Human saliva contains a large number of proteins and peptides whose composition may alter as a consequence 。f disease. To date. however‘ the proteins and peptides that routinely populate thi s ora l fluid a re largely unknown To provide a catalogue of saliva proteins, we have surveyed the unstimulated human whole saliva by us ing shotgun proteornics. For the shotgun approach, whole saliva proteins were digested into peptides with ChemDigestD ‘ and the resul ting peptide fragments were separated by RP-HPLC, followed by each fraction was t ryptic digestion. ChemDigestD-Trypsin digested peptides were analyzed by tandem mass spectrometry(MS/MS) us ing a nano-LC eq 버 pped quadrupole-time of f1ight mass spectrometer, and the obtained spectra were searched against human protein seq uence data base using MASCOT. Shotgun proteomics allowed a total of 291 human pr。 teins to be confidently assigned . The largest group(17 .2%) of the identified proteins sorted into functional catego ries was included in t he signal t ransduction funct ion except for the hypothetical 0 1' unknown function. This work provides a valuable s ta rting point for the analysis of human salivary proteins and their biological functions and candidates from human whole sali va that may prove to be of diagnostic and therapeutic significance

The human ELAV(embryonic lethal abnormal vision)-like protein HuR stabilizes a certain group of cellular mRNAs that contain AU- ri ch elements in theil‘ 3’• untranslated region, Dysregulation of mRNA s tability may be relevant in tumor biology and may lead to abnormal expression of several proteins in malignant tumors, The aim of this study is to identify the differentially expressed proteins according to the functi onal activi ty of HuR Methods : We used stabl e expression of small interfering RNA(siRNA) of HuR gene to inhibit the expression of HuR in human ora l car cinoma cell lines‘ KB cell line and YD10B cell line‘ and compared the proteomic changes between s iRNA-treated and cont rol cell line using two-dimensional gel electrophoresis , Flow cytometry caliber scan(FACS) was employed to investigate the effects of HuR on cell apoptosis and proliferation , Results: Seventeen differentially expressed proteins between the two cell lines were identified by electrospray ioruzati on quadrupole time-of- fl ight mass spectrometry(ESI-Q-TOF MS) and database searching, Among them there are eleven proteins which are significant ly up- regulated in siRNA- treated cell line, which include heat shock protein 10(influencing nucl eocytoplas rnic transpo 다， cell dedifferentiation , and inhibition of apoptosis) , keratin 19(basal cell differ ent iat ion) ‘ and nucleoside diphosphate kinase B(G protein activator), etc Enolase 1 and Ml of pyruvate kinase a re the representatives of signifi cantly down-regulated proteins in siRNA-treated cell 11l1e Conclusion : Our data suggest that HuR participate in mRNAs stability of proteins that have the counter effects in the carcinogenesis of oral ca ncer , And the functiona l proteomics a re needed to elucidate the detailed interactions between HuR and t hese molec ules

Al t hough substance P(SP) , a potent pro- inflammatory peptide, is involved in inflammation and immune responses‘ t he eff'ect of SP on t he expression of macrophage inflammatory protein 3a (MIP- 3α CCL20) in periodontal liga ment(PDL) cell s a re unknown, Equally as enigmatic is the link between SP, t he stress protein heme oxygenase- l(HO-l) ‘ and CCL20 procluction, We investigated whether SP induces the release of chemokine CCL20 from immortal ized PDL(IPDL) ceJJ s‘ and fur ther c l a꺼 SP mediated pathways, We also examined the relationship between HO-l a ncl CCL20 by t reating PDL cells with SP, Incubating IPDL cells with SP increased expression of CCL20 mRNA a nd CCL20 protein in a dose-time dependent manner Highly selective p38 and ERKl/2 inhibitors abrogated SP-induced expression of CCL20 in IPDL cell s, SP is a lso responsible for ini t iating phosphorylation of I/C B, degradation of Iκ B‘ ancl activat ion of NF'-/C B, SP induced expression of HO-l in both a concentration- and time-dependent man nel ‘ and CCL20 refl ected s imilar patterns, The inductive effects o[ SP on HO- l and CCL20 wer e enhanced by HO- j inducer hemin and the membrane-permeable cGMP analog 8-bromo-cGMP, Conversely, this pathway was inJübited by t he 1-10난 inhi bitor zinc protoporphyrin IX(ZnPP IX) and the selective inl뼈itor of guanylate cyc1ase‘ lH-[l , 2, 4Joxad iazole[4‘ 3-aJquinoxal in-l-one (ODQ) , We report herein the pathway that connects SP along with other modulators 。f neuroimmunoregulationto the induction of HO-l and t he inflammatory mediator MIP-3a /CCL20 in IPDL cell s‘ which play an important role in the development 01' periodontitis or inflamrnation during orthodontic tooth movem

