Pituitary adenomas (PAs) are well known as a common intracranial benign tumor, and a portion of PAs are refractory to current therapeutic methods. ErbB receptors family signaling pathway regulates the expression of PAs activation associated gene. Inhibition of epidermal growth factor receptor (EGFR) can inhibit proliferation of PAs. Leucine-rich repeats and immunoglobulin-like domains protein 1 ( LRIG1), a negative mediated gene of ErbB receptors family, plays a role in many tumors. However, there are seldom researches about the functional role of LRIG1 in PAs. The aim of this study is to explore the potential effect of LRIG1 and its regulating mechanism in PAs. First, we investigated the role of LRIG1 in cell migration, invasion of PAs with transfected LRIG1 or control. Then, we explored its impact on cell proliferation and apoptosis of PAs in vivo. To study the regulating mechanism of LRIG1, we examined the expression of molecular factor of PI3K/AKT and Ras/Raf/ERK pathway using Western blotting in vitro and RT-PCR in vitro and in vivo. It was found that LRIG1 over-expression inhibited cell migration, invasion and proliferation, and promoted apoptosis of PAs in vivo and in vitro. Furthermore, LRIG1 suppressed the expression of signaling of PI3K/AKT and Ras/Raf/ERK pathways in PAs. LRIG1, as a negative mediated gene of tumor, can inhibit biological function of PAs via inhibiting PI3K/AKT and Ras/Raf/ERK pathways, and it might be a new target for gene therapy of PAs.
Animals ; Apoptosis ; genetics ; Brain Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MAP Kinase Signaling System ; genetics ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; Oncogene Protein v-akt ; biosynthesis ; Phosphatidylinositol 3-Kinases ; genetics ; Pituitary Neoplasms ; genetics ; pathology ; Xenograft Model Antitumor Assays ; raf Kinases ; biosynthesis ; genetics

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

The spike (S) glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Four fragments coding spike proteins (S1, S2, RL and RS) from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus (Tiantan strain), and recombinant vaccinia viruses expressing four segments of spike proteins were generated (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS), respectively. Their expression location in cell and level were characterized using indirect immune fluorescence assay (IFA) and Western-Blot, respectively. The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification (glycosylation), and S1, RL and RS were mainly distributed in the cell membrane, while the S2 was mainly distributed in the cytoplasm. Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.
Base Sequence ; Blotting, Western ; Coronavirus NL63, Human ; chemistry ; Fluorescent Antibody Technique, Indirect ; Humans ; Membrane Glycoproteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Plasmids ; Recombinant Proteins ; biosynthesis ; Spike Glycoprotein, Coronavirus ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVETo explore whether overexpression of P-selectin glycoprotein ligand-1 (PSGL-1) promotes the adhesive ability of endothelial progenitor cells and functionally facilitates neovascularization in mouse model of hindlimb ischemia.

METHODSRat endothelial progenitor cells were transfected with recombinant adenovirus vector encoding human PSGL-1. The mRNA and protein expression levels of PSGL-1 were measured by RT-PCR and Western blot, respectively. The effect of overexpression of PSGL-1 in endothelial progenitor cells was analyzed by adherence assay. Histological examination of skeletal muscle sections retrieved from the mouse ischemic hindlimbs was performed, and the hindlimb blood flow was measured by laser Doppler flow meter.

RESULTSAdenovirus vector expressing of PSGL-1 gene was successfully constructed with high titer of 3.1 × 10¹¹ pfu/ml. After transfection, PSGL-1 gene was highly expressed in the transfected endothelial progenitor cells. In vitro assay showed that overexpression of PSGL-1 enhanced the adhesive properties of endothelial progenitor cells. When the transfected endothelial progenitor cells were transplanted into the ischemic hindlimb of nude mice, the number of new capillary vessels was (41.0 ± 2.2)/HPF compared to that of (21.0 ± 2.5)/HPF in the negative control group and (10.0 ± 1.6)/HPF in the blank control group (P < 0.01). Furthermore, the blood flow was increased in the experimental group (119.1% ± 7.0%), whereas in the negative control group, it was (93.3% ± 3.0%) and in the blank control group it was (76.3% ± 12.0%), P < 0.01.

CONCLUSIONSOverexpression of PSGL-1 enhances the adhesive and angiogenic properties of endothelial progenitor cells. The approach may provide an effective therapeutic strategy to improve the efficiency of cell-based proangiogenic therapy.

Adenoviridae ; genetics ; Animals ; Cell Adhesion ; Cells, Cultured ; Endothelial Cells ; cytology ; Hindlimb ; blood supply ; Humans ; Ischemia ; therapy ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; Mice, Nude ; Neovascularization, Physiologic ; physiology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Stem Cell Transplantation ; Stem Cells ; cytology ; metabolism ; Transfection

OBJECTIVETo construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.

METHODSS2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.

RESULTSThe pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.

CONCLUSIONSThe expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.

Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; Membrane Fusion ; drug effects ; Membrane Fusion Proteins ; biosynthesis ; isolation & purification ; Membrane Glycoproteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; SARS Virus ; genetics ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; biosynthesis ; genetics

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

Xanthohumol (XH), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis in B16 melanoma cells, with little cytotoxicity at the effective concentrations. Decreased melanin content was accompanied by reduced tyrosinase enzyme activity, protein and mRNA expression. The levels of tyrosinase-related protein 1 and 2 mRNAs were decreased by XH. XH also inhibited alpha-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. XH downregulated the protein and mRNA expression of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of key melanogenic enzymes. These results suggest that XH might act as a hypo-pigmenting agent through the downregulation of MITF in the cAMP-dependent melanogenic pathway.
1-Methyl-3-isobutylxanthine/pharmacology ; Animals ; Cell Line ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Antagonism ; Forskolin/pharmacology ; *Humulus ; Intramolecular Oxidoreductases/antagonists & inhibitors/biosynthesis ; Melanins/antagonists & inhibitors/*biosynthesis ; Melanocytes/*drug effects/*metabolism ; Melanoma, Experimental ; Membrane Glycoproteins/antagonists & inhibitors/biosynthesis ; Mice ; Microphthalmia-Associated Transcription Factor/antagonists & inhibitors ; Monophenol Monooxygenase/antagonists & inhibitors/biosynthesis/genetics ; Oxidoreductases/antagonists & inhibitors/biosynthesis ; Propiophenones/*pharmacology ; Signal Transduction/drug effects ; alpha-MSH/metabolism

OBJECTIVESTo investigate the regulative effect of the nonsteroidal anti-inflammatory drug NS398 on the RECK gene in the prostate carcinoma strain DU145.

METHODSDU145 was treated with various concentrations of NS398 for 48 hours. The mRNA level was measured by RT PCR technique and the expression of the RECK protein determined by Western blot.

RESULTSThe mRNA level of the RECK gene was obviously higher, while the MMP9 level markedly lower in the treated group than in the control, and so was the expression of the RECK protein.

CONCLUSIONNS398 induces the expression of the RECK gene, which might be the mechanism of its anti-tumor effect.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Blotting, Western ; Cattle ; Cell Line, Tumor ; GPI-Linked Proteins ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Nitrobenzenes ; pharmacology ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfonamides ; pharmacology

OBJECTIVE: To determine on the expression of prostate stem cell antigen (PCSA )and Oct-4 and detect their clinicopathological significance in benign and malignant lesions of the stomach. METHODS: EnVision immunohistochemistry for assaying the expression of PSCA and Oct-4 was used in paraffin-embedded sections from specimens of primary foci (n = 58) and metastatic foci of regional lymph nodes (n = 36) of gastric cancer, peritumoral tissues (n = 20), and benign lesions of the stomach (n = 80). RESULTS The positive rates of PSCA and Oct-4 were significantly higher in gastric cancer than those in peritumoral tissues and benign lesions (P<0.05 or P<0.01).The positive cases of PSCA and Oct-4 in peritumoral tissues and benign lesions showed mild- to severe-atypical hyperplasia. No difference of the positive rate of PSCA and Oct-4 was found between the primary foci and the metastatic foci of gastric cancer(P > 0.05). The positive rates of PSCA and Oct-4 were significantly lower in infiltrating depth T(1) to approximately T(2), non-metastasis of lymph nodes, metastasis of lymph nodes N1 site, and non-metastasis of distant organs than those in infiltrating depth T(3) to approximately T(4), metastasis of lymph nodes, metastasis of lymph node N(2) to approximately N(3) site, and metastasis of distant organs(P < 0.05 or P < 0.01).Conclusion The expressive levels of PSCA and Oct-4 might be related to the invasive potential, metastasis of lymph nodes, and distant organs, it suggested the expressions levels of PSCA and Oct-4 might be important markers for reflecting the biological behaviors and prognosis of gastric cancer.
Adenocarcinoma ; metabolism ; pathology ; Adult ; Aged ; Antigens, Neoplasm ; Biomarkers, Tumor ; Female ; GPI-Linked Proteins ; Humans ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Middle Aged ; Neoplasm Proteins ; biosynthesis ; genetics ; Octamer Transcription Factor-3 ; biosynthesis ; genetics ; Prognosis ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVETo construct the eukaryotic expression recombinant pSG. SS. C3d3. YL-Fbeta and analyze the expression of mouse fertilin beta subunit in HEK293 cells.

METHODSThe cDNA fragment expressing the disintegrin domain of mouse fertilin beta was obtained by PCR, and then inserted into the eukaryotic plasmid pSG. SS. C3d3. YL to get recombinant plasmid pSG. SS. C3d3. YL-Fbeta, which was transfected into the HEK293 cell line to express the target protein Fbeta after identified by restriction enzyme digestion. And then Fbeta was detected by indirect immunofluorescence through confocal laser scanning microscopy, Western blot, immunohistochemistry staining and flow cytometry assay.

RESULTSThe recombinant vector pSG.SS.C3d3. YL-Fbeta could express Fbeta in HEK293 cells.

CONCLUSIONThe expression of Fbeta in eukaryotic cells provides a foundation for further researches on the effect of high F, expression on fertilization process.

ADAM Proteins ; biosynthesis ; genetics ; Animals ; Blotting, Western ; Cells, Cultured ; Eukaryotic Cells ; metabolism ; Fertilins ; Gene Expression ; Genetic Vectors ; Humans ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Transfection