Objective To subclone express and identify the immune mapped protein 1 IMP1 which encodes a surface an?tigen of Toxoplasma gondii. Methods The cDNA of T. gondii RH strain was synthesized by reverse transcription PCR the IMP1 open reading frame ORF was amplified by PCR using the T. gondii RH strain cDNA as template the PCR products were identified by TA?cloning and sequencing then the IMP1 ORF was subcloned into the NdeⅠand Xho I sites of the vector pET28b and the positive recombinant pET28b?IMP1 was identified by double?digesting and sequencing. The protein of 6 × His tagged IMP1 was inducibly expressed in E. coli strain BL21 DE3 with isopropylβ?D?1?thiogalactopyranoside IPTG and the induction time concentration of IPTG and temperature gradients to optimize protein expression conditions were determined. After the cells carried IMP1 were induced by the optimized conditions and harvested the resulting bacteria were suspended in resuspension buffer and lysed by sonication and the supernatants were loaded onto the Ni2+Chelating Sepharose Fast Flow col?umn for affinity chromatography of the N?terminal 6 × His tagged IMP1 protein. Finally the fusion IMP1 proteins were identified by Western blotting. Results The ORF sequence of IMP1 was successfully subcloned from the cDNA of Toxoplasma Gondii RH strain and the amplified product was sequenced and identified based on which the IMP1 ORF gene was inserted into the prokaryotic expression vector pET28b and the recombinant pET28b?IMP1 was constructed successfully. The double?digesting and sequencing results indicated the validity of the recombinant vector. And the optimized conditions for the expression of IMP 1 was determined namely 0.3 mmol/L IPTG induction for 9 h at 20℃. Furthermore IMP1 protein was expressed solubly and che?lated on Ni2+sepharose beads with high affinity thus this protein could be purified efficiently by affinity chromatography. The pure fusion protein was confirmed with fine immunocompetence by SDS?PAGE and Western blotting. Conclusions IMP1 pro? tein can be high efficiently expressed by the E. coli prokaryotic expression systems the protein of IMP1 is soluble and has stable characters. The study may lay a useful foundation for the following works including in vivo expression of IMP1 crystal structure study of IMP1 and anti?toxoplasmosis subunit vaccine development.

Objective To explore the cultural method of oral keratinocytes and make preparations for further investigation in using oral keratinocytes as a new choice of seed cells for the reconstruction of tissue-engineered urethra. Methods Oral keratinocytes of rabbits were isolated in vitro and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3) or a culture dish without i3T3 respectively. Cell morphology was observed and cell growth was detected at intervals.Meanwhile,oral keratinocytes obtained from in vitro culture were performed immunofluorescence staining with broad-spectrum keratin antibody(AE1/AE3) and keratin 19 antibody(K19).The percentage of positive cells of passage 2 reactive to AE1/AE3 was assessed by flow cytometry. ResultsOral keratinocytes seeded onto a feeder layer of i3T3 exhibited finer morphous,better amplification capability,and could be passed for 7 or 8 generations.However,those cultured without i3T3 took on various morphous and could only be subcultured 2 generations before ageing.It was indicated by immunofluorescence staining that oral keratinocytes obtained from in vitro culture were positive for AE1/AE3 staining and 40% were positive for K19 staining.The result of flow cytometry revealed that the amout of positive keratinocytes reactive to AE1/AE3 was more than 95% of total cellular score. Conclusion Oral keratinocytes of rabbits can be cocultured with i3T3 in vitro and magnitude quantity can be attained,laying a favourable foundation for oral keratinocytes as a new choice of seed cells for urethral reconstruction with tissue engineering.

AIM:To study the effects of adiponectin on H 2 O2-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells.METHODS:Cell viability was determined by MTT assay .H2 O2-induced cell in-jury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed .The level of tau phosphorylation as well as the activities of protein phosphatase 2A (PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting . RESULTS: Adiponectin significantly attenuated H 2 O2-induced cell injury (P<0.01).Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimula-tion with H2O2(P<0.01).Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin (P<0.01).Adiponectin increased the phosphorylation level of GSK-3βat Ser9 site under H2O2stimulation (P<0.01).CON-CLUSION:Adiponectin protects SH-SY5Y cells against H2O2-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.

