Objective To screen melanocortin 4 receptor (Mc4R) gene mutation by direct DNA sequencing in order to explore the mutation situation of Mc4R gene in simple obese children in Nanjing.Methods One hundred and five simple obese children(obesity group) and 127 healthy children(healthy control group) were examined for mutations of Mc4R gene.Body mass index(BMI)cutoff points for overweight and obesity adopted Chinese children and adolescents,recommended by China Working Group of Obesity,and all children had no other hereditary and metabolic abnormality.Touch-down PCR was performed to amplify the full length Mc4R gene,then direct DNA sequencing was used to analyze the Mc4R gene.The differences of biochemical index levels between obesity group and healthy control group were analyzed ,including alanine transaminase,aspartate transaminas,total protein,albumin,globulin,albumin/globulin,triglyceride,cholesterol,high density lipoprotein,cortisol,insulin and C peptide.Results There were significant differences of biochemical index levels between obesity group and healthy control group,including triglyceride,insulin level after dining 2 h,C peptide and BMI(Pa

MicroRNAs (miRNAs) are recently discovered major regulators of gene expression, which play a pivotal role in a wide spectrum of biological processes including antiviral defence. There is growing evidence that some viruses either encode their own viral miRNAs or subvert cellular miRNAs. The host-and virus-encoded miRNAs and their targets together thus form a novel regulatory layer of interactions between the host and the virus. A better understanding of host-virus interaction mediated by miRNAs would not only enable us to unravel the molecular basis of viral pathogenesis, but also enable us to develop better therapeutic strategies.

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride ( CoCl2 ) on cave-olin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcino-ma A549 cells.METHODS:The concentrations of Cav-1 and hypoxia-inducible factor ( HIF)-1αin pleural effusion of the patients with lung cancer ( MPE) or tuberculous pleurisy ( TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1αinhibitor YC-1.The concentrations of Cav-1 and HIF-1αin the cell supernatants were measured by ELISA.The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respec-tively.RESULTS:The levels of Cav-1 and HIF-1αin MPE were significantly higher than those in TBPE.There was a highly positive correlation between Cav-1 and HIF-1αlevels in the pleural effusion.CoCl2 induced the generation of Cav-1 and HIF-1αin A549 cells in a concentration-and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration-or time-dependent inhibition was ob-served.HIF-1αinhibitor YC-1 concentration-dependently inhibited the generation of HIF-1αand Cav-1 induced by CoCl2 in A549 cells.CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1.CONCLUSION:The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells.A possible role for HIF-1αis indicated in Cav-1 generation.

Acinetobacter baumannii ; classification ; genetics ; isolation & purification ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Phylogeny

Objective To investigate the antimicrobial resistance of clinical isolates from a hospital in Chongqing during one year according to CHINET project.Methods Disc diffusion test (K-B method) was employed to study the antimicrobial resistance. WHONET5 was used for data analysis.Results In one year period from 2004 to 2005,690 non-duplicate isolates were collect- ed.Enterobacter isolates showed the lowest resistance rate to imipenem and meropenem.About 37.5% of E.coli and 31.4% of K.pneumoniae isolates produced ESBLs,respectively.All ESBLs-producing strains were susceptible to imipenem and mer- openem.About 37.2%,39.4% and 48.9% of P.aeruginosa isolates were resistant to imipenem,meropenem and ceftazidime, respectively.Pandrug-resistant (PDR) P.aeruginosa was isolated from our hospital.All strains of A.baumannii were sus- ceptible to imipenem and meropenem.About 37.7% of A.baumannii were resistant to cefoperazone-sulbactam.Twenty-nine strains showed the same resistant pattern among non-susceptible strains of A.baumannii,mainly derived from 2 clones by PFGE analysis.Conclusions The surveillance results suggest that prevalent strain resistant to cefoperazone-sulbactam may pres- ent in some ICUs.Resistance rate to cefoperazone-sulbactam increased significantly.

