OBJECTIVETo investigate the effect of removing EDTA-soluble phosphate protein in dentin on the later remineralization for the purpose of better understanding of mechanism of dentin phosphate proteins on dentin mineralization.

METHODSTo remove soluble phosphate protein by EDTA dissolution, then the remineralization rate was monitored by a constant composition crystal growth technique. The results were compared with those from the normal dentin and the dentin partially demineralized by acetic acid.

RESULTSFaster remineralization rates were found with dentin demineralized by EDTA (0.5 and 2 h) compared with normal dentin powder, while a slower rate was found with dentin demineralized by acetic acid. The increase of remineralization rate by removing phosphate protein from dentin was 100% more at 200 min after the start of the reaction.

CONCLUSIONEDTA-soluble phosphate protein in dentin has a great potential to inhibit remineralization.

Dental Cementum ; chemistry ; metabolism ; Dentin ; chemistry ; Edetic Acid ; Humans ; Phosphoproteins ; analysis ; physiology ; Tooth Demineralization ; metabolism ; Tooth Remineralization

BACKGROUNDThe molecular mechanisms underlying the endometriosis are still not completely understood. In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets, we carried out a phosphoproteomics analysis of human endometrium.

METHODSA large-scale differential phosphoproteome analysis, using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry, was performed in endometrium tissues from 8 women with or without endometriosis.

RESULTSThe phosphorylation profiling of endometrium from endometriosis patients had been obtained, and found that identified 516 proteins were modified at phosphorylation level during endometriosis. Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity. Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways, which might be deregulated during the development of endometriosis.

CONCLUSIONSThat large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.

Adolescent ; Adult ; Chromatography, Affinity ; Endometriosis ; metabolism ; Endometrium ; metabolism ; Female ; Humans ; Phosphoproteins ; analysis ; Phosphorylation ; Proteomics ; methods ; Tandem Mass Spectrometry ; Young Adult

OBJECTIVETo investigate the host-defense role of short palate, lung, and nasal epithelium clone 1 (SPLUNC1) in Streptococcus pneumoniae (SP) infection and the effect of resveratrol (Res) on SPLUNC1 expression, and to provide new thoughts for the treatment of diseases caused by SP infection.

METHODSAccording to the multiplicity of infection (MOI), BEAS-2B cells with SP infection were divided into control group, MOI20 SP group, and MOI50 SP group. According to the different concentrations of Res, the BEAS-2B cells with MOI20 SP infection pretreated by Res were divided into 12.5Res+SP group, 25Res+SP group, and 50Res+SP group (the final concentrations of Res were 12.5, 25, and 50 μmol/L, respectively). Cell Counting Kit-8 was used to measure cell activity and determine the optimal concentration and action time of SP and Res. In the formal experiment, the cells were divided into control group, Res group, SP group, and Res+SP group. Real-time PCR and ELISA were used to measure the mRNA and protein expression of SPLUNC1.

RESULTSOver the time of SP infection, cell activity tended to decrease. Compared with the control group and the MOI20 SP group, the MOI50 SP group had a reduction in cell activity. Compared with the MOI20 SP group, the 25Res+SP group had increased cell activity and the 50Res+SP group had reduced cell activity (P<0.05). MOI20 SP bacterial suspension and 25 μmol/L Res were used for the formal experiment. Over the time of SP infection, the mRNA expression of SPLUNC1 in BEAS-2B cells firstly increased and then decreased in the SP group and the Res+SP group (P<0.05). Compared with the SP group, the Res+SP group had significant increases in the mRNA and protein expression of SPLUNC1 at all time points (P<0.05). Compared with the control group, the Res group had no significant changes in the mRNA and protein expression of SPLUNC1 (P>0.05).

CONCLUSIONSSP infection can induce SPLUNC1 expression and the host-defense role of SPLUNC1. Res can upregulate SPLUNC1 expression during the development of infection and enhance cell protection in a concentration- and time-dependent manner.

Bronchi ; metabolism ; Cells, Cultured ; Cytoprotection ; Epithelial Cells ; metabolism ; Glycoproteins ; analysis ; genetics ; physiology ; Humans ; Phosphoproteins ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Stilbenes ; pharmacology ; Streptococcus pneumoniae ; pathogenicity

OBJECTIVEThe purpose of this study was to assess weather the immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells (BEMC).

METHODSImmortalized mouse brain endothelial cell line, Bend.3 cells were cultured in transwell inserts and their restrictive characteristics were assessed by transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) permeability assays. Western blot and direct fluorescent staining methods were used to detect the tight junction protein expression and F-actin distribution.