BACKGROUND: The association of genetic variation in the IRAK-1 gene with sepsis outcome has been proved. However, few studies have addressed the impact of the IRAK-4 gene variants on sepsis risk. This study aimed to determine whether the polymorphisms in the IRAK-4 gene are associated with susceptibility to and prognosis of severe sepsis in the Chinese Han ethnic population.METHODS: In this case-control study, 192 patients with severe sepsis hospitalized in the emergency department of Zhongshan Hospital from February 2006 to December 2009 and 192 healthy volunteers were enrolled. Exclusion criteria included metastatic tumors, autoimmune diseases, AIDS or treatment with immunosuppressive drugs. This study was approved by the ethical committee of Zhongshan Hospital, Fudan University. Sepsis patients were divided into a survival group (n=124) and a non-survival group (n=68) according to the 30-day mortality. Primer 3 software was used to design PCR and sequencing primers. Genomic DNA was extracted from peripheral blood mononuclear cells. Seven tagSNPs in IRAK-4 were selected according to the data of the Chinese Han population in Beijing from the Hapmap project and genotyped by direct sequencing. The chi-square test was used to evaluate the differences in genotype and allele frequencies between the two groups.RESULTS: The distributions of all tagSNPs were consistent with Hardy-Weinberg equilibrium. The allele and genotype frequencies of rs4251545 (G/A) were significantly different between the severe sepsis and healthy control groups (P=0.015, P=0.035, respectively). Carriers of the rs4251545A had a higher risk for severe sepsis compared with carriers of the rs4251545G (OR=1.69, 95％ CI: 1.10-2.58). The allele and genotype frequencies of all SNPs were not significantly different between the survival group and non-survival group.CONCLUSION: These findings indicate that the variants in IRAK-4 are significantly associated with susceptibility to severe sepsis in the Chinese Han ethnic population.

To study the anti-coagulant effect and influence of danggui Sini decoction (DSD) on rat's plasma endogenous metabolites by animal experiment and ¹H-NMR based metabolomics method. After intragastric administration of Danggui Sini Decoction for 7 days, Plasma thrombin time (TT) was measured. Rat plasma metabolic fingerprint in two groups was analyzed using ¹H-NMR, based on which the principal component analysis( PCA) and orthogonal partial least-squares discriminant analysis(OPLS-DA) models for metabonomic analysis. Potential biomarkers were screened by using variable importance in the projection (VIP) and T test. DSD could prolong TT of the rat significantly (P < 0.05). Compared with control group, six kinds of endogenous metabolites in DSD group change significantly (P < 0.05), among which isobutyrate, carnitine and phenylalanine content had an upward trend (P < 0.01) and lysine, Histidine and cholesterol content had a downward trend (P < 0.05). It is likely that carnitine, phenylalanine, Histidine and cholesterol are the potential metabolic markers in the anti-coagulant process and DSD affects the platelet aggregation and the expression of tissue factor and fiber protease by regulating the energy, amino acid and lipid metabolism.
Animals ; Anticoagulants ; chemistry ; Blood Coagulation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; Female ; Male ; Metabolomics ; methods ; Proton Magnetic Resonance Spectroscopy ; methods ; Rats

OBJECTIVETo study the relationship of expression of mucin 1 and E-cadherin with recurrence of pleomorphic adenoma in salivary gland, and to investigate the signal to predict the recurrence potential of the tumor.

METHODS; The capsule of tumor was observed by microscope. The expression of mucin 1 and E-cadherin in 33 cases of primary adenoma, 12 cases of recurrent pleomorphic adenomas and 7 cases of malignant pleomorphic adenomas were detected by immunohistochemistry.

RESULTSThere was no significant difference about the status of capsule and the positive rate of mucin 1 expression between primary and recurrent pleomorphic adenoma (P > 0.05). The abnormal distribution of mucin 1 expression was observed in recurrent pleomorphic adenoma (6/8), which was characterized by the positive staining of the whole cytomembrane. On the other hand, positive staining of the primary pleomorphic adenoma was observed on the top of the membrane (19/21). The difference was statistically significant (P < 0.05). The staining pattern in malignant pleomorphic adenoma was similar with the recurrent ones except higher ratio of positive expression. No significant different was observed among the three kind of tumors on the expression rate of E-cadherin (P > 0.05).

CONCLUSIONThe status of capsule didn't have much actual usage in predicting the recurrence of pleomorphic adenoma. There was no significant relationship between the expression of E-cd and the recurrence of the tumor. The abnormal distribution of mucin 1 expression contributes to the invasiveness of the tumor and can be used as the predictive signal for recurrence of pleomorphic adenoma.

Adenocarcinoma ; Adenoma, Pleomorphic ; physiopathology ; Cadherins ; metabolism ; Humans ; Immunohistochemistry ; Mucin-1 ; metabolism ; Neoplasm Recurrence, Local ; Salivary Gland Neoplasms ; physiopathology

Objective To assess the health status of rodent laboratory animals by pathological diagnosis, our lab has being take apart in investigating the quality of laboratory animals in Beijing area for years and offer some advices for standardized breeding to ensure accurate results of scientific research.This paper focuses on the analysis of laboratory rodent samples that collected in October 2014.Methods We collected the heart, liver, spleen, lung, kidney, large intestine and small intestine, and put these organs into 10%Calcium formaldehyde solution for fixation, and then prepared into two different sections for optical microscopy observation including all paraffin specimens stained with H&E and the frozen sections stained with Oil Red-O and PAS.Results The vast majority of laboratory rodents were up to standard, but there still a problem in individual units.The main problem is liver and lung disease.The rate of Hepatocyte swellingis 6%(mouse), 2.5% (rat), 8.2% (guinea pig), moreover part of them were lipidosis, according to Oil Red-O stain.the mainly problem of lung is congestion ,edema and Interstitial pneumonia ,the detectable rate of pulmonarydiseases is 15.5%(guinea pig).Conclusions The vast majority of laboratory rodents were pathologically diagnosed as healthy animals.The liver disease may be caused by improper feeding.And disease of lung may led by haze, unqualified bedding and low temperature.

