The recombination clones contained CFP, LTB-ST foreign gene were screening by PCR using individual bacterial colonies as template, the aimed band was amplified from positive clones, the result was as well as plasmid PCR. The selecting of agrobacterium transformed with recombination plasmid could also use this method of PCR screening of individual bacterial colonies. The result of individual bacterial colonies PCR was as well as that of PCR using bacterial solution as template. It showed that the method individual bacterial colonies PCR was an efficient, easy one that characterized recombination clones.

Objective To investigate the expression of pro-inflammatory cytokines in lumbar dorsal root ganglions (DRG) of rats model of high-dose fentanyl induced hyperalgesia. Methods 64 male SD rats were divided into 2 groups (n = 32), fentanyl group and normal saline (NS) group. The rats were injected with fentanyl (60 μg/kg) or NS 4 times in total subcutaneously with a 15-minute interval. Mechanical and thermal nociception were measured via the tail pressure test (tail flick thresholds, TFT) and paw withdrawal test (paw withdrawal latency, PWL) at 1 day before, at 1, 2, 3 and 4 hour and on 1 ~ 7 day after administration. 4 rats were sacrificed and the lumbar DRG were harvested to analyze the expression of PGE2 , IL-1β, IL-6 and TNF-αvia ELISA. Results There were no significant changes of TFT, PWL and the expression of pro-inflammatory cytokines in DRG compared to baseline of rats in NS group. The value of TFT , PWL in fentanyl group were above the baseline at the 1 ~ 4 hour and below the baseline at 1~3 day after fentanyl injections. PGE2 , IL-1β, TNF-α and IL-6 increased on 1,3,5,7 day after fentanyl injections significantly. Conclusions High-dose fentanyl induced significant hyperalgesia and up-regulation of pro-inflammatory cytokines in DRG. The expression pro-inflammatory cytokines peaked later and were more protracted than the change of behavior test and show no direct relationship between the two.

To study the protective effect and the mechanism of asiatic acid (AA) from Potentilla chinensis on alcohol hepatic injury in rats. Male Wistar rats were randomly divided into six groups: the normal control group, the AA control group (8 mg · kg(-1) AA), the model group (5.0-9.0 g · kg(-1) alcohol) and high, medium and low-dose AA-treated groups (alcohol + 8, 4, 2 mg · kg(-1) AA). Each group was orally administered with the corresponding drugs once a day for 24 weeks. Approximately 1. 5 hours after the final administration, all rats were killed, and their blood samples and hepatic tissues were collected. The AST and ALT in rat serum and the contents of MPO, TNF-α, IL-1β, SOD, GSH-Px, GSH-Rd and MDA in hepatic tissues were detected. The expressions of NF-κB, TLR4, CD14, MyD88, TRIF and protein expression in hepatic tissues were measured by western blot. The pathological changes in liver tissues were observed by histological examination. The results showed that compared with the model group, the AA-treated groups showed significant decreases in serum ALT, AST and MDA and increases in the activities of SOD, GSH-Px, GSH-Rd and MPO. Moreover, AA markedly inhibited the expressions of TNF-α, IL-1β, TLR4, CD14, MyD88 and NF-κB. The histological examination showed alleviated hepatic issue ijury to varying degrees. In short, asiatic acid (AA) from P. chinensis could protect alcohol-induced hepatic injury in rats. Its mechanism may be related to the inhibition of NF-κB inactivation and the reduction of inflammatory response.
Animals ; Liver ; drug effects ; pathology ; Liver Diseases, Alcoholic ; prevention & control ; Male ; NF-kappa B ; physiology ; Pentacyclic Triterpenes ; pharmacology ; Potentilla ; chemistry ; Protective Agents ; pharmacology ; Rats ; Rats, Wistar ; Toll-Like Receptor 4 ; antagonists & inhibitors

Endoscopy ; Female ; Humans ; Middle Aged ; Nose ; surgery ; Oculomotor Muscles ; surgery

