L^?1 by a vector distance of 7 m at Moss Point and from 365 ± 151 mg L^?1 to 3.6 ± 7.6 mg L^?1 by a vector distance of 6 m for Bayou Segnette. Of seven trials, only one failed to achieve effluent CBOD₅ levels below a National Pollutant Discharge Elimination System (NPDES) standard level of 25 mg L^?1.     

Magnetostructural correlations and catecholase-like activities of μ-alkoxo-μ-carboxylato double bridged dinuclear and tetranuclear copper(II) complexes

Two μ-alkoxo-μ-carboxylato bridged dinuclear copper(II) complexes, [Cu₂(L¹)(μ-HCO₂)] (1) ((H₃L¹ = 1,3-bis(5-bromosalicylideneamino)-2-propanol)), [Cu₂(L²)(μ-HCO₂)] · dmf (2) (H₃L² = 1,3-bis(3,5-chlorosalicylideneamino-2-propanol)), and two μ-alkoxo-μ-dicarboxylato doubly bridged tetranuclear copper(II) complexes, [{Cu₂(L³)}₂(μ-O₂C–C(CH₃)₂–CO₂)] · 5H₂O · 3CH₃OH (3) ((H₃L³ = 1,3-bis(salicylid-deneamino)-2-propanol)) and [{Cu₂(L³)}₂(μ- O₂CCH₂–C₆H₄–CH₂CO₂)] · 2H₂O (4) have been prepared and characterized. The single crystal X-ray analysis shows that the structures of complexes 1 and 2 are dimeric with two adjacent copper(II) atoms bridged by μ-alkoxo-μ-carboxylato ligands with the Cu⋯Cu distances and Cu–O(alkoxo)–Cu angles are 3.511 Å and 132.85° for 1, 3.517 Å and 131.7° for 2, respectively. Complexes 3 and 4 consist of μ-alkoxo-μ-dicarboxylato doubly bridged tetranuclear Cu(II) complexes with mean Cu–Cu distances and Cu–O–Cu angles of 3.158 Å and 108.05° for 3 and 3.081 Å and 104.76° for 4, respectively. Magnetic measurements reveal that 1 and 2 are strong antiferromagnetically coupled with 2J = −156 and −152 cm⁻¹, respectively, while 3 and 4 exhibit ferromagnetic coupling with 2J = 86 and 155.2 cm⁻¹, respectively. The 2J values of 1–4 are linearly correlated to the Cu–O–Cu angles. Dependence of the pH at 25 °C on the reaction rate of oxidation of 3,4-di-tert-butylcatechol (3,5-dtbc) to the corresponding quinone catalyzed by 1–4 was studied. Complexes 1–4 exhibit high catecholase-like activity at pH 9.0 and 25 °C for oxidation of 3,5-di-tert-butylcatechol.