Rare earth thiocyanate complexes with tripiperidinophosphine oxide: synthesis and characterization. Crystal structure of Pr(SCN)3(tpppO)3

The synthesis and characterization of rare-earth (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and Y) thiocyanate adducts with tripiperidinophosphine oxide (tpppO) with general formula (RE)(SCN)₃(tpppO)₃ are reported. Conductance measurements in acetonitrile indicate the non-electrolytic nature of the complexes. Infrared absorption spectra evidence that the SCN⁻ ion coordinates through the nitrogen atom (isothiocyanate form) and that tpppO coordinates through the phosphoryl oxygen. X-ray powder patterns suggest the existence of three different crystal forms: (1) La; (2) an isomorphous series including Ce, Nd and Pr; and (3) another isomorphous series, including Sm, Gd, Eu, Ho, Er, Tb, Lu and Y. The visible spectra of the Nd adduct and the calculated parameters β = 0.98, b^1/2 = 0.072 and δ = 1.06 indicate that the metal-ligand bonds are essentially electrostactic. The emission spectra of the Eu compound showed ⁵D₀ → ⁷F_J bands (J = 0, 1, 2), suggesting a C_3v symmetry for the coordination polyhedron. The lifetime of the ⁵D₀ state is 1.28 ms. The emission spectra of the Tb complex presented ⁵D₄ → ⁷F_J bands (J = 4, 5, 6) and the Dy complex showed the ⁴F_9/2 → ⁶H_13/2 band. The structure of the Pr complex showed that the coordination polyhedron is a trigonal antiprism, with the isothiocyanate anions in one base and three tpppO ligands in the other. Thermal analyses (TG-DTG) were carried out for the Ce, Nd and Gd adducts. Mass losses start between 250 and 334 °C. The final residues at 1300 °C are the corresponding phosphates.     

Rotation of lipids in membranes: molecular dynamics simulation, 31P spin-lattice relaxation, and rigid-body dynamics

Molecular dynamics simulations and ³¹P-NMR spin-lattice (R₁) relaxation rates from 0.022 to 21.1 T of fluid phase dipalmitoylphosphatidylcholine bilayers are compared. Agreement between experiment and direct prediction from simulation indicates that the dominant slow relaxation (correlation) times of the dipolar and chemical shift anisotropy spin-lattice relaxation are ∼10 ns and 3 ns, respectively. Overall reorientation of the lipid body, consisting of the phosphorus, glycerol, and acyl chains, is well described within a rigid-body model. Wobble, with D_⊥ = 1-2 × 10⁸ s⁻¹, is the primary component of the 10 ns relaxation; this timescale is consistent with the tumbling of a lipid-sized cylinder in a medium with the viscosity of liquid hexadecane. The value for D_|| the diffusion constant for rotation about the long axis of the lipid body, is difficult to determine precisely because of averaging by fast motions and wobble; it is tentatively estimated to be 1 × 10⁷ s⁻¹. The resulting D_||/D_⊥ ≈ 0.1 implies that axial rotation is strongly modulated by interactions at the lipid/water interface. Rigid-body modeling and potential of mean force evaluations show that the choline group is relatively uncoupled from the rest of the lipid. This is consistent with the ratio of chemical shift anisotropy and dipolar correlation times reported here and the previous observations that ³¹P-NMR lineshapes are axially symmetric even in the gel phase of dipalmitoylphosphatidylcholine.     

Synthesis and luminescent properties of gadolinium aluminates phosphors

In this work, aluminum-gadolinium oxides with different phases were prepared by the non-hydrolytic sol-gel route, using lower temperatures than those employed in methods such as solid-state reaction and Pechini method. The influence of heating treatment on sample structure was investigated. The formation process and the local structure of the samples are discussed on the basis of thermal, X-ray diffraction, photoluminescence (PL) spectroscopy, and infrared spectroscopy analyses. The quantum efficiency of Eu³⁺ in the different phases obtained in this studied was evaluated. Initial crystallization and the GdAlO₃ phase were observed at temperatures around 400 °C. PL data of all the samples revealed the characteristic transition bands arising from the ⁵D₀ → ⁵F_J (J = 0, 1, 2, 3, and 4) manifolds under maximum excitation at 275, 393, and 467 nm in all cases. The ⁵D₀ → ⁷F₂ transition often dominates the emission spectra, indicating that the Eu³⁺ ion occupies a site without inversion center.     

