Neokestose suppresses the growth of human melanoma A2058 cells via inhibition of the nuclear factor­κB signaling pathway.

Neokestose has superior prebiotic effects compared with the commercial fructooligosaccharides (FOS). In addition, the branched structure of neokestose, a type of neo­FOS, confers improved chemical stability compared with conventional FOS; therefore, the investigation of the branched structure by the present study may be of high biomedical value. The present study aimed to determine whether neokestose may suppress growth of the A2058 melanoma cell line. The cells were initially treated with neokestose; subsequently, in vitro cytotoxicity was assessed using MTT, and cell cycle progression and apoptosis were detected using flow cytometry. The protein expression levels of cyclin D1, phosphorylated (p)­inhibitor of κB (IκB) and nuclear factor­κB (NF­κB) were determined using western blotting. Treatment with neokestose led to a dose­dependent inhibition of cell viability. Flow cytometry data indicated that neokestose increased the sub­G1 cell population, and induced early and late apoptosis. Western blot analysis revealed that neokestose treatment reduced the expression levels of p­IκB and cyclin D1. These findings suggest that neokestose treatment may induce suppression of A2058 melanoma cell viability via inhibition of the NF­κB pathway. The present findings support the requirement for further investigation into the potential use of neokestose as an additional or chemopreventive therapeutic agent for the treatment of melanoma.

Antineoplastic effect of a novel chemopreventive agent, neokestose, on the Caco-2 cell line via inhibition of expression of nuclear factor-κB and cyclooxygenase-2.

Neokestose is a 6G-fructooligosaccharide (FOS) and an important prebiotic. When FOS are ingested by patients with colorectal cancer, they may come into contact with cancer cells prior to being fermented by bifidobacteria in the colon. In the present study, the effects of neokestose on cell proliferation, cell cycle and apoptosis of the colorectal cancer cell line Caco-2 were investigated to evaluate its anti-cancer effect. An MTT assay showed that neokestose-treated Caco-2 cells exhibited a significant and dose-dependent loss of viability. Flow cytometric analysis indicated that the sub-G1 population of Caco-2 cells was significantly increased following treatment with neokestose, and the percentage of Caco-2 cells in the stage of late apoptosis was also significantly increased in a dose-dependent manner. Western blot analysis showed that the overexpression of nuclear factor-κB, a central molecule responsible for the transition from inflammation to cancer, and cyclooxygenase-2, an important enzyme in colorectal tumorigenesis, in colorectal carcinoma cells was inhibited by neokestose. Accordingly, the present study provided in vitro evidence that neokestose may be used as a dietary chemopreventive agent, whose application is more rational than that of COX-2 inhibitors or aspirin for preventing colorectal cancer.

Molecular structural property and potential energy dependence on nonequilibrium-thermodynamic state point of liquid n-hexadecane under shear.

Extensive computer experiments have been conducted in order to shed light on the macroscopic shear flow behavior of liquid n-hexadecane fluid under isobaric-isothermal conditions through the nonequilibrium molecular dynamic methodology. With respect to shear rates, the accompanying variations in structural properties of the fluid span the microscopic range of understanding from the intrinsic to extrinsic characteristics. As drawn from the average value of bond length and bond angle, the distribution of dihedral angle, and the radius distribution function of intramolecular and intermolecular van der Waals distances, these intrinsic structures change with hardness, except in the situation of extreme shear rates. The shear-induced variation of thermodynamic state curve along with the shear rate studied is shown to consist of both the quasiequilibrium state plateau and the nonequilibrium-thermodynamic state slope. Significantly, the occurrence of nonequilibrium-thermodynamic state behavior is attributed to variations in molecular potential energies, which include bond stretching, bond bending, bond torsion, and intra- and intermolecular van der Waals interactions. To unfold the physical representation of extrinsic structural deformation, under the aggressive influence of a shear flow field, the molecular dimension and appearance can be directly described via the squared radius of gyration and the sphericity angle, R(g)(2) and ϕ, respectively. In addition, a specific orientational order S(x) defines the alignment of the molecules with the flow direction of the x-axis. As a result, at low shear rates, the overall molecules are slightly stretched and shaped in a manner that is increasingly ellipsoidal. Simultaneously, there is an obvious enhancement in the order. In contrast to high shear rates, the molecules spontaneously shrink themselves with a decreased value of R(g)(2), while their shape and order barely vary with an infinite value of ϕ and S(x). It is important to note that under different temperatures and pressures, these three parameters are integrated within a molecular description in response to thermodynamic state variable of density and rheological material function of shear viscosity.