RESULTSThe TEER in Bend.3 cells increased with the prolonged culture time and increased to 82.3+/-6.0 Omega cm2 10 days after culture, which was significantly higher than that 3 days after culture (37.3+/-3.1 Omega cm2; P<0.05). There were significant differences in the permeability rates for HRP 3 and 10 days after culture (4.3+/-0.20)% vs (2.2+/-0.05)% (P<0.05). Western blot indicated high level expression of tight junction proteins occludin and ZO-1 in Bend.3 cells 10 days after culture. F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence 10 days after culture.

CONCLUSIONSBend.3 cells have similar barrier characteristics to BEMC, and their barrier function may reach to the best effect 10 days after culture.

Actins ; analysis ; Animals ; Blood-Brain Barrier ; Cell Line ; Electric Impedance ; Endothelial Cells ; metabolism ; Horseradish Peroxidase ; metabolism ; Membrane Proteins ; analysis ; Mice ; Phosphoproteins ; analysis ; Zonula Occludens-1 Protein

To evaluate the expression of P120ctn in non-Hodgkin's lymphoma (NHL) and to explore its clinical significance, immunohistochemistry stain method was applied to comparatively investigate the protein expression of P120ctn in paraffin-embedded lymph node tissue slices from 40 cases of NHL and 10 cases of reactive hyperplasia of lymph node. The results showed that P120ctn was not detected in reactive hyperplasia of lymph node, but was detected in 55% (22/40) cases of NHL. P120ctn expression increased with the tumor malignancy of NHL, there was a significant difference between the expression rates of P120ctn in low grade (16.7%, 2/12) and intermediate to high grade malignant (71.4%, 20/28) NHL (P < 0.001). Moreover, P120ctn was also detected in vascular endothelial cells of NHL. It is concluded that the level of P120ctn expression is closely related to the malignant grade of NHL, it suggests that P120ctn possibly plays an important role in the malignant proliferation of lymphoma with a certain significance in diagnosis and therapy of lymphoma.
Adolescent ; Adult ; Aged ; Catenins ; Cell Adhesion Molecules ; analysis ; biosynthesis ; Child ; Female ; Humans ; Immunohistochemistry ; Lymph Nodes ; metabolism ; pathology ; Lymphoma, Non-Hodgkin ; metabolism ; pathology ; Male ; Middle Aged ; Phosphoproteins ; analysis ; biosynthesis

OBJECTIVETo characterize the DNA damage property represented by the distinct whole nucleus stain pattern of gammaH2AX induced by N-methyl-No-nitro-N-nitrosoguanidine (MNNG).

METHODSMNNG-induced gammaH2AX foci formation in human amnion FL cells was observed by immunofluorescent microscopy. DNA double-stranded breaks (DSBs) were detected by neutral comet assay. General DNA damages were detected by alkaline comet assay.

RESULTA distinct whole nucleus stain pattern of gammaH2AX was induced by high concentration MNNG (10 mg/L). 1 mg/L MNNG also induced this type of stain pattern in a small fraction of cells, although the effect was transient. Neutral comet assay did not detect any significant DSBs formation in this type of cells, while alkaline comet assay revealed the presence of DNA damage.

CONCLUSIONAlthough normal gammaH2AX foci were regarded as a biomarker for DSBs, the whole nucleus stain pattern might represent DNA damage other than DSBs.

Amnion ; cytology ; Cell Nucleus ; drug effects ; metabolism ; Comet Assay ; DNA Breaks, Double-Stranded ; drug effects ; DNA Damage ; Histones ; analysis ; Humans ; Methylnitronitrosoguanidine ; pharmacology ; Microscopy, Fluorescence ; Phosphoproteins ; analysis

OBJECTIVETo To establish a method of phosphoprotein affinity profiling for identifying phosphoproteomic differences between Thp-1 cells with or without lipopolysaccharide (LPS) stimulation, aiming to screen potential regulators involved in LPS pathway.

METHODSThp-1 cells were stimulated with 100 ng/ml PMA for 48 h to induce differentiation into mature macrophages, which, after culture for another 48 h in the absence of PMA, were either treated with 100 ng/ml LPS for 30 min or left untreated. After desalting procedure with ultrafiltration, the phosphoproteins enriched by phosphoprotein metal affinity column (PMAC) of both groups were run on 2-D electrophoresis to find the spots with different phosphorylation status. Finally, some of these spots were identified by mass spectrometry (MS) and subsequent bioinformatic analysis.

RESULTSCompared to untreated Thp-1 cells, LPS stimulated Thp-1 cells showed 29 spots with reproducible alterations on the 2-D map, including 8 representing up-regulated spots, 7 new spots, 10 down-regulated spots, and 4 absent spots. The newly emerged and absent protein spots were subjected to MS analysis, and 4 of them were identified to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, phosphorylation of proteasome C2 subunit was dramatically up-regulated in LPS-stimulated cells, as was consistent with previous reports; the phosphorylation of Z-DNA-binding protein 1 has not been reported so far and needs further confirmation.

