Four screening methods, colorimetric assay, blood-plate hemolysis method, surface tension activ- ity and oil spreading technique were introduced to isolate strains producing bio-demulsifiers from 6 different bacteria source samples. The results of various screening methods were evaluated in this paper. Seventeen demulsifying strains were obtained, which are qualified in demulsification test of kerosene model emulsions. Among them, 5 strains showed high demulsifying ability, achieving 70% plus demulsifying ratio within 24 hours. Petroleum-contaminated soil, excess sludge from biological process treating oilfield produced water and sludge from municipal wastewater treatment plant were the best among all tested sources. Due to the determination limit, the colorimetric assay and blood-plate hemolysis method are not competent to screen bio-demulsifiers strains. The measurement of surface tension and oil spreading method were easy but accu- rate methods to isolate bio-demulsifiers strains. Although demulsification test of model emulsion is an effec- tive technique to target strains with the capability of breaking emulsions, it is sophisticated and time-con- suming. Thus it is recommended to use surface tension and oil spreading methods in pre-screening and vali- date the results in demulsification test with kerosene model emulsions.

Objective To evaluate the expression and significance of differential expression gene Hepsin in the prostate cancer (PC) screened by the cDNA microarray technique.Methods The techniques of semiquantitative RT-PCR and Western blotting were used to detect the mRNA and protein expression of Hepsin. Specimens of 40 cases of PC, 15 benign prostatic hyperplasia (BPH) and 6 normal prostate tissues were examined by the immunohistochemical stain.Results Hepsin was more expressed in PC tissue than normal prostate tissue (P=0.026) and was confirmed by Western blot analysis. Immunohistochemical test confirmed this and demonstrated that Hepsin did not expressed in normal prostate but expressed in PC and BPH and there was a significant difference in Hepsin expression level between PC and BPH tissues ( P=0.000).Conclusion Hepsin high expressed in PC may be a new molecular marker in early diagnosis of PC and a new target for gene therapy of PC.

OBJECTIVETo study the risk factors related to the survival rate, recurrence and metastasis of malignant fibrous histiocytoma of bone.

METHODSFrom July 1997 and July 2010, 56 patients with malignant fibrous histiocytoma of bone were treated. Univariate and multivariate analysis were performed to determine the probable risk factors including gender, age, tumor location, tumor size and so on.

RESULTSForty-four cases were followed up ranged from 2 weeks to 78 months (medium 33.3). The 5-year overall survival rate was 50.1%, local recurrence rate 40.9% with a median time of 12 months (3 to 60 months) and metastatic rate 27.5% (11/40) with a median time of 6.5 months (2 to 23 months). Univariate analysis indicated that gender, condition of presentation (primary case or recurrence case), tumor location, surgical margin and surgical stage were significantly related to survival rate (P < 0.05), and tumor location and surgical margin were related to local recurrence rate (P < 0.05), and important vessel or nerve invasion was related to metastatic rate (P < 0.05). Multivariate analysis showed that surgical margin and surgical stage were independent risk factors for survival rate, of which surgical margin was the independent risk factor for recurrence rate.

CONCLUSIONSSurgical margin and surgical stage are independent risk factors for survival rate, of which surgical margin is the independent risk factor for recurrence rate.

Adolescent ; Adult ; Aged ; Bone Neoplasms ; diagnosis ; pathology ; Female ; Histiocytoma, Malignant Fibrous ; diagnosis ; pathology ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Neoplasm Recurrence, Local ; pathology ; Prognosis ; Retrospective Studies ; Survival Rate ; Young Adult

OBJECTIVESTo study the change of ability to transform from 5'-deoxy-fluorouracil monophosphate (5'-DFUR) to fluorouracil (5-FU) in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase (TP) gene. And to discuss the anti-cancer activity of 5'-DFUR to SW480 and LOVO cells.

METHODSTP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6.3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5'-DFUR both in 2 cells and medium were detected by high performance liquid chromatography (HPLC). The 50% inhibitory concentration (IC50) of 5'-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTT assay.

RESULTSThe colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were (695 ± 171) folds (t = -7.00, P = 0.002) and (282 ± 87) folds (t = -5.61, P = 0.030), respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5'-DFUR in the medium cultured SW480-TP and LOVO-TP were increased compared with their parent cells, respectively (t = 19.406-66.921, P < 0.01), whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5'-DFUR on SW480-TP and LOVO-TP were (587 ± 17) µmol/L and (1088 ± 89) µmol/L respectively, and there were significantly less than their parent cells (t = -32.59 and -8.52, P < 0.01).

CONCLUSIONSThe stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5'-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.

Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Floxuridine ; metabolism ; Fluorouracil ; metabolism ; Humans ; Thymidine Phosphorylase ; genetics ; Transcription, Genetic ; Transfection

Objective To investigate the effects of dominant-negative truncation mutant?NTCF4, lacking the N-terminal form of TCF4 gene,on biological characteristics of renal cancer cell line GRC-I and explore the molecular mechanisms.Methods GRC-I cell was transfected with pCDNA3-?NTCF4 eukary- otie expression plasmid,pCDNA3 empty vector to construct the stable cell line GRC-I/?NTCF4 and GRC-I/ Mock respectively.The morphological changes of stable cells were observed and the cells growth curve was detected through light microscope.The cellular proliferation activities were determined using the MTT assay. The protein expression of Wnt pathway downstream target gene C-Myc and Cox-2 was evaluated by immuno- cytoehemieal method and Western Blot analysis.Results After the dominant-negative?NTCF4 gene was permanently expressed,the GRC-I/?NTCF4 stable cells morphologically showed that appearance changed from circular to long-spindle shape,growth rate decreased with less karyosehisis found,malignant pheno- types reversed to normal renal tubular cells.MTT assay revealed that the proliferation activities of GRC-1/?NTCF4 cells were inhibited by 11.2%-35.5% compared with GRC-I cells (P＜0.05),while the GRC- I/Mock cells have no difference with the control cells.Immunocytochemical analysis and Western Blot showed that the C-Myc and Cox-2 protein expression level of GRC-I/?ANTCF4 cells were significantly sup- pressed in comparison with that of GRC-I/Mock and GRC-I cells.Conclusions The dominant-negative truncation mutant?NTCF4 could partially inhibit the growth of renal cancer cells and down-regulate the pro- tein expression of Wnt pathway target gene C-Myc and Cox-2.These findings provide a experimental founda- tion for applying cell signal therapy to renal cell cancer by blocking the Wnt signaling pathway.

OBJECTIVETo study the clinical features and surgical treatment of osteoid osteoma and improve the diagnostic therapeutic level.

METHODSClinical data of 35 patients (25 males and 10 females) with osteoid osteoma diagnosed and treated between January 1997 to October 2009 were retrospectively reviewed. The average age was 21 years (ranged, 6 to 49 years). The average interval time between onset of symptoms and diagnosis was 12 months (ranged, 1 to 24 months). The most common sites were the tibia (13 patients) and the femurs (7 patients). The most common presenting complaints for patients with osteoid osteoma was pain which usually responded to NSAIDs and was generally more severe at night. The imaging manifestations revealed a circular or oval nidus. All the patients underwent surgical treatment. The tumors were treated with curettage or excision with autograft or allograft in 19 patients, simple surgical resection in 9 patients, curettage or excision with autograft or allograft and fixation in 7 patients.

RESULTThe mean follow-up period was 49 months (ranged,2 months to 12 years). The symptom of pain disappeared after operation. There were no evidence of recurrence. Tibial pathological fracture happened in one patient 4 months postoperatively, and the patient got healing after plate-screw internal fixation. One patient with sinus formation 5 years postoperatively got wound healing after sinus resection, intramedullary nail removal and debridement.

CONCLUSIONAccording to the typical clinical presentation, radiographic findings, the diagnosis of osteoid osteoma is not difficult. Once the diagnosis is confirmed, the operation should be carried out as early as possible to relieve the symptoms, improve the quality of life and prevent long-term complications.

Adolescent ; Adult ; Bone Neoplasms ; diagnosis ; surgery ; Child ; Female ; Humans ; Male ; Middle Aged ; Osteoma, Osteoid ; diagnosis ; surgery

OBJECTIVETo investigate the inhibiting impact of 5'-deoxy-5-fluorouridine (5'-DFUR) on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA.

METHODSTP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP, and the transfection efficiency was analyzed by flow cytometry. TP mRNA and protein expressions were detected by RT-PCR and Western blotting respectively. The IC50 of 5'-DFUR on TP-transfected LOVO and parental cell were evaluated by MTT assay. The volumes of 5-FU converted from 5'-DFUR in media, where TP-transfected and parental LOVO were cultured, were detected by HPLC.

