The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 degrees C was 8.5 and the optimum temperature at pH 8.5 was 55 degrees C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.
Bacillus subtilis ; classification ; enzymology ; Enzyme Activation ; Enzyme Stability ; Peptide Hydrolases ; chemistry ; isolation & purification ; Species Specificity ; Substrate Specificity

OBJECTIVETo evaluate the impact of hyphema secondary to high intraocular pressure on corneal pathology in rabbits.

METHODSThirty adult New Zealand rabbit were randomized into 3 equal groups, and in each rabbit, one eye served as the experimental eye with the other as the control eye. In the experimental eye, autoblood was injected into the anterior chamber to induce high intraocular pressure maintained for 3, 5, or 8 days. Only saline was injected into the control eye. After the injections, the cornea was observed with slit-lamp microscopy, and at 3, 5, or 8 days, the experimental and control eyes were taken from the 3 groups for microscopic examination of the corneas to detect the occurrence of cornea bloodstain with prolonged high intraocular pressure. Corneal edema, elastic fibers changes, growth of new blood vessels, changes of eosinophils, fibroblasts, lymphocytes and plasma cells, as well as the pathological changes of the corneal layers were observed and compared between the experimental and control eyes.

RESULTSMaintenance of high intraocular pressure for 8 days resulted in the most severe corneal edema and thickening, and histopathologically, the corneal stroma showed widened space between the elastic fibers and obvious fiber distortion. Neovascularization was seen in the marginal cornea where eosinophil infiltration occurred with a small number of lymphocytes, plasma cells and fiber cells. All the three groups showed more obvious edema in the posterior than in the anterior cornea.

CONCLUSIONProlonged hyphema with ocular hypertension results in aggravation of corneal edema, and corneal blood staining does not occur until 8 days of high intraocular pressure but corneal elastic fiber disruption can be seen, suggesting the impending irreversible pathological changes of cornea.

Animals ; Cornea ; pathology ; Edema ; pathology ; Female ; Hyphema ; complications ; pathology ; Male ; Ocular Hypertension ; complications ; pathology ; Rabbits ; Random Allocation

OBJECTIVETo establish a solvent desorption Gas chromatographic method for detecting the isoflurane in air of workplaces.

METHODSThis method is based on "Standardization of methods for determination of toxic substances in workplace air".

RESULTSThis method presents the linear relation with the minimum detectable limit 1.0 µg/ml and the minimum detectable concentration 0.07 mg/m(3). The precision (RSD) was 0.5% ∼ 5.0%, the mean dsorption efficiencies were 96.7% ∼ 98.9%, the absorption efficiencies were 92.1% ∼ 100%, the breakthrough volume was 3.7 mg isoflurane/100 mg active carbon. Other volatile organic solvents (Sevoflurane, Enflurane and Ethyl Alcohol) did not interfere the detection. The sample could be stored in the active carbon tube at least for 10 days.

CONCLUSIONThis method is meet the requirement of GBZ/T 210.4-2008 "Guide for establishing occupational health standards-Part4: Determination methods of air chemicals in workplace" and is feasible for determining the isoflurane in the air of workplaces.

Air Pollutants, Occupational ; analysis ; Chromatography, Gas ; methods ; Isoflurane ; analysis ; Workplace

OBJECTIVETo investigate the clinical features and surgical strategy for pediatric intractable epilepsy due to posterior quadrantic cortical dysplasia and to assess the surgical outcomes.

METHODSThe clinical features and preoperative evaluation results of 14 children with intractable epilepsy due to posterior quadrantic cortical dysplasia were retrospectively analyzed. The localization values of video-electroencephalography and intraoperative monitoring and the indications, advantages and disadvantages of temporoparietooccipital disconnection were evaluated.

RESULTSThe 14 children had different seizure types, of which spasm was the most common one. The lesions of cortical dysplasia involved the central cerebral region in 2 cases. After temporoparietooccipital disconnection in 14 patients, 13 cases were seizure-free; only one case still had seizures, but the frequency dropped by more than 50%.

CONCLUSIONSTemporoparietooccipital disconnection is a safe and effective surgical procedure for children with intractable epilepsy due to posterior quadrantic cortical dysplasia.

Child ; Child, Preschool ; Electroencephalography ; Epilepsy ; etiology ; physiopathology ; surgery ; Evoked Potentials, Somatosensory ; Female ; Humans ; Infant ; Male ; Malformations of Cortical Development ; complications

OBJECTIVETo investigate six trace elements including Cr, Zn, Mn, Se, Cu and V of 13 traditional Chinese medicines (TCM) commonly used in diet therapy of diabetes mellitus.

