OBJECTIVE: To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide (LPS). METHODS: Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope. RESULTS: SPLUNC1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells. CONCLUSION SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.
Cell Line, Tumor ; Glycoproteins ; isolation & purification ; pharmacology ; Humans ; Membrane Proteins ; chemistry ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Phosphoproteins ; isolation & purification ; pharmacology ; Pseudomonas aeruginosa ; drug effects ; Respiratory Mucosa ; chemistry ; immunology ; Respiratory System ; chemistry ; immunology ; Transfection

The S1 gene hypervariable region I (HVR I) of 22 infectious bronchitis virus (IBV) strains isolated in Guangxi during the period of 1985-2007 were sequenced and compared to that of the other IBV reference strains and the pigeon coronavirus isolates. A phylogenetic tree based on nucleotide sequences of HVR I of all the IBV showed that they were classified into 5 distinct Clusters. 16 out of 22 IBV isolates were grouped into Cluster I, and had higher homology with pigeon coronavirus isolates but lower homology with the Massachusetts (Mass) type vaccine strains. There were 4 and 3 amino-acid residues inserted at the sites of 33-34 and 34-35 respectively within HVR I in 15 isolates, except in isolate GX-NN6 there had 4 amino-acid residues inserted at the both sites; isolates GX-YL1 and GX-NN2 had close relationship with Mass type vaccine strains, and they shared Cluster II; isolates GX-G and GX-XD of Cluster III had close relationship with the Japanese strain JP Miyazaki 89 which was isolated at the same period; isolates GX-YL6 and GX-NN7 of Cluster V had close relationship with the European strain 4/91. The results showed that there were high phylogenetic diversity among the IBVs prevailed in the field in Guangxi resulting from the commonly occurred mutation or insertion within the S1 gene HVR I of the viruses, and majority of the isolates had lower homology with the commonly used Mass type vaccine strains. There was much higher homology among viruses isolated in the same period of time, but without distinct difference in geographical origins.
Amino Acid Sequence ; Animals ; Chickens ; virology ; Genetic Variation ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Membrane Glycoproteins ; chemistry ; genetics ; Molecular Sequence Data ; Phylogeny ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; chemistry ; genetics

Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).
Animals ; Cloning, Molecular ; DNA Primers ; Escherichia coli/genetics/metabolism ; Flavoproteins/chemistry/genetics ; Kinetics ; Membrane Glycoproteins/chemistry/*genetics/isolation & purification ; *NADPH Oxidase ; Neutrophils/physiology ; Polymerase Chain Reaction/methods ; Recombinant Fusion Proteins/chemistry/isolation & purification/metabolism ; Recombinant Proteins/chemistry/isolation & purification ; Restriction Mapping ; Sequence Deletion

OBJECTIVETo explore the effect of Herba Epimedii flavone (HEF) on the osteoblast metabolism in vitro.

METHODOsteoblast were obtained from new born rat calvaria by digestive enzymes. MTF, PNPP and RT-PCR were used to observe the proliferation, activity of ALP and mRNA expression of OPG and RANKL of cultured osteoblasts in vitro.

RESULTIt was found that HEF had the effect on stimulating cell proliferation, activity of ALP and the mRNA expression of OPG of cultured osteoblasts (P < 0.01, P < 0.05).

CONCLUSIONHEF can promote the proliferation, the differentiation and the expression of OPG mRNA of the osteoblasts cultured in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Carrier Proteins ; biosynthesis ; genetics ; Cell Proliferation ; drug effects ; Cells, Cultured ; Epimedium ; chemistry ; Flavones ; isolation & purification ; pharmacology ; Glycoproteins ; biosynthesis ; genetics ; Membrane Glycoproteins ; biosynthesis ; genetics ; Osteoblasts ; cytology ; metabolism ; Osteoprotegerin ; Plants, Medicinal ; chemistry ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics

BACKGROUNDTo investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike (S) protein of SARS-associated coronavirus (SARS-CoV).

METHODSTwo different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli (E.coli) using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Vero cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls.

RESULTSThe recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E.coli. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients' sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells.

CONCLUSIONBoth S260-600 and S397-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.

Animals ; Binding, Competitive ; Blotting, Western ; Cercopithecus aethiops ; Escherichia coli ; genetics ; metabolism ; Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Mice ; NIH 3T3 Cells ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Binding ; Receptors, Cell Surface ; metabolism ; Recombinant Proteins ; isolation & purification ; metabolism ; SARS Virus ; genetics ; metabolism ; Spike Glycoprotein, Coronavirus ; Vero Cells ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism