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Sample records for spe b-induced apoptosis

  1. Bax is upregulated by p53 signal pathway in the SPE B-induced apoptosis.

    PubMed

    Lee, Wei-Ting; Chang, Chia-Wen

    2010-10-01

    We identify integrin α(v)β(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S (SPE B mutant, glycine at residue 308 is changed to serine), which interacts with Fas only, in our previous study. Here, we explore the signal pathways that regulate proapoptotic protein expression after SPE B stimulation. We find that both SPE B and G308S can stimulate the serine phosphorylation of p53, and p53 phosphorylation is inhibited by the anti-Fas antibody but not by anti-α(V)β(3) antibody. p38 inhibitor and siRNA decrease the activation and translocation of p53 into the nucleus, which executes its transcription activity. These results indicate that after SPE B treatment, p53 is activated and p38 is the upstream of p53. p38 siRNA also decreases the binding of p53 to the bax promoter and interferes with the association of p53 and STAT1. p53, p38, and STAT1 siRNAs downregulate SPE B-induced Bax expression. This shows that SPE B activates the bax promoter via p38/p53 signal pathways through the Fas receptor, and that STAT1 acts as a coactivator of p53. In addition, p38 and p53 siRNAs inhibit SPE B-induced apoptosis. This is consistent with the findings that SPE B upregulates Bax expression through p38/p53 signal pathways that enhance cell apoptosis. PMID:20567883

  2. Ophiopogonin B induces apoptosis, mitotic catastrophe and autophagy in A549 cells.

    PubMed

    Chen, Meijuan; Guo, Yuanyuan; Zhao, Ruolin; Wang, Xiaoxia; Jiang, Miao; Fu, Haian; Zhang, Xu

    2016-07-01

    Ophiopogonin B (OP-B), a saponin compound isolated from Radix Ophiopogon japonicus, was verified to inhibit cell proliferation in numerous non-small cell lung cancer (NSCLC) cells in our previous study. However, the precise mechanisms of action have remained unclear. In the present study, we mainly investigated the effects of OP-B on adenocarcinoma A549 cells to further elaborate the underlying mechanisms of OP-B in different NSCLC cell lines. Detection by high content screening (HCS) and TUNEL assay verified that OP-B induced apoptosis in this cell line, while detection of Caspase-3, Bcl-2 and Bax showed that OP-B induced cell death was caspase and mitochondrial independent. Further experiments showed that OP-B induced cell cycle arrest in the S and G2/M phases by inhibiting the expression of Myt1 and phosphorylation of Histone H3 (Ser10), which resulted in mitotic catastrophe in the cells. Transmission electron microscopy (TEM) observation of cell micro-morphology combined with detection of Atgs by western blot analysis showed that OP-B induced autophagy in this cell line. Autophagy inhibition by the lysosome inhibitor CQ or Beclin1-siRNA knockdown both attenuated cell viability, demonstrated that autophagy also being the vital reason resulted in cell death. More importantly, the xenograft model using A549 cells provided further evidence of the inhibition of OP-B on tumor proliferation. Immunohistochemistry detection of LC3 and Tunel assay both verified that high dose of OP-B (75 mg/kg) induced autophagy and apoptosis in vivo, and western blot detection of p-Histone H3 (Ser10), Survivin and XIAP further indicated the molecular mechanism of OP-B in vivo. As our findings revealed, multiple types of cell death overlapped in OP-B treated A549 cells, it displayed multitarget characteristics of the compounds extracted from the Chinese herbal, which may be used as candidate anticancer medicine in clinic. PMID:27175570

  3. LincRNA-p21 acts as a mediator of ING1b-induced apoptosis

    PubMed Central

    Tran, U M; Rajarajacholan, U; Soh, J; Kim, T-s; Thalappilly, S; Sensen, C W; Riabowol, K

    2015-01-01

    ING1b is a tumor suppressor that affects transcription, cell cycle control and apoptosis. ING1b is deregulated in disease, and its activity is closely linked to that of p53. In addition to regulating protein-coding genes, we found that ING1b also influences the expression of large intergenic non-coding RNAs (lincRNAs). In particular, lincRNA-p21 was significantly induced after DNA-damage stress or by ING1b overexpression. Furthermore, lincRNA-p21 expression in response to DNA damage was significantly attenuated in cells lacking ING1b. LincRNA-p21 is also a target of p53 and can trigger apoptosis in mouse cell models. We found that this function of lincRNA-p21 is conserved in human cell models. Moreover, ING1b and p53 could function independently to influence lincRNA-p21 expression. However, their effects become more additive under conditions of stress. In particular, ING1b regulates lincRNA-p21 levels by binding to its promoter and is required for induction of lincRNA-p21 by p53. The ability of ING1b to cause apoptosis is also impaired in the absence of lincRNA-p21. Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21. Our findings suggest that ING1b induces lincRNA-p21 expression independently of histone 3 lysine 4 trimethylation mark recognition and that lincRNA-p21 functions downstream of ING1b. Thus, regulation at the level of lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis under specific stress conditions. PMID:25741593

  4. p51/p63 Inhibits ultraviolet B-induced apoptosis via Akt activation.

    PubMed

    Ogawa, E; Okuyama, R; Ikawa, S; Nagoshi, H; Egawa, T; Kurihara, A; Yabuki, M; Tagami, H; Obinata, M; Aiba, S

    2008-01-31

    The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member DeltaNp51B/DeltaNp63alpha (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, DeltaNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of DeltaNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that DeltaNp51B blocks keratinocyte apoptosis. Although the exogenous expression of DeltaNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH(2)-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes. PMID:17653081

  5. Mangiferin induces apoptosis in multiple myeloma cell lines by suppressing the activation of nuclear factor kappa B-inducing kinase.

    PubMed

    Takeda, Tomoya; Tsubaki, Masanobu; Kino, Toshiki; Yamagishi, Misa; Iida, Megumi; Itoh, Tatsuki; Imano, Motohiro; Tanabe, Genzoh; Muraoka, Osamu; Satou, Takao; Nishida, Shozo

    2016-05-01

    Mangiferin is a naturally occurring glucosyl xanthone, which induces apoptosis in various cancer cells. However, the molecular mechanism underlying mangiferin-induced apoptosis has not been clarified thus far. Therefore, we examined the molecular mechanism underlying mangiferin-induced apoptosis in multiple myeloma (MM) cell lines. We found that mangiferin decreased the viability of MM cell lines in a concentration-dependent manner. We also observed an increased number of apoptotic cells, caspase-3 activation, and a decrease in the mitochondrial membrane potential. In addition, mangiferin inhibited the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated inhibitor kappa B (IκB) and increased the expression of IκB protein, whereas no changes were observed in the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase 1/2 (JNK1/2), and mammalian target of rapamycin (mTOR). The molecular mechanism responsible for mangiferin-induced inhibition of nuclear translocation of NF-κB was a decrease in the expression of phosphorylated NF-κB-inducing kinase (NIK). Moreover, mangiferin decreased the expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, and Bcl-xL proteins. Knockdown of NIK expression showed results similar to those observed with mangiferin treatment. Our results suggest that mangiferin induces apoptosis through the inhibition of nuclear translocation of NF-κB by suppressing NIK activation in MM cell lines. Our results provide a new insight into the molecular mechanism of mangiferin-induced apoptosis. Importantly, since the number of reported NIK inhibitors is limited, mangiferin, which targets NIK, may be a potential anticancer agent for the treatment of MM. PMID:26996543

  6. Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells

    PubMed Central

    Ha, Sun-Hyung; Lee, Ji-Min; Kwon, Kyung-Min; Kwak, Choong-Hwan; Abekura, Fukushi; Park, Jun-Young; Cho, Seung-Hak; Lee, Kichoon; Chang, Young-Chae; Lee, Young-Choon; Choi, Hee-Jung; Chung, Tae-Wook; Ha, Ki-Tae; Chang, Hyeun-Wook; Kim, Cheorl-Ho

    2016-01-01

    Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: β1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis. PMID:27144558

  7. Hesperidin Attenuates Ultraviolet B-Induced Apoptosis by Mitigating Oxidative Stress in Human Keratinocytes.

    PubMed

    Madduma Hewage, Susara Ruwan Kumara; Piao, Mei Jing; Kang, Kyoung Ah; Ryu, Yea Seong; Han, Xia; Oh, Min Chang; Jung, Uhee; Kim, In Gyu; Hyun, Jin Won

    2016-05-01

    Human skin cells undergo pathophysiological processes via generation of reactive oxygen species (ROS) upon excessive exposure to ultraviolet B (UVB) radiation. This study investigated the ability of hesperidin (C28H34O15) to prevent apoptosis due to oxidative stress generated through UVB-induced ROS. Hesperidin significantly scavenged ROS generated by UVB radiation, attenuated the oxidation of cellular macromolecules, established mitochondrial membrane polarization, and prevented the release of cytochrome c into the cytosol. Hesperidin downregulated expression of caspase-9, caspase-3, and Bcl-2-associated X protein, and upregulated expression of B-cell lymphoma 2. Hesperidin absorbed wavelengths of light within the UVB range. In summary, hesperidin shielded human keratinocytes from UVB radiation-induced damage and apoptosis via its antioxidant and UVB absorption properties. PMID:26797112

  8. Hesperidin Attenuates Ultraviolet B-Induced Apoptosis by Mitigating Oxidative Stress in Human Keratinocytes

    PubMed Central

    Hewage, Susara Ruwan Kumara Madduma; Piao, Mei Jing; Kang, Kyoung Ah; Ryu, Yea Seong; Han, Xia; Oh, Min Chang; Jung, Uhee; Kim, In Gyu; Hyun, Jin Won

    2016-01-01

    Human skin cells undergo pathophysiological processes via generation of reactive oxygen species (ROS) upon excessive exposure to ultraviolet B (UVB) radiation. This study investigated the ability of hesperidin (C28H34O15) to prevent apoptosis due to oxidative stress generated through UVB-induced ROS. Hesperidin significantly scavenged ROS generated by UVB radiation, attenuated the oxidation of cellular macromolecules, established mitochondrial membrane polarization, and prevented the release of cytochrome c into the cytosol. Hesperidin downregulated expression of caspase-9, caspase-3, and Bcl-2-associated X protein, and upregulated expression of B-cell lymphoma 2. Hesperidin absorbed wavelengths of light within the UVB range. In summary, hesperidin shielded human keratinocytes from UVB radiation-induced damage and apoptosis via its antioxidant and UVB absorption properties. PMID:26797112

  9. Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation

    PubMed Central

    Cohen-Mazor, Meital; Mazor, Rafi; Kristal, Batya; Kistler, Erik B.; Ziv, Inbal; Chezar, Judith; Sela, Shifra

    2015-01-01

    Heparin is known to have anti-inflammatory effects, yet the mechanisms are not completely understood. In this study, we tested the hypothesis that heparin has a direct effect on activated polymorphonuclear leukocytes (PMNLs), changing their activation state, and can explain its anti-inflammatory effect. To test our hypothesis, we designed both in vitro and ex vivo studies to elucidate the mechanism by which heparin modulates PMNL functions and therefore the inflammatory response. We specifically tested the hypothesis that priming of PMNLs renders them more susceptible to heparin. Amplified levels of CD11b and increased rate of superoxide release manifested PMNL priming. Increase in cell priming resulted in a dose-dependent increase in heparin binding to PMNLs followed by augmented apoptosis. Blocking antibodies to CD11b inhibited heparin binding and abolished the apoptotic response. Moreover, heparin caused a significant dose-dependent decrease in the rate of superoxide release from PMNLs, which was blunted by blocking antibodies to CD11b. Altogether, this study shows that the interaction of heparin with the PMNL CD11b results in cell apoptosis and explains heparin's anti-inflammatory effects. PMID:26819958

  10. The role of cholesterol in UV light B-induced apoptosis.

    PubMed

    George, Kimberly S; Elyassaki, Walid; Wu, Qiong; Wu, Shiyong

    2012-01-01

    Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of programmed cell death under various stimuli. However, the relationship between cholesterol level modification and the activation of apoptotic signaling cascades upon UVB light has not been established. In this report, we demonstrate that upon UVB irradiation cholesterol levels in membrane rafts of skin cells increase, which leads to Fas-receptor (Fas) aggregation in the rafts. Utilizing a continuous velocity floatation technique, we show that Fas accumulated in the lipid rafts of human melanoma M624 cells after UVB irradiation. The subsequent events of death-inducing signaling complex formation were also detected in the lipid raft fractions. Depletion of cholesterol by methyl-β-cyclodextrin reduces Fas aggregation, while overloading increases. Disruption of lipid rafts also prevents Fas death domain-associated protein (Daxx) from dissociating from Fas in the lipid rafts, which is accompanied with a reduced apoptotic, but increased nonapoptotic death of UVB-irradiated human keratinocytes, HaCaT cells. Results indicate that cholesterol located in the plasma membrane of skin cells is required for lipid raft domain formation and activation of UVB-induced apoptosis. PMID:22077874

  11. Glaucocalyxin B induces apoptosis and autophagy in human cervical cancer cells.

    PubMed

    Pan, Ying; Bai, Jieyu; Shen, Fangfang; Sun, Li; He, Quanzhong; Su, Bing

    2016-08-01

    Glaucocalyxin (Gln), an ent‑kaurane diterpenoid isolated from the Chinese traditional medicine, Rabdosia japonica, represents a novel class of anticancer drugs. GlnA is one of the three major forms of Gln and has demonstrated potent anticancer effects in a variety of cancer types. GlnB has only one structural difference from GlnA, an acetylated hydroxyl group at C14. This acetyl group results in high liposolubility and may enhance the antitumor activity of ent‑kaurane diterpenoid GlnB. However, few studies have reported the role of GlnB in cancer. The present study investigated the effect of GlnB in cervical cancer proliferation and cell death. Treatment with GlnB inhibits the proliferation of HeLa and SiHa cervical cancer cell lines in a dose‑dependent manner, as assessed by 3‑(4,5‑dimethylthiazol-2‑yl)-2,5 diphenyl tetrazolium bromide assays. In addition, GlnB increases the apoptotic cell population of HeLa and SiHa cells, as determined by fluorescence‑activated cell sorting analysis and enhanced poly (ADP‑ribose) polymerase 1 cleavage by western blotting. GlnB also induces increased light chain 3 II/I protein cleavage in both cells, indicating the induction of autophagy. Furthermore, GlnB treatment increased the expression of phosphatase and tensin homolog and decreased the expression of phosphorylated‑protein kinase B (Akt) in HeLa and SiHa cells, as assessed by western blotting. Taken together, the present results demonstrated that GlnB inhibited the proliferation of human cervical cancer cells in vitro through the induction of apoptosis and autophagy, which may be mediated by the phosphatidylinositol‑4,5‑bisphosphate 3‑kinase/Akt signaling pathway. PMID:27356884

  12. Eriocalyxin B-induced apoptosis in pancreatic adenocarcinoma cells through thiol-containing antioxidant systems and downstream signalling pathways.

    PubMed

    Li, L; Yue, G G L; Pu, J X; Sun, H D; Fung, K P; Leung, P C; Han, Q B; Lau, C B S; Leung, P S

    2014-01-01

    Thiol-containing antioxidant systems play an important role in regulating cellular redox homeostasis. Several anti-cancer agents act by targeting these systems by inducing the production of reactive oxygen species (ROS). Our earlier studies have shown that Eriocalyxin B (EriB), a diterpenoid isolated from Isodon eriocalyx, possesses anti-pancreatic tumour activities in vitro and in vivo. The present study further demonstrated that only thiol-containing antioxidants, N-acetylcysteine (NAC) or dithiothreitol (DTT), inhibited EriB-induced cytotoxicity and apoptosis. EriB suppressed the glutathione and thioredoxin antioxidant systems, thus increasing the intracellular ROS levels and regulating the MAPK, NFκB pathways. Treatment with EriB depleted the intracellular thiol-containing proteins in CAPAN-2 cells. In vivo studies also showed that EriB treatment (2.5 mg/kg) reduced the pancreatic tumour weights significantly in nude mice with increased superoxide levels. Taken together, our results shed important new light on the molecular mechanisms of EriB acting as an apoptogenic agent and its therapeutic potential for pancreatic cancer. PMID:24894173

  13. Macranthoside B Induces Apoptosis and Autophagy Via Reactive Oxygen Species Accumulation in Human Ovarian Cancer A2780 Cells.

    PubMed

    Shan, Yu; Guan, Fuqin; Zhao, Xingzeng; Wang, Ming; Chen, Yu; Wang, Qizhi; Feng, Xu

    2016-01-01

    Macranthoside B (MB), a saponin compound in Lonicera macranthoides, can block cell proliferation and induce cell death in several types of cancer cells; however, the precise mechanisms by which MB exerts its anticancer effects remain poorly understood. MB blocked A2780 human ovarian carcinoma cell proliferation both dose- and time-dependently. MB induced apoptosis, with increased poly (ADP-ribose) polymerase (PARP) and caspase-3/9 cleavage. MB also caused autophagy in A2780 cells, with light chain 3 (LC3)-II elevation. Inhibiting MB-induced autophagy with the autophagy inhibitor 3-methyladenine (3-MA) significantly decreased apoptosis, with a reduction of growth inhibition; inhibiting MB-induced apoptosis with the pan-caspase inhibitor Z-VAD-FMK did not decrease autophagy but elevated LC3-II levels, indicating that MB-induced autophagy is cytotoxic and may be upstream of apoptosis. Furthermore, MB increased intracellular reactive oxygen species (ROS) levels, with activated 5' adenosine monophosphate-activated protein kinase (AMPK), decreased mammalian target of rapamycin (mTOR) and P70S6 kinase phosphorylation, and increased PARP and caspase-3/9 cleavage, and LC3-II elevation; treatment with the ROS scavenger N-acetyl cysteine and the AMPK inhibitor Compound C diminished this effect. Therefore, the ROS/AMPK/mTOR pathway mediates the effect of MB on induction of apoptosis via autophagy in human ovarian carcinoma cells. PMID:26943028

  14. Jungermannenone A and B induce ROS- and cell cycle-dependent apoptosis in prostate cancer cells in vitro

    PubMed Central

    Guo, Yan-xia; Lin, Zhao-min; Wang, Mei-juan; Dong, Yi-wen; Niu, Huan-min; Young, Charles YF; Lou, Hong-xiang; Yuan, Hui-qing

    2016-01-01

    Aim: Jungermannenone A and B (JA, JB) are new ent-kaurane diterpenoids isolated from Chinese liverwort Jungermannia fauriana, which show anti-proliferation activities in cancer cells. In this study we investigated the mechanisms underlying the anticancer action of JA and JB in PC3 human prostate cancer cells in vitro. Methods: A panel of 9 human cancer cell lines was tested. Cell proliferation was assessed with a real-time cell analyzer and MTT assay. Cell apoptosis, cell cycle distribution and ROS levels were measured using cytometry. Mitochondrial damage was examined by transmission electron microscopy. DNA damage was detected with comet assay. Apoptotic, DNA damage- and cell cycle-related proteins were analyzed using Western blotting. The expression of DNA repair genes was measured with qRT-PCR. Results: Both JA and JB exerted potent anti-proliferative action against the 9 cancer cell lines, and PC3 cells were more sensitive with IC50 values of 1.34±0.09 and 4.93±0.20 μmol/L, respectively. JA (1.5 μmol/L) and JB (5 μmol/L) induced PC3 cell apoptosis, which was attenuated by the caspase inhibitor Z-VAD. Furthermore, both JA and JB caused mitochondrial damage and ROS accumulation in PC3 cells, whereas vitamin C blocked the ROS accumulation and attenuated the cytotoxicity of JA and JB. Moreover, both JA and JB induced DNA damage, accompanied by downregulated DNA repair proteins Ku70/Ku80 and RDA51. JA induced marked cell cycle arrest at the G0/G1 phase, which was related to c-Myc suppression, whereas JB enforced the cell cycle blockade in the G2/M phase, which associated with activation of the JNK signaling. Conclusion: Both JA and JB induce prostate cancer apoptosis via ROS accumulation and induction of cell cycle arrest. PMID:27133304

  15. Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways

    SciTech Connect

    Li, Lin; Yue, Grace G.L.; Lau, Clara B.S.; Sun, Handong; Fung, Kwok Pui; Leung, Ping Chung; Han, Quanbin; Leung, Po Sing

    2012-07-01

    Pancreatic cancer is difficult to detect early and responds poorly to chemotherapy. A breakthrough in the development of new therapeutic agents is urgently needed. Eriocalyxin B (EriB), isolated from the Isodon eriocalyx plant, is an ent-kaurane diterpenoid with promise as a broad-spectrum anti-cancer agent. The anti-leukemic activity of EriB, including the underlying mechanisms involved, has been particularly well documented. In this study, we demonstrated for the first time EriB's potent cytotoxicity against four pancreatic adenocarcinoma cell lines, namely PANC-1, SW1990, CAPAN-1, and CAPAN-2. The effects were comparable to that of the chemotherapeutic camptothecin (CAM), but with much lower toxicity against normal human liver WRL68 cells. EriB's cytoxicity against CAPAN-2 cells was found to involve caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Moreover, the p53 pathway was found to be activated by EriB in these cells. Furthermore, in vivo studies showed that EriB inhibited the growth of human pancreatic tumor xenografts in BALB/c nude mice without significant secondary adverse effects. These results suggest that EriB should be considered a candidate for pancreatic cancer treatment. -- Highlights: ► We study Eriocalyxin B (EriB)'s cytotoxic effects on pancreatic cancer cell lines. ► EriB inhibits cell proliferation via mediation of apoptosis and cell cycle arrest. ► The effects are involved in caspase-dependent apoptosis and p53 pathway. ► In vivo study also shows EriB inhibits the growth of human pancreatic tumor. ► EriB can be a good candidate for chemotherapy in pancreatic cancer.

  16. Involvement of the extrinsic and intrinsic pathways in ultraviolet B-induced apoptosis of corneal epithelial cells.

    PubMed

    Ubels, John L; Glupker, Courtney D; Schotanus, Mark P; Haarsma, Loren D

    2016-04-01

    The goal of this study was to elucidate the pathway by which UVB initiates efflux of K(+) and subsequently apoptosis in human corneal limbal epithelial (HCLE) cells. The initial focus of the study was on the extrinsic pathway involving Fas. HCLE cells transfected with Fas siRNA were exposed to 80-150 mJ/cm(2) UVB and incubated in culture medium with 5.5 mM K(+). Knockdown of Fas resulted in limited reduction in UVB-induced caspase-8 and -3 activity. Patch-clamp recordings showed no difference in UVB-induced normalized K(+) currents between Fas transfected and control cells. Knockdown of caspase-8 had no effect on the activation of caspase-3 following UVB exposure, while a caspase-8 inhibitor completely eliminated UVB activation of caspase-3. This suggests that caspase-8 is a robust enzyme, able to activate caspase-3 via residual caspase-8 present after knockdown, and that caspase-8 is directly involved in the UVB activation of caspase-3. Inhibition of caspase-9 significantly decreased the activation of caspases-8 and -3 in response to UVB. Knockdown of Apaf-1, required for activation of caspase-9, resulted in a significant reduction in UVB-induced activation of caspases-9, -8, and -3. Knockdown of Apaf-1 also inhibited intrinsic and UVB-induced levels of apoptosis, as determined by DNA fragmentation measured by TUNEL assay. In UVB exposed cultures treated with caspase-3 inhibitor, the percentage of apoptotic cells was reduced to control levels, confirming the necessity of caspase-3 activation in DNA fragmentation. The lack of effect of Fas knockdown on K(+) channel activation, as well as the limited effect on activation of caspases-8 and -3, strongly suggest that Fas and the extrinsic pathway is not of primary importance in the initiation of apoptosis in response to UVB in HCLE cells. Inhibition of caspase-8 and -3 activation following inhibition of caspase-9, as well as reduction in activation of caspases-9, -8, and -3 and DNA fragmentation in response to Apaf

  17. Licochalcone B induces apoptosis of human oral squamous cell carcinoma through the extrinsic- and intrinsic-signaling pathways.

    PubMed

    Oh, Hana; Yoon, Goo; Shin, Jae-Cheon; Park, Seon-Min; Cho, Seung-Sik; Cho, Jin Hyoung; Lee, Mee-Hyun; Liu, Kangdong; Cho, Young Sik; Chae, Jung-Il; Shim, Jung-Hyun

    2016-04-01

    Licochalcone B (Lico B), which belongs to the retrochalcone family, is isolated from the roots of Chinese licorice. Lico B has been reported to have several other useful pharmacological properties, such as anti-inflammatory, antibacterial, antioxidant, antiulcer, anticancer, and anti-metastasis activities. We elucidated the underlying mechanism by which Lico B can induce apoptosis in oral squamous cell carcinoma (OSCC). Our results showed that exposure of OSCC cells (HN22 and HSC4) to Lico B significantly inhibited cell proliferation in a time- and concentration-dependent manner. Lico B caused cell cycle arrest at G1 phase along with downregulation of cyclin D1 and upregulation of p21 and p27 proteins. Lico B also facilitated the diffusion of phospholipid phosphatidylserine (PS) from inner to outer leaflets of the plasma membrane with chromatin condensation, DNA fragmentation, accumulated sub-G1 population in a concentration-dependent manner. Moreover, Lico B promoted the generation of reactive oxygen species (ROS), which, in turn, can induce CHOP, death receptor (DR) 4 and DR5. Lico B treatment induced downregulation of anti-apoptotic proteins (Bid and Bcl-xl and Mcl-1), and up-regulation of pro-apoptotic protein (Bax). Lico B also led to the loss of mitochondrial membrane potential (MMP), resulting in cytochrome c release. As can be expected from the above results, the apoptotic protease activating factor-1 (Apaf-1) and survivin were oppositely expressed in favor of apoptotic cell death. This notion was supported by the fact that Lico B activated multi-caspases with cleavage of poly (ADP-ribose) polymerase (PARP) protein. Therefore, it is suggested that Lico B is a promising drug for the treatment of human oral cancer via the induction of apoptotic cell death. PMID:26847145

  18. Paeoniflorin attenuates ultraviolet B-induced apoptosis in human keratinocytes by inhibiting the ROS-p38-p53 pathway.

    PubMed

    Kong, Lingwen; Wang, Shangshang; Wu, Xiao; Zuo, Fuguo; Qin, Haihong; Wu, Jinfeng

    2016-04-01

    Ultraviolet (UV) light is one of the most harmful environmental factors that contribute to skin damage. Exposure to UV induces extensive generation of reactive oxygen species (ROS), and results in photoaging and skin cancer development. One approach to protecting human skin against UV radiation is the use of antioxidants. In recent years, naturally occurring herbal compounds have gained considerable attention as protective agents for UV exposure. Paeoniflorin (PF) is a novel natural antioxidant, which is isolated from peony root (Radix Paeoniae Alba). The present study evaluated the protective effects of PF on UV‑induced skin damage in vitro, and demonstrated that the effects were mediated via the ROS‑p38‑p53 pathway. The results of the present study demonstrated that treatment with PF (25, 50, and 100 µM) significantly increased the percentage of viable keratinocytes after UV‑B exposure. In addition, cell death analysis indicated that PF treatment markedly reduced UV‑B‑radiation‑induced apoptosis in keratinocytes, which was accompanied by increased procaspase 3 expression and decreased cleaved caspase 3 expression. Treatment with PF markedly reduced the production of ROS, and inhibited the activation of p38 and p53 in human keratinocytes, thus suggesting that the ROS‑p38‑p53 pathway has a role in UV‑B‑induced skin damage. In conclusion, the present study reported that PF was able to attenuate UV‑B‑induced cell damage in human keratinocytes. Notably, these effects were shown to be mediated, at least in part, via inhibition of the ROS-p38-p53 pathway. PMID:26936104

  19. Negative feedback regulation of NF-κB-inducing kinase is proteasome-dependent but does not require cellular inhibitors of apoptosis.

    PubMed

    Gray, Carolyn M; McCorkell, Kelly A; Chunduru, Srinivas K; McKinlay, Mark A; May, Michael J

    2014-07-18

    Non-canonical NF-κB signaling is controlled by the precise regulation of NF-κB inducing kinase (NIK) stability. NIK is constitutively ubiquitylated by cellular inhibitor of apoptosis (cIAP) proteins 1 and 2, leading to its complete proteasomal degradation in resting cells. Following stimulation, cIAP-mediated ubiquitylation of NIK ceases and NIK is stabilized, allowing for inhibitor of κB kinase (IKK)α activation and non-canonical NF-κB signaling. Non-canonical NF-κB signaling is terminated by feedback phosphorylation of NIK by IKKα that promotes NIK degradation; however, the mechanism of active NIK protein turnover remains unknown. To address this question, we established a strategy to precisely distinguish between basal degradation of newly synthesized endogenous NIK and induced active NIK in stimulated cells. Using this approach, we found that IKKα-mediated degradation of signal-induced activated NIK occurs through the proteasome. To determine whether cIAP1 or cIAP2 play a role in active NIK turnover, we utilized a Smac mimetic (GT13072), which promotes degradation of these E3 ubiquitin ligases. As expected, GT13072 stabilized NIK in resting cells. However, loss of the cIAPs did not inhibit proteasome-dependent turnover of signal-induced NIK showing that unlike the basal regulatory mechanism, active NIK turnover is independent of cIAP1 and cIAP2. Our results therefore establish that the negative feedback control of IKKα-mediated NIK turnover occurs via a novel proteasome-dependent and cIAP-independent mechanism. PMID:24942881

  20. Aspafilioside B induces G2/M cell cycle arrest and apoptosis by up-regulating H-Ras and N-Ras via ERK and p38 MAPK signaling pathways in human hepatoma HepG2 cells.

    PubMed

    Liu, Wei; Ning, Rui; Chen, Rui-Ni; Huang, Xue-Feng; Dai, Qin-Sheng; Hu, Jin-Hua; Wang, Yu-Wen; Wu, Li-Li; Xiong, Jing; Hu, Gang; Guo, Qing-Long; Yang, Jian; Wang, Hao

    2016-05-01

    We recently establish that aspafilioside B, a steroidal saponin extracted from Asparagus filicinus, is an active cytotoxic component. However, its antitumor activity is till unknown. In this study, the anticancer effect of aspafilioside B against HCC cells and the underlying mechanisms were investigated. Our results showed that aspafilioside B inhibited the growth and proliferation of HCC cell lines. Further study revealed that aspafilioside B could significantly induce G2 phase cell cycle arrest and apoptosis, accompanying the accumulation of reactive oxygen species (ROS), but blocking ROS generation with N-acetyl-l-cysteine (NAC) could not prevent G2/M arrest and apoptosis. Additionally, treatment with aspafilioside B induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase. Moreover, both ERK inhibitor PD98059 and p38 inhibitor SB203580 almost abolished the G2/M phase arrest and apoptosis induced by aspafilioside B, and reversed the expression of cell cycle- and apoptosis-related proteins. We also found that aspafilioside B treatment increased both Ras and Raf activation, and transfection of cells with H-Ras and N-Ras shRNA almost attenuated aspafilioside B-induced G2 phase arrest and apoptosis as well as the ERK and p38 activation. Finally, in vivo, aspafilioside B suppressed tumor growth in mouse xenograft models, and the mechanism was the same as in vitro study. Collectively, these findings indicated that aspafilioside B may up-regulate H-Ras and N-Ras, causing c-Raf phosphorylation, and lead to ERK and p38 activation, which consequently induced the G2 phase arrest and apoptosis. This study provides the evidence that aspafilioside B is a promising therapeutic agent against HCC. PMID:25683703

  1. Stellettin B Induces G1 Arrest, Apoptosis and Autophagy in Human Non-small Cell Lung Cancer A549 Cells via Blocking PI3K/Akt/mTOR Pathway

    PubMed Central

    Wang, Ran; Zhang, Qian; Peng, Xin; Zhou, Chang; Zhong, Yuxu; Chen, Xi; Qiu, Yuling; Jin, Meihua; Gong, Min; Kong, Dexin

    2016-01-01

    Until now, there is not yet antitumor drug with dramatically improved efficacy on non-small cell lung cancer (NSCLC). Marine organisms are rich source of novel compounds with various activities. We isolated stellettin B (Stel B) from marine sponge Jaspis stellifera, and demonstrated that it induced G1 arrest, apoptosis and autophagy at low concentrations in human NSCLC A549 cells. G1 arrest by Stel B might be attributed to the reduction of cyclin D1 and enhancement of p27 expression. The apoptosis induction might be related to the cleavage of PARP and increase of ROS generation. Moreover, we demonstrated that Stel B induced autophagy in A549 cells by use of various assays including monodansylcadaverine (MDC) staining, transmission electron microscopy (TEM), tandem mRFP-GFP-LC3 fluorescence microscopy, and western blot detection of the autophagy markers of LC3B, p62 and Atg5. Meanwhile, Stel B inhibited the expression of PI3K-p110, and the phosphorylation of PDK1, Akt, mTOR, p70S6K as well as GSK-3β, suggesting the correlation of blocking PI3K/Akt/mTOR pathway with the above antitumor activities. Together, our findings indicate the antitumor potential of Stel B for NSCLC by targeting PI3K/Akt/mTOR pathway. PMID:27243769

  2. Xerophilusin B induces cell cycle arrest and apoptosis in esophageal squamous cell carcinoma cells and does not cause toxicity in nude mice.

    PubMed

    Yao, Ran; Chen, Zhaoli; Zhou, Chengcheng; Luo, Mei; Shi, Xuejiao; Li, Jiagen; Gao, Yibo; Zhou, Fang; Pu, Jianxin; Sun, Handong; He, Jie

    2015-01-23

    Esophageal cancer is the eighth most common cancer in the world and ranks as the sixth leading cause of cancer-related mortality. Esophageal cancer has a poor prognosis partially due to its low sensitivity to chemotherapy agents, and the development of new therapeutic agents is urgently needed. Here, the antitumor activity of a natural ent-kaurane diterpenoid, xerophilusin B (1), which was isolated from Isodon xerophilus, a perennial herb frequently used in Chinese folk medicine for tumor treatment, was investigated. Compound 1 exhibited antiproliferative effects against esophageal squamous cell carcinoma (ESCC) cell lines in a time- and dose-dependent manner with lower toxicity against normal human and murine cell lines. In vivo studies demonstrated that 1 inhibited tumor growth of a human esophageal tumor xenograft in BALB/c nude mice without significant secondary adverse effects, indicating its safety in treating ESCC. Furthermore, 1 induced G2/M cell cycle arrest and promoted apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway in ESCC cell lines. In conclusion, the observations herein reported showed that 1 is a potential chemotherapeutic agent for ESCC and merits further preclinical and clinical investigation for cancer drug development. PMID:25555195

  3. SPE (tm) electrolyzers for space propulsion

    NASA Technical Reports Server (NTRS)

    Shane, E. M.

    1990-01-01

    Viewgraphs on SPE electrolyzers for space propulsion are presented. Topics covered include: SPE electrochemical cell reactions; SPE fuel cell/electrolyzer features; SPE cell life capability; SPE cell voltage stability; state-of-the-art SPE cell structure; electrolysis cell stack; electrolysis system; integrated propulsion test article-electrolyzer module components; and oxygen generator module.

  4. MicroRNA let-7b induces lens epithelial cell apoptosis by targeting leucine-rich repeat containing G protein-coupled receptor 4 (Lgr4) in age-related cataract.

    PubMed

    Dong, Yuchen; Zheng, Yajuan; Xiao, Jun; Zhu, Chao; Zhao, Meisheng

    2016-06-01

    Owing to a rapidly aging population, vision impairment caused by age-related cataract has become very common. Age-related cataract has also become one of the principal causes of blindness, and apoptosis of lens epithelial cells contributes to non-congenital cataract development. Previous studies have reported that microRNA let-7b (let-7b) is upregulated in cataractous lens epithelial cells, and the expression level of let-7b is positively associated with N, C and P cataract scores. However, the role of let-7b in the development of age-related cataract remains unclear. Here, we observed that the expression level of let-7b in the anterior lens capsules of age-related cataract was significantly higher than that in the normal anterior lens capsules. We performed ultraviolet (UV) irradiation to induce lens epithelial cell apoptosis. The results showed that the expression level of let-7b in lens epithelial cells which were treated by UV irradiation was significantly higher than that in the control, and let-7b promoted UV irradiation-induced apoptosis. Furthermore, we showed that leucine-rich repeat containing G protein-coupled receptor 4 (Lgr4) was a direct target of let-7b, and let-7b modulated lens epithelial cell apoptosis by directly targeting Lgr4. These findings will offer new insights into our understanding of the molecular mechanisms underlying the pathogenesis of cataract. PMID:27179410

  5. Human papillomavirus type 16 E6 activates NF-kappaB, induces cIAP-2 expression, and protects against apoptosis in a PDZ binding motif-dependent manner.

    PubMed

    James, Michael A; Lee, John H; Klingelhutz, Aloysius J

    2006-06-01

    Infection with human papillomavirus (HPV) is a critical factor in the pathogenesis of most cervical cancers and some aerodigestive cancers. The HPV E6 oncoprotein from high-risk HPV types contributes to the immortalization and transformation of cells by multiple mechanisms, including degradation of p53, transcriptional activation of human telomerase reverse transcriptase (hTERT), and degradation of several proteins containing PDZ domains. The ability of E6 to bind PDZ domain-containing proteins is independent of p53 degradation or hTERT activation but does correlate with oncogenic potential (R. A. Watson, M. Thomas, L. Banks, and S. Roberts, J. Cell Sci. 116:4925-4934, 2003) and is essential for induction of epithelial hyperplasia in vivo (M. L. Nguyen, M. M. Nguyen, D. Lee, A. E. Griep, and P. F. Lambert, J. Virol. 77:6957-6964, 2003). In this study, we found that HPV type 16 E6 was able to activate NF-kappaB in airway epithelial cells through the induction of nuclear binding activity of p52-containing NF-kappaB complexes in a PDZ binding motif-dependent manner. Transcript accumulation for the NF-kappaB-responsive antiapoptotic gene encoding cIAP-2 and binding of nuclear factors to the proximal NF-kappaB binding site of the cIAP-2 gene promoter are induced by E6 expression. Furthermore, E6 is able to protect cells from TNF-induced apoptosis. All of these E6-dependent phenotypes are dependent on the presence of the PDZ binding motif of E6. Our results imply a role for targeting of PDZ proteins by E6 in NF-kappaB activation and protection from apoptosis in airway epithelial cells. PMID:16699010

  6. rs78378222 polymorphism in the 3'-untranslated region of TP53 contributes to development of age-associated cataracts by modifying microRNA-125b-induced apoptosis of lens epithelial cells.

    PubMed

    Zhao, Yang; Li, Xiao; Zhu, Siquan

    2016-09-01

    MicroRNAs (miRNAs) negatively regulate the expression of the target genes by binding to 'seed sequences' in the 3'‑untranslated region (3'‑UTR) mRNA transcripts, and the variants within or nearby 'seed sequences' may compromise or enhance miRNA/mRNA interaction leading to either 'loss‑of‑function' or 'gain‑of‑function' effects. Cataracts are the leading cause of blindness worldwide and are characterized by progressive aggregation and precipitation of lens proteins, and the development of age‑related cataracts is associated with dysregulated cellular activities of lens epithelial cells. Luciferase assays and online miRNA databases were used to validate that tumor protein p53 (TP53) is the target gene of miR‑125b. Furthermore, reverse transcription‑quantitative polymerase chain reaction and western blotting were conducted to detect expression levels of miR‑125b and TP53 in different groups of cells transfected with miR‑125b mimics or inhibitors. In addition, flow cytometry analysis and the MTT assay were conducted to detect the effects of miR‑125b on apoptosis and cell viability. The current study demonstrated that the rs78378222 polymorphism minor allele introduces a novel potential miR‑125b binding site in the TP53 3'‑UTR with a consecutive 8‑bp perfect match, creating a 'gain‑of‑function' variant and affecting the regulation of TP53 expression. A luciferase assay demonstrated that transfection of lens epithelial cells with wild type TP53 3'‑UTR significantly reduced the luciferase activity of the miR‑125b overexpressing cells compared with scramble controls. In addition, the luciferase activity of miR‑125b overexpressing cells transfected with the construct containing the rs78378222 polymorphism minor allele was also reduced compared with cells transfected with the wild type 3'‑UTR. Furthermore, it was demonstrated that the expression level of miR‑125 was comparable in epithelial cells from patients with age

  7. Biological Response to SPE Exposures

    NASA Technical Reports Server (NTRS)

    Wilson, J. W.; Cucinotta, F. A.; Kim, M.; Shinn, J. L.; Jones, T. D.; Chang, C. K.

    2004-01-01

    It has long been recognized that a single solar particle event (SPE) can produce, over a short period of time, exposures on the order of LD50 for humans under normal physiological conditions. It is further recognized that recovery from injury over the period of exposure would greatly increase the chances of survival (dose rate effects) although such effects were left unquantified. In the present report we use the bioresponse model derived from a broad range of animal and human exposure data for evaluation of troop readiness in tactical nuclear warfare to evaluate the biological risk posed by the solar event of 4 August 1972. The astronaut blood forming organ (BFO) exposure in deep space would have been 2.2 Sv (1.6 Gy) in a space suit, 1.8 Sv (1.3 Gy) in an aluminum pressure vessel, and 0.7 Sv (0.5 Gy) in an equipment room compared to an X-ray mortality threshold of 1.5 Gy (assuming high dose rate). We find BFO dose rate effectiveness factors for this SPE on the order of 3 to 4, greatly reducing the mortality risks for this event. There is an approximate 3 percent chance that an even larger event may occur for which exposures could be 2-4 times higher. Assured survival of the astronaut requires added shelter shielding and a warning system for this event. The required mass of the shelter shield can be greatly reduced by using hydrogenous materials such as polymers, water, food, and other biological materials in its construction. Limitations of the current bioresponse model arise from the exposures taking place in the microgravity environment wherein the immune system is already challenged and the effective mortality threshold may be reduced by a factor of two. Such microgravity effects could greatly affect astronaut risks.

  8. Current SPE Hydrodynamic Modeling and Path Forward

    SciTech Connect

    Knight, Earl E.; Rougier, Esteban

    2012-08-14

    Extensive work has been conducted on SPE analysis efforts: Fault effects Non-uniform weathered layer analysis MUNROU: material library incorporation, parallelization, and development of non-locking tets Development of a unique continuum-based-visco-plastic strain-rate-dependent material model With corrected SPE data path is now set for a multipronged approach to fully understand experimental series shot effects.

  9. Recent advances in SPE (tm) water electrolyzer

    NASA Technical Reports Server (NTRS)

    Mcelroy, James F.

    1993-01-01

    A new cell structure has been introduced into the SPE Water Electrolyzer which has improved overall characteristics significantly. Weight, reliability, and efficiency are the characteristics that are improved the most, with volume having a second order improvement. This paper discusses the capabilities of the new cell structure and the impact it would have in various space applications.

  10. SPE water electrolyzers in support of the lunar outpost

    NASA Technical Reports Server (NTRS)

    Mcelroy, J. F.

    1992-01-01

    During the 1970s, the SPE water electrolyzer, which uses ion exchange membranes as its sole electrolyte, was developed for nuclear submarine metabolic oxygen production. These developments included SPE water electrolyzer operation at up to 3,000 psia and at current densities in excess of 1,000 amps per square foot. The SPE water electrolyzer system is now fully qualified for both the U.S. and U.K. Navies with tens of thousands of system hours accumulated at sea. During the 1980s, the basic SPE water electrolyzer cell structure developed for the Navies was incorporated into several demonstrations for NASA's Space Station Program. Among these were: the SPE regenerative fuel cell for electrical energy storage; the SPE water electrolyzer for metabolic oxygen production; and the high pressure SPE water electrolyzer for reboost propulsion reactant production. In the 1990s, one emphasis will be the development of SPE water electrolyzers for the Lunar Outposts Currently defined potential Lunar Outpost applications for the SPE water electrolyzer include: SPE water electrolyzers for metabolic oxygen and potable water production from reclaimed water; and SPE water electrolyzers operating at high pressure as part of stationary and mobile surface energy storage systems.

  11. SPE propulsion electrolyzer for NASA's integrated propulsion test article

    NASA Astrophysics Data System (ADS)

    1991-08-01

    Hamilton Standard has delivered a 3000 PSI SPE Propulsion Electrolyzer Stack and Special Test Fixture to the NASA Lyndon B. Johnson Space Center (JSC) Integrated Propulsion Test Article (IPTA) program in June 1990, per contract NAS9-18030. This prototype unit demonstrates the feasibility of SPE-high pressure water electrolysis for future space applications such as Space Station propulsion and Lunar/Mars energy storage. The SPE-Propulsion Electrolyzer has met or exceeded all IPTA program goals. It continues to function as the primary hydrogen and oxygen source for the IPTA test bed at the NASA/JSC Propulsion and Power Division Thermochemical Test Branch.

  12. SPE propulsion electrolyzer for NASA's integrated propulsion test article

    NASA Technical Reports Server (NTRS)

    1991-01-01

    Hamilton Standard has delivered a 3000 PSI SPE Propulsion Electrolyzer Stack and Special Test Fixture to the NASA Lyndon B. Johnson Space Center (JSC) Integrated Propulsion Test Article (IPTA) program in June 1990, per contract NAS9-18030. This prototype unit demonstrates the feasibility of SPE-high pressure water electrolysis for future space applications such as Space Station propulsion and Lunar/Mars energy storage. The SPE-Propulsion Electrolyzer has met or exceeded all IPTA program goals. It continues to function as the primary hydrogen and oxygen source for the IPTA test bed at the NASA/JSC Propulsion and Power Division Thermochemical Test Branch.

  13. Comparison of Reduced Displacement Potentials from Spe Free Field Measurements: SPE-4PRIME Versus Previous Events

    NASA Astrophysics Data System (ADS)

    Patton, H. J.; Rougier, E.

