Mechanical properties of native and cross-linked type I collagen fibrils

Micromechanical bending experiments using atomic force microscopy were performed to study the mechanical properties of native and carbodiimide-cross-linked single collagen fibrils. Fibrils obtained from a suspension of insoluble collagen type I isolated from bovine Achilles tendon were deposited on a glass substrate containing microchannels. Force-displacement curves recorded at multiple positions along the collagen fibril were used to assess the bending modulus. By fitting the slope of the force-displacement curves recorded at ambient conditions to a model describing the bending of a rod, bending moduli ranging from 1.0 GPa to 3.9 GPa were determined. From a model for anisotropic materials, the shear modulus of the fibril is calculated to be 33 ± 2 MPa at ambient conditions. When fibrils are immersed in phosphate-buffered saline, their bending and shear modulus decrease to 0.07-0.17 GPa and 2.9 ± 0.3 MPa, respectively. The two orders of magnitude lower shear modulus compared with the Young's modulus confirms the mechanical anisotropy of the collagen single fibrils. Cross-linking the collagen fibrils with a water-soluble carbodiimide did not significantly affect the bending modulus. The shear modulus of these fibrils, however, changed to 74 ± 7 MPa at ambient conditions and to 3.4 ± 0.2 MPa in phosphate-buffered saline.     

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).     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Substrate modulus directs neural stem cell behavior

Although biochemical signals that modulate stem cell self-renewal and differentiation were extensively studied, only recently were the mechanical properties of a stem cell's microenvironment shown to regulate its behavior. It would be desirable to have independent control over biochemical and mechanical cues, to analyze their relative and combined effects on stem-cell function. We developed a synthetic, interfacial hydrogel culture system, termed variable moduli interpenetrating polymer networks (vmIPNs), to assess the effects of soluble signals, adhesion ligand presentation, and material moduli from 10-10,000 Pa on adult neural stem-cell (aNSC) behavior. The aNSCs proliferated when cultured in serum-free growth media on peptide-modified vmIPNs with moduli of ≥100 Pa. In serum-free neuronal differentiation media, a peak level of the neuronal marker, β-tubulin III, was observed on vmIPNs of 500 Pa, near the physiological stiffness of brain tissue. Furthermore, under mixed differentiation conditions with serum, softer gels (∼100-500 Pa) greatly favored neurons, whereas harder gels (∼1,000-10,000 Pa) promoted glial cultures. In contrast, cell spreading, self-renewal, and differentiation were inhibited on substrata with moduli of ∼10 Pa. This work demonstrates that the mechanical and biochemical properties of an aNSC microenvironment can be tuned to regulate the self-renewal and differentiation of aNSCs.     

A sensitive liquid chromatography–electrospray tandem mass spectrometric method for lancemaside A and its metabolites in plasma and a pharmacokinetic study in mice

A high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for simultaneous determination of lancemaside A (3-O-β-d-glucuronopyranosyl-3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) and its metabolites in mouse plasma. When lancemaside A (60 mg/kg) was orally administered to mice, echinocystic acid was detected in the blood. T_max and C_max of the echinocystic acid were 6.5 ± 1.9 h and 56.7 ± 29.1 ppb. Orally administered lancemaside A was metabolized to lancemaside X (3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) by intestinal microflora in mice, which was metabolized to echinocystic acid by intestinal microflora and/or intestinal tissues. Human intestinal microflora also metabolized lancemaside A to echinocystic acid via lancemaside X. These results suggest that the metabolism by intestinal microflora may play an important role in pharmacological effects of orally administered lancemaside A.     

Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains

Cuticle tanning in insects involves simultaneous cuticular pigmentation and hardening or sclerotization. The dynamic mechanical properties of the highly modified and cuticle-rich forewings (elytra) from Tribolium castaneum (red flour beetle) wild-type and body color mutant strains were investigated to relate body coloration and elytral mechanical properties. There was no statistically significant variation in the storage modulus E′ among the elytra from jet, cola, sooty and black mutants or between the mutants and the wild-type GA-1 strain: E′ averaged 5.1 ± 0.6 GPa regardless of body color. E′ is a power law function of oscillation frequency for all types. The power law exponent, n, averaged 0.032 ± 0.001 for elytra from all genotypes except black; this small value indicated that the elytra are cross-linked. Black elytra, however, displayed a significantly larger n of 0.047 ± 0.004 and an increased loss tangent (tan δ), suggesting that metabolic differences in the black mutant strain result in elytra that are less cross-linked and more pigmented than the other types. These results are consistent with the hypothesis that black elytra have a β-alanine-deficient and dopamine-abundant metabolism, leading to greater melanin (black pigment) production, probably at the expense of cross-linking of cuticular proteins mediated by N-β-alanyldopamine quinone.     

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k_c, from 23.1 ± 3.5 to 10.6 ± 4.5 k_BT) and the apparent area compressibility modulus (K_app, from 176 ± 35 to 113 ± 25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N terminus to create the Hsp90α(NT) column or via the C terminus to create the Hsp90α(CT) column. Binding to the exposed C terminus on the Hsp90α(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A₁ (CA1) and novobiocin (NOVO). The calculated K_d values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Nonlinear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex: 22.2 ± 8.8 μM⁻¹ s⁻¹ and 2.7 ± 0.6 s⁻¹, respectively. Binding to the exposed N terminus on the Hsp90α(CT) column was characterized using frontal chromatography. The K_d values of the N-terminus ligands geldanamycin (GM, 90 ± 50 nM), 17-allylamino-17-demethoxygeldanamycin (17-AAG, 210 ± 50 nM), and radicicol (RAD, 20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC₅₀ values for inhibition of ATPase activity on the Hsp90α(CT) column. The IC₅₀ for GM was 2.80 ± 0.18 μM, and the relative IC₅₀ values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition.     

Spectrophotometric determination of formation constants for the Cu-ethylenediamine-halogen (chloride and bromide) system and their catalytic effect on the oxidative coupling of 2,6-di-tert-butyl-phenol

In order to explain the mechanism of the dimerization of 2,6-di-tert-butyl-phenol when catalyzed by the copper-ethylenediamine complexes, a spectrophotometric study of the speciation of copper(II) complexes in methanol of Cu(II), ethylendiamine and Cl⁻ or Br⁻ was carried out at 303 K. The formation constants obtained for the copper chloride system are: log β₁₀₁ = 2.90 ± 0.03, log β₁₀₂ = 6.39 ± 0.03 and log β₁₀₃ = 8.62 ± 0.04, for the copper bromide system are log β₁₀₁ = 3.01 ± 0.10, log β₁₀₂ = 5.50 ± 0.08, for the copper-ethylendiamine complexes are log β₁₁₀ = 6.13 ± 0.05 and log β₁₂₀ = 10.54 ± 0.08, and for the ternary copper-ethylenediamine chloride or bromide systems are log β₁₁₁ = 10.21 ± 0.03 and log β₁₁₁ = 10.07 ± 0.03, respectively. Knowing the speciation of the copper-ethylenediamine-halide systems, the kinetic studies can be correlated with the species in solution. Comparative studies of the oxidation reaction of 2,6-di-tert-butyl-phenol using different copper(II) complexes with chloride or bromide and ethylenediamine as catalyst are reported. Their catalytic activity in the oxidation of 2,6-di-tert-butyl-phenol was monitored in methanol solution, following the corresponding quinone formation, at 418 nm (ε = 3.95 × 10⁴ mol⁻¹ L cm⁻¹ at 303 K). The results indicate that the most active species are [Cu(en)X]⁺, where X is bromide or chloride, Both complexes have similar activity.     

