Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.
Crops, Agricultural ; chemistry ; Phosphoproteins ; chemistry ; Phosphorylation ; Plant Proteins ; chemistry ; Pollen ; chemistry ; Proteome ; Tandem Mass Spectrometry ; Zea mays ; chemistry

OBJECTIVETo investigate the relationship between dentine phosphoprotein (DPP) and remineralization of demineralized dentine.

METHODS(1) Soluble DPP was extracted with 1 mol/L NaCl from demineralized dentine and was evaluated. (2) Soluble DPP was removed with 0.1 mol/L NaCl or was not removed from demineralized dentine sections in human tooth roots. Then all sections were subjected to remineralization treatment, and remineralization degrees were compared by atomic absorption spectrum, SEM and microradiography.

RESULTS(1) Soluble DPP was extracted with 1 mol/L NaCl. (2) Removal of soluble DPP resulted in significantly lower calcium concentration in remineralization solution (P < 0.01), less mean light-absorbed value in demineralized dentin sections by microradiography (P < 0.01).

CONCLUSIONSSoluble DPP may have an inhibiting effect on remineralization of demineralized dentine, this study suggests that the remove of soluble DPP from root caries lesions may enhance their remineralization potential.

Adolescent ; Child ; Dentin ; chemistry ; Humans ; In Vitro Techniques ; Phosphoproteins ; isolation & purification ; physiology ; Tooth Demineralization ; Tooth Remineralization

Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.
Animal ; Clathrin/*metabolism ; Clathrin-Coated Vesicles/*chemistry ; Liver/*chemistry ; Nerve Tissue Proteins/*isolation & purification ; Phosphoproteins/*isolation & purification ; Rats

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

OBJECTIVETo investigate the effect of dentin phosphoprotein (DPP) in inducing dentinal mineralization.

METHODSHuman DPP was combined with EAH-Sepharose 4B beads and its function of inducing mineralization was studied in mineralization system in-vitro. The mineral formed on the surface of the beads was analyzed by scanning electron microscopy (SEM) and the structure was analyzed by X-ray diffraction and plasma emission spectrum.

RESULTSThere was mineral formed on the beads with combined DPP and the mineral was calcium phosphates whose ratio of calcium to phosphate was 1.33. The diffractogram of the formed mineral was more similar to hydroxyapatite than to other calcium phosphates.

CONCLUSIONWhen tightly combined with certain support substance, human DPP can induce mineralization.

Calcium Phosphates ; chemistry ; metabolism ; Dentin ; chemistry ; metabolism ; Dentinogenesis ; Humans ; Microscopy, Electron, Scanning ; Minerals ; chemistry ; metabolism ; Phosphoproteins ; chemistry ; metabolism ; Tooth Calcification ; X-Ray Diffraction

The secondary structures of fusion protein pp150/MDBP, including alpha-helix, beta-sheet, turn regions, were analyzed by Garnier-Robson's and Chou-Fasman's methods; the antigenic epitopes of B cells were analysed by using hydrophilicity plot. The results showed that the fusion protein pp150/MDBP might have less alpha-helix, but be rich in beta-sheet and turn regions. The epitopes recognized by B cells may be at 7-56 amino acid residues or adjacent to 137-192 amino acid residues.
Amino Acid Sequence ; Binding Sites ; Cytomegalovirus ; chemistry ; Epitopes ; Humans ; Molecular Sequence Data ; Phosphoproteins ; chemistry ; immunology ; Protein Structure, Secondary ; Viral Fusion Proteins ; chemistry ; immunology ; Viral Matrix Proteins ; chemistry ; immunology

BACKGROUNDAnkylosing spondylitis (AS) is the most common rheumatic condition that is slowly progressive and predominantly affects adolescents. Pathological bone formation associated with AS is an important cause of disability. The aim of the study was to investigate the possible involvement of the genes related to endochondral ossification and ectopia ossification in genetic susceptibility to AS in a Chinese Han population.

METHODSSixty-eight single nucleotide polymorphisms (SNPs) from 13 genes were genotyped in discovery cohorts including 300 AS patients and 180 healthy controls. The rs10019009 in dentin matrix protein 1 (DMP1) gene shown as association with AS after multiple testing corrections in discovery cohorts was replicated in a validation independent cohort of 620 AS patients and 683 healthy controls. The rs10019009 was assessed with bioinformatics including phylogenetic context, F-SNP and FastSNP functional predictions, secondary structure prediction, and molecular modeling. We performed a functional analysis of rs10019009 via reverse transcription-polymerase chain reaction, alkaline phosphatase (ALP) activity in human osteosarcoma U 2 OS cells.

RESULTSInterestingly, the SNP rs10019009 was associated with AS in both the discovery cohort (P = 0.0012) and validation cohort (P = 0.0349), as well as overall (P = 0.0004) in genetic case-control association analysis. After a multivariate logistic regression analysis, the effect of this genetic variant was observed to be independent of linkage disequilibrium. Via bioinformatics analysis, it was found that the amino acid change of the rs10019009 led to changes of SNP function, secondary structure, tertiary conformation, and splice mode. Finally, functional analysis of rs10019009 in U 2 OS cells demonstrated that the risk T allele of the rs10019009 increased enzymatic activity of ALP, compared to that of the nonrisk allele (P = 0.0080).

CONCLUSIONSThese results suggested that the DMP1 gene seems to be involved in genetic predisposition to AS, which may contribute to the ectopic mineralization or ossification in AS. In addition, DMP1 gene may be a promising intervention target for AS in the future.

Adult ; China ; ethnology ; Extracellular Matrix Proteins ; chemistry ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Logistic Models ; Male ; Phosphoproteins ; chemistry ; genetics ; Polymorphism, Single Nucleotide ; Spondylitis, Ankylosing ; etiology ; genetics

Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.
Animal ; Antibodies, Monoclonal ; Calpain/chemistry ; Caspases/chemistry ; Clathrin-Coated Vesicles/metabolism* ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/metabolism ; Escherichia coli/genetics ; Female ; Glutathione Transferase/genetics* ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins/metabolism ; Nerve Tissue Proteins/metabolism ; Nerve Tissue Proteins/chemistry* ; Phosphoproteins/metabolism ; Phosphoproteins/genetics ; Phosphoproteins/chemistry* ; Protein Binding ; Rabbits ; Recombinant Fusion Proteins/metabolism ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/chemistry* ; src Homology Domains

OBJECTIVETo investigate the anti-atherosclerosis mechanism of Astragalus mongholicus polysaccharides (APS) by examining its effect on gene expression profiles of the dendritic cells (DCs) from healthy donors.

METHODSPeripheral blood DCs from healthy donors were incubated with 200 mg/L APS overnight, and changes in the gene expression profiles were investigated using microarray technique and RT-PCR.

RESULTSCompared with the control cells, APS-treated DCs showed significantly up-regulated expressions of CD36 (0.97 ± 0.23 vs 5.45 ± 1.14) and IL-27 (1.08 ± 0.22 vs 2.97 ± 0.61) and down-regulated expression of expression of IFI16 (0.98 ± 0.18 vs 0.46 ± 0.11).

CONCLUSIONSAPS can promote the maturation and differentiation of DCs by up-regulating CD36 and IL-27 and down-regulating IFI16, and thus positively affects the occurrence and progression of the atherosclerosis.

Astragalus Plant ; chemistry ; CD36 Antigens ; metabolism ; Cell Differentiation ; Dendritic Cells ; drug effects ; Humans ; Interleukins ; metabolism ; Nuclear Proteins ; metabolism ; Phosphoproteins ; metabolism ; Polysaccharides ; pharmacology ; Transcriptome

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