    2015-12-01

    Since 2010, the U. S. Department of Energy has funded a series of chemical tests at the National Nuclear Security Site (NNSS) in Climax Stock granite as part of the Source Physics Experiment (SPE) with the aim of gaining a better understanding of the generation and propagation of seismic energy from underground explosions in hard rock media. To date, four tests have been conducted in the same borehole with yields of 100, 1000, 900 and 100 kg at different depths of burials. The nominal scaled depths of burial are 938, 363, 376 and 1556 m/kt1/3 compared to standard containment practices of ~120 m/kt1/3. A quite dense array of free field accelerometers were installed around the borehole, both on and off shot depth. Acceleration data were corrected for shock-generated baseline-shifts, and free field ground velocity waveforms were obtained. This work concentrates on the qualitative analysis of the reduced displacement potentials and the explosion source spectra for the last shot of the series (SPE-4Prime) and the comparison of the obtained results against the previous events. Finally, the results obtained from the experimental data are compared to the Mueller-Murphy empirical explosion model both using the Heard and Ackerman and Denny and Johnson cavity radius scaling laws.

  14. New Insights into the Explosion Source from SPE

    NASA Astrophysics Data System (ADS)

    Patton, H. J.

    2015-12-01

    Phase I of the Source Physics Experiments (SPE) is a series of chemical explosions at varying depths and yields detonated in the same emplacement hole on Climax stock, a granitic pluton located on the Nevada National Security Site. To date, four of the seven planned tests have been conducted, the last in May 2015, called SPE-4P, with a scaled depth of burial of 1549 m/kt1/3 in order to localize the source in time and space. Surface ground motions validated that the source medium did not undergo spallation, and a key experimental objective was achieved where SPE-4P is the closest of all tests in the series to a pure monopole source and will serve as an empirical Green's function for analysis against other SPE tests. A scientific objective of SPE is to understand mechanisms of rock damage for generating seismic waves, particularly surface and S waves, including prompt damage under compressive stresses and "late-time" damage under tensile stresses. Studies have shown that prompt damage can explain ~75% of the seismic moment for some SPE tests. Spallation is a form of late-time damage and a facilitator of damage mechanisms under tensile stresses including inelastic brittle deformation and shear dilatancy on pre-existing faults or joints. As an empirical Green's function, SPE-4P allows the study of late-time damage mechanisms on other SPE tests that induce spallation and late-time damage, and I'll discuss these studies. The importance for nuclear monitoring cannot be overstated because new research shows that damage mechanisms can affect surface wave magnitude Ms more than tectonic release, and are a likely factor related to anomalous mb-Ms behavior for North Korean tests.

  15. SPE (tm) regenerative hydrogen/oxygen fuel cells for extraterrestrial surface and microgravity applications

    NASA Technical Reports Server (NTRS)

    Mcelroy, J. F.

    1990-01-01

    Viewgraphs on SPE regenerative hydrogen/oxygen fuel cells for extraterrestrial surface and microgravity applications are presented. Topics covered include: hydrogen-oxygen regenerative fuel cell energy storage system; electrochemical cell reactions; SPE cell voltage stability; passive water removal SPE fuel cell; fuel cell performance; SPE water electrolyzers; hydrophobic oxygen phase separator; hydrophilic/electrochemical hydrogen phase separator; and unitized regenerative fuel cell.

  16. Momilactone B induces apoptosis and G1 arrest of the cell cycle in human monocytic leukemia U937 cells through downregulation of pRB phosphorylation and induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1.

    PubMed

    Park, Cheol; Jeong, Na Young; Kim, Gi-Young; Han, Min Ho; Chung, Ill-Min; Kim, Wun-Jae; Yoo, Young Hyun; Choi, Yung Hyun

    2014-04-01

    Momilactone B, a terpenoid phytoalexin present in rice bran, has been shown to exhibit several biological activities. The present study was conducted using cultured human leukemia U937 cells to elucidate the possible mechanisms by which momilactone B exerts its anticancer activity, which to date has remained poorly understood. Momilactone B treatment of U937 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by chromatin condensation, DNA fragmentation, the cleavage of poly(ADP-ribose) polymerase and Annexin V-FITC staining. Flow cytometric analysis revealed that momilactone B resulted in G1 arrest in cell cycle progression, which was associated with the dephosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB with the E2F transcription factor family proteins. Treatment with momilactone B also increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1 in a p53-independent manner, without any noticeable changes in G1 cyclins and cyclin-dependent kinases (Cdks), except a slight decrease in cyclin E. Moreover, in vitro kinase assay indicated that momilactone B significantly decreased Cdk4- and Cdk6-associated kinase activities through a notably increased binding of p21 to Cdk4 and Cdk6. Our results demonstrated that momilactone B caused G1 cell cycle arrest and apoptosis in U937 cells through the induction of p21 expression, inhibition of Cdk/cyclin-associated kinase activities, and reduced phosphorylation of pRB, which may be related to anticancer activity. PMID:24503697

  17. SPE(R)-OBOGS: On-board oxygen generating sustem

    NASA Technical Reports Server (NTRS)

    Mcelroy, J.; Smith, W.

    1995-01-01

    Regulations require oxygen usage by commercial airline flight crews during check out and during certain aircraft configurations. This oxygen is drawn from a high pressure onboard pressure cylinder storage system. In a typical aircraft oxygen cylinder removal for oxygen ground servicing is conducted every 4 to 6 weeks. An on board oxygen generating system has been developed to eliminate the need for oxygen ground servicing. The SPE-OBOGS supplies oxygen during flight in a 'trickle charge' mode to replenish the consumed oxygen at pressures up to 1850 psi. The Electrochemical cell stack is the fundamental SPE-OBOGS system component. The same basic proton exchange membrane technology, previously used for the Gemini program fuel cells and currently used in nuclear submarines as oxygen generators, is used in the SPE-OBOGS. An in-serivce evaluation of the SPE-OBOGS is in the planning stage and a zero gravity version is being promoted for on orbit space suit oxygen system recharge. Summary results of the SPE-OBOGS development will be addressed.

  18. Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages

    SciTech Connect

    Gammelsrud, A.; Solhaug, A.; Dendelé, B.; Sandberg, W.J.; Ivanova, L.; Kocbach Bølling, A.; Lagadic-Gossmann, D.; Refsnes, M.; Becher, R.; Eriksen, G.; Holme, J.A.

    2012-05-15

    The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte–macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1beta (IL-1β) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1β and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B. -- Highlights: ► The mycotoxin EnnB induced cell cycle arrest, cell death and inflammation. ► The G0/G1-arrest was linked to a reduced ability to internalize receptors. ► EnnB caused lysosomal damage, leakage of cathepsin B and caspase-1 cleavage. ► Caspase-1 was partly involved in both apoptosis and release of IL-1

  19. Theoretical performance of hydrogen-bromine rechargeable SPE fuel cell

    NASA Technical Reports Server (NTRS)

    Savinell, Robert F.; Fritts, S. D.

    1987-01-01

    A mathematical model was formulated to describe the performance of a hydrogen-bromine fuel cell. Porous electrode theory was applied to the carbon felt flow-by electrode and was coupled to theory describing the solid polymer electrolyte (SPE) system. Parametric studies using the numerical solution to this model were performed to determine the effect of kinetic, mass transfer, and design parameters on the performance of the fuel cell. The results indicate that the cell performance is most sensitive to the transport properties of the SPE membrane. The model was also shown to be a useful tool for scale-up studies.

  20. Spermiogenesis initiation in Caenorhabditis elegans involves a casein kinase 1 encoded by the spe-6 gene.

    PubMed Central

    Muhlrad, Paul J; Ward, Samuel

    2002-01-01

    Immature spermatids from Caenorhabditis elegans are stimulated by an external activation signal to reorganize their membranes and cytoskeleton to form crawling spermatozoa. This rapid maturation, termed spermiogenesis, occurs without any new gene expression. To better understand this signal transduction pathway, we isolated suppressors of a mutation in the spe-27 gene, which is part of the pathway. The suppressors bypass the requirement for spe-27, as well as three other genes that act in this pathway, spe-8, spe-12, and spe-29. Eighteen of the suppressor mutations are new alleles of spe-6, a previously identified gene required for an early stage of spermatogenesis. The original spe-6 mutations are loss-of-function alleles that prevent major sperm protein (MSP) assembly in the fibrous bodies of spermatocytes and arrest development in meiosis. We have isolated the spe-6 gene and find that it encodes a predicted protein-serine/threonine kinase in the casein kinase 1 family. The suppressor mutations appear to be reduction-of-function alleles. We propose a model whereby SPE-6, in addition to its early role in spermatocyte development, inhibits spermiogenesis until the activation signal is received. The activation signal is transduced through SPE-8, SPE-12, SPE-27, and SPE-29 to relieve SPE-6 repression, thus triggering the formation of crawling spermatozoa. PMID:12019230

  1. An improved SPE method for fractionation and identification of phospholipids.

    PubMed

    Fauland, Alexander; Trötzmüller, Martin; Eberl, Anita; Afiuni-Zadeh, Somaieh; Köfeler, Harald; Guo, Xinghua; Lankmayr, Ernst

    2013-02-01

    This work reports an efficient and universal SPE method developed for separation and identification of phospholipids derived from complex biological samples. For the separation step, sequential combination of silica gel-aminopropyl-silica gel SPE cartridges is applied. This setup enables separation of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, and sphingomyelin into four fractions according to the polarity of their headgroups. Sample acquisition of the SPE fractions is performed by a high-resolution LC-MS system consisting of a hybrid linear IT Fourier transform ion cyclotron resonance mass spectrometer coupled to RP-HPLC. The unequivocal advantage of our SPE sample preparation setup is avoidance of analyte peak overlapping in the determination step done by RP-HPLC. Overlapping phospholipid signals would otherwise exert adverse ion suppression effects. An additional benefit of this method is the elimination of polar and nonpolar (e.g. neutral lipids) contaminants from the phospholipid fractions, which highly reduces contamination of the LC-MS system. The method was validated with fermentation samples of organic waste, where 78 distinct phospholipid and sphingomyelin species belonging to six lipid classes were successfully identified. PMID:23349108

  2. Assay Development for the Discovery of Semaphorin 3B Inducing Agents from Natural Product Sources

    PubMed Central

    Yong, Yeonjoong; Pan, Li; Ren, Yulin; Fatima, Nighat; Ahmed, Safia; Chang, Leng Chee; Zhang, Xiaoli; Kinghorn, A. Douglas; Swanson, Steven M.; Carcache de Blanco, Esperanza J.

    2014-01-01

    Semaphorins are a class of membrane-bound and secreted proteins. They have been found to regulate basic cell functions such as axonal growth cone guidance and recent studies have focused on their effect on tumor progression. Semaphorin 3B (Sema 3B) particularly is a secreted protein that has been known to modulate proliferation and apoptosis, processes that are critical for tumor progression and development. In spite of its importance, there is yet no high-throughput screening assay available to detect or quantify the expression of Sema 3B for natural product anticancer drug discovery purposes. Therefore, the development of a new high-throughput bioassay for the discovery of Sema 3B inducing agents from natural product sources is described herein. A wide variety of pure compounds and extracts from plants and microorganisms has been found suitable for screening using this Sema 3B assay to detect and quantify the effect of Sema 3B inducing agents and thereby identify new selective bioactive Sema 3B lead compounds for anticancer drug discovery and development. Also, this new bioassay procedure is based on a high-throughput platform using an enzyme-linked immunosorbent assay that involves the optimization of sensitivity and selectivity levels as well as accuracy, reproducibility, robustness, and cost effectiveness. PMID:25016954

  3. Reciprocal, temporal expression of SpeA and SpeB by invasive M1T1 group a streptococcal isolates in vivo.

    PubMed

    Kazmi, S U; Kansal, R; Aziz, R K; Hooshdaran, M; Norrby-Teglund, A; Low, D E; Halim, A B; Kotb, M

    2001-08-01

    The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including speA, speB, speF, speG, and smeZ. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of the speA-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that speA gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of the speA gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced speA gene expression and suppressed speB gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of

  4. Reciprocal, Temporal Expression of SpeA and SpeB by Invasive M1T1 Group A Streptococcal Isolates In Vivo

    PubMed Central

    Kazmi, Shahana U.; Kansal, Rita; Aziz, Ramy K.; Hooshdaran, Massoumeh; Norrby-Teglund, Anna; Low, D. E.; Halim, Abdel-Baset; Kotb, Malak

    2001-01-01

    The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including speA, speB, speF, speG, and smeZ. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of the speA-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that speA gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of the speA gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced speA gene expression and suppressed speB gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of

  5. SPE (tm) water electrolyzers in support of mission from planet Earth

    NASA Astrophysics Data System (ADS)

    McElroy, J. F.

    1991-09-01

    During the 1970's, the Solid Polymer Electrolyte (SPE) water electrolyzer, which uses ion exchange membranes as its sole electrolyte, was developed for nuclear submarine metabolic oxygen production. SPE water electrolyzer developments included operation at up to 3,000 psia and at current densities in excess of 1,000 amps per square foot. The SPE water electrolyzer system has accumulated tens of thousands of system hours with the Navies of both the United States and the United Kingdom. During the 1980's, the basic SPE water electrolyzer cell structure developed for the Navies was incorporated into several demonstrators for NASA's Space Station Program. Among these were: (1) the SPE regenerative fuel cell for electrical energy storage; (2) the SPE water electrolyzer for metabolic oxygen production; and (3) the high pressure SPE water electrolyzer for reboost propellant production. In the 1990's, emphasis will be the development of SPE water electrolyzers for Mission from Planet Earth. Currently defined potential applications for the SPE water electrolyzer include: (1) SPE water electrolyzers operating at high pressure as part of a regenerative fuel cell extraterrestrial surface energy storage system; (2) SPE water electrolyzers for propellant production from extraterrestrial indigenous materials; and (3) SPE water electrolyzers for metabolic oxygen and potable water production from reclaimed water.

  6. SPE (tm) water electrolyzers in support of mission from planet Earth

    NASA Technical Reports Server (NTRS)

    Mcelroy, J. F.

    1991-01-01

    During the 1970's, the Solid Polymer Electrolyte (SPE) water electrolyzer, which uses ion exchange membranes as its sole electrolyte, was developed for nuclear submarine metabolic oxygen production. SPE water electrolyzer developments included operation at up to 3,000 psia and at current densities in excess of 1,000 amps per square foot. The SPE water electrolyzer system has accumulated tens of thousands of system hours with the Navies of both the United States and the United Kingdom. During the 1980's, the basic SPE water electrolyzer cell structure developed for the Navies was incorporated into several demonstrators for NASA's Space Station Program. Among these were: (1) the SPE regenerative fuel cell for electrical energy storage; (2) the SPE water electrolyzer for metabolic oxygen production; and (3) the high pressure SPE water electrolyzer for reboost propellant production. In the 1990's, emphasis will be the development of SPE water electrolyzers for Mission from Planet Earth. Currently defined potential applications for the SPE water electrolyzer include: (1) SPE water electrolyzers operating at high pressure as part of a regenerative fuel cell extraterrestrial surface energy storage system; (2) SPE water electrolyzers for propellant production from extraterrestrial indigenous materials; and (3) SPE water electrolyzers for metabolic oxygen and potable water production from reclaimed water.

  7. Cysteine protease SpeB expression in group A streptococci is influenced by the nutritional environment but SpeB does not contribute to obtaining essential nutrients.

    PubMed

    Podbielski, A; Woischnik, M; Kreikemeyer, B; Bettenbrock, K; Buttaro, B A

    1999-11-01

    Group A streptococcal (GAS) cysteine protease is a major virulence factor involved in the pathogenesis of purulent and invasive infections. The secreted enzyme cleaves a number of different bacterial and host proteins which could contribute to different stages of the infective processes. It has been proposed that, among these functions, SpeB plays a role in obtaining nutrients during late growth phases. In the present study, speB mutants of various GAS serotypes were found to exhibit unaltered growth characteristics in several complex and chemically defined media (CDM). When amino acid-depleted CDM was prepared, neither SpeB activity on whole proteins added to the medium during incubation nor the addition of SpeB-digested proteins was able to support bacterial growth. SpeB also was unable to liberate iron from iron-containing protein sources added to iron-deficient CDM. However, SpeB levels in culture supernatants changed in response to the protein and glucose content of the media. Using a speB promoter-luciferase reporter, speB expression levels were found to correspond to peptide concentrations in the culture media. The effect appeared to be specific for peptides since addition of peptides derived from various proteins had an affect on expression, while addition of the whole proteins had no effect. Addition of glucose to CDM had no effect on speB expression, while glucose addition to complex medium decreased speB expression. Overall, SpeB did not appear to be directly involved in providing the bacteria with nutritional factors but expression of the speB gene responded to ratios of peptides and carbohydrates in the culture medium. PMID:10753062

  8. Data Release Report for Source Physics Experiments 2 and 3 (SPE-2 and SPE-3) Nevada National Security Site

    SciTech Connect

    Townsend, Margaret; Obi, Curtis

    2015-04-30

    The second Source Physics Experiment shot (SPE-2) was conducted in Nevada on October 25, 2011, at 1900:00.011623 Greenwich Mean Time (GMT). The explosive source was 997 kilograms (kg) trinitrotoluene (TNT) equivalent of sensitized heavy ammonium fuel oil (SHANFO) detonated at a depth of 45.7 meters (m). The third Source Physics Experiment shot (SPE-3) was conducted in Nevada on July 24, 2012, at 1800:00.44835 GMT. The explosive source was 905 kg TNT equivalent of SHANFO detonated at a depth of 45.8 m. Both shots were recorded by an extensive set of instrumentation that includes sensors both at near-field (less than 100 m) and far-field (100 m or greater) distances. The near-field instruments consisted of three-component accelerometers deployed in boreholes at 15, 46, and 55 m depths around the shot and a set of single-component vertical accelerometers on the surface. The far-field network was composed of a variety of seismic and acoustic sensors, including short-period geophones, broadband seismometers, three-component accelerometers, and rotational seismometers at distances of 100 m to 25 kilometers. This report coincides with the release of these data for analysts and organizations that are not participants in this program. This report describes the second and third Source Physics Experiment shots and the various types of near-field and farfield data that are available.This revised document includes reports on baseline shift corrections for the SPE-2 and SPE-3 shots that were missing from the original January 2015 version.

  9. Apoptosis induced by weisiensin B isolated from Rabdosia weisiensis C.Y. Wu in K562.

    PubMed

    Liu, Guo-An; Chang, Jin-Chun; Feng, Xiao-Lu; Ding, Lan

    2015-04-01

    The ent-kaurane diterpenoid weisinensis B shows significant cytotoxicity to human chronic myeloid leukemia K562 cells. It inhibits cell growth at low concentration and kills cells at high concentration. The compound induced cell apoptosis and necrosis mainly associated with G2/M phase cell cycle arrest and the ROS generation is the early event in weisiensin B induced cell apoptosis. PMID:26012257

  10. NEAR FIELD MODELING OF SPE1 EXPERIMENT AND PREDICTION OF THE SECOND SOURCE PHYSICS EXPERIMENTS (SPE2)

    SciTech Connect

    Antoun, T; Xu, H; Vorobiev, O; Lomov, I

    2011-10-20

    Motion along joints and fractures in the rock has been proposed as one of the sources of near-source shear wave generation, and demonstrating the validity of this hypothesis is a focal scientific objective of the source physics experimental campaign in the Climax Stock granitic outcrop. A modeling effort has been undertaken by LLNL to complement the experimental campaign, and over the long term provide a validated computation capability for the nuclear explosion monitoring community. The approach involves performing the near-field nonlinear modeling with hydrodynamic codes (e.g., GEODYN, GEODYN-L), and the far-field seismic propagation with an elastic wave propagation code (e.g., WPP). the codes will be coupled together to provide a comprehensive source-to-sensor modeling capability. The technical approach involves pre-test predictions of each of the SPE experiments using their state of the art modeling capabilities, followed by code improvements to alleviate deficiencies identified in the pre-test predictions. This spiral development cycle wherein simulations are used to guide experimental design and the data from the experiment used to improve the models is the most effective approach to enable a transition from the descriptive phenomenological models in current use to the predictive, hybrid physics models needed for a science-based modeling capability for nuclear explosion monitoring. The objective of this report is to describe initial results of non-linear motion predictions of the first two SPE shots in the Climax Stock: a 220-lb shot at a depth of 180 ft (SPE No.1), and a 2570-lb shot at a depth of 150 ft (SPE No.2). The simulations were performed using the LLNL ensemble granite model, a model developed to match velocity and displacement attenuation from HARDHAT, PILE DRIVER, and SHOAL, as well as Russian and French nuclear test data in granitic rocks. This model represents the state of the art modeling capabilities as they existed when the SPE campaign was

  11. SPE ® water electrolyzers in support of Mission from Planet Earth

    NASA Astrophysics Data System (ADS)

    McElroy, J. F.

    Although the Mission from Planet Earth is still in the early planning stage, several unique and potentially enabling uses of the SPE water electrolyzer have been identified. The maturity of the SPE water electrolyzer cells gained from the Naval applications should give mission planners the confidence to take advantage of the leveraging effects of the SPE cell technology. Although the inherent capabilities of this technology have been proven, significant development effort remains to package these cells for the Mission from Planet Earth applications.

  12. SPE-8, a protein-tyrosine kinase, localizes to the spermatid cell membrane through interaction with other members of the SPE-8 group spermatid activation signaling pathway in C. elegans

    PubMed Central

    2014-01-01

    Background The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid. Results Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background. Conclusions These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid. PMID:25022984

  13. Data Release Report for Source Physics Experiments 2 and 3 (SPE-2 and SPE-3) Nevada National Security Site

    SciTech Connect

    Townsend, Margaret; Obi, Curtis

    2015-01-26

    The second Source Physics Experiment shot (SPE-2) was conducted in Nevada on October 25, 2011, at 1900:00.011623 Greenwich Mean Time (GMT). The explosive source was 997 kilograms (kg) trinitrotoluene (TNT) equivalent of sensitized heavy ammonium fuel oil (SHANFO) detonated at a depth of 45.7 meters (m). The third Source Physics Experiment shot (SPE-3) was conducted in Nevada on July 24, 2012, at 1800:00.44835 GMT. The explosive source was 905 kg TNT equivalent of SHANFO detonated at a depth of 45.8 m. Both shots were recorded by an extensive set of instrumentation that includes sensors both at near-field (less than 100 m) and far-field (100 m or greater) distances. The near-field instruments consisted of three-component accelerometers deployed in boreholes at 15, 46, and 55 m depths around the shot and a set of single-component vertical accelerometers on the surface. The far-field network was composed of a variety of seismic and acoustic sensors, including short-period geophones, broadband seismometers, three-component accelerometers, and rotational seismometers at distances of 100 m to 25 kilometers. This report coincides with the release of these data for analysts and organizations that are not participants in this program. This report describes the second and third Source Physics Experiment shots and the various types of near-field and far-field data that are available.

  14. Premature Sperm Activation and Defective Spermatogenesis Caused by Loss of spe-46 Function in Caenorhabditis elegans

    PubMed Central

    Liau, Wei-Siang; Nasri, Ubaydah; Elmatari, Daniel; Rothman, Jason; LaMunyon, Craig W.

    2013-01-01

    Given limited resources for motility, sperm cell activation must be precisely timed to ensure the greatest likelihood of fertilization. Like those of most species, the sperm of C. elegans become active only after encountering an external signaling molecule. Activation coincides with spermiogenesis, the final step in spermatogenesis, when the spherical spermatid undergoes wholesale reorganization to produce a pseudopod. Here, we describe a gene involved in sperm activation, spe-46. This gene was identified in a suppressor screen of spe-27(it132ts), a sperm-expressed gene whose product functions in the transduction of the spermatid activation signal. While spe-27(it132ts) worms are sterile at 25°C, the spe-46(hc197)I; spe-27(it132ts)IV double mutants regain partial fertility. Single nucleotide polymorphism mapping, whole genome sequencing, and transformation rescue were employed to identify the spe-46 coding sequence. It encodes a protein with seven predicted transmembrane domains but with no other predicted functional domains or homology outside of nematodes. Expression is limited to spermatogenic tissue, and a transcriptional GFP fusion shows expression corresponds with the onset of the pachytene stage of meiosis. The spe-46(hc197) mutation bypasses the need for the activation signal; mutant sperm activate prematurely without an activation signal in males, and mutant males are sterile. In an otherwise wild-type genome, the spe-46(hc197) mutation induces a sperm defective phenotype. In addition to premature activation, spe-46(hc197) sperm exhibit numerous defects including aneuploidy, vacuolization, protruding spikes, and precocious fusion of membranous organelles. Hemizygous worms [spe-46(hc197)/mnDf111] are effectively sterile. Thus, spe-46 appears to be involved in the regulation of spermatid activation during spermiogenesis, with the null phenotype being an absence of functional sperm and hypomorphic phenotypes being premature spermatid activation and numerous

  15. Method of making MEA for PEM/SPE fuel cell

    DOEpatents

    Hulett, Jay S.

    2000-01-01

    A method of making a membrane-electrode-assembly (MEA) for a PEM/SPE fuel cell comprising applying a slurry of electrode-forming material directly onto a membrane-electrolyte film. The slurry comprises a liquid vehicle carrying catalyst particles and a binder for the catalyst particles. The membrane-electrolyte is preswollen by contact with the vehicle before the electrode-forming slurry is applied to the membrane-electrolyte. The swollen membrane-electrolyte is constrained against shrinking in the "x" and "y" directions during drying. Following assembly of the fuel cell, the MEA is rehydrated inside the fuel cell such that it swells in the "z" direction for enhanced electrical contact with contiguous electrically conductive components of the fuel cell.

  16. The Streptococcal Cysteine Protease SpeB Is Not a Natural Immunoglobulin-Cleaving Enzyme

    PubMed Central

    Persson, Helena; Vindebro, Reine

    2013-01-01

    The human bacterial pathogen Streptococcus pyogenes has developed a broad variety of virulence mechanisms to evade the actions of the host immune defense. One of the best-characterized factors is the streptococcal cysteine protease SpeB, an important multifunctional protease that contributes to group A streptococcal pathogenesis in vivo. Among many suggested activities, SpeB has been described to degrade various human plasma proteins, including immunoglobulins (Igs). In this study, we show that SpeB has no Ig-cleaving activity under physiological conditions and that only Igs in a reduced state, i.e., semimonomeric molecules, are cleaved and degraded by SpeB. Since reducing conditions outside eukaryotic cells have to be considered nonphysiological and IgG in a reduced state lacks biological effector functions, we conclude that SpeB does not contribute to S. pyogenes virulence through the proteolytic degradation of Igs. PMID:23569114

  17. Osteopontin Facilitates Ultraviolet B-induced Squamous Cell Carcinoma Development

    PubMed Central

    Chang, Pi-Ling; Hsieh, Yu-Hua; Wang, Chao-Cheng; Juliana, M. Margaret; Tsuruta, Yuko; Timares, Laura; Elmets, Craig; Ho, Kang-Jey

    2014-01-01

    Background Osteopontin (OPN) is a matricellular glycoprotein that is markedly expressed in cutaneous squamous cell carcinomas (cSCCs) and in actinic keratoses implicating its role in photocarcinogenesis. Objective To determine whether OPN facilitates the development of cSCC and its function. Methods cSCCs development was compared between wild-type (WT) and OPN-null mice subjected to UVB irradiation for 43 weeks. UVB-induced OPN expression was determined by Western blot, immunoprecipitation, ELISA, and semi-quantitative RT-PCR. Epidermal layer and TUNEL analyses assessed if OPN mediates UVB-induced epidermal hyperplasia or suppresses UVB-induced apoptosis of basal keratinocytes, respectively. In vitro experiments determined whether OPN enhances cell survival of UVB-induced apoptosis and its potential mechanisms. Immunohistochemical analyses of epidermis assessed the expression of CD44 and focal adhesion kinase (FAK), molecules that mediate OPN survival function. Results Compared to female WT mice, OPN-null mice did not develop cSCCs. UVB irradiation stimulated OPN protein expression in the dorsal skin by 11 h and remains high at 24 to 48h.OPN did not mediate UVB-induced epidermal hyperplasia; instead, it protected basal keratinocytes from undergoing apoptosis upon UVB exposure. Likewise, the addition of OPN suppressed UVB-induced OPN-null cSCC cell apoptosis, the activation of caspase-9 activity, and increased phosphorylation of FAK at Y397. Furthermore, the expression of CD44 and FAK in WT mice epidermis was greater than that of OPN-null mice prior to and during early acute UVB exposure. Conclusion These data support the hypothesis that chronic UVB-induced OPN expression protects the survival of initiated basal keratinocytes and, consequently, facilitates cSCC develop. PMID:24888687

  18. OSTEOCYTE APOPTOSIS

    PubMed Central

    Jilka, Robert L.; Noble, Brendon; Weinstein, Robert S.

    2012-01-01

    Apoptotic death of osteocytes was recognized over 15 years ago, but its significance for bone homeostasis has remained elusive. A new paradigm has emerged that invokes osteocyte apoptosis as a critical event in the recruitment of osteoclasts to a specific site in response to skeletal unloading, fatigue damage, estrogen deficiency and perhaps in other states where bone must be removed. This is accomplished by yet to be defined signals emanating from dying osteocytes, which stimulate neighboring viable osteocytes to produce osteoclastogenic cytokines. The osteocyte apoptosis caused by chronic glucocorticoid administration does not increase osteoclasts; however, it does negatively impact maintenance of bone hydration, vascularity, and strength. PMID:23238124

  19. SPE-39 family proteins interact with the HOPS complex and function in lysosomal delivery.

    PubMed

    Zhu, Guang-dan; Salazar, Gloria; Zlatic, Stephanie A; Fiza, Babar; Doucette, Michele M; Heilman, Craig J; Levey, Allan I; Faundez, Victor; L'hernault, Steven W

    2009-02-01

    Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis. PMID:19109425

  20. SPE-39 Family Proteins Interact with the HOPS Complex and Function in Lysosomal Delivery

    PubMed Central

    Zhu, Guang-dan; Salazar, Gloria; Zlatic, Stephanie A.; Fiza, Babar; Doucette, Michele M.; Heilman, Craig J.; Levey, Allan I.

    2009-01-01

    Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39–like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis. PMID:19109425

  1. Development of an SPE/CE method for analyzing HAAs

    USGS Publications Warehouse

    Zhang, L.; Capel, P.D.; Hozalski, R.M.

    2007-01-01

    The haloacetic acid (HAA) analysis methods approved by the US Environmental Protection Agency involve extraction and derivatization of HAAs (typically to their methyl ester form) and analysis by gas chromatography (GC) with electron capture detection (ECD). Concerns associated with these methods include the time and effort of the derivatization process, use of potentially hazardous chemicals or conditions during methylation, poor recoveries because of low extraction efficiencies for some HAAs or matrix effects from sulfate, and loss of tribromoacetic acid because of decarboxylation. The HAA analysis method introduced here uses solid-phase extraction (SPE) followed by capillary electrophoresis (CE) analysis. The method is accurate, reproducible, sensitive, relatively safe, and easy to perform, and avoids the use of large amounts of solvent for liquid-liquid extraction and the potential hazards and hassles of derivatization. The cost of analyzing HAAs using this method should be lower than the currently approved methods, and utilities with a GC/ECD can perform the analysis in-house.

  2. Effect of SpeB and EndoS from Streptococcus pyogenes on Human Immunoglobulins

    PubMed Central

    Collin, Mattias; Olsén, Arne

    2001-01-01

    Streptococcus pyogenes secretes a specific immunoglobulin G (IgG)-protease, SpeB, as well as the IgG glycan-hydrolyzing enzyme EndoS. Here we show that SpeB also degrades IgA, IgM, IgD, and IgE. We also show that EndoS only hydrolyzes the glycan moiety on native but not denatured IgG. Thus, SpeB has a broad immunoglobulin-degrading activity, while EndoS is highly specific for IgG. PMID:11598100

  3. Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins.

    PubMed

    Collin, M; Olsén, A

    2001-11-01

    Streptococcus pyogenes secretes a specific immunoglobulin G (IgG)-protease, SpeB, as well as the IgG glycan-hydrolyzing enzyme EndoS. Here we show that SpeB also degrades IgA, IgM, IgD, and IgE. We also show that EndoS only hydrolyzes the glycan moiety on native but not denatured IgG. Thus, SpeB has a broad immunoglobulin-degrading activity, while EndoS is highly specific for IgG. PMID:11598100

  4. NFκB induces overexpression of bovine FcRn

    PubMed Central

    Cervenak, Judit; Doleschall, Márton; Bender, Balázs; Mayer, Balázs; Schneider, Zita; Doleschall, Zoltán; Zhao, Yaofeng; Bősze, Zsuzsanna; Hammarström, Lennart; Oster, Wolfgang; Kacskovics, Imre

    2013-01-01

    Among the many functions of the neonatal Fc receptor (FcRn) for IgG, it binds to IgG-opsonized antigen complexes and propagates their traffic into lysosomes where antigen processing occurs. We previously reported that transgenic (Tg) mice and rabbits that carry multiple copies and overexpress FcRn have augmented humoral immune responses. Nuclear factor-kappa B (NFκB) is a critical molecule in the signaling cascade in the immune response. NFκB induces human FcRn expression and our previous in silico analysis suggested NFκB binding sites in the promoter region of the bovine (b) FcRn α-chain gene (FCGRT). Here, we report the identification of three NFκB transcription binding sites in the promoter region of this gene using luciferase reporter gene technology, electromobility shift assay and supershift analysis. Stimulation of primary bovine endothelial cells with the Toll-like receptor-4 ligand lipopolysaccharide (LPS), which mediates its effect via NFκB, resulted in rapid upregulation of the bFcRn expression and a control gene, bovine E-selectin. This rapid bFcRn gene induction was also observed in the spleen of bFcRn Tg mice treated with intraperitoneally injected LPS, analyzed by northern blot analysis. Finally, NFκB-mediated bFcRn upregulation was confirmed at the protein level in macrophages isolated from the bFcRn Tg mice using flow cytometry with a newly developed FcRn specific monoclonal antibody that does not cross-react with the mouse FcRn. We conclude that NFκB regulates bFcRn expression and thus optimizes its functions, e.g., in the professional antigen presenting cells, and contributes to the much augmented humoral immune response in the bFcRn Tg mice. PMID:24492342

  5. Pre-shot simulations of far-field ground motion for the Source Physics Experiment (SPE) Explosions at the Climax Stock, Nevada National Security Site: SPE2

    SciTech Connect

    Mellors, R J; Rodgers, A; Walter, W; Ford, S; Xu, H; Matzel, E; Myers, S; Petersson, N A; Sjogreen, B; Hauk, T; Wagoner, J

    2011-10-18

    The Source Physics Experiment (SPE) is planning a 1000 kg (TNT equivalent) shot (SPE2) at the Nevada National Security Site (NNSS) in a granite borehole at a depth (canister centroid) of 45 meters. This shot follows an earlier shot of 100 kg in the same borehole at a depth 60 m. Surrounding the shotpoint is an extensive array of seismic sensors arrayed in 5 radial lines extending out 2 km to the north and east and approximately 10-15 to the south and west. Prior to SPE1, simulations using a finite difference code and a 3D numerical model based on the geologic setting were conducted, which predicted higher amplitudes to the south and east in the alluvium of Yucca Flat along with significant energy on the transverse components caused by scattering within the 3D volume along with some contribution by topographic scattering. Observations from the SPE1 shot largely confirmed these predictions although the ratio of transverse energy relative to the vertical and radial components was in general larger than predicted. A new set of simulations has been conducted for the upcoming SPE2 shot. These include improvements to the velocity model based on SPE1 observations as well as new capabilities added to the simulation code. The most significant is the addition of a new source model within the finite difference code by using the predicted ground velocities from a hydrodynamic code (GEODYN) as driving condition on the boundaries of a cube embedded within WPP which provides a more sophisticated source modeling capability linked directly to source site materials (e.g. granite) and type and size of source. Two sets of SPE2 simulations are conducted, one with a GEODYN source and 3D complex media (no topography node spacing of 5 m) and one with a standard isotropic pre-defined time function (3D complex media with topography, node spacing of 5 m). Results were provided as time series at specific points corresponding to sensor locations for both translational (x,y,z) and rotational

  6. Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

    PubMed

    Lee, Eric A; Angka, Leonard; Rota, Sarah-Grace; Hanlon, Thomas; Mitchell, Andrew; Hurren, Rose; Wang, Xiao Ming; Gronda, Marcela; Boyaci, Ezel; Bojko, Barbara; Minden, Mark; Sriskanthadevan, Shrivani; Datti, Alessandro; Wrana, Jeffery L; Edginton, Andrea; Pawliszyn, Janusz; Joseph, Jamie W; Quadrilatero, Joe; Schimmer, Aaron D; Spagnuolo, Paul A

    2015-06-15

    Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function. PMID:26077472

  7. SPE (trademark) Oxygen Generator Assembly (OGA). (Refurbishment of the technology demonstrator LFSPE oxygen generation subsystem)

    NASA Technical Reports Server (NTRS)

    Roy, Robert J.

    1995-01-01

    The SPE Oxygen Generator Assembly (OGA) has been modified to correct operational deficiencies present in the original system, and to effect changes to the system hardware and software such that its operating conditions are consistent with the latest configuration requirements for the International Space Station Alpha (ISSA). The effectiveness of these changes has recently been verified through a comprehensive test program which saw the SPE OGA operate for over 740 hours at various test conditions, including over 690 hours, or approximately 460 cycles, simulating the orbit of the space station. This report documents the changes made to the SPE OGA, presents and discusses the test results from the acceptance test program, and provides recommendations for additional development activities pertinent to evolution of the SPE OGA to a flight configuration. Copies of the test data from the acceptance test program are provided with this report on 3.5 inch diskettes in self-extracting archive files.

  8. Characterization of plasma protein binding dissociation with online SPE-HPLC

    PubMed Central

    Li, Ping; Fan, Yiran; Wang, Yunlong; Lu, Yaxin; Yin, Zheng

    2015-01-01

    A novel parameter of relative recovery (Rre) was defined and determined by online SPE-HPLC to characterize plasma protein binding (PPB) kinetics of highly plasma binding drugs. The proportional relationship of Rre with koff of PPB has been established with a new SPE model. A rapid, easy to use method could potentially be used to categorize PK properties of the drug candidates in the decision process of drug discovery and development. PMID:26460813

  9. Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis.

    PubMed

    Valdés-Santiago, Laura; Cervantes-Chávez, José Antonio; Winkler, Robert; León-Ramírez, Claudia G; Ruiz-Herrera, José

    2012-03-01

    Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell. PMID:22222500

  10. Role of group A Streptococcus HtrA in the maturation of SpeB protease.

    PubMed

    Cole, Jason N; Aquilina, John A; Hains, Peter G; Henningham, Anna; Sriprakash, Kadaba S; Caparon, Michael G; Nizet, Victor; Kotb, Malak; Cordwell, Stuart J; Djordjevic, Steven P; Walker, Mark J

    2007-12-01

    The serine protease high-temperature requirement A (HtrA) (DegP) of the human pathogen Streptococcus pyogenes (group A Streptococcus; GAS) is localized to the ExPortal secretory microdomain and is reportedly essential for the maturation of cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Here, we utilize HSC5 (M5 serotype) and the in-frame isogenic mutant HSC5DeltahtrA to determine whether HtrA contributes to the maturation of other GAS virulence determinants. Mutanolysin cell wall extracts and secreted proteins were arrayed by 2-DE and identified by MALDI-TOF PMF analysis. HSC5DeltahtrA had elevated levels of cell wall-associated M protein, whilst the supernatant had higher concentrations of M protein fragments and a reduced amount of mature SpeB protease, compared to wild-type (WT). Western blot analysis and protease assays revealed a delay in the maturation of SpeB in the HSC5DeltahtrA supernatant. HtrA was unable to directly process SpeB zymogen (proSpeB) to the active form in vitro. We therefore conclude that HtrA plays an indirect role in the maturation of cysteine protease SpeB. PMID:18072207

  11. Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.

    PubMed

    Tornieri, Karine; Zlatic, Stephanie A; Mullin, Ariana P; Werner, Erica; Harrison, Robert; L'hernault, Steven W; Faundez, Victor

    2013-12-20

    Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion. PMID:23918659

  12. Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes

    PubMed Central

    Tornieri, Karine; Zlatic, Stephanie A.; Mullin, Ariana P.; Werner, Erica; Harrison, Robert; L'Hernault, Steven W.; Faundez, Victor

    2013-01-01

    Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome–lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion. PMID:23918659

  13. The Majority of 9,729 Group A Streptococcus Strains Causing Disease Secrete SpeB Cysteine Protease: Pathogenesis Implications

    PubMed Central

    Raghuram, Anjali; Cantu, Concepcion; Hartman, Meredith H.; Jimenez, Francisco E.; Lee, Susan; Ngo, Ashley; Rice, Kelsey A.; Saddington, Deborah; Spillman, Hannaka; Valson, Chandni; Flores, Anthony R.; Beres, Stephen B.; Long, S. Wesley; Nasser, Waleed; Musser, James M.

    2015-01-01

    Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy. PMID:26416912

  14. The majority of 9,729 group A streptococcus strains causing disease secrete SpeB cysteine protease: pathogenesis implications.

    PubMed

    Olsen, Randall J; Raghuram, Anjali; Cantu, Concepcion; Hartman, Meredith H; Jimenez, Francisco E; Lee, Susan; Ngo, Ashley; Rice, Kelsey A; Saddington, Deborah; Spillman, Hannaka; Valson, Chandni; Flores, Anthony R; Beres, Stephen B; Long, S Wesley; Nasser, Waleed; Musser, James M

    2015-12-01

    Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy. PMID:26416912

  15. Inhibitory effect of SPE-39 due to tyrosine phosphorylation and ubiquitination on the function of Vps33B in the EGF-stimulated cells.

    PubMed

    Ishii, Ayumi; Kamimori, Kanae; Hiyoshi, Mineyoshi; Kido, Hiroshi; Ohta, Takeshi; Konishi, Hiroaki

    2012-07-30

    Although SPE-39 is a binding protein to Vps33B that is one of the subunit in the mammalian HOPS complex, the elements of SPE-39 function remain unknown. Here, we show that tyrosine phosphorylation of SPE-39 following EGF stimulation plays a role in the stability of SPE-39 itself. Ubiquitination of the C-terminal region of SPE-39 was also elevated in response to EGF stimulation, and this process was regulated by the phosphorylation of Tyr-11 in SPE-39. However, association of Vps33B with SPE-39 inhibited the elevation of ubiquitination of SPE-39 following EGF stimulation, which might be responsible for the stabilization of SPE-39. Furthermore, an opposing functional relationship between SPE-39 and Vps33B on the downregulation of the EGF receptor was observed in EGF-stimulated COS-7 cells. PMID:22677173

  16. Large-N Seismic Deployment at the Source Physics Experiment (SPE) Site

    NASA Astrophysics Data System (ADS)

    Chen, T.; Snelson, C. M.; Mellors, R. J.; Pitarka, A.

    2015-12-01

    The Source Physics Experiment (SPE) is multi-institutional and multi-disciplinary project that consists of a series of chemical explosion experiments at the Nevada National Security Site. The goal of SPE is to understand the complicated effect of earth structures on source energy partitioning and seismic wave propagation, develop and validate physics-based monitoring, and ultimately better discriminate low-yield nuclear explosions from background seismicity. Deployment of a large number of seismic sensors is planned for SPE to image the full 3-D wavefield with about 500 three-component sensors and 500 vertical component sensors. This large-N seismic deployment will operate near the site of SPE-5 shot for about one month, recording the SPE-5 shot, ambient noise, and additional controlled-sources. This presentation focuses on the design of the large-N seismic deployment. We show how we optimized the sensor layout based on the geological structure and experiment goals with a limited number of sensors. In addition, we will also show some preliminary record sections from deployment. This work was conducted under Contract No. DE-AC52-06NA25946 with the U.S. Department of Energy.

  17. The Transurethral Suprapubic endo-Cystostomy (T-SPeC): A Novel Suprapubic Catheter Insertion Device

    PubMed Central

    Egerdie, R. Blair; Albala, David M.; Flynn, Brian J.