METHODThe samples were prepared through digesting in a microwave oven and determined with Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

RESULTThe correlative coefficients of the calibration curves were above 0.999. Standard addition recovery was 88.7% - 110.0% and RSD < or = 7.0%. Under the given analytical conditions, the results obtained showed that the contents of the six trace elements varied in these TCM samples, for element Cr, ranging between 0-11.82 microg x g(-1), Mn in 4.36-564.3 microg x g(-1), Cu in 0.55-13.34 microg x g(-1), Zn in 3.19-86.23 microg x g(-1) Se in 0-0.39 microg x g(-1) and V in 0.05-12.04 microg x g(-1), respectively.

CONCLUSIONThe possible relationship between the content of the six trace elements and 13 TCM samples for dietetic therapy of diabetes mellitus was also discussed.

Chromium ; analysis ; Copper ; analysis ; Diet, Diabetic ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Manganese ; analysis ; Mass Spectrometry ; methods ; Medicine, Chinese Traditional ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Selenium ; analysis ; Spectrophotometry, Atomic ; Trace Elements ; analysis ; Vanadium ; analysis ; Zinc ; analysis

OBJECTIVETo explore the role of NF-E2-related factor 2(Nrf2) and its related factors in the progression of nonalcoholi steatohepatitis (NASH) by investigating the alterations of lipid metabolism and liver histopathology as well as the changes of mRNA and protein expression levels of Nrf2 and its related factors in rats during NASH progression.

METHODSMale SD rats were randomly divided into normal group and model group, which were administrated with high fat diet to establish nonalcoholic steatohepatitis model. The rats from both groups were randomly killed at the end of 4, 12 weeks respectively. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) were detected in the serum and liver tissue; Changes in fat deposition in liver tissue were determined by oil red O staining. HE staining were used to observe the pathological changes of liver tissue and to calculate nonalcoholic fatty liver disease (NAFLD) activity score (hepatic steatosis, inflammation and ballooning degeneration of liver cells). The expression of Nrf2 in liver was detected by immunohistochemical staining. The mRNA and protein levels of Nrf2 and related factors in liver were determined by Realtime PCR and Western blot, respectively.

RESULTSAfter 4 weeks of high fat diet, the levels of ALT, AST, TC in rat serum and TC, TG, LDL-C in liver were significantly increased compared with that of the normal group (P < 0.01, P < 0.05). After 4 weeks of high fat diet, the levels of ALT, AST, TC, TG in serum and TC, TG, LDL- C in liver increased further (P < 0.01, P < 0.05). Until the 12th week, the content of HDL-C in liver was significantly lower than that of the normal group (P < 0.05). At the end of the 4th or the 12th week, lipid droplets in the model rat liver cells were heavily dyed red and hepatic steatosis increased severely, with ballooning degeneration of liver cells. With the extension of high fat diet feeding time, fat deposition in the liver tissue, hepatic steatosis, NAFLD score, Nrl2 expression were significantly increased (P < 0.01). Expression levels of mRNA and protein of Nrf2, heme oxyenase 1(HO1), NADPH quinone oxidoreductase 1(NQO1), γ-glutamylcysteine synthethase (γ-GCS), glutathione S-transferase (GST) in the model rats increased or decreased at the end of the 4th or the 12th week differentially, (P < 0.01, P < 0.05) with the more significant changes at the end of the 4th week than the 12th week.

CONCLUSIONNrf2 and its related factors may be involved in the occurrence and development of nonalcoholic fatty liver disease, which may play an important role in the process of NASH formation.

Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Cholesterol ; metabolism ; Diet, High-Fat ; Dipeptides ; metabolism ; Disease Progression ; Glutathione Transferase ; metabolism ; Heme Oxygenase (Decyclizing) ; metabolism ; Lipid Metabolism ; Liver ; pathology ; Male ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Non-alcoholic Fatty Liver Disease ; metabolism ; Rats ; Rats, Sprague-Dawley ; Triglycerides ; metabolism

Objective To investigate the clinical efficacy of electroacupuncture and (or) massotherapy plus behavior therapy for cervical spondylosis.Methods Three hundred patients with cervical spondylosis were randomly allocated to three groups (A, B and C) by random number generated from calculator, 100 cases each. In addition to cupping and behavior therapy, group A received electroacupuncture; group B, massotherapy; group C, electroacupuncture and massotherapy. The clinical symptom and sign score was recorded in the three groups before and after treatment. The clinical therapeutic effects were compared.Results There were no statistically significant differences in the clinical control rate, marked control rate and total efficacy rate among the three groups after four and eight weeks of treatment and at the 4-month and 6-month follow-ups after treatment (P>0.05). In the three groups, there was a statistically significant difference in the clinical symptom and sign score between before treatment and after four and eight weeks of treatment or at the 4-month and 6-month follow-ups after treatment (P<0.01) and between after four weeks of treatment and after eight weeks of treatment or at the 4-month and 6-month follow-ups after treatment (P<0.01). There was no statistically significant differences in the clinical symptom and sign score among the three groups after four and eight weeks of treatment and at the 4-month and 6-month follow-ups after treatment (P>0.05). Conclusions Electroacupuncture and (or) massotherapy plus behavior therapy is an effective protocol for preventing and treating cervical spondylosis. It is characterized by simplicity, convenience, easiness and cheapness and can be provide for clinical application.

