Cordyceps militaris is well known as a traditional herbal ingredient, which has been used for patients suffering from cancer in oriental medicine. In this study we have investigated the biochemical mechanisms of anti-proliferative effects by C. militaris extract(CBE) in human breast cancer MDA-MB-231 cells. It was found that CBE treatment induced chromatin condensation, mitochondrial energization, annexin V staining and sub-G1 phase DNA content. These indicators of apoptosis correlate with the mitochondrial dependent pathway, which results in the activation of caspase-3 activity. Both the cytotoxic effect by CBE treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the observed cytotoxic effect. Co-treatment of CBE and LY294002, resulted in significantly induction of apoptosis. These results indicate that caspase-3 is a key regulator of apoptosis in response to CBE in human breast cancer MDA-MB-231 through down regulation of Akt, and that the C. militaris extract may therefore have therapeutic potential against human breast cancer.

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

Sulfur is commonly used in Asia as an herbal medicine to treat inflammation and cancer , a nd potent chemo preventi ve effects have been demons tra ted in various in vivo and in vitromodels for s ul fur-containing compounds found in natura l1y occurring product s. Here, we 1'eport the growth inhibitory and apoptosis-related effects of a n ewly developedhigh-puri ty edible sulfur(ES) on immo1'tali zed human o1'al ke1'atinocytes(IHOKs) and on oral cancer cells representing two stages of oral cancer (HN4‘ HN12) based on an 3-(4,5-Dimethylthiazol-2-yl) -2.5- diphe n yltetrazolium bromide(MTI) assay, Western blotting, cell cycle analysis, and nuclear staining. The puri ty of the ES used in thi s study was ve1'ified by high performance liquid chromatog1'aphy (HPLC) , ami no acid analysis and energy dispersive spectroscopy(EDS). ES inhibited the prolife1'ation of imrnortalized and ma lig nant o1'al kerati nocytes in a dose- and time-dependent manne1' FITC-Annex.in V staining, DNA fragmentation t esting. and Hoechst 33258 s taining revealed that ES inhibits cell growth via apoptosis. ES bl ocked cell-cycle prog1'ession at t he sub-Gl phase‘ wi th decreased expression of cyclins Dl, D2‘ and E, and their activating partn ers cdk2‘ cdk4‘ and cdkfì, and a concomitant induction of p53 and p21/WAF1. Furthe1'more, ES treatment in creased the cytosolic level of cytochrome c and resulted in caspase- 3 activation‘ and thi s effect was co1'1'elated with Bax up-regulation and Bcl-2 down-1'egulation Taken together‘ these data suggest that ES is a potential chemopreventive and chemotherapeut ic agent fo r oral ca ncer

Al though the changes in tooth morphology and hardness by hydrogen peroxide(H20 z) have been r‘epor‘.ted .‘ the pαr。o야t뻐ec야tive role of heme oxygenase-l(HO-l) against the cytotoxic effects of H202 has not been clarifïed i n human pulp cells ln this st udy. we investigated whether HO-l is involved in Hz0 2-induced cytoLox icity a nd examined the production 0 1' dentin sia lophosphoprotein(DSPP) and other mineralization markers‘ in hllman pu lp cells H202 decreased cell viabi lity, but increased HO-l and DSPP expression in a concentra tion and time dependent manner . Inhibitors of gllanylate cyclase. PI3K, ERK. and p38 MAP kinase blocked H202-indllced cytotoxicity and the expression of HO-l and DSPP mRNAs in pulp cells. These data suggest that the induction of HO-l by H202 in plllp cells plays a protective role against the cytotoxic effects 0 1' HzOz and stimulates DSPP expression‘ reslllting in prematllre odontoblast dilTerentiation throllgh pathways that involve cGMP‘ p38. and ERK.

