A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons

The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)₂ of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.

Development of a functional cell-based HTS assay for the identification mGluR4 modulators / 生物工程学报

To identify metabotropic glutamate receptor 4 (mGluR4) modulators by Ca2+ influx assay, we developed the functional cell-based high throughput-screening (HTS) assay. The human mGluR4 cDNA was transfected into HEK-293 stably expressing promiscuous G-protein (Ga alpha15) cells. Recombinant stable mGluR4 cell line was selected under Zeocin and validated by Ca2+ influx assay. The assay was optimized on loading time of Fluo Calcium Indicator, Dimethyl sulfoxide (DMSO) tolerance and sodium hydroxide (NaOH) tolerance using agonist (L-Glutamic acid (L-Glu)) of mGluR4. The rank order of the agonist potency for the stable human mGluR4 cell line was L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) > L-Serine-O-phosphate (L-SOP) > L-Glu, and of the antagonist potency was (RS)-alpha-Methylserine-O-phosphate (MSOP) > (RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG). Z' factor value of the cell line in 96- and 384-well plate format was 0.80 and 0.65. Our data indicate a successful development of functional human mGluR4 recombinant stable cell line that was suitable for high throughput screening to identify mGluR4 agonist/antagonist.

Activation of periphery group III metabotropic glutamate receptors inhibits formalin-induced activation of spinal p38-MAPK in rats / 中国应用生理学杂志

<p><b>AIM</b>To explore the effects of periphery injection of L-SOP on the activation of p38MAPK in spinal cord in formalin pain model in rats.</p><p><b>METHODS</b>Fourty-eight male Wistar rats were divided randomly into four groups (n=12): NS group and three different dose of L-SOP groups. For each group, 6 rats used to observe flinching and licking time every as nociception behavior 3 minutes in 1 hour after formalin injected and the other 6 rats used to observe the activation of p38(P-p38) by Western blotting.</p><p><b>RESULTS</b>All the three different groups of L-SOP could inhibit nociception behavior in the tonic phase,and 250 nmoVl/L and 500 nmol/L groups could suppress not only in the tonic phase but also in the acute phase. 250 nmol/L and 500 nmol/L groups could reduce activated or phosphorylated p38MAPK in spinal cord.</p><p><b>CONCLUSION</b>Periphery injection of L-SOP can reduce nociceptive behavior and phosphorylated p38MAPK in the spinal cord in formalin-induced hyperalgia, it is suggested that there is functional expression of mGluRs III in the periphery and is involved in the processing of peripheral noxious informations.</p>

The preparation of bioglass/collagen/phosphoserine biomemetic composite scaffold and a study on its cytocompatibility / 生物医学工程学杂志

In the present study, novel biomemetic composite scaffolds using the sol-gel derived bioactive glass (BG), collagen (COL), and phosphoserine (PS) were prepared by freeze-drying. MC3T3-E1 were cultivated in vitro, collected and seeded onto the surface of BG, BG-COL and BG-COL-PS. Cell attachment and proliferation were observed. The cell proliferation was tracked by MTT method 1,3,5 d after seeding. MTT showed that the cells can adhere to and proliferate well on the surface of the scaffolds, and the cell proliferation result of scaffold BG-COL-PS was better than those of scaffolds BG and BG-COL. Therefore, the scaffold BG-COL-PS can be a potential scaffold for tissue engineer.

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1alpha were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1alpha were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.

Phosphorylation of glycogen synthase kinase-3beta at serine-9 by phospholipase Cgamma1 through protein kinase C in rat 3Y1 fibroblasts

Phospholipase Cgamma1 (PLCgamma1) plays an important role in controlling cellular proliferation and differentiation. PLCgamma1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mechanisms underlying PLCgamma1-induced cell proliferation are not fully understood. Here we show that overexpression of PLCgamma1 highly phosphorylated glycogen synthase kinase-3beta (GSK-3beta) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3beta phosphorylation by PLCgamma1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLCgamma1-transfected cells as compared with vector-transfected cells. Since GSK-3beta is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLCgamma1-mediated GSK-3beta phosphorylation may function as a regulation of cyclin D1 accumulation in PLCgamma1-overexpressing cells.

Molecular Cloning of Human 14-3-3 cDNA with CD3 Zeta Peptide Containing Phosphosering Tag / 대한면역학회지

14-3-3 proteins are highly conserved proteins of about 29 kDa and have a minimum of seven isoforms. 14- 3-3 proteins interact with many signalling proteins by the recognition of phosphoserine. For the identification of proteins which react with ITAM (immunoreceptor tyrosine-based activation motif) of CD3 zeta chain, labeled synthetic peptides representing the CD3 zeta chain structual motifs (ITAMs) with a tag of PKC substrate sequence were used for western blotting. One major protein band of approximately 29 kDa was identified in lysate of Jurkat T cell, B cells and HeLa cells. Screening of lamda gt 11 library derived from HeLa cell gave two clones of 14-3-3 protein cDNA. Inspection of their nucleotide sequences identified these two full length cDNA clones as the 29 kDa human homologue of rat 14-3-3 gamma and the human 14-3-3 zeta protein. The human 14-3-3 gamma isoform also showed high homology with other species in amino acid and nucleotide sequence. Although 14-3-3 proteins are phosphoserine-binding proteins, there may be another way of interaction between ITAMs of CD3 and 14-3-3 proteins.

Production of Monoclonal Antibody Against Human 14 - 3 - 3 Zeta Isoform Expressed in Escherichia coli / 대한면역학회지

14-3-3 proteins are cytoplasmic proteins of about 29 kDa and have a minimum of seven isoforms. This protein is important in signal transduction with the ability of binding with phosphoserine of many signalling proteins. We expressed 14-3-3 protein tagged with 6 histidine residues in E. coli and purified the protein by nickel affinity chromatography. Using this purified protein as an antigen, we made rabbit antisera and mouse monoclonal antibodies to 14-3-3 zeta isoform. We subcloned cDNA of 14-3-3 zeta isoform derived from HeLa cell lamda gt 11 library into an E. coli expression vector which is designed to express heterologous protein with N- terminal 6 hidtidine tag. BALB/c mice were immunized with purified 14-3-3 protein and the hybridoma clones which produce monoclonal antibodies angainst 14-3-3 protein were selected. These monoclonal antibodies reacted with the recombinant protein expressed in E. coli as well as the 29-kDa native protein in various cell lines. However, they did not immunoprecipitate 14-3-3 protein. The monoclonal antibodies produced in this study can be valuable tools for the identification of the 14-3-3 in signal transduction study.

The Effect of Disodium Etidronate on Phosphatase Activity in Experimental bone Formation of Rats / 대한정형외과학회잡지

The bone formation of periarticular connective tissue after head injury and total hip arthroplasty is included in the category of heterotopic ossification. Induction of a new bone formation in the soft tissue is related to various materials such as bone morphogenic protein. The alkaline phosphatase and acid phosphatase act as important factors in the formation and absorption of the bone. The acid phospatase has the important function of acting as the control with specific activity of phosphatase in vivo. Cholecalciferol induces absorption of the calcium in the alimentary tract and bone resorption and increment of bone calcification, whereas disodium etidronate inhibits the deposition and dissolution of calcium salt and formation of heterotopic bone. This paper reports on the relationship of alkaline phosphatase and various phosphoaminoacid phosphatase which affect the cellular differentiation and remodelling in the heterotopic ossification, with the effect of cholecalciferol and disodium etidronate on the heterotopic bone induction in rats. The following results were obtained: 1. The contents of the calcium in the implanted bone matrix increased markedly from two to five weeks. There was no changes in the calcium content by cholecalciferol or in the administration of small doses of disodium etidronate (5mg/kg). However, in the administration of large dose of disodium etidronate (25mg/kg), calcium mobilization was totally suppressed for the whole period of the experiment. 2. The protein content in the implanted bone matrix did not much change for the whole period of the experiment and the administratinn of cholecalciferol or disodium etidronate also had no effect on the protein content. 3. The activities of alkaline phosphatase in the implanted bone matrix peaked at two weeks in control or cholecalciferol group, whereas disodium etidronate admninstration caused the highest activity in the third week. 4. The activity of acid phosphatase in the implanted bone matrix increased in first and third weeks by cholecalciferol treatment. Disoidum etidronate inhibited the activity of the acid phosphatase in the first, fourth & sixth weeks of implantation. 5. The activity of phosphoserine phosphatase increased due to cholecalciferol treatment, but was significantly inhibited by disodium etidronate (25mg/kg) treatment. 6. The activity of phosphothreonine phosphatase in the implanted bone matrix slightly increased due to cholecalciferol treatment, whereas the activity decreased significantly for the whole period of the experiment by disodium etidronate (25mg/kg) treatment. 7. The activity of phosphotyrosine phosphatase in the implanted bone matrix was not change much for the whole period of the experiment and the administration of cholecalciferol or disodium etidronate had no effect on the activity of phosphotyrosine phosphatase. In conclusion, the disodium etidronate (25mg/kg) almost completely inhibited the molilization of calcium and the activities of acid phosphatase, phosphoserine and phosphothreonine phosphatases. Therefore, it can be suggested that the above phosphatases are closely related to the action mechanism of disodium etidronate.