Objective To establish a chromogenic assay for blood-plasma collagenase Ⅳ in order to evaluation the reference range of collagenase Ⅳ in the plasma of healthy individuals.Methods The assay was based on measurement of terminal amino group with succinylated gelatin as substrates and TNBS as chromogenic reagent.The optical density of each reaction was determined at 405 nm using a Sunrise microplate reader.Chromatographic and detection conditions were optimized and performance of the methed was evaluated by recovery experiments and precision experiments.It was compared with ELISA.The levels of collagenase Ⅳ in 112 health persons'plasma were determined and the data were analyzed by SPSS statistical software.Results The whole determing time was within 1.5 h,the linear range of this method was 1.5-10.0 mg/L,and the minimum detection limit was 0.965 mg/L.They were well correlated with ELISA results (R~2=0.999 7,P＜0.01).The within-run CV was less than 3.16%and between-run CV was less than 9.81%.The 95%confidence interval of collagenase Ⅳ in healthy plasma was 33.38-49.80 mg/L.Conclusion This chromogenic assay for blood-plasma collagenase Ⅳ can be used for measurement of collagenase Ⅳ in blood-plasma and the reference range of collagenase Ⅳ in healthy plasma was established.

Objective A high-performance liquid chromatography(HPLC)method was modified and used to determine the inhibitory effects of GM6001 and inhibitor A on tumor necrosis factor-α converting enzyme(TACE).Methods TACE and polypeptides substrate were incubated for 15 min at 37°C.Ala-Dpa was added as internal standard of quantitative analysis.Then the solution was analyzed by HPLC.The 55% methanol aqueous solution was used as the mobile phase.The wavelength of detector was 353 nm.The ratio of the peak area of remaining substrate to that of internal standard was determined.And the amounts of inverted substrate could be obtained from calibration curve.The TACE activity could be calculated.Results The relative peak areas of substrate were linearly increased depending on the growth of substrate concentration.The correlation coefficient was 0.996 8 and linear range was from 10 to 400 μmol/L.Precision experiments indicated that the precision was improved obviously by using internal standard method in the determination of TACE activity by HPLC.The values of 50% inhibitory concentration IC50 of GM6001 and inhibitor A determined by the newly proposed method were 317.5 and 175.8 nmol/L,respectively.Conclusion The HPLC method assaying TACE activity with Ala-Dpa as internal standard is more accurate,and more practical for screening of TACE inhibitors.

The crystal structural data of TACE, MMP-1, MMP-2, MMP-3 and MMP-9 were obtained from PDB database, and then their catalytic domains' properties including conformation, molecular surface hydrophobicity and electrostatic potential were analyzed and compared by using Insight II molecular modeling software. It was found that the conformation and molecular surface hydrophobicity of catalytic domains of TACE and MMPs were not obviously different, but the molecular surface electrostatic potential of catalytic domain of TACE and MMPs had obvious differences. The findings are helpful in the Rational Drug Design of TACE selective inhibitor.

Objective To determine the dose-response relationship of sufentanil blunting responses to double-lumen endotracheal intubation when combined with propofol given by target-controlled infusion (TCI) in patients with pulmonary tuberculosis.Methods One hundred patients of both sexes with pulmonary tuberculosis,of American Society of Anesthesiologists physical status Ⅰ or Ⅱ,aged 24-58 yr,with body mass index ＜30 kg/m2,with Mallampati grade Ⅰ or Ⅱ,undergoing thoracic surgery under general anesthesia,were divided into Ⅰ-Ⅴ groups (n =20 each) using a random number table.Anesthesia was induced with iv sufentanil 0.35 μg/kg (group Ⅰ),0.40 μg/kg (group Ⅱ),0.45 μg/kg (group Ⅲ),0.50 μg/kg (group Ⅳ) and 0.55 μg/kg (group Ⅴ),and propofol TCI (target plasma concentration 3.5 μg/ml) and iv vecuronium 0.15 mg/kg.The patients were endotracheally intubated and mechanically ventilated.The response to double-lumen endotracheal intubation was defined as positive when mean arterial pressure increased by＞ 20％ of the baseline value and/or heart rate ＞ 90 bpm within 5 min after intubation.The median effective dose (ED50),ED95 and 95％ confidence interval (95％ CI) of sufentanil blunting the responses to double-lumen endotracheal intubation were calculated by Probit analysis.Results The ED50 (95％ CI) and ED95 (95％ CI) of sufentanil blunting the responses to intubation were 0.411 (0.370-0.441) μg/kg and 0.635 (0.556-0.888) μg/kg,respectively,when combined with propofol given by TCI.Conclusion When combined with propofol given by TCI (target plasma concentration 3.5 μg/ml),the ED50 and ED95 of sufentanil blunting the responses to double-lumen endotracheal intubation are 0.411 and 0.635 μg/kg,respectively,in patients with pulmonary tuberculosis.

The crystal structural data of TACE, MMP-1, MMP-2, MMP-3 and MMP-9 were obtained from PDB database, and then their catalytic domains' properties including conformation, molecular surface hydrophobicity and electrostatic potential were analyzed and compared by using Insight Ⅱ molecular modeling software. It was found that the conformation and molecular surface hydrophobicity of catalytic domains of TACE and MMPs were not obviously different, but the molecular surface electrostatic potential of catalytic domain of TACE and MMPs had obvious differences.The findings are helpful in the Rational Drug Design of TACE selective inhibitor.

Objective To evaluate the effect of mild hypothermia on the expression of phosphorylated eukaryotic translation initiation factor 2α (p-eIf2αt) in the hippocampus in a mouse model of cerebral ischemia-reperfusion (I/R).Methods Sixty pathogen-free healthy male C57BL6 mice,aged 8-12 weeks,weighing 20-30 g,were divided into 3 groups (n =20 each) using a random number table:sham operation group (group S),group I/R and mild hypothermia group (group H).Cerebral I/R was induced by 15-min occlusion of bilateral common carotid arteries followed by reperfusion in chloral hydrate-anesthetized mice.Surface cooling was started immediately after reperfusion to maintain the rectal temperature at 32-34 ℃ for 3 h in group H.The neurologic deficit score was evaluated at 24 h of reperfusion.The mice were then sacrificed,brains were immediately removed,and hippocampi were isolated for examination of pathologic changes of hippocampal CA1 region and for determination of neuroapoptosis (by TUNEL) and expression of peIf2α (by Western blot).The apoptosis rate was calculated.Results Compared with group S,the neurologic deficit scores at 24 h of reperfusion and apoptosis rate of hippocampal neurons were significantly increased,and the expression of p-eIf2α was up-regulated in I/R and H groups (P＜0.05).Compared with group I/R,the neurologic deficit scores at 24 h of reperfusion and apoptosis rate of hippocampal neurons were significantly decreased,and the expression of p-eIf2α was down-regulated in group H (P＜ 0.05).Conclusion Mild hypothermia reduces endoplasmic reticulum stress and inhibits neuroapoptosis through inhibiting the expression of p-eIf2α in the hippocampus in a mouse model of cerebral I/R.