Objective To investigate the effect of c-fos gene silencing on differentially expressed proteins (DEPs) in human bronchial epithelial (HBE) cells after exposure to fine particulate matter (PM2.5).Methods HBE cells and c-fos-silenced HBE cells were exposed to 50 μg/mL PM2.5, LC-MS/MS and tandem mass tag (TMT) labeling methods were combined with bioinformatics methods, and DEPs and interaction networks were identified.Results In the HBE group, 414 DEPs were screened, of which 227 were up-regulated and 187 down-regulated. In the c-fos silenced HBE group, 480 DEPs were screened, including 240 up-regulated proteins and 240 down-regulated proteins. KEGG annotations showed that DEPs in the HBE group are mainly concentrated in the glycolysis/gluconeogenesis pathway and those in the c-fos silenced group are concentrated mainly in endoplasmic reticulum and the processing of proteins. Additionally, the abnormal expression of GPRC5C, DKK4, and UBE2C was identified in top 15 DEPs. After constructing the protein interaction network, 20 Hub proteins including HNRNPA2B1, HNRNPL, RPS15A, and RPS25 were screened from the HBE group and the c-fos silenced HBE group.Conclusion c-fos gene affected the expression of cancer-related proteins. Our results provided a scientific basis for further study of PM2.5-induced carcinogenesis mechanism.

The present study investigated the chemical constituents from the stems of Buddleja lindleyana. Ten compounds were isolated from the 95% EtOH extract of B. lindleyana stems by means of some techniques including polyamide, silica gel, MCI, Sephadex LH-20 column chromatography, and semi-preparative high-performance liquid chromatography(HPLC). Their structures were identified by spectral analysis and single-crystal X-ray diffraction as buddledin F(1), 6-O-4″-hydroxy-3″-methoxy-benzoyl ajugol(2), negundoin G(3),(+)-dihydrocubebin(4), 7-O-ethylguaiacylglycerol(5),(-)-jatrointelignan B(6), threo-1,2-bis-(4-hydroxy-3-methoxyphenyl)-propane-1,3-diol(7), vomifoliol(8), hinokinin(9), and isovanillic acid(10). Compound 1 was a new sesquiterpene named buddledin F. Compounds 3-8 were isolated from the Buddleja plant for the first time. The anti-inflammatory activities of compounds 1-10 in vitro were investigated, and the results failed to show the inhibitory activities of these compounds on the production of inflammatory factor NO.
Buddleja/chemistry* ; Sesquiterpenes/pharmacology* ; Chromatography, High Pressure Liquid

Objective::To study the effect of total alkaloids of Nandina domestica in attenuating arsenic trioxide, and analyze chemical constituents of alkaloids extract of Nandina, in order to provide the theoretical basis for studying the effect of N. domestica in attenuating arsenic trioxide with alkaloids extract of N. domestica as effective fraction. Method::The model of acute liver injury induced by arsenic trioxide was used to compare the effects on heart and liver functions of mice between arsenic trioxide alone and total alkaloids of N. domestica combined with arsenic trioxide. The detoxification of total alkaloids on arsenic trioxide was evaluated based on biochemical parameters and pathological report. Peakview (Version1.2, AB SCIEX) software was used to process the mass spectrometry data of total alkaloids of N. domestica. The structures of determined compounds were identified by molecular weight of compound (molecular formula), secondary fragments, chromatographic peak retention and literature information. Result::Among biochemical indicators, creatine kinase(CK), lactate dehydrogenase(LDH), blood urea nitrogen(BUN), malondialdehyde(MDA), alanine aminotransferase(ALT) and amino transferase of aspartate(AST) of the arsenic trioxide group were increased, while elimination rates of Na⁺ -K⁺ -adenosine triphosphate(ATP), superoxide dismutase(SOD), catalase(CAT) and serum creatinine(SCr) were decreased, compared with those of the combination group. CK and LDH of the alkaloids extract group were more obviously increased than those of the N. domestica extract group, but with no remarkable difference. In histomorphometric examination, edema of mouse heart cells was improved, and some kidney and liver damages in rats were alleviated. Totally 25 alkaloids of alkaloid extract were identified. Among them, 18 were known, and 7 were unknown, including 3 structural types, in which apomorphine alkaloids were mostly. Conclusion::Heart, kidney and liver damage degrees of the combination group were significantly alleviated compared with the arsenic trioxide group. The total alkaloids fraction extracted and purified have a significant attenuation in arsenic trioxide toxicity. The detoxification of total alkaloids extract was equal to that of N. domestica extract. Furthermore, apomorphine alkaloids, such as nantenine and domesticine, can be used as index components to establish a quality control method for total alkaloids.