Excitotoxic effects of glutamate on cochlear organotypic cultures / 华中科技大学学报（医学）（英德文版）

Glutamate (Glu) is the major afferent excitatory neurotransmitter in the auditory system, and excessive Glu may play an important role in cochlear dysfunction. It is unclear how excessive Glu plays roles in cochlear dysfunction in cochlear organotypic cultures. In this study neonatal rat cochlear organotypic cultures were prepared, and then the cochlear tissues were incubated with a new medium containing specific concentrations of Glu (0.1, 0.5, 1, 10 or 20 mmol/L) for 24 h, or incubated with the medium containing a concentration of 20 mmol/L Glu for 6, 12, 24 or 72 h, respectively. It was found that when the cochlear tissues were cultured for 24 h, the inner hair cells (IHCs) were damaged at the concentration of 0.5 mmol/L Glu, and with the increases of the concentrations, the injury was gradually aggravated, and 20 mmol/L Glu resulted in the significant loss of IHCs. In the 20 mmol/L Glu groups, the stereocilia bundles were missing or disarrayed on a few IHCs after culture for 6 h and the damage effect was time-dependent. The missing of IHCs was more significant in the basal turn of the cochlea than in the middle turn of the cochlea under the same concentration of Glu exposure. These results suggest that excessive exogenous Glu affects the morphology of IHCs, but not affects the outer hair cells (OHCs) in cochlear organotypic cultures, and the excitotoxic effects are different on IHCs of different parts of the cochlea under the same concentration of Glu exposure.

Excitotoxic effects of glutamate on cochlear organotypic cultures / 华中科技大学学报（医学）（英德文版）

Glutamate (Glu) is the major afferent excitatory neurotransmitter in the auditory system, and excessive Glu may play an important role in cochlear dysfunction. It is unclear how excessive Glu plays roles in cochlear dysfunction in cochlear organotypic cultures. In this study neonatal rat cochlear organotypic cultures were prepared, and then the cochlear tissues were incubated with a new medium containing specific concentrations of Glu (0.1, 0.5, 1, 10 or 20 mmol/L) for 24 h, or incubated with the medium containing a concentration of 20 mmol/L Glu for 6, 12, 24 or 72 h, respectively. It was found that when the cochlear tissues were cultured for 24 h, the inner hair cells (IHCs) were damaged at the concentration of 0.5 mmol/L Glu, and with the increases of the concentrations, the injury was gradually aggravated, and 20 mmol/L Glu resulted in the significant loss of IHCs. In the 20 mmol/L Glu groups, the stereocilia bundles were missing or disarrayed on a few IHCs after culture for 6 h and the damage effect was time-dependent. The missing of IHCs was more significant in the basal turn of the cochlea than in the middle turn of the cochlea under the same concentration of Glu exposure. These results suggest that excessive exogenous Glu affects the morphology of IHCs, but not affects the outer hair cells (OHCs) in cochlear organotypic cultures, and the excitotoxic effects are different on IHCs of different parts of the cochlea under the same concentration of Glu exposure.

Four-component reaction of N-sulfonylimines, (cyanomethylene)triphenylphosphorane, nitromethane, and formaldehyde for the synthesis of 3-substituted 2-methylene-4-nitrobutanenitriles.

An efficient four-component synthesis of 3-substituted 2-methylene-4-nitrobutanenitriles has been developed from N-sulfonylimines, (cyanomethylene)triphenylphosphorane, nitromethane, and formaldehyde in the absence of catalysts and additives at room temperature.

Catalytic kinetic spectrophotometry for the determination of trace amount of oxalic acid in biological samples with oxalic acid-rhodamine B-potassium dichromate system.

A new catalytic kinetic spectrophotometric method was proposed for determining trace oxalic acid based on the catalytic effect of oxalate on the oxidation of potassium dichromate with rhodamin B in 0.10 M of sulfuric acid. Good linearity is obtained over the concentration range 0.40-6.0 microg/mL of oxalic acid. After the reactions of the catalytic and non-catalytic systems were terminated by using 2.00 mL of 4 M sodium hydroxide solution, they can be stable for 3 h at room temperature. The apparent activation energy of the catalytic reaction is 12.44 kJ/mol. The effect of 50 coexisting substances was observed. The method was used to determine trace oxalic acid in tea, spinach and urine samples with satisfactory results.

Expression and activity of the nucleotide-binding domains of the human ABCA1 transporter.

The aim of this study was the expression and production in Escherichia coli of the nucleotide-binding domains (NBDs) of the human ABCA1 transporter, in a soluble, non-denatured form. To increase the protein solubility, and avoid expression in E. coli inclusion bodies, we extended the length of the expressed NBD domains, to include proximal domains. The corresponding cDNA constructs were used to express the N-terminal His-tagged WT and mutant proteins, which were purified by Ni(2+)-affinity chromatography. Optimal expression of soluble proteins was obtained for constructs including the NBD, the downstream 80-residue domain, and about 20 upstream residues. The size homogeneity of WT and mutant NBDs was determined by Dynamic Light Scattering, and ATP-binding constants and ATPase activities were measured. The NBD1 and NBD2 domains bound ATP with comparable affinity. The ATPase activity of WT His-NBD1 was about three times higher than that of NBD2 and amounted to 5913 compared to 1979 nmol Pi/micromol NBD/min for WT His-NBD2. All engineered mutants had comparable ATPase activity to the corresponding WT protein. The optimisation of the length of the expressed proteins, based upon the boundary prediction of NBDs and neighbour domains, enables the expression and purification of soluble ABCA1 NBDs, with high ATPase activity. This approach should prove useful for the study of the structural and functional properties of the NBDs and other domains of the ABC transporters.