Objective To clone the genes coding for cysteine proteases (CPs, TvCPs) from Trichomonas vaginalis and to analyze their genetic variations with the related sequences from NCBI database (GenBank) and T. vaginalis Genome Project database from The Institute for Genomic Research (TIGR). Method TvCP genes were amplified using PCR, and inserted into vector pET28b or pBS-T. The recombinant plasmids were then transformed to Escherichia coli BL21 or Top10 strain. The recombinant plasmids were used for sequencing. Homologous TvCP genes were blasted based on NCBI GenBank and TIGR T. vaginalis Genome Project database. The sequences of cloned TvCP genes were aligned and clustered by Clustal X (1.83 version) with retrieved sequences. Comparisons of amino acids among cathepsin L-like TvCPs, human L-like cathepsins and papaya papain were performed using DNAstar software, and their phylogenic tree was constructed based on neighbor-joining method using Clustal X. Results Two TvCP3 clones and one TvCP2 had a high identity of more than 99% with their responding TvCPs. Three clones of TvCP4 genes, GZ-CP4-clone 1-3, belonged to two members of a family showing a high percentage identity of more than 97.5% with the sequences of TvCP4 genes from databases (GenBank and TIGR) both at amino acid and nucleotide levels. Nine homologous TvCP4 pro-enzymes with 304 amino acids and other two members with deletions of N-terminal sequence existed in T. vaginalis sharing a similarity of 62.3-96.7% amino acids, which may evolve by means of gene replication and deletion. TvCP1-4, TvCP12, TvCP25 and CP65 had an identity of 61-88.2% at amino acid levels. So far, all reported sequences of C1 family from T. vaginalis belonged to capanthesin L-like subfamily with the same enzymatic active sites, conserved cysteine residues and similar structural features such as ERFNIN-like motif in pro-enzyme region, suggesting that they might result from gene duplication and mutations. Conclusion TvCPs belong to cathepsin L-like family with genetic diversity, but they have the same active amino acid residues, cysteine residues and similar structural characteristics, suggesting that they may derive from one ancestor.

Objective To evaluate the factors influencing early diagnosis and prognosis in patients with pancreatic carcinoma.Methods The clinical data of 280 patients who had complete follow-up data with pancreatic carcinoma treated from January 2002 to January 2007 were reviewed retrospectively.The medical history and follow-up data were collected from all patients.Survival rate was calculated by the life table method and the Kaplan-Meier estimation.Log-rank test was used for univariate prognostic analysis and Cox regression was used for multivariate prognostic analysis.Results 91.8%of the patients were more than 40 years old and the peak age was 50～73 years old;the major presentations were abdominal pain and jaundice.Major imaging tests included B-ultrasound and CT,the sensitivity was 70.6%,95.3%,respectively;89.3%of patients had combined B-ultrasound and CT examination.The sensitivity of CA19-9 was 81.1%.The median survival time was(7.0±0.5)months.Overall survival rates at 1～5 year survival rates were 28%,9%,6%,2%,and 1%.Univariate analysis suggested that age＞65 years old,CA19-9＞mean value,TNM Ⅲ or Ⅳ stage,lymph nodes invasion,vascular invasion,and metastasis of two or more organs,non-surgical treatment,KPS score＜60 points,weight loss≥5 kg were poor prognostic factors;Cox multivariate analysis showed that treatment modalities,age,TNM stage,KPS score and ascites were independent risk factors for dismal prognosis.Conclusions The age,ascites,tumor stage and treatment modalities affected the prognosis of patients with pancreatic cancer.Early diagnosis and treatment was important to improve the survival time of patients with pancreatic cancer.

Objective To study the influence of the small interfering RNA (siRNA) interference TMPRSS4 expression on human pancreatic cancer SW1990 cell's proliferation and invasion. Methods The four eukaryotic expression vector of TMPRSS4 gene were synthesized in vitro and were transfected transiently into human pancreatic cancer SW1990 cells. TMPRSS4 mRNA expression of transfected cells was detected by real-time RT-PCR. The most efficient eukaryotic expression vector was used to be transfected into SW1990 cells. By using G418, cell strain that can silence TMPRSS4 gene stably was screened. The TMPRSS4 mRNA expression of the stable cell strain was detected by real time PCR TMPRSS4 protein expression was detected by western blot. The proliferation ability of transfected SW1990 cells was detected by CCK-8 method. By Transwell, the invasion change of SW1990 cell was detected. Results A stable cell strain, SW1990/psi TMPRSS4, was successfully constructed, in which the expression level of TMPRSS4 could be reduced stably by RNA interference. Cell transfection efficiency was 82.9%. Compared with the control group, the TMPRSS4 mRNA and protein levels were reduced by 80.1% and 60% ,and number of penetrating cells was 118.6 ±13.4 in SW1990/psi TMPRSS4 group, which was significantly lower than those in the negative control group (157.4 ± 12.9) and control group (157.0±9.5, P <0.01). Cells invasion inhibitory rate was 24.5% in SW1990/psi TMPRSS4 group. The cell proliferation was not significantly different among all the groups. Conclusions A stable cell strain is screened successfully in which the expression level of TMPRSS4 can be reduced stably. The down-regulation of TMPRSS4 gene expression level can inhibit the invasion of SW1990 cells, but has no effect on cell proliferation.