Systematic multiscale parameterization of heterogeneous elastic network models of proteins

We present a method to parameterize heterogeneous elastic network models (heteroENMs) of proteins to reproduce the fluctuations observed in atomistic simulations. Because it is based on atomistic simulation, our method allows the development of elastic coarse-grained models of proteins under different conditions or in different environments. The method is simple and applicable to models at any level of coarse-graining. We validated the method in three systems. First, we computed the persistence length of ADP-bound F-actin, using a heteroENM model. The value of 6.1 ± 1.6 μm is consistent with the experimentally measured value of 9.0 ± 0.5 μm. We then compared our method to a uniform elastic network model and a realistic extension algorithm via covariance Hessian (REACH) model of carboxy myoglobin, and found that the heteroENM method more accurately predicted mean-square fluctuations of α-carbon atoms. Finally, we showed that the method captures critical differences in effective harmonic interactions for coarse-grained models of the N-terminal Bin/amphiphysin/Rvs (N-BAR) domain of amphiphysin, by building models of N-BAR both bound to a membrane and free in solution.     

Mucus secretion and cytoskeletal modifications in cultured nasal epithelial cells exposed to wall shear stresses

The nasal epithelium is continuously subjected to wall shear stresses (WSS) induced by respiratory airflows. An in vitro experimental model was developed to expose nasal epithelial cells cultured under air-liquid interface conditions to steady airflow-induced WSS. Mucus secretion from epithelial goblet cells was quantified using an enzyme-linked lectinosorbent assay, and modifications of the cytoskeletal structure were qualitatively evaluated from fluorescent stains of actin and β-tubulin fibers. The results show increased mucus secretion from cells subjected to WSS of 0.1 and 1.0 dyne/cm² for more than 15 min in comparison with unstressed cells. The integrity levels of β-tubulin fibers were significantly lower in cells subjected to WSS than in unstressed cells. The increased mucus secretion in response to WSS was approximately the same in Taxol-free and Taxol-treated cultures, which indicates that there is no direct connection between β-tubulin fragmentation and mucus secretion. The stressed cells regained their normal cytoskeletal appearance 24 h after the exposure to WSS. The results of this study suggest that WSS have an important role in the mechanical regulation of the nasal surface epithelium function.     

Elasticity, strength, and water permeability of bilayers that contain raft microdomain-forming lipids

Bilayers composed of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (CHOL) are commonly used as systems to model the raft-lipid domain structure believed to compartmentalize particular cell membrane proteins. In this work, micropipette aspiration of giant unilamellar vesicles was used to test the elasticities, water permeabilities, and rupture tensions of single-component PC, binary 1:1 PC/CHOL, and 1:1 SM/CHOL, and ternary 1:1:1 PC/SM/CHOL bilayers, one set of measurements with dioleoyl PC (DOPC; C18:1/C18:1 PC) and the other with stearoyloleoyl PC (SOPC; C18:0/C18:1 PC). Defining the elastic moduli (K_A), the initial slopes of the increase in tension (σ) versus stretch in lipid surface area (α_e) were determined for all systems at low (15°C) and high (32-33°C) temperatures. The moduli for the single-component PC and binary phospholipid/CHOL bilayers followed a descending hierarchy of stretch resistance with SM/CHOL > SOPC/CHOL > DOPC/CHOL > PC. Although much more resistant to stretch than the single-component PC bilayers, the elastic response of vesicle bilayers made from the ternary phospholipid/CHOL mixtures showed an abrupt softening (discontinuity in slope), when immediately subjected to a steady ramp of tension at the low temperature (15°C). However, the discontinuities in elastic stretch resistance at low temperature vanished when the bilayers were held at ∼1 mN/m prestress for long times before a tension ramp and when tested at the higher temperature 32-33°C. The elastic moduli of single-component PC and DOPC/CHOL bilayers changed very little with temperature, whereas the moduli of the binary SOPC/CHOL and SM/CHOL bilayers diminished markedly with increase in temperature, as did the ternary SOPC/SM/CHOL system. For all systems, increasing temperature increased the water permeability but decreased rupture tension. Concomitantly, the measurements of permeability exhibited a prominent correlation with the rupture tension across all the systems. Together, these micromechanical tests of binary and ternary phospholipid/CHOL bilayers demonstrate that PC hydrocarbon chain unsaturation and temperature are major determinants of the mechanical and permeation properties of membranes composed of raft microdomain-forming lipids.     

Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects

Background

α-Klotho (αKl) regulates mineral metabolism such as calcium ion (Ca²⁺) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type αKl has been demonstrated, less is known regarding the physiological importance of soluble-type αKl (sαKl) in circulation.

Objectives

The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum sαKl, and (2) to determine reference values for sαKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects.

Results

We successively developed an ELISA to measure serum sαKl in healthy volunteers (n = 142, males 66) of ages (61.1 ± 18.5 year). The levels (mean ± SD) in these healthy control adults were as follows: total calcium (Ca; 9.46 ± 0.41 mg/dL), Pi (3.63 ± 0.51 mg/dL), blood urea nitrogen (BUN; 15.7 ± 4.3 mg/dL), creatinine (Cre; 0.69 ± 0.14 mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)₂D; 54.8 ± 17.7 pg/mL), intact parathyroid hormone (iPTH; 49.2 ± 20.6 pg/mL), calcitonin (26.0 ± 12.3 pg/mL) and intact fibroblast growth factor (FGF23; 43.8 ± 17.6 pg/mL).Serum levels of sαKl ranged from 239 to 1266 pg/mL (mean ± SD; 562 ± 146 pg/mL) in normal adults. Although sαKl levels were not modified by gender or indices of mineral metabolism, sαKl levels were inversely related to Cre and age. However, sαKl levels in normal children (n = 39, males 23, mean ± SD; 7.1 ± 4.8 years) were significantly higher (mean ± SD; 952 ± 282 pg/mL) than those in adults (mean ± SD; 562 ± 146, P < 0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that sαKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum sαKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of α-klotho gene. In this patient, sαKl level was notably lower than those of age-matched controls.

Conclusion

We established a detection system to measure human serum sαKl for the first time. Age, Ca and Pi seem to influence serum sαKl levels in a normal population. This detection system should be an excellent tool for investigating sαKl functions in mineral metabolism.     

Steroidal saponins from the leaves of Beaucarnea recurvata

Thirteen steroidal saponins were isolated from the leaves of Beaucarnea recurvata Lem. Their structures were established using one- and two-dimensional NMR spectroscopy and mass spectrometry. Six of them were identified as: 26-O-β-d-glucopyranosyl (25S)-furosta-5,20(22)-diene 1β,3β,26-triol 1-O-α-l-rhamnopyranosyl-(1 → 2) β-d-fucopyranoside, 26-O-β-d-glucopyranosyl (25S)-furosta-5,20(22)-diene 1β,3β,26-triol 1-O-α-l-rhamnopyranosyl-(1 → 2)-4-O-acetyl-β-d-fucopyranoside, 26-O-β-d-glucopyranosyl (25R)-furosta-5,20(22)-diene-23-one-1β,3β,26-triol 1-O-α-l-rhamnopyranosyl-(1 → 2) β-d-fucopyranoside, 26-O-β-d-glucopyranosyl (25S)-furosta-5-ene-1β,3β,22α,26-tetrol 1-O-α-l-rhamnopyranosyl-(1 → 4)-6-O-acetyl-β-d-glucopyranoside, 26-O-β-d-glucopyranosyl (25S)-furosta-5-ene-1β,3β,22α,26-tetrol 1-O-α-l-rhamnopyranosyl-(1 → 2) β-d-fucopyranoside, and 24-O-β-d-glucopyranosyl (25R)-spirost-5-ene-1β,3β,24-triol 1-O-α-l-rhamnopyranosyl-(1 → 2)-4-O-acetyl-β-d-fucopyranoside. The chemotaxonomic classification of B. recurvata in the family Ruscaceae was discussed.     

