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Background Topotecan is an anti-cancer chemotherapy drug with common side effects, including hepatotoxicity. In this study, we aim to investigate the mechanisms of topotecan-induced hepatocellular injury beyond conventional DNA damage. Materials and methods Methyl Thiazolyl Tetrazolium (MTT) assay was used to detect the inhibitory effect of topotecan on cell proliferation. Western blot was used to detect protein expression. Flow cytometry assay was performed to determine apoptosis rate under topotecan treatment. ASCT2 overexpression was addressed using adenovirus vector. qRT-PCR and western blot assay were used to detect the expression of ASCT2. Glutamine uptake, intracellular glutathione (GSH) and reactive oxygen species (ROS) level were detected by glutamine detection kit, GSH detection kit and ROS detection kit respectively. Results MTT results showed that topotecan had an inhibitory effect on cell proliferation and induced apoptosis in both L02 and HepG2 cell lines. Topotecan inhibited the expression of glutamine transporter ASCT2 and the uptake of glutamine in both L02 and HepG2 cell lines. The uptake of glutamine and the GSH level was increased in both L02 and HepG2 cell lines after ASCT2 overexpression. The ROS level was inhibited by ASCT2 overexpression upon topotecan treatment in both L02 and HepG2 cell lines. Topotecan-induced hepatocellular apoptosis and proliferation inhibition were attenuated by ASCT2 overexpression in both L02 and HepG2 cell lines. Conclusion Topotecan-induced hepatocytes death is dependent on ASCT2 down-regulation, which causes oxidative stress via inhibiting GSH production.

The aggregation-induced emission (AIE) luminogens are now showing strong potential in mimicking the energy donor of fluorescence resonance energy transfer (FRET) system. Herein, one highly efficient FRET system 1-NiR is successfully fabricated in aqueous solution based on an AIE active compound 1 and fluorescence dyes (Nile red (NiR)). 1 acts as the energy donor and NiR acts as the acceptor in the FRET system with the optimum concentrations ratio [1]/[NiR] = 100. Besides, the AIE(1) itself displays excellent selectivity for Cu2+ ions at 525 nm with the detection limit of 1.32 × 10−7 M. While through the FRET system of 1-NiR system, the detection limit of Cu2+ can be further decreased to 9.12 nM by monitoring the fluorescence at 630 nm. As a result, using an AIE probe to detect Cu2+ based on FRET mechanism is a promising strategy.

To evaluate the spatial distribution of chemicals and their contribution to processing quality, wheat grains were layered into nine fractions from the surface to the center layer. The content of total starch, amylose and amylopectin gradually increased from the surface layer to inner layer, while albumin, globulin and total protein decreased in this direction. Contents of gliadin, glutenin, high molecular weight glutenin subuints (HMW-GS) and glutenin macropolymers (GMP) increased from the surface layer, and peaked at the second or the third pearling layer, and then decreased till the center layer. The mean particle diameter of starch granules increased gradually from the surface to inner except at P9. Bread baking performance was the best for flour at the mid layers, while was the worst for flour from the surface layer, especially in terms of taste and texture. Biscuit making performance was the best at the center layers, while poorest at the surface layer.

Monosialotetrahexosylganglioside (GM1), one of glycosphingolipids containing sialic acid, plays particularly important role in fighting against paralysis, dementia and other diseases caused by brain and nerve damage. In this work, a simple and highly efficient method with high yield was developed for isolation and purification of GM1 from pig brain. The method consisted of an extraction by chloroform–methanol–water and a two-step chromatographic separation by DEAE–Sepharose Fast Flow anion-exchange medium and Sephacryl S-100 HR size-exclusion medium. The purified GM1 was proved to be homogeneous and had a purity of >98.0% by high-performance anion-exchange and size-exclusion chromatography. The molecular weight was 30.0 kDa by high-performance size-exclusion chromatography and 1546.9 Da by electrospray ionization mass spectrometry. The chromogenic reaction by resorcinol–hydrochloric acid solution indicated that the purified GM1 showed a specific chromogenic reaction of sialic acid. Through this isolation and purification program, ~1.0 mg of pure GM1 could be captured from 500 g wet pig brain tissue and the yield of GM1 was around 0.022%, which was higher than the yields by other methods. The method may provide an alternative for isolation and purification of GM1 in other biological tissues. Copyright © 2015 John Wiley & Sons, Ltd.

The interaction between TEM-1 β-lactamase and antibiotics is very important in the hydrolysis of antibiotics. In the present study, the recognition and binding of TEM-1 β-lactamase with three β-lactam antibiotics, including penicillin G, cefalexin and cefoxitin, was investigated by fluorescence and ultraviolet-visible absorption spectra in combination with molecular docking in the temperature range of 278-288 K and under simulated physiological conditions. The results demonstrated that the fluorescence emissions of TEM-1 β-lactamase were extinguished by static quenching and the energy of TEM-1 β-lactamase was transferred in a non-radioactive manner. The binding of TEM-1 β-lactamase with the three antibiotics was a spontaneously exothermic process, with binding constants of 1.41×107, 7.81×106 and 5.43×104 at 278 K. Furthermore, binding was driven by enthalpy change and the binding forces between them were mainly hydrogen bonding and Van der Waals forces. A TEM-1 β-lactamase only bound with one antibiotic at a time and the binding capacity between them was closely associated with the functional groups and flexibility in the antibiotics. In addition, a conformational change occurred in the TEM-1 β-lactamases when they bound with the three antibiotics and TEM-1 β-lactamase-antibiotic complexes were formed. The present study provided an insight into the recognition and binding of TEM-1 β-lactamase with β-lactam antibiotics, which may be helpful for designing a novel substrate for TEM-1 β-lactamase and developing novel antibiotics that are resistant to the enzyme.

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was first studied through intrinsic fluorescence emission spectra and fluorescence phase diagram and the results showed that both of them separately followed a two-state model. As the simplest sample of the unfolding of protein molecules induced by denaturants, an equation was presented to show the effect of the denaturant concentrations in denaturation solution on the residual activity ratios of bovine heart cytochrome c in their two-state unfolding. There are two characteristic unfolding parameters K and m in this equation. The former is the thermodynamic equilibrium constant of the unfolding of bovine heart cytochrome c induced by denaturants, the latter is the number of denaturant molecules associated with a bovine heart cytochrome c molecule during the unfolding procedure, and through them the distribution and transition of native and completely unfolded bovine heart cytochrome c conformations under different concentrations of urea or guanidine hydrochloride in denaturation solution can be accurately described.