    2013-01-01

    Abstract Background and Purpose Current methods of suprapubic cystostomy (SPC) catheter insertion may be difficult for patients in poor health and can result in significant morbidity and mortality. These include a highly invasive open procedure, as well as the use of the percutaneous trocar punch methods, commonly associated with short-term SPC. We present the first human experience with the Transurethral Suprapubic endo-Cystostomy (T-SPeC®) device, a novel disposable device used for introducing a suprapubic catheter via a retrourethral (inside-to-out) approach similar to the Lowsley technique. Patients and Methods Four men at St. Mary's General Hospital in Kitchener Ontario, Canada, received the T-SPeC device (model T7) under general anesthesia. Results Patients had no complications from catheterization using the T-SPeC T7 Surgical System. The mean surgical time of the four procedures was 9.7 minutes, with a range of 7.9 to 13.5 minutes, including instrument preparation and cystoscopy. All four procedures were highly accurate and rapid. There were no complications and minimal blood loss from the procedure. Conclusions We found that the T-SPeC device allows for efficient and safe insertion of a suprapubic catheter in an outpatient setting and may be a useful addition to the urologic armamentarium. The T-SPeC Surgical System facilitates rapid and precise suprapubic catheter placement. PMID:23488708

  18. Par-4/NF-κB Mediates the Apoptosis of Islet β Cells Induced by Glucolipotoxicity.

    PubMed

    QiNan, Wu; XiaGuang, Gan; XiaoTian, Lei; WuQuan, Deng; Ling, Zhang; Bing, Chen

    2016-01-01

    Apoptosis of islet β cells is a primary pathogenic feature of type 2 diabetes, and ER stress and mitochondrial dysfunction play important roles in this process. Previous research has shown that prostate apoptosis response-4 (Par-4)/NF-κB induces cancer cell apoptosis through endoplasmic reticulum (ER) stress and mitochondrial dysfunction. However, the mechanism by which Par-4/NF-κB induces islet β cell apoptosis remains unknown. We used a high glucose/palmitate intervention to mimic type 2 diabetes in vitro. We demonstrated that the high glucose/palmitate intervention induced the expression and secretion of Par-4. It also causes increased expression and activation of NF-κB, which induced NIT-1 cell apoptosis and dysfunction. Overexpression of Par-4 potentiates these effects, whereas downregulation of Par-4 attenuates them. Inhibition of NF-κB inhibited the Par-4-induced apoptosis. Furthermore, these effects occurred through the ER stress cell membrane and mitochondrial pathway of apoptosis. Our findings reveal a novel role for Par-4/NF-κB in islet β cell apoptosis and type 2 diabetes. PMID:27340675

  19. Par-4/NF-κB Mediates the Apoptosis of Islet β Cells Induced by Glucolipotoxicity

    PubMed Central

    QiNan, Wu; XiaGuang, Gan; XiaoTian, Lei; WuQuan, Deng; Ling, Zhang; Bing, Chen

    2016-01-01

    Apoptosis of islet β cells is a primary pathogenic feature of type 2 diabetes, and ER stress and mitochondrial dysfunction play important roles in this process. Previous research has shown that prostate apoptosis response-4 (Par-4)/NF-κB induces cancer cell apoptosis through endoplasmic reticulum (ER) stress and mitochondrial dysfunction. However, the mechanism by which Par-4/NF-κB induces islet β cell apoptosis remains unknown. We used a high glucose/palmitate intervention to mimic type 2 diabetes in vitro. We demonstrated that the high glucose/palmitate intervention induced the expression and secretion of Par-4. It also causes increased expression and activation of NF-κB, which induced NIT-1 cell apoptosis and dysfunction. Overexpression of Par-4 potentiates these effects, whereas downregulation of Par-4 attenuates them. Inhibition of NF-κB inhibited the Par-4-induced apoptosis. Furthermore, these effects occurred through the ER stress cell membrane and mitochondrial pathway of apoptosis. Our findings reveal a novel role for Par-4/NF-κB in islet β cell apoptosis and type 2 diabetes. PMID:27340675

  20. Spacecraft Solar Particle Event (SPE) Shielding: Shielding Effectiveness as a Function of SPE model as Determined with the FLUKA Radiation Transport Code

    NASA Technical Reports Server (NTRS)

    Koontz, Steve; Atwell, William; Reddell, Brandon; Rojdev, Kristina

    2010-01-01

    Analysis of both satellite and surface neutron monitor data demonstrate that the widely utilized Exponential model of solar particle event (SPE) proton kinetic energy spectra can seriously underestimate SPE proton flux, especially at the highest kinetic energies. The more recently developed Band model produces better agreement with neutron monitor data ground level events (GLEs) and is believed to be considerably more accurate at high kinetic energies. Here, we report the results of modeling and simulation studies in which the radiation transport code FLUKA (FLUktuierende KAskade) is used to determine the changes in total ionizing dose (TID) and single-event environments (SEE) behind aluminum, polyethylene, carbon, and titanium shielding masses when the assumed form (i. e., Band or Exponential) of the solar particle event (SPE) kinetic energy spectra is changed. FLUKA simulations have fully three dimensions with an isotropic particle flux incident on a concentric spherical shell shielding mass and detector structure. The effects are reported for both energetic primary protons penetrating the shield mass and secondary particle showers caused by energetic primary protons colliding with shielding mass nuclei. Our results, in agreement with previous studies, show that use of the Exponential form of the event

  1. Theoretical performance of hydrogen-bromine rechargeable SPE fuel cell. [Solid Polymer Electrolyte

    NASA Technical Reports Server (NTRS)

    Savinell, R. F.; Fritts, S. D.

    1988-01-01

    A mathematical model was formulated to describe the performance of a hydrogen-bromine fuel cell. Porous electrode theory was applied to the carbon felt flow-by electrode and was coupled to theory describing the solid polymer electrolyte (SPE) system. Parametric studies using the numerical solution to this model were performed to determine the effect of kinetic, mass transfer, and design parameters on the performance of the fuel cell. The results indicate that the cell performance is most sensitive to the transport properties of the SPE membrane. The model was also shown to be a useful tool for scale-up studies.

  2. Glufosinate ammonium clean-up procedure from water samples using SPE

    NASA Astrophysics Data System (ADS)

    Tayeb M., A.; Ismail B., S.; Mardiana-Jansar, K.; Ta, Goh Choo; Agustar, Hani Kartini

    2015-09-01

    For the determination of glufosinate ammonium residue in soil and water samples, different solid phase extraction (SPE) sorbent efficiency was studied. Four different SPE sorbents i.e.: CROMABOND PS-H+, CROMABOND PS-OH-, ISOLUTE ENV+, Water Sep-Pak and OASIS HLB were used. Sample clean-up performance was evaluated using high performance liquid chromatography (Agilent 1220 infinity LC) with fluorescence detector. Detection of FMO-derivatives was done at λ ex = 260 nm and λ em= 310 nm. OASIS HLB column was the most suitable for the clean-up in view of the overall feasibility of the analysis.

  3. Cleavage of Antigen-Bound Immunoglobulin G by SpeB Contributes to Streptococcal Persistence in Opsonizing Blood

    PubMed Central

    Eriksson, Anna; Norgren, Mari

    2003-01-01

    Group A streptococci (GAS) express a superantigen, SpeB, having cysteine protease activity. SpeB exhibits several properties that might contribute to virulence, the most recently discovered being the ability to cleave immunoglobulin G (IgG) in a manner similar to that of papain. In the present study, we confirmed this latter finding and found that the irreversible inhibition of SpeB protease activity completely abolishes IgG cleavage. SpeB cleavage of IgG was not species restricted since SpeB cleaved both human, rabbit, and mouse IgG. In order to investigate the nature of the SpeB cleavage of IgG, antibodies were immobilized prior to exposure to SpeB, either by unspecific binding of the Fc to GAS surface proteins or by antigen-specific binding. Analysis of the IgG molecules by SDS-PAGE showed that SpeB could cleave antigen-bound antibodies, while the IgG bound to IgG-binding proteins was protected from cleavage. In a phagocytosis assay using whole blood, the M49 GAS strain NZ131 showed a significantly higher survival than its isogenic speB mutant. Furthermore, the addition of extracellular supernatant derived from an overnight culture of native NZ131 increased the survival of its isogenic speB derivative. This indicates that SpeB's ability to cleave off the Fc part of antigen-bound IgG contributes to GAS escape from opsonophagocytosis while not interfering with the formation of a host-like coat by unspecific IgG binding. PMID:12496168

  4. Cleavage of antigen-bound immunoglobulin G by SpeB contributes to streptococcal persistence in opsonizing blood.

    PubMed

    Eriksson, Anna; Norgren, Mari

    2003-01-01

    Group A streptococci (GAS) express a superantigen, SpeB, having cysteine protease activity. SpeB exhibits several properties that might contribute to virulence, the most recently discovered being the ability to cleave immunoglobulin G (IgG) in a manner similar to that of papain. In the present study, we confirmed this latter finding and found that the irreversible inhibition of SpeB protease activity completely abolishes IgG cleavage. SpeB cleavage of IgG was not species restricted since SpeB cleaved both human, rabbit, and mouse IgG. In order to investigate the nature of the SpeB cleavage of IgG, antibodies were immobilized prior to exposure to SpeB, either by unspecific binding of the Fc to GAS surface proteins or by antigen-specific binding. Analysis of the IgG molecules by SDS-PAGE showed that SpeB could cleave antigen-bound antibodies, while the IgG bound to IgG-binding proteins was protected from cleavage. In a phagocytosis assay using whole blood, the M49 GAS strain NZ131 showed a significantly higher survival than its isogenic speB mutant. Furthermore, the addition of extracellular supernatant derived from an overnight culture of native NZ131 increased the survival of its isogenic speB derivative. This indicates that SpeB's ability to cleave off the Fc part of antigen-bound IgG contributes to GAS escape from opsonophagocytosis while not interfering with the formation of a host-like coat by unspecific IgG binding. PMID:12496168

  5. Spacecraft Solar Particle Event (SPE) Shielding: Shielding Effectiveness as a Function of SPE Model as Determined with the FLUKA Radiation Transport Code

    NASA Astrophysics Data System (ADS)

    Koontz, S. L.; Atwell, W. A.; Reddell, B.; Rojdev, K.

    2010-12-01

    In the this paper, we report the results of modeling and simulation studies in which the radiation transport code FLUKA (FLUktuierende KAskade) is used to determine the changes in total ionizing dose (TID) and single-event effect (SEE) environments behind aluminum, polyethylene, carbon, and titanium shielding masses when the assumed form (i.e., Band or Exponential) of the solar particle event (SPE) kinetic energy spectra is changed. FLUKA simulations are fully three dimensional with an isotropic particle flux incident on a concentric spherical shell shielding mass and detector structure. FLUKA is a fully integrated and extensively verified Monte Carlo simulation package for the interaction and transport of high-energy particles and nuclei in matter. The effects are reported of both energetic primary protons penetrating the shield mass and secondary particle showers caused by energetic primary protons colliding with shielding mass nuclei. SPE heavy ion spectra are not addressed. Our results, in agreement with previous studies, show that use of the Exponential form of the event spectra can seriously underestimate spacecraft SPE TID and SEE environments in some, but not all, shielding mass cases. The SPE spectra investigated are taken from four specific SPEs that produced ground-level events (GLEs) during solar cycle 23 (1997-2008). GLEs are produced by highly energetic solar particle events (ESP), i.e., those that contain significant fluences of 700 MeV to 10 GeV protons. Highly energetic SPEs are implicated in increased rates of spacecraft anomalies and spacecraft failures. High-energy protons interact with Earth’s atmosphere via nuclear reaction to produce secondary particles, some of which are neutrons that can be detected at the Earth’s surface by the global neutron monitor network. GLEs are one part of the overall SPE resulting from a particular solar flare or coronal mass ejection event on the sun. The ESP part of the particle event, detected by spacecraft

  6. LC-SPE-NMR Identification of Co-products from Biomass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hyphenated technique of LC-SPE-NMR was utilized to isolate and identify cinnamic acids and investigate their derivatives from the chloroform extract from Coastal bermudagrass. This method has shown to be useful in handling impure mixtures of extractants from biomass material. It overcomes the ...

  7. Determination of trace organic pollutants in aqueous samples using GC/MS and SPE techniques

    SciTech Connect

    Yoo, L.J.; Yamamoto, M.; Fitzsimmons, S.; Shen, Y.

    1996-11-01

    This study evaluates the advantage of using GC/MS (ion trap) and solid phase extraction (SPE) for the determination of semi-volatile organics which cover priority pollutants, such as polycyclic aromatic hydrocarbons, pesticides, phthalates, and synthetic organic analytes. SPE of trace organic compounds using reverse phase sorbent is attractive compared to the more traditional methods that utilize liquid-liquid extraction or microextraction for the removal of these pollutants from aqueous samples. GC/MS method involving SPE for sample preparation reduces manual labor, speed sample processing,and substantially reduces the volume of solvent required. Also, the application of axial modulation ion trap mass spectrometry improved sensitivity in GC/MS analysis and the method accuracy and precision of semi-volatile organics from GC/MS (ion trap) are very competitive with electron capture detector and photo ionization detector. Systematic studies were done to determine the factors that effect the optimum disk sampling/elution conditions to achieve the quality control requirements for the compliance monitoring. The recoveries of phthalates, polycyclic aromatic hydrocarbons (PAH`s) and most of the organic pesticides, which have very hydrophobic nature and high boiling points, are very acceptable. Consequently GC/MS analysis using solid phase extraction (SPE) techniques can be applied as the primary analytical method and final conformation tool for the routine monitoring samples such as ground water, surface water and reclaimed water for the determination of trace organic pollutants with improved sensitivity, reduced extraction time and monitoring expense.

  8. A High-Quality Teacher for Every Classroom. SPeNSE Summary Sheet.

    ERIC Educational Resources Information Center

    Westat, Inc., Rockville, MD.

    This report from the Study of Personnel Needs in Special Education (SPeNSE) focuses on working conditions that affect special education teachers and how teachers acquire needed professional skills. The report found: (1) 80% of special education teachers serve students with two or more primary disabilities; (2) almost one-fourth of students served…

  9. Surface Signature Characterization at SPE through Ground-Proximal Methods: Methodology Change and Technical Justification

    SciTech Connect

    Schultz-Fellenz, Emily S.

    2015-09-09

    A portion of LANL’s FY15 SPE objectives includes initial ground-based or ground-proximal investigations at the SPE Phase 2 site. The area of interest is the U2ez location in Yucca Flat. This collection serves as a baseline for discrimination of surface features and acquisition of topographic signatures prior to any development or pre-shot activities associated with SPE Phase 2. Our team originally intended to perform our field investigations using previously vetted ground-based (GB) LIDAR methodologies. However, the extended proposed time frame of the GB LIDAR data collection, and associated data processing time and delivery date, were unacceptable. After technical consultation and careful literature research, LANL identified an alternative methodology to achieve our technical objectives and fully support critical model parameterization. Very-low-altitude unmanned aerial systems (UAS) photogrammetry appeared to satisfy our objectives in lieu of GB LIDAR. The SPE Phase 2 baseline collection was used as a test of this UAS photogrammetric methodology.

  10. Cucurbitacin B induced ATM-mediated DNA damage causes G2/M cell cycle arrest in a ROS-dependent manner.

    PubMed

    Guo, Jiajie; Wu, Guosheng; Bao, Jiaolin; Hao, Wenhui; Lu, Jinjian; Chen, Xiuping

    2014-01-01

    Cucurbitacins are a class of triterpenoids widely distributed in plant kingdom with potent anti-cancer activities both in vitro and in vivo by inducing cycle arrest, autophagy, and apoptosis. Cucurbitacin B (Cuc B), could induce S or G2/M cell cycle arrest in cancer cells while the detailed mechanisms remain to be clear. This study was designed to precisely dissect the signaling pathway(s) responsible for Cuc B induced cell cycle arrest in human lung adenocarcinoma epithelial A549 cells. We demonstrated that low concentrations of Cuc B dramatically induced G2/M phase arrest in A549 cells. Cuc B treatment caused DNA double-strand breaks (DSBs) without affecting the signal transducer and activator of transcription 3 (STAT3), the potential molecular target for Cuc B. Cuc B triggers ATM-activated Chk1-Cdc25C-Cdk1, which could be reversed by both ATM siRNA and Chk1 siRNA. Cuc B also triggers ATM-activated p53-14-3-3-σ pathways, which could be reversed by ATM siRNA. Cuc B treatment also led to increased intracellular reactive oxygen species (ROS) formation, which was inhibited by N-acetyl-l-cysteine (NAC) pretreatment. Furthermore, NAC pretreatment inhibited Cuc B induced DNA damage and G2/M phase arrest. Taken together, these results suggested that Cuc B induces DNA damage in A549 cells mediated by increasing intracellular ROS formation, which lead to G2/M cell phase arrest through ATM-activated Chk1-Cdc25C-Cdk1 and p53-14-3-3-σ parallel branches. These observations provide novel mechanisms and potential targets for better understanding of the anti-cancer mechanisms of cucurbitacins. PMID:24505404

  11. Cucurbitacin B Induced ATM-Mediated DNA Damage Causes G2/M Cell Cycle Arrest in a ROS-Dependent Manner

    PubMed Central

    Guo, Jiajie; Wu, Guosheng; Bao, Jiaolin; Hao, Wenhui; Lu, Jinjian; Chen, Xiuping

    2014-01-01

    Cucurbitacins are a class of triterpenoids widely distributed in plant kingdom with potent anti-cancer activities both in vitro and in vivo by inducing cycle arrest, autophagy, and apoptosis. Cucurbitacin B (Cuc B), could induce S or G2/M cell cycle arrest in cancer cells while the detailed mechanisms remain to be clear. This study was designed to precisely dissect the signaling pathway(s) responsible for Cuc B induced cell cycle arrest in human lung adenocarcinoma epithelial A549 cells. We demonstrated that low concentrations of Cuc B dramatically induced G2/M phase arrest in A549 cells. Cuc B treatment caused DNA double-strand breaks (DSBs) without affecting the signal transducer and activator of transcription 3 (STAT3), the potential molecular target for Cuc B. Cuc B triggers ATM-activated Chk1-Cdc25C-Cdk1, which could be reversed by both ATM siRNA and Chk1 siRNA. Cuc B also triggers ATM-activated p53-14-3-3-σ pathways, which could be reversed by ATM siRNA. Cuc B treatment also led to increased intracellular reactive oxygen species (ROS) formation, which was inhibited by N-acetyl-l-cysteine (NAC) pretreatment. Furthermore, NAC pretreatment inhibited Cuc B induced DNA damage and G2/M phase arrest. Taken together, these results suggested that Cuc B induces DNA damage in A549 cells mediated by increasing intracellular ROS formation, which lead to G2/M cell phase arrest through ATM-activated Chk1-Cdc25C-Cdk1 and p53-14-3-3-σ parallel branches. These observations provide novel mechanisms and potential targets for better understanding of the anti-cancer mechanisms of cucurbitacins. PMID:24505404

  12. cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds.

    PubMed

    Song, Xiaomin; Wang, Jing; Wu, Fang; Li, Xu; Teng, Maikun; Gong, Weimin

    2005-01-01

    SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal alpha-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far. PMID:15821865

  13. Time-Dependent Moment Tensors of the First Four Source Physics Experiments (SPE) Explosions

    NASA Astrophysics Data System (ADS)

    Yang, X.

    2015-12-01

    We use mainly vertical-component geophone data within 2 km from the epicenter to invert for time-dependent moment tensors of the first four SPE explosions: SPE-1, SPE-2, SPE-3 and SPE-4Prime. We employ a one-dimensional (1D) velocity model developed from P- and Rg-wave travel times for Green's function calculations. The attenuation structure of the model is developed from P- and Rg-wave amplitudes. We select data for the inversion based on the criterion that they show consistent travel times and amplitude behavior as those predicted by the 1D model. Due to limited azimuthal coverage of the sources and the mostly vertical-component-only nature of the dataset, only long-period, diagonal components of the moment tensors are well constrained. Nevertheless, the moment tensors, particularly their isotropic components, provide reasonable estimates of the long-period source amplitudes as well as estimates of corner frequencies, albeit with larger uncertainties. The estimated corner frequencies, however, are consistent with estimates from ratios of seismogram spectra from different explosions. These long-period source amplitudes and corner frequencies cannot be fit by classical P-wave explosion source models. The results motivate the development of new P-wave source models suitable for these chemical explosions. To that end, we fit inverted moment-tensor spectra by modifying the classical explosion model using regressions of estimated source parameters. Although the number of data points used in the regression is small, the approach suggests a way for the new-model development when more data are collected.

  14. Analysis of the Source Physics Experiment SPE4 Prime Using State-Of Parallel Numerical Tools.

    NASA Astrophysics Data System (ADS)

    Vorobiev, O.; Ezzedine, S. M.; Antoun, T.; Glenn, L.

    2015-12-01

    This work describes a methodology used for large scale modeling of wave propagation from underground chemical explosions conducted at the Nevada National Security Site (NNSS) fractured granitic rock. We show that the discrete natures of rock masses as well as the spatial variability of the fabric of rock properties are very important to understand ground motions induced by underground explosions. In order to build a credible conceptual model of the subsurface we integrated the geological, geomechanical and geophysical characterizations conducted during recent test at the NNSS as well as historical data from the characterization during the underground nuclear test conducted at the NNSS. Because detailed site characterization is limited, expensive and, in some instances, impossible we have numerically investigated the effects of the characterization gaps on the overall response of the system. We performed several computational studies to identify the key important geologic features specific to fractured media mainly the joints characterized at the NNSS. We have also explored common key features to both geological environments such as saturation and topography and assess which characteristics affect the most the ground motion in the near-field and in the far-field. Stochastic representation of these features based on the field characterizations has been implemented into LLNL's Geodyn-L hydrocode. Simulations were used to guide site characterization efforts in order to provide the essential data to the modeling community. We validate our computational results by comparing the measured and computed ground motion at various ranges for the recently executed SPE4 prime experiment. We have also conducted a comparative study between SPE4 prime and previous experiments SPE1 and SPE3 to assess similarities and differences and draw conclusions on designing SPE5.

  15. Glycyrrhizic acid prevents ultraviolet-B-induced photodamage: a role for mitogen-activated protein kinases, nuclear factor kappa B and mitochondrial apoptotic pathway.

    PubMed

    Afnan, Quadri; Kaiser, Peerzada J; Rafiq, Rather A; Nazir, Lone A; Bhushan, Shashi; Bhardwaj, Subhash C; Sandhir, Rajat; Tasduq, Sheikh A

    2016-06-01

    Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV-B-induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV-B-induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV-B-mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV-B-mediated increase in intracellular reactive oxygen species (ROS) and down-regulated the release of pro-inflammatory cytokines interleukin (IL)-1α, -1β and -6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2). GA inhibited UV-B-mediated activation of p38 and JNK MAP kinases, COX-2 expression and nuclear translocation of NF-κB. Furthermore, GA inhibited UV-B-mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA-treated HaCaT cells also exhibited elevated antiapoptotic Bcl-2 protein, concomitant with reduced caspase-3 cleavage and decreased PARP-1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV-B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX-2, NF-κB and ICAM-1. Based on the above findings, we conclude that GA protects against UV-B-mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF-κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage. PMID:26836460

  16. An Overview of the Source Physics Experiments (SPE) at the Nevada National Security Site (NNSS)

    NASA Astrophysics Data System (ADS)

    Snelson, C. M.; Barker, D. L.; White, R. L.; Emmitt, R. F.; Townsend, M. J.; Graves, T. E.; Becker, S. A.; Teel, M. G.; Lee, P.; Antoun, T. H.; Rodgers, A.; Walter, W. R.; Mellors, R. J.; Brunish, W. M.; Bradley, C. R.; Patton, H. J.; Hawkins, W. L.; Corbell, B. H.; Abbott, R. E.; SPE Working Group

    2011-12-01

    Modeling of explosion phenomenology has been primarily empirically based when looking at the seismic, infrasound, and acoustic signals. In order to detect low-yield nuclear explosions under the Comprehensive Nuclear Test-Ban Treaty (CTBT), we must be able to understand and model the explosive source in settings beyond where we have empirical data. The Source Physics Experiments (SPE) at the Nevada National Security Site are the first step in this endeavor to link the empirically based with the physics-based modeling to develop this predictive capability. The current series of tests is being conducted in a granite body called the Climax Stock. This location was chosen for several reasons, including the site's expected "simple geology"-the granite is a fairly homogeneous body. In addition, data are available from underground nuclear tests that were conducted in the same rock body, and the nature of the geology has been well-documented. Among the project goals for the SPE is to provide fully coupled seismic energy to the seismic and acoustic seismic arrays so that the transition between the near and far-field data can be modeled and our scientists can begin to understand how non-linear effects and anisotropy control seismic energy transmission and partitioning. The first shot for the SPE was conducted in May 2011 as a calibration shot (SPE1) with 220 lb (100 kg) of chemical explosives set at a depth of 180 ft (55 m). An array of sensors and diagnostics recorded the shot data, including accelerometers, geophones, rotational sensors, short-period and broadband seismic sensors, Continuous Reflectometry for Radius vs. Time Experiment (CORRTEX), Time of Arrival (TOA), Velocity of Detonation (VOD) as well as infrasound sensors. The three-component accelerometer packages were set at depths of 180 ft (55 m), 150 ft (46 m), and 50 ft (15 m) in two rings around ground zero (GZ); the inner ring was at 10 m and the outer ring was 20 m from GZ. Six sets of surface accelerometers

  17. PHARMACOLOGIC PROBING OF AMPHOTERICIN B-INDUCED RENAL DYSFUNCTION IN THE NEONATAL RAT

    EPA Science Inventory

    Pharmacologic Probing of Amphotericin B-Induced Renal Dysfunction in the Neonatal Rat. Gray, J.A., and Kavlock, R.J. (1988). Toxicol. Appl. Pharmacol. 93, 360-368. Acetazolamide, furosemide, chlorothiazide, and amiloride pharmacologic agents that act primarily in the proximal tub...

  18. Antimicrobial Peptide Lactoferricin B-Induced Rapid Leakage of Internal Contents from Single Giant Unilamellar Vesicles.

    PubMed

    Moniruzzaman, Md; Alam, Jahangir Md; Dohra, Hideo; Yamazaki, Masahito

    2015-09-29

    Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to

  19. Sphingosine in apoptosis signaling.

    PubMed

    Cuvillier, Olivier

    2002-12-30

    The sphingolipid metabolites ceramide, sphingosine, and sphingosine 1-phosphate contribute to controlling cell proliferation and apoptosis. Ceramide and its catabolite sphingosine act as negative regulators of cell proliferation and promote apoptosis. Conversely, sphingosine 1-phosphate, formed by phosphorylation of sphingosine by a sphingosine kinase, has been involved in stimulating cell growth and inhibiting apoptosis. As the phosphorylation of sphingosine diminishes apoptosis, while dephosphorylation of sphingosine 1-phosphate potentiates it, the role of sphingosine as a messenger of apoptosis is of importance. Herein, the effects of sphingosine on diverse signaling pathways implicated in the apoptotic process are reviewed. PMID:12531549

  20. Hindlimb Suspension and SPE-Like Radiation Impairs Clearance of Bacterial Infections

    PubMed Central

    Li, Minghong; Holmes, Veronica; Zhou, Yu; Ni, Houping; Sanzari, Jenine K.; Kennedy, Ann R.; Weissman, Drew

    2014-01-01

    A major risk of extended space travel is the combined effects of weightlessness and radiation exposure on the immune system. In this study, we used the hindlimb suspension model of microgravity that includes the other space stressors, situational and confinement stress and alterations in food intake, and solar particle event (SPE)-like radiation to measure the combined effects on the ability to control bacterial infections. A massive increase in morbidity and decrease in the ability to control bacterial growth was observed using 2 different types of bacteria delivered by systemic and pulmonary routes in 3 different strains of mice. These data suggest that an astronaut exposed to a strong SPE during extended space travel is at increased risk for the development of infections that could potentially be severe and interfere with mission success and astronaut health. PMID:24454913

  1. EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis.

    PubMed

    Collin, Mattias; Svensson, Mikael D; Sjöholm, Anders G; Jensenius, Jens C; Sjöbring, Ulf; Olsén, Arne

    2002-12-01

    The human pathogen Streptococcus pyogenes primarily infects the upper respiratory tract and skin, but occasionally it disseminates and causes severe invasive disease with high mortality. This study revealed that the activity of extracellular EndoS, which hydrolyzes the functionally important N-linked oligosaccharides on opsonizing immunoglobulin G (IgG), contributes to increased survival of S. pyogenes in human blood ex vivo. The inability to kill the bacteria is due to reduced binding of IgG to Fc receptors and impaired classical pathway-mediated activation of complement. In addition, the activity of extracellular SpeB, which cleaves IgG into Fc and Fab fragments, also increases bacterial survival. This suggests that S. pyogenes expresses two enzymes, EndoS and SpeB, which modulate IgG by different mechanisms in order to evade the adaptive immune system. PMID:12438337

  2. EndoS and SpeB from Streptococcus pyogenes Inhibit Immunoglobulin-Mediated Opsonophagocytosis

    PubMed Central

    Collin, Mattias; Svensson, Mikael D.; Sjöholm, Anders G.; Jensenius, Jens C.; Sjöbring, Ulf; Olsén, Arne

    2002-01-01

    The human pathogen Streptococcus pyogenes primarily infects the upper respiratory tract and skin, but occasionally it disseminates and causes severe invasive disease with high mortality. This study revealed that the activity of extracellular EndoS, which hydrolyzes the functionally important N-linked oligosaccharides on opsonizing immunoglobulin G (IgG), contributes to increased survival of S. pyogenes in human blood ex vivo. The inability to kill the bacteria is due to reduced binding of IgG to Fc receptors and impaired classical pathway-mediated activation of complement. In addition, the activity of extracellular SpeB, which cleaves IgG into Fc and Fab fragments, also increases bacterial survival. This suggests that S. pyogenes expresses two enzymes, EndoS and SpeB, which modulate IgG by different mechanisms in order to evade the adaptive immune system. PMID:12438337

  3. Comparison of Radiation Transport Codes, HZETRN, HETC and FLUKA, Using the 1956 Webber SPE Spectrum

    NASA Technical Reports Server (NTRS)

    Heinbockel, John H.; Slaba, Tony C.; Blattnig, Steve R.; Tripathi, Ram K.; Townsend, Lawrence W.; Handler, Thomas; Gabriel, Tony A.; Pinsky, Lawrence S.; Reddell, Brandon; Clowdsley, Martha S.; Singleterry, Robert C.; Norbury, John W.; Badavi, Francis F.; Aghara, Sukesh K.

    2009-01-01

    Protection of astronauts and instrumentation from galactic cosmic rays (GCR) and solar particle events (SPE) in the harsh environment of space is of prime importance in the design of personal shielding, spacec raft, and mission planning. Early entry of radiation constraints into the design process enables optimal shielding strategies, but demands efficient and accurate tools that can be used by design engineers in every phase of an evolving space project. The radiation transport code , HZETRN, is an efficient tool for analyzing the shielding effectiveness of materials exposed to space radiation. In this paper, HZETRN is compared to the Monte Carlo codes HETC-HEDS and FLUKA, for a shield/target configuration comprised of a 20 g/sq cm Aluminum slab in front of a 30 g/cm^2 slab of water exposed to the February 1956 SPE, as mode led by the Webber spectrum. Neutron and proton fluence spectra, as well as dose and dose equivalent values, are compared at various depths in the water target. This study shows that there are many regions where HZETRN agrees with both HETC-HEDS and FLUKA for this shield/target configuration and the SPE environment. However, there are also regions where there are appreciable differences between the three computer c odes.

  4. The Crystal Structure of Escherichia coli Spermidine Synthase SpeE Reveals a Unique Substrate-binding Pocket

    SciTech Connect

    Zhou, X.; Chua, T; Tkaczuk, K; Bujnicki, J; Sivaraman, J

    2010-01-01

    Polyamines are essential in all branches of life. Biosynthesis of spermidine, one of the most ubiquitous polyamines, is catalyzed by spermidine synthase (SpeE). Although the function of this enzyme from Escherichia coli has been thoroughly characterized, its structural details remain unknown. Here, we report the crystal structure of E. coli SpeE and study its interaction with the ligands by isothermal titration calorimetry and computational modelling. SpeE consists of two domains - a small N-terminal {beta}-strand domain, and a C-terminal catalytic domain that adopts a canonical methyltransferase (MTase) Rossmann fold. The protein forms a dimer in the crystal and in solution. Structural comparison of E. coli SpeE to its homologs reveals that it has a large and unique substrate-binding cleft that may account for its lower amine substrate specificity.

  5. Inactivation of the cysteine protease SpeB affects hyaluronic acid capsule expression in group A streptococci.

    PubMed

    Woischnik, M; Buttaro, B A; Podbielski, A

    2000-04-01

    The human pathogen Streptococcus pyogenes expresses several virulence factors that are required for the pathogens survival within the host and the concomitant development of disease. To examine the influence of one virulence factor, the extracellular cysteine protease SpeB, on the expression of other virulence factors, the speB structural gene of a serotype M3 and M49 strain was inactivated. Morphologic examination, quantification of extracellular hyaluronic acid capsule, and Northern blot analysis of the isogenic speB -mutants revealed a strain-dependent decrease of hyaluronic acid capsule production and an increase in superoxide dismutase transcription. The transcription of streptolysin O (slo), di- and oligo-peptide permease (dpp, opp), hyaluronidase (hyl), streptokinase (ska) and streptococcal pyrogenic exotoxin A (speA) was unaffected. PMID:10764613

  6. Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB.

    PubMed

    Aziz, Ramy K; Pabst, Michael J; Jeng, Arthur; Kansal, Rita; Low, Donald E; Nizet, Victor; Kotb, Malak

    2004-01-01

    A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2-D gel analysis of the secreted M1T1 proteome, coupled with MALDI-TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA- M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase-shift to a SpeB-/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte-stimulating capacity. The proteome of the SpeB-in vivo phase-shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage-encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB- phase-shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation. PMID:14651616

  7. Substrate-induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

    DOE PAGESBeta

    Filippova, Ekaterina V.; Weigand, Steven J.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F.

    2015-09-26

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Twomore » hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. As a result, our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.« less

  8. Substrate-induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

    SciTech Connect

    Filippova, Ekaterina V.; Weigand, Steven J.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F.

    2015-09-26

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. As a result, our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.

  9. Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG.

    PubMed

    Filippova, Ekaterina V; Weigand, Steven; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F

    2015-11-01

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites. PMID:26410587

  10. Possible involvement of a tetrahydrobiopterin in photoreception for UV-B-induced anthocyanin synthesis in carrot.

    PubMed

    Takeda, Junko; Nakata, Rieko; Ueno, Hiroshi; Murakami, Akio; Iseki, Mineo; Watanabe, Masakatsu

    2014-01-01

    Our previous studies of action spectra for UV-B-induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV-B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV-B light sensing. UV-B-induced phenylalanine ammonia-lyase (PAL) activity was considerably suppressed by N-acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N-acetylserotonin-treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV-B-induced PAL activity. These results suggest the occurrence of an unidentified UV-B photoreceptor (other than UVR8, the tryptophan-based UV-B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After reexamining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV-B sensors. PMID:24943195

  11. COP1 is required for UV-B-induced nuclear accumulation of the UVR8 photoreceptor.

    PubMed

    Yin, Ruohe; Skvortsova, Mariya Y; Loubéry, Sylvain; Ulm, Roman

    2016-07-26

    The UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) promotes UV-B acclimation and tolerance in Arabidopsis thaliana UVR8 localizes to both cytosol and nucleus, but its main activity is assumed to be nuclear. UV-B photoreception stimulates nuclear accumulation of UVR8 in a presently unknown manner. Here, we show that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is required for UV-B-induced nuclear accumulation of UVR8, but bypassing the COP1 requirement for UVR8 nuclear accumulation did not rescue the cop1 mutant UV-B phenotype. Using a glucocorticoid receptor (GR)-based fusion protein system to conditionally localize GR-UVR8 to the nucleus, we have demonstrated that both photoactivation and nuclear localization of UVR8 are required for UV-B-induced photomorphogenic responses. In contrast, there was no UV-B response when UV-B-activated UVR8 was artificially retained in the cytosol. In agreement with a predominantly nuclear activity, constitutively active UVR8(W285A) accumulated in the nucleus also in the absence of UV-B. Furthermore, GR-COP1 expression lines suggested that UV-B-activated UVR8 can be coimported into the nucleus by COP1. Our data strongly support localization of UVR8 signaling in the nucleus and a dual role for COP1 in the regulation of UV-B-induced UVR8 nuclear accumulation and in UVR8-mediated UV-B signaling. PMID:27407149

  12. Signal Transducer and Activator of Transcription-5 Mediates Neuronal Apoptosis Induced by Inhibition of Rac GTPase Activity*

    PubMed Central

    Stankiewicz, Trisha R.; Loucks, F. Alexandra; Schroeder, Emily K.; Nevalainen, Marja T.; Tyler, Kenneth L.; Aktories, Klaus; Bouchard, Ron J.; Linseman, Daniel A.

    2012-01-01

    In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition. STAT1 expression was induced by ToxB; however, CGNs from STAT1 knock-out mice succumbed to ToxB-induced apoptosis as readily as wild-type CGNs. STAT3 displayed enhanced tyrosine phosphorylation following treatment with ToxB, and a reputed inhibitor of STAT3, cucurbitacin (JSI-124), reduced CGN apoptosis. Unexpectedly, JSI-124 failed to block STAT3 phosphorylation, and CGNs were not protected from ToxB by other known STAT3 inhibitors. In contrast, STAT5A tyrosine phosphorylation induced by ToxB was suppressed by JSI-124. In addition, roscovitine similarly inhibited STAT5A phosphorylation and protected CGNs from ToxB-induced apoptosis. Consistent with these results, adenoviral infection with a dominant negative STAT5 mutant, but not wild-type STAT5, significantly decreased ToxB-induced apoptosis of CGNs. Finally, chromatin immunoprecipitation with a STAT5 antibody revealed increased STAT5 binding to the promoter region of prosurvival Bcl-xL. STAT5 was recruited to the Bcl-xL promoter region in a ToxB-dependent manner, and this DNA binding preceded Bcl-xL down-regulation, suggesting transcriptional repression. These data indicate that a novel JAK/STAT5 proapoptotic pathway significantly contributes to neuronal apoptosis induced by the inhibition of Rac GTPase. PMID:22378792

  13. Signal transducer and activator of transcription-5 mediates neuronal apoptosis induced by inhibition of Rac GTPase activity.

    PubMed

    Stankiewicz, Trisha R; Loucks, F Alexandra; Schroeder, Emily K; Nevalainen, Marja T; Tyler, Kenneth L; Aktories, Klaus; Bouchard, Ron J; Linseman, Daniel A

    2012-05-11

    In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition. STAT1 expression was induced by ToxB; however, CGNs from STAT1 knock-out mice succumbed to ToxB-induced apoptosis as readily as wild-type CGNs. STAT3 displayed enhanced tyrosine phosphorylation following treatment with ToxB, and a reputed inhibitor of STAT3, cucurbitacin (JSI-124), reduced CGN apoptosis. Unexpectedly, JSI-124 failed to block STAT3 phosphorylation, and CGNs were not protected from ToxB by other known STAT3 inhibitors. In contrast, STAT5A tyrosine phosphorylation induced by ToxB was suppressed by JSI-124. In addition, roscovitine similarly inhibited STAT5A phosphorylation and protected CGNs from ToxB-induced apoptosis. Consistent with these results, adenoviral infection with a dominant negative STAT5 mutant, but not wild-type STAT5, significantly decreased ToxB-induced apoptosis of CGNs. Finally, chromatin immunoprecipitation with a STAT5 antibody revealed increased STAT5 binding to the promoter region of prosurvival Bcl-xL. STAT5 was recruited to the Bcl-xL promoter region in a ToxB-dependent manner, and this DNA binding preceded Bcl-xL down-regulation, suggesting transcriptional repression. These data indicate that a novel JAK/STAT5 proapoptotic pathway significantly contributes to neuronal apoptosis induced by the inhibition of Rac GTPase. PMID:22378792

  14. Anion exchange SPE and liquid chromatography-tandem mass spectrometry in GHB analysis.

    PubMed

    Elian, Albert A; Hackett, Jeffery

    2011-12-01

    In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d(6) as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r(2)>0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL. PMID:22055831

  15. Large-N Over the Source Physics Experiment (SPE) Phase I and Phase II Test Beds

    NASA Astrophysics Data System (ADS)

    Snelson, C. M.; Carmichael, J. D.; Mellors, R. J.; Abbott, R. E.

    2014-12-01

    One of the current challenges in the field of monitoring and verification is source discrimination of low-yield nuclear explosions from background seismicity, both natural and anthropogenic. Work is underway at the Nevada National Security Site to conduct a series of chemical explosion experiments using a multi-institutional, multi-disciplinary approach. The goal of this series of experiments, called the Source Physics Experiments (SPE), is to refine the understanding of the effect of earth structures on source phenomenology and energy partitioning in the source region, the transition of seismic energy from the near field to the far field, and the development of S waves observed in the far field. To fully explore these problems, the SPE series includes tests in both hard and soft rock geologic environments. The project comprises a number of activities, which range from characterizing the shallow subsurface to acquiring new explosion data from both the near field (<100 m) and the far field (>100 m). SPE includes a series of planned explosions (with different yields and depths of burials), which are conducted in the same hole and monitored by a diverse set of sensors recording characteristics of the explosions, ground-shock, seismo-acoustic energy propagation. This presentation focuses on imaging the full 3D wavefield over hard rock and soft rock test beds using a large number of seismic sensors. This overview presents statistical analyses of optimal sensor layout required to estimate wavefield discriminants and the planned deployment for the upcoming experiments. This work was conducted under Contract No. DE-AC52-06NA25946 with the U.S. Department of Energy. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  16. Retention behaviour of some high-intensity sweeteners on different SPE sorbents.

    PubMed

    Zygler, Agata; Wasik, Andrzej; Namieśnik, Jacek

    2010-10-15

    The objective of this paper is to provide information about application of solid-phase extraction (SPE) for isolation of nine high-intensity sweeteners (acesulfame-K, alitame, aspartame, cyclamate, dulcin, neotame, saccharin, sucralose and neohesperidin dihydrochalcone) from aqueous solutions. The influence of several types of LC-MS compatible buffers (different pH values and compositions) on their recovery has been studied and discussed. A number of commercially available SPE cartridges, such as Chromabond C18ec, Strata-X RP, Bakerbond Octadecyl, Bakerbond SDB-1, Bakerbond SPE Phenyl, Oasis HLB, LiChrolut RP-18, Supelclean LC-18, Discovery DSC-18 and Zorbax C18 were tested in order to evaluate their applicability for the isolation of analytes. Very high recoveries (better than 92%) of all studied compounds were obtained using formic acid-N,N-diisopropylethylamine buffer adjusted to pH 4.5 and C(18)-bonded silica sorbents. Behaviour of polymeric sorbents strongly depends on their structure. Strata-X RP behaves much like a C(18)-bonded silica sorbent. Recoveries obtained using Oasis HLB were comparable with those observed for silica-based sorbents. The only compound less efficiently (83%) retained by this sorbent was cyclamate. Bakerbond SDB-1 shows unusual selectivity towards aspartame and alitame. Recoveries of these two sweeteners were very low (26 and 42%, respectively). It was also found that aspartame and alitame can be selectively separated from the mixture of sweeteners using formic acid-triethylamine buffer at pH 3.5. PMID:20875571

  17. Calpains, mitochondria, and apoptosis

    PubMed Central

    Smith, Matthew A.; Schnellmann, Rick G.

    2012-01-01

    Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca2+-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca2+ overload causes mitochondrial calpain 1 cleavage of the Na+/Ca2+ exchanger leading to mitochondrial Ca2+ accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-α-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca2+ overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system. PMID:22581845

  18. IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes.

    PubMed

    von Pawel-Rammingen, Ulrich; Björck, Lars

    2003-02-01

    The Gram-positive bacterium Streptococcus pyogenes is a major human pathogen causing substantial morbidity and mortality in society. S. pyogenes has evolved numerous molecular mechanisms to avoid the various actions of the human immune system and has established means to modulate both adaptive and innate immune responses. S. pyogenes produces and secretes proteolytic enzymes, which have an important impact on the ability of the bacteria to survive in the human host. Prominent among these are two immunoglobulin-degrading enzymes: the newly discovered streptococcal cysteine proteinase, IdeS, and the classical cysteine proteinase of S. pyogenes, SpeB. PMID:12615219

  19. Simultaneous determination of seven pesticides in waters using multi-walled carbon nanotube SPE and NACE.

    PubMed

    Asensio-Ramos, María; Hernández-Borges, Javier; Ravelo-Pérez, Lidia M; Rodríguez-Delgado, Miguel Angel

    2008-11-01

    In this work, NACE with UV detection is combined with SPE using multi-walled carbon nanotubes (MWCNT) as stationary phase to determine a group of seven pesticides (pirimicarb, pyrifenox, penconazol, carbendazim, cyromazine, pyrimethanil and cyprodinil) in mineral water samples using ametryn as internal standard. The optimized BGE, consisting of a mixture of MeOH and ACN (1:2 v/v) with 90 mM SDS and 20.5 mM HClO(4), was satisfactory to get a good resolution of the seven compounds in less than 13 min. On-line preconcentration was carried out by electrokinetic injection of the sample dissolved in 78:22 v/v MeOH/ACN, 1.11 mM HClO(4). Repeatability was studied for the same day (n=4), for nine different days (n=36) and for four different capillaries. RSD values were appropriate in all cases, i.e. in the range 4.3-9.4% between different capillaries. MWCNT of 10-15 nm od, 2-6 nm id and 0.1-10 mum length were used as SPE materials for the preconcentration of these pesticides from water samples. SPE parameters influencing the enrichment were optimized and the most favorable conditions were as follows: the amount of stationary phase, eluent, sample pH and sample volume were 40 mg MWCNT, 10 mL ACN and 10 mL dichloromethane containing 5% v/v formic acid, pH 8.0, and 750 mL, respectively. Mean recovery values ranged between 53 and 94% for Milli-Q water and between 47 and 93% for mineral waters (RSD values were in the range 2-16%). The method allowed the determination of these pesticides at concentrations below the maximum residue limits established by the European Union legislation (LOD in the range 27-58 ng/L). When the cost, amount and type of the carbon nanotubes used in this work are compared with those carbon nanotubes previously used in the literature it is clear that the proposed materials can be used as economical stationary phases, even cheaper than conventional SPE cartridges. PMID:18956435

  20. Srv mediated dispersal of streptococcal biofilms through SpeB is observed in CovRS+ strains.

    PubMed

    Connolly, Kristie L; Braden, Amy K; Holder, Robert C; Reid, Sean D

    2011-01-01

    Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds. PMID:22163320

  1. Srv Mediated Dispersal of Streptococcal Biofilms Through SpeB Is Observed in CovRS+ Strains

    PubMed Central

    Connolly, Kristie L.; Braden, Amy K.; Holder, Robert C.; Reid, Sean D.

    2011-01-01

    Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds. PMID:22163320

  2. Modulation of apoptosis by V protein mumps virus

    PubMed Central

    2011-01-01

    Background The Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway. Findings We studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process. Conclusions The reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals. PMID:21569530

  3. Geologic Assessment of the Damage Zone from the Second Test at Source Physics Experiment-Nevada (SPE-N)

    SciTech Connect

    Townsend, M. J.; Huckins-Gang, H. E.; Prothro, L. B.; Reed, D. N.

    2012-12-01

    The National Center for Nuclear Security, established by the U.S. Department of Energy, National Nuclear Security Administration (NNSA), is conducting a series of explosive tests at the Nevada National Security Site that are designed to increase the understanding of certain basic physical phenomena associated with underground explosions. These tests will aid in developing technologies that might be used to detect underground nuclear explosions in support of verification activities for the Comprehensive Nuclear-Test-Ban Treaty. The initial project is a series of explosive tests, known collectively as the Source Physics Experiment-Nevada (SPE-N), being conducted in granitic rocks. The SPE-N test series is designed to study the generation and propagation of seismic waves. The results will help advance the seismic monitoring capability of the United States by improving the predictive capability of physics-based modeling of explosive phenomena. The first SPE N (SPE-N-1) test was conducted in May 2011, using 100 kg of explosives at the depth of 54.9 m in the U 15n source hole. SPE-N-2 was conducted in October 2011, using 1,000 kg of explosives at the depth of 45.7 m in the same source hole. The SPE-N-3 test was conducted in the same source hole in July 2012, using the same amount and type of explosive as for SPE-N-2, and at the same depth as SPE-N-2, within the damage zone created by the SPE-N-2 explosion to investigate damage effects on seismic wave propagation. Following the SPE-N-2 shot and prior to the SPE-N-3 shot, the core hole U-15n#10 was drilled at an angle from the surface to intercept the SPE-N-2 shot point location to obtain information necessary to characterize the damage zone. The objective was to determine the position of the damage zone near the shot point, at least on the northeast, where the core hole penetrated it, and obtain information on the properties of the damaged medium. Geologic characterization of the post-SPE-N-2 core hole included

  4. Development of colorimetric solid phase extraction (C-SPE) for in-flight monitoring of spacecraft water supplies

    NASA Astrophysics Data System (ADS)

    Gazda, Daniel Bryan

    2004-12-01

    Colorimetric solid phase extraction (C-SPE) is a sorption-spectrophotometric technique that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water samples. In C-SPE, a syringe is used to meter a known volume of sample through a membrane impregnated with a selective colorimetric reagent along with any additives required to optimize the complexation of the reagent and analyte. As the sample is passed through the membrane, analytes are extracted and complexed, leading to a detectable change in the optical characteristics of the membrane. The analyte-reagent complex is then quantified directly on the membrane, using a hand-held diffuse reflectance spectrophotometer. This dissertation focuses on the development, ground testing, and microgravity flight demonstration of C-SPE methods to meet the near- and long-term water quality monitoring needs of NASA. To this end, the ability of C-SPE to function in a microgravity environment was tested through performance evaluations of methods for the determination of the biocidal agents silver(I) and iodine on the KC-135 microgravity simulator. The biocidal iodine platform was investigated further to determine which iodine species is responsible for the C-SPE signal. Through systematic comparisons of C-SPE results and UV-Visible absorbance studies it was determined that biocidally active I2 is the iodine species complexed by poly(vinylpyrrolidone). The application of C-SPE to additional target water quality parameters is demonstrated through the determination of nickel(II), a metal leachate found in archived water samples from the International Space Station, using dimethylglyoxime. This method introduced a new variation of C-SPE, the quantification of trace analytes based on the collection of an insoluble, colored precipitate. The nickel(II) method was then combined with the method for biocidal silver(I) and a new method to measure sample pH to create a

  5. cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus.

    PubMed

    Wu, Fang; Yan, Ming; Li, Yikun; Chang, Shaojie; Song, Xiaomin; Zhou, Zhaocai; Gong, Weimin

    2003-12-19

    SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized "hydrophobic probe" for proteins. This binding activity indicated another biological function of SPE-16. PMID:14680830

  6. CATHARE2 calculation of SPE-3 test small break loca on PMK facility

    SciTech Connect

    Laugier, E.; Radet, J.

    1995-09-01

    Bind and post test calculations with CATHARE2 have been performed concerning the SPE-4 exercise organized under the auspices of IAEA on the hungarian PMK-2 facility, a one loop scaled model of VVER 440/213 Nuclear Power Plant. The SPE-4 test is a cold leg SBLOCA associated to a {open_quotes}bleed and feed{close_quotes} procedure applied in the secondary circuit. The present paper is devoted to the analysis of the post test calculation. For the first part of the transient (until the end of the SIT activations), the primary and secondary pressures are rather well predicted, leading to a good agreement with the experimental trips, as scram, flow coast down, SIT beginning and end of activation. Nevertheless, some discrepancy with the experiment may be due to an over prediction of the thermal exchanges from the primary to the secondary circuits. For the second part of the transient, the predicted primary circuit repressurization is shifted after the SITs are off, while in the experiment this event immediately follows the end of SIT activation. The delay in the calculation leads to underpredict primary and secondary pressures, thus anticipating the timing of events, such as LPIS and emergency feedwater activation.

  7. IRTF/SPeX Observations of the Unusual Kepler Light Curve System KIC8462852

    NASA Astrophysics Data System (ADS)

    Lisse, C. M.; Sitko, M. L.; Marengo, M.

    2015-12-01

    We have utilized the NASA/IRTF 3 m SpeX instrument’s high-resolution spectral mode to observe and characterize the near-infrared flux emanating from the unusual Kepler light curve system KIC 8462852. By comparing the resulting 0.8–4.2 μm spectrum to a mesh of model photospheric spectra, the 6 emission line analyses of the Rayner et al. catalog, and the 25 system collections of debris disks we have observed to date using SpeX under the Near InfraRed Debris disk Survey, we have been able to additionally characterize the system. Within the errors of our measurements, this star looks like a normal solar abundance main-sequence F1V to F3V dwarf star without any obvious traces of significant circumstellar dust or gas. Using Connelley & Greene’s emission measures, we also see no evidence of significant ongoing accretion onto the star nor any stellar outflow away from it. Our results are inconsistent with large amounts of static close-in obscuring material or the unusual behavior of a YSO system, but are consistent with the favored episodic giant comet models of a Gyr old stellar system favored by Boyajian et al. We speculate that KIC 8462852, like the ∼1.4 Gyr old F2V system η Corvi, is undergoing a late heavy bombardment, but is only in its very early stages.

  8. Systematic optimization of an SPE with HPLC-FLD method for fluoroquinolone detection in wastewater.

    PubMed

    He, Ke; Blaney, Lee

    2015-01-23

    This paper describes a selective and ultra-sensitive analytical method for simultaneous determination of 11 fluoroquinolone (FQ) antibiotics in environmental and wastewater samples. The method employs offline solid-phase extraction (SPE) and reversed-phase high performance liquid chromatography with fluorescence detection (HPLC-FLD). A weak cation exchange SPE protocol was developed with a novel loading volume optimization algorithm and a methanol cleanup step to remove background organic matter. Various parameters were optimized to recover FQs from water/wastewater and analyte recovery was generally greater than 80%. Chromatographic separation of the 11 FQs was achieved on a 150 mm pentafluorophenyl column using a gradient elution scheme with methanol, acetonitrile, and 20mM phosphate buffer (pH=2.4). Excitation and emission wavelengths were individually optimized for each FQ using fluorescence spectroscopy; the excitation and emission wavelengths were 276-296 nm and 444-506 nm, respectively. Instrumental quantitation limits were 20-100 pg of mass injected. Of the 11 FQs investigated, seven (i.e., ciprofloxacin, difloxacin, enrofloxacin, fleroxacin, norfloxacin, moxifloxacin, and ofloxacin) were detected during a four-month sampling campaign of wastewater and wastewater-impacted surface water. Concentrations of FQs in raw wastewater, wastewater effluent, and wastewater-impacted surface water were 5-1292, 2-504, and 4-187ng/L, respectively. PMID:25200119

  9. IRTF/SPeX Observations of the Unusual Kepler Light Curve System KIC8462852

    NASA Astrophysics Data System (ADS)

    Lisse, C. M.; Sitko, M. L.; Marengo, M.

    2015-12-01

    We have utilized the NASA/IRTF 3 m SpeX instrument's high-resolution spectral mode to observe and characterize the near-infrared flux emanating from the unusual Kepler light curve system KIC 8462852. By comparing the resulting 0.8-4.2 μm spectrum to a mesh of model photospheric spectra, the 6 emission line analyses of the Rayner et al. catalog, and the 25 system collections of debris disks we have observed to date using SpeX under the Near InfraRed Debris disk Survey, we have been able to additionally characterize the system. Within the errors of our measurements, this star looks like a normal solar abundance main-sequence F1V to F3V dwarf star without any obvious traces of significant circumstellar dust or gas. Using Connelley & Greene's emission measures, we also see no evidence of significant ongoing accretion onto the star nor any stellar outflow away from it. Our results are inconsistent with large amounts of static close-in obscuring material or the unusual behavior of a YSO system, but are consistent with the favored episodic giant comet models of a Gyr old stellar system favored by Boyajian et al. We speculate that KIC 8462852, like the ˜1.4 Gyr old F2V system η Corvi, is undergoing a late heavy bombardment, but is only in its very early stages.

  10. Recent improvements in SPE3D: a VR-based surgery planning environment

    NASA Astrophysics Data System (ADS)

    Witkowski, Marcin; Sitnik, Robert; Verdonschot, Nico

    2014-02-01

    SPE3D is a surgery planning environment developed within TLEMsafe project [1] (funded by the European Commission FP7). It enables the operator to plan a surgical procedure on the customized musculoskeletal (MS) model of the patient's lower limbs, send the modified model to the biomechanical analysis module, and export the scenario's parameters to the surgical navigation system. The personalized patient-specific three-dimensional (3-D) MS model is registered with 3-D MRI dataset of lower limbs and the two modalities may be visualized simultaneously. Apart from main planes, any arbitrary MRI cross-section can be rendered on the 3-D MS model in real time. The interface provides tools for: bone cutting, manipulating and removal, repositioning muscle insertion points, modifying muscle force, removing muscles and placing implants stored in the implant library. SPE3D supports stereoscopic viewing as well as natural inspection/manipulation with use of haptic devices. Alternatively, it may be controlled with use of a standard computer keyboard, mouse and 2D display or a touch screen (e.g. in an operating room). The interface may be utilized in two main fields. Experienced surgeons may use it to simulate their operative plans and prepare input data for a surgical navigation system while student or novice surgeons can use it for training.

  11. Design and test status for life support applications of SPE oxygen generation systems. [Solid Polymer Electrolyte

    NASA Technical Reports Server (NTRS)

    Titterington, W. A.; Erickson, A. C.

    1975-01-01

    An advanced six-man rated oxygen generation system has been fabricated and tested as part of a NASA/JSC technology development program for a long lived, manned spacecraft life support system. Details of the design and tests results are presented. The system is based on the Solid Polymer Electrolyte (SPE) water electrolysis technology and its nominal operating conditions are 2760 kN/sq m (400 psia) and 355 K (180 F) with an electrolysis module current density capability up to 350 mA/sq cm (326 ASF). The system is centered on a 13-cell SPE water electrolysis module having a single cell active area of 214 sq cm (33 sq in) and it incorporates instrumentation and controls for single pushbutton automatic startup/shutdown, component fault detection and isolation, and self-contained sensors and controls for automatic safe emergency shutdown. The system has been tested in both the orbital cyclic and continuous mode of operation. Various parametric tests have been completed to define the system capability for potential application in spacecraft environmental systems.

  12. Efficient conversion of myricetin from Ampelopsis grossedentata extracts and its purification by MIP-SPE.

    PubMed

    Zhong, Shian; Kong, Yanyue; Zhou, Ling; Zhou, Chengyun; Zhang, Xiaona; Wang, Yan

    2014-01-15

    In this study, we developed an efficient conversion process of dihydromyricetin to myricetin from Ampelopsis grossedentata extracts. The content of myricetin increased from 2.38% to 85.57%, demonstrating the successful dehydrogenation of dihydromyricetin. Molecularly imprinted polymers (MIPs) were prepared by surface imprinting method using silica microspheres as the support matrices and myricetin as template. The MIPs were applied for the selective adsorption of myricetin. The chemical structure of the MIPs was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Static, dynamic and selective adsorption experiments showed that the MIPs exhibited good adsorption ability, rather fast template rebinding kinetics, and appreciate selectivity over structurally related compounds. Accordingly, the MIPs were applied as the selective sorbent in SPE to purify myricetin obtained through dehydrogenation, followed by HPLC-UV analysis. The recoveries of myricetin and dihydromyricetin were 92.7% and 55.6%, respectively. This study demonstrates the feasibility of using the developed MIP-SPE method to purify and enrich myricetin in the natural products. PMID:24321759

  13. Using SPE-LC-ESI-MS/MS Analysis to Assess Disperse Dyes in Environmental Water Samples.

    PubMed

    Zocolo, Guilherme Julião; Pilon dos Santos, Glauco; Vendemiatti, Josiane; Vacchi, Francine Inforçato; Umbuzeiro, Gisela de Aragão; Zanoni, Maria Valnice Boldrin

    2015-09-01

    We have optimized an SPE-LC-ESI-MS/MS method and used it to monitor disperse azo dyes in environmental aquatic samples. Calibration curves constructed for nine disperse dyes-Red 1, Violet 93, Blue 373, Orange 1, Orange 3, Orange 25, Yellow 3, Yellow 7 and Red 13-in aqueous solution presented good linearity between 2.0 and 100.0 ng mL(-1). The method provided limits of detection and quantification around 2.0 and 8.0 ng L(-1), respectively. For dyes at concentrations of 25.0 ng mL(-1), the intra- and interday analyses afforded relative standard deviation lower than 6 and 13%, respectively. The recovery values obtained for each target analyte in Milli-Q water, receiving waters and treated water samples spiked with the nine studied dyes at concentrations of 8.0, 25.0 and 50.0 ng L(-1) (n = 3) gave average recoveries greater than 70%, with RSD <20%. Statistical evaluation aided method validation. The validated method proved to be useful for analysis of organic extracts from effluents and receiving water samples after an SPE extraction step. More specifically, the method enabled detection of the dyes Disperse Red 1, Disperse Blue 373 and Disperse Violet 93 at concentrations ranging from 84 to 3452 ng L(-1) in the treated effluent (TE), affluent and points collected upstream and downstream of the drinking water treatment plant of a textile dye industry in Brazil. PMID:25637135

  14. Simple SPE-GC method for anethole determination in human serum.

    PubMed

    Dawidowicz, Andrzej L; Dybowski, Michal P

    2014-02-01

    Recently, much attention has been given to congener analysis, which can be used to check the possibility of postoffence drinking claims in forensic toxicology. In this type of analysis, the information given by the defendant regarding the type, quantity, and time of consumption of a specific alcoholic beverage is used to calculate theoretically expected congener concentration in the blood and this is compared with the analytically determined concentrations in the blood sample. Many alcoholic drinks aromatized with essential oils of plants and fruits contain a specific congener, for example, anethole in aniseed drinks. The present study describes the GC procedure of anethole analysis in human plasma using SPE as the sample preparation method. The procedure involves the protein precipitation process, which generally degrades the protein-analyte complex, and SPE isolation of anethole from the examined materials. This analytical approach is proposed as a method of choice for the estimation of anethole concentration in human fluids after the consumption of alcoholic beverages and other foods containing the substance. The described method is characterized by a low LOD (8.33 ng/g) and a very high recovery (average recovery 98.37%) of the analyte. PMID:24302657

  15. Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides.

    PubMed

    Selman, Maurice H J; Hemayatkar, Mahdi; Deelder, André M; Wuhrer, Manfred

    2011-04-01

    Solid-phase extraction microtips are important devices in modern bioanalytics, as they allow miniaturized sample preparation for mass spectrometric analysis. Here we introduce the use of cotton wool for the preparation of filter-free HILIC SPE microtips. To this end, pieces of cotton wool pads (approximately 500 μg) were packed into 10 μL pipet tips. The performance of the tips was evaluated for microscale purification of tryptic IgG Fc N-glycopeptides. Cotton wool HILIC SPE microtips allowed the removal of salts, most nonglycosylated peptides, and detergents such as SDS from glycoconjugate samples. MALDI-TOF-MS glycopeptide profiles were very repeatable with different tips as well as reused tips, and very similar profiles were obtained with different brands of cotton wool pads. In addition, we used cotton HILIC microtips to purify N-glycans after N-glycosidase F treatment of IgG and transferrin followed by MALDI-TOF-MS detection. In conclusion, we establish cotton wool microtips for glycan and glycopeptide purification with subsequent mass spectrometric detection. PMID:21366235

  16. Role of CsrR, Hyaluronic Acid, and SpeB in the Internalization of Streptococcus pyogenes M Type 3 Strain by Epithelial Cells

    PubMed Central

    Jadoun, Jeries; Eyal, Osnat; Sela, Shlomo

    2002-01-01

    Internalization of group A streptococcus by human epithelial cells has been extensively studied during the past 6 years. It is now clear that multiple mechanisms are involved in this process. We have previously demonstrated that the CsrR global regulator controls the internalization of an invasive M type 3 strain through regulation of the has (hyaluronic acid synthesis) operon, as well as another, unknown gene(s). Recently, it was reported that the CsrR-regulated cysteine protease (SpeB) is also involved in bacterial uptake. In this study we have examined the roles of CsrR, hyaluronic acid capsule, and SpeB in streptococcal internalization. We have constructed isogenic mutants of the M3 serotype deficient in the csrR, hasA, and speB genes and tested their ability to be internalized by HEp-2 epithelial cells. Inactivation of csrR abolished internalization, while inactivation of either hasA or speB increased the internalization efficiency. Mutation in csrR derepressed hasA transcription and lowered the activity of SpeB, while no effect on speB transcription was observed. The speB mutant expressed smaller amounts of capsule, while the hasA mutant transcribed more csrR and speB mRNAs. Thus, it seems that complex interactions between CsrR, SpeB, and capsule are involved in modulation of group A streptococcus internalization. PMID:11796571

  17. Role of CsrR, hyaluronic acid, and SpeB in the internalization of Streptococcus pyogenes M type 3 strain by epithelial cells.

    PubMed

    Jadoun, Jeries; Eyal, Osnat; Sela, Shlomo

    2002-02-01

    Internalization of group A streptococcus by human epithelial cells has been extensively studied during the past 6 years. It is now clear that multiple mechanisms are involved in this process. We have previously demonstrated that the CsrR global regulator controls the internalization of an invasive M type 3 strain through regulation of the has (hyaluronic acid synthesis) operon, as well as another, unknown gene(s). Recently, it was reported that the CsrR-regulated cysteine protease (SpeB) is also involved in bacterial uptake. In this study we have examined the roles of CsrR, hyaluronic acid capsule, and SpeB in streptococcal internalization. We have constructed isogenic mutants of the M3 serotype deficient in the csrR, hasA, and speB genes and tested their ability to be internalized by HEp-2 epithelial cells. Inactivation of csrR abolished internalization, while inactivation of either hasA or speB increased the internalization efficiency. Mutation in csrR derepressed hasA transcription and lowered the activity of SpeB, while no effect on speB transcription was observed. The speB mutant expressed smaller amounts of capsule, while the hasA mutant transcribed more csrR and speB mRNAs. Thus, it seems that complex interactions between CsrR, SpeB, and capsule are involved in modulation of group A streptococcus internalization. PMID:11796571

  18. Serum Bile Acids Are Associated with Pathological Progression of Hepatitis B-Induced Cirrhosis.

    PubMed

    Wang, Xiaoning; Xie, Guoxiang; Zhao, Aihua; Zheng, Xiaojiao; Huang, Fengjie; Wang, Yixing; Yao, Chun; Jia, Wei; Liu, Ping

    2016-04-01

    Recent metabonomic studies have identified an important role of bile acids in patients with liver cirrhosis. Serum bile acids, such as glycocholate (GCA), glycochenodeoxycholate (GCDCA), taurocholate (TCA), and taurochenodeoxycholate (TCDCA), increased significantly in liver cirrhosis patients. Our recently published urinary metabonomic study showed that glycocholate 3-glucuronide, taurohyocholate, TCA, glycolithocholate 3-sulfate, and glycoursodeoxycholate (GUDCA) were markedly increased in hepatitis B-induced cirrhotic patients (n = 63) compared with healthy controls (n = 31). The urinary levels of GUDCA were able to differentiate among three stages of cirrhotic patients with Child-Pugh (CP) score A, B, and C. In this study, we recruited two new cohorts of patients with hepatitis-B-induced cirrhosis and healthy control subjects and quantitatively profiled their serum bile acids using ultra-performance liquid chromatography triple quadrupole mass spectrometry. Serum bile acid profile and corresponding differential bile acids were characterized, in addition to the blood routine, liver, and renal function tests. The alterations of bile acids contributing to the intergroup variation between healthy controls and cirrhotic patients and among pathological stages of CP grade A, B and C were also investigated. Five bile acids, GCA, GCDCA, TCA, TCDCA, and GUDCA, were significantly altered among different stages of liver cirrhosis (n = 85), which was validated with an independent cohort of cirrhotic patients (n = 53). Our results show that dynamic alteration of serum bile acids is indicative of an exacerbated liver function, highlighting their potential as biomarkers for staging the liver cirrhosis and monitoring its progression. PMID:25964117

  19. Cassia tora Linn Cream Inhibits Ultraviolet-B-Induced Psoriasis in Rats

    PubMed Central

    Singhal, Manmohan; Kansara, Niraj

    2012-01-01

    The aim of present study was to determine the antipsoriatic activity of newly formulated O/W creams of methanolic extract of Cassia tora L. leaves by using ultraviolet-B-induced psoriasis in rat. The plant Cassia tora L. is traditionally claimed to be useful in the treatment of a number of skin diseases. However, there are no established scientific reports for its antipsoriatic activity. Methanolic Cassia tora L. leaves extract was used to prepare various concentrations of O/W creams and tested for acute dermal toxicity study. The different O/W creams showed good physical characteristics and passed the sensitivity, irritation, grittiness and bleeding test. The results of acute dermal toxicity showed that the creams were safe up to the dose of 2000 mg/kg. In case of psoriasis model, histopathological analysis revealed that there were absence of Munro's microabscess, elongation of rete ridges, and capillary loop dilation in the section in Test 2 (0.1%) and standard group. O/W creams and methanolic extract of Cassia tora L. leaves exhibited significant reduction in percentage of relative epidermal thickness and spleen index as compared to positive control. We concluded that topical O/W creams and crude extract containing methanolic extract of Cassia tora L. leaves have potent antipsoriatic activity in ultraviolet-B-induced psoriasis in rat. PMID:22536527

  20. Galangin (3,5,7-Trihydroxyflavone) Shields Human Keratinocytes from Ultraviolet B-Induced Oxidative Stress

    PubMed Central

    Madduma Hewage, Susara Ruwan Kumara; Piao, Mei Jing; Kim, Ki Cheon; Cha, Ji Won; Han, Xia; Choi, Yung Hyun; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Most skin damage caused by ultraviolet B (UVB) radiation is owing to the generation of reactive oxygen species. Phytochemicals can act as antioxidants against UVB-induced oxidative stress. This study investigated the protective effects of the flavone galangin against UVB-induced oxidative damage in human keratinocytes. Galangin efficiently scavenged free radicals and reduced UVB-induced damage to cellular macromolecules, such as DNA, lipids, and proteins. Furthermore, galangin rescued cells undergoing apoptosis induced by UVB radiation via recovering mitochondrial polarization and down-regulating apoptotic proteins. These results showed that galangin protects human keratinocytes against UVB radiation-induced cellular damage and apoptosis via its antioxidant effects. PMID:25767685

  1. The Source Physics Experiments (SPE) at the Nevada National Security Site (NNSS): An Overview

    NASA Astrophysics Data System (ADS)

    Snelson, C. M.; Chipman, V.; White, R. L.; Emmitt, R.; Townsend, M.; Barker, D.; Lee, P.

    2012-12-01

    Understanding the changes in seismic energy as it travels from the near field to the far field is the ultimate goal in monitoring for explosive events of interest. This requires a clear understanding of explosion phenomenology as it relates to seismic, infrasound, and acoustic signals. Although there has been much progress in modeling these phenomena, this has been primarily based in the empirical realm. As a result, the logical next step in advancing the seismic monitoring capability of the United States is to conduct field tests that can expand the predictive capability of the physics-based modeling currently under development. The Source Physics Experiment at the Nevada National Security Site (SPE) is the first step in this endeavor to link the empirically based with the physics-based modeling. This is a collaborative project between National Security Technologies (NSTec), Lawrence Livermore National Laboratory (LLNL), Los Alamos National Laboratory (LANL), Sandia National Laboratories (SNL), the Defense Threat Reduction Agency (DTRA), and the Air Force Technical Applications Center (AFTAC). The test series require both the simple and complex cases to fully characterize the problem, which is to understand the transition of seismic energy from the near field to the far field; to understand the development of S-waves in explosives sources; and how anisotropy controls seismic energy transmission and partitioning. The current series is being conducted in a granite body called the Climax Stock. This location was chosen for several reasons, including the fairly homogenous granite; the location of previous nuclear tests in the same rock body; and generally the geology has been well characterized. The simple geology series is planned for 7 shots using conventional explosives in the same shot hole surrounded by Continuous Reflectometry for Radius vs. Time Experiment (CORRTEX), Time of Arrival (TOA), Velocity of Detonation (VOD), down-hole accelerometers, surface

  2. Near-infrared spectral monitoring of Pluto/Charon with IRTF/SpeX

    NASA Astrophysics Data System (ADS)

    Grundy, W. M.; Buie, M. W.; Spencer, J. R.; Young, L. A.; Young, E. F.

    2003-05-01

    The non-uniform distribution of Pluto's surface ices, along with the planet's high obliquity, 6 day rotation period, and 250 year orbital period combine to produce variations in Pluto's reflectance spectrum over a broad range of times scales. Volatile transport can also influence the spectrum over time, as ices sublimate and re-condense elsewhere on Pluto's surface. In an effort to detect and to distinguish between the various factors influencing Pluto's spectral reflectance over time, we have been monitoring the system spectroscopically over the past 2 decades. The SpeX instrument, commissioned in 2000 at NASA's IRTF, is an ideal tool for this purpose. Using it, we have obtained good quality spectra of the Pluto\\slash Charon system on 15 nights since 2000, covering the 0.8 to 2.4 μ m wavelength range at spectral resolutions ˜1000\

  3. Utilising copper screen-printed electrodes (CuSPE) for the electroanalytical sensing of sulfide.

    PubMed

    Thakur, Bhawana; Bernalte, Elena; Smith, Jamie P; Foster, Christopher W; Linton, Patricia E; Sawant, Shilpa N; Banks, Craig E

    2016-02-21

    A mediatorless sulfide electrochemical sensing platform utilising a novel nanocopper-oxide screen-printed electrodes (CuSPE) is reported for the first time. The state-of-the-art screen-printed electrochemical sensors demonstrate their capability to quantify sulfide within both the presence and absence of an array of interferents with good levels of sensitivity and repeatability. The direct sensing (using linear sweep voltammetry) of sulfide utilising the CuSPEs provides a mediatorless approach for the detection of sulfide, yielding useful analytical signatures that can be successfully quantified. The proposed novel protocol using the CuSPEs is successfully applied to the sensing of sulfide within drinking water exhibiting a high level of recovery. PMID:26815001

  4. An Overview of the Source Physics Experiment at the Nevada National Security Site (SPE-N)

    SciTech Connect

    Snelson, C. M., Chipman, V. D., White, R. L., Emmitt, R. F., Townsend, M. J., Barker, D., Lee, P.

    2012-07-11

    Understanding the changes in seismic energy as it travels from the near field to the far field is the ultimate goal in monitoring for explosive events of interest. This requires a clear understanding of explosion phenomenology as it relates to seismic, infrasound, and acoustic signals. Although there has been much progress in modeling these phenomena, this has been primarily based in the empirical realm. As a result, the logical next step in advancing the seismic monitoring capability of the United States is to conduct field tests that can expand the predictive capability of the physics-based modeling currently under development. The Source Physics Experiment at the Nevada National Security Site (SPE-N) is the first step in this endeavor to link the empirically based with the physics-based modeling. This is a collaborative project between National Security Technologies (NSTec), Lawrence Livermore National Laboratory (LLNL), Los Alamos National Laboratory (LANL), Sandia National Laboratories (SNL), the Defense Threat Reduction Agency (DTRA), and the Air Force Technical Applications Center (AFTAC). The test series require both the simple and complex cases to fully characterize the problem, which is to understand the transition of seismic energy from the near field to the far field; to understand the development of S-waves in explosives sources; and how anisotropy controls seismic energy transmission and partitioning. The current series is being conducted in a granite body called the Climax Stock. This location was chosen for several reasons, including the fairly homogenous granite; the location of previous nuclear tests in the same rock body; and generally the geology has been well characterized. The simple geology series is planned for 7 shots using conventional explosives in the same shot hole surrounded by Continuous Reflectometry for Radius vs. Time Experiment (CORRTEX), Time of Arrival (TOA), Velocity of Detonation (VOD), down-hole accelerometers, surface

  5. Optimization of an SPE and GC/MS Method for Analyzing Endocrine Disrupting Compounds in Water

    NASA Astrophysics Data System (ADS)

    Thomas, S. M.; Bodour, A.; Murray, K. E.

    2006-12-01

    Endocrine disrupting compounds (EDCs) are compounds that interrupt hormonal functions in the body. The literature reports the presence of EDCs in all environmental matrices (air, water and soil) at concentrations of at least 1 nanogram/liter (ng/l), which may be high enough to induce adverse health effects. Therefore, reliable analytical methods for detecting trace amounts of EDCs in water is very important for investigating and controlling their concentrations in the environment. This study investigated a method for analyzing four known or suspected EDCs (chlorpyrifos, musk HHCB, diethyl phthalate, and butylated hydroxyanisole) in water samples. The analytical method was based on the USGS wastewater method developed by Zaugg et al. (2001), but modified, using solid phase extraction (SPE) followed by gas chromatography and mass spectrometry (GC/MS) analysis. The EDCs were extracted using 60mg Water Oasis hydrophilic-lipophillic balance (HLB) extraction cartridges. The SPE efficiency was investigated by using different initial extraction volumes and different EDC concentrations. The lowest concentration was 1ng/l and the lowest extraction volume was 100mL. Results of the study indicate that the initial oven temperature conditions and rate of temperature increases affects the peak signal to noise ratio and the sample run-time in the GC/MS. An increase in gas flow rate did not show any significant changes and hence was maintained at 1ml/min. Preliminary data suggests that the percent recovery of the compounds obtained using this method either met or exceeded those presented by Zaugg et al. (2001) as the USGS wastewater method.

  6. Full Waveform Inversion Methods for Source and Media Characterization before and after SPE5

    NASA Astrophysics Data System (ADS)

    Phillips-Alonge, K. E.; Knox, H. A.; Ober, C.; Abbott, R. E.

    2015-12-01

    The Source Physics Experiment (SPE) was designed to advance our understanding of explosion-source phenomenology and subsequent wave propagation through the development of innovative physics-based models. Ultimately, these models will be used for characterizing explosions, which can occur with a variety of yields, depths of burial, and in complex media. To accomplish this, controlled chemical explosions were conducted in a granite outcrop at the Nevada Nuclear Security Test Site. These explosions were monitored with extensive seismic and infrasound instrumentation both in the near and far-field. Utilizing this data, we calculate predictions before the explosions occur and iteratively improve our models after each explosion. Specifically, we use an adjoint-based full waveform inversion code that employs discontinuous Galerkin techniques to predict waveforms at station locations prior to the fifth explosion in the series (SPE5). The full-waveform inversions are performed using a realistic geophysical model based on local 3D tomography and inversions for media properties using previous shot data. The code has capabilities such as unstructured meshes that align with material interfaces, local polynomial refinement, and support for various physics and methods for implicit and explicit time-integration. The inversion results we show here evaluate these different techniques, which allows for model fidelity assessment (acoustic versus elastic versus anelastic, etc.). In addition, the accuracy and efficiency of several time-integration methods can be determined. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  7. Insect ornithine decarboxylase (ODC) complements SPE1 knock-out of yeast Saccharomyces cerevisiae.

    PubMed

    Choi, Soon-Yong; Park, Hee Yun; Paek, Aron; Kim, Gil Seob; Jeong, Seong Eun

    2009-12-31

    Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth, differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. Mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%, respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of > 4% of the wild-type strain in polyaminefree media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore, its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system. PMID:19937472

  8. Signal processing to evaluate parameters affecting SPE for multi-residue analysis of personal care products.

    PubMed

    Pietrogrande, Maria Chiara; Basaglia, Giulia; Dondi, Francesco

    2009-05-01

    This paper discusses the development of a comprehensive method for the simultaneous analysis of personal care products (PCPs) based on SPE and GC-MS. The method was developed on 29 target compounds to represent PCPs belonging to different chemical classes: surfactants in detergents (alkyl benzenes), fragrances in cosmetics (nitro and polycyclic musks), antioxidants and preservatives (phenols), plasticizers (phthalates) displaying a wide range of volatility, polarity, water solubility. In addition to the conventional C(18) stationary phase, a surface modified styrene divinylbenzene polymeric phase (Strata X SPE cartridge) has been investigated as suitable for the simultaneous extraction of several PCPs with polar and non-polar characteristics. For both sorbents different solvent compositions and eluting conditions were tested and compared in order to achieve high extraction efficiency for as many sample components as possible. Comparison of the behavior of the two cartridges reveals that, overall, Strata-X provides better efficiency with extraction recovery higher than 70% for most of the PCPs investigated. The best results were obtained under the following operative conditions: an evaporation temperature of 40 degrees C, elution on Strata-X cartridge using a volume of 15 mL of ethyl acetate (EA) as solvent and operating with slow flow rate (-10 KPa). In addition to the conventional method based on peak integration, a chemometric approach based on the computation of the experimental autocovariance function (EACVF(tot)) was applied to the complex GC-MS signal: the percentage recovery and information on peak abundance distribution can be evaluated for each procedure step. The PC-based signal processing proved very helpful in assisting the development of the analytical procedure, since it saves labor and time and increases result reliability in handling GC complex signals. PMID:19399858

  9. Application of Nanofiber-packed SPE for Determination of Urinary 1-Hydroxypyrene Level Using HPLC

    PubMed Central

    Ifegwu, Okechukwu Clinton; Anyakora, Chimezie; Chigome, Samuel; Torto, Nelson

    2014-01-01

    It is always desirable to achieve maximum sample clean-up, extraction, and pre-concentration with the minimum possible organic solvent. The miniaturization of sample preparation devices was successfully demonstrated by packing 10 mg of 11 electrospun polymer nanofibers into pipette tip micro column and mini disc cartridges for efficient pre-concentration of 1-hydroxypyrene in urine samples. 1-hydroxypyrene is an extensively studied biomarker of the largest class of chemical carcinogens. Excretory 1-hydroxypyrene was monitored with HPLC/fluorescence detector. Important parameters influencing the percentage recovery such as fiber diameter, fiber packing amount, eluent, fiber packing format, eluent volume, surface area, porosity, and breakthrough parameters were thoroughly studied and optimized. Under optimized condition, there was a near perfect linearity of response in the range of 1–1000 μg/L with a coefficient of determination (r2) between 0.9992 and 0.9999 and precision (% RSD) ≤7.64% (n = 6) for all the analysis (10, 25, and 50 μg/L). The Limit of detection (LOD) was between 0.022 and 0.15 μg/L. When compared to the batch studies, both disc packed nanofiber sorbents and pipette tip packed sorbents exhibited evident dominance based on their efficiencies. The experimental results showed comparable absolute recoveries for the mini disc packed fibers (84% for Nylon 6) and micro columns (80% for Nylon 6), although the disc displayed slightly higher recoveries possibly due to the exposure of the analyte to a larger reacting surface. The results also showed highly comparative extraction efficiencies between the nanofibers and conventional C-18 SPE sorbent. Nevertheless, miniaturized SPE devices simplified sample preparation, reducing back pressure, time of the analysis with acceptable reliability, selectivity, detection levels, and environmental friendliness, hence promoting green chemistry. PMID:24812483

  10. Coupling hydrodynamic and wave propagation modeling for waveform modeling of SPE.

    NASA Astrophysics Data System (ADS)

    Larmat, C. S.; Steedman, D. W.; Rougier, E.; Delorey, A.; Bradley, C. R.

    2015-12-01

    The goal of the Source Physics Experiment (SPE) is to bring empirical and theoretical advances to the problem of detection and identification of underground nuclear explosions. This paper presents effort to improve knowledge of the processes that affect seismic wave propagation from the hydrodynamic/plastic source region to the elastic/anelastic far field thanks to numerical modeling. The challenge is to couple the prompt processes that take place in the near source region to the ones taking place later in time due to wave propagation in complex 3D geologic environments. In this paper, we report on results of first-principles simulations coupling hydrodynamic simulation codes (Abaqus and CASH), with a 3D full waveform propagation code, SPECFEM3D. Abaqus and CASH model the shocked, hydrodynamic region via equations of state for the explosive, borehole stemming and jointed/weathered granite. LANL has been recently employing a Coupled Euler-Lagrange (CEL) modeling capability. This has allowed the testing of a new phenomenological model for modeling stored shear energy in jointed material. This unique modeling capability has enabled highfidelity modeling of the explosive, the weak grout-filled borehole, as well as the surrounding jointed rock. SPECFEM3D is based on the Spectral Element Method, a direct numerical method for full waveform modeling with mathematical accuracy (e.g. Komatitsch, 1998, 2002) thanks to its use of the weak formulation of the wave equation and of high-order polynomial functions. The coupling interface is a series of grid points of the SEM mesh situated at the edge of the hydrodynamic code domain. Displacement time series at these points are computed from output of CASH or Abaqus (by interpolation if needed) and fed into the time marching scheme of SPECFEM3D. We will present validation tests and waveforms modeled for several SPE tests conducted so far, with a special focus on effect of the local topography.

  11. A Naturally Occurring Mutation in ropB Suppresses SpeB Expression and Reduces M1T1 Group A Streptococcal Systemic Virulence

    PubMed Central

    Hollands, Andrew; Aziz, Ramy K.; Kansal, Rita; Kotb, Malak; Nizet, Victor; Walker, Mark J.

    2008-01-01

    Epidemiological studies of group A streptococcus (GAS) have noted an inverse relationship between SpeB expression and invasive disease. However, the role of SpeB in the course of infection is still unclear. In this study we utilize a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on hemolysis of RBC's, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS. PMID:19116661

  12. A naturally occurring mutation in ropB suppresses SpeB expression and reduces M1T1 group A streptococcal systemic virulence.

    PubMed

    Hollands, Andrew; Aziz, Ramy K; Kansal, Rita; Kotb, Malak; Nizet, Victor; Walker, Mark J

    2008-01-01

    Epidemiological studies of group A streptococcus (GAS) have noted an inverse relationship between SpeB expression and invasive disease. However, the role of SpeB in the course of infection is still unclear. In this study we utilize a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on hemolysis of RBC's, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS. PMID:19116661

  13. Apoptosis in Anthracycline Cardiomyopathy

    PubMed Central

    Shi, Jianjian; Abdelwahid, Eltyeb; Wei, Lei

    2011-01-01

    Apoptosis is a tightly regulated physiologic process of programmed cell death that occurs in both normal and pathologic tissues. Numerous in vitro or in vivo studies have indicated that cardiomyocyte death through apoptosis and necrosis is a primary contributor to the progression of anthracycline-induced cardiomyopathy. There are now several pieces of evidence to suggest that activation of intrinsic and extrinsic apoptotic pathways contribute to anthracycline-induced apoptosis in the heart. Novel strategies were developed to address a wide variety of cardiotoxic mechanisms and apoptotic pathways by which anthracycline influences cardiac structure and function. Anthracycline-induced apoptosis provides a very valid representation of cardiotoxicity in the heart, an argument which has implications for the most appropriate animal models of damaged heart plus diverse pharmacological effects. In this review we describe various aspects of the current understanding of apoptotic cell death triggered by anthracycline. Differences in the sensitivity to anthracycline-induced apoptosis between young and adult hearts are also discussed. PMID:22212952

  14. Role of AtMSH7 in UV-B-induced DNA damage recognition and recombination.

    PubMed

    Lario, Luciana Daniela; Botta, Pablo; Casati, Paula; Spampinato, Claudia Patricia

    2015-06-01

    The mismatch repair (MMR) system maintains genome integrity by correcting replication-associated errors and inhibiting recombination between divergent DNA sequences. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in this DNA repair system vary among organisms. Plants have an extra mismatch recognition protein, MutSγ, which is a heterodimer: MSH2-MSH7. To further understand the role of MSH7 in vivo, we present data from this protein in Arabidopsis thaliana. First, we generated transgenic plants that express β-glucuronidase (GUS) under the control of the MSH7 promoter. Histochemical staining of the transgenic plants indicated that MSH7 is preferentially expressed in proliferating tissues. Then, we identified msh7 T-DNA insertion mutants. Plants deficient in MSH7 show increased levels of UV-B-induced cyclobutane pyrimidine dimers relative to wild-type (WT) plants. Consistent with the patterns of MSH7 expression, we next analysed the role of the protein during somatic and meiotic recombination. The frequency of somatic recombination between homologous or homeologous repeats (divergence level of 1.6%) was monitored using a previously described GUS recombination reporter assay. Disruption of MSH7 has no effect on the rates of somatic homologous or homeologous recombination under control conditions or after UV-B exposure. However, the rate of meiotic recombination between two genetically linked seed-specific fluorescent markers was 97% higher in msh7 than in WT plants. Taken together, these results suggest that MSH7 is involved in UV-B-induced DNA damage recognition and in controlling meiotic recombination. PMID:25465032

  15. Spaceflight Associated Apoptosis

    NASA Technical Reports Server (NTRS)

    Ichiki, Albert T.; Gibson, Linda A.; Allebban, Zuhair

    1996-01-01

    Lymphoid tissues have been shown to atrophy in rats flown on Russian spaceflights. Histological examination indicated evidence for cell degradation. Lymphoid tissues from rats flown on Spacelab Life Sciences-2 mission were analyzed for apoptosis by evidence of fragmented lymphocytes, which could be engulfed by macrophages, or DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Apoptosis was not detected in the thymus and spleen collected inflight or from the synchronous ground rats but was detected in the thymus, spleen and inguinal lymph node of the flight animals on recovery. These results indicate that the apoptosis observed in the lymphatic tissues of the rats on recovery could have been induced by the gravitational stress of reentry, corroborating the findings from the early space-flight observations.

  16. Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes.

    PubMed

    Neely, Melody N; Lyon, William R; Runft, Donna L; Caparon, Michael

    2003-09-01

    The Rgg family of transcription regulators is widely distributed among gram-positive bacteria; however, how the members of this family control transcription is poorly understood. In the pathogen Streptococcus pyogenes, the Rgg family member RopB is required for transcription of the gene that encodes the secreted SpeB cysteine protease. Expression of the protease follows distinct kinetics that involves control of transcription in response to the growth phase. In this study, the contribution of RopB to growth phase control was examined. The gene encoding the protease (speB) and ropB are transcribed divergently from a 940-bp intergenic region. Primer extension analyses, in conjunction with reporter fusion studies, revealed that the major region controlling the transcription of both speB and ropB is adjacent to ropB and that the promoters for the two genes likely overlap. Furthermore, it was found that RopB is a DNA-binding protein that specifically binds to sequences in this control region. The interrelationship between ropB and speB expression was further reflected in the observation that transcription of ropB itself is subject to growth phase control. However, while expression of ropB from a promoter expressed during the early logarithmic phase of growth could complement a ropB deletion mutant, ectopic expression of ropB did not uncouple the expression of speB from its growth phase signal. These data implicate other factors in growth phase control and suggest that regulation of ropB expression itself is not the central mechanism of control. PMID:12923089

  17. Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation

    PubMed Central

    Ender, Miriam; Andreoni, Federica; Zinkernagel, Annelies Sophie; Schuepbach, Reto Andreas

    2013-01-01

    Background The family of 4 related protease-activated receptors (PAR-1, 2, 3 & 4) expressed by mammalian cells allow to sense for and react to extracellular proteolytic activity. Since major human bacterial pathogens secret a wide array of protease(-s) we investigated whether they interfere with human PAR function. Methodology/Principal Findings Supernatants from cultures of major human bacterial pathogens were assayed for the presence of protease(-s) capable to cleave overexpressed human PAR-1, 2, 3 and 4 reporter constructs. Group A streptococcus (GAS) was found to secret a PAR-1-cleaving protease. Experiments involving genetical and pharmacological gain and loss of function identified streptococcal pyrogenic exotoxin B SpeB as the protease responsible. On the host’s side analysis of overexpressed PAR-1 carrying alanine substitutions and deletions showed the amino acid residue leucine44 on PAR-1’s extracellular N-terminus to be the only cleavage site. Complementary studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin. Conclusions/Significance Our results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine44 SpeB rendered endothelial cells unresponsive to thrombin and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination. PMID

  18. Role of RopB in Growth Phase Expression of the SpeB Cysteine Protease of Streptococcus pyogenes

    PubMed Central

    Neely, Melody N.; Lyon, William R.; Runft, Donna L.; Caparon, Michael

    2003-01-01

    The Rgg family of transcription regulators is widely distributed among gram-positive bacteria; however, how the members of this family control transcription is poorly understood. In the pathogen Streptococcus pyogenes, the Rgg family member RopB is required for transcription of the gene that encodes the secreted SpeB cysteine protease. Expression of the protease follows distinct kinetics that involves control of transcription in response to the growth phase. In this study, the contribution of RopB to growth phase control was examined. The gene encoding the protease (speB) and ropB are transcribed divergently from a 940-bp intergenic region. Primer extension analyses, in conjunction with reporter fusion studies, revealed that the major region controlling the transcription of both speB and ropB is adjacent to ropB and that the promoters for the two genes likely overlap. Furthermore, it was found that RopB is a DNA-binding protein that specifically binds to sequences in this control region. The interrelationship between ropB and speB expression was further reflected in the observation that transcription of ropB itself is subject to growth phase control. However, while expression of ropB from a promoter expressed during the early logarithmic phase of growth could complement a ropB deletion mutant, ectopic expression of ropB did not uncouple the expression of speB from its growth phase signal. These data implicate other factors in growth phase control and suggest that regulation of ropB expression itself is not the central mechanism of control. PMID:12923089

  19. [Sphingolipid and apoptosis].

    PubMed

    Wang, Jing; Hu, Xiao-Song; Shi, Jie-Ping

    2003-07-01

    Over the last decade, considerable progress has been made in the study of sphingolipids with the development of biological techniques. Sphingolipids play important roles in diverse physiological process, including cytoskeleton migration, angiogenesis, embryonic development and signal transduction. Except for this, the lastest evidence has suggested that sphingolipids and their metabolite (ceramide, sphingosine, sphingosine 1-phosphate) can induce apoptosis in a wide variety of tumor cell lines such as LoVo HT29, Bel7402, A549, CNE2 cells. This paper is attempted to review the recent advances of investigation into the relationship between sphingolipids and apoptosis. PMID:14628466

  20. [Apoptosis during embryo development].

    PubMed

    Jezek, Davor; Kozina, Viviana

    2009-10-01

    The development of human embryo includes two essential processes, i.e., rapid mitotic activity of cells and gradual differentiation of tissues and organs. The latter process is very often characterized by extensive migration of cells from their site of origin to the site of definitive location, inductive action of the neighboring germ layers and programmed cell death (apoptosis). This paper describes examples of proliferative and apoptotic processes during the development of human embryo. The development of trilaminar germ disk, skin, gonads, central and peripheral nerve system as well as limbs provides instructive examples of how apoptosis regulates the development and differentiation of cells. PMID:19999545

  1. Granzyme B-activated p53 interacts with Bcl-2 to promote cytotoxic lymphocyte-mediated apoptosis.

    PubMed

    Ben Safta, Thouraya; Ziani, Linda; Favre, Loetitia; Lamendour, Lucille; Gros, Gwendoline; Mami-Chouaib, Fathia; Martinvalet, Denis; Chouaib, Salem; Thiery, Jerome

    2015-01-01

    Granzyme B (GzmB) plays a major role in CTLs and NK cell-mediated elimination of virus-infected cells and tumors. Human GzmB preferentially induces target cell apoptosis by cleaving the proapoptotic Bcl-2 family member Bid, which, together with Bax, induces mitochondrial outer membrane permeabilization. We previously showed that GzmB also induces a rapid accumulation of the tumor-suppressor protein p53 within target cells, which seems to be involved in GzmB-induced apoptosis. In this article, we show that GzmB-activated p53 accumulates on target cell mitochondria and interacts with Bcl-2. This interaction prevents Bcl-2 inhibitory effect on both Bax and GzmB-truncated Bid, and promotes GzmB-induced mitochondrial outer membrane permeabilization. Consequently, blocking p53-Bcl-2 interaction decreases GzmB-induced Bax activation, cytochrome c release from mitochondria, and subsequent effector caspases activation leading to a decreased sensitivity of target cells to both GzmB and CTL/NK-mediated cell death. Together, our results define p53 as a new important player in the GzmB apoptotic signaling pathway and in CTL/NK-induced apoptosis. PMID:25404359

  2. Simultaneous determination of most prescribed antibiotics in multiple urban wastewater by SPE-LC-MS/MS.

    PubMed

    Rossmann, Julia; Schubert, Sara; Gurke, Robert; Oertel, Reinhard; Kirch, Wilhelm

    2014-10-15

    A rapid analytical method was developed for the application of a long-term monitoring (>one year) of the most prescribed and often in hospitals used antibiotics in diverse wastewaters of an urban sewage treatment plant (STP). Additionally to the selected multi-class antibiotics amoxicillin, penicillin V and piperacillin (penicillins), cefotaxime and cefuroxime (cephalosporins), azithromycin, clarithromycin and roxithromycin (macrolids), ciprofloxacin and levofloxacin-ofloxacin (fluoroquinolones), clindamycin (lincosamide), doxycycline (tetracycline), sulfamethoxazole (sulfonamide) and trimethoprim (dihydrofolate reductase inhibitor), the bioactive metabolite clindamycin-sulfoxide, the reserve antibiotic vancomycin (glycopeptide) and as tracer of the STP the anticonvulsant carbamazepine and the antifungal fluconazole were involved. The analytical method combines a low-sample-volume solid phase extraction (SPE), followed by a chromatographic separation using a reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) technique, respectively, coupled to a triple quadrupole mass spectrometer. Detection was performed with multiple reaction monitoring (MRM) measured with positive electrospray ionization (ESI+). The extraction efficiency of different SPE cartridges and optimized pH-values of the preparation procedure were tested. Finally, the extraction of antibiotics was realized with the Oasis HLB cartridge and a pH adjustment at 3.5. An external calibration curve in diluted blank urine was used for quality control of the sample set of daily composite samples of the STP for the duration of one year monitoring. The squared coefficient of determination (r(2)) in the concentration range (20-20,000ng/L or 100-100,000ng/L) of the calibration curves for the method was higher than 0.99 for all determined substances. The limit of quantification (LoQ) ranged between 0.8ng/L (azithromycin) and 245.1ng/L (vancomycin). Furthermore, a standard addition was used

  3. [Estrogens determination of livestock dung based on UE-SPE-HPLC/FLD].

    PubMed

    Fu, Yin-Jie; Ling, Wan-Ting; Dong, Chang-Xun; Liu, Juan; Gao, Yan-Zheng; Pan, Yu-Lan

    2013-11-01

    A method for detecting the estrogens estriol, 17beta-estradiol, ethinyl estradiol, and bisphenol A in livestock dung was established by the combination of ultrasonic extraction (UE), solid phase extraction (SPE) purification, and high performance liquid chromatography (HPLC) with fluorescence detector (FLD). The dung samples were extracted with ethyl acetate ultrasonication for 30 min, and purified with C18 solid phase extraction column and related solvents. The test four estrogens in the dung samples were isolated with Inertsil ODS-SP-C18 reversed-phase columns (150 mm x 4.6 mm, 5 microm), and the isolated estrogens were detected with HPLC/FLD. The mobile phase of HPLC for the detection was methanol/acetonitrile/water (volume ratio of 20:30:50), with a flow rate of 0.8 mL x min(-1). The excitation and emission wavelengths of FLD were 280 and 310 nm, respectively, the HPLC column temperature was 40 degrees C, and the injection volume was 20 microL. Good linearity (correlation coefficient greater than 0.9995) was observed by the HPLC/FLD detection when the test four estrogens concentrations were in the range of 1.00-1000.00 microg x L(-1). The detection limit of estriol, bisphenol A, 17beta-estradiol, and ethinyl estradiol was 3.35, 5.01, 2.13, and 1.12 microg x kg(-1), respectively. When the added estrogens concentrations of pig, cow, and chicken dung samples were 0.05, 0.40, and, 1.00 microg x kg(-1), the average recovery of the four estrogens was 75.1%-91.1%, 78.4%-117.0%, and 78.6%-97.8%, respectively, with the relatively standard deviations (RSD, n = 6) all less than 6%. By adopting the established SPE-HPLC/FLD method to detect the estrogens in real pig, cow, and chicken dung samples from parts of the large-scale livestock raising farms in Nanjing of East China, the detection reproducibility was high, and the detection limit was low, being available and effective for the detection of the estrogens in livestock dung. PMID:24564161

  4. Apoptosis in colorectal cancer.

    PubMed

    Stoian, M; State, N; Stoica, V; Radulian, G

    2014-06-15

    Apoptosis is an inborn process that has been preserved during evolution; it allows the cells to systematically inactivate, destroy and dispose of their own components thus leading to their death. This programme can be activated by both intra and extracellular mechanisms. The intracellular components involve a genetically defined development programme while the extracellular aspects regard endogenous proteins, cytokines and hormones as well as xenobiotics, radiations, oxidative stress and hypoxia. The ability of a cell to enter apoptosis as a response to a "death" signal depends on its proliferative status, the position in the cell cycle and also on the controlled expression of those genes that have the capacity of promoting and inhibiting cell death. The fine regulation of these parameters needs to be maintained in order to ensure the physiological environment required for the induction of apoptosis. Any malfunction in any of the steps of controlled cellular death can lead to dysfunctions and, as a consequence, to different pathological conditions. The importance of apoptosis lies in its active nature and in the potential of controlling biological systems. PMID:25408720

  5. The Development and Optimization of Techniques for Monitoring Water Quality on-Board Spacecraft Using Colorimetric Solid-Phase Extraction (C-SPE)

    SciTech Connect

    Hill, April Ann

    2007-12-01

    The main focus of this dissertation is the design, development, and ground and microgravity validation of methods for monitoring drinking water quality on-board NASA spacecraft using clorimetric-solid phase extraction (C-SPE). The Introduction will overview the need for in-flight water quality analysis and will detail some of the challenges associated with operations in the absence of gravity. The ability of C-SPE methods to meet these challenges will then be discussed, followed by a literature review on existing applications of C-SPE and similar techniques. Finally, a brief discussion of diffuse reflectance spectroscopy theory, which provides a means for analyte identification and quantification in C-SPE analyses, is presented. Following the Introduction, four research chapters are presented as separate manuscripts. Chapter 1 reports the results from microgravity testing of existing C-SPE methods and procedures aboard NASA's C-9 microgravity simulator. Chapter 2 discusses the development of a C-SPE method for determining the total concentration of biocidal silver (i.e., in both dissolved and colloidal forms) in water samples. Chapter 3 presents the first application of the C-SPE technique to the determination of an organic analyte (i.e., formaldehyde). Chapter 4, which is a departure from the main focus of the thesis, details the results of an investigation into the effect of substrate rotation on the kinetics involved in the antigen and labeling steps in sandwich immunoassays. These research chapters are followed by general conclusions and a prospectus section.

  6. A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus.

    PubMed Central

    Schwartz, B; Hittelman, A; Daneshvar, L; Basu, H S; Marton, L J; Feuerstein, B G

    1995-01-01

    Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination. Images Figure 3 Figure 10 PMID:7492339

  7. An N-Terminal Signal Peptide Of Vfr Protein Negatively Influences RopB-Dependent SpeB Expression and Attenuates Virulence in Streptococcus pyogenes

    PubMed Central

    Shelburne, Samuel A.; Olsen, Randall J.; Makthal, Nishanth; Brown, Nicholas G.; Sahasrabhojane, Pranoti; Watkins, Ebru M.; Palzkill, Timothy; Musser, James M.; Kumaraswami, Muthiah

    2015-01-01

    SUMMARY Streptococcal pyrogenic exotoxin B (SpeB) is an extracellular cysteine protease that is a critical virulence factor made by the major human pathogen group A Streptococcus (GAS). speB expression is dependent on the regulator of proteinase B (RopB) and is upregulated with increasing cell density and during infection. Because computer modeling suggested significant structural similarity between RopB and peptide-sensing regulatory proteins made by other Gram-positive bacteria, we hypothesized that speB expression is influenced by RopB-peptide interactions. Inactivation of the gene (vfr) encoding the virulence factor related (Vfr) protein resulted in increased speB transcript level during the exponential growth phase, whereas provision of only the amino-terminal region of Vfr comprising the secretion signal sequence in trans restored a wild-type speB expression profile. Addition of the culture supernatant from a Vfr signal peptide-expressing GAS strain restored wild-type speB transcript level to a vfr-inactivated isogenic mutant strain. A distinct peptide in the Vfr secretion signal sequence specifically bound to recombinant RopB. Finally, overexpression of the Vfr secretion signal sequence significantly decreased speB transcript level and attenuated GAS virulence in two mouse models of invasive infection. Taken together, these data delineate a previously unknown small peptide-mediated regulatory system that controls GAS virulence factor production. PMID:22040048

  8. Characterisation of antimicrobial extracts from dandelion root (Taraxacum officinale) using LC-SPE-NMR.

    PubMed

    Kenny, O; Brunton, N P; Walsh, D; Hewage, C M; McLoughlin, P; Smyth, T J

    2015-04-01

    Plant extracts have traditionally been used as sources of natural antimicrobial compounds, although in many cases, the compounds responsible for their antimicrobial efficacy have not been identified. In this study, crude and dialysed extracts from dandelion root (Taraxacum officinale) were evaluated for their antimicrobial properties against Gram positive and Gram negative bacterial strains. The methanol hydrophobic crude extract (DRE3) demonstrated the strongest inhibition of microbial growth against Staphylococcus aureus, methicillin-resistant S. aureus and Bacillus cereus strains. Normal phase (NP) fractionation of DRE3 resulted in two fractions (NPF4 and NPF5) with enhanced antimicrobial activity. Further NP fractionation of NPF4 resulted in two fractions (NPF403 and NPF406) with increased antimicrobial activity. Further isolation and characterisation of compounds in NPF406 using liquid chromatography solid phase extraction nuclear magnetic resonance LC-SPE-NMR resulted in the identification of 9-hydroxyoctadecatrienoic acid and 9-hydroxyoctadecadienoic acid, while the phenolic compounds vanillin, coniferaldehyde and p-methoxyphenylglyoxylic acid were also identified respectively. The molecular mass of these compounds was confirmed by LC mass spectroscopy (MS)/MS. In summary, the antimicrobial efficacy of dandelion root extracts demonstrated in this study support the use of dandelion root as a source of natural antimicrobial compounds. PMID:25644491

  9. Asteroid 21 Lutetia at 3 μm: Observations with IRTF SpeX

    NASA Astrophysics Data System (ADS)

    Rivkin, Andrew S.; Clark, Beth E.; Ockert-Bell, Maureen; Volquardsen, Eric; Howell, Ellen S.; Bus, Schelte J.; Thomas, Cristina A.; Shepard, Michael

    2011-11-01

    We present observations of Asteroid 21 Lutetia collected 2003-2008 using the SpeX instrument on the NASA Infrared Telescope Facility (IRTF) covering 2-4 μm. We also reevaluate NSFCam observations obtained in 1996 (Rivkin, A.S., Lebofsky, L.A., Clark, B.E., Howell, E.S., Britt, D.T. [2000]. Icarus 145, 351-368). Taken together, these show deeper 3-μm band depths (of order 3-5%) in the southern hemisphere of Lutetia, and shallower band depths (of order 2% or less) in the north. Such variation is consistent with observations at shorter wavelength by previous workers (Nedelcu, D.A. et al. [2007]. Astron. Astrophys. 470, 1157-1164; Lazzarin, M. et al. [2010]. Mon. Not. R. Astron. Soc. 408, 1433-1437), who observed hemispheric-level variations from C-like spectra to X-like spectra. While the shallowness of absorption bands on Lutetia hinders identification of its surface composition, goethite appears plausible as a constituent in its southern hemisphere (Beck, P., Quirico, E., Sevestre, D., Montes-Hernandez, G., Pommerol, A., Schmitt, B. [2011]. Astron. Astrophys. 526, A85-A89). Mathematical models of space weathered goethite are most consistent with Lutetia's southern hemisphere spectrum, but more work and further observations are necessary to confirm this suggestion.

  10. Determination of phthalates in fruit jellies by dispersive SPE coupled with HPLC-MS.

    PubMed

    Ma, Yuwei; Hashi, Yuki; Ji, Feng; Lin, Jin-Ming

    2010-02-01

    In this study, a simple, rapid and sensitive method for the determination of five phthalates including dimethyl phthalate, diethyl phthalate, dipropyl phthalate, benzyl butyl phthalate, and dicyclohexyl phthalate in fruit jellies by LC coupled with MS has been developed. Samples were pretreated by a dispersive SPE method, termed QuEChERS, which is an acronym for quick, easy, cheap, effective, rugged, and safe. The standard calibration curves were linear for all the analytes over the concentration range of 10-250 ng/mL, and the correlation coefficients ranged from 0.9976 to 0.9991. The LODs and LOQs were in the ranges of 0.09-3.68 ng/mL and 0.28-11.25 ng/mL, respectively. The accuracy of this method was evaluated by measuring the recovery from spiked samples. The recoveries of all five phthalates from samples spiked at three different concentrations (0.01, 0.03, and 0.05 mg/kg), were in the ranges of 83.5-103.9%, 86.7-95.8%, and 87.1-95.2%, respectively. The RSD values for the samples spiked at 0.01, 0.03, and 0.05 mg/kg ranged from 2.0-7.6%, 1.4-6.4%, and 1.2-3.8%, respectively. The method has been used for the analysis of real samples and BBP and DEP were found in real samples. PMID:19998379

  11. Data Release Report for Source Physics Experiment 1 (SPE-1), Nevada National Security Site

    SciTech Connect

    Townsend, Margaret; Mercadente, Jennifer

    2014-04-28

    The first Source Physics Experiment shot (SPE-1) was conducted in May 2011. The explosive source was a ~100-kilogram TNT-equivalent chemical set at a depth of 60 meters. It was recorded by an extensive set of instrumentation that includes sensors both at near-field (less than 100 meters) and far-field (more than 100 meters) distances. The near-field instruments consisted of three-component accelerometers deployed in boreholes around the shot and a set of singlecomponent vertical accelerometers on the surface. The far-field network comprised a variety of seismic and acoustic sensors, including short-period geophones, broadband seismometers, three-component accelerometers, and rotational seismometers at distances of 100 meters to 25 kilometers. This report coincides with the release of these data for analysts and organizations that are not participants in this program. This report describes the first Source Physics Experiment and the various types of near-field and far-field data that are available.

  12. Development of methodology for determination of pesticides residue in water by SPE/HPLC/DAD.

    PubMed

    Cappelini, Luciana Teresa Dias; Cordeiro, Daniela; Brondi, Silvia Helena Govoni; Prieto, Kátia Roberta; Vieira, Eny Maria

    2012-01-01

    To boost crop yield, sugarcane growers are using increasing amounts of pesticides to combat insects and weeds. But residues of these compounds can pollute water resources, such as lakes, rivers and aquifers. The present paper reports the results of a study of water samples from the Feijão River, which is the source of drinking water for the city of São Carlos, São Paulo, Brazil. The samples were evaluated for the presence of four leading pesticides--ametryn, atrazine, diuron and fipronil--used on sugarcane, the dominant culture in the region. The samples were obtained from three points along the river: the headwaters, along the middle course of the river and just before the municipal water intake station. The pesticides were extracted from the water samples by solid-phase extraction (SPE) and then analyzed by liquid chromatography with diode array detection (LC-DAD). The analytical method was validated by traditional methods, obtaining recovery values between 90 and 95%, with precision deviations inferior to 2.56%, correlation coefficients above 0.99 and detection and quantification limits varying from 0.02 to 0.05 mg L(-1) and 0.07 to 0.17 mg L(-1), respectively. No presence of residues of the pesticides was detected in the samples, considering the detection limits of the method employed. PMID:23393971

  13. Brevenal inhibits pacific ciguatoxin-1B-induced neurosecretion from bovine chromaffin cells.

    PubMed

    Mattei, César; Wen, Peter J; Nguyen-Huu, Truong D; Alvarez, Martha; Benoit, Evelyne; Bourdelais, Andrea J; Lewis, Richard J; Baden, Daniel G; Molgó, Jordi; Meunier, Frédéric A

    2008-01-01

    Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and beta-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera. PMID:18941627

  14. Brevenal Inhibits Pacific Ciguatoxin-1B-Induced Neurosecretion from Bovine Chromaffin Cells

    PubMed Central

    Mattei, César; Alvarez, Martha; Benoit, Evelyne; Bourdelais, Andrea J.; Lewis, Richard J.; Baden, Daniel G.; Molgó, Jordi; Meunier, Frédéric A.

    2008-01-01

    Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and β-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera. PMID:18941627

  15. Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage.

    PubMed

    Tam, John; Beilhartz, Greg L; Auger, Anick; Gupta, Pulkit; Therien, Alex G; Melnyk, Roman A

    2015-02-19

    Clostridium difficile causes life-threatening diarrhea through the actions of its homologous toxins TcdA and TcdB on human colonocytes. Therapeutic agents that block toxin-induced damage are urgently needed to prevent the harmful consequences of toxin action that are not addressed with current antibiotic-based treatments. Here, we developed an imaging-based phenotypic screen to identify small molecules that protected human cells from TcdB-induced cell rounding. A series of structurally diverse compounds with antitoxin activity were identified and found to act through one of a small subset of mechanisms, including direct binding and sequestration of TcdB, inhibition of endosomal maturation, and noncompetitive inhibition of the toxin glucosyltransferase activity. Distinct classes of inhibitors were used further to dissect the determinants of the toxin-mediated necrosis phenotype occurring at higher doses of toxin. These findings validate and inform novel targeting strategies for discovering small molecule agents to treat C. difficile infection. PMID:25619932

  16. Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity

    PubMed Central

    LaFrance, Michelle E.; Farrow, Melissa A.; Chandrasekaran, Ramyavardhanee; Sheng, Jinsong; Rubin, Donald H.; Lacy, D. Borden

    2015-01-01

    Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection. PMID:26038560

  17. Ram ion scattering caused by Space Shuttle v x B induced differential charging

    NASA Technical Reports Server (NTRS)

    Katz, I.; Davis, V. A.

    1987-01-01

    Observations of secondary, high-inclination ions streams have been reported in the literature. The authors of these previous papers attributed the source of the secondary ions to a disturbed region in the plasma about 10 m from the Space Shuttle Orbiter. A new theory has been developed which shows how v x B induced differential charging on the plasma diagnostics package (PDP) can scatter the ram ion flux. Some of these ions are reflected back to the PDP and may be the sorce of the observed ion distributions. The effect is unique to large spacecraft; it occurs only when the magnitude of the induced v x B potentials are much larger than the electron thermal energy and of the order of the ion ram energy. That the ion streams observed at large angles must have been reflected from the PDP surface is demonstrated with three-dimensional sheath and particle trajectory calculations using the low earth orbit version of the NASA Charging Analyzer Program (NASCAP/LEO).

  18. Simultaneous detection of antibiotics and other drug residues in the dissolved and particulate phases of water by an off-line SPE combined with on-line SPE-LC-MS/MS: Method development and application.

    PubMed

    Tlili, Ines; Caria, Giovanni; Ouddane, Baghdad; Ghorbel-Abid, Ibtissem; Ternane, Riadh; Trabelsi-Ayadi, Malika; Net, Sopheak

    2016-09-01

    Due to their widespread use in human and animal healthcare, antibiotics and other drug residues are ubiquitous in the aquatic environment. Given their potential impacts on ecosystem functioning and public health, the quantification of environmental drug residues has become a necessity. Various analysis techniques have been found to be suitable for reliable detection of such compounds. However, quantification can be difficult because these compounds are present at trace or ultra-trace levels. Consequently, the accuracy of environmental analyses depends on both the efficiency and the robustness of the extraction and quantification method. In this work, an off-line solid-phase extraction (SPE) combined with on-line SPE-LC-MS/MS was applied to the simultaneous extraction and quantification of 26 pharmaceutical products, including 18 antibiotics, dissolved in a water phase. Optimal conditions were determined and then applied to assess the contamination level of the targeted drug residues in water collected from four sites in Northern France: a river, the input and output of an aerated lagoon, and a wastewater treatment plant. Drug residues associated with suspended solid matter (SSM) were also quantified in this work using pressurized liquid extraction (PLE) combined with an on-line SPE-LC-MS/MS system in order to complete an assessment of the degree of total background pollution. PMID:27151499

  19. CORRTEX Diagnostic Deployment for the SPE-III experiment, 24 July 2012: Fielding Report and Preliminary Data Analysis

    SciTech Connect

    Sandoval, Thomas D.; Schultz-Fellenz, Emily S.

    2012-08-29

    The Continuous Reflectometry for Radius vs Time Experiments (CORRTEX) diagnostic system was deployed for the third explosives test in the Source Physics Experiment (SPE) sequence to monitor and verify several conditions of the experiment including the detonation velocity of the explosive package and functioning of explosive initiators. Six distance-marked coaxial cables were installed on the SPE-III explosives canister, and key locations documented through along-cable length measurements and photography. CORRTEX uses electrical-pulse time-domain reflectometry to continuously record the two-way transit time (TWTT) of the cables. As the shock front of the detonation advances, the coaxial cable is shorted or destroyed, and the resulting TWTT also decreases. Interpretation of these changes as a function of TWTT can be converted to positional measurements using known parameters of the cables.

  20. Absence of SpeB production in virulent large capsular forms of group A streptococcal strain 64.

    PubMed

    Raeder, R; Harokopakis, E; Hollingshead, S; Boyle, M D

    2000-02-01

    Passage in human blood of group A streptococcal isolate 64p was previously shown to result in the enhanced expression of M and M-related proteins. Similarly, when this isolate was injected into mice via an air sac model for skin infection, organisms recovered from the spleens showed both increased expression of M and M-related proteins and increased skin-invasive potential. We show that these phenotypic changes were not solely the result of increased transcription of the mRNAs encoding the M and M-related gene products. Rather, the altered expression was associated with posttranslational modifications of the M and M-related proteins that occur in this strain, based on the presence or absence of another virulence protein, the streptococcal cysteine protease SpeB. The phenotypic variability also correlates with colony size variation. Large colonies selected by both regimens expressed more hyaluronic acid, which may explain differences in colony morphology. All large-colony variants were SpeB negative and expressed three distinct immunoglobulin G (IgG)-binding proteins in the M and M-related protein family. Small-colony variants were SpeB positive and bound little IgG through their M and M-related proteins because these proteins, although made, were degraded or altered in profile by the SpeB protease. We conclude that passage in either human blood or a mouse selects for a stable, phase-varied strain of group A streptococci which is altered in many virulence properties. PMID:10639442

  1. A new piece of the Shigella Pathogenicity puzzle: spermidine accumulation by silencing of the speG gene [corrected].

    PubMed

    Barbagallo, Marialuisa; Di Martino, Maria Letizia; Marcocci, Lucia; Pietrangeli, Paola; De Carolis, Elena; Casalino, Mariassunta; Colonna, Bianca; Prosseda, Gianni

    2011-01-01

    The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a

  2. Absence of SpeB Production in Virulent Large Capsular Forms of Group A Streptococcal Strain 64

    PubMed Central

    Raeder, Roberta; Harokopakis, Evlambia; Hollingshead, Susan; Boyle, Michael D. P.

    2000-01-01

    Passage in human blood of group A streptococcal isolate 64p was previously shown to result in the enhanced expression of M and M-related proteins. Similarly, when this isolate was injected into mice via an air sac model for skin infection, organisms recovered from the spleens showed both increased expression of M and M-related proteins and increased skin-invasive potential. We show that these phenotypic changes were not solely the result of increased transcription of the mRNAs encoding the M and M-related gene products. Rather, the altered expression was associated with posttranslational modifications of the M and M-related proteins that occur in this strain, based on the presence or absence of another virulence protein, the streptococcal cysteine protease SpeB. The phenotypic variability also correlates with colony size variation. Large colonies selected by both regimens expressed more hyaluronic acid, which may explain differences in colony morphology. All large-colony variants were SpeB negative and expressed three distinct immunoglobulin G (IgG)-binding proteins in the M and M-related protein family. Small-colony variants were SpeB positive and bound little IgG through their M and M-related proteins because these proteins, although made, were degraded or altered in profile by the SpeB protease. We conclude that passage in either human blood or a mouse selects for a stable, phase-varied strain of group A streptococci which is altered in many virulence properties. PMID:10639442

  3. Mortalin, Apoptosis, and Neurodegeneration

    PubMed Central

    Londono, Carolina; Osorio, Cristina; Gama, Vivian; Alzate, Oscar

    2012-01-01

    Mortalin is a highly conserved heat-shock chaperone usually found in multiple subcellular locations. It has several binding partners and has been implicated in various functions ranging from stress response, control of cell proliferation, and inhibition/prevention of apoptosis. The activity of this protein involves different structural and functional mechanisms, and minor alterations in its expression level may lead to serious biological consequences, including neurodegeneration. In this article we review the most current data associated with mortalin’s binding partners and how these protein-protein interactions may be implicated in apoptosis and neurodegeneration. A complete understanding of the molecular pathways in which mortalin is involved is important for the development of therapeutic strategies for cancer and neurodegenerative diseases. PMID:24970131

  4. Induction of T-cell apoptosis by human herpesvirus 6.

    PubMed Central

    Inoue, Y; Yasukawa, M; Fujita, S

    1997-01-01

    The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection. PMID:9094650

  5. Spe3, which encodes spermidine synthase, is required for full repression through NRE(DIT) in Saccharomyces cerevisiae.

    PubMed Central

    Friesen, H; Tanny, J C; Segall, J

    1998-01-01

    We previously identified a transcriptional regulatory element, which we call NRE(DIT), that is required for repression of the sporulation-specific genes, DIT1 and DIT2, during vegetative growth of Saccharomyces cerevisiae. Repression through this element is dependent on the Ssn6-Tup1 corepressor. In this study, we show that SIN4 contributes to NRE(DIT)-mediated repression, suggesting that changes in chromatin structure are, at least in part, responsible for regulation of DIT gene expression. In a screen for additional genes that function in repression of DIT (FRD genes), we recovered alleles of TUP1, SSN6, SIN4, and ROX3 and identified mutations comprising eight complementation groups of FRD genes. Four of these FRD genes appeared to act specifically in NRE(DIT)mediated repression, and four appeared to be general regulators of gene expression. We cloned the gene complementing the frd3-1 phenotype and found that it was identical to SPE3, which encodes spermidine synthase. Mutant spe3 cells not only failed to support complete repression through NRE(DIT) but also had modest defects in repression of some other genes. Addition of spermidine to the medium partially restored repression to spe3 cells, indicating that spermidine may play a role in vivo as a modulator of gene expression. We suggest various mechanisms by which spermidine could act to repress gene expression. PMID:9725830

  6. Forward Genetics Identifies a Requirement for the Izumo-like Immunoglobulin Superfamily spe-45 Gene in Caenorhabditis elegans Fertilization.

    PubMed

    Singaravelu, Gunasekaran; Rahimi, Sina; Krauchunas, Amber; Rizvi, Anam; Dharia, Sunny; Shakes, Diane; Smith, Harold; Golden, Andy; Singson, Andrew

    2015-12-21

    Fertilization is a conserved process in all sexually reproducing organisms whereby sperm bind and fuse with oocytes. Despite the importance of sperm-oocyte interactions in fertilization, the molecular underpinnings of this process are still not well understood. The only cognate ligand-receptor pair identified in the context of fertilization is sperm-surface Izumo and egg-surface Juno in the mouse [1]. Here we describe a genetic screening strategy to isolate fertilization mutants in Caenorhabditis elegans in order to generate a more complete inventory of molecules required for gamete interactions. From this screening strategy, we identified, cloned, and characterized spe-45, a gene that encodes an Izumo-like immunoglobulin superfamily protein. Mammalian Izumo is required for male fertility and has the same basic mutant phenotype as spe-45. Worms lacking spe-45 function produce morphologically normal and motile sperm that cannot fuse with oocytes despite direct contact in the reproductive tract. The power of this screen to identify proteins with ancient sperm functions suggests that characterization of additional mutants from our screen may reveal other deeply conserved components in fertility pathways and complement studies in other organisms. PMID:26671668

  7. The cell envelope-associated protein, LytR, regulates the cysteine protease SpeB in Streptococcus pyogenes.

    PubMed

    Minami, Masaaki; Ichikawa, Mariko; Ohta, Michio; Hasegawa, Tadao

    2012-05-01

    The LytR family of cell envelope-associated transcriptional attenuators in bacteria has been brought into focus of scientific interest on the expression of various virulence factors, as well as bacterial cell envelope maintenance. However, this protein of Streptococcus pyogenes has been only described as cell surface-associated protein, and its function is completely unknown. We created lytR mutant strains from two independent S. pyogenes strains to analyze the function of LytR. The protease assay in culture supernatant showed that lytR mutant had the higher cysteine protease activity than wild-type. Two-dimensional gel electrophoresis and western blotting analysis revealed that the amount of cysteine protease, SpeB in lytR mutant was more compared with that in wild-type. The level of speB mRNA in lytR mutant also increased compared with that of wild-type. The membrane integrity and potential in lytR mutant also were decreased compared with that of wild-type. Murine infection model showed that less survival was detected in mice inoculated with lytR mutant than that with wild-type, and the size of wound lesion of mice with lytR mutant was larger than that with wild-type. Our data suggest that the lytR regulates the expression of SpeB in S. pyogenes with relation to membrane integrity. PMID:22515297

  8. Colorimetric-Solid Phase Extraction Technology for Water Quality Monitoring: Evaluation of C-SPE and Debubbling Methods in Microgravity

    NASA Technical Reports Server (NTRS)

    Hazen-Bosveld, April; Lipert, Robert J.; Nordling, John; Shih, Chien-Ju; Siperko, Lorraine; Porter, Marc D.; Gazda, Daniel B.; Rutz, Jeff A.; Straub, John E.; Schultz, John R.; McCoy, J. Torin

    2007-01-01

    Colorimetric-solid phase extraction (C-SPE) is being developed as a method for in-flight monitoring of spacecraft water quality. C-SPE is based on measuring the change in the diffuse reflectance spectrum of indicator disks following exposure to a water sample. Previous microgravity testing has shown that air bubbles suspended in water samples can cause uncertainty in the volume of liquid passed through the disks, leading to errors in the determination of water quality parameter concentrations. We report here the results of a recent series of C-9 microgravity experiments designed to evaluate manual manipulation as a means to collect bubble-free water samples of specified volumes from water sample bags containing up to 47% air. The effectiveness of manual manipulation was verified by comparing the results from C-SPE analyses of silver(I) and iodine performed in-flight using samples collected and debubbled in microgravity to those performed on-ground using bubble-free samples. The ground and flight results showed excellent agreement, demonstrating that manual manipulation is an effective means for collecting bubble-free water samples in microgravity.

  9. Determination of abamectin in citrus fruits using SPE combined with dispersive liquid-liquid microextraction and HPLC-UV detection.

    PubMed

    Rezaee, Mohammad; Mashayekhi, Hossein Ali; Saleh, Abolfazl; Abdollahzadeh, Yaser; Naeeni, Mohammad Hosein; Fattahi, Nazir

    2013-08-01

    A new pretreatment method, SPE combined with dispersive liquid-liquid microextraction, was proposed for the determination of abamectin in citrus fruit samples for the first time. In this method, fruit samples were extracted by ultrasound-assisted extraction followed by SPE. Then, the SPE was used as a disperser solvent in the next dispersive liquid-liquid microextraction step for further purification and enrichment of abamectin. The effects of various parameters on the extraction efficiency of the proposed method were investigated and optimized. Good linearity of abamectin was obtained from 0.005 to 10.0 mg/kg for B1a and from 0.05 to 10.0 mg/kg for B1b with correlation coefficient (r(2)) of 0.998 for B1a and 0.991 for B1b, respectively. The LODs were 0.001 and 0.008 mg/kg (S/N = 3) for B1a and B1b, respectively. The relative recoveries at three spiked levels were ranged from 87 to 96% with the RSD less than 11% (n = 3). The method has been successfully applied to the determination of abamectin in citrus fruit samples. PMID:23913592

  10. Analysis of drugs of abuse by online SPE-LC high resolution mass spectrometry: communal assessment of consumption.

    PubMed

    Heuett, Nubia V; Ramirez, Cesar E; Fernandez, Adolfo; Gardinali, Piero R

    2015-04-01

    An online SPE-LC-HRMS method was developed to monitor the consumption of 18 drugs of abuse (DOAs) including amphetamines, opioids, cocainics, cannabinoids, lysergics, and their corresponding metabolites in a well characterized college campus setting via wastewater analysis. Filtered and diluted (10×) sewage water samples (5 mL inj.) were automatically pre-concentrated and analyzed in 15 min using a Thermo EQuan MAX online SPE system equipped with a HyperSep™ Retain PEP (20×2.1 mm×12 μm) SPE column and a Hypersil Gold™ aQ (150×2.1 mm×3 μm) analytical column. A Q Exactive™ Hybrid Quadrupole-Orbitrap HRMS was used in full scan mode (R=140,000) for positive identification, and quantitation of target compounds. Method detection limits for all analytes ranged between 0.6 and 1.7 ng/L in sewage. A total of 14 DOAs were detected from two different locations (dorms and main college campus) within a one-year period. Most frequently detected drugs throughout the entire study were amphetamine (>96%) and THC's metabolite 11-nor-9-carboxy-Δ-9-THC (>100%) with maximum concentrations of 5956 and 2413 ng/L respectively. Daily doses per 1000 people were determined in order to assess consumption of THC, amphetamine, heroin and cocaine, in both dorms and main campus. PMID:25553546

  11. Mangiferin, a novel nuclear factor kappa B-inducing kinase inhibitor, suppresses metastasis and tumor growth in a mouse metastatic melanoma model.

    PubMed

    Takeda, Tomoya; Tsubaki, Masanobu; Sakamoto, Kotaro; Ichimura, Eri; Enomoto, Aya; Suzuki, Yuri; Itoh, Tatsuki; Imano, Motohiro; Tanabe, Genzoh; Muraoka, Osamu; Matsuda, Hideaki; Satou, Takao; Nishida, Shozo

    2016-09-01

    Advanced metastatic melanoma, one of the most aggressive malignancies, is currently without reliable therapy. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone and exerts many beneficial biological activities. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we evaluated the effect of mangiferin on metastasis and tumor growth in a mouse metastatic melanoma model. We found that mangiferin inhibited spontaneous metastasis and tumor growth. Furthermore, mangiferin suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated NF-κB-inducing kinase (NIK), inhibitor of kappa B kinase (IKK), and inhibitor of kappa B (IκB) and increases the expression of IκB protein in vivo. In addition, we found that mangiferin inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs) in vivo. Mangiferin treatment also increased the expression of cleaved caspase-3, cleaved Poly ADP ribose polymerase-1 (PARP-1), p53 upregulated modulator of apoptosis (PUMA), p53, and phosphorylated p53 proteins, and decreased the expression of Survivin and Bcl-associated X (Bcl-xL) proteins in vivo. These results indicate that mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation, thereby inhibiting metastasis and tumor growth. Importantly, the number of reported NIK selective inhibitors is limited. Taken together, our data suggest that mangiferin may be a potential therapeutic agent with a new mechanism of targeting NIK for the treatment of metastatic melanoma. PMID:27417526

  12. NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

    PubMed

    Mao, Xianrong; Phanavanh, Bounleut; Hamdan, Hamdan; Moerman-Herzog, Andréa M; Barger, Steven W

    2016-04-01

    The control of NFκB in CNS neurons appears to differ from that in other cell types. Studies have reported induction of NFκB in neuronal cultures and immunostaining in vivo, but others have consistently detected little or no transcriptional activation by NFκB in brain neurons. To test if neurons lack some component of the signal transduction system for NFκB activation, we transfected cortical neurons with several members of this signaling system along with a luciferase-based NFκB-reporter plasmid; RelA was cotransfected in some conditions. No component of the NFκB pathway was permissive for endogenous NFκB activity, and none stimulated the activity of exogenous RelA. Surprisingly, however, the latter was inhibited by cotransfection of NFκB-inducing kinase (NIK). Fluorescence imaging of RelA indicated that co-expression of NIK sequestered RelA in the cytoplasm, similar to the effect of IκBα. NIK-knockout mice showed elevated expression of an NFκB-reporter construct in neurons in vivo. Cortical neurons cultured from NIK-knockout mice showed elevated expression of an NFκB-reporter transgene. Consistent with data from other cell types, a C-terminal fragment of NIK suppressed RelA activity in astrocytes as well as neurons. Therefore, the inhibitory ability of the NIK C-terminus was unbiased with regard to cell type. However, inhibition of NFκB by full-length NIK is a novel outcome that appears to be specific to CNS neurons. This has implications for unique aspects of transcription in the CNS, perhaps relevant to aspects of development, neuroplasticity, and neuroinflammation. Full-length NIK was found to inhibit (down arrow) transcriptional activation of NFκB in neurons, while it elevated (up arrow) activity in astrocytes. Deletion constructs corresponding to the N-terminus or C-terminus also inhibited NFκB in neurons, while only the C-terminus did so in astrocytes. One possible explanation is that the inhibition in neurons occurs via two different

  13. Ganglioside GQ1b induces dopamine release through the activation of Pyk2.

    PubMed

    Zhang, Zhao; Chu, Shi-Feng; Mou, Zheng; Gao, Yan; Wang, Zhen-Zhen; Wei, Gui-Ning; Chen, Nai-Hong

    2016-03-01

    Growing evidence indicates that GQ1b, one of the gangliosides members, contributes to synaptic transmission and synapse formation. Previous studies have shown that GQ1b could enhance depolarization induced neurotransmitter release, while the role of GQ1b in asynchronous release is still largely unknown. Here in our result, we found low concentration of GQ1b, but not GT1b or GD1b (which were generated from GQ1b by plasma membrane-associated sialidases), evoked asynchronous dopamine (DA) release from both clonal rat pheochromocytoma PC12 cells and rat striatal slices significantly. The release peaked at 2min after GQ1b exposure, and lasted for more than 6min. This effect was caused by the enhancement of intracellular Ca(2+) and the activation of Pyk2. Inhibition of Pyk2 by PF-431396 (a dual inhibitor of Pyk2 and FAK) or Pyk2 siRNA abolished DA release induced by GQ1b. Moreover, Pyk2 Y402, but not other tyrosine site, was phosphorylated at the peaking time. The mutant of Pyk2 Y402 (Pyk2-Y402F) was built to confirm the essential role of Y402 activation. Further studies revealed that activated Pyk2 stimulated ERK1/2 and p-38, while only the ERK1/2 activation was indispensable for GQ1b induced DA release, which interacted with Synapsin I directly and led to its phosphorylation, then depolymerization of F-actin, thus contributed to DA release. In conclusion, low concentration of GQ1b is able to enhance asynchronous DA release through Pyk2/ERK/Synapsin I/actin pathway. Our findings provide new insights into the role of GQ1b in neuronal communication, and implicate the potential application of GQ1b in neurological disorders. PMID:26704905

  14. NGFI-B targets mitochondria and induces cardiomyocyte apoptosis in restraint-stressed rats by mediating energy metabolism disorder

    PubMed Central

    Wang, XinXing; Liu, XiaoHua; Kong, RuiRui; Zhan, Rui; Wang, XiaoMing; Leng, Xue; Gong, JingBo; Duan, Meng; Wang, LiQun; Wu, Lei

    2009-01-01

    NGFI-B/Nur77/TR3, originally identified as an immediate-early gene rapidly induced by serum and growth factors, is a member of the steroid hormone nuclear receptor superfamily with no identified endogenous ligand. NGFI-B induces apoptosis in a number of cell lineages exposed to proapoptotic stimuli by directly targeting the mitochondria, inducing cytochrome c release. The present study was designed to determine the role of NGFI-B in cardiomyocytes of restraint-stressed rats. The NGFI-B content was increased in mitochondria and reduced in plasma as apoptosis increased. Analysis showed that NGFI-B induces cardiomyocyte apoptosis in restraint-stressed rats by mediating mitochondrial energy metabolism disorder. Several novel mitochondrial proteins, which correlate with NGFI-B, were reported in cardiomyocyte apoptosis of restraint-stressed rats. Five proteins associated with NGFI-B participate directly in mitochondrial energy metabolism. Studies of mitochondrial respiratory efficiency and ATP synthase activity strongly support the findings. These results provide significant information for comprehensively understanding the cellular mechanism of cardiovascular diseases. PMID:19412742

  15. Optimization of a pre-MEKC separation SPE procedure for steroid molecules in human urine samples.

    PubMed

    Olędzka, Ilona; Kowalski, Piotr; Dziomba, Szymon; Szmudanowski, Piotr; Bączek, Tomasz

    2013-01-01

    Many steroid hormones can be considered as potential biomarkers and their determination in body fluids can create opportunities for the rapid diagnosis of many diseases and disorders of the human body. Most existing methods for the determination of steroids are usually time- and labor-consuming and quite costly. Therefore, the aim of analytical laboratories is to develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Due to the fact that there is little literature data on concentrations of steroid hormones in urine samples, we have made attempts at the electrophoretic determination of these compounds. For this purpose, an extraction procedure for the optimized separation and simultaneous determination of seven steroid hormones in urine samples has been investigated. The isolation of analytes from biological samples was performed by liquid-liquid extraction (LLE) with dichloromethane and compared to solid phase extraction (SPE) with C18 and hydrophilic-lipophilic balance (HLB) columns. To separate all the analytes a micellar electrokinetic capillary chromatography (MECK) technique was employed. For full separation of all the analytes a running buffer (pH 9.2), composed of 10 mM sodium tetraborate decahydrate (borax), 50 mM sodium dodecyl sulfate (SDS), and 10% methanol was selected. The methodology developed in this work for the determination of steroid hormones meets all the requirements of analytical methods. The applicability of the method has been confirmed for the analysis of urine samples collected from volunteers--both men and women (students, amateur bodybuilders, using and not applying steroid doping). The data obtained during this work can be successfully used for further research on the determination of steroid hormones in urine samples. PMID:24232737

  16. Analysis of cannabis in oral fluid specimens by GC-MS with automatic SPE.

    PubMed

    Choi, Hyeyoung; Baeck, Seungkyung; Kim, Eunmi; Lee, Sooyeun; Jang, Moonhee; Lee, Juseon; Choi, Hwakyung; Chung, Heesun

    2009-12-01

    Methamphetamine (MA) is the most commonly abused drug in Korea, followed by cannabis. Traditionally, MA analysis is carried out on both urine and hair samples and cannabis analysis in urine samples only. Despite the fact that oral fluid has become increasingly popular as an alternative specimen in the field of driving under the influence of drugs (DUID) and work place drug testing, its application has not been expanded to drug analysis in Korea. Oral fluid is easy to collect and handle and can provide an indication of recent drug abuse. In this study, we present an analytical method using GC-MS to determine tetrahydrocannabinol (THC) and its main metabolite 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid. The validated method was applied to oral fluid samples collected from drug abuse suspects and the results were compared with those in urine. The stability of THC and THC-COOH in oral fluid stored in different containers was also investigated. Oral fluid specimens from 12 drug abuse suspects, submitted by the police, were collected by direct expectoration. The samples were screened with microplate ELISA. For confirmation they were extracted using automated SPE with mixed-mode cation exchange cartridge, derivatized and analyzed by GC-MS using selective ion monitoring (SIM). The concentrations ofTHC and THC-COOH in oral fluid showed a large variation and the results from oral fluid and urine samples from cannabis abusers did not show any correlation. Thus, detailed information about time interval between drug use and sample collection is needed to interpret the oral fluid results properly. In addition, further investigation about the detection time window ofTHC and THC-COOH in oral fluid is required to substitute oral fluid for urine in drug testing. PMID:20120601

  17. Composition and evolution of Triton's icy surface between 2002-2014 from SpeX/IRTF

    NASA Astrophysics Data System (ADS)

    Holler, Bryan J.; Young, Leslie A.; Grundy, William M.; Olkin, Cathy B.