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

There is no universally agreed classification of human error, nor is there one in prospect. Thus, a taxonomy is usually made for a specific purpose. To seek the types of human errors in the environment of man-machine interface under the railway industry, we develop a cognitive information processing model incorporating the human's mental states. Using the model, this study investigates the types of human errors about the railway workers. Thus, a survey is conducted for railway safety personnel-locomotive engineers, station employees, and train commanders- in Korean railway company. Through the survey that is designed to investigate four types of human errors from the Questionnaires composed of thirty Questions, we analyze the types of human errors related to railway safety according to affiliated offices, operation shifts, age, and working years. Finally, from the insights of the results some guidelines for the railway safety management are presented.

Through so that accident of semiconductor industry deduces unsafe factor of the person center on unsafe behaviour that incident history and questionnaire and I made starting point that extract very important factor. It served as a momentum that make up base that analyzes factors that happen based on factor that extract factor cause classification for the first factor, the second factor and the third factor and presents model of human error. Factor for whole defines factor component for human factor and to cause analysis 1 stage in human factor and step that wish to do access of problem and it do analysis cause of data of 1 step. Also, see significant difference that analyzes interrelation between leading persons about human mistake in semiconductor industry and connect interrelation of mistake by this. Continuously, dictionary road map to human error theoretical background to basis traditional accidental cause model and modern accident cause model and leading persons. I wish to present model and new model in semiconductor industry by backbone that leading persons of existing scholars who present model of existent human error deduce relation. Finally, I wish to deduce backbone of model of pre-suppression about accident leading person of the person center.

The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gonadotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp. 2: 0, 0.5, 1.0 or 10 IU) or both (Exp. 3: 1 IU FSH + 1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with 1.9 μM. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.

Human saliva conta ins a la rge number of proteins and peptides whose composition may alter as a conseq uence of disease. '1'0 date‘ however. the proteins and peptides that routinely populate t his oral fluid are largely unknown, '1'0 provid e a ca ta logue 이， sali va protei ns. we have surveyed the unstimulated human whole saliva by using shotgun proteomics. F'or the shotgun a pproach‘ whole sali va proteins were digested into peptides with ChemDigestD and the res ulting pe ptide fragments were sepa rated by RP- HPLC, followed by each fraction was tryptic di gestion ChemDiges tD- Trypsin diges ted pe pt ides were analyzed by tandem mass spectrometry (MS!MS) using a nano-LC equi pped quadru po le-time of fli ght rnass spect rometer, and the obtained spectra were searched against human protein sequence da tabase us ing MASCOT Shotgun proteomics a llowed a total of 291 human proteins to be confidently assigned. The largest gro u p (17 , 2%) of the identifi ed proteins sorted into functional categories was included in the s ignal transducti on function except for the hypothet ical or unknown functio n, This work provides a valuable starting point for the ana lys is of human sa l i va ry protei ns a nd theil‘ biological functions and candidates from human whole saliva that may prove to be of diagn ost ic and t herapeutic s ignif‘ Ica nce

We have developed a new passaging technique for the expansion of human embryonic stem cells (hESCs) that involves simply pipetting portions of hESCs acquired from colonies, reducing the laborious and time-consuming steps in the expansion of hESCs. Compared to general mechanical methods of passaging, our pipetting method allowed hESCs colonies to be broken into small fragments, which showed significantly higher attachment rates onto feeder cell layers. This technique produced three times the number of hESCs colonies than conventional mechanical methods. In addition, this pipetting method allowed us to distinguish differentiated hESCs from undifferentiated hESCs during hESCs colony pipetting. The hESCs cultured by pipetting method displayed normal human chromosomes for over 60 passages. According to RT-PCR and immunohistochemical analysis, the hESCs successfully maintained their undifferentiated state and pluripotency which was also confirmed by teratoma formation in vivo. Therefore, the pipetting method described in this study is a useful tool to efficiently and quickly expand hESCs on a large scale without enzyme treatment.

Vitrification has been suggested to be an effective method for the cryopreservation of human ES cells. However, the efficiency of vitrification with different vehicles remains a matter of ongoing controversy. The objective of this study was to assess the efficiency of cryopreservation in human ES cells by vitrification using different vehicles. A human ES cell line and a variety of vehicles, including microdroplet (MD), openpulled straw (OPS) and electron microscopic grid (EMgrid), were employed in an attempt to assess vitrification efficiency. In order to evaluate the survivability and the undifferentiated state of the postvitrified human ES cells, we conducted alkaline phosphatase staining and characterization via both RTPCR and immunofluorescence assays. The survival rates of the postvitrified human ES cells using MD, OPS and EMgrid were determined to be 61.5%, 66.6% and 53.8%, respectively. There also exist significant differences between slowfreezing and vitrification (p<0.01). However, no significant differences were detected between the vehicle types. Finally, the pluripotency of human ES cells after thawing was verified by teratoma formation. Cryopreservation using vitrification is more effective than slowfreezing, and the efficiency of vehicles proved effective with regard to the preservation of human ES cells.

Extensive oral mucosa loss from a variety of conditions is associated with significant functional morbidity and mortality. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-β1(TGF-β1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms expressed different levels of TGF-β1. When NHOK reached confluency in serum free medium(KBM), in test medium containing 1.2 mM Ca++ KBM NHOK were allowed to form multi-layers and differentiate. The purpose of this study were to investigate the mRNA level of TGF-β1, FGF-2, and TIMP-1 by RT-PCR analysis and also to evaluate the expression of TGF-β1 and involucrin in keratinocytes at different times of the onset of differentiation. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. Cultured NHOK in preconfluency under KBM medium expressed a significantly higher level of TGF-β1 relative to those grown in multi-layer forms, while the level of TGF-β1 mRNA gradually reduced to its lowest level at 7 days of growing cells in test medium. Cultured NHOK in preconfluency of KBM medium expressed a lower level of FGF-2 and TIMP-1 relative to those grown in multi-layer forms, while the level of FGF-2 and TIMP-1 mRNA showed the highest level at 3 days at gradually reduced to its lowest level at 7 days of growing cells in test medium. As a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. It suggested that the expression of TGF-β1 mRNA be consistent with the expression of FGF-2 and TIMP-1 mRNA in NHOK grown in high calcium medium during the terminal differentiation. But differentiated NHOK expressing higher involucrin mRNA could show constant espression of TGF-β1, FGF-2 and TIMP-1.

Gemcitabine (Gemzar, 2,2-difluorodeoxycytidine, or dFdC) is an analog of cytosine arabinoside with anti-tumor activity in a few human cancers (lung, ovary, pancreatic and breast cancers). However, the mechanism of apoptosis by this compound in carcinoma has not been fully elucidated. In the present study, we investigated that gemcitabine alone and combination with cisplatin or 5-FU are cancer toxicity using lung cancer cell line A549 by MTT, FACS analysis, and Western blot assay. Also, to confirm enhanced antitumor activity in vivo using an xenograft tumor model. The MTT assay showed higher cytotoxic effect in combination with gemcitabine-cisplatin or gemcitabine-5-FU than gemcitabine alone. FACS analysis showed that gemcitabine-cisplatin combination increased hypodiploid DNA to 70.84 %. The induction of apoptosis showed more increase in combination with gemcitabine-cisplatin or gemcitabine-5-FU than gemcitabine alone. The Western results showed higher expression of p53 and p21WAF/CIP1 protein in combination treatment with gemcitabine-cisplatin or gemcitabine-5-FU than gemcitabine treatment alone. But, Bcl-2 protein expression decreased. In vivo experiments showed that more decreased tumor size and more increased survival rate on combination with gemcitabine- cisplatin or gemcitabine-5-FU combination than gemcitabine alone. In conclusion, this study suggests that gemcitabine combined with cisplatin or 5-FU are the synergistic effect of anticancer therapy on lung cancer.

The purpose of this study was to improve the professional's development of skill in qualitative analysis of humanMovement fromthe various subdisciplines of physical education. The conclusions were as follows; First, Physical educators and coaches can impr