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

OBJECTIVETo investigate whether the sonic hedgehog signaling pathway is involved in the development of human fetal prostate, and to evaluate the changing staining patterns of its molecules, sonic hedgehog (SHH), patchedl (PTC1), smoothened (SMO), and GLI1, in the human fetal prostate at various gestation stages.

METHODSFifteen human fetal prostate specimens at various developmental stages (10 - 39 weeks) were included in this study. SHH, PTC1, SMO and GLI1 were detected in all the specimens by immunohistochemical technique. All the slides were observed and assessed under the light microscope.

RESULTSSHH, PTC1, SMO and GLI1 could be detected in human fetal prostate tissues, and their expression formed two surges, the former at week 16, and the latter at week 28. The staining of SHH and SMO was distributed only in the ductal epithelium but not in the stroma. The expression of PTC1 and GLI1 could be found mainly in the epithelium, with minimal staining in the stroma.

CONCLUSIONThe sonic hedgehog signaling pathway is involved in the development of the human fetal prostate. The high expression of its molecules at early gestation stages might be associated with the induction of prostatic buds, while their abundant expression at later gestation stages might be related to the prostate ductal branching, growth, differentiation and morphogenesis.

Gene Expression Regulation, Developmental ; physiology ; Hedgehog Proteins ; biosynthesis ; Humans ; Male ; Oncogene Proteins ; biosynthesis ; Patched Receptors ; Prostate ; embryology ; metabolism ; Receptors, Cell Surface ; biosynthesis ; Receptors, G-Protein-Coupled ; biosynthesis ; Signal Transduction ; physiology ; Smoothened Receptor ; Trans-Activators ; biosynthesis ; Zinc Finger Protein GLI1

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

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

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

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

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

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

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC1 ) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth.
Chorionic Villi/metabolism ; Female ; Gene Expression ; Humans ; Nerve Growth Factors/*biosynthesis ; Neuropeptides/*biosynthesis ; Neurotransmitter Agents/*biosynthesis ; Pituitary Adenylate Cyclase-Activating Polypeptide ; Placenta/*metabolism ; Pregnancy ; Pregnancy Trimester, First ; Pregnancy Trimester, Second ; RNA, Messenger ; Receptors, Cell Surface/*biosynthesis ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I