CONCLUSIONPhosphoprotein affinity profiling is an attractive method for screening novel regulators involved in LPS signaling pathways and can be widely used in systemic study of signal transduction.

Cell Line ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Mass Spectrometry ; Phosphoproteins ; analysis ; metabolism ; Phosphorylation ; Proteomics ; methods ; Signal Transduction

OBJECTIVETo study the changes of blood-brain barrier-tight junction (BBB-TJ) proteins ZO-1, occludin and actin following hypoxia-ischemia (HI) in order to explore the possible mechanism of permeability increasing of blood-brain barrier (BBB) induced by HI.

METHODSBBB models were established by co-culture of cell ECV304 and astrocytes (AS) in vitro, then randomly assigned to control and HI groups. Transmission electron microscope was used to observe the changes of BBB-TJ. The distribution of actin was determined by direct-immunofluorescence microscope. Definite permeability of BBB models by 125I-BSA was detected by gamma events-per-unit-time meter. Expression of actin, ZO-1 and occludin was detected by Western blot.

RESULTSAfter 10-day culture, endothelial cells connected tightly, with plenty of TJ which was smooth, continuous and of high density, in the BBB models. After 5 hrs of HI, the TJ was opened with intercellular gaps formation. The direct immunofluorescence showed that the peripheral filament bands became blurred, the cell-cell junction loosened and fissure appeared in the HI group. The permeability of 125I-BSA in the HI group increased significantly compared with the control group (P<0.01). Expression of ZO-1 decreased markedly, while expression of actin and occludin was not different in the HI group compared with the control group.

CONCLUSIONSThe changes in occludin distribution and decreased expression of ZO-1 lead the reorganization of BBB-actin protein, which may be one of the mechanisms of permeability increasing of BBB following HI.

Actins ; analysis ; physiology ; Animals ; Blood-Brain Barrier ; Female ; Hypoxia ; metabolism ; Ischemia ; metabolism ; Male ; Membrane Proteins ; analysis ; physiology ; Occludin ; Permeability ; Phosphoproteins ; analysis ; physiology ; Rats ; Rats, Sprague-Dawley ; Tight Junctions ; ultrastructure ; Zonula Occludens-1 Protein

Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-microm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2.
Aged ; Amino Acid Sequence ; Biological Markers/*analysis ; Carcinoma/*diagnosis/metabolism/pathology ; Chromatography, High Pressure Liquid ; Cluster Analysis ; Female ; Humans ; Image Processing, Computer-Assisted ; Male ; Middle Aged ; Molecular Sequence Data ; Molecular Weight ; Phosphoproteins/analysis/metabolism ; Proteome/analysis ; Proteomics ; Reproducibility of Results ; Ribosomal Proteins/analysis/metabolism ; *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; *Tandem Mass Spectrometry ; Thyroid Gland/metabolism/pathology ; Thyroid Neoplasms/*diagnosis/metabolism/pathology

PRAS40 (proline-rich Akt substrate 40 kD) associates with mammalian target of rapamycin complex 1(mTORC1), serine 183 site (Ser183) of PRAS40 can be phosphorylated by mTORC1. To prepare the phosphorylated PRAS40 (Ser183) antibody, We chosen 10-amino acid including Ser183 as antigen peptide through antigenicity and hydrophobicity analysis, hinged on keyhole limpet hemocyanin (KLH), and used the KLH-peptide to immunize rabbits. After antibody serum titer detection by enzyme linked immunosorbent assay (ELISA), the antibody was purified with rProtein A sepharose fast flow and dephosphorylated antigen membrane. The antibody titrate reached 1:10 000 after purification and its special property was enhanced with absorption treatment of dephosphorylated antigen membrane. In addition, we used rabbit anti-PRAS40 antibody and the phosphorylated PRAS40 (Ser183) antibody to detect PRAS40 expression in several cell lines, including the normal cells HL7702, HEK293, tumor cells HepG2, A549 and S180. There were no quite difference among these cells; otherwise, we observed the decreased phosphorylation level of Ser183 after amino acid withdrawal treatment. Therefore, the polyclonal phosphorylated PRAS40 (Ser183) antibody was specific to PRAS40 (Ser183) site and could be used for the function study of PRAS40.
Adaptor Proteins, Signal Transducing ; Animals ; Antibodies ; analysis ; Cell Line, Tumor ; Humans ; Male ; Mechanistic Target of Rapamycin Complex 1 ; Multiprotein Complexes ; Phosphoproteins ; chemistry ; immunology ; Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rabbits ; Serine ; metabolism ; TOR Serine-Threonine Kinases