Objective To evaluate the feasibility and effectiveness of combination chemotherapy with etoposide and cisplatin(EP)regimen on the patients with high-risk,chemorefractory and recurrent gestational trophoblastic neoplasia(GTN).Methods Thirty-nine patients with gestational trophoblastic tumors were analyzed retrospectively,25 of 39 patients were of high-risk,9 patients were chemorefractory and 5 patients were recurrent.All 39 patients were administrated with EP regimen,and 10 patients were assisted with surgery.All the patients were followed up.Clinical response,toxicity,the occurrence of secondary tumors of all patients,and the fertility of 30 patients whose fertility function was preserved were investigated. Results Thirty-nine GTN patients underwent a total of 221 cycles of the EP regimen.The average number of courses for each patient was 5.7.The total complete remission rate of the regimen was 74%(29/39). Twenty-five patients with high-risk GTN received a total of 139 cycles and the average number of courses was 5.6.Nineteen patients achieved complete remission and 6 patients showed drug-resistant.The complete remission rate of the high-risk group was 76%(19/25).Nine patients with chemorefractory GTN obtained a total of 55 cycles and the average number of courses was 6.1.Six patients achieved complete remission and 3 patients showed drug-resistant again.The complete remission rate of the chemorefractory group was 6/9. Five patients with recurrent GTN received 27 cycles and the average number of courses was 5.4.Four patients achieved complete remission,1 patient showed drug-resistance and died.Bone marrow toxicity, gastrointestinal reaction and alopecia were the main side effects of the EP regimen,but the bone marrow toxicity was slight and no grade Ⅳ side effect occurred.No fatal effect was found.Eight of 30 patients whose fertility faction was preserved had become pregnant after recovery,with a total of 8 pregnancies.Among them,2 were terminated by induced abortion,and 6 underwent normal term delivery and gained 6 infants who had no congenital malformation.All the 6 children had normal growth and development after childbirth. None of the women developed secondary tumors.Conclusion The EP regimen is effective and safe for the treatment of high-risk,chemorefractory and recurrent GTN.

Objective To detect the immunogenicity of VirB9,a protein of type Ⅳ secretion system of Brucella.Methods Full length VirB9 gene was cloned into plasmid pET32a and expressed in Escherichia (E.) coli BL21 (DE3).Expression of recombinant protein was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and the recombinant fusion protein was purified by affinity chromatography on Ni2+-conjugated chelateing sepharose.The purity of the purified protein was ascertained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE) and the concentration was measured by bicinchoninic acid (BCA) protein assay kit.Animal model was established by immunizing BAL B/c mice with live vaccine strain S19 of Brucella and the mice immunized with phosphate buffered saline (PBS) as control.The blood of immunized mice was acquired after 4 weeks.Antibody against VirB9 in S19 immunized mice was detected by Rose Bengal plate agglutination test and serum tube agglutination test; IgG antibody titers against VirB9 in immunized mice were determined by enzyme linked immunosorbent assay(ELISA).At the 35th day,the immunized mice and control mice were killed and spleens were collected.The splenocytes were harvested and stimulated with each of VirB9,concanvalin A(ConA) or medium in triplicate.Production of gamma interferon (IFN-γ) was determined by enzyme-linked immunospot assay (Elispot).Results The full length of VirB9 gene was cloned into pET32a.The recombinant VirB9 protein was expressed at 43 × 103 in relative molecular mass and the purity of the purified recombinant VirB9 protein was above 97％ in SDS-PAGE and the concentration was 1.6 g/L in BCA protein assay.The antibody of VirB9 was detected in all S19 immunized mice but not PBS immunized mice by Rose Bengal plate agglutination test.The antibody titer in all S19 immunized mice was ＞ 1 ∶ 800 or ＞ 1 ∶ 3200 by tube agglutination test and ELISA,respectively.Meanwhile,the protein stimulated stronger IFN-γresponse in immunized mice than that in the control mice(147 cells Vs 38 cells).Conclusion VirB9 can stimulate humoral and cellular immunity and it might be an appropriate target for developing subunit vaccine against Brucella.

Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.

Objective To investigate the effects of different concentration and inhalation duration time of isoflurane on cognitive performance and the expression of GABAR1 and NMDAR2B in aged SD rat cerebral temporal lopes.Methods Aged male SD rats (9 months) were randomly divided into control group (n=10) and test group (n=80).The control group received air at room tempreture.Test groups were divided into four groups: group S1 (1.5%-2 h),group S2 (2.5%-2 h),group S3 (1.5%-4 h),group S4 (2.5%-4 h)according to isoflurane concentration and inhalation duration time.Every group was equally divided into two groups and Morris water maze test was performed day 1 and day 7 after isoflurane inhalation.Then the right temporal lobe was gathered and the mRNA transcription and protein expression of GABAR1 and NMDAR2B were detected by RT-PCR and Immunofluorescence technique.Results One day after isoflurane inhalation, accompanied with increased isoflurane concentration and inhalation duration, the spatial memory ability of every test group decreased continually, and the mRNA transcription and protein expression of GABAR1 increased and the mRNA transcription and protein expression of NMDAR2B decreased compared with control group (P<0.01).Seven days after isoflurane inhalation, the spatial memory ability of group S4 decreased, the mRNA transcription and protein expression of both GABAR1 increased, the mRNA transcription and protein expression of NMDAR2B decreased compared with control group and the other test groups (P<0.01).There was no significant difference between the control group and groups S1, S2, S3.Conclusion Continuous inhalation of isoflurane has great effects on spatial memory ability.And impaired spatial memory by isoflurane inhalation of high concentration with long duration is present in a long time.Thoses are related with the mRNA transcription and protein expressions of GABAR1 and NMDAR2B in cerebral temporal lope.

OBJECTIVETo study the effects of Compound Danshen Dripping Pill (CDDP) on the structure and functions of sternohyoid muscle in metabolic syndrome (MS) rats, and to study whether it has therapeutic effects on obstructive sleep apnea-hypopnea syndrome (OSAHS).

METHODSTwenty-one healthy male SD rats were randomly divided into three groups, i.e., the normal control group (n = 6), the MS group (n = 8), and the CDDP group (n = 7). Rats in the normal control group were routinely fed. High lipid forage was given to rats in the rest two groups. Nine weeks later, CDDP (at the dose of 375 mg/kg) was additionally given to rats in the CDDP group by gastrogavage, and then rats in the CDDP group and the MS group were fed with the same high lipid forage for 12 successive weeks. The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) in the sternohyoid muscle were detected in the three groups. The capillary density, capillary-to-fiber ratio (C/F), the section area of type I muscle fiber were detected using myosin-ATPase histochemical assay. The contractile changes of isometric stemohyoid muscles were determined under electric stimulation by different frequencies.

RESULTSThe contents of MDA were obviously lower in the CDDP group than in the MS group, while the activities of SOD, the capillary density, C/F, the section area of type I muscle fiber, the tension of stemohyoid muscle at 10 -60 Hz, and the 1-5 min tension percentages of the stemohyoid muscle were higher in the CDDP group than in the MS group (P < 0.05, P < 0.01).

CONCLUSIONCDDP could improve oxidative stress induced intramuscularly microcirculation disturbance and changes of muscular fiber structures of the upper airway muscles, and elevate their contractile functions, thus possibly contributing to favorable effects on OSAHS.

Animals ; Capillaries ; Drugs, Chinese Herbal ; pharmacology ; Male ; Metabolic Syndrome ; metabolism ; Muscle Contraction ; drug effects ; Neck Muscles ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Phenanthrolines ; pharmacology ; Rats ; Rats, Sprague-Dawley