Objective To investigate the effects of apigenin on the migration of vascular smooth muscle cells (VSMC) induced by platelet derived growth factor (PDGF)-BB and the possible molecular mechanism. Methods VSMCs were isolated from thoracic aortas of male Sprague-Dawley rats using enzyme digestion method. Migration of VSMCs was determined by transwell assay. Western blotting was carried out to evaluate phosphorylation of c-jun N-terminal kinase (JNK). Results Treatment with PDGF-BB (20 ng·mL-1 ) significantly promote VSMC migration,the number of migrated cells was 2. 46 times than that of control group. However,after 12. 5 μmol·L-1 apigenin pretreatment,the number of migrated cells was 46. 5% of the PDGF-BB group. Various dose of apigenin can significantly inhibit VSMC migration induced by PDGF-BB,12. 5 μmol · L-1 apigenin treatment significantly inhibited PDGF-BB phosphorylation of JNK. Conclusion Apigenin can suppress the migration of VSMC induced by PDGF-BB. These beneficial effects on VSMC were at least partly mediated by the inhibition of activity of JNK.

Objective:To choose the best vector for the expression of CS3 fimbriae. Methods: The CS3 operon was cloned into different plasmid vectors such as pUC19 and pTrc99A. The expression of CS3 was monitored by whole-cell ELISA and SDS-PAGE analysis. The assembly of CS3 fimbriae was detected with electron microscopy. Results:The expression level of CS3 fimbriae using plasmid pUC19 as carrying vector was the highest, and the insertion orientation of CS3 gene into the plasmid has a little effect on its expression level. The expression of CS3 fimbriae was confirmed by SDS-PAGE analysis and electron microscopy.Conclusions:The promotor of CS3 itself played the key role in the expression of CS3 fimbriae and the copy number of plasmid was the main factor to affect the expression level.

Objective:To prepare a kind of G418 resistance fibroblast feeder layers.Methods: In order to prepare a stock primary embryonic fibroblast (EMFI) cells, the pregnant Smad3ex8+/- mice containing heterozygous neo gene were killed 14 days after coitus to get the embryo. EMFI cells were tested by PCR to identify their gene types. Only one EMFI cell which contained heterozygous neo gene was chosen and treated with mitomycin C.The mitomycin C-treated EMFI feeders were stored at -70℃. Results: The EMFI feeder layers could support the growth of embryonic stem cell line TC-1 efficiently and could live in G418 resistance medium for 1-2 weeks.

Breast cancer susceptibility gene 1(BRCA1) plays an important role in breast cancer development and progression. BRCA1 encodes a 1863-amino acid protein with two BRCA1 C-terminal (BRCT) domains at its C-terminus, BRCT1 and BRCT2. Many cancer-predisposing mutations are located in the BRCT domains, which have been shown to induce chromatin unfolding by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To map the important region of BRCT domain (amino acid residues 1642-1736), six deletion mutant constructs were made. The chromatin structure assay showed that amino acid residues 1691-1721 are involved in the induction of chromatin unfolding. To further localize the critical amino acid residues, ten alanine scanning mutant constructs were made. The chromatin structure assay demonstrated that the 1707IAGGK1711 region is critical for the chromatin unfolding activity. Based on the mapped important region, Blast analysis identified a novel homologous protein. Mapping of the BRCT1 domain may aid in the presymptomatic risk assessment and provide a valuable tool for further study on the BRCT1 structure and function.
BRCA1 Protein ; chemistry ; physiology ; Base Sequence ; Chromatin ; chemistry ; Cloning, Molecular ; Female ; Genes, BRCA1 ; Humans ; Molecular Sequence Data ; Mutation ; Protein Folding ; Structure-Activity Relationship

OBJECTIVETo study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment.

METHODSAn eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed.

RESULTSA stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed.

CONCLUSIONExogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estradiol ; pharmacology ; Estrogen Antagonists ; pharmacology ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Humans ; Tamoxifen ; analogs & derivatives ; pharmacology ; Transfection

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Bacteriophage lambda ; genetics ; DNA, Recombinant ; genetics ; Escherichia coli ; genetics ; Genetic Engineering ; Plasmids ; genetics ; Rec A Recombinases ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

We have studied the proteomic changes of the serum of the Smad3 targeted deficient mice using 2-DE and PMF approaches. 7 proteins expressed at different level in wild type mice and the Smad3 deficient mice were identified. These results would benefit the research on diagnosis and therapy of osteoarthritis and provided clues to studying the important function of Smad3 mediated TGF-beta signals during the skeletal development.
Animals ; DNA-Binding Proteins ; blood ; deficiency ; genetics ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Genotype ; Mice ; Mice, Knockout ; Peptide Mapping ; Proteome ; analysis ; Smad3 Protein ; Trans-Activators ; blood ; deficiency ; genetics

The transgenic mice that express Cre recombinase in a tissue specific manner is a powerful tool in generating the conditional gene knockout mice. The rat insulin promoter was cloned target the expression of Cre in pancreatic tissue. The Cre gene was modified by adding the nuclear localization signal and the sequence for initiation by eukaryotic ribosomes at 5' terminal of the Cre gene. Cre gene was linked to the intron of human growth factor gene. This construct was introduced into the mouse eggs using microinjection. Seven mice were identified as founders carrying the Cre gene by PCR. The results of RT-PCR showed that the transgenic mouse from one founder could transcribe the foreign gene in pancreas. The Southern blot analysis indicated that the Cre recombinase expressed in pancreas of the transgenic mouse was functional.
Animals ; Blotting, Southern ; Female ; Insulin ; genetics ; Integrases ; genetics ; Mice ; Mice, Transgenic ; Pancreas ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; analysis ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Proteins ; genetics

Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.
Breast Neoplasms ; genetics ; metabolism ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; metabolism ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Humans ; Protein Interaction Domains and Motifs ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Regulatory Factor X Transcription Factors ; Signal Transduction ; Transcription Factors ; genetics ; metabolism ; X-Box Binding Protein 1

OBJECTIVETo study the clinicopathologic features, immunotype and cytogenetics of Chinese mantle cell lymphoma (MCL).

METHODS114 MCL cases were collected from hematopathology lab of department of pathology, Peking University, HSC. Routine HE stain and immune stain were used to investigate the clinicopathologic features and immune type. Breaks of CCND1 and IgH/CCND1 fusion genes were detected by FISH.

RESULTSThe ratio of male to female was 3.56:1 (89:25) with the median age of 60 years old (20 - 83 years old). 78 cases (68.42%, 78/114) primarily showed lymph node involvement, including 49 cases (49/78, 62.82%) jugular node involvement; 36 cases (31.58%, 36/114) showed extra-nodal involvement. 23 cases (23/114, 20.18%)showed bone marrow involvement. The expressions of CD3ε, CD20, CD79a, PAX5, CD5, cyclinD1 and Bcl-2 were 0% (0/114), 99.12% (113/114), 96.43% (27/28), 97.56% (40/41), 67.89% (74/109), 100% (114/114) and 94.12% (48/51), respectively. Break of CCND1 gene was found in 20 cases (80%, 20/25), the fusion gene of IgH-CCND1 in 16 cases (80%, 16/20), the break of IgH gene in 9 cases (100%, 9/9)and its fusion gene in 8 cases (88.89%, 8/9). We followed up 75 cases with a period of 2-57 months. The median survival was 40.78 months. The survivals at 1 year, 2 year and 3 year were 84.13% (53/63), 68.09% (32/47) and 37.5% (12/32), respectively. The median survival of group with more than 40% expression of Ki-67 was 36 months, the group with less than 40% expression of Ki67 57 months (P = 0.003). 7 of 13 patients accepted Rituximab plus traditional chemotherapy attained CR, 3 cases PR. 11 of 44 cases accepted traditional chemotherapy attained CR, 9 cases PR (P = 0.052).

CONCLUSIONMost of Chinese MCL occurred in older male, multi-lymphadenopathy and bone marrow involvement were common in MCL as a aggressive tumor. High expression of Ki-67 was an adverse prognostic indicator. Rituximab could improve the survival. Change of CCND1 gene was the most common cytogenetic abnormality.

Adult ; Aged ; Aged, 80 and over ; Chromosome Aberrations ; Cyclin D1 ; genetics ; Cytogenetics ; Female ; Humans ; Ki-67 Antigen ; genetics ; Lymphoma, Mantle-Cell ; genetics ; pathology ; Male ; Middle Aged ; Prognosis ; Young Adult