LC-Q-TOF-MS and LC-IT-MS in positive and negative ion mode were applied to simultaneously characterize the constituents in Suanzaoren tang. Analysis was performed on an Agilent Zorbax SB-C18, Rapid Resolution HT column(4.6 mmx 50 mm, 1. 8 micro m) with gradient elution of acetonitrile(A) -aqueous solution containing 0. 05% formic acid(B) at a flow rate of 0. 6 mL min(-1) and the column temperature was 30 degreesC. By comparing MS fragmentation, accurate molecular weight, literature date and standard compounds information, a total of48 compounds were successfully identified or speculated. The origins of these compounds were assigned to the corresponding Chinese medicine. Thirty-one compounds were reported in Suanzaoren tang for the first time. LC-Q-TOF-MS combined with LC-IT-MS is a simple and rapid tool for the identification of constituents of Suanzaoren tang, and the results could provide evidence for the research on quality combined and effective constituents of Suanzaoren tang.
Chromatography, Liquid ; Drugs, Chinese Herbal ; chemistry ; Mass Spectrometry ; Organic Chemicals ; analysis

Objective]To investigate the change of spinal pro?inflammatory cytokines in a rat model of fentanyl induced acute hyperalgesia.[Methods]64 male SD rats were divided into 2 groups(n=32),fentanyl group and NS group. The rats were subcutaneously injected with fentanyl (60 μg/kg) or normal saline (1.2 mL/kg) 4 times with 15?minute intervals. Mechanical nociceptive thresholds and thermal nociceptive latency were measured via the tail pressure test(Tail flick thresholds,TFT) and paw withdrawal test(Paw withdrawal latency,PWL)on the day before,at 1,2,3,and 4 hour and on 1~5 day after injection. 4 rats were killed concomitantly and the lumber spinal cord were harvested to analysis the expression of NF-κB,PGE2,TNF-α,IL-1β,and IL-6.[Results]There were no significant changes of TFT,PWL and the expression of spinal inflammatory cytokines such as NF-κB, PGE2,IL-1β,and TNF-αcompared to baseline of rats treated with normal saline. The value of TFT ,PWL in fentanyl group raised to the highest(above the baseline)at the 1st hour after fentanyl injections and decreased thereafter,reached the lowest at the 1st day, raised increasinglyand up to baseline on the 3 day after injection. NF-κB,PGE2,IL-1β,and TNF-αincreased at the 4th hour,on 1 and 2 day and IL-6 increased at the 4 hour and onthe 1 day after fentanyl injections.[Conclusion]Subcutaneously injection of fentanyl induced significant mechanical and thermal hyperalgesia and increased spinal pro?inflammatory cytokines parallelly , indicated that fentanyl induced acute hyperalgesia is associated with spinal inflammatory reaction in rats.

Objective To explore the risk factors affecting prognosis of children with Tourette syndrome(TS).Methods The follow-up visits were conducted on the clinical data of 98 cases with TS(85 male,13 female;aged 4-16 years old)from 1997 to 2005 in Wuhan children's hospital.All cases were consistent with the diagnostic criteria of TS in the 4th edition of Diagnostic and Statistical Manual of Mental Disorders(DSM-Ⅳ).The investigations were performed by the investigators who received special training using the unified questionnaire with the methods of direct inquiry or by telephone.The factors included sex,age,severity of TS,the primary symptoms,family history,coexisting diseases,basic diseases,perinatal abnormity and family-social relations.The prognosis of TS and these factors were analyzed by linear regression and stepwise regression with SPSS 12.0 software.Results About 16 cases lost follow-up and the other 82 cases with follow-up(72 male and 10 female)received retrospective review.They were 14 to 25 years old with complete data,and 50 cases healed,32 cases not healed.Results from non-conditional simple variant Logistic regression showed that such cases were associated with the following factors:age,family history of TS,severity of TS,coexisting diseases,basic diseases and perinatal abnormity(Pa0.05).Out of 6 suspicious factors,there were coexisting diseases(OR=84.088,95%CI 10.850-651.682),severity of TS(OR=13.956,95% CI 2.412-80.762),and family history of TS(OR=27.127,95% CI 1.047-702.831)of risk factors.Conclusion The long-term prognosis of children with TS may be related with coexisting disease,severity of TS and family history respectively.