RESULTSThe stable transfectants passed 5 generations were obtained and the transfection rate was 95%. Compared with parental cell, the RQ values of mRNA expression in TP-transfected LOVO was (282.5±86.8) folds higher significantly (P<0.01), also the TP protein expression of TP-transfected LOVO was obviously up-regulated as compared to parental cells. The IC50 value of 5'-DFUR of TP-transfectants was (1087.7±89.1) μmol/L, less than (1607.3±56.8) μmol/L of parental cells significantly (P<0.01), while there was no significant difference between parental cells and vector-transfectants [(1699.5±38.7) μmol/L, P>0.05]. HPLC revealed that when medium was added with 0, 500, 1000, and 2000 μmol/L of 5'-DFUR respectively, 0, 2.10, 3.13, and 7.19 μmol/L of 5-FU was found in the parental cells culture, while 0, 22.16, 30.94 and 40.02 μmol/L of 5-FU was found in TP-transfectants culture, but no 5-FU was found in the vector-transfectants culture.

CONCLUSIONTP cDNA transfection into LOVO can up-regulate the TP mRNA and protein expressions, increase the 5-FU converted from 5'-DFUR, and enhance the cytotoxic effect of 5'-DFUR on the LOVO cells.

Cell Line, Tumor ; Colonic Neoplasms ; pathology ; DNA, Complementary ; genetics ; Floxuridine ; pharmacology ; Humans ; Thymidine Phosphorylase ; genetics ; Transfection

Carnosic acid (CA) is the main phenolic diterpenoid active ingredient in plants such as rosemary and sage, and has antiviral, antioxidant, anti-inflammatory effects and so on, however, its antiviral activity against influenza virus infections was not reported. In this study, antiviral activities against influenza A virus infections of three main bioactive ingredients from rosemary, including rosmarinic acid, CA and ursolic acid, were evaluated using virus titer titration assay, and CA showed remarkable inhibition on influenza H5N1 replication in A549 cells. The antiviral activity of CA was further confirmed and its mechanism of action was investigated using the indirect immunofluorescence assay (IFA), Western blot and real-time fluorescence quantification polymerase chain reaction (qRT-PCR). The results showed that the 50% effective concentration (EC₅₀) of CA against influenza H5N1 in A549 cells and MDCK cells were 4.30 and 3.64 μmol·L^-1, respectively. Meanwhile, CA also showed inhibition on influenza virus 2009panH1N1 (EC₅₀: 10.1 μmol·L^-1) and H3N2 (EC₅₀: 12.8 μmol·L^-1) replications in A549 cells. Mechanistic studies showed that antiviral activity of CA is related to its induction of heme oxygenase-1 (HO-1) in A549 cells and suppression on production of reactive oxygen in H5N1-infected cells.

OBJECTIVETo construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC).

METHODSPoly A(+) RNA was isolated from RCC lines 786-O (tester) and renal cell (RC) lines HK-2 (driver), respectively. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit (Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F'. All positive clones picked out were digested and some of which were sequenced.

RESULTSThe SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly, 2 represented unknown genes and the other 48 derived from 36 known genes.

CONCLUSIONThe quality of the SSH library of human RCC is reliable and its construction is the basis for further screening differentially expressed genes of RCC.

Adenocarcinoma, Clear Cell ; genetics ; Cell Line, Tumor ; Gene Library ; Humans ; Kidney Neoplasms ; genetics ; Nucleic Acid Hybridization ; methods

OBJECTIVETo investigate the influence of the amount of portal blood stasis removal on endotoxemia and liver function after liver transplantation.

METHODSForty-seven patients who received liver transplantation from February 2006 to November 2007 were divided into 2 groups according to the amount of portal blood stasis removal during operation: group A (n = 26) 50 ml and group B (n = 21) 200 ml of portal blood stasis removal respectively. The levels of plasma endotoxin, D-lactate, tumor necrosis factor-alpha, interleukin-6, liver function and blood coagulation were examined and analyzed.

RESULTSUnder the condition of no significant difference in sex, age, primary liver diseases and Child-pugh's classification, cold ischemic time, total operation and anhepatic time, operation methods, volume of blood loss and transfusion, and all preoperative observations. Most of observations showed the restoration of the patients in group B was better than that in group A. The plasma levels of endotoxin, D-lactate, tumor necrosis factor-alpha, interleukin-6, alanine aminotransferase, aspartate aminotransferase, prothrombin time and activated partial thromboplastin time in group B were significantly lower than those in group A (P < 0.05). The level of plasma prealbumin in group B was significantly higher than that in group A (P < 0.05).

CONCLUSIONSThe removal of 200 ml portal blood stasis leads to a better results than that of 50 ml, and it can help alleviate endotoxemia and facilitate the restoration of the liver function after liver transplantation.

Adult ; Aged ; Bloodletting ; methods ; Endotoxemia ; prevention & control ; Female ; Humans ; Liver ; physiopathology ; Liver Transplantation ; Male ; Middle Aged ; Portal Vein ; surgery ; Postoperative Complications ; prevention & control ; Reperfusion Injury ; prevention & control