Complexation study of europium(III) and curium(III) with urea in aqueous solution investigated by time-resolved laser-induced fluorescence spectroscopy

The complex formation of europium(III) and curium(III) with urea in aqueous solution has been studied at I = 0.1 M (NaClO₄), room temperature and trace metal concentrations in the pH-range of 1-8 at various ligand concentrations using time-resolved laser-fluorescence spectroscopy. While for curium(III) the luminescence maximum is red shifted upon complexation, in case of europium(III) emission wavelengths remain unaltered but a significant change in peak splitting occurs. Both heavy metals form weak complexes of the formulae ML³⁺ and MLOH²⁺ with urea. Stability constants were determined to be log β₁₁₀ = −0.12 ± 0.05 and log β_11-1 = −6.86 ± 0.15 for europium(III) and log β₁₁₀ = −0.28 ± 0.12 and log β_11-1 = −7.01 ± 0.15 for curium(III).     

Changes in the hyperelastic properties of endothelial cells induced by tumor necrosis factor-alpha

Mechanical properties of living cells can be determined using atomic force microscopy (AFM). In this study, a novel analysis was developed to determine the mechanical properties of adherent monolayers of pulmonary microvascular endothelial cells (ECs) using AFM and finite element modeling, which considers both the finite thickness of ECs and their nonlinear elastic properties, as well as the large strain induced by AFM. Comparison of this model with the more traditional Hertzian model, which assumes linear elastic behavior, small strains, and infinite cell thickness, suggests that the new analysis can predict the mechanical response of ECs during AFM indentation better than Hertz's model, especially when using force-displacement data obtained from large indentations (>100 nm). The shear moduli and distensibility of ECs were greater when using small indentations (<100 nm) compared to large indentations (>100 nm). Tumor necrosis factor-α induced changes in the mechanical properties of ECs, which included a decrease in the average shear moduli that occurred in all regions of the ECs and an increase in distensibility in the central regions when measured using small indentations. These changes can be modeled as changes in a chain network structure within the ECs.     

Triterpenoidal saponins of Pulsatilla koreana roots

Sixteen (1-16) triterpenoidal saponins were isolated from the roots of Pulsatilla koreana, of which four were determined as the previously unknown 23-hydroxy-3β-[(O-α-L-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid 28-O-β-D-glucopyranosyl ester (1), 23-hydroxy-3β-[(O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid 28-O-β-D-glucopyranosyl ester (2), 3β-[(O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid 28-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl ester (3), and 3β-[(O-α-L-rhamnopyranosyl-(1 → 2)-O-[β-D-glucopyranosyl-(1 → 4)]-α-L-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1 → 4)-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl ester (4), respectively, based on spectroscopic analysis. The inhibition of the lipopolysaccharide-induced nitric oxide production of sixteen isolated compounds was evaluated in RAW 264.7 cells at concentrations ranging from 1 μM to 100 μM.     

Mechanical behavior of bovine nasal cartilage under static and dynamic loading

Abnormal mechanical loading may trigger cartilage degeneration associated with osteoarthritis. Tissue response to load has been the subject of several in vitro studies. However, simple stimuli were often applied, not fully mimicking the complex in vivo conditions. Therefore, a rolling/plowing explant test system (RPETS) was developed to replicate the combined in vivo loading patterns. In this work we investigated the mechanical behavior of bovine nasal septum (BNS) cartilage, selected as tissue approximation for experiments with RPETS, under static and dynamic loading. Biphasic material properties were determined and compared with those of other cartilaginous tissues. Furthermore, dynamic loading in plowing modality was performed to determine dynamic response and experimental results were compared with analytical models and Finite Elements (FE) computations. Results showed that BNS cartilage can be modeled as a biphasic material with Young's modulus E=2.03±0.7 MPa, aggregate modulus H_A=2.35±0.7 MPa, Poisson's ratio ν=0.24±0.07, and constant hydraulic permeability k₀=3.0±1.3×10⁻¹⁵ m⁴ (N s)⁻¹. Furthermore, dynamic analysis showed that plowing induces macroscopic reactions in the tissue, proportionally to the applied loading force. The comparison among analytical, FE analysis and experimental results showed that predicted tangential forces and sample deformation lay in the range of variation of experimental results for one specific experimental condition. In conclusion, mechanical properties of BNS cartilage under both static and dynamic compression were assessed, showing that this tissue behave as a biphasic material and has a viscoelastic response to dynamic forces.