Syntheses and properties of new metal-carbon bonded heteroleptic complexes of ruthenium(II) containing terpyridine coligand

Reactions of 2-(arylazo)aniline, HL [H represents the dissociable protons upon orthometallation and HL is p-RC₆H₄N = NC₆H₄-NH₂; R = H for HL¹; CH₃ for HL² and Cl for HL³] with Ru(R₁-tpy)Cl₃ (where R₁-tpy is 4′-(R₁)-2,2′,6′′,2′′-terpyridine and R₁ = H or 4-N,N-dimethylaminophenyl or 4-methylphenyl) afford a group of complexes of type [Ru(L)(R₁-tpy)]·ClO₄ each of which contains C,N,N coordinated L⁻ as a tridentate ligand along with a terpyridine. Structure of one such complex has been determined by X-ray crystallography. All the Ru(II) complexes are diamagnetic, display characteristic ¹H NMR signals and intense dπ(Ru^II) → π∗(tpy) MLCT transitions in the visible region. Cyclic voltammetric studies on [Ru(L)(R₁-tpy)]·ClO₄ complexes show Ru(II)-Ru(III) oxidation within 0.63-0.67 V versus SCE.     

Enzymatically derived aldouronic acids from Cryptomeria japonica arabinoglucuronoxylan

An arabinoglucuronoxylan was extracted from the holocellulose of sugi (Cryptomeria japonica) wood with 10% KOH and subjected to hydrolysis by partially purified xylanase fraction from a commercial cellulase preparation “Meicelase”. Neutral sugars liberated were analyzed by size exclusion chromatography showing the presence of xylooligosaccharides up to xylohexaose. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were identified by monosaccharide analysis, comparison of their volume distribution coefficients (Dvs) with those of the authentic samples in anion exchange chromatography and ¹H and ¹³C NMR spectroscopy, resulting in the characterization of eight aldouronic acids including acids consisting of two 4-O-Me-α-D-GlcAp residues and 3-5 D-Xyl residues.

1.

Fr. 1-S1: (aldohexaouronic acid, MeGlcA³Xyl₅), O-β-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)-(1 → 2)]-O-β-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

2.

Fr. 1-S2: (aldopentaouronic acid, MeGlcA³Xyl₄), O-β-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)-(1 → 2)]-O-β-D-Xylp-(1 → 4)-O-β-Xylp-(1 → 4)-D-Xyl

3.

Fr. 2-S1: (aldotetraouronic acid, MeGlcA³Xyl₃), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

4.

Fr. 3-S1: (aldotetraouronic acid, GlcA³Xyl₃), O-(α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-Xylp-(1 → 4)-D-Xyl,

5.

Fr. 4-S1: (aldotriouronic acid, GlcA²Xyl₂), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-D-Xyl

6.

Fr. 4-S2: (MeGlc⁴MeGlcA³Xyl₅), O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

7.

Fr. 6-S1: (MeGlcA⁴MeGlcA³Xyl₄), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-[(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

8.

Fr. 7-S1: (MeGlcA³MeGlc²Xyl₃), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-[(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-D-Xyl

Fr. 4-S2 was a new acidic oligosaccharide. The distribution pattern of these vicinal uronic acid units along the D-xylan chain was discussed.     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

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.     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

A biotin-hydrogel-coated quartz crystal microbalance biosensor and applications in immunoassay and peptide-displaying cell detection

A biotin-coated quartz crystal microbalance (QCM) chip was prepared by dip-coating a long-chain alkanethiol-modified crystal with precoupled dextran-biotin hydrogels. The resulting biotin chip was used to affinity-immobilize streptavidin (SAv) and was then further employed for various biosensor assays. First, the SAv chip allowed efficient on-line binding of biotinylated bovine serum albumin (bBSA), followed by a sensitive and specific response toward anti-bovine serum albumin (BSA) antibodies. Three consecutive immunoassays were reproducibly demonstrated with a single chip. The apparent binding kinetics with k_on = 5.9 μM⁻¹ h⁻¹, k_off = 10.1 h⁻¹, and K_D = 1.71 μM was readily resolved by fitting the real-time sensorgrams. Second, the capability of the SAv chip to selectively recognize recombinant Escherichia coli with flagella displaying an artificial SAv binding peptide, Strep-tag II, was demonstrated by QCM analysis and verified by scanning transmission electron microscope (STEM) image analysis with biotin-coated gold nanoparticles as the label. Finally, the affinity of the cell-displayed Strep-tag II peptide to surface-coated SAv, K_D = 6.8 × 10⁸ CFU/ml, was resolved on-line using equilibrium binding kinetics by QCM. This study presents an easy, economical, and reliable method of preparing high-performance SAv-coated biotin chips with potential for application in real-time repetitive immunoassays, on-line binding kinetics studies, and high-affinity peptide screening.     