Objective To study the exact function of human gene 5 transactivated by pre-S1 protein of hepatitis B virus(PS1TP5)by investigating the gene expression of PS1TP5 in yeast cells. Methods Reverse transcription-polymerase chain reaction(RT-PCR)was performed to amplify the gene of PS1TP5 using the mRNA of HepG2 cells as template and the gene was cloned into pGEM-T vector.The gene of PS1TP5 was cut from pGEM-T-PS1TP5 vector and cloned into yeast expressive plasmid pGBKT7,then pGBKT7-PS1TP5 was transformed into yeast cell AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western hybridization.Results PS1TP5 gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The results of SDS-PAGE and Western assay showed that the relative molecular weight of the expressed product was about 36 950,and PS1TP5 protein existed in yeast cells.Conclusion The findings suggest that PS1TP5 can be successfully expressed in yeast cell.

BACKGROUNDThe hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein.

METHODSThe reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.

RESULTSForty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class II lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 (LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327).

CONCLUSIONSGenes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S1 protein.

Amino Acid Sequence ; Base Sequence ; DNA, Complementary ; chemistry ; Gene Library ; Hepatitis B Surface Antigens ; genetics ; physiology ; Humans ; Leukocytes ; metabolism ; Molecular Sequence Data ; Plasmids ; Protein Interaction Mapping ; Protein Precursors ; genetics ; physiology ; Recombinant Fusion Proteins ; biosynthesis ; Transcriptional Activation ; Two-Hybrid System Techniques ; Yeasts ; genetics

Authors raised that staging based strategies and practice of integrative medicine (IM) by combining syndrome typing and disease identification, and choosing suitable measures in accordance with different persons and seasonal conditions after more than ten years' clinical practice and researches. Radical operation as prior (as evil eliminating) and strengthening vital qi in perioerative period are best strategy for promoting rapid rehabilitation of early stage prostate cancer patients. Strengthening body resistance to eliminate evil was used in treating advanced prostate cancer patients. For example, a comprehensive treatment program for hormone-dependent patients was combined with endocrinotherapy and Chinese herbs for synergisic efficacy-enhancing actions. In this way, these patients' quality of life (QOL) were improved and time to castration resistant prostate cancer (CRPC) was delayed, even some patients were clinically cured. There are lack of effective medicines and methods for CRPC patients. Greatly tonifying original qi is mainly used for improving their clinical symptoms and prolonging survivals. Practice has proved staging based strategies and practice of IM has favorable advantages in treating prostate cancer, especially showing prospect in prolonging survival and postponing progression of advanced prostate cancer patients. Besides, it also could provide beneficial considerations and inspiration for combination of syndrome typing and disease identification.
Disease Progression ; Humans ; Male ; Medicine, Chinese Traditional ; Neoplasm Staging ; Prostatic Neoplasms, Castration-Resistant ; diagnosis ; Quality of Life

OBJECTIVETo evaluate the efficacy of three-dimensional anal and endorectal ultrasound in identifying the internal opening and tracing the tract of the anorectal fistula.

METHODSFrom November 2008 to January 2010, 127 patients suffering anorectal fistula were managed with three-dimensional endoanal and endorectal ultrasound. The internal opening, the tract of the fistula and fistula trace were identified by the ultrasonography with three-dimensional imaging. All results were confirmed and compared with findings from the operation.

RESULTSThe internal opening of the fistula was specified in 116 patients, the accuracy rate was 91.3% (116/127). The internal opening of the fistula was located above the dentate line in 112 patients, and located in rectal ampulla in 4 patients. The main fistula tract was identified in all the patients, the accuracy rate was 100%. In this group, the fistula tunneled as follows: trans-sphincteric in 47 patients, intersphincteric in 75 cases, supra sphincteric in 2 cases, extra sphincteric in 3 patients. Secondary extension was found in 37 patients, the accuracy rate was 100% (37/37).

CONCLUSIONSThree-dimensional anal and endorectal ultrasound is an effective way for localizing the internal opening and the tract of anorectal fistula. It can provide valuable information for curative operation.

Adult ; Aged ; Endosonography ; methods ; Female ; Humans ; Imaging, Three-Dimensional ; Male ; Middle Aged ; Rectal Fistula ; diagnostic imaging ; Young Adult

OBJECTIVETo study the clinical effects of AO distal humerus plate (DHP) for the treatment of distal humeral fractures in elderly osteoporotic patients.

METHODSFrom September 2008 to January 2010, 18 elderly osteoporotic patients with distal humeral fractures were treated with open reduction and internal fixation with DHP. There were 3 males and 15 females, ranging in age from 62 to 83 years (averaged, 71.4 years). According to AO classification, 1 patient was Type A2, 3 patients were Type A3; 2 patients were Type B1, 2 patients were Type B2; 3 patients were Type C1, 4 patients were Type C2, 3 patients were Type C3. The surgical approaches were either bilateral or via olecranon process of ulna. Early mobilization was initiated after surgery. Functional results were evaluated according to the Mayo elbow performance score (MEPS).

RESULTSThe average duration of follow-up was 14.6 months (ranged from 11 to 24 months). All the patients had a complete healing of their fractures. There was no infection, hardware failure or loss of reduction after the operations. The average Mayo elbow performance score (MEPS) was (92.2 +/- 9.6) (65 to 100 points), among which the score of pain was (42.5 +/- 5.8) (30 to 45 points), the score of motion range was (19.2 +/- 1.9) (15 to 20 points), the score of stability was (9.2 +/- 1.9) (5 to 10 points), and the score of function was (21.4 +/- 2.9) (15 to 25 points). According to MEPS, 11 patients got excellent results, 5 good and 2 fair.

CONCLUSIONTreatment of distal humeral fractures in elderly osteoporotic patients with AO distal humeral plates can obtain immediate stabilization which facilitates early mobilization of the elbow.

Aged ; Aged, 80 and over ; Bone Plates ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Humeral Fractures ; surgery ; Male ; Middle Aged ; Osteoporotic Fractures ; surgery

OBJECTIVETo evaluate the correlation between different therapies and survival of liver metastasis from colorectal cancer ( LMCC) , and to compare the clinical outcome of synchronous liver metastasis (SLM) with that of metachronous liver metastasis (MLM).

METHODSThe clinical data of 363 patients with LMCC were retrospectively reviewed with focus on the correlation between different therapy and survival.

RESULTSOf these 363 patients, 160 had SLM and 203 had MLM. Between the SLM and MLM group, there was no significant difference in age, or gender or primary cancer site (P > 0. 05 ), but significant differences were observed in condition of liver metastasis including liver lobe involved, focus number, maximum focus diameters and level of serum CEA and CA199 before therapy(P <0. 05). Ninety-one patients underwent curative hepatic resection, 22 of them in the SLM group and 69 in the MLM group. Mortality rate related to operation was 4. 5% (1/22) in SLM group and 2. 9% (2/69) in MLM group( P < 0.05). All patients were followed until 31/6/2005. The 3-year survival rate was 5. 2% with a median survival time of 10 +/- 1 months for the SLM group, and it wasl6. 4% and 17 +/- 1 months for the MLM group (P<0.01). Regarding to the treatment modalities, the 3-year survival rate was 30. 2% with a median survival time of 26 months for curative hepatic resection group, and it was 0% - 16. 7% and 10 - 17 months for non-operation groups treated by intervention, chemotherapy, radiofrequency therapy, percutaneous ethanol injection and Chinese traditional drugs (P <0. 05, P <0. 01 ).

CONCLUSIONCurative hepatic resection is still the first choice for liver metastasis from colorectal cancer improving the survival significantly. Other non-operative methods also can improve phase II resection rate. Metachronous liver metastasis has higher resection rate and better survival than the synchronous liver one.

Antineoplastic Agents, Phytogenic ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; CA-19-9 Antigen ; blood ; Carcinoembryonic Antigen ; blood ; Chemoembolization, Therapeutic ; Colonic Neoplasms ; blood ; pathology ; therapy ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Hepatectomy ; methods ; Humans ; Liver Neoplasms ; blood ; secondary ; therapy ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Phytotherapy ; methods ; Rectal Neoplasms ; blood ; pathology ; therapy ; Retrospective Studies ; Survival Analysis

OBJECTIVETo establish a stable method for obtaining large quantity of highly purified immature dendritic cells (imDCs) in vitro, and identify the morphology, function and surface markers of the cells.

METHODSCD117(+) hemopoietic stem cells (HSCs) were isolated and purified from the bone marrow of healthy C57 mice by magnetic affinity cell sorting. After cell expansion by treatment with stem cell factor (SCF) and interleukin-3 (IL-3), the HSCs were induced for directional differentiation into imDCs by treatment with GM-CSF, IL-4 and IL-10. The imDCs obtained were identified by morphological and functional observation under inverted microscope, scanning electron microscope and transmission electron microscope, followed by detection of the expressions of the surface markers using flow cytometry.

RESULTSAfter 3, 5 and 7 days of culture in the presence of SCF+IL-3, the cells were expanded by 10.34-/+1.43, 22.65-/+2.71 and 54.39-/+3.08 folds, respectively. The HSCs were successfully induced to differentiate into imDCs with phagocytotic activity. The dendrites of the imDCs were short small, and appearing spinous. The expressions of surface markers were detected from the cells showing the phenotype of CD11c(+), I-A/I-E(low), CD40(-), CD80(-), CD86(-).

CONCLUSIONThe method described allows steadily acquisition of large quanty of highly purified imDCs and of their effective identification in vitro.

Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Separation ; methods ; Cells, Cultured ; Dendritic Cells ; cytology ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Proto-Oncogene Proteins c-kit