    2015-11-01

    We observed Triton in the near-infrared (0.7-2.5 μm) over 63 nights using the SpeX instrument at NASA's Infrared Telescope Facility (IRTF) between 2002 and 2014. Triton’s spectrum has absorption features due to N2, CO, CH4, CO2, and H2O in this wavelength range. We calculated the equivalent width (or fractional band depth for H2O) of select absorption bands in each of the 63 night-averaged spectra. Longitudinal distributions for the volatile ices (N2, CO, CH4) show large rotational amplitude, while the non-volatile ices (CO2, H2O) show little amplitude over one Triton rotation. Absorption from N2 and CH4 increased over the period of the observations, whereas absorption from the non-volatile ices remained constant. The sub-solar latitude on Triton is currently at -42 degrees south, so some areas of Triton are visible for a full rotation. Combined with our findings, this suggests that the southern latitudes are dominated by non-volatile ices, with larger concentrations of volatile ices found in the observable region north of the equator. Changing viewing geometry over the period of the observations explains the increase in volatile absorption: As the sub-solar point moves northwards, more of the volatile-rich northern regions are coming directly into view. Geological evidence from Voyager 2 pointed to a southern hemisphere dominated by volatile ices; significant changes have occurred over the intervening quarter century.

  18. Simple and fast determination of perfluorinated compounds in Taihu Lake by SPE-UHPLC-MS/MS.

    PubMed

    Zhu, Pengfei; Ling, Xia; Liu, Wenwei; Kong, Lingcan; Yao, Yuyang

    2016-09-15

    A simple and fast analytical method for determination of eleven Polyfluorinated Compounds (PFCs) in source water was developed in the present work. The water sample was prepared without filtered through microfiltration membrane and 500mL of source water was enriched by the solid phase extraction (SPE). The targent compounds were analyzed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The optimized analytical method was validated in terms of recovery, precision and method detection limits (MDLs). The recovery values after correction with the corresponding labeled standard were between 97.3 and 113.0% for samples spiked at 5ng/L, 10ng/L and 20ng/L. All PFCs showed good linearity and the linear correlation coefficient was over 0.99. The precisions were 1.0-9.0% (n=6). As the result of the enrichment, the MDL values ranged from 0.03 to 1.9ng/L and were enough for analysis of the trace levels of PFCs in the Taihu Lake. The method was further validated in determining the source water and the results showed that PFHxS, PFHxA, PFOA and PFOS were the primary PFCs in Taihu Lake which might be different from the other researches. The method can be used for determination of PFCs in water with a stable recovery, good reproducibility, low detection limit, less solvent consumption, time saving and labor saving. To our knowledge, this is the first method that describes the effect of the filter membrane on the determination of PFCs in water which might acquire more accurate concentration of PFCs in Taihu Lake. PMID:27454901

  19. The SpeX Prism Library Analysis Toolkit: Design Considerations and First Results

    NASA Astrophysics Data System (ADS)

    Burgasser, Adam J.; Aganze, Christian; Escala, Ivana; Lopez, Mike; Choban, Caleb; Jin, Yuhui; Iyer, Aishwarya; Tallis, Melisa; Suarez, Adrian; Sahi, Maitrayee

    2016-01-01

    Various observational and theoretical spectral libraries now exist for galaxies, stars, planets and other objects, which have proven useful for classification, interpretation, simulation and model development. Effective use of these libraries relies on analysis tools, which are often left to users to develop. In this poster, we describe a program to develop a combined spectral data repository and Python-based analysis toolkit for low-resolution spectra of very low mass dwarfs (late M, L and T dwarfs), which enables visualization, spectral index analysis, classification, atmosphere model comparison, and binary modeling for nearly 2000 library spectra and user-submitted data. The SpeX Prism Library Analysis Toolkit (SPLAT) is being constructed as a collaborative, student-centered, learning-through-research model with high school, undergraduate and graduate students and regional science teachers, who populate the database and build the analysis tools through quarterly challenge exercises and summer research projects. In this poster, I describe the design considerations of the toolkit, its current status and development plan, and report the first published results led by undergraduate students. The combined data and analysis tools are ideal for characterizing cool stellar and exoplanetary atmospheres (including direct exoplanetary spectra observations by Gemini/GPI, VLT/SPHERE, and JWST), and the toolkit design can be readily adapted for other spectral datasets as well.This material is based upon work supported by the National Aeronautics and Space Administration under Grant No. NNX15AI75G. SPLAT code can be found at https://github.com/aburgasser/splat.

  20. The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity.

    PubMed

    Hytönen, J; Haataja, S; Gerlach, D; Podbielski, A; Finne, J

    2001-01-01

    The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin-binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins. PMID:11136470

  1. SMG1 and NIK regulate apoptosis induced by Smac mimetic compounds

    PubMed Central

    Cheung, H H; St Jean, M; Beug, S T; Lejmi-Mrad, R; LaCasse, E; Baird, S D; Stojdl, D F; Screaton, R A; Korneluk, R G

    2011-01-01

    Smac mimetic compounds (SMCs) are experimental small molecules that induce tumour necrosis factor alpha (TNFα)-dependent cancer cell death by targeting the inhibitor of apoptosis proteins. However, many cancer cell lines are resistant to SMC-mediated apoptosis despite the presence of TNFα. To add insight into the mechanism of SMC-resistance, we used functional siRNA-based kinomic and focused chemical screens and identified suppressor of morphogenesis in genitalia-1 (SMG1) and NF-κB-inducing kinase (NIK) as novel protective factors. Both SMG1 and NIK prevent SMC-mediated apoptosis likely by maintaining FLICE inhibitory protein (c-FLIP) levels to suppress caspase-8 activation. In SMC-resistant cells, the accumulation of NIK upon SMC treatment enhanced the activity of both the classical and alternative nuclear factor-κB pathways, and increased c-FLIP mRNA levels. In parallel, persistent SMG1 expression in SMC-resistant cells repressed SMC-mediated TNFα-induced JNK activation and c-FLIP levels were sustained. Importantly, SMC-resistance is overcome by depleting NIK and SMG1, which appear to facilitate the downregulation of c-FLIP in response to SMC and TNFα treatment, leading to caspase-8-dependent apoptosis. Collectively, these data show that SMG1 and NIK function as critical repressors of SMC-mediated apoptosis by potentially converging on the regulation of c-FLIP metabolism. PMID:21490678

  2. Impacts of Salmonella enterica Serovar Typhimurium and Its speG Gene on the Transcriptomes of In Vitro M Cells and Caco-2 Cells

    PubMed Central

    Wang, Ke-Chuan; Huang, Chih-Hung; Huang, Ching-Jou

    2016-01-01

    Microfold or membranous (M) cells are specialized intestinal epithelial cells responsible for host immunity. The speG mutant of Salmonella Typhimurium (S. Typhimurium) is a nonreplicating strain within human cells to be a candidate vaccine vector for interacting with M cells. We conducted this study to identify the genes are differently expressed between in vitro M cells and Caco-2 cells, and to determine whether S. Typhimurium and speG affect the transcriptomes of both cell types. In vitro M cells and Caco-2 cells were infected with wild-type (WT) S. Typhimurium, its ΔspeG mutant, or none for 1 h for RNA microarrays; the transcriptomes among the 6 pools were pairwisely compared. Genetic loci encoding scaffold (e.g., HSCHR7_CTG4_4, HSCHR9_CTG9_35), long noncoding RNA, membrane-associated protein (PITPNB), neuron-related proteins (OR8D1, OR10G9, and NTNG2), and transporter proteins (MICU2 and SLC28A1) were significantly upregulated in uninfected M cells compared with uninfected Caco-2 cells; and their encoding proteins are promising M-cell markers. Significantly upregulated HSCHR7_CTG4_4 of uninfected in vitro M cells were speG-independently downregulated by S. Typhimurium infection that is a remarkable change representing an important but unreported characteristic of M cells. The immune responses of in vitro M cells and Caco-2 cells can differ and reply on speG or not, with speG-dependent regulation of KYL4, SCTR, IL6, TNF, and CELF4 in Caco-2 cells, JUN, KLF6, and KCTD11 in M cells, or speG-independent modulation of ZFP36 in both cells. This study facilitates understanding of the immune responses of in vitro M cells after administering the S. Typhimurium ΔspeG mutant as a future vaccine vector. PMID:27064787

  3. Geologic Assessment of the Damage Zone from the Second Test at Source Physics Experiment-Nevada (SPE-N)

    SciTech Connect

    ,

    2012-09-18

    The National Center for Nuclear Security (NCNS), established by the U.S. Department of Energy, National Nuclear Security Administration, is conducting a series of explosive tests at the Nevada National Security Site (NNSS; formerly the Nevada Test Site) that are designed to increase the understanding of certain basic physical phenomena associated with underground explosions. These tests will aid in developing technologies that might be used to detect underground nuclear explosions in support of verification activities for the Comprehensive Nuclear-Test-Ban Treaty (CTBT). The initial NCNS project is a series of explosive tests, known collectively as the Source Physics Experiment at the NNSS (SPE-N), being conducted in granitic rocks at the Climax stock in northern Yucca Flat. The SPE-N test series is designed to study the generation and propagation of seismic waves. The data will be used to improve the predictive capability of calculational models for detecting and characterizing underground explosions. The first SPE-N test (SPE-N-1) was a “calibration” shot conducted in May 2011, using 100 kilograms (kg) of explosives at the depth of 54.9 meters (m) (180 feet [ft]) in the U-15n source hole. SPE-N-2 was conducted in October 2011, using 1,000 kg of explosives at the depth of 45.7 m (150 ft) in the same source hole. Following the SPE-N-2 test, the core hole U-15n#10 was drilled at an angle from the surface to intercept the SPE-N-2 shot point location to obtain information necessary to characterize the damage zone. The desire was to determine the position of the damage zone near the shot point, at least on the northeast side, where the core hole penetrated it. The three-dimensional shape and symmetry of the damage zone are unknown at this time. Rather than spherical in shape, the dimensions of the damage zone could be influenced by the natural fracture sets in the vicinity. Geologic characterization of the borehole included geophysical logging, a directional survey

  4. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

    PubMed

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-07-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  5. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    PubMed Central

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  6. Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

    PubMed

    Blackburn, J; Kawasaki, K; Porntaveetus, T; Kawasaki, M; Otsuka-Tanaka, Y; Miake, Y; Ota, M S; Watanabe, M; Hishinuma, M; Nomoto, T; Oommen, S; Ghafoor, S; Harada, F; Nozawa-Inoue, K; Maeda, T; Peterková, R; Lesot, H; Inoue, J; Akiyama, T; Schmidt-Ullrich, R; Liu, B; Hu, Y; Page, A; Ramírez, Á; Sharpe, P T; Ohazama, A

    2015-01-01

    Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKβ, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkβ). K5-Ikkβ mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkβ mice. The supernumerary incisors in K5-Ikkβ mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions. PMID:25376721

  7. Role of Calpain in Apoptosis

    PubMed Central

    Momeni, Hamid Reza

    2011-01-01

    Apoptosis, a form of programmed cell death that occurs under physiological as well as pathological conditions, is characterized by morphological and biochemical features. While the importance of caspases in apoptosis is established, several noncaspase proteases (Ca2+-dependent proteases) such as calpain may play a role in the execution of apoptosis. The calpain family consists of two major isoforms, calpain I and calpain II which require µM and mM Ca2+ concentrations to initiate their activity. An increase in intracellular Ca2+ level is thought to trigger a cascade of biochemical processes including calpain activation. Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates, leading to the breakdown of cellular architecture and finally apoptosis. The activation of calpain has been implicated in neuronal apoptosis following spinal cord injuries and neurodegenerative diseases. This review focuses on calpain with an emphasis on its key role in the proteolysis of cellular protein substrates following apoptosis. PMID:23507938

  8. SpeB modulates fibronectin-dependent internalization of Streptococcus pyogenes by efficient proteolysis of cell-wall-anchored protein F1.

    PubMed

    Nyberg, Patrik; Rasmussen, Magnus; Von Pawel-Rammingen, Ulrich; Björck, Lars

    2004-05-01

    SpeB is a cysteine proteinase and virulence determinant secreted by the important human pathogen Streptococcus pyogenes. Recent investigations have suggested a role for SpeB in streptococcal entry into human cells. However, conflicting data concerning the contribution of SpeB to internalization have been presented. Protein F1 is a cell-wall-attached fibronectin (Fn)-binding protein that is present in a majority of streptococcal isolates and is important for internalization. This study shows that protein F1 is efficiently degraded by SpeB, and that removal of protein F1 from the bacterial surface leads to reduced internalization. Whereas M1 protein and protein H, two additional surface proteins of S. pyogenes that bind human plasma proteins, are protected from proteolytic degradation by their ligands, protein F1 is readily cleaved by SpeB also when in complex with Fn. This finding, and the connection between the presence of Fn at the bacterial surface and entry into human cells, suggest that SpeB plays a role in the regulation of the internalization process. PMID:15133117

  9. Pathophysiological Significance of Hepatic Apoptosis

    PubMed Central

    Wang, Kewei; Lin, Bingliang

    2013-01-01

    Apoptosis is a classical pathological feature in liver diseases caused by various etiological factors such as drugs, viruses, alcohol, and cholestasis. Hepatic apoptosis and its deleterious effects exacerbate liver function as well as involvement in fibrosis/cirrhosis and carcinogenesis. An imbalance between apoptotic and antiapoptotic capabilities is a prominent characteristic of liver injury. The regulation of apoptosis and antiapoptosis can be a pivotal step in the treatment of liver diseases. PMID:27335822

  10. Cucurbitacin B inhibits proliferation and induces apoptosis via STAT3 pathway inhibition in A549 lung cancer cells

    PubMed Central

    ZHANG, MENG; BIAN, ZHI-GANG; ZHANG, YI; WANG, JIA-HE; KAN, LIANG; WANG, XIN; NIU, HUI-YAN; HE, PING

    2014-01-01

    Natural products are a great source of cancer chemotherapeutic agents. The present study was conducted to investigate whether cucurbitacin B (CuB), one of the most potent and widely used cucurbitacins, inhibits proliferation and induces apoptosis in the A549 lung cancer cell line. Furthermore, CuB induced apoptosis of A549 cells in a concentration-dependent manner, as determined by fluorescence microscopy, flow cytometry and transmission electron microscopy. The present study also demonstrated that CuB dose-dependently inhibited lung cancer cell proliferation, with cell cycle inhibition and cyclin B1 downregulation. Apoptosis induced by CuB was shown to be associated with cytochrome c release, B-cell lymphoma 2 downregulation and signal transducer and activator of transcription 3 pathway inhibition. CuB may prove to be a useful approach for the chemotherapy of lung cancer. PMID:25242136

  11. Cucurbitacin B inhibits proliferation and induces apoptosis via STAT3 pathway inhibition in A549 lung cancer cells.

    PubMed

    Zhang, Meng; Bian, Zhi-Gang; Zhang, Yi; Wang, Jia-He; Kan, Liang; Wang, Xin; Niu, Hui-Yan; He, Ping

    2014-12-01

    Natural products are a great source of cancer chemotherapeutic agents. The present study was conducted to investigate whether cucurbitacin B (CuB), one of the most potent and widely used cucurbitacins, inhibits proliferation and induces apoptosis in the A549 lung cancer cell line. Furthermore, CuB induced apoptosis of A549 cells in a -concentration-dependent manner, as determined by fluorescence microscopy, flow cytometry and transmission electron microscopy. The present study also demonstrated that CuB dose-dependently inhibited lung cancer cell proliferation, with cell cycle inhibition and cyclin B1 downregulation. Apoptosis induced by CuB was shown to be associated with cytochrome c release, B-cell lymphoma 2 downregulation and signal transducer and activator of transcription 3 pathway inhibition. CuB may prove to be a useful approach for the chemotherapy of lung cancer. PMID:25242136

  12. JNK Signaling in Apoptosis

    PubMed Central

    Dhanasekaran, Danny N.; Reddy, E. Premkumar

    2011-01-01

    Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation pro-apoptotic genes via the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and anti-apoptotic proteins through distinct phosphorylation events. This review analyzes our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis PMID:18931691

  13. Direct vasoconstriction as a possible cause for amphotericin B-induced nephrotoxicity in rats.

    PubMed Central

    Sawaya, B P; Weihprecht, H; Campbell, W R; Lorenz, J N; Webb, R C; Briggs, J P; Schnermann, J

    1991-01-01

    In anesthetized rats we tested the hypothesis that amphotericin B (AmB) reduces glomerular filtration rate (GFR) by activating the tubuloglomerular feedback (TGF) mechanism. Infusion of 1 mg/kg AmB over 50 min was followed by a reduction in kidney GFR (from 0.47 +/- 0.03 to 0.39 +/- 0.02 ml/min per 100 g body wt during the second hour after infusion; P less than 0.05) and by an increase in urine flow and urinary chloride excretion. Single-nephron GFR (SNGFR) measured in proximal (TGF interrupted) or distal tubules (TGF intact) decreased to a similar degree from 33.4 +/- 1.8 and 30.6 +/- 1.2 nl/min in the control period to 19.7 +/- 1.9 and 21.2 +/- 1.6 nl/min during the second hour after AmB infusion (P less than 0.05). Distal chloride concentrations and TGF responses to changes in loop of Henle flow rate were not significantly altered by AmB. AmB at 10(-5) M reduced the diameter of isolated perfused afferent arterioles from rabbit kidneys. In isometrically contracting rings of rabbit aorta and renal artery in vitro AmB produced endothelium-independent constriction, with half-maximal contraction (EC50) being achieved by 1.8 x 10(-6) and 2.6 x 10(-6) M in intact vessels and 1.3 x 10(-6) and 1.7 x 10(-6) M in endothelium-denuded vessels respectively. Tension development did not occur in Ca-free media or in the presence of Ca channel blockers. Pretreatment with ouabain or Bay K 8644 potentiated the effect of AmB. The vasoconstrictive effect of AmB was counteracted by aminophylline and atrial natriuretic peptide. We conclude that the AmB-induced reduction in GFR is not caused by TGF activation and that AmB has a direct vasoconstrictor effect that is probably initiated by depolarization-induced opening of Ca channels. This effect may be an important component of the nephrotoxic actions of AmB. Images PMID:1710234

  14. The Methoxyflavonoid Isosakuranetin Suppresses UV-B-Induced Matrix Metalloproteinase-1 Expression and Collagen Degradation Relevant for Skin Photoaging.

    PubMed

    Jung, Hana; Lee, Eunjoo H; Lee, Tae Hoon; Cho, Man-Ho

    2016-01-01

    Solar ultraviolet (UV) radiation is a main extrinsic factor for skin aging. Chronic exposure of the skin to UV radiation causes the induction of matrix metalloproteinases (MMPs), such as MMP-1, and consequently results in alterations of the extracellular matrix (ECM) and skin photoaging. Flavonoids are considered as potent anti-photoaging agents due to their UV-absorbing and antioxidant properties and inhibitory activity against UV-mediated MMP induction. To identify anti-photoaging agents, in the present study we examined the preventative effect of methoxyflavonoids, such as sakuranetin, isosakuranetin, homoeriodictyol, genkwanin, chrysoeriol and syringetin, on UV-B-induced skin photo-damage. Of the examined methoxyflavonoids, pretreatment with isosakuranetin strongly suppressed the UV-B-mediated induction of MMP-1 in human keratinocytes in a concentration-dependent manner. Isosakuranetin inhibited UV-B-induced phosphorylation of mitogen-activated protein kinase (MAPK) signaling components, ERK1/2, JNK1/2 and p38 proteins. This result suggests that the ERK1/2 kinase pathways likely contribute to the inhibitory effects of isosakuranetin on UV-induced MMP-1 production in human keratinocytes. Isosakuranetin also prevented UV-B-induced degradation of type-1 collagen in human dermal fibroblast cells. Taken together, our findings suggest that isosakuranetin has the potential for development as a protective agent for skin photoaging through the inhibition of UV-induced MMP-1 production and collagen degradation. PMID:27598131

  15. Preparation of malathion MIP-SPE and its application in environmental analysis.

    PubMed

    Zuo, Hai Gen; Zhu, Jian Xin; Zhan, Chun Rui; Shi, Lei; Xing, Ming; Guo, Ping; Ding, Yuan; Yang, Hong

    2015-07-01

    Malathion is an organophosphorous insecticide for controlling insects on fruits and vegetables, miscellaneous household insects, and animal parasites. It is important to develop highly efficient and selective pre-treatment method for analyzing malathion residues in environment and samples from agricultural products based on the molecularly imprinted polymers (MIPs). In this study, we developed a tailor-made MIP method with highly specific recognization to the template. The MIPs were prepared using malathion as a template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinker, azodiisobutyronitrile (AIBN) as an initiator, and the acetonitrile-chloroform (1:1, v/v) as a porogen. The molecular recognization mechanism of malathion and MAA was evaluated by molecular simulation, ultraviolet spectrometry (UV), and (1)H-nuclear magnetic resonance ((1)H-NMR). MAA interacted specifically with malathion by hydrogen bond with a ratio of 2:1. The MIPs exhibit a high affinity, recognition specificity, and efficient adsorption performance for malathion. The Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), surface area and porosimeter analyzer, thermogravimetric/differential thermal analyzer (TG/DTA) were used to characterize the properties of MIP. The malathion residues in soil, tap water, and cabbage were cleaned up by MIP-SPE, detected quantitatively using GC-FPD, and confirmed by GC-MS/MS. The limits of tap water, soil, and cabbage were confined to 0.001 mg L(-1), 0.004 and 0.004 mg kg(-1), respectively. The spiked recoveries of malathion were 96.06-111.49% (with RSD being 5.7-9.2%), 98.13-103.83% (RSD, 3.5-8.7%), and 84.94-93.69% (RSD, 4.7-5.8%) for tap water, soil, and cabbage samples, respectively. Thus, the method developed here can be used effectively in assessing malathion residues in multiple environmental samples. The aim of the study was to provide an efficient, selective, and

  16. Discrete and continuum simulations of near-field ground motion from Source Physics Experiments (SPE) (Invited)

    NASA Astrophysics Data System (ADS)

    Ezzedine, S. M.; Vorobiev, O.; Herbold, E. B.; Glenn, L. A.; Antoun, T.

    2013-12-01

    This work is focused on analysis of near-field measurements (up to 100 m from the source) recorded during Source Physics Experiments in a granitic formation. One of the main goals of these experiments is to investigate the possible mechanisms of shear wave generation in the nonlinear source region. SPE experiments revealed significant tangential motion (up to 30 % of the magnitude in the radial direction) at many locations. Furthermore, azimuthal variations in radial velocities were also observed which cannot be generated by a spherical source in isotropic materials. Understanding the nature of this non-radial motion is important for discriminating between the natural seismicity and underground explosions signatures. Possible mechanisms leading to such motion include, but not limited to, heterogeneities in the rock such as joints, faults and geologic layers as well as surface topography and vertical motion at the surface caused by material spall and gravity. We have performed a three dimensional computational studies considering all these effects. Both discrete and continuum methods have been employed to model heterogeneities. In the discrete method, the joints and faults were represented by cohesive contact elements. This enables us to examine various friction laws at the joints which include softening, dilatancy, water saturation and rate-dependent friction. Yet this approach requires the mesh to be aligned with joints, which may present technical difficulties in three dimensions when multiple non-persistent joints are present. In addition, the discrete method is more computationally expensive. The continuum approach assumes that the joints are stiff and the dilatancy and shear softening can be neglected. In this approach, the joints are modeled as weakness planes within the material, which are imbedded into and pass through many finite elements. The advantage of this approach is that it requires neither sophisticated meshing algorithms nor contact detection

  17. Biomarkers of apoptosis

    PubMed Central

    Ward, T H; Cummings, J; Dean, E; Greystoke, A; Hou, J M; Backen, A; Ranson, M; Dive, C

    2008-01-01

    Within the era of molecularly targeted anticancer agents, it has become increasingly important to provide proof of mechanism as early on as possible in the drug development cycle, especially in the clinic. Selective activation of apoptosis is often cited as one of the major goals of cancer chemotherapy. Thus, the present minireview focuses on a discussion of the pros and cons of a variety of methodological approaches to detect different components of the apoptotic cascade as potential biomarkers of programmed cell death. The bulk of the discussion centres on serological assays utilising the technique of ELISA, since here there is an obvious advantage of sampling multiple time points. Potential biomarkers of apoptosis including circulating tumour cells, cytokeratins and DNA nucleosomes are discussed at length. However, accepting that a single biomarker may not have the power to predict proof of concept and patient outcome, it is clear that in the future more emphasis will be placed on technologies that can analyse panels of biomarkers in small volumes of samples. To this end the increased throughput afforded by multiplex ELISA technologies is discussed. PMID:19238626

  18. Use of two SPE-GC/MS methods for field isolation and quantitation of polycyclic aromatic hydrocarbons

    SciTech Connect

    Furlong, E.T.; Koleis, J.C.; Gates, P.M.

    1995-12-31

    Increasing sensitivity and subsequently improving method-detection limits for a variety of organic contaminant compounds is an ongoing effort in environmental analytical chemistry. Several field-deployable isolation techniques have been developed for concentrating hydrophobic organic contaminants from large (> 1 liter) volumes of aqueous samples. The capacity of large-volume solid-phase extraction (LV-SPE) was used as part of a simple, efficient, field-operable technique for isolating microgram-per-liter to nanogram-per-liter quantities of polycyclic aromatic hydrocarbons (PAH) in 4- to 8-liter ground-water samples from a petroleum- contaminated aquifer near Bemidji, Minn.

  19. Development of colorimetric solid Phase Extraction (C-SPE) for in-flight Monitoring of spacecraft Water Supplies

    SciTech Connect

    Daniel Bryan Gazda

    2004-12-19

    Although having recently been extremely successful gathering data on the surface of Mars, robotic missions are not an effective substitute for the insight and knowledge about our solar system that can be gained though first-hand exploration. Earlier this year, President Bush presented a ''new course'' for the U.S. space program that shifts NASA's focus to the development of new manned space vehicles to the return of humans to the moon. Re-establishing the human presence on the moon will eventually lead to humans permanently living and working in space and also serve as a possible launch point for missions into deeper space. There are several obstacles to the realization of these goals, most notably the lack of life support and environmental regeneration and monitoring hardware capable of functioning on long duration spaceflight. In the case of the latter, past experience on the International Space Station (ISS), Mir, and the Space Shuttle has strongly underscored the need to develop broad spectrum in-flight chemical sensors that: (1) meet current environmental monitoring requirements on ISS as well as projected requirements for future missions, and (2) enable the in-situ acquisition and analysis of analytical data in order to further define on-orbit monitoring requirements. Additionally, systems must be designed to account for factors unique to on-orbit deployment such as crew time availability, payload restrictions, material consumption, and effective operation in microgravity. This dissertation focuses on the development, ground testing, and microgravity flight demonstration of Colorimetric Solid Phase Extraction (C-SPE) as a candidate technology to meet the near- and long-term water quality monitoring needs of NASA. The introduction will elaborate further on the operational and design requirements for on-orbit water quality monitoring systems by discussing some of the characteristics of an ''ideal'' system. A description of C-SPE and how the individual

  20. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  1. Comparison of molecularly imprinted solid-phase extraction (MISPE) with classical solid-phase extraction (SPE) for the detection of benzodiazepines in post-mortem hair samples.

    PubMed

    Anderson, Robert A; Ariffin, Marinah M; Cormack, Peter A G; Miller, Eleanor I

    2008-01-15

    This preliminary study compares the benzodiazepine results for 10 post-mortem scalp hair samples using a classical solid-phase extraction (SPE) and a molecularly imprinted solid-phase extraction (MISPE) system. The hair samples selected for testing were from drug-related deaths where a positive benzodiazepine blood result was obtained. Samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water and dichloromethane, incubated overnight in methanol/25% aqueous ammonium hydroxide (20:1), extracted by SPE or MISPE and subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Both extraction methods detected diazepam, nordiazepam, oxazepam, temazepam and nitrazepam in the samples. Diazepam was detected in a greater number of samples using MISPE due to both its lower limit of detection (LOD) and higher extraction recovery as a result of excellent molecular recognition of the template (diazepam) imparted by the imprinting process. The selective recognition of two diazepam analogues, nordiazepam and oxazepam, was demonstrated using MISPE since they were also detected in a greater number of samples. In contrast, another diazepam analogue, temazepam, was detected in a greater number of samples using SPE since the LOD using this extraction was lower than with MISPE. Nitrazepam was detected in one sample using both extraction methods. Overall the MISPE and SPE hair results were in good qualitative agreement. For the samples, where both extraction methods detected nordiazepam, temazepam and oxazepam, the concentrations were always higher for SPE. This is probably due to the MIP procedure producing extracts with fewer matrix interferences than the extracts produced using the classical SPE method. MISPE could be used as a complementary method to classical SPE for the analysis of benzodiazepine positive hair samples collected from chronic users. PMID:17467213

  2. miR-125b induces cellular senescence in malignant melanoma

    PubMed Central

    2014-01-01

    Background Micro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory. Methods We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization. Results In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours. Conclusions Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma. PMID:24762088

  3. Small angle X-ray scattering data and structure factor fitting for the study of the quaternary structure of the spermidine N-acetyltransferase SpeG

    PubMed Central

    Weigand, Steven; Filippova, Ekaterina V.; Kiryukhina, Olga; Anderson, Wayne F.

    2015-01-01

    Here we describe the treatment of the small-angle X-ray Scattering (SAXS) data used during SpeG quaternary structure study as part of the research article “Substrate induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG” published in Journal of Molecular Biology [1]. These data were collected on two separate area detectors as separate dilution series of the SpeG and the SpeG with spermine samples along with data from their companion buffers. The data were radially integrated, corrected for incident beam variation, and scaled to absolute units. After subtraction of volume-fraction scaled buffer scattering and division by the SpeG concentration, multiple scattering curves free of an inter-molecular structure factor were derived from the dilution series. Rather than extrapolating to infinite dilution, the structure factor contribution was estimated by fitting to the full set of data provided by dividing the scattering curves of a dilution series by the curve from the most dilute sample in that series. PMID:26793756

  4. Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a Model for In Vivo Gene Expression†

    PubMed Central

    Loughman, Jennifer A.; Caparon, Michael

    2006-01-01

    For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo. PMID:16385029

  5. Small angle X-ray scattering data and structure factor fitting for the study of the quaternary structure of the spermidine N-acetyltransferase SpeG.

    PubMed

    Weigand, Steven; Filippova, Ekaterina V; Kiryukhina, Olga; Anderson, Wayne F

    2016-03-01

    Here we describe the treatment of the small-angle X-ray Scattering (SAXS) data used during SpeG quaternary structure study as part of the research article "Substrate induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG" published in Journal of Molecular Biology [1]. These data were collected on two separate area detectors as separate dilution series of the SpeG and the SpeG with spermine samples along with data from their companion buffers. The data were radially integrated, corrected for incident beam variation, and scaled to absolute units. After subtraction of volume-fraction scaled buffer scattering and division by the SpeG concentration, multiple scattering curves free of an inter-molecular structure factor were derived from the dilution series. Rather than extrapolating to infinite dilution, the structure factor contribution was estimated by fitting to the full set of data provided by dividing the scattering curves of a dilution series by the curve from the most dilute sample in that series. PMID:26793756

  6. Broad-Spectrum Antibiotic or G-CSF as Potential Countermeasures for Impaired Control of Bacterial Infection Associated with an SPE Exposure during Spaceflight

    PubMed Central

    Li, Minghong; Holmes, Veronica; Ni, Houping; Sanzari, Jenine K.; Romero-Weaver, Ana L.; Lin, Liyong; Carabe-Fernandez, Alejandro; Diffenderfer, Eric S.; Kennedy, Ann R.; Weissman, Drew

    2015-01-01

    A major risk for astronauts during prolonged space flight is infection as a result of the combined effects of microgravity, situational and confinement stress, alterations in food intake, altered circadian rhythm, and radiation that can significantly impair the immune system and the body’s defense systems. We previously reported a massive increase in morbidity with a decrease in the ability to control a bacterial challenge when mice were maintained under hindlimb suspension (HS) conditions and exposed to solar particle event (SPE)-like radiation. HS and SPE-like radiation treatment alone resulted in a borderline significant increase in morbidity. Therefore, development and testing of countermeasures that can be used during extended space missions in the setting of exposure to SPE radiation becomes a serious need. In the present study, we investigated the efficacy of enrofloxacin (an orally bioavailable antibiotic) and Granulocyte colony stimulating factor (G-CSF) (Neulasta) on enhancing resistance to Pseudomonas aeruginosa infection in mice subjected to HS and SPE-like radiation. The results revealed that treatment with enrofloxacin or G-CSF enhanced bacterial clearance and significantly decreased morbidity and mortality in challenged mice exposed to suspension and radiation. These results establish that antibiotics, such as enrofloxacin, and G-CSF could be effective countermeasures to decrease the risk of bacterial infections after exposure to SPE radiation during extended space flight, thereby reducing both the risk to the crew and the danger of mission failure. PMID:25793272

  7. [Apoptosis and its biomedical significance].

    PubMed

    Ortega-Camarillo, C; Díaz-Flores, M; Avalos-Rodríguez, A; Vergara-Onofre, M; Rosales-Torres, A M

    2001-01-01

    Cell death can occur through apoptotic or necrotic death pathways. Membrane disruption leads to inflammation, a typical feature of necrosis. Apoptosis constitutes a genetically controlled physiologic process of cell removal. It is characterized by cell shrinkage, chromatin condensation, and DNA cleavage. Apoptotic cells are rapidly recognized and engulfed by phagocytes thus inhibiting an inflammatory response following necrosis. Apoptosis has been proposed as a basic event to protect tissue homeostasis. This paper analyzes the genetic, biochemical, and morphologic characteristics related to apoptosis, as well as its relationship to certain illnesses. PMID:11766462

  8. Mitochondria and apoptosis: emerging concepts

    PubMed Central

    Li, Mark Xiang

    2015-01-01

    As mitochondria are the powerhouses of the cell, their damage during the cell suicide process of apoptosis is essentially responsible for cellular demise in most cells. A key family of proteins, the B-cell lymphoma-2 (BCL-2) family, determines the integrity of mitochondria in the face of apoptotic insult. A comprehensive understanding of the molecular details of how apoptosis is initiated and how it culminates is essential if apoptosis is to fulfil its undoubted potential as a therapeutic target to treat diseases ranging from cancer to neurodegenerative conditions. Recent advances have provided significant insight into the control of this fundamental process while prompting a re-evaluation of what was considered dogma in the field. Emerging evidence also points to a potential overarching control network that governs not only apoptosis but other fundamental mitochondrial processes, including mitochondrial fission/fusion and quality control. PMID:26097715

  9. Development and validation of an on-line multidimensional SPE-LC-MS/MS method for the quantitation of Tetrandrine in blood samples.

    PubMed

    Caglar, Sena; Morello, Rosa; Boos, Karl-Siegfried

    2015-04-15

    On-line solid-phase extraction (SPE) is becoming an increasingly widespread technique in the clean-up of complex matrices such as body fluids, prior to chromatographic analysis. The use of small SPE columns instead of disposable SPE cartridges allows multiple injections and complete automation. In addition, it decreases the cost of consumables and improves the quality of the overall analysis. Coupling of SPE with HPLC combines sample preparation and separation in one system. In this paper a validated on-line multidimensional (MD) SPE-LC-MS/MS method is described for the determination of Tetrandrine (model drug) in human blood samples. The developed method showed the applicability of direct injection of plasma samples to an on-line MD-SPE-LC-MS/MS system to determine small molecules i.e. drugs. The experimental design is unique. Quantification was through tandem mass spectrometry with positive electrospray ionization (ESI) and multiple reactions monitoring (MRM). The limit of detection was calculated as 31.98 ng/mL. The linear range of the method was between 40.0 and 800.0 ng/mL. Pharmacokinetic parameters are usually determined by analysis of drug concentrations in plasma rather than whole blood. Parameters determined using plasma data may be misleading if concentrations of drug differ between plasma and red blood cells. We successfully applied the developed method for the determination of the distribution coefficient of the model drug Tetrandrine between human red blood cells and blood plasma proteins. The determination of distribution coefficient study results demonstrated that the developed method can provide direct and accurate measurement of RBC partitioning in a model drug and could be applied for screening of other compounds for potential high RBC partition, predicting potential drug toxicity and investigating mechanisms associated with RBC partitions. PMID:25746132

  10. Beyond Apoptosis in Lupus

    PubMed Central

    Colonna, Lucrezia; Lood, Christian; Elkon, Keith B.

    2014-01-01

    Purpose of review Systemic lupus erythematosus (SLE) is characterized by autoantibodies directed against nuclear autoantigens normally concealed from immune recognition in healthy individuals. Here we summarize recently identified mechanisms of abnormal cell death leading to exposure and aberrant processing of nucleoprotein self antigens, and discuss their role in the SLE pathogenesis. Recent findings During the past few years, the unveiling of several new forms of cell death has expanded our understanding beyond the simple view of “apoptotic” versus “necrotic” cell death. SLE patients show abnormalities in cell death at several levels, including increased rates of apoptosis, necrosis, and autophagy, as well as reduced clearance of dying cells. These abnormalities lead to an increased autoantigen burden and also antigen modifications, such as nucleic acid oxidation that increase the inflammatory properties of self antigens. Recent investigations have highlighted the role of opsonins in determining the immunogenic versus tolerogenic characteristics of self antigens. Summary Dysregulation of different forms of programmed cell death contributes to increased exposure, availability, and immunogenic characteristic of intracellular self antigens, which all participate in development of lupus autoimmunity. As our understanding of abnormalities of cell death in SLE advances, potential therapeutic opportunities await human implementation. PMID:25036095

  11. Cytoskeleton and apoptosis.

    PubMed

    Ndozangue-Touriguine, Olivia; Hamelin, Jocelyne; Bréard, Jacqueline

    2008-07-01

    Apoptosis is a genetically programmed and physiological mode of cell death that leads to the removal of unwanted or abnormal cells. Cysteine-proteases called caspases are responsible for the apoptotic execution phase which is characterized by specific biochemical events as well as morphological changes. These changes, which lead to the orderly dismantling of the apoptotic cell, include cell contraction, dynamic membrane blebbing, chromatin condensation, nuclear disintegration, cell fragmentation followed by phagocytosis of the dying cell. They involve major modifications of the cytoskeleton which are largely mediated by cleavage of several of its components by caspases. For example, dynamic membrane blebbing is due to the increased contractility of the acto-myosin system following myosin light chain (MLC) phosphorylation. MLC phosphorylation is a consequence of the cleavage of a Rho GTPase effector, the kinase ROCK I, by caspase-3. This cleavage induces a constitutive kinase activity by removal of an inhibitory domain. Chromatin condensation is facilitated by the processing of lamins by caspases. Collapse of the cytokeratin network is mediated by cleavage of keratin 18. On another hand, the actin cytoskeleton rearrangement needed in the phagocyte for engulfment of the dying cell is due to the activation of the small GTPase Rac, a GTPase of the Rho family that induces actin polymerisation and formation of lamellipodia. In addition to mediating the morphological modifications of the apoptotic cell, several proteins of the cytoskeleton such as actin and keratins are also involved in the regulation of apoptotic signaling. PMID:18462707

  12. Effects of different light conditions on repair of UV-B-induced damage in carpospores of Chondrus ocellatus Holm

    NASA Astrophysics Data System (ADS)

    Ju, Qing; Xiao, Hui; Wang, You; Tang, Xuexi

    2015-05-01

    We evaluated the effects of ultraviolet-B (UV-B) radiation and different light conditions on the repair of UV-B-induced damage in carpospores of Chondrus ocellatus Holm (Rhodophyta) in laboratory experiments. Carpospores were treated daily with different doses of UV-B radiation for 48 days, when vertical branches had formed in all treatments; after each daily treatment, the carpospores were subjected to photosynthetically active radiation (PAR), darkness, red light, or blue light during a 2-h repair stage. Carpospore diameters were measured every 4 days. We measured the growth and cellular contents of cyclobutane pyrimidine dimers (CPDs), chlorophyll a, phycoerythrin, and UV-B-absorbing mycosporine-like amino acids (MAAs) in carpospores on Day 48. Low doses of UV-B radiation (36 and 72 J/m2) accelerated the growth of C. ocellatus. However, as the amount of UV-B radiation increased, the growth rate decreased and morphological changes occurred. UV-B radiation significant damaged DNA and photosynthetic pigments and induced three kind of MAAs, palythine, asterina-330, and shinorine. PAR conditions were best for repairing UV-B-induced damage. Darkness promoted the activity of the DNA darkrepair mechanism. Red light enhanced phycoerythrin synthesis but inhibited light repair of DNA. Although blue light, increased the activity of DNA photolyase, greatly improving remediation efficiency, the growth and development of C. ocellatus carpospores were slower than in other light treatments.

  13. Liver myofibroblasts up-regulate monocyte CD163 expression via PGE2 during hepatitis B induced liver failure

    PubMed Central

    2014-01-01

    Background Although patients with liver failure exhibit a generalized inflammatory-imbalance status, substantial evidence indicates that this immunosuppressive or anti-inflammatory state may be deleterious. Increased expression of CD163 (known to be involved in several anti-inflammatory functions of the immune system) in patients with liver failure is significantly correlated with a fatal outcome. However, little is known of the regulatory mechanisms that influence the expression of CD163. Methods We assessed the expression of CD163 on monocytes from both circulating cells and the liver tissues of patients with hepatitis B induced liver failure using flow cytometry and isolated the myofibroblasts from diseased livers. The ability of human liver myofibroblasts to regulate CD163 expression on monocytes was studied in vitro. Results We showed that CD163+ monocytes were enriched primarily in diseased livers and that they were associated with liver myofibroblasts in the same area. Accordingly, liver myofibroblasts were significantly superior to normal skin fibroblasts in inducing the expression of CD163 on monocytes in vitro. Moreover, we found that liver myofibroblasts triggered the activation of monocytes by secreting PGE2. Inhibition of PGE2 production in liver myofibroblasts using NS-398 markedly reduced CD163 expression in vitro. Conclusion These results suggest that liver myofibroblasts play a direct role in regulating the expression of CD163 on monocytes in human liver tissues and thereby may regulate monocyte function during hepatitis B induced liver failure. PMID:24597777

  14. Involvement of Inositol Biosynthesis and Nitric Oxide in the Mediation of UV-B Induced Oxidative Stress

    PubMed Central

    Lytvyn, Dmytro I.; Raynaud, Cécile; Yemets, Alla I.; Bergounioux, Catherine; Blume, Yaroslav B.