Triterpenoid saponins from a cytotoxic root extract of Sideroxylonfoetidissimum subsp. gaumeri

Evaluation of the cytotoxicity of an ethanolic root extract of Sideroxylonfoetidissimum subsp. gaumeri (Sapotaceae) revealed activity against the murine macrophage-like cell line RAW 264.7. Systematic bioassay-guided fractionation of this extract gave an active saponin-containing fraction from which four saponins were isolated. Use of 1D (¹H, ¹³C, DEPT135) and 2D (COSY, TOCSY, HSQC, and HMBC) NMR, mass spectrometry and sugar analysis gave their structures as 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, and the known compound, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-protobassic acid. Two further saponins were obtained from the same fraction, but as a 5:4 mixture comprising 3-O-(β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid and 3-O-(β-d-apiofuranosyl-(1 → 3)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, respectively. This showed greater cytotoxicity (IC₅₀ = 11.9 ± 1.5 μg/ml) towards RAW 264.7 cells than the original extract (IC₅₀ = 39.5 ± 4.1 μg/ml), and the saponin-containing fraction derived from it (IC₅₀ = 33.7 ± 6.2 μg/ml).     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

Diffusion of Exchangeable Water in Cortical Bone Studied by Nuclear Magnetic Resonance

The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D_a = (7.8 ± 1.5) × 10⁻⁷ cm²/s) measured at 40°C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D_a measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D_a perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (∼30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase.     

Concentration dependent effects of dextran on the physical properties of acid milk gels

The effect of dextran from Leuconostoc mesenteroides (DEX₅₀₀), added to milk prior to acidification with glucono-δ-lactone (GDL) or Streptococcus thermophilus DSM20259, was studied with respect to polysaccharide concentration. The incorporation of 5–30 g/kg DEX₅₀₀ significantly affected gelation behavior. Increasing DEX₅₀₀ concentrations resulted in a linear increase of gel stiffness (GDL gels: R² = 0.96; microbial acidification: R² = 0.94; P < 0.05) and 30 g/kg DEX₅₀₀ resulted in a 2-fold higher stiffness compared to gels without polysaccharide. The respective stirred gels depicted a significant reduction in syneresis, which decreased from 30.4% (0 g/kg DEX₅₀₀) to 22.0% (30 g/kg DEX₅₀₀) for chemically acidified gels after 1 d of storage. Physical characteristics of DEX₅₀₀ in aqueous solution were helpful to explain its behavior in the complex system milk.     

Potential respiration is a better respiratory predictor than biomass in young Artemia salina

These experiments test whether respiration can be predicted better from biomass or from potential respiration, a measurement of the mitochondrial and microsomal respiratory electron transport systems. For nearly a century Kleiber's law or a similar precursor have argued the importance of biomass in predicting respiration. In the last decade, a version of the Metabolic Theory of Ecology has elaborated on Kleiber's Law adding emphasis to the importance of biomass in predicting respiration. We argue that Kleiber's law works because biomass packages mitochondria and microsomal electron transport complexes. On a scale of five orders of magnitude we have shown previously that potential respiration predicts respiration as well as biomass in marine zooplankton. Here, using cultures of the branchiopod, Artemia salina and on a scale of less than 2 orders of magnitude, we investigated the power of biomass and potential respiration in predicting respiration. We measured biomass, respiration and potential respiration in Artemia grown in different ways and found that potential respiration (Ф) could predict respiration (R), both in µlO₂ h⁻¹ (R = 0.924Φ + 0.062, r² = 0.976), but biomass (as mg dry mass) could not (R = 27.02DM + 8.857, r² = 0.128). Furthermore the R/Ф ratio appeared independent of age and differences in the food source.     

Cytotoxic triterpenoid saponins from the stem bark of Antonia ovata

Phytochemical investigation of the MeOH extract of the stem bark of Antonia ovata led to the isolation of four triterpenoid saponins, along with eleven known compounds. Their structures were established by extensive 1D and 2D NMR, as well as HR-MS analysis and acid hydrolysis. All isolated saponins contained the same tetrasaccharide chain O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-glucopyranosyl-(1 → 2)]-β-d-glucuropyranoside linked to C-3 of esterified derivatives of R₁-barrigenol, A₁-barrigenol, barringtogenol C, or camelliagenin. Biological evaluation of the compounds against KB cell line revealed a potent cytotoxic activity with IC₅₀ values ranging from 3.1 to 6.6 μM. The known compounds were found to be inactive at 10 μg/ml concentration.