Background Diabetic complication is associated with lipid peroxidation.Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of a variety of biological aldehydes,including lipid-derived aldehydes (LDAs),and thus protect organs and tissues from toxic LDAs.Understanding the activity of ALDH in different ocular tissues in diabetic subjects is very important for prevention and treatment of diabetic ocular complications.Objective This research aimed to investigate the activity and expression of ALDH in different ocular tissues in diabetic rats and to explore the mechanism of ALDH in diabetes-induced eye disease.Methods Twenty-eight healthy SPF male Sprague-Dawley(SD) rats weighted 170-180 g were randomly divided into the normal control group and diabetic group.The diabetic animal model was established by intraperitonial injection of 4％ streptozotocin at 65 mg/kg.Isometric citric acid buffer was injected in the rats of the normal control group.The rats were sacrificed in each group 2 and 4 months after the establishment of the diabetic models,and eyeballs were obtained for the preparation of corneal,lens and retinal homogenates.ALDH activity was detected using a multifunctional microplate reader SpectraMax M5,and ALDH content was measured by ELISA at the wavelength of 450 nm with the SpectraMax M5 ELISA reader.Results The blood glucose level in diabetic rats was significantly elevated at various time points compared with the normal control group(P=0.000),and body weights were evidently lower in the diabetic group than in the normal group (P =0.000).The activities of ALDH (A340) in corneal,lens and retinal tissues in the diabetic group were increased in comparison with the normal control group (F =396.601,P=0.000),and showed an enhancement with the lapsing of time (F =53.139,P =0.000).In addition,the highest level of ALDH was found in the cornea and the lowest level in the lens(F =6973.000,P=0.000).The expression level of ALDH in the corneal,lens and retinal homogenates was significantly higher in the diabetic group compared with the normal control group (F=312.985,P =0.000) and showed a considerable increase over the course (F =19.203,P=0.000).The highest expression level was seen in the cornea and the lowest was in the lens,with a significant difference among these three kinds of tissues (F =3243.000,P =0.000).Conclusions ALDH can protect ocular tissue from the damage of lipid peroxidation.Thess results suggest that ALDH plays a role in preventing diabetes-related ocular complications.

Objective To evaluate the effects of 131 adrenoceptor anfisense on blood pressure and?1 adrenoceptor mRNA and protein levels in 2 kidney 1 clip(2K1C)rats.Method 2KIC hypertensive rots were produced by clipping renal artery of SD rats.Liposome/AS-ODNs 2.0 were tested intravenously in rats with 2KIC hypertension.Animals were divided into 5 groups(n=18 in each group):?1-AS-ODN group,?1-IN-ODN group,2K1C group,Sham group and SD group.Blood pressure was measured by tail-cuff method,the levels of myocardial?adreneceptor mRNA and protein were tested by RT-PCR and binding assay.Results On the basis of the magnitude and duration of hypotension,?1-AS-ODN decreased blood pressure by 39 mmHg at the most for 4 weeks.Compared with the 2KIC group,?1-AS-ODN did not significantly change the levels of myocardial?1 adrenoceptor mRNA but significantly decreased the levels of myocardial?1 adrenoceptor protein at 2,7,30 days (P

OBJECTIVETo screen the potential hepatotoxic components in Chinese herb medicine Aucklandiae Radix.

METHODSThe potential hepatotoxic components were screened using HepG2 cells labeled with fluorescein diacetate from 25 fractions of Aucklandiae Radix, in which the hepatotoxic compounds were further identified with GC-MS.

RESULTSTen potential hepatotoxic fractions were screened. The identification results by GC-MS indicated that the main compounds in C09 were dehydrocostuslactone, santamarine (or magnolialide) and reynosin, and in C11 were α-costol and elemol.

CONCLUSIONDehydrocostuslactone, santamarine (or magnolialide), reynosin, α-costol and elemol are potential hepatotoxic compounds in Aucklandiae Radix.

Drugs, Chinese Herbal ; chemistry ; Gas Chromatography-Mass Spectrometry ; methods ; Lactones ; analysis ; toxicity ; Sesquiterpenes ; analysis ; toxicity