    2016-01-01

    The involvement of NO-signaling in ultraviolet B (UV-B) induced oxidative stress (OS) in plants is an open question. Inositol biosynthesis contributes to numerous cellular functions, including the regulation of plants tolerance to stress. This work reveals the involvement of inositol-3-phosphate synthase 1 (IPS1), a key enzyme for biosynthesis of myo-inositol and its derivatives, in the response to NO-dependent OS in Arabidopsis. Homozygous mutants deficient for IPS1 (atips1) and wild-type plants were transformed with a reduction- grx1-rogfp2 and used for the dynamic measurement of UV-B-induced and SNP (sodium nitroprusside)-mediated oxidative stresses by confocal microscopy. atips1 mutants displayed greater tissue-specific resistance to the action of UV-B than the wild type. SNP can act both as an oxidant or repairer depending on the applied concentration, but mutant plants were more tolerant than the wild type to nitrosative effects of high concentration of SNP. Additionally, pretreatment with low concentrations of SNP (10, 100 μM) before UV-B irradiation resulted in a tissue-specific protective effect that was enhanced in atips1. We conclude that the interplay between nitric oxide and inositol signaling can be involved in the mediation of UV-B-initiated oxidative stress in the plant cell. PMID:27148278

  15. Source spectra of the first four Source Physics Experiments (SPE) explosions from the frequency-domain moment-tensor inversion

    DOE PAGESBeta

    Yang, Xiaoning

    2016-01-01

    In this study, I used seismic waveforms recorded within 2 km from the epicenter of the first four Source Physics Experiments (SPE) explosions to invert for the moment-tensor spectra of these explosions. I employed a one-dimensional (1D) Earth model for Green's function calculations. The model was developed from P- and Rg-wave travel times and amplitudes. I selected data for the inversion based on the criterion that they had consistent travel times and amplitude behavior as those predicted by the 1D model. Due to limited azimuthal coverage of the sources and the mostly vertical-component-only nature of the dataset, only long-period, volumetricmore » components of the moment-tensor spectra were well constrained.« less

  16. Application of an ampholine-functionalized hybrid organic-inorganic silica material for the SPE of aromatic amines.

    PubMed

    Chen, Yihui; Wang, Tingting; Ma, Junfeng; Liang, Zhen; Chen, Mingliang; Fang, Jianghua; Gao, Haoqi; Zhang, Lihua; Zhang, Yukui

    2014-01-01

    An SPE cartridge based on an ampholine-functionalized hybrid organic-inorganic silica sorbent has been adopted for the analysis of aromatic amines including 4-aminobiphenyl, benzidine, 2-naphthylamine, p-chloroaniline, 2,4,5-trimethylaniline, and 3,3'-dichlorobenzidine. Crucial variables governing the extraction efficiency of the material such as the pH of sample, sample loading volume, solvent used for elution, and elution volume have been thoroughly optimized. The adsorption capacities for the six aromatic amines ranged from 0.17 to 1.82 μg/mg. The recoveries of aromatic amines spiked in textile samples ranged from 78.9 to 103.0%, with RSDs of 1.1-11.9% (n = 3). Moreover, the extraction efficiency of the ampholine-functionalized hybrid organic-inorganic silica sorbent was at least comparable with that of Oasis WCX. PMID:24178632

  17. Development and Application of a Novel SPE-Method for Bioassay-Guided Fractionation of Marine Extracts.

    PubMed

    Cutignano, Adele; Nuzzo, Genoveffa; Ianora, Adrianna; Luongo, Elvira; Romano, Giovanna; Gallo, Carmela; Sansone, Clementina; Aprea, Susanna; Mancini, Francesca; D'Oro, Ugo; Fontana, Angelo

    2015-09-01

    The biological diversity of marine habitats is a unique source of chemical compounds with potential use as pharmaceuticals, cosmetics and dietary supplements. However, biological screening and chemical analysis of marine extracts pose specific technical constraints and require adequate sample preparation. Here we report an improved method on Solid Phase Extraction (SPE) to fractionate organic extracts containing high concentration of salt that hampers the recovery of secondary metabolites. The procedure uses a water suspension to load the extracts on a poly(styrene-divynylbenzene)-based support and a stepwise organic solvent elution to effectively desalt and fractionate the organic components. The novel protocol has been tested on MeOH-soluble material from three model organisms (Reniera sarai, Dendrilla membranosa and Amphidinium carterae) and was validated on a small panel of 47 marine samples, including sponges and protists, within discovery programs for identification of immuno-stimulatory and anti-infective natural products. PMID:26378547

  18. Development and Application of a Novel SPE-Method for Bioassay-Guided Fractionation of Marine Extracts

    PubMed Central

    Cutignano, Adele; Nuzzo, Genoveffa; Ianora, Adrianna; Luongo, Elvira; Romano, Giovanna; Gallo, Carmela; Sansone, Clementina; Aprea, Susanna; Mancini, Francesca; D’Oro, Ugo; Fontana, Angelo

    2015-01-01

    The biological diversity of marine habitats is a unique source of chemical compounds with potential use as pharmaceuticals, cosmetics and dietary supplements. However, biological screening and chemical analysis of marine extracts pose specific technical constraints and require adequate sample preparation. Here we report an improved method on Solid Phase Extraction (SPE) to fractionate organic extracts containing high concentration of salt that hampers the recovery of secondary metabolites. The procedure uses a water suspension to load the extracts on a poly(styrene-divynylbenzene)-based support and a stepwise organic solvent elution to effectively desalt and fractionate the organic components. The novel protocol has been tested on MeOH-soluble material from three model organisms (Reniera sarai, Dendrilla membranosa and Amphidinium carterae) and was validated on a small panel of 47 marine samples, including sponges and protists, within discovery programs for identification of immuno-stimulatory and anti-infective natural products. PMID:26378547

  19. WDM compatible and electrically tunable SPE-OCDMA system based on the temporal self-imaging effect.

    PubMed

    Tainta, S; Amaya, W; Erro, M J; Garde, M J; Sales, S; Muriel, M A

    2011-02-01

    A coding/decoding setup for a spectral phase encoding optical code-division multiple access (SPE-OCDMA) system has been developed. The proposal is based on the temporal self-imaging effect and the use of an easily tunable electro-optic phase modulator to achieve line-by-line coding of the transmitted signal, thus assuring compatibility with WDM techniques. Modulation of the code is performed at the same rate as the data, avoiding the use of high-bandwidth electro-optic modulators. As proof of concept of the technique, experimental results are presented for a back-to-back coder/decoder setup transmitting a 10 GHz unmodulated optical pulse train within an 80 GHz optical window and using 8-chip Hadamard codes. PMID:21283203

  20. Determination of acrylamide in coffee and coffee products by GC-MS using an improved SPE clean-up.

    PubMed

    Soares, C; Cunha, S; Fernandes, J

    2006-12-01

    An improved gas chromatography-mass spectrometry (GC-MS) method to determine acrylamide (AA) in coffee and coffee products was developed. The method was based on two main purification steps: the first with ethanol and Carrez solutions in order to precipitate polysaccharides and proteins, respectively; and the second with a layered solid-phase extraction (SPE) column which proved to be efficient in the elimination of the main chromatographic interferences. The method is applicable to a wide range of coffee products. Twenty-six samples of different coffee products were analysed. The levels of AA were in the range 11.4-36.2 microg l-1 for 'espresso coffee' and 200.8-229.4 microg l-1 for coffee blends with cereals. The results indicate that the presence of cereals significantly increased the levels of AA. PMID:17118870

  1. Source spectra of the first four Source Physics Experiments (SPE) explosions from the frequency-domain moment-tensor inversion

    SciTech Connect

    Yang, Xiaoning

    2016-01-01

    In this study, I used seismic waveforms recorded within 2 km from the epicenter of the first four Source Physics Experiments (SPE) explosions to invert for the moment-tensor spectra of these explosions. I employed a one-dimensional (1D) Earth model for Green's function calculations. The model was developed from P- and Rg-wave travel times and amplitudes. I selected data for the inversion based on the criterion that they had consistent travel times and amplitude behavior as those predicted by the 1D model. Due to limited azimuthal coverage of the sources and the mostly vertical-component-only nature of the dataset, only long-period, volumetric components of the moment-tensor spectra were well constrained.

  2. Seismic source functions from free-field ground motions recorded on SPE: Implications for source models of small, shallow explosions

    NASA Astrophysics Data System (ADS)

    Rougier, Esteban; Patton, Howard J.

    2015-05-01

    Reduced displacement potentials (RDPs) for chemical explosions of the Source Physics Experiments (SPE) in granite at the Nevada Nuclear Security Site are estimated from free-field ground motion recordings. Far-field P wave source functions are proportional to the time derivative of RDPs. Frequency domain comparisons between measured source functions and model predictions show that high-frequency amplitudes roll off as ω- 2, but models fail to predict the observed seismic moment, corner frequency, and spectral overshoot. All three features are fit satisfactorily for the SPE-2 test after cavity radius Rc is reduced by 12%, elastic radius is reduced by 58%, and peak-to-static pressure ratio on the elastic radius is increased by 100%, all with respect to the Mueller-Murphy model modified with the Denny-Johnson Rc scaling law. A large discrepancy is found between the cavity volume inferred from RDPs and the volume estimated from laser scans of the emplacement hole. The measurements imply a scaled Rc of ~5 m/kt1/3, more than a factor of 2 smaller than nuclear explosions. Less than 25% of the seismic moment can be attributed to cavity formation. A breakdown of the incompressibility assumption due to shear dilatancy of the source medium around the cavity is the likely explanation. New formulas are developed for volume changes due to medium bulking (or compaction). A 0.04% decrease of average density inside the elastic radius accounts for the missing volumetric moment. Assuming incompressibility, established Rc scaling laws predicted the moment reasonable well, but it was only fortuitous because dilation of the source medium compensated for the small cavity volume.

  3. Reversed Phase SPE and GC-MS Study of Polycyclic Aromatic Hydrocarbons in Water Samples from the River Buriganga, Bangladesh

    PubMed Central

    Nawaz, Md. Saddam; Ferdousi, Farhana Khanam; Alam, A. M. Shafiqul

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are semivolatile organic compounds (SVOCs) categorized as persistent organic pollutants (POPs). PAHs are ubiquitous in terrestrial, atmospheric, and particularly aquatic environments throughout the world and have been detected in lakes, ground waters, and rivers. This research work involved the analysis of five PAHs, anthracene, fluorene, naphthalene, phenanthrene, and pyrene, in water sample collected from the river Buriganga, Bangladesh. The extraction of water samples was carried out by reversed phase solid-phase extraction (RP-SPE) technique with C-18 SPE cartridges. A solvent mixture of dichloromethane and hexane (1 : 2) with a flow rate of 0.5 mL/min was used as eluent. Percentage recoveries of five PAHs for this technique were in the range of 81.47 ± 1.16 to 98.60 ± 0.61%. PAHs quantification was achieved by using an ion trap gas chromatography mass spectrometer (GC-MS) interfaced to gas chromatography (GC) equipped with a fused silica capillary column. Helium was used as carrier gas with a flow rate of 1.0 mL/min. The commonly detected PAH compounds in the river water were anthracene, naphthalene, and phenanthrene at the concentration ranges of 0.451 to 3.201, 0.033 to 3.1131, and 0.320 to 2.546 μg/mL, respectively. The results reflect that PAHs presented in this river water were mostly from petrogenic and pyrogenic sources. PMID:27340687

  4. Simultaneous online SPE-HPLC-MS/MS analysis of docetaxel, temsirolimus and sirolimus in whole blood and human plasma.

    PubMed

    Navarrete, Alicia; Martínez-Alcázar, M Paz; Durán, Ignacio; Calvo, Emiliano; Valenzuela, Belén; Barbas, Coral; García, Antonia

    2013-03-15

    Docetaxel and temsirolimus are some of the most used drugs in a wide range of solid tumors. In preclinical studies, mTOR inhibitors such as temsirolimus have demonstrated synergistic cytotoxic effects with taxanes providing the rationale for combination studies. These anticancer agents exhibit a narrow therapeutic concentration range and due to their high inter- and intra-individual pharmacokinetic variability, therapeutic dose monitoring by highly sensitive methods as LC-MS/MS are important for clinical research. Therefore, the aim of this study was to develop and validate a sensitive, fast and convenient method for the simultaneous identification and quantification of docetaxel, temsirolimus and its main metabolite, sirolimus, using paclitaxel, another anticancer drug, as the internal standard. These analytes were quantified by an integrated online solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) system. Separation was performed on a Zorbax eclipse XDB-C8 (150mm×4.6mm, 5μm) column. The mass spectrometer tandem quadruple detector was equipped with jet stream electrospray ionization, monitored in multiple reactions monitoring (MRM) and operated in positive mode. A combination of protein precipitation with methanol/zinc sulphate (70:30) (v/v) and online SPE using a Zorbax eclipse plus C8 (12.5mm×4.6mm, 5μm) cartridge was used to extract the compounds. This method allows the use of the same reagents, sample treatment and analytical technique independently of whether the samples are whole blood or plasma. The method has been successfully validated and applied to real samples. It is a suitable method for dose adjustment and for evaluating potential drug interactions during combined treatments. PMID:23422405

  5. Reversed Phase SPE and GC-MS Study of Polycyclic Aromatic Hydrocarbons in Water Samples from the River Buriganga, Bangladesh.

    PubMed

    Nawaz, Md Saddam; Ferdousi, Farhana Khanam; Rahman, Mohammad Arifur; Alam, A M Shafiqul

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are semivolatile organic compounds (SVOCs) categorized as persistent organic pollutants (POPs). PAHs are ubiquitous in terrestrial, atmospheric, and particularly aquatic environments throughout the world and have been detected in lakes, ground waters, and rivers. This research work involved the analysis of five PAHs, anthracene, fluorene, naphthalene, phenanthrene, and pyrene, in water sample collected from the river Buriganga, Bangladesh. The extraction of water samples was carried out by reversed phase solid-phase extraction (RP-SPE) technique with C-18 SPE cartridges. A solvent mixture of dichloromethane and hexane (1 : 2) with a flow rate of 0.5 mL/min was used as eluent. Percentage recoveries of five PAHs for this technique were in the range of 81.47 ± 1.16 to 98.60 ± 0.61%. PAHs quantification was achieved by using an ion trap gas chromatography mass spectrometer (GC-MS) interfaced to gas chromatography (GC) equipped with a fused silica capillary column. Helium was used as carrier gas with a flow rate of 1.0 mL/min. The commonly detected PAH compounds in the river water were anthracene, naphthalene, and phenanthrene at the concentration ranges of 0.451 to 3.201, 0.033 to 3.1131, and 0.320 to 2.546 μg/mL, respectively. The results reflect that PAHs presented in this river water were mostly from petrogenic and pyrogenic sources. PMID:27340687

  6. Photo-protective effect of americanin B against ultraviolet B-induced damage in cultured human keratinocytes.

    PubMed

    Zheng, Jian; Piao, Mei Jing; Kim, Ki Cheon; Yao, Cheng Wen; Cha, Ji Won; Shin, Jennifer H; Yoo, Suk Jae; Hyun, Jin Won

    2014-11-01

    Excessive ultraviolet (UV) radiation, a constituent of sunlight, can induce multiple types of skin damage. We recently demonstrated that americanin B, a lignin compound, protected cells against hydrogen peroxide (H2O2)-induced damage by exerting antioxidant effects and inhibiting apoptosis. In this study, we investigated the ability of americanin B to protect against cell injury induced by UVB (280-320nm), the most harmful UV wavelengths, in human HaCaT keratinocytes. Americanin B absorbed UVB, eliminated UVB-induced intracellular reactive oxygen species (ROS), and decreased the extent of UVB-induced oxidative modification of lipids, proteins, and DNA. In addition, americanin B inhibited UVB-induced apoptosis, as indicated by reductions in apoptotic body formation and DNA fragmentation. Furthermore, americanin B reversed the depolarization of the mitochondrial membrane induced by UVB exposure. These protective activities were associated with down-regulation of apoptosis-promoting proteins, Bax, caspase-9, and caspase-3 and up-regulation of an apoptosis inhibitor, Bcl-2. These results suggest that americanin B can protect human keratinocytes against UVB-induced cell damage. PMID:25461549

  7. Spontaneous apoptosis in human thymocytes.

    PubMed Central

    Tiso, M.; Gangemi, R.; Bargellesi Severi, A.; Pizzolitto, S.; Fabbi, M.; Risso, A.

    1995-01-01

    Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 PMID:7639336

  8. Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

    SciTech Connect

    Mader, Jamie S.; Richardson, Angela; Salsman, Jayme; Top, Deniz; Antueno, Roberto de; Duncan, Roy; Hoskin, David W. . E-mail: d.w.hoskin@dal.ca

    2007-07-15

    Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.

  9. Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

    SciTech Connect

    González-Páez, Gonzalo E.; Wolan, Dennis W.

    2012-09-05

    Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50} values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.

  10. Effect of SPE-like Proton or Photon Radiation on the Kinetics of Mouse Peripheral Blood Cells and Radiation Biological Effectiveness Determinations

    PubMed Central

    Romero-Weaver, A.L.; Wan, X.S.; Diffenderfer, E.S.; Lin, L.

    2013-01-01

    Abstract Exploration missions outside low-Earth orbit are being planned; therefore, it is critical to understand the risk astronauts would be exposed to in the space environment, especially during extravehicular activities (EVAs). Reductions in white blood cell (WBC) numbers can occur as a result of exposure to solar particle event (SPE) radiation. The aim of the present study was to determine the duration of the effects on blood cell numbers from exposure to a single whole-body dose of SPE-like proton radiation or photon radiation as well as to determine the radiation biological effectiveness (RBE) values at those times when radiation exposure causes blood cell numbers to experience the most critical effects when using mice as a model. Our results indicate that both types of radiation cause significant reductions in the numbers of all blood cell types at different times post-irradiation. The RBE values were not significantly different from 1.0. These results indicate that the risk estimations for astronauts from exposure of mice to SPE-like proton radiation are comparable to those previously made for doses of standard reference radiations, suggesting that countermeasures should be developed for the decreases in blood cell counts observed following the exposure of mice to SPE radiation. Key Words: Proton radiation—Gamma radiation—Blood cell counts—Solar particle event. Astrobiology 13, 570–577. PMID:23980767

  11. Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

    PubMed Central

    González-Páez, Gonzalo E.; Wolan, Dennis W.

    2012-01-01

    Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 Å resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC50 values for trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products. PMID:22645124

  12. Simultaneous analysis of multiple classes of antimicrobials in environmental water samples using SPE coupled with UHPLC-ESI-MS/MS and isotope dilution.

    PubMed

    Tran, Ngoc Han; Chen, Hongjie; Do, Thanh Van; Reinhard, Martin; Ngo, Huu Hao; He, Yiliang; Gin, Karina Yew-Hoong

    2016-10-01

    A robust and sensitive analytical method was developed for the simultaneous analysis of 21 target antimicrobials in different environmental water samples. Both single SPE and tandem SPE cartridge systems were investigated to simultaneously extract multiple classes of antimicrobials. Experimental results showed that good extraction efficiencies (84.5-105.6%) were observed for the vast majority of the target analytes when extraction was performed using the tandem SPE cartridge (SB+HR-X) system under an extraction pH of 3.0. HPLC-MS/MS parameters were optimized for simultaneous analysis of all the target analytes in a single injection. Quantification of target antimicrobials in water samples was accomplished using 15 isotopically labeled internal standards (ILISs), which allowed the efficient compensation of the losses of target analytes during sample preparation and correction of matrix effects during UHPLC-MS/MS as well as instrument fluctuations in MS/MS signal intensity. Method quantification limit (MQL) for most target analytes based on SPE was below 5ng/L for surface waters, 10ng/L for treated wastewater effluents, and 15ng/L for raw wastewater. The method was successfully applied to detect and quantify the occurrence of the target analytes in raw influent, treated effluent and surface water samples. PMID:27474294

  13. Plant microtubules reorganization under the indirect UV-B exposure and during UV-B-induced programmed cell death

    PubMed Central

    Krasylenko, Yuliya A.; Yemets, Alla I.; Blume, Yaroslav B.

    2013-01-01

    The role of microtubules in cellular pathways of UV-B signaling in plants as well as in related structural cell response become into focus of few last publications. As microtubules in plant cell reorient/reorganize (become randomized, fragmented or depolymerized) in a response to direct UV-B exposure, these cytoskeletal components could be involved into UV-B signaling pathways as highly responsive players. In the current addendum, indirect UV-B-induced microtubules reorganization in cells of shielded Arabidopsis thaliana (GFP-MAP4) primary roots and the correspondence of microtubules depolymerization with the typical hallmarks of the programmed cell death in Nicotiana tabacum BY-2 (GFP-MBD) cells are discussed. PMID:23438586

  14. Granzyme B-Induced Neurotoxicity Is Mediated via Activation of PAR-1 Receptor and Kv1.3 Channel

    PubMed Central

    Wang, Tongguang; Lee, Myoung-Hwa; Choi, Elliot; Pardo-Villamizar, Carlos A.; Lee, Sung Bin; Yang, In Hong; Calabresi, Peter A.; Nath, Avindra

    2012-01-01

    Increasing evidence supports a critical role of T cells in neurodegeneration associated with acute and subacute brain inflammatory disorders. Granzyme B (GrB), released by activated T cells, is a cytotoxic proteinase which may induce perforin-independent neurotoxicity. Here, we studied the mechanism of perforin-independent GrB toxicity by treating primary cultured human neuronal cells with recombinant GrB. GrBactivated the protease-activated receptor (PAR)-1 receptor on the neuronal cell surface leading to decreased intracellular cyclic AMP levels. This was followed by increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane. Similar expression of Kv1.3 was also seen in neurons of the cerebral cortex adjacent to active inflammatory lesions in patients with multiple sclerosis. Kv1.3 expression was followed by activation of Notch-1 resulting in neurotoxicity. Blocking PAR-1, Kv1.3 or Notch-1 activation using specific pharmacological inhibitors or siRNAs prevented GrB-induced neurotoxicity. Furthermore, clofazimine protected against GrB-induced neurotoxicity in rat hippocampus, in vivo. These observations indicate that GrB released from T cells induced neurotoxicity by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1. These pathways provide novel targets to treat T cell-mediated neuroinflammatory disorders. Kv1.3 is of particular interest since it is expressed on the cell surface, only under pathological circumstances, and early in the cascade of events making it an attractive therapeutic target. PMID:22952817

  15. SpeI restriction enzyme displays greater discriminatory power than XbaI enzyme does in a pulsed-field gel electrophoresis study on 146 clinical Burkholderia pseudomallei isolates.

    PubMed

    Chua, Kek Heng; See, Kah Heng; Thong, Kwai Lin; Puthucheary, S D

    2011-01-01

    Restriction enzymes SpeI and XbaI were used in a pulsed-field gel electrophoresis (PFGE) study for molecular characterization of 146 clinical Burkholderia pseudomallei isolates. The PFGE parameters were optimized to enable comparable, reproducible, and robust results. The optimized parameters for both SpeI and XbaI restriction enzymes used in this study were 200 V and a pulse time of 5 to 65 s for a 28-h runtime. Using SpeI, 9 different clusters were identified, whereas 6 clusters were identified by XbaI digestion, which exhibited 85% similarity to SpeI. SpeI (discrimination index [D]=0.854) showed higher discriminatory power than XbaI did (D=0.464). PMID:21617308

  16. Apoptosis deregulation in myeloproliferative neoplasms

    PubMed Central

    Tognon, Raquel; Nunes, Natália de Souza; de Castro, Fabíola Attié

    2013-01-01

    ABSTRACT Philadelphia-chromosome negative chronic myeloproliferative neoplasms are clonal hematologic diseases characterized by hematopoietic progenitor independence from or hypersensitivity to cytokines. The cellular and molecular mechanisms involved in the pathophysiology of myeloproliferative neoplasms have not yet been fully clarified. Pathophysiologic findings relevant for myeloproliferative neoplasms are associated with genetic alterations, such as, somatic mutation in the gene that codifies JAK-2 (JAK V617F). Deregulation of the process of programmed cellular death, called apoptosis, seems to participate in the pathogenesis of these disorders. It is known that expression deregulation of pro- and anti-apoptotic genes promotes cell resistance to apoptosis, culminating with the accumulation of myeloid cells and establishing neoplasms. This review will focus on the alterations in apoptosis regulation in myeloproliferative neoplasms, and the importance of a better understanding of this mechanism for the development of new therapies for these diseases. PMID:24488400

  17. Molecular mechanisms of hepatic apoptosis

    PubMed Central

    Wang, K

    2014-01-01

    Apoptosis is a prominent feature of liver diseases. Causative factors such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response, can induce apoptotic cell death via membrane receptors and intracellular stress. Apoptotic signaling network, including membrane death receptor-mediated cascade, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, lysosomal permeabilization, and mitochondrial dysfunction, is intermixed each other, but one mechanism may dominate at a particular stage. Mechanisms of hepatic apoptosis are complicated by multiple signaling pathways. The progression of liver disease is affected by the balance between apoptotic and antiapoptotic capabilities. Therapeutic options of liver injury are impacted by the clear understanding toward mechanisms of hepatic apoptosis. PMID:24434519

  18. Liquid Metering Centrifuge Sticks (LMCS): A Centrifugal Approach to Metering Known Sample Volumes for Colorimetric Solid Phase Extraction (C-SPE)

    NASA Technical Reports Server (NTRS)

    Gazda, Daniel B.; Schultz, John R.; Clarke, Mark S.

    2007-01-01

    Phase separation is one of the most significant obstacles encountered during the development of analytical methods for water quality monitoring in spacecraft environments. Removing air bubbles from water samples prior to analysis is a routine task on earth; however, in the absence of gravity, this routine task becomes extremely difficult. This paper details the development and initial ground testing of liquid metering centrifuge sticks (LMCS), devices designed to collect and meter a known volume of bubble-free water in microgravity. The LMCS uses centrifugal force to eliminate entrapped air and reproducibly meter liquid sample volumes for analysis with Colorimetric Solid Phase Extraction (C-SPE). C-SPE is a sorption-spectrophotometric platform that is being developed as a potential spacecraft water quality monitoring system. C-SPE utilizes solid phase extraction membranes impregnated with analyte-specific colorimetric reagents to concentrate and complex target analytes in spacecraft water samples. The mass of analyte extracted from the water sample is determined using diffuse reflectance (DR) data collected from the membrane surface and an analyte-specific calibration curve. The analyte concentration can then be calculated from the mass of extracted analyte and the volume of the sample analyzed. Previous flight experiments conducted in microgravity conditions aboard the NASA KC-135 aircraft demonstrated that the inability to collect and meter a known volume of water using a syringe was a limiting factor in the accuracy of C-SPE measurements. Herein, results obtained from ground based C-SPE experiments using ionic silver as a test analyte and either the LMCS or syringes for sample metering are compared to evaluate the performance of the LMCS. These results indicate very good agreement between the two sample metering methods and clearly illustrate the potential of utilizing centrifugal forces to achieve phase separation and metering of water samples in microgravity.

  19. Pre-Shot Simulations of Far-Field Ground Motions for the Source Physics Experiment (SPE) Explosions at the Climax Stock, Nevada National Security Site

    SciTech Connect

    Rodgers, A J; Wagoner, J; Petersson, N A; Sjogreen, B

    2010-11-07

    The Source Physics Experiment (SPE) will involve a series of explosions in various geologic and emplacement conditions to validate numerical simulation methods to predict behavior of seismic wave excitation and propagation for nuclear test monitoring. The first SPE's currently underway involve explosions in the Climax Stock (granitic geology) at the Nevada National Security Site (NNSS). Detailed geologic data and published material properties for the major lithologic units of the NNSS and surrounding region were used to build three-dimensional models for seismic wave propagation simulations. The geologic structure near the SPE shot point is quite varied including granitic, carbonate, tuff and alluvium lithologies. We performed preliminary ground motion simulations for a near-source domain covering 8 km x 8 km at the surface centered on the shot point to investigate various source and propagation effects using WPP, LLNL's anelastic seismic wave finite difference code. Simulations indicate that variations in wave propagation properties of the sub-surface will generate strongly path-dependent response once the energy has left the relatively small granitic geology of the near-surface Climax Stock near the SPE shot point. Rough topography to the north and west of SPE shot point causes additional complexity in the signals including energy on the transverse components. Waves propagate much faster through the granitic and carbonate formations and slower through the tuff and alluvium. Synthetic seismograms for a pure explosion source in a 3D geologic structure show large amplitudes on transverse component. For paths to the south sampling the granite, tuff and alluvium lithologies transverse component amplitudes are as high as 50% of that on the vertical and radial components.

  20. Genetic inactivation of an extracellular cysteine protease (SpeB) expressed by Streptococcus pyogenes decreases resistance to phagocytosis and dissemination to organs.

    PubMed

    Lukomski, S; Burns, E H; Wyde, P R; Podbielski, A; Rurangirwa, J; Moore-Poveda, D K; Musser, J M

    1998-02-01

    Streptococcal pyrogenic exotoxin B (SpeB), a conserved cysteine protease expressed by virtually all Streptococcus pyogenes strains, has recently been shown to be an important virulence factor (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574-2580, 1997). Genetic inactivation of SpeB significantly decreased the lethality of a serotype M49 strain for mice and abolished the lethality of a serotype M3 strain after intraperitoneal (i.p.) injection. In the present study, a wild-type M3 isolate and an M3 speB mutant derivative were used to investigate the mechanism responsible for altered virulence. Following i.p. injection, the mutant and wild-type strains induced virtually identical cellular inflammatory responses, characterized largely by an influx of polymorphonuclear leukocytes (PMNs). In addition, the mutant and wild-type strains rapidly entered the blood and were recovered from all organs examined. However, significantly fewer (P < 0.05) CFUs of the isogenic mutant derivative than of the wild-type parent strain were recovered from blood and organs. PMNs effectively cleared the M3 speB mutant from the peritoneum by 22 h, thereby sparing the host. In contrast, the wild-type M3 strain continued to replicate intraperitoneally and had the ability to kill phagocytes. This process allowed the wild-type strain to continuously disseminate, resulting in host death. Our results indicate that genetic inactivation of the cysteine protease decreased the resistance of the mutant to phagocytosis and impaired its subsequent dissemination to organs. These results provide insight into the detrimental effect of SpeB inactivation on virulence. PMID:9453640

  1. Genetic Inactivation of an Extracellular Cysteine Protease (SpeB) Expressed by Streptococcus pyogenes Decreases Resistance to Phagocytosis and Dissemination to Organs

    PubMed Central

    Lukomski, Slawomir; Burns, Eugene H.; Wyde, Philip R.; Podbielski, Andreas; Rurangirwa, Jacqueline; Moore-Poveda, Donna K.; Musser, James M.

    1998-01-01

    Streptococcal pyrogenic exotoxin B (SpeB), a conserved cysteine protease expressed by virtually all Streptococcus pyogenes strains, has recently been shown to be an important virulence factor (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574–2580, 1997). Genetic inactivation of SpeB significantly decreased the lethality of a serotype M49 strain for mice and abolished the lethality of a serotype M3 strain after intraperitoneal (i.p.) injection. In the present study, a wild-type M3 isolate and an M3 speB mutant derivative were used to investigate the mechanism responsible for altered virulence. Following i.p. injection, the mutant and wild-type strains induced virtually identical cellular inflammatory responses, characterized largely by an influx of polymorphonuclear leukocytes (PMNs). In addition, the mutant and wild-type strains rapidly entered the blood and were recovered from all organs examined. However, significantly fewer (P < 0.05) CFUs of the isogenic mutant derivative than of the wild-type parent strain were recovered from blood and organs. PMNs effectively cleared the M3 speB mutant from the peritoneum by 22 h, thereby sparing the host. In contrast, the wild-type M3 strain continued to replicate intraperitoneally and had the ability to kill phagocytes. This process allowed the wild-type strain to continuously disseminate, resulting in host death. Our results indicate that genetic inactivation of the cysteine protease decreased the resistance of the mutant to phagocytosis and impaired its subsequent dissemination to organs. These results provide insight into the detrimental effect of SpeB inactivation on virulence. PMID:9453640

  2. Monitoring apoptosis in real time.

    PubMed

    Green, Allan M; Steinmetz, Neil D

    2002-01-01

    Many therapeutically active anticancer treatments exert their effect by the induction of apoptosis and necrosis. Serial biopsies in breast cancer patients have suggested that response to therapy correlates with early posttreatment increases in tumor apoptotic index. Radiolabeled technetium Tc 99m-recombinant human (rh) annexin V provides a noninvasive technique for imaging treatment-induced cell death. Annexin V is a naturally occurring human protein that binds avidly to membrane-associated phosphatidylserine (PS). PS is normally found only on the inner leaflet of the cell membrane double layer, but it is actively transported to the outer layer as an early event in apoptosis and becomes available for annexin binding. Annexin also gains access to PS as a result of the membrane fragmentation associated with necrosis. In vitro studies of apoptosis using fluorescein annexin have shown good correlation with assessments of apoptosis documented by nuclear DNA degradation and caspase activation. In vivo localization of intravenously administered Tc 99m-annexin V has been demonstrated in numerous preclinical models of apoptosis, including anti-Fas-mediated hepatic apoptosis, rejection of allogeneic heterotopic cardiac allografts, cyclophosphamide treatment of murine lymphoma, cyclophosphamide-induced apoptosis in bone marrow, and leukocyte apoptosis associated with abscess formation. Scintigraphic studies in humans using Tc 99m-rh annexin V have demonstrated the feasibility of imaging cell death in acute myocardial infarction, in tumors with a high apoptotic index, and in response to anti-tumor chemotherapy of non-small cell lung cancer, small-cell lung cancer, breast cancer, lymphoma, and sarcoma. Increased localization of Tc 99m-rh annexin V within 1 to 3 days of chemotherapy has been noted in some, but not all, subjects with these tumors. To date, most subjects showing increased Tc 99m-rh annexin V uptake after the first course of chemotherapy have shown objective

  3. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    PubMed

    Kozjak-Pavlovic, Vera; Dian-Lothrop, Elke A; Meinecke, Michael; Kepp, Oliver; Ross, Katharina; Rajalingam, Krishnaraj; Harsman, Anke; Hauf, Eva; Brinkmann, Volker; Günther, Dirk; Herrmann, Ines; Hurwitz, Robert; Rassow, Joachim; Wagner, Richard; Rudel, Thomas

    2009-10-01

    The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection. PMID:19851451

  4. The Source Physics Experiments (SPE): A Physics-Based Approach to Discriminate Low-Yield Nuclear Events (Invited)

    NASA Astrophysics Data System (ADS)

    Snelson, C. M.; Chipman, V.; White, R. L.; Emmitt, R.; Townsend, M.

    2013-12-01

    Discriminating low-yield nuclear explosions is one of the current challenges in the field of monitoring and verification. Work is currently underway in Nevada to address this challenge by conducting a series of experiments using a physics-based approach. This has been accomplished by using a multifaceted, multi-disciplinary approach that includes a range of activities, from characterizing the shallow subsurface to acquiring new explosion data both in the near field (< 100 m from the source) to the far field (> 100 m to 10 s km from the source). The Source Physics Experiment (SPE) is a collaborative project between National Security Technologies, LLC, Lawrence Livermore National Laboratory, Los Alamos National Laboratory, Sandia National Laboratories, the Defense Threat Reduction Agency, and the Air Force Technical Applications Center. The goal of the SPE is to understand the transition of seismic energy from the near field to the far field; to understand the development of S-waves in explosives sources; and to understand how anisotropy controls seismic energy transmission and partitioning. To fully explore these problems, the SPE test series includes tests in both simple and complex geology cases. The current series is being conducted in a highly fractured granite body. This location was chosen, in part, because it was the location of previous nuclear tests in the same rock body and because generally the geology has been well characterized. In addition to historic data, high-resolution seismic reflection, cross-hole tomography, core samples, LIDAR, hyperspectral, and fracture mapping data have been acquired to further characterize and detect changes after each of the shot across the test bed. The complex geology series includes 7 planned shots using conventional explosives in the same shot hole surrounded by Continuous Reflectometry for Radius vs. Time Experiment (CORRTEX), Time of Arrival, Velocity of Detonation, down-hole accelerometers, surface accelerometers

  5. Pancreatic carcinogenesis: apoptosis and angiogenesis.

    PubMed

    Onizuka, Shinya; Kawakami, Shunsuke; Taniguchi, Ken; Fujioka, Hikaru; Miyashita, Kosei

    2004-04-01

    Apoptosis and angiogenesis are critical biologic processes that are altered during carcinogenesis. Both apoptosis and angiogenesis may play an important role in pancreatic carcinogenesis. Despite numerous advances in the diagnosis and treatment of pancreatic cancer, its prognosis remains dismal and a new therapeutic approach is much needed. Recent research has revealed that apoptosis and angiogenesis are closely interrelated. Several reports show that a tumor suppresser gene that is expressed in pancreatic carcinoma and related to malignant potential can induce apoptosis and also inhibit angiogenesis. At present, it is generally accepted that tumor growth in cancers, including pancreatic cancer, depends on angiogenesis. We have identified 2 new angiogenesis inhibitors from a conditioned medium of human pancreatic carcinoma cell line (BxPC-3): antiangiogenic antithrombin III (aaAT-III) and vitamin D binding protein-macrophage activating factor (DBP-maf). These molecules were able to regress tumors in severe combined immunodeficiency disease (SCID) mice, demonstrating potent inhibition of endothelial cell proliferation. Moreover, the angiogenesis inhibitors induced tumor dormancy in the animal model. These results suggest that antiangiogenic therapy using angiogenesis inhibitors may become a new strategy for treatment of pancreatic cancer in the near future. PMID:15084979

  6. Fluorescence Lifetime Imaging of Apoptosis

    PubMed Central

    Xiao, Annie; Gibbons, Anne E.; Luker, Kathryn E.; Luker, Gary D.

    2015-01-01

    Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools to analyze cell signaling and function at single cell resolution in standard two-dimensional cell cultures, but these reporters rarely have been applied to three-dimensional environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in absorption of light complicate this analysis method in three-dimensional settings. Here we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time, to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from two-dimensional cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity. PMID:26771007

  7. APOPTOSIS IN WHOLE MOUSE OVARIES

    EPA Science Inventory

    Apoptosis in Whole Mouse Ovaries
    Robert M. Zucker Susan C. Jeffay and Sally D. Perreault
    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711.

  8. Calmodulin antagonists induce platelet apoptosis.

    PubMed

    Wang, Zhicheng; Li, Suping; Shi, Quanwei; Yan, Rong; Liu, Guanglei; Dai, Kesheng

    2010-04-01

    Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis. PMID:20172594

  9. Application of HPLC-DAD after SPE/QuEChERS with ZrO2-based sorbent in d-SPE clean-up step for pesticide analysis in edible oils.

    PubMed

    Tuzimski, Tomasz; Rejczak, Tomasz

    2016-01-01

    In this study, the solid-phase extraction/quick, easy, cheap, effective, rugged and safe (SPE/QuEChERS) technique was adapted to develop a simple sample treatment for multi-residue pesticide analysis of edible oils. The proposed method is based on liquid-liquid partitioning with acetonitrile followed by dispersive solid phase extraction using zirconia-coated silica particles for extract purification. To evaluate the described method, 21 pesticides belonging to different chemical classes were analysed using high performance liquid chromatography with diode-array detection (HPLC-DAD). For validation purposes, recovery studies were performed at 75 ng g(-1), 125 ng g(-1), 250 ng g(-1), 500 ng g(-1) and 1000 ng g(-1) levels. Recoveries were over the range of 50-130% for most of the analytes, with relative standard deviations less than 15% being observed. HPLC-DAD provided suitable linearity, precision and accuracy. The validated method was successfully applied to the analysis of edible oil samples selected from the market. PMID:26212943

  10. Real-Time Data Management, IP Telemetry, Data Integration, and Data Center Operations for the Source Physics Experiment (SPE), Nevada National Security Site

    NASA Astrophysics Data System (ADS)

    Plank, G.; Slater, D.; Torrisi, J.; Presser, R.; Williams, M.; Smith, K. D.

    2012-12-01

    The Nevada Seismological Laboratory (NSL) manages time-series data and high-throughput IP telemetry for the National Center for Nuclear Security (NCNS) Source Physics Experiment (SPE), underway on the Nevada National Security Site (NNSS). During active-source experiments, SPE's heterogeneous systems record over 350 channels of a variety of data types including seismic, infrasound, acoustic, and electro-magnetic. During the interim periods, broadband and short period instruments record approximately 200 channels of continuous, high-sample-rate seismic data. Frequent changes in sensor and station configurations create a challenging meta-data environment. Meta-data account for complete operational histories, including sensor types, serial numbers, gains, sample rates, orientations, instrument responses, data-logger types etc. To date, these catalogue 217 stations, over 40 different sensor types, and over 1000 unique recording configurations (epochs). Facilities for processing, backup, and distribution of time-series data currently span four Linux servers, 60Tb of disk capacity, and two data centers. Bandwidth, physical security, and redundant power and cooling systems for acquisition, processing, and backup servers are provided by NSL's Reno data center. The Nevada System of Higher Education (NSHE) System Computer Services (SCS) in Las Vegas provides similar facilities for the distribution server. NSL staff handle setup, maintenance, and security of all data management systems. SPE PIs have remote access to meta-data, raw data, and CSS3.0 compilations, via SSL-based transfers such as rsync or secure-copy, as well as shell access for data browsing and limited processing. Meta-data are continuously updated and posted on the Las Vegas distribution server as station histories are better understood and errors are corrected. Raw time series and refined CSS3.0 data compilations with standardized formats are transferred to the Las Vegas data server as available. For better

  11. Determination of etoxazole residues in fruits and vegetables by SPE clean-up and HPLC-DAD.

    PubMed

    Malhat, Farag; Badawy, Hany; Barakat, Dalia; Saber, Ayman

    2013-01-01

    A method for determination of etoxazole residues in apples, strawberries and green beans was developed and validated. The analyte was extracted with acetonitrile from foodstuff and a charcoal-celite cartridge was used for clean-up of raw extracts. Reversed phase high performance liquid chromatography with photodiode array detector (HPLC-DAD) was used for the determination and quantification of etoxazole residues in the studied samples. The calibration graphs of etoxazole in a solvent or three blank matrixes were linear within the tested intervals 0.01-2 mg L(-1), with correlation coefficient of determination >0.999. The combined solid phase extraction (SPE) clean-up and the chromatographic method steps were sensitive and reliable for simultaneous determination of etoxazole residues in the studied samples. The average recoveries of etoxazole in the tested foodstuffs were between 93.4 to 102% at spiking levels of 0.01, 0.10, and 0.50 mg kg(-1), with relative standard deviations ranging from 2.8 to 4.7%, in agreement with directives for method validation in residue analyses. The limit of detection (LOD) of the HPLC-DAD system was 100 pg. The limit of quantification of the entire method was 0.01 mg kg(-1). PMID:23431971

  12. Rapid Determination of Dichlofluanid Residues in Vegetables Using Dispersive-SPE Sample Preparation Combined with Gas Chromatography-Mass Spectrometry.

    PubMed

    Zhou, Xue; Cao, Shurui; Li, Xianliang; Xi, Cunxian; Ding, Xiaowen; Xu, Fen; Hu, Jiangtao; Chen, Zhiqiong

    2016-05-01

    A method for rapid determination of dichlofluanid residue in vegetables using dispersive solid-phase extraction (dispersive-SPE) sample preparation combined with gas chromatography-mass spectrometry (GC-MS) was developed. Samples were extracted with actone-ethyl acetate (1:1, V/V), and then detected by GC-MS with an external standard method after being purified by optimized primary secondary amine, graphitized carbon black and anhydrous magnesium sulphate (MgSO4). It turned out that dichlofluanid showed a good linearity (y= 2.7E+ 5x- 2710.5) over the range of 0.02-2.00 mg/L with a correlation coefficient of 0.9994. The limit of detection was 0.13 μg/kg (S/N = 3) and the limit of quantification was 0.43 µg/kg (S/N = 10). The recoveries of the dichlofluanid were in the range of 73.3-106.7, 83.3-116.7 and 83.3∼106.7% with the spiked levels of 0.01, 0.02 and 0.05 mg/kg, and the relative standard deviations were in the range of 4.1-22.3%. Compared with the reported literature, the method is more simple, rapid, sensitive, reliable and can be applied to many vegetables. PMID:26921896

  13. Investigation of structural heterogeneity at the SPE site using combined P–wave travel times and Rg phase velocities

    DOE PAGESBeta

    Rowe, Charlotte A.; Patton, Howard J.

    2015-10-01

    Here, we present analyses of the 2D seismic structure beneath Source Physics Experiments (SPE) geophone lines that extended radially at 100 m spacing from 100 to 2000 m from the source borehole. With seismic sources at only one end of the geophone lines, standard refraction profiling methods cannot resolve seismic velocity structures unambiguously. In previous work, we demonstrated overall agreement between body-wave refraction modeling and Rg dispersion curves for the least complex of the five lines. A more detailed inspection supports a 2D reinterpretation of the structure. We obtained Rg phase velocity measurements in both the time and frequency domains,more » then used iterative adjustment of the initial 1D body-wave model to predict Rg dispersion curves to fit the observed values. Our method applied to the most topographically severe of the geophone lines is supplemented with a 2D ray-tracing approach, whose application to P-wave arrivals supports the Rg analysis. In addition, midline sources will allow us to refine our characterization in future work.« less

  14. Glycyrrhizic acid pretreatment prevents sepsis-induced acute kidney injury via suppressing inflammation, apoptosis and oxidative stress.

    PubMed

    Zhao, Hongyu; Liu, Zhenning; Shen, Haitao; Jin, Shuai; Zhang, Shun

    2016-06-15

    Glycyrrhizic acid (GA), an active ingredient in licorice, has multiple pharmacological activities. The aim of our study was to investigate the molecular mechanism involved in the protective effects of GA in lipopolysaccharide (LPS) stimulated rat mesangial cells (HBZY-1) and septic rats. Sepsis model was established by injection of 5mg/kg LPS in rats or incubation with 1μg/ml LPS for 24h in HBZY-1 cells. A variety of molecular biological experiments were carried out to assess the effects of GA on inflammation, apoptosis, and oxidative stress. First we found that GA alleviated sepsis-induced kidney injury in vivo. Furthermore, GA suppressed inflammatory response in vivo and in vitro. Additionally, GA inhibited cell apoptosis and the changes in expressions of apoptosis related proteins induced by LPS. Moreover, GA markedly inhibited oxidative stress induced by LPS via activation of ERK signaling pathway. Finally GA could inhibit the activation of NF-κ B induced by LPS. Our present study indicates that GA has a protective effect against sepsis-induced inflammatory response, apoptosis, and oxidative stress damage, which provides a molecular basis for a new medical treatment of septic acute kidney injury. PMID:27063444

  15. Nitric Oxide Functions as a Signal in Ultraviolet-B-Induced Baicalin Accumulation in Scutellaria baicalensis Suspension Cultures

    PubMed Central

    Zhang, Jin-Jie; Li, Xue-Qin; Sun, Jun-Wei; Jin, Song-Heng

    2014-01-01

    Stress induced by ultraviolet-B (UV-B) irradiation stimulates the accumulation of various secondary metabolites in plants. Nitric oxide (NO) serves as an important secondary messenger in UV-B stress-induced signal transduction pathways. NO can be synthesized in plants by either enzymatic catalysis or an inorganic nitrogen pathway. The effects of UV-B irradiation on the production of baicalin and the associated molecular pathways in plant cells are poorly understood. In this study, nitric oxide synthase (NOS) activity, NO release and the generation of baicalin were investigated in cell suspension cultures of Scutellaria baicalensis exposed to UV-B irradiation. UV-B irradiation significantly increased NOS activity, NO release and baicalin biosynthesis in S. baicalensis cells. Additionally, exogenous NO supplied by the NO donor, sodium nitroprusside (SNP), led to a similar increase in the baicalin content as the UV-B treatment. The NOS inhibitor, Nω-nitro-l-arginine (LNNA), and NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) partially inhibited UV-B-induced NO release and baicalin accumulation. These results suggest that NO is generated by NOS or NOS-like enzymes and plays an important role in baicalin biosynthesis as part of the defense response of S. baicalensis cells to UV-B irradiation. PMID:24646913

  16. Hydrogen peroxide activates NFkappaB and the interleukin-6 promoter through NFkappaB-inducing kinase.

    PubMed

    Zhang, J; Johnston, G; Stebler, B; Keller, E T

    2001-06-01

    Aging is associated not only with oxidant stress, but also with increased interleukin-6 (IL-6) levels. To determine if oxidative stress could contribute to the age-associated increase IL-6 expression, we exposed LNCaP prostate carcinoma cells and HeLa cervical carcinoma cells to H2O2 as an oxidant challenge. We found that H2O2 induced IL-6 expression through activation of the IL-6 promoter. Furthermore, H2O2-induced activation of the promoter was mediated through nuclear factor-kappaB (NFkappaB) secondary to H2O2-induced phosphorylation and degradation of IkappaBalpha. NFkappaB-inducing kinase (NIK) is upstream of the IkappaB kinase complex that induces IkappaBalpha degradation. Accordingly, we explored if H2O2 induces IL-6 expression through NIK. In addition to H2O2 inducing NIK autophosphorylation, transfection of LNCaP cells with a dominant negative NIK diminished H2O2-mediated NFkappaB and IL-6 promoter activity. Taken together, these results demonstrate that H2O2 induces the IL-6 promoter by activating NFkappaB through NIK. These data provide a candidate mechanism through which oxidant challenge induces IL-6 gene expression with age. PMID:11491660

  17. Nuclear factor kappa B-inducing kinase and Ikappa B kinase-alpha signal skeletal muscle cell differentiation.

    PubMed

    Canicio, J; Ruiz-Lozano, P; Carrasco, M; Palacin, M; Chien, K; Zorzano, A; Kaliman, P

    2001-06-01

    Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK), IkappaB kinase (IKK)-alpha and -beta, and IkappaBalpha are common elements that signal NF-kappaB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappaB DNA-binding activity that correlated in time with the activation of IKKalpha, IKKbeta, and NIK. Moreover, the activation of IKKalpha, IKKbeta, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IkappaBalpha(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappaB activation and myoblast differentiation, indicating that phosphorylation of IkappaBalpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II. PMID:11279241

  18. Cell intrinsic role of NF-κB-inducing kinase in regulating T cell-mediated immune and autoimmune responses

    PubMed Central

    Li, Yanchuan; Wang, Hui; Zhou, Xiaofei; Xie, Xiaoping; Chen, Xiang; Jie, Zuliang; Zou, Qiang; Hu, Hongbo; Zhu, Lele; Cheng, Xuhong; Brightbill, Hans D; Wu, Lawren C.; Wang, Linfang; Sun, Shao-Cong

    2016-01-01

    NF-κB inducing kinase (NIK) is a central component of the noncanonical NF-κB signaling pathway. Although NIK has been extensively studied for its function in the regulation of lymphoid organ development and B-cell maturation, the role of NIK in regulating T cell functions remains unclear and controversial. Using T cell-conditional NIK knockout mice, we here demonstrate that although NIK is dispensable for thymocyte development, it has a cell-intrinsic role in regulating the homeostasis and function of peripheral T cells. T cell-specific NIK ablation reduced the frequency of effector/memory-like T cells and impaired T cell responses to bacterial infection. The T cell-conditional NIK knockout mice were also defective in generation of inflammatory T cells and refractory to the induction of a T cell-dependent autoimmune disease, experimental autoimmune encephalomyelitis. Our data suggest a crucial role for NIK in mediating the generation of effector T cells and their recall responses to antigens. Together, these findings establish NIK as a cell-intrinsic mediator of T cell functions in both immune and autoimmune responses. PMID:26912039

  19. Nitric oxide functions as a signal in ultraviolet-B-induced baicalin accumulation in Scutellaria baicalensis suspension cultures.

    PubMed

    Zhang, Jin-Jie; Li, Xue-Qin; Sun, Jun-Wei; Jin, Song-Heng

    2014-01-01

    Stress induced by ultraviolet-B (UV-B) irradiation stimulates the accumulation of various secondary metabolites in plants. Nitric oxide (NO) serves as an important secondary messenger in UV-B stress-induced signal transduction pathways. NO can be synthesized in plants by either enzymatic catalysis or an inorganic nitrogen pathway. The effects of UV-B irradiation on the production of baicalin and the associated molecular pathways in plant cells are poorly understood. In this study, nitric oxide synthase (NOS) activity, NO release and the generation of baicalin were investigated in cell suspension cultures of Scutellaria baicalensis exposed to UV-B irradiation. UV-B irradiation significantly increased NOS activity, NO release and baicalin biosynthesis in S. baicalensis cells. Additionally, exogenous NO supplied by the NO donor, sodium nitroprusside (SNP), led to a similar increase in the baicalin content as the UV-B treatment. The NOS inhibitor, Nω-nitro-l-arginine (LNNA), and NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) partially inhibited UV-B-induced NO release and baicalin accumulation. These results suggest that NO is generated by NOS or NOS-like enzymes and plays an important role in baicalin biosynthesis as part of the defense response of S. baicalensis cells to UV-B irradiation. PMID:24646913

  20. Activation of p53 contributes to pseudolaric acid B-induced senescence in human lung cancer cells in vitro

    PubMed Central

    Yao, Guo-dong; Yang, Jing; Li, Qiang; Zhang, Ye; Qi, Min; Fan, Si-miao; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2016-01-01

    Aim: Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi Gordon, has shown to exert anti-tumor effects via inducing cell cycle arrest followed by apoptosis in several cancer cell lines. Here we reported that PAB induced a mitotic catastrophe in human lung cancer A549 cells, which resulted in senescence without apoptosis or necrosis. Methods: Three human lung cancer cell lines (A549, H460 and H1299 cells) were examined. Cell growth inhibition was assessed with MTT assay. Cell cycle distribution was determined using a flow cytometer. Cell nuclear morphology was observed under a fluorescence microscope. Senescent cells were detected using SA-β-Gal staining. Apoptotic and senescent protein expression was examined using Western blot analysis. The expression of p53 and p21 in the cells was downregulated by siRNAs. Results: Treatment with PAB (5–80 μmol/L) inhibited the growth of A549 cells in dose- and time-dependent manners. Prolonged treatment with PAB (20 μmol/L) caused G2/M arrest at day 1 followed by mitotic catastrophe from day 2, which eventually resulted in cell senescence between days 3 and 4 without cell death (apoptosis or necrosis). Knockdown of p53 expression with siRNA significantly suppressed PAB-induced senescence in A549 cells (p53 wild). Furthermore, PAB-induced senescence was also observed in human lung cancer H460 cells (p53 wild), but not in human lung cancer H1299 cells (p53 null). Conclusion: The anti-tumor action of PAB against human lung cancer A549 cells in vitro involves the induction of senescence through activation of the p53 pathway. PMID:27041461

  1. Use of diffusion-ordered NMR spectroscopy and HPLC-UV-SPE-NMR to identify undeclared synthetic drugs in medicines illegally sold as phytotherapies.

    PubMed

    Silva, Lorena M A; Filho, Elenilson G A; Thomasi, Sérgio S; Silva, Bianca F; Ferreira, Antonio G; Venâncio, Tiago

    2013-09-01

    The informal (and/or illegal) e-commerce of pharmaceutical formulations causes problems that governmental health agencies find hard to control, one of which concerns formulas sold as natural products. The purpose of this work was to explore the advantages and limitations of DOSY and HPLC-UV-SPE-NMR. These techniques were used to identify the components of a formula illegally marketed in Brazil as an herbal medicine possessing anti-inflammatory and analgesic properties. DOSY was able to detect the major components present at higher concentrations. Complete characterization was achieved using HPLC-UV-SPE-NMR, and 1D and 2D NMR analyses enabled the identification of known synthetic drugs. These were ranitidine and a mixture of orphenadrine citrate, piroxicam, and dexamethasone, which are co-formulated in a remedy called Rheumazim that is used to relieve severe pain, but it is prohibited in Brazil because of a lack of sufficient pharmacokinetic and pharmacodynamic information. PMID:23818305

  2. Effect of carvedilol on cardiomyocyte apoptosis in a rat model of myocardial infarction: A role for toll-like receptor 4

    PubMed Central

    Liu, Qingwei; Zhang, Jianhua; Xu, Yan; Huang, Ying; Wu, Changhao

    2013-01-01

    Objectives: Toll-like receptor 4 (TLR4) is crucial in cardiomyocyte apoptosis induced by myocardial infarction (MI) and carvedilol has been reported to have anti-apoptotic effects. We hypothesized that the effects of this agent are in part mediated through TLR4 signaling pathways. Materials and Methods: A total of 48 rats were randomized to the following groups before surgery: sham-operated group (n = 8), MI group (n = 10) and three carvedilol-treatment groups (n = 30, 2 mg/kg, 10 mg/kg and 30 mg/kg). Sham and MI groups were given vehicle and carvedilol groups received different dose carvedilol, by direct gastric gavage for 7 days. On the 4th day of drug or vehicle administration, MI model was produced by ligating the left anterior descending coronary artery. On day 3 after MI, apoptosis was assessed by TdT-UTP nick-end assay; the levels of expression of Bax, Bcl-2, TLR4 and nuclear factor-κB (NF-κB) in infarcted myocardium were analyzed by immunohistochemistry. Results: Carvedilol ameliorated MI-induced apoptosis in a dose-dependent manner. In parallel, carvedilol also decreased the ratio of Bax to Bcl-2, the expression of TLR4 and NF-κB induced by MI. The extent of apoptosis and Bax-Bcl-2 ratio was strongly correlated with the TLR4 levels. Conclusion: This study suggests that the short-term administration of carvedilol can significantly alleviate cardiomyocyte apoptosis in the infarcted myocardium probably by inhibiting the excessive expression of TLR4 and NF-κB induced by infarction. PMID:24130379

  3. Pin1 in Neuronal Apoptosis

    PubMed Central

    Becker, Esther B.E.; Bonni, Azad

    2009-01-01

    While the role of the prolyl isomerase Pin1 in dividing cells has long been recognized, Pin1’s function in postmitotic neurons is poorly understood. We have identified a novel mechanism by which Pin1 mediates activation of the mitochondrial cell death machinery specifically in neurons. This perspective presents a sophisticated signaling pathway that triggers neuronal apoptosis upon JNK-mediated phosphorylation of the BH3-only protein BIMEL at serine 65. Pin1 is enriched at the mitochondria in neurons together with BIMEL and components of a neuron-specific JNK signaling complex and functions as a molecular switch that couples the phosphorylation of BIMEL by JNK to apoptosis specifically in neurons. We discuss how these findings relate to our understanding of the development of the nervous system and the pathogenesis of neurologic disorders. PMID:17568190

  4. Inhibitors of apoptosis catch ubiquitin.

    PubMed

    Rajalingam, Krishnaraj; Dikic, Ivan

    2009-01-01

    IAP (inhibitor of apoptosis) proteins are a class of anti-apoptotic regulators characterized by the presence of BIR (baculoviral IAP repeat) domains. Some of the IAPs also possess a RING (really interesting new gene) domain with E3 ubiquitin ligase activity. In this issue of the Biochemical Journal, Blankenship et al. unveil the presence of an UBA (ubiquitin-associated domain) in several IAPs. UBAs in c-IAPs (cellular IAPs) bind to monoubiquitin and ubiquitin chains and are implicated in degradation of c-IAPs by promoting their interaction with proteasomes as well as in regulation of TNF-alpha (tumour necrosis factor-alpha)-induced apoptosis. These novel observations establish IAPs as ubiquitin-interacting proteins and opens up new lines of investigation. PMID:19061481

  5. An SPE-assisted BODIPY fluorometric paper sensor for the highly selective and sensitive determination of Cd²⁺ in complex sample: rice.

    PubMed

    Zhang, Yu; Li, Hui; Niu, Li-Ya; Yang, Qing-Zheng; Guan, Ya-Feng; Feng, Liang

    2014-06-21

    By using sensing technology, the individual component analysis at trace level in complex samples remains problematic simply because of various interfering species. For example, the determination of Cd(2+) in rice is difficult due to the co-existing interfering metal cations at thousands or even millions of times higher concentrations. In this study, a heavy-metal ion sensitive BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-based fluorometric paper sensor with assistance of solid phase extraction (SPE) was developed for the highly selective and sensitive determination of trace Cd(2+) in rice. SPE column packed with prepared sulfonated PS-DVB microspheres was employed to enrich trace Cd(2+) and meanwhile remove most interfering heavy-metal ions in simulated complex rice sample with oxalic acid as eluent, which was theoretically selected on the basis of f values. Mn(2+), as a major coexistent heavy-metal ion, could not be easily removed by SPE, but showed little fluorescent response for BODIPY fluorometric paper sensor even in excess amounts. Combining the separation and enrichment capability of SPE column with the selectivity of BODIPY-based fluorometric paper sensor, we were able to detect trace Cd(2+) in complex samples. The response of fluorometric paper sensor was linearly related with Cd(2+) concentrations in the range of 0.5-4 μM, with a detection limit of 0.5 μM. Twelve real rice samples spiked with Cd(2+) were analysed using this method and the results are in good agreement with ICP-MS measurements. PMID:24802241

  6. Determination of polychlorinated biphenyls and organochlorine pesticides in small volumes of human blood by high-throughput on-line SPE-LVI-GC-HRMS.

    PubMed

    Wittsiepe, Jürgen; Nestola, Marco; Kohne, Matthias; Zinn, Peter; Wilhelm, Michael

    2014-01-15

    A fully automated and robust method featuring on-line solid-phase extraction (SPE) and large volume injection (LVI) gas chromatographic (GC) high resolution mass spectrometry (HRMS) is used to determine polychlorinated biphenyls (PCBs) and organochlorine pesticides, such as penta- and hexachlorobenzene (PeCBz, HxCBz), hexachlorocyclohexane isomers (HCH) and 4,4'-dichlorodiphenyldichloroethene (a metabolite of dichlorodiphenyltrichloroethane (DDT)), with only 200μl of human blood, serum or plasma. After spiking the sample with (13)C-labeled internal standards and precipitating the proteins, the sample is passed through a 10mm×2.0mm ID SPE cartridge filled with C18 material that adsorbs the analytes. After washing and drying, the cartridge is extracted with hexane/dodecane (99/1, v/v); the extract is directly injected into a LVI where GC/HRMS analysis follows. The fully automated system utilizes a robotic autosampler and a modular SPE system including two high-pressure syringe pumps, an automatic SPE cartridge exchanger unit and 6 switchable valves. All sample preparation steps are performed within 20min during the GC run of a previous sample, limiting the throughput with only the GC runtime. The contents are quantified using the isotope dilution method. Due to laboratory air contamination problems, we achieved LOQs of 0.017 (PeCBz), 0.009 (HxCBz), 0.007 (HCH), 0.016 (DDE), while for the six indicator PCBs, we achieved values of 0.030 (PCB-28), 0.044 (PCB-52), 0.024 (PCB-101), 0.009 (PCB-138), 0.015 (PCB-153) and 0.008 (PCB-180)μg/l serum. Under clean laboratory air conditions, these values may be improved. This method is recommended when high throughput is desirable and/or only small amounts of material are available, such as during studies involving children. PMID:24361859

  7. Apoptosis in irradiated murine tumors.

    PubMed

    Stephens, L C; Ang, K K; Schultheiss, T E; Milas, L; Meyn, R E

    1991-09-01

    Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors. PMID:1886987

  8. Direct rapid analysis of multiple PPCPs in municipal wastewater using ultrahigh performance liquid chromatography-tandem mass spectrometry without SPE pre-concentration.

    PubMed

    Yu, Ke; Li, Bing; Zhang, Tong

    2012-08-13

    Ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was utilized to develop a rapid, sensitive and reliable method without solid phase extraction (SPE) pre-concentration for trace analysis of 11 pharmaceuticals and personal care products (PPCPs) in influent and effluent from municipal wastewater treatment plants (WWTPs). This method not only shortened the analysis time but also reduced analysis cost significantly by omitting SPE process and avoiding the consumption of SPE cartridge. Detection parameters for UHPLC-MS/MS analysis were optimized, including sample pH, eluent, mobile phase (solvent and additive), column temperature, and flow rate. Under the optimal conditions, all analytes were well separated and detected within 8.0min by UHPLC-MS/MS. The method quantification limits (MQLs) for the 11 PPCPs ranged from 0.040 to 88ngL(-1) and from 0.030 to 90ngL(-1) for influent and effluent, respectively. The matrix effect was systematically investigated and quantified for different types of samples. The analysis of influent and effluent samples of two WWTPs in Hong Kong revealed the presence of 11 PPCPs, including acyclovir, benzophenone-3, benzylparaben, carbamazepine, ethylparaben, fluconazole, fluoxetine, methylparaben, metronidazole, propylparaben, and ranitidine. Their concentrations ranged from 9.1 to 1810ngL(-1) in influent and from 6.5 to 823ngL(-1) in effluent samples collected from Hong Kong WWTPs. PMID:22790701

  9. Application of Colorimetric Solid Phase Extraction (C-SPE) to Monitoring Nickel(II) and Lead(II) in Spacecraft Water Supplies

    NASA Technical Reports Server (NTRS)

    Diaz, Neil C.; Gazda, Daniel B.; Fritz, James S.; Porter, Marc D.; Rutz, Jeff; Mudgett, Paul; Schultz, John

    2004-01-01

    Archived water samples collected on the International Space Station (ISS) and returned to Earth for analysis have, in a few instances, contained trace levels of heavy metals. Building on our previous advances using Colorimetric Solid Phase Extraction (C-SPE) as a biocide monitoring technique, we are devising methods for the low level monitoring of nickel(II), lead(II) and other heavy metals. C-SPE is a sorption-spectrophotometric platform based on the extraction of analytes onto a membrane impregnated with a colorimetric reagent that are then quantified on the surface of the membrane using a diffuse reflectance spectrophotometer. Along these lines, we have determined nickel(II) via complexation with dimethylglyoxime (DMG) and begun to examine the analysis of lead(II) by its reaction with 2,5- dimercapto-1,3,4-thiadiazole (DMTD) and 4-(2- pyridylazo)-resorcinol (PAR). These developments are also extending a new variant of C-SPE in which immobilized reagents are being incorporated into this methodology in order to optimize sample reaction conditions and to introduce the colorimetric reagent. This paper describes the status of our development of these two new methods.

  10. Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-NMR system for the rapid identification of antioxidants in complex plant extracts.

    PubMed

    Pukalskas, Audrius; van Beek, Teris A; de Waard, Pieter

    2005-05-13

    A rapid method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining an HPLC with on-line radical scavenging using DPPH* as a model radical and an HPLC-DAD-SPE-NMR system. Using this method a commercial rosemary extract was investigated. All major compounds present in the extract were collected on SPE cartridges after their separation. Advantages of on-line SPE peak trapping are the possibility to perform HPLC with non-deuterated solvents, a concentration effect and being able to record NMR spectra in pure 100% deuterated solvents. After comparing DAD and DPPH scavenging chromatograms, 1H NMR spectra of compounds having radical scavenging activities were recorded. Afterwards all compounds were collected and infused into an ESI-MS. The five main active compounds - carnosol, carnosic acid carnosaldehyde, 12-methoxycarnosic acid and epiisorosmanol could be identified from the combined UV, NMR and mass spectral data without actually isolating them. It was possible to record on-line an HMBC spectrum of carnosic acid. Also one compound was tentatively identified as epirosmanol methyl ether. PMID:15941042

  11. Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

    PubMed

    Kagawa, T F; Cooney, J C; Baker, H M; McSweeney, S; Liu, M; Gubba, S; Musser, J M; Baker, E N

    2000-02-29

    Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes. PMID:10681429

  12. Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: An integrin-binding cysteine protease

    PubMed Central

    Kagawa, Todd F.; Cooney, Jakki C.; Baker, Heather M.; McSweeney, Sean; Liu, Mengyao; Gubba, Siddeswar; Musser, James M.; Baker, Edward N.

    2000-01-01

    Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-Å resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes. PMID:10681429

  13. Development of Chromatographic Fingerprints of Eurycoma longifolia (Tongkat Ali) Roots Using Online Solid Phase Extraction-Liquid Chromatography (SPE-LC).

    PubMed

    Zaini, Nor Nasriah; Osman, Rozita; Juahir, Hafizan; Saim, Norashikin

    2016-01-01

    E. longifolia is attracting interest due to its pharmacological properties and pro-vitality effects. In this study, an online SPE-LC approach using polystyrene divinyl benzene (PSDVB) and C18 columns was developed in obtaining chromatographic fingerprints of E. longifolia. E. longifolia root samples were extracted using pressurized liquid extraction (PLE) technique prior to online SPE-LC. The effects of mobile phase compositions and column switching time on the chromatographic fingerprint were optimized. Validation of the developed method was studied based on eurycomanone. Linearity was in the range of 5 to 50 µg∙mL(-1) (r² = 0.997) with 3.2% relative standard deviation of peak area. The developed method was used to analyze 14 E. longifolia root samples and 10 products (capsules). Selected chemometric techniques: cluster analysis (CA), discriminant analysis (DA), and principal component analysis (PCA) were applied to the fingerprint datasets of 37 selected peaks to evaluate the ability of the chromatographic fingerprint in classifying quality of E. longifolia. Three groups were obtained using CA. DA yielded 100% correlation coefficient with 19 discriminant compounds. Using PCA, E. longifolia root samples were clearly discriminated from the products. This study showed that the developed online SPE-LC method was able to provide comprehensive evaluation of E. longifolia samples for quality control purposes. PMID:27144555

  14. Simultaneous determination of PPCPs, EDCs, and artificial sweeteners in environmental water samples using a single-step SPE coupled with HPLC-MS/MS and isotope dilution.

    PubMed

    Tran, Ngoc Han; Hu, Jiangyong; Ong, Say Leong

    2013-09-15

    A high-throughput method for the simultaneous determination of 24 pharmaceuticals and personal care products (PPCPs), endocrine disrupting chemicals (EDCs) and artificial sweeteners (ASs) was developed. The method was based on a single-step solid phase extraction (SPE) coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and isotope dilution. In this study, a single-step SPE procedure was optimized for simultaneous extraction of all target analytes. Good recoveries (≥ 70%) were observed for all target analytes when extraction was performed using Chromabond(®) HR-X (500 mg, 6 mL) cartridges under acidic condition (pH 2). HPLC-MS/MS parameters were optimized for the simultaneous analysis of 24 PPCPs, EDCs and ASs in a single injection. Quantification was performed by using 13 isotopically labeled internal standards (ILIS), which allows correcting efficiently the loss of the analytes during SPE procedure, matrix effects during HPLC-MS/MS and fluctuation in MS/MS signal intensity due to instrument. Method quantification limit (MQL) for most of the target analytes was below 10 ng/L in all water samples. The method was successfully applied for the simultaneous determination of PPCPs, EDCs and ASs in raw wastewater, surface water and groundwater samples collected in a local catchment area in Singapore. In conclusion, the developed method provided a valuable tool for investigating the occurrence, behavior, transport, and the fate of PPCPs, EDCs and ASs in the aquatic environment. PMID:23708627

  15. An optimized and validated SPE-LC-MS/MS method for the determination of caffeine and paraxanthine in hair.

    PubMed

    De Kesel, Pieter M M; Lambert, Willy E; Stove, Christophe P

    2015-11-01

    Caffeine is the probe drug of choice to assess the phenotype of the drug metabolizing enzyme CYP1A2. Typically, molar concentration ratios of paraxanthine, caffeine's major metabolite, to its precursor are determined in plasma following administration of a caffeine test dose. The aim of this study was to develop and validate an LC-MS/MS method for the determination of caffeine and paraxanthine in hair. The different steps of a hair extraction procedure were thoroughly optimized. Following a three-step decontamination procedure, caffeine and paraxanthine were extracted from 20 mg of ground hair using a solution of protease type VIII in Tris buffer (pH 7.5). Resulting hair extracts were cleaned up on Strata-X™ SPE cartridges. All samples were analyzed on a Waters Acquity UPLC® system coupled to an AB SCIEX API 4000™ triple quadrupole mass spectrometer. The final method was fully validated based on international guidelines. Linear calibration lines for caffeine and paraxanthine ranged from 20 to 500 pg/mg. Precision (%RSD) and accuracy (%bias) were below 12% and 7%, respectively. The isotopically labeled internal standards compensated for the ion suppression observed for both compounds. Relative matrix effects were below 15%RSD. The recovery of the sample preparation procedure was high (>85%) and reproducible. Caffeine and paraxanthine were stable in hair for at least 644 days. The effect of the hair decontamination procedure was evaluated as well. Finally, the applicability of the developed procedure was demonstrated by determining caffeine and paraxanthine concentrations in hair samples of ten healthy volunteers. The optimized and validated method for determination of caffeine and paraxanthine in hair proved to be reliable and may serve to evaluate the potential of hair analysis for CYP1A2 phenotyping. PMID:26452792

  16. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    PubMed Central

    Ramani, Shilpa; Chelliah, Jayabaskaran

    2007-01-01

    Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed. PMID:17988378

  17. UV-B-Induced CPD Photolyase Gene Expression is Regulated by UVR8-Dependent and -Independent Pathways in Arabidopsis.

    PubMed

    Li, Nan; Teranishi, Mika; Yamaguchi, Hiroko; Matsushita, Tomonao; Watahiki, Masaaki K; Tsuge, Tomohiko; Li, Shao-Shan; Hidema, Jun

    2015-10-01

    Plants have evolved various mechanisms that protect against the harmful effects of UV-B radiation (280-315 nm) on growth and development. Cyclobutane pyrimidine dimer (CPD) photolyase, the repair enzyme for UV-B-induced CPDs, is essential for protecting cells from UV-B radiation. Expression of the CPD photolyase gene (PHR) is controlled by light with various wavelengths including UV-B, but the mechanisms of this regulation remain poorly understood. In this study, we investigated the regulation of PHR expression by light with various wavelengths, in particular low-fluence UV-B radiation (280 nm, 0.2 µmol m(-2) s(-1)), in Arabidopsis thaliana seedlings grown under light-dark cycles for 7 d and then adapted to the dark for 3 d. Low-fluence UV-B radiation induced CPDs but not reactive oxygen species. AtPHR expression was effectively induced by UV-B, UV-A (375 nm) and blue light. Expression induced by UV-A and blue light was predominantly regulated by the cryptochrome-dependent pathway, whereas phytochromes A and B played a minor but noticeable role. Expression induced by UV-B was predominantly regulated by the UVR8-dependent pathway. AtPHR expression was also mediated by a UVR8-independent pathway, which is correlated with CPD accumulation induced by UV-B radiation. These results indicate that Arabidopsis has evolved diverse mechanisms to regulate CPD photolyase expression by multiple photoreceptor signaling pathways, including UVR8-dependent and -independent pathways, as protection against harmful effects of UV-B radiation. PMID:26272552

  18. Topical application of spent coffee ground extracts protects skin from ultraviolet B-induced photoaging in hairless mice.

    PubMed

    Choi, Hyeon-Son; Park, Eu Ddeum; Park, Yooheon; Han, Sung Hee; Hong, Ki Bae; Suh, Hyung Joo

    2016-06-01

    The aim of this study was to evaluate the protective effect of spent coffee ground (SCG) on ultraviolet (UV) B-induced photoaging in hairless mice. The oil fraction (OSCG) and ethanol extract (ESCG) of SCG were prepared from SCG. OSCG contained a much higher level of caffeine (547.32 ± 1.68 μg mg(-1)) when compared to the sum of its chlorogenic acid derivatives (∼119 μg mg(-1)), and pyrazines were the major aromatic compounds in OSCG. OSCG effectively inhibited the UVB-induced increase in intracellular reactive oxygen species in HaCaT cells. Topical application of OSCG or ESCG significantly reduced the UVB-induced wrinkle formation in mice dorsal skin. The combined application of OSCG and ESCG (OEH) led to a decrease in the wrinkle area by over 35% when compared with the UVB-treated control (UVBC). Epidermal thickness was also reduced by 40%. This result was connected to the significant reduction in transdermal water loss (27%) and erythema formation (48%) that result from UVB irradiation. Polarization-sensitive optical coherence tomography (PS-OCT) and antibody-based histological analyses showed that OSCG and ESCG effectively suppressed the UVB-induced decrease in collagen content. The level of type 1 collagen (COL1) in the OEH group was enhanced by around 40% compared with the UVB control group (UVBC). This was attributed to the down-regulation of matrix metalloproteinases (MMP2, 9, and 13), which are known to be responsible for collagen destruction. Our results indicate that topical treatment with OSCG/ESCG protects mouse skin from UVB-induced photoaging by down-regulating MMPs; therefore, suggesting the potential of SCG extracts as a topical anti-photoaging agent. PMID:27195822

  19. Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

    PubMed Central

    Connolly, Kristie L.; Roberts, Amity L.; Holder, Robert C.; Reid, Sean D.

    2011-01-01

    Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which

  20. Dispersal of Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease severity in a murine model.

    PubMed

    Connolly, Kristie L; Roberts, Amity L; Holder, Robert C; Reid, Sean D

    2011-01-01

    Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which

  1. Inverse relation between disease severity and expression of the streptococcal cysteine protease, SpeB, among clonal M1T1 isolates recovered from invasive group A streptococcal infection cases.

    PubMed

    Kansal, R G; McGeer, A; Low, D E; Norrby-Teglund, A; Kotb, M

    2000-11-01

    The streptococcal cysteine protease (SpeB) is one of the major virulence factors produced by group A streptococci (GAS). In this study we investigated if differences exist in SpeB production by clonally related M1T1 clinical isolates derived from patients with invasive infections. Twenty-nine of these isolates were from nonsevere cases and 48 were from severe cases, including streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) cases. The expression and amount of the 28-kDa SpeB protein produced were determined by quantitative Western blotting, and protease activity was measured by a fluorescent enzymatic assay. A high degree of variation in SpeB expression was seen among the isolates, and this variation seemed to correlate with the severity and/or clinical manifestation of the invasive infection. The mean amount of 28-kDa SpeB protein and cysteine protease activity produced by isolates from nonsevere cases was significantly higher than that from STSS cases (P = 0.001). This difference was partly due to the fact that 41% of STSS isolates produced little or no SpeB compared to only 14% of isolates recovered in nonsevere cases. Moreover, the cysteine protease activity among those isolates that expressed SpeB was significantly lower for STSS isolates than for isolates from nonsevere cases (P = 0.001). Increased SpeB production was also inversely correlated with intact M protein expression, and inhibition of cysteine protease activity blocked the cleavage of the surface M protein. Together, the data support the existence of both an "on-off" and a posttranslational regulatory mechanism(s) controlling SpeB production, and they suggest that isolates with the speB gene in the "off" state are more likely to spare the surface M protein and to be isolated from cases of severe rather than nonsevere invasive infection. These findings may have important implications for the role of SpeB in host-pathogen interactions via regulation of the expression of GAS

  2. Inverse Relation between Disease Severity and Expression of the Streptococcal Cysteine Protease, SpeB, among Clonal M1T1 Isolates Recovered from Invasive Group A Streptococcal Infection Cases

    PubMed Central

    Kansal, Rita G.; McGeer, Allison; Low, Donald E.; Norrby-Teglund, Anna; Kotb, Malak

    2000-01-01

    The streptococcal cysteine protease (SpeB) is one of the major virulence factors produced by group A streptococci (GAS). In this study we investigated if differences exist in SpeB production by clonally related M1T1 clinical isolates derived from patients with invasive infections. Twenty-nine of these isolates were from nonsevere cases and 48 were from severe cases, including streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) cases. The expression and amount of the 28-kDa SpeB protein produced were determined by quantitative Western blotting, and protease activity was measured by a fluorescent enzymatic assay. A high degree of variation in SpeB expression was seen among the isolates, and this variation seemed to correlate with the severity and/or clinical manifestation of the invasive infection. The mean amount of 28-kDa SpeB protein and cysteine protease activity produced by isolates from nonsevere cases was significantly higher than that from STSS cases (P = 0.001). This difference was partly due to the fact that 41% of STSS isolates produced little or no SpeB compared to only 14% of isolates recovered in nonsevere cases. Moreover, the cysteine protease activity among those isolates that expressed SpeB was significantly lower for STSS isolates than for isolates from nonsevere cases (P = 0.001). Increased SpeB production was also inversely correlated with intact M protein expression, and inhibition of cysteine protease activity blocked the cleavage of the surface M protein. Together, the data support the existence of both an “on-off” and a posttranslational regulatory mechanism(s) controlling SpeB production, and they suggest that isolates with the speB gene in the “off” state are more likely to spare the surface M protein and to be isolated from cases of severe rather than nonsevere invasive infection. These findings may have important implications for the role of SpeB in host-pathogen interactions via regulation of the expression of

  3. Quercitrin protects against ultraviolet B-induced cell death in vitro and in an in vivo zebrafish model.

    PubMed

    Yang, Hye-Mi; Ham, Young-Min; Yoon, Weon-Jong; Roh, Seong Woon; Jeon, You-Jin; Oda, Tatsuya; Kang, Sung-Myung; Kang, Min-Cheol; Kim, Eun-A; Kim, Daekyung; Kim, Kil-Nam

    2012-09-01

    Chronic exposure of skin to ultraviolet (UV) B radiation induces oxidative stress, which in turn, plays a crucial role in the induction of skin aging. The search for strategies to reverse skin aging is being constantly pursued. Here, the cytoprotective effect of quercitrin (QR) on UVB-induced cell injury in HaCaT human keratinocytes and in the zebrafish was investigated. Intracellular reactive oxygen species (ROS) generated by the exposure of HaCaT cells to UVB radiation were significantly decreased after treatment with QR, and significantly so with QR at 50 μM. As a result, QR reduced UVB-induced cell death and apoptosis in HaCaT cells. QR similarly reduced UVB-induced ROS generation and cell death in live zebrafish. PMID:22727929

  4. Optogenetic apoptosis: light-triggered cell death.

    PubMed

    Hughes, Robert M; Freeman, David J; Lamb, Kelsey N; Pollet, Rebecca M; Smith, Weston J; Lawrence, David S

    2015-10-01

    An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax. PMID:26418181

  5. Apoptosis and Molecular Targeting Therapy in Cancer

    PubMed Central

    Hassan, Mohamed; Watari, Hidemichi; AbuAlmaaty, Ali; Ohba, Yusuke; Sakuragi, Noriaki

    2014-01-01

    Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction. PMID:25013758

  6. Death-Defining Immune Responses After Apoptosis

    PubMed Central

    Campisi, L.; Cummings, R. J.; Blander, J. Magarian

    2014-01-01

    Apoptosis is a programmed form of cell death whereby characteristic internal cellular dismantling is accompanied by the preservation of plasma membrane integrity. Maintaining this order during apoptosis prevents the release of cellular contents and ensures a noninflammatory death. Here, we consider examples of apoptosis in different contexts and discuss how the same form of cell death could have different immunological consequences. Multiple parameters such as cell death as a result of microbial infection, the nature of the inflammatory microenvironment, the type of responding phagocytic cells and the genetic background of the host organism all differentially influence the immunological consequences of apoptosis. PMID:24903539

  7. VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation

    PubMed Central

    Zhang, Yan-Ting; Xu, Li-Hui; Lu, Qun; Liu, Kun-Peng; Liu, Pei-Yan; Ji, Fang; Liu, Xiao-Ming; Ouyang, Dong-Yun; He, Xian-Hui

    2014-01-01

    Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway. PMID:24691407

  8. Stereoselective suppressive effects of protopanaxadiol epimers on UV-B-induced reactive oxygen species and matrix metalloproteinase-2 in human dermal keratinocytes.

    PubMed

    Oh, Sun-Joo; Lee, Sihyeong; Kho, Ye Eun; Kim, Kyunghoon; Jin, Chang Duck; Lim, Chang-Jin

    2015-01-01

    This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin. PMID:25405256

  9. Formation of cofilin-actin rods following cucurbitacin-B-induced actin aggregation depends on Slingshot homolog 1-mediated cofilin hyperactivation.

    PubMed

    Zhang, Yan-Ting; Ouyang, Dong-Yun; Xu, Li-Hui; Zha, Qing-Bing; He, Xian-Hui

    2013-10-01

    Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e., activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod-like aggregates, which were highly colocalized with actin rods, indicating the formation of cofilin-actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase Slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB-induced cofilin hyperactivation and cofilin-actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol-containing reactive oxygen species (ROS) scavenger N-acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB-induced actin aggregation, cofilin hyperactivation and cofilin-actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB-induced formation of cofilin-actin rods was mediated by SSH1-dependent but CIN-independent cofilin hyperactivation. PMID:23695982

  10. Low-dose spiruchostatin-B, a potent histone deacetylase inhibitor enhances radiation-induced apoptosis in human lymphoma U937 cells via modulation of redox signaling.

    PubMed

    Rehman, Mati Ur; Jawaid, Paras; Zhao, Qing Li; Li, Peng; Narita, Koichi; Katoh, Tadashi; Shimizu, Tadamichi; Kondo, Takashi

    2016-06-01

    Spiruchostatin B (SP-B), is a potent histone deacetylase (HDAC) inhibitor, in addition to HDAC inhibition, the pharmacological effects of SP-B are also attributed to its ability to produce intracellular reactive oxygen species (ROS), particularly H2O2. In this study, we investigated the effects of low dose (non-toxic) SP-B on radiation-induced apoptosis in human lymphoma U937 cells in vitro. The treatment of cells with low-dose SP-B induced the acetylation of histones, however, does not induce apoptosis. Whereas, the combined treatment with SP-B and radiation significantly enhanced the radiation-induced apoptosis, suggesting the potential role of this combined treatment for future radiation therapy. Interestingly, the enhancement of apoptosis was accompanied by significant increased in the ROS generation. Pre-treatment with an antioxidant, N-acetyl-l-cysteine (NAC) significantly inhibited the enhancement of apoptosis induced by combined treatment, indicating that ROS play an essential role. It was also found that SP-B combined with radiation caused the activation of death receptor and intrinsic apoptotic pathways, via modulation of ROS-mediated signaling. Moreover, SP-B also significantly enhanced the radiation-induced apoptosis in other lymphoma cell lines such as Molt-4 and HL-60. Taken together, our findings suggest that the low-dose SP-B enhances radiation-induced apoptosis via modulation of redox signaling because of its ability to serve as an intracellular ROS generating agent, mainly (H2O2 or [Formula: see text]). This study provides further insights into the mechanism of action of SP-B with radiation and demonstrates that SP-B can be used as a future novel sensitizer for radiation therapy. PMID:27108737

  11. Therapeutic approaches to the modulation of apoptosis.

    PubMed

    Murphy, Finbarr J; Seery, Liam T; Hayes, Ian

    2003-01-01

    The appreciation of the role of apoptosis in the vast majority of diseases affecting humans has revolutionized the discovery and development of drugs targeting inflammation and oncology. Novel therapeutic approaches to modulate disease by regulating apoptosis are currently being tested in preclinical and clinical settings. Enthusiasm for some of these therapies is reflected in the fact that they have received U.S. Food and Drug Administration approval in record time. Approaches include the traditional use of small molecules to target specific players in the apoptosis cascade. They also include radical new approaches such as using antisense molecules to inhibit production of the Bcl-2 protein or antibodies that ligate either death receptors, such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), or the MHC (HLA-DR), resulting in the initiation of apoptosis of target cells. Antibodies targeting cell-specific antigens are being used in conjunction with radioactive isotopes to deliver a more specific chemotherapy, particularly in the case of B-cell lymphomas. Other therapies target mitochondria, a key organelle in the apoptosis cascade. This diverse range of therapies includes photodynamic therapy, retinoic acid and arsenic trioxide, all of which induce apoptosis by generating reactive oxygen species. As our understanding of apoptosis increases, further opportunities will arise for tailor-made therapies that will result in improved clinical outcome without the devastating side effects of current interventions. PMID:14585079

  12. Apoptosis in mammalian oocytes: a review.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals. PMID:25958165

  13. CHCHD2 connects mitochondrial metabolism to apoptosis

    PubMed Central

    Liu, Yong; Zhang, Yanping

    2015-01-01

    As the powerhouse of cells and gatekeeper for apoptosis, mitochondria control life and death. CHCHD2, a mitochondrial protein previously known to regulate metabolism, has recently been identified as an apoptosis inhibitor. New data suggest a model in which CHCHD2 performs a prosurvival function by acting as both a reactive oxygen species scavenger and BCL-XL activator. PMID:27308501

  14. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY.

    EPA Science Inventory

    The role of apoptosis in neurodegeneration in developing animals and in adults has become increasingly apparent in the past ten years. Normal apoptosis occurs in the CNS from the embryonic stage through senescence, with different cells in each region of the nervous system having ...

  15. Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation

    PubMed Central

    Mo, Hao; Henriksson, Marie

    2006-01-01

    The Myc transcription factor plays a central role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Myc is a potent oncoprotein that is deregulated in a wide variety of human tumors and is therefore an attractive target for novel cancer therapies. Using a cellular screening approach, we have identified low-molecular-weight compounds, Myc pathway response agents (MYRAs), that induce apoptosis in a c-Myc-dependent manner and inhibit Myc-driven cellular transformation. MYRA-A inhibits Myc transactivation and interferes with the DNA-binding activity of Myc family proteins but has no effect on the E-box-binding protein USF. In contrast, MYRA-B induces Myc-dependent apoptosis without affecting Myc transactivation or Myc/Max DNA binding. Our data show that cellular screening assays can be a powerful strategy for the identification of candidate substances that modulate the Myc pathway. These compounds can be useful tools for studying Myc function and may also be of therapeutic potential as leads for drug development. PMID:16606833

  16. The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase.

    PubMed

    Guenebeaud, Céline; Goldschneider, David; Castets, Marie; Guix, Catherine; Chazot, Guillaume; Delloye-Bourgeois, Céline; Eisenberg-Lerner, Avital; Shohat, Galit; Zhang, Mingjie; Laudet, Vincent; Kimchi, Adi; Bernet, Agnès; Mehlen, Patrick

    2010-12-22

    The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65β of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65β and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation. PMID:21172653

  17. Honokiol, a chemopreventive agent against skin cancer, induces cell cycle arrest and apoptosis in human epidermoid A431 cells.

    PubMed

    Chilampalli, Chandeshwari; Guillermo, Ruth; Kaushik, Radhey S; Young, Alan; Chandrasekher, Gudiseva; Fahmy, Hesham; Dwivedi, Chandradhar

    2011-11-01

    Honokiol is a plant lignan isolated from bark and seed cones of Magnolia officinalis. Recent studies from our laboratory indicated that honokiol pretreatment decreased ultraviolet B-induced skin cancer development in SKH-1 mice. The aim of the present investigation was to study the effects of honokiol on human epidermoid squamous carcinoma A431 cells and to elucidate possible mechanisms involved in preventing skin cancer. A431 cells were pretreated with different concentrations of honokiol for a specific time period and investigated for effects on apoptosis and cell cycle analysis. Treatment with honokiol significantly decreased cell viability and cell proliferation in a concentration- and time-dependent manner. Honokiol pretreatment at 50 μmol/L concentration induced G0/G1 cell cycle arrest significantly (P < 0.05) and decreased the percentage of cells in the S and G2/M phase. Honokiol down-regulated the expression of cyclin D1, cyclin D2, Cdk2, Cdk4 and Cdk6 proteins and up-regulated the expression of Cdk's inhibitor proteins p21 and p27. Pretreatment of A431 cells with honokiol leads to induction of apoptosis and DNA fragmentation. These findings indicate that honokiol provides its effects in squamous carcinoma cells by inducing cell cycle arrest at G0/G1 phase and apoptosis. PMID:21908486

  18. Development and optimization of SPE-HPLC-UV/ELSD for simultaneous determination of nine bioactive components in Shenqi Fuzheng Injection based on Quality by Design principles.

    PubMed

    Wang, Lu; Qu, Haibin

    2016-03-01

    A method combining solid phase extraction, high performance liquid chromatography, and ultraviolet/evaporative light scattering detection (SPE-HPLC-UV/ELSD) was developed according to Quality by Design (QbD) principles and used to assay nine bioactive compounds within a botanical drug, Shenqi Fuzheng Injection. Risk assessment and a Plackett-Burman design were utilized to evaluate the impact of 11 factors on the resolutions and signal-to-noise of chromatographic peaks. Multiple regression and Pareto ranking analysis indicated that the sorbent mass, sample volume, flow rate, column temperature, evaporator temperature, and gas flow rate were statistically significant (p < 0.05) in this procedure. Furthermore, a Box-Behnken design combined with response surface analysis was employed to study the relationships between the quality of SPE-HPLC-UV/ELSD analysis and four significant factors, i.e., flow rate, column temperature, evaporator temperature, and gas flow rate. An analytical design space of SPE-HPLC-UV/ELSD was then constructed by calculated Monte Carlo probability. In the presented approach, the operating parameters of sample preparation, chromatographic separation, and compound detection were investigated simultaneously. Eight terms of method validation, i.e., system-suitability tests, method robustness/ruggedness, sensitivity, precision, repeatability, linearity, accuracy, and stability, were accomplished at a selected working point. These results revealed that the QbD principles were suitable in the development of analytical procedures for samples in complex matrices. Meanwhile, the analytical quality and method robustness were validated by the analytical design space. The presented strategy provides a tutorial on the development of a robust QbD-compliant quantitative method for samples in complex matrices. PMID:26825340

  19. Orally administered fructose increases the numbers of peripheral lymphocytes reduced by exposure of mice to gamma or SPE-like proton radiation

    NASA Astrophysics Data System (ADS)

    Romero-Weaver, A. L.; Ni, J.; Lin, L.; Kennedy, A. R.

    2014-07-01

    Exposure of the whole body or a major portion of the body to ionizing radiation can result in Acute Radiation Sickness (ARS), which can cause symptoms that range from mild to severe, and include death. One of the syndromes that can occur during ARS is the hematopoietic syndrome, which is characterized by a reduction in bone marrow cells as well as the number of circulating blood cells. Doses capable of causing this syndrome can result from conventional radiation therapy and accidental exposure to ionizing radiation. It is of concern that this syndrome could also occur during space exploration class missions in which astronauts could be exposed to significant doses of solar particle event (SPE) radiation. Of particular concern is the reduction of lymphocytes and granulocytes, which are major components of the immune system. A significant reduction in their numbers can compromise the immune system, causing a higher risk for the development of infections which could jeopardize the success of the mission. Although there are no specific countermeasures utilized for the ARS resulting from exposure to space radiation(s), granulocyte colony-stimulating factor (G-CSF) has been proposed as a countermeasure for the low number of neutrophils caused by SPE radiation, but so far no countermeasure exists for a reduced number of circulating lymphocytes. The present study demonstrates that orally administered fructose significantly increases the number of peripheral lymphocytes reduced by exposure of mice to 2 Gy of gamma- or SPE-like proton radiation, making it a potential countermeasure for this biological end-point.

  20. Orally Administered Fructose Increases the Numbers of Peripheral Lymphocytes Reduced by Exposure of Mice to Gamma or SPE-like Proton Radiation.

    PubMed

    Romero-Weaver, A L; Ni, J; Lin, L; Kennedy, A R

    2014-07-01

    Exposure of the whole body or a major portion of the body to ionizing radiation can result in Acute Radiation Sickness (ARS), which can cause symptoms that range from mild to severe, and include death. One of the syndromes that can occur during ARS is the hematopoietic syndrome, which is characterized by a reduction in bone marrow cells as well as the number of circulating blood cells. Doses capable of causing this syndrome can result from conventional radiation therapy and accidental exposure to ionizing radiation. It is of concern that this syndrome could also occur during space exploration class missions in which astronauts could be exposed to significant doses of solar particle event (SPE) radiation. Of particular concern is the reduction of lymphocytes and granulