OBJECTIVETo investigate the expression of Shh and its receptors Ptc1 and Ptc2 mRNA in the cap stage of mouse molar and discuss its role in early tooth morphogenesis.

METHODSThe embryonic mouse heads of early tooth development (E10.5 - E15.5) were obtained and 5 micro m serial sections were made. Immunohistochemical staining of PCNA was carried out by SP method. The expression pattern of Shh, Ptc1, and Ptc2 mRNA was analysed by in situ hybridization.

RESULTSE14.5, outer dental epithelium, inner dental epithelium, stellate reticulum and underlying dental mesenchyme were PCNA positive. Most of the enamel knot cells were PCNA negative. A few of the enamel knot cells were PCNA positive. Shh, Ptc1, and Ptc2 mRNA were strongly expressed in outer dental epithelium, inner dental epithelium, stellate reticulum and the enamel knot.

CONCLUSIONIn the cap stage, Shh as a paracrine and autocrine signaling molecule might stimulate epithelium and mesenchyme proliferation.

Animals ; Hedgehog Proteins ; Mice ; Molar ; metabolism ; Patched Receptors ; Patched-1 Receptor ; RNA, Messenger ; biosynthesis ; Receptors, Cell Surface ; biosynthesis ; genetics ; Tooth Germ ; growth & development ; metabolism ; Trans-Activators ; biosynthesis

GPR43 (G protein-coupled receptor 43) is a recently discovered short-chain free fatty acid receptor which plays important role in adipogenesis. Here we explored the transcriptional expression rule of GPR43 in porcine adipose tissue and primary cultured adipocytes. Partial cDNA of GPR43 was successfully cloned from swine by RT-PCR and the expression profile of GPR43 mRNA was studied from different types, different growing stages, and different sites of porcine adipose tissue as well as porcine primary cultured adipocytes. The results showed that porcine GPR43 shared high homology with human (89%), mouse (84%) and rat (83%). The expression level of GPR43 mRNA was significantly higher in adipose tissue of obese pigs than that of lean pigs, and also the expression level gradually increased with age. Further, the abundance of GPR43 mRNA level was higher in subcutaneous fat than in visceral fat. In addition, during the adipocytes differentiation, the expression of GPR43 mRNA increased in a time-dependent manner. These data indicated that GPR43 gene expression was relate to the site of adipose tissue, economic type, and age of pig as well as differentiating state of adipocytes, implying that GPR43 can be a potential factor to regulate adipogenesis.
Adipocytes ; cytology ; metabolism ; Adipose Tissue ; metabolism ; Animals ; Cells, Cultured ; DNA, Complementary ; biosynthesis ; genetics ; Gene Expression Profiling ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Receptors, G-Protein-Coupled ; biosynthesis ; genetics ; Swine ; Transcription, Genetic

ATR-Fc is a fusion protein consisting of extracellular domain of human anthrax toxin receptor (ATR) and a fragment (hinge, CH2, and CH3 domains) of the Fc of human IgG1. The aim of ATR-Fc expression is to get an antibody-like molecule binding to protective antigen (PA), a component of anthrax toxins, this fusion protein may compete with cell surface receptor for PA binding, and block the transport of lethal factor (LF) and edema factor (EF) into cells, thereby act as an antitoxin to prevent and treat anthrax infection. A DNA fragment encoding N-terminal amino acids 1-227 of ATR and human IgG1 Fc was inserted into the Hind III and Not I sites of pcDNA3.1 to generate the eukaryotic vector pcDNA3.1/ATR-Fc for expression of ATR-Fc fusion protein. Using lipofectine-mediated gene transfer technique, pcDNA3.1/ATR-Fc was transfected into CHO-K1 cells. After selected with G418, a recombinant CHO cell line, ATR-Fc-1D5, whose expression level was about 10 - 15 microg/(10(6) cells x d), was established. The recombinant protein expressed by the ATR-Fc-1D5 cells was purified with protein A chromatography. The experimental results demonstrated a direct and specific interaction between ATR-Fc and PA assessed by ELISA.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo investigate the characterization of Notch gene involved in genetic regulatory networks in dental pulp stem cells.

METHODSThe pulp tissue was separated from mouse teeth and digested by collagenase type I. Single-cell suspensions of dental pulp were seeded into 6-well plates with alpha modification of Eagle's medium supplemented with ES cell qualified Fetal Bovine Serum. Colony-forming efficiency was assessed in 14ds culture. Transcripts for Notch were detected by reverse transcription-PCR by using total RNA isolated from cells.

RESULTSThere were clonogenic cells in dental pulp cell and the incidence of colony-forming cells derived from mouse dental pulp cells was 1.6-2.5 colonies/10(4) plate. Mouse-specific Notch mRNA expressed in colony-forming cells.

CONCLUSIONNotch mRNA expressing in colony-forming cells provided a more detailed understanding of mouse dental pulp stem cell biology.

Animals ; Cell Differentiation ; Cells, Cultured ; Dental Pulp ; cytology ; metabolism ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Receptors, Notch ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Stem Cells ; metabolism

Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction. In this study, we obtained the soluble mZP3 fusion protein and identified its immunoreactivity. mZP3 cDNA was cloned into plasmid pMAL-p2x, and the recombinant plasmid was transformed into Escherichia coli BL21. To get the soluble mZP3 fusion protein, we tried to optimize the expression conditions, including additives, IPTG concentrations, temperatures and induction duration. Then, Western blotting and ELISA were used to identify the immunoreactivity of the purified protein. Based on the optimization experiments, we concluded that the best soluble expression conditions for the mZP3 fusion protein involved incubation to an A600 of 0.6, addition of glucose to a final concentration of 0.02 mol/L, addition of IPTG to a final concentration of 0.6 mmol/L and then further incubation for 4 h at 25 degrees C. Western blotting and ELISA showed that the mZP3 fusion protein retained immunoreactivity. The fusion protein can be used as solubility antigens for developing the immunocontraception vaccines of mZP3 and detecting the immune effects of the vaccine.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Vaccines, Contraceptive ; immunology ; Zona Pellucida Glycoproteins

Animals ; Endothelium, Vascular ; metabolism ; Eukaryotic Cells ; metabolism ; Genetic Therapy ; Neoplasms ; blood supply ; therapy ; Neovascularization, Pathologic ; Plasmids ; Receptors, Cell Surface ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Swine

OBJECTIVESTo study the fibrinolytic activity and the expression of uPAR and Annexin II in leukemic cell lines and their alterations on all-trans retinoic acid ( ATRA) treatment.

METHODSThe fibrinolytic activity was measured by chromogenic assay in NB4, SHI-1, K562, Jurkat and Raji cell lines. The protein expression of uPAR and Annexin II on cells surface and the mRNA expression of uPAR and Annexin II in cells of these cell lines were detected using flow cytometry and RT-PCR method respectively.

RESULTSThe plasmin activity in supernatant was increased significantly after incubation of SHI-1 and NB4 cells with plasminogen. The plasmin activity of NB4 cells was obviously decreased by ATRA. The plasmin activity of NB4 and SHI-1 cells was significantly decreased by uPAR monoclonal antibodies. The expressions of uPAR and Annexin II and their mRNA in SHI-1 and NB4 cells were higher than that in other cell lines. ATRA could remarkably decrease the expressions of Annexin II and uPAR and their mRNA in NB4 cells.

CONCLUSIONIn leukemia cell lines, NB4 and SHI-1 cells have stronger fibrinolytic activity. Both Annexin II and uPAR on the leukemic cell membranes might contribute to this activity. The high fibrinolytic activity can be corrected by ATRA by down-regulating Annexin I and uPAR mRNA and protein expression in NB4 cells.

Annexin A2 ; biosynthesis ; genetics ; Cell Line, Tumor ; Fibrinolysis ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; RNA, Messenger ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Receptors, Urokinase Plasminogen Activator ; Reverse Transcriptase Polymerase Chain Reaction ; Tretinoin ; pharmacology

OBJECTIVEIn order to explore pathogenic mechanism of laryngeal carcinoma, the involved genes were identified in larynx carcinogenesis by comparing the gene expression profile in matched primary normal epithelial cells and primary laryngeal carcinoma cells from the same patients.

METHODSA cDNA microarray analysis consisting of 11,431 human genes revealed significant changes in the expression of 35 genes, with 8 genes being up-regulated and 27 being down-regulated. The epithelial membrane protein-1 (EMP-1) is one of the down-regulated genes. EMP-1 expression in various kinds of laryngeal carcinoma was determined by semi-quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

RESULTSThe EMP-1 mRNA levels in all laryngeal carcinoma cells was significantly lower than that in the matched primary normal epithelial cells (P < 0.05) and were not correlated to the stage and differentiation of laryngeal carcinoma (P > 0.05).

CONCLUSIONSThe EMP-1 expression was correlated to larynx carcinogenesis and may be helpful to elucidate the pathogenic mechanism in laryngeal carcinoma.

Adult ; Aged ; Female ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; Oligonucleotide Array Sequence Analysis ; Receptors, Cell Surface ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Fas transduces apoptotic signals upon cross-linking with the Fas ligand (FasL), which is experimentally replaced by agonistic anti-Fas monoclonal antibodies (mAb). Of eight human malignant hematopoietic cell lines (HL-60, KG-1, THP-1, K562, U937, Jurkat, IM-9, RPMI-8226) examined by flow cytometric analysis, all, except K562, were found to be positive for surface Fas antigen. However, despite surface Fas expression, the agonistic anti-Fas mAb (7C11) induced apoptosis in only three of seven Fas-expressing cell lines (KG-1, Jurkat and IM-9). This Fas-resistance did not correlated with high levels of mRNA either for DcR3, a decoy receptor for FasL, or for FAP-1, a Fas-associated phosphatase that can block the apoptotic function of Fas. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not show consistent differences in the expression of Bcl-2 and Bax between Fas-sensitive and Fas-resistant cell lines examined. These findings indicated that the presence or absence of mRNA expression of DcR3, FAP-1, Bcl-2 and Bax did not always correlate with relative sensitivity to Fas-mediated apoptosis. Treatment of cells with cycloheximide converted the phenotype of resistant cell lines from Fas-resistant to Fas-sensitive, and enhanced the sensitivity of Fas-sensitive cell lines. These results suggest that the Fas-resistance is dependent on the presence of labile proteins that determine resistance to Fas-mediated apoptosis and the apoptotic machinery is already in place in Fas-resistant cell lines.
Antigens, CD95/*metabolism ; Apoptosis/drug effects/*genetics ; Carrier Proteins/biosynthesis/genetics ; Comparative Study ; Cycloheximide/pharmacology ; Gene Expression Regulation, Neoplastic ; Hematologic Neoplasms/*genetics/metabolism ; Human ; Membrane Glycoproteins/*metabolism ; Protein Synthesis Inhibitors/pharmacology ; Protein-Tyrosine-Phosphatase/biosynthesis/genetics ; Proto-Oncogene Proteins/biosynthesis/genetics ; Proto-Oncogene Proteins c-bcl-2/biosynthesis/genetics ; Receptors, Cell Surface/biosynthesis/genetics ; Signal Transduction ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured

OBJECTIVETo investigate the protective mechanisms of Radix Salviae miltiorrhizae (RSM) on chronic alcoholic liver injury in mice.

METHODSThe chronic alcoholic liver injury mouse model was established. The morphologic change of hepatic tissue was observed with hematoxylin-eosin (HE) staining; the levels of toll-like receptor-4 (TLR-4) mRNA in hepatic tissue and hemeoxygenase-1 (HO-1) mRNA were determined using reverse transcription polymerase chain reaction (RT-PCR) technique; and the expression of TLR-4 protein was determined by immunohistochemistry method.

RESULTSRSM could alleviate the fatty degeneration and adiponecrosis of hepatic cells induced by alcohol, down-regulate the expressions of TLR-4 mRNA and HO-1 mRNA, and significantly decrease the number of TLR-4 positive cells.

CONCLUSIONRSM could prevent liver injury from alcohol by way of influencing TLR-4 signal transcription.

Animals ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Hepatitis, Alcoholic ; drug therapy ; pathology ; Liver ; metabolism ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Membrane Proteins ; Mice ; Phytotherapy ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Salvia miltiorrhiza ; Toll-Like Receptor 4 ; Toll-Like Receptors