This paper is aimed to investigate the effect of titanium (Ti) particles and tumor necrosis factor alpha (TNF-alpha) on the expressions of MMP-1, 2, 3 in human synovial cells, so as to explore the possible mechanism of osteolysis post-operation of metal-on-metal total joint arthroplasty in human synovial cells induced by Ti particles. In vitro cell cultures, human synovial cells were treated by Ti particles and/or TNF-alpha. The total RNA was isolated at 2 hours after the treatment. The gene expression of MMP-1, 2, 3 was analyzed by Semi-quantitative Reverse-transcriptional PCR and quantitative real-time PCR. Cell supernatant was collected at 12, 24, 48 hours after the treatment and Gelatin zymography was performed to detect the activity of MMP-2. Compared to those in the control group (untreated), Ti particles and TNF-alpha increased the gene expression of MMP-1, 2, 3 respectively (P < 0.05), and the effect of combination of the two was even more significant (P < 0.01). The trend of activities of MMP-2 is similar with gene expression. Ti particles and TNF-alpha increased MMP-2 activities by 1.3 times and 1.5 times respectively (P < 0.05), and the combination of the two increased by 1.7 times (P < 0.01). Ti particles and TNF-alpha-induced the stimulation of MMP-1, 2, 3 expressions and MMP-2 activities in human knee joint synovial cells may be involved in aseptic loosening after metal-on-metal arthroplasty through increasing the degradation of bone matrix and declining of osseous support structure mechanics.
Cells, Cultured ; Humans ; Joint Prosthesis ; Knee Joint ; cytology ; Matrix Metalloproteinase 1 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 3 ; genetics ; metabolism ; Matrix Metalloproteinases ; genetics ; metabolism ; Particle Size ; Prosthesis Failure ; adverse effects ; RNA ; genetics ; metabolism ; Synovial Membrane ; cytology ; enzymology ; Titanium ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the gene mutation frequencies and patterns of β-thalassemia (β-thal) in the minority populations of Guizhou province.

METHODSThree thousand and five hundred couples in the reproductive age were screened by using automatic hemocyte analyzer and hemoglobin autoanalyzer-variant. The diagnostic criteria for β-thal were: the mean corpuscular volume (MCV) was ≤ 82 fl, and the HbA(2) level was ≥ 3.5%. A total of 194 positive samples were detected and further identified by PCR-reverse dot blot (PCR-RDB) assay for 18 common β -thal mutations in Chinese population. Those subjects with positive phenotypes but without the 18 common β-thal mutations were subjected to DNA sequence analysis of the β-globin gene.

RESULTSOne hundred and eighty-nine samples with gene mutations were observed from the 3500 samples, with the incidence of β-thal being 5.4%. A total of 10 different β-thal mutations were identified from the 189 diagnosed samples. The five most common mutations were as the following: CD17 (43.9%), CD41-42 (38.6%), IVS-II-654(10.1%), -28 (2.6%) and CD71-72 (1.6%). In addition, a novel β-globin gene mutation (-CD53) allele was detected. One rare mutation of IntM was observed.

CONCLUSIONThe minority population in Guizhou province is of high risk of β-thal. It is recommended that more attention should be paid to detect the carriers of β-thal in the population in reproductive age by hematologic screening and common gene diagnosis in the area with high risk of β-thal.

Adult ; Base Sequence ; China ; ethnology ; DNA Mutational Analysis ; Ethnic Groups ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Young Adult ; beta-Globins ; genetics ; beta-Thalassemia ; genetics

The oncogenic isoform of the p63 protein, delta NP63, plays an important role in the pathogenesis of many epithelial carcinomas, and emerging evidences suggest that delta NP63 is a promising drug target. However, the functions of delta NP63 in transitional cell carcinoma of bladder (TCCB) are poorly defined. In this study, a delta NP63 shRNA expression vector was transfected into TCCB cell line 5637 and cell cycling, cell proliferation and protein expression were assessed by flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-dimethyl tetrazolium bromide (MTT) assay, and immunohistochemistry, respectively. The delta NP63 shRNA expression vector was also injected into 5637 cell xenograft tumors in nude mice, and tumor size was measured, tumor tissue morphology was assessed by immunohistopathology and transmission electron microscopy. In the in vitro study, delta NP63 shRNA transfection caused successful delta NP63 gene silencing and resulted in significant arrest of cell cycling and cellular proliferation (p<0.05) as well as cyclin D1 expression. In the nude mouse xenograft model, delta NP63 shRNA greatly inhibited tumor growth, induced tumor cell apoptosis (p<0.05) and resulted in cyclin D1 downregulation. Our data suggest that delta NP63 may play an oncogenic role in TCCB progression through promoting cell survival and proliferation. Intratumoral administration of delta NP63-specific shRNA suppressed tumor delta NP63 expression and cellular proliferation while promoted tumor cellular apoptosis, and therefore inhibited tumor growth and improved survival of xenograft-bearing mice, which was not accompanied by significant signs of systemic toxicity.
Animals ; Carcinoma, Transitional Cell/*genetics/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1/biosynthesis ; Disease Progression ; Female ; Humans ; Mice ; Mice, Nude ; Microscopy, Electron, Transmission ; Models, Biological ; Neoplasm Transplantation ; Trans-Activators/*biosynthesis/*physiology ; Tumor Suppressor Proteins/*biosynthesis/*physiology ; Urinary Bladder Neoplasms/*genetics/metabolism

This experiment was aimed to create A20 gene site-specific zinc finger DNA-binding protein. The sequence of A20 gene promoter was analyzed with bioinformatics means and submitted to ZF Tools Server at TSRI. Using the database of the web site, we determined the A20 gene valid target sites and designed the amino acid sequence of zinc finger protein predicted to be bound to the target site. And then, the structure of the protein sequence was analyzed and homology was modeled with various bioinformatics means. Based on the characteristic of this protein, the prokaryotic expression vector pTYB11-ZFP was constructed and expressed. Thus, the artificial zinc finger protein that recognized A20 specific sequence was designed, and expressed in Escherichia coli. The results indicate that it is feasible to design engineered artificial Zinc finger proteins by means of bioinformatics.
Amino Acid Sequence ; Base Sequence ; DNA-Binding Proteins ; chemistry ; genetics ; Humans ; Molecular Sequence Data ; Protein Binding ; Protein Engineering ; methods ; Transcription Factors ; chemistry ; genetics ; Zinc Fingers ; genetics

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Adaptor Proteins, Signal Transducing ; Carrier Proteins ; chemistry ; Chemical Precipitation ; DNA-Binding Proteins ; chemistry ; GATA1 Transcription Factor ; chemistry ; Genetic Vectors ; Glutathione Transferase ; chemistry ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; chemistry ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Renaturation ; Proto-Oncogene Proteins ; chemistry ; Recombinant Fusion Proteins ; genetics ; metabolism

Bone morphogenetic protein 2 (BMP2), which belongs to the transforming growth factor-beta (TGF-beta) superfamily, is a multifunctional molecule with distinct abilities to induce bone formation. BMP2 has been identified to have eminent pharmaceutical importance for clinical application. We previously constructed stable cell line in Chinese hamster ovary cells (CHO) that highly expressed recombinant human BMP2 (rhBMP2). For large-scale production of the recombinant protein used in clinical application, it is critical to have both high expression and stability of the protein. In the present study, the stability of the cell line (rCHO(hBMP2)-C8) with the highest expression, as well as the stability of rhBMP2 protein were investigated systematically. We cultured the rCHO (hBMP2)-C8 cell line in the presence or absence of MTX for two months, the cell growth and rhBMP2 production characteristics were examined during the culture; we found the duration that the rCHO(hBMP2)-C8 cell line could secret rhBMP2 continually into the serum-free medium. Moreover, we detected the temperature sensitivity of rhBMP2 in culture medium. This study will contribute to our understanding for further producing rhBMP2 by large-scale culture technology.
Animals ; Bone Morphogenetic Protein 2 ; biosynthesis ; genetics ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media ; Genetic Vectors ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; genetics

BACKGROUNDBronchoplasty plus pulmonary arterioplasty has become one of the standard surgical operation for central-type lung cancer. The aim of this study is to review the surgical experience of bronchoplasty and pulmonary arterioplasty in treatment of central-type lung cancer.

METHODSFrom 1987 to 2005, 56 patients with central-type lung cancer underwent bronchoplasty and pulmonary arterioplasty. There were 45 males and 11 females with a mean age of 56 years. According to pTNM classification, 18 cases were in stage IIB, and 32 in stage IIIA and 6 in stage IIIB. Histologically, there were 35 cases of squamous cell carcinoma, 14 cases of adenocarcinoma, 4 cases of small cell lung cancer and 3 cases of carcinoid. The surgical procedures included sleeve resection of bronchus for 30 cases, wedge resection of bronchus for 26 cases, and sleeve resection of pulmonary artery for 16 cases and wedge resection of pulmonary artery for 40 cases.

RESULTSOne patient died in the perioperative period. The overall 1-, 3-, and 5-year survival rate was 79.6% (43/54), 48.1% (25/52) and 34.0% (17/50), respectively.

CONCLUSIONSThe results suggest that bronchoplasty and pulmonary arterioplasty can decrease the proportions of total pneumonectomy and exploratory thoracotomy and expand the indication of operation. Bronchoplasty and pulmonary arterioplasty can be achieved with satisfactory outcome for central-type lung cancer, especially for those patients with advanced lesions or poor pulmonary function.