Increasing hybridoma viability and antibody repertoire after the cell fusion by the use of human plasma as an alternative supplement.

Prion diseases such as Bovine Spongiform Encephalopathy (BSE) and new variant Creutzfeldt-Jakob disease (nvCJD) have caused a major safety concern in cell cultures using fetal calf serum (FCS). In this study, we found that screened and tested human plasma (HP) obtained from blood centers may be an ideal alternate nutrient substitute to FCS for culturing hybridoma. In addition to the inherent safety, a ten-fold increase in the fusion efficiency has been observed if the HP was used as the nutrient supplement instead of FCS. Subsequently, a broader antibody repertoire may be recovered. The HP supplement was found to promote the growth of hybridoma cells but no impact on antibody secretion. Interestingly, this effect of enrichment was only observed for HP, but not plasma from other animals. Unidentified murine hybridoma cloning factors other than IL-6 may specifically reside in human blood.

Linear viscoelasticity and thermorheological simplicity of n-hexadecane fluids under oscillatory shear via non-equilibrium molecular dynamics simulations.

A small amplitude oscillatory shear flows with the classic characteristic of a phase shift when using non-equilibrium molecular dynamics simulations for n-hexadecane fluids. In a suitable range of strain amplitude, the fluid possesses significant linear viscoelastic behavior. Non-linear viscoelastic behavior of strain thinning, which means the dynamic modulus monotonously decreased with increasing strain amplitudes, was found at extreme strain amplitudes. Under isobaric conditions, different temperatures strongly affected the range of linear viscoelasticity and the slope of strain thinning. The fluid's phase states, containing solid-, liquid-, and gel-like states, can be distinguished through a criterion of the viscoelastic spectrum. As a result, a particular condition for the viscoelastic behavior of n-hexadecane molecules approaching that of the Rouse chain was obtained. Besides, more importantly, evidence of thermorheologically simple materials was presented in which the relaxation modulus obeys the time-temperature superposition principle. Therefore, using shift factors from the time-temperature superposition principle, the estimated Arrhenius flow activation energy was in good agreement with related experimental values. Furthermore, one relaxation modulus master curve well exhibited both transition and terminal zones. Especially regarding non-equilibrium thermodynamic states, variations in the density, with respect to frequencies, were revealed.

Phytochelatin synthase is regulated by protein phosphorylation at a threonine residue near its catalytic site.

Heavy metals are toxic to most living organisms and cause health problems by contaminating agricultural products. In plants, phytochelatin synthase (PCS, EC 2.3.2.15) uses glutathione (GSH) as its substrate to catalyze the synthesis of heavy metal-binding peptides, known as phytochelatins (PC). PCS has been described as a constitutive enzyme that may be controlled by post-translational modifications. However, the detailed mechanism of its catalytic activity is not clear. In this study, in vitro experiments demonstrate that PCS activity increased following phosphorylation by casein kinase 2 (CK2) and decreased following treatment with alkaline phosphatase. Site-directed mutagenesis experiments at amino acids on AtPCS1 indicate that Thr 49 is the site for phosphorylation. This is further supported by fact that the mutant AtPCS1(T49A) cannot be phosphorylated, and its activity is significantly lower than that of the wild-type enzyme. In the modeled three-dimensional structure of AtPCS1, Arg 183 is within close proximity to Thr 49. The mutant AtPCS1(R183A) can be phosphorylated, but it shows much lower catalytic activity than the wild-type protein. This result suggested that Arg 183 may play an important role in the catalytic mechanism of AtPCS1. The possibility of the presence of a second substrate-binding site as a result of the interaction of these two amino acids is discussed. In addition, the activity of AtPCS1 was also found to be modulated by the C-terminal domain. The N-terminal catalytic domain of AtPCS1 was expressed (AtPCS1-N), and its catalytic activity was found to be even more sensitive to Cd or phosphorylation status than was the full-length enzyme.

Master curves and radial distribution functions for shear dilatancy of liquid n-hexadecane via nonequilibrium molecular dynamics simulations.

Shear dilatancy, a significant nonlinear behavior of nonequilibrium thermodynamics states, has been observed in nonequilibrium molecular dynamics (NEMD) simulations for liquid n-hexadecane fluid under extreme shear conditions. The existence of shear dilatancy is relevant to the relationship between the imposed shear rate gamma and the critical shear rate gamma(c). Consequently, as gammagamma(c), the intermolecular distance is lengthened substantially by strong shear deformation breaking the equilibrium thermodynamic state so that shear dilatancy takes place. Notably, a characteristic shear rate gamma(m), which depends on the root mean square molecular velocity and the average free molecular distance, is found in nonequilibrium thermodynamics state curves. Studies of the variations in the intermolecular radial distribution function (RDF) with respect to the shear rate provide a direct measure of the variation in the degree of intermolecular separation. Additionally, the variations of the RDF curve in the microscopic regime are consistent with those of the nonequilibrium thermodynamic state in the macroscopic world. By inspecting the overall shape of the RDF curve, it can be readily corroborated that the fluid of interest exists in the liquid state. More importantly, both primary characteristic values, the equilibrium thermodynamic state variable and a particular shear rate of gamma(p), are determined cautiously, with gamma(p) depending on the gamma(m) value and the square root of pressure. Thereby, the nonequilibrium thermodynamic state curves can be normalized as temperature-, pressure-, and density-invariant master curves, formulated by applying the Cross constitutive equation. Clearly, gamma(c) occurs at which a reduced shear rate gamma/gamma(p) approaches 0.1. Furthermore, the trends in the rates of shear dilatancy in both the constant-pressure and constant-volume NEMD systems under isothermal conditions conform to the cyclic rule of pressure, as a function of density and shear rate.

Material functions of liquid n-hexadecane under steady shear via nonequilibrium molecular dynamics simulations: temperature, pressure, and density effects.

Computer experiments of rheology regarding the effects of temperature (T), pressure (P), and density (rho) on steady shear flow material functions, which include viscosity (eta) and first and second normal stress coefficients (psi(1) and psi(2)) depending on shear rate (gamma), have been conducted via nonequilibrium molecular dynamics simulations for liquid n-hexadecane. Straightforwardly, using both characteristic values of a zero-shear-rate viscosity and critical shear rate, eta-gamma flow curves are well normalized to achieve the temperature-, pressure-, and density-invariant master curves, which can be formulary described by the Carreau-Yasuda rheological constitutive equation. Variations in the rate of shear thinning, obviously exhibiting in eta-gamma, psi(1)-gamma, and -psi(2)-gamma relationships, under different T, P, and rho values, are concretely revealed through the power-law model's exponent. More importantly, at low shear rates, the fluid explicitly possesses Newtonian fluidic characteristics according to both manifestations; first and second normal stress differences decay to near zero, while nonequilibrium states are close to equilibrium ones. Significantly, the tendency to vary of the degree of shear thinning in rheology is qualitatively contrary to that of shear dilatancy in thermodynamics. In addition, a convergent transition point is evidently observed in the -psi(2)/psi(1)-gamma curves undergoing dramatic variations, which should be associated with shear dilatancy, as addressed analytically.

Shear thinning and shear dilatancy of liquid n-hexadecane via equilibrium and nonequilibrium molecular dynamics simulations: Temperature, pressure, and density effects.

Equilibrium and nonequilibrium molecular dynamics (MD) simulations have been performed in both isochoric-isothermal (NVT) and isobaric-isothermal (NPT) ensemble systems. Under steady state shearing conditions, thermodynamic states and rheological properties of liquid n-hexadecane molecules have been studied. Between equilibrium and nonequilibrium states, it is important to understand how shear rates (gamma) affect the thermodynamic state variables of temperature, pressure, and density. At lower shear rates of gamma<1 x 10(11) s(-1), the relationships between the thermodynamic variables at nonequilibrium states closely approximate those at equilibrium states, namely, the liquid is very near its Newtonian fluid regime. Conversely, at extreme shear rates of gamma>1 x 10(11) s(-1), specific behavior of shear dilatancy is observed in the variations of nonequilibrium thermodynamic states. Significantly, by analyzing the effects of changes in temperature, pressure, and density on shear flow system, we report a variety of rheological properties including the shear thinning relationship between viscosity and shear rate, zero-shear-rate viscosity, rotational relaxation time, and critical shear rate. In addition, the flow activation energy and the pressure-viscosity coefficient determined through Arrhenius and Barus equations acceptably agree with the related experimental and MD simulation results.

Characterization of the high molecular weight Cd-binding complex in water hyacinth (Eichhornia crassipes) when exposed to Cd.

Water hyacinth ( Eichhornia crassipes) is a rapid-growing freshwater vascular plant that has been used to remove heavy metals in contaminated water. But the transportation and distribution of the absorbed heavy metal in the plant are not clear. In this study, water hyacinth was exposed to cadmium (Cd, 10 microM, pulse) and then transferred to a Cd-free solution (chase). The Cd content in the tissues was measured, and the Cd-binding complexes were isolated and identified. We found that (1) in two days, up to 80% of the Cd in the solution was absorbed by the plant, and the Cd could not be released back to the growth solution in the chase period; (2) approximately 1 mg of Cd was accumulated in the water hyacinth/g of dry weight in this condition; (3) invading Cd was bound rapidly as the low-molecular-weight (LMW) complex serving as the transient form for further sequestration; (4) the LMW complex in water hyacinth contained no phytochelatins and was different from the LMW complex in fission yeast; (5) the Cd absorbed in the plant was essentially stored in the high-molecular-weight (HMW) form after 1 week; (6) a small fraction of the absorbed Cd was found in the upper part of the plant (stem and leaves) in the form of complexes; (7) the HMW complex was composed of phytochelatins PC 3 and PC 4 primarily, with only a small amount of PC 2; (8) a rare PC-related peptide was found in the HMW complex that might be derived from PC 3. These observations contribute to the further understanding of water hyacinth in serving as an efficient and reliable accumulator for heavy metals.

Preparation of monoclonal antibody bank against whole water-soluble proteins from rapid-growing bamboo shoots.

An antibody bank against the whole proteins in a proteome is a useful tool for biological research. Using the standard cell fusion method, and a modified screening protocol, we produced an mAb bank against the total water-soluble proteins extracted from the rapid-growing green bamboo shoots. An improved two-stage strategy was employed to enrich those poor immunogenic or lower expressed proteins. Totally, we obtained a bank of 192 mAb which were identified as distinctive to each other by 2-DE and immunostaining.

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots.

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters.

Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis.

Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the alpha-glucan in plant cells. When compared to its isoform in an animal cell, glycogen phosphorylase, a peptide containing 78 amino acids (L78) is inserted in the centre of the low-affinity type starch phosphorylase (L-SP). We found that the amino acid sequence of L78 had several interesting features including the presence of a PEST region, which serves as a signal for rapid degradation. Indeed, most L-SP molecules isolated from mature sweet potato roots were nicked in the middle of a molecule, but still retained their tertiary or quaternary structures, as well as full catalytic activity. The nicking sites on the L78 were identified by amino acid sequencing of these peptides, which also enabled us to propose a proteolytic process for L-SP. Enzyme kinetic studies of L-SP in the direction of starch synthesis indicated that the Km decreased during the proteolytic process when starch was used as the limiting substrate, but the Km for the other substrate (Glc-1-P) increased. On the other hand, the maximum velocities (Vmax) increased for both substrates. Mobility of the nicked L-SP was retarded on a native polyacrylamide gel containing soluble starch, indicating the increased affinity for starch. Results in this study suggested that L78 and its proteolytic modifications might play a regulatory role on the catalytic behaviour of L-SP in starch biosynthesis.