Objective To prepare specific polyclonal antibodies to outer membrane protein (Opr) D_2 of Pseudomonas aeruginosa (PA),and explore the relationship between loss of OprD_2 and imipenem resistance.Methods The genomic DNA of PA was ex- tracted with phenol:chloroform.OprD_2 coding gene was amplified by PCR and prokaryotic expression vector pRSET-OprD_2 was constructed.OprD_2 protein was expressed by IPTG induction in E.coli BL21(DE3),and purified with SDS-PAGE.The new protein band was recovered and used as antigens to subcutaneously immunize two New Zealand rabbits to prepare poly- clonal antibody.The specificity of the antibody was determined by Western blot.The expression of OprD_2 in 32 clinical isolates of PA was detected with the prepared polyclonal antibody by Western blot.Results The vector pRSET-OprD_2 has been success- fully expressed in E.coli BL21 (DE3).The polyclonal anti-OprD_2 antibody with high specificity has been successfully pre- pared.Present results show that of the 27 imipenem-resistant PA clinical isolates,OprD2 protein was low-expressed in 5 iso- lates (18.5%) and normally expressed in 2 isolates (7.4%) but not expressed in 20 isolates (74.1%).Conclusions The loss or low-expression of OprD_2 is one of the essential mechanisms accounting for imipenem resistance in clinical isolates of PA.

Objective To review the experience of modified sternal elevation in management of pectus excavatum deformities. Methods A retrospective study was carried out on 268 children with pectus excavatum deformities from January 2002 to December 2005.Of these patients,213 were boys and 55 were girls.Their age ranged from 2 to 16 years and 2 months[mean,(4.48?2.74) years)].Among then,69 cases were aged from 2 to 3 years,130 cases from 3 to 6 years and 69 cases over 6 years.268 patients with PE underwent modified sternal elevation and fixation with the home made stainless steel strut.The lung cysts,esophageal hatal hernia and congenital heart diseases were surgical treated simultaneously.Results There was no death postoperative.Postoperative compli- cations included pneumonia in 1 case,subcutaneous fluid in 2.the foUow-up period was 1-5 years.One patient was found having light notch in sternum.All patients had satisfactory results.In 165 cases stainless steel strut have been taken off postoperatively and no recurrence occurred.Conclusion The modified sternal elevation procedure for pectus excavatum results in an excellent cosmetic outcome.

This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Arctium ; chemistry ; Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Cytokines ; metabolism ; Female ; Furans ; isolation & purification ; pharmacology ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-6 ; metabolism ; Ionomycin ; pharmacology ; Lectins, C-Type ; metabolism ; Lignans ; isolation & purification ; pharmacology ; Lymphocyte Activation ; drug effects ; Mice ; Mice, Inbred BALB C ; Plants, Medicinal ; chemistry ; T-Lymphocytes ; cytology ; drug effects ; immunology ; Tetradecanoylphorbol Acetate ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the intervention of astragalus injection in the kidney injury of infants with congenital heart disease after cardiopulmonary bypass, thus providing a new method for protection of the kidney injury in them.

METHODSForty infants undergoing cardiac surgery with cardiopulmonary bypass were randomly assigned to the test group and the control group, twenty in each group. Astragalus Injection (at the dose of 2 mL/kg) was added in the perfusion fluid before giving to infants in the test group before bypass, while the normal saline of the same volume was added in the perfusion fluid before giving to infants in the control group (P < 0.01). The concentrations of serum tumor necrosis factor-alpha (TNF)-alpha, interleukin-6 (IL-6), cystatin C (CysC), and N-acetyl-beta-D-glucosaminidase (NAG) were detected with ELISA at the following time points, i.e., before bypass (T1), by the end of the surgery (T2), 2 h after surgery (T3), 6 h after surgery (T4), and 24 h after surgery (T5).

RESULTSThe serum CysC concentrations were not significantly higher after CPB (P > 0.05). The urinary NAG level increased significantly in the control group after surgery (P < 0.05), but no obvious increase of the urinary NAG level was found in the test group after surgery (P > 0.05). It was obviously lower than that of the control group (P < 0.05). After CPB serum TNF-alpha and IL-6 levels increased significantly in the control group (P < 0.05), while they were lower in the test group than in the control group (P < 0.01).

CONCLUSIONSCPB may result in the renal tubular injury in infants with congenital heart disease. The application of Astragalus Injection before the CPB plays a role in protecting renal tubular functions.

Acetylglucosaminidase ; urine ; Astragalus Plant ; Cardiopulmonary Bypass ; adverse effects ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Heart Defects, Congenital ; blood ; drug therapy ; urine ; Humans ; Infant ; Interleukin-10 ; blood ; Kidney Function Tests ; Male ; Phytotherapy ; Postoperative Period ; Tumor Necrosis Factor-alpha ; blood

Objective To explore the effects and safety of hemoperfusion(HP) therapy on tetramine poisoning in infants.Methods Thirty-five infants with tetramine poisoning were divided into two groups: HP group(n=18) and non HP group(n=17).The changes of blood tetramine concentration and clinical symptom improving of both groups after the treatment were observed together with the adverse effects of HP group.Results The average blood tetramine concentration of HP group was higher than that of non HP group(342.2?333.4 vs 117.9?50.8 ?g/L,P

OBJECTIVETo explore the effect of the phytoestrogen daidzein on the growth and development of the testis and epididymis in male SD rats.

METHODSThirty 10-week old (early adult) and 30 4-week old (pubertal) male SD rats were included in the study, each age group equally divided into 5 subgroups: normal control, positive control, low-dose, medium-dose and high-dose. The normal and positive control groups were given 1 ml distilled water and the same amount of distilled water containing diethylstilbestrol (DES) at the dose of 0.1 mg/kg, and the low-, medium- and high-dose groups administered daidzein in the dose of 2mg/kg, 20 mg/ kg and 100 mg/kg, respectively, all by gavage for 90 days. Observations were made on the changes in body weight and testicular and epididymal indexes, as well as on the structural changes of the testis and epididymis by H&E staining.

RESULTSThe early adult rats showed no significant differences in body weight and testicular and epididymal indexes between the claiclzein groups and the control (P > 0.05), nor did the pubertal rats in epididymal index (P > 0.05). The testicular index differed significantly between the high-dose group (3.21 +/- 0.07) and the normal control (3.71 +/- 0.45) (P < 0.05). The body weight reduced markedly in the high-dose group (P < 0.05), but with no significant differences between the normal control and the other two dose groups (P > 0.05). No obvious changes were observed in epididymal morphology in all the daidzein groups of the early adult and pubertal rats, but high-dose daidzein resulted in smaller testes and impaired spermatogenesis.

CONCLUSIONThe phytoestrogen daidzein, administered in a high dose, could delay the growth and development of the testis and induce structural changes of testicular tissues in pubertal SD rats.

Animals ; Dose-Response Relationship, Drug ; Epididymis ; drug effects ; growth & development ; Isoflavones ; pharmacology ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; drug effects ; growth & development

BACKGROUND: Increasing studies have shown that cell-sheet engineering is a more promising method for the treatment of myocardial infarction. OBJECTIVE: To construct sandwich-like myocardial cell sheets with electrospun chitosan-collagen membrane as support membrane and with adipose-derived stem cells as seed cells. METHODS: In this study, we prepared electrospun chitosan-collagen membranes at different mass ratio (7:3, 5:5) as a support membrane. Adipose-derived stem cells at passage 4 that were cultured in a thermo-sensitive petri dish for 3 days were seeded onto the two kinds of electrospun membranes at 20 ℃ for 15 minutes until the cell sheets rolled up. Then, the electrospun membranes and cell sheets with the cell layers facing up were placed together onto another thermo-sensitive petri dish in which adipose-derived stem cells were confluent completely. After 30 minutes of culture, the cell sheets were cultured in myocardial cell medium. Two weeks later, cell morphology was observed using multi-photon microscopy. Immunocytochemistry, western blot and flow cytometry were used to detect the expression of TnI and Cx43 in the myocardium. Real-time quantitative PCR was used to detect cardiomyocyte-specific gene expression. RESULTS AND CONCLUSION: From the results of scanning electronic microscopy (SEM) and propidium iodide (PI) staining, we could found that ADSCs grew easily on the chitosan-collagen (5:5) membrane, and there were more dead cells on the chitosan-collagen (7:3) membrane than on the chitosan-collagen (5:5) membrane. After 2 weeks of differentiation with the myocardial cell medium, higher troponin I and Cx43 protein expressions were observed on the chitosan-collagen (5:5) membrane (P＜0.05). Moreover, the mRNA levels of α-skA, β-MHC, TnI, Cx43, ANP, GATA-4 and Nkx2.5 were higher in the chitosan-collagen (5:5) membrane group than the chitosan-collagen (7:3) membrane group. All these results indicate that the chitosan-collagen (5:5) membrane is better for the growth and differentiation of adipose-derived stem cells, as well as for sandwich-like cell-sheet construction.

Objective A resistin binding peptide (RBP) was selected by phage display in our previous work. Studies had shown that RBP could antagonize the role of resistin on the lipid metabolism and endocrine function of adipose tissue, but whether RBP affects the insulin secretion of pancreatic cells is still unknown. The aim of this study is to assess the effect of RBP on basal insulin secretion in RINm5F insulinoma cells. Methods The cell viability was measured by 3-[4,5-dimethyhhiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay. The supernatants were assayed for insulin content by enzyme linked immunosorbent assay (ELISA). Reverse transcriptase-PCR assay and Western blotting were used to determine the expression of glucose transporter 2 (GLUT2) involved in insulin secretion. Cytosolic Ca2+, the trigger of insulin exocytosis, was analyzed with the fluorescent probe FURA-3/AM. Results RBP did no effect on the cell viability with a concentration of 10-8-10-12mol/L of 2 hours intervention. But it stimulated basal insulin secretion of RINm5F cells, accompanied by up-regulated increased expression of GLUT2 and elevated concentration of cytosolic Ca2+. Conclusion RBP could stimulate basal insulin secretion without affecting the cell viability.

A device to produce low temperature plasma ( LTP) was designed and constructed to serve as the ion source of a high resolution mass spectrometry, and was applied to qualitatively analyze the steroid samples. In comparison with conventional electrospray ionization mass spectrometry, low temperature plasma mass spectrometry ( LTP-MS) had some advantages such as simple sample pretreatment and less interference. Mass spectrometry and tandem mass spectrometry were used to characterize the steroid samples in this research, and it was found that the structural stability of each steroid sample was presented in its mass spectrum, while in the tandem mass spectra there were more fragments of H2 O lost. And then the fragmentation process of typical steroid samples in collision induced dissociation ( CID ) was discussed based on theoretical calculation. In addition, by comparing tandem mass spectrometry and the fragmentation process, a pair of isomers of testosterone and dehydroepiandrosterone could be distinguished successfully.

OBJECTIVE Evidience appears that parthenolide (PN) induces anti-tumor effects by NF-κB signal pathway. MCL3 the derivative of PN,is sesquiterpene lactone synthesized by the group of Professor Pan Xiandao.The study was to explore the anti-tumor activity and mechanism of MCL3 in glioma. METHODS The effect of MCL3 on the proliferation of glioma cell lines was examined by MTT assay. Apoptotic activity was investigated by flow cytometry. The Transwell cell invasion assay was used to determine the effect of MCL3 on the G422 cell invasive ability.The effect of MCL3 on the angio-genesis was analyzed by a capillary-like tube formation assay. The subcutaneously transplanted and orthotopic G422 cell xenograft models were used to detect the effect of MCL3 on tumor growth in vivo. The pathological changes were analyzed by H&E staining. Protein level related to the NF-κB signal pathway was dertimined by Western blotting. The effect of MCL3 on the NF-κB transcriptional activity was examined by a dual-luciferase reporter assay.RESULTS The anti-proliferative activity was observed following treatment with MCL3 for 96 h in G422, U-87 MG, U251 and Hs683 cell lines, and the IC50 was 8.94 μmol·L-1,6.44 μmol·L-1,14.8 μmol·L-1,18.9 μmol·L-1,respectively.The percentage of apop-totic cells increased in MCL3-treated G422 cells,and the apoptosis rate was 26.4%(the apoptosis rate was 5.68% in control group).MCL3 could inhibit the invasion in G422 cells,and the invasive inhibition rate was 43.63%(P<0.01)at 10.0 μmol·L-1.MCL3 inhibited tube formation of EA.hy926 cells,and the inhibitory rate was 81.67%(P<0.01)at 10.0 μmol·kg-1.At 40.00 mg·kg-1,MCL3 supressed tumor growth by 79.03% (P<0.01) in tumor weight in subcutaneously transplanted G422 xenograft models, and by 69.97% (P<0.01) in volume in orthopotic G422 xenograft models. H&E staining demonstrated that MCL3 could decrease tumor angiogenesis and invasion, increased necrosis of tumor cells. The dual-luciferase reporter assay showed that MCL3 inhibited NF-κB transcriptional actvity, and the inhibition rate was 50.07%(P<0.05)at 10.0 μmol·L-1compared with control.Moreover,MCL3 inhibited the phos-phorylation of NF-κB in nuclear mediated by supression of phosphorylated IKKα/β and IκB,and decreased the expression of IL-6 regulated by NF-κB.Eventually,the phosphorylation of State3 decreased following the administration of MCL3, resulting in the downregulation of State3 taget genes, including HIF, VEGF,FAK,MMP-2,MMP-9,Bcl-2 and Bcl-xL.CONCLUSION The anti-tumor effect of MCL3 was partly due to the inhibition of NF-κB/IL-6/State3 pathway in glioma.