We have generated transgenic mice that expressed mouse extracellular superoxide dismutase (EC-SOD) in their skin. In particular, the expression plasmid DNA containing human keratin K14 promoter was used to direct the keratinocyte-specific transcription of the transgene. To compare intron-dependent and intron-independent gene expression, we constructed two vectors. The vector B, which contains the rabbit -globin intron 2, was not effective for mouse EC-SOD overexpression. The EC-SOD transcript was detected in the skin, as determined by Northern blot analysis. Furthermore, EC-SOD protein was detected in the skin tissue, as demonstrated by Western blot analysis. To evaluate the expression levels of EC-SOD in various tissues, we purified EC-SOD from the skin, lungs, brain, kidneys, livers, and spleen of transgenic mice and measured its activities. EC-SOD activities in the transgenic mice skin were approximately 7 fold higher than in wild-type mice. These results suggest that the mouse overexpressing vector not only induces keratinocyte-specific expression of EC-SOD, but also expresses successfully functional EC-SOD. Thus, these transgenic mice appeared to be useful for the expression of the EC-SOD gene and subsequent analysis of various skin changes, such as erythema, inflamation, photoaging, and skin tumors.

To study the signaling effect of insulin-like growth factor-Ⅰ(IGF-1), transgenic mice containing IGF-1 Receptor (IGF-1R) cDNA fused to metallothionein promoter were produced by DNA microinjection into the pronucleus of mouse zygote. Three founders were produced with transgenic mice containing IGF-1R gene. Transgenic mice lines contained approximately 4～20 copies of transgenes per cell and transmission of this gene into the progeny with Mendelian manner were determined. The founder mice were mated with normal mice to produce F1 mice and then F2 mice. Transmission rates of IGF-1R transgene in the progeny mice were 25～60% in F1 generation and 40～50% in F2 generation. The mRNA expression of IGF-1R transgene in liver was analyzed using RT-PCR for IGF-1R gene in liver. When body weights of transgenic pups were measured during 4, 10 and 14 weeks after birth, IGF-1R transgenic mice grew faster than non transgenic littermates. This study indicated that growth regulation by IGF-1 signaling through IGF-1R can be elucidated using IGF-1R transgenic mice.

This study was conducted to examine the effect of IRES controlled reporter gene on screening and production of recombinant human erythropoietin (EPO) proteins from cultured CHO cells. The cDNA was cloned for EPO from human liver cDNA. Using site-directed mutagenesis, we generated recombinant human EPO (rhEPO) with two additional N-glycosylations (Novel erythropoiesis-stimulating protein: NESP). Wild type hEPO and NESP were cloned into expression vectors with GFP reporter gene under regulatory control of CMV promoter and IRES so that the vectors could express both rhEPO and GFP. The expression vectors were transfected to cultured CHO-K1 cells. Under microscopy, expression of GFP was visible. Using supernatant of the culture, ELISA assay, immunocytochemistry and in vitro assay using EPO dependant cell line were performed to estimate biological activity to compare the production characteristics (secretion levels, etc.) between rhEPO and NESP. The activity of NESP protein, obtained by mutagenesis, was described and compared with its rhEPO counterpart produced under same conditions. Although NESP had less secretion level in CHO cell line, the biological activity of NESP was greater than that of rhEPO. These results are consistent with previous researches. We also demonstrated that rhEPO and GFP proteins expressed simultaneously from transfected CHO cell line. Therefore we conclude that use of GFP reporter gene under IRES control could be used to screen and produce rhEPO in cultured CHO cells.