Objective To explore the neuron injury in rat hippocampus induced by Aβ25-35 and the cyclophilin A (CyPA) expression changes in these neurons. Methods Sixty healthy Wister rats were equally randomized into experimental group and control group (n=30); AD rat models in the experimental group were established by injection of Aβ25-35 into the bilateral hippocampus of rats,and rats of the control group were received NS injection. The morphological features of neurons in the CA1 area of hippocampus were observed by HE staining; the neuron apoptosis was determined with TUNEL staining; the mRNA and protein expressions of CyPA were detected by PT-PCR and Western blotting,respectively. Results Aβ25-35 caused damage and apoptosis of neurons in the CA1 area of hippocampus; with time being prolonged,the cell injury aggravated and apoptosis increased in the CA1 area ofhippocampus; significant differences were noted as compared with those in control group 1,7 and 14 d after the inducement (P＜0.05).After injection of Aβ25-35 into the hippocampus of rat,the mRNA and protein expressions of CyPA were obviously changed:in early stage,the expressions increased,and then,the expressions decreased gradually; significant differences were noted as compared with those in control group 1 and 7 d after the inducement (P＜0.05); the protein expression of CyPA in the experimental group 14 d after the inducement was significantly decreased as compared with that in the control group (P＜0.05). Conclusion Aβ25-35 plays a neurotoxicity role through aggravating the apoptosis of neurons; and the increment of CyPA expressions maybe play an endogenously protective role in these damage.

Objective To explore the effect of cyclophilin A (CyPA) on apoptosis of PC 12 cells induced by Aβ25-35 and its potential mechanism. Methods PC 12 cells were divided into normal control group (0 μmol/L Aβ25-35), Aβ25-35 inducement group (10 μmol/L Aβ25-35) and drug protection groups (0.1, 1,10 and 100 nmol/L CyPA+10 μmol/L Aβ25-35). Cells in the drug protection groups were pretreated by CyPA of different concentrations for 30 min, and then co-cultured with Aβ25-35 We evaluated the survival rate of PC12 cells with MTT assay, analyzed the apoptosis of PC12 cells with Hoechst33258 staining, and detected the mRNA expressions of Bcl-2 and Bax with PT-PCR and the protein levels of Bcl-2 and Bax with Western blotting. Results Cells pretreated wth CyPA of 1, 10 and 100 nmol/L enjoyed an obvious elevation of survival rate of PC 12 cells, a significant reduction of apoptosis induced by Aβ25-35,an obvious increase of mRNA expression of Bcl-2 and protein level of Bcl-2, and a statistical decrease of mRNA expression of Bax and protein level of Bax as compared with those cells of the Aβ25-35 inducement group (P<0.05);and these effects were dose-dependent. Conclusion CyPA could resist the toxic role of Aβ25-35 on PC 12 cells and reduce the apoptosis in a dose-dependent manner by up-regulation of anti-apoptosis gene Bcl-2 and down-regulation of apoptosis gene Bax.

OBJECTIVETo investigate the safety and feasibility of testis-sparing surgery (TSS) in the treatment of testicular tumor.

METHODSWe retrospectively analyzed the clinical data of 8 cases of benign testicular tumor treated by TSS in our hospital from October 2005 to March 2012.

RESULTSThe 8 patients, aged 18-67 (mean 45) years, were preoperatively diagnosed with benign testicular tumor and all underwent partial testis resection. Rapid intraoperative pathology showed the incisal margins to be negative. Postoperative pathological examination confirmed Sertoli cell tumor in 3 cases, adenomatoid tumor in another 3, and mature teratoma in the other 2. The patients were followed up for 6 months to 7 years (mean 4 years), which revealed no relapse and metastasis, nor significant differences from the baseline in the testosterone level, IIEF score, and routine semen parameters.

CONCLUSIONTestis-sparing surgery is one of the effective options for the management of benign testicular tumor, which can maximally preserve the testis tissue and protect the patient's sexual function.

Adolescent ; Adult ; Aged ; Humans ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Organ Sparing Treatments ; methods ; Retrospective Studies ; Sertoli Cell Tumor ; pathology ; surgery ; Teratoma ; pathology ; surgery ; Testicular Neoplasms ; pathology ; surgery ; Testis