Characterization of the molecular properties and allergenicity (IgE-binding capacity) of ß-lactoglobulin by heat treatment using asymmetric-flow field-flow fractionation and ultra-performance liquid chromatography quadrupole time of flight mass chromatography.

In this study, the heat product (90 °C, 10 min) of ß-lactoglobulin (ß-LG) was analyzed by asymmetric-flow field-flow fractionation (AF4) to observe the effect of heat treatment. The changes in molar mass (M) and molar size induced by heat treatment were characterized by AF4, and changes in molar shape were observed by transmission electron microscopy (TEM). The results showed that ß-LG dissociated and aggregated into four fractions with different M values, sizes, and shapes after heat treatment. The vast aggregations with the highest allergenicity (IgE-binding capacity) might enhance the allergenicity of ß-LG. However, the number of characterized epitope peptides was decreased due to heat treatment. The above results provide some references for related studies of ß-LG and its allergenicity. Further separation and characterization of the high-allergenicity fractions and peptides will help to eliminate allergens in dairy products and reduce the occurrence of allergic reactions.

Preparation and In Vitro Evaluation of a MRI Contrast Agent Based on Aptamer-Modified Gadolinium-Loaded Liposomes for Tumor Targeting.

The aim of this study was to prepare aptamer-modified liposomes loaded with gadolinium (Gd) to enhance the effective diagnosis for tumor by MRI. A modified GBI-10 (GBI-10m) was used to prepare targeted liposomes (GmLs). Liposomes with GBI-10 aptamer (GLs) and without aptamer (non-targeted liposomes (NLs)) were also prepared as controls. The particle size and zeta potential of GmLs, GLs, and NLs were all assayed. A clinical 3.0 T MR scanner was employed to assess the imaging efficiency and measure the longitudinal relaxivity (r 1) of the above liposomes. Confocal laser scanning microscopy and flow cytometry were used to analyze and compare the targeting effects of GmLs, GLs, and NLs to MDA-MB-435s cells at 37°C. The particle size of the prepared liposomes was scattered at 100-200 nm, and their values of r 1 were ∼4 mM-1 s-1. The images of confocal laser scanning microscopy showed that GmLs in the cytoplasm were significantly more than GLs and both GmLs and GLs were more than NLs. The fluorescence intensity of GmLs was increased by about two times than that of GLs and three times than that of NLs by flow cytometry. Therefore, the GmLs in this initial study were suggested to be a potential MRI contrast agent at 37°C for diagnosing tumors with the protein of tenascin-C over-expressed.

Effects of comprehensive function of factors on retention behavior of microparticles in gravitational field-flow fractionation.

Gravitational field-flow fractionation (GrFFF) is a useful technique for separation and characterization for micrometer-sized particles. Elution behavior of micrometer-sized particles in GrFFF was researched in this study. Particles in GrFFF channel are subject to hydrodynamic lift forces (HLF), fluid inertial forces and gravity, which drive them to different velocities by carrier flow, resulting in a size-based separation. Effects of ionic strength, flow rate and viscosity as well as methanol were investigated using polystyrene latex beads as model particles. This study is devoted to experimental verification of the effect of every factor and their comprehensive function. All experiments were performed to show isolated influence of every variable factor. The orthogonal design test was used to evaluate various factors comprehensively. Results suggested that retention ratio of particles increases with increasing flow rate or the viscosity of carrier liquid by adjusting external forces acting on particles. In addition, retention ratio increases as ionic strength decreases because of decreased electrostatic repulsion between particles and channel accumulation wall. As far as methanol, there is no general trend due to the change of both density and viscosity. On the basis of orthogonal design test it was found that viscosity of carrier liquid plays a significant role in determining resolution of micrometer-sized particles in GrFFF.

In vitro study of novel gadolinium-loaded liposomes guided by GBI-10 aptamer for promising tumor targeting and tumor diagnosis by magnetic resonance imaging.

Novel gadolinium-loaded liposomes guided by GBI-10 aptamer were developed and evaluated in vitro to enhance magnetic resonance imaging (MRI) diagnosis of tumor. Nontargeted gadolinium-loaded liposomes were achieved by incorporating amphipathic material, Gd (III) [N,N-bis-stearylamidomethyl-N'-amidomethyl] diethylenetriamine tetraacetic acid, into the liposome membrane using lipid film hydration method. GBI-10, as the targeting ligand, was then conjugated onto the liposome surface to get GBI-10-targeted gadolinium-loaded liposomes (GTLs). Both nontargeted gadolinium-loaded liposomes and GTLs displayed good dispersion stability, optimal size, and zeta potential for tumor targeting, as well as favorable imaging properties with enhanced relaxivity compared with a commercial MRI contrast agent (CA), gadopentetate dimeglumine. The use of GBI-10 aptamer in this liposomal system was intended to result in increased accumulation of gadolinium at the periphery of C6 glioma cells, where the targeting extracellular matrix protein tenascin-C is overexpressed. Increased cellular binding of GTLs to C6 cells was confirmed by confocal microscopy, flow cytometry, and MRI, demonstrating the promise of this novel delivery system as a carrier of MRI contrast agent for the diagnosis of tumor. These studies provide a new strategy furthering the development of nanomedicine for both diagnosis and therapy of tumor.

Mechanism underlying carbon tetrachloride-inhibited protein synthesis in liver.

AIM: To study the mechanism underlying carbon tetrachloride (CCl(4)) -induced alterations of protein synthesis in liver. METHODS: Male Sprague-Dawley rats were given CCl(4) (1 mL/100 g body weight) and (3)H-leucine incorporation. Malondialdehyde (MDA) level in the liver, in vitro response of hepatocyte nuclei nucleotide triphosphatase (NTPase) to free radicals, and nuclear export of total mRNA with 3'-poly A(+) were measured respectively. Survival response of HepG2 cells to CCl(4) treatment was assessed by methyl thiazolyl tetrazolium. Km and Vmax values of nuclear envelope NTPase activity in liver of rats treated with CCl(4) were assayed by a double-reciprocal plot. RESULTS: The protein synthesis was inhibited while the MDA level was significantly increased in liver of rats treated with CCl(4). In addition, CCl(4) decreased the NTPase binding capacity of nuclear envelope (Km value) in cultured HepG2 cells. Moreover, in vitro ferrous radicals from Fenton's system suppressed the NTPase activity of liver nuclear envelope in a dose-dependent manner. Down-regulation of the nuclear envelope NTPase activity indicated a lower energy provision for nucleocytoplasmic transport of mRNA molecules, an evidence in CCl(4)-treated HepG2 cells correspondingly supported by the nuclear sequestration of poly (A)(+) mRNA molecules in morphological hybridization research. CONCLUSION: Inhibition of mRNA transport, suggestive of decreased NTPase activity of the nuclear envelope, may be involved in carbon tetrachloride-inhibited protein synthesis in liver.

Immobilization of phospholipid-avidin on fused-silica capillaries for chiral separation in open-tubular capillary electrochromatography.

Phospholipid-coated fused-silica capillaries with immobilized avidin were applied in the chiral separation of D,L-tryptophan, D,L-PTH-serine, and D,L-PTH-threonine at pH 7.4 by open-tubular CEC. Liposomes prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap biotinyl), or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Biotinyl) with different amounts of phosphatidylserine were assessed as phospholipid coating materials. The stability of the coating and the success of the coating procedure were evaluated in terms of the repeatability of the enantiomer migration times and the resolution of enantiomers. The coating procedure itself significantly affected the migration times and resolution of the enantiomers. Reliable chiral separations with high separation efficiencies were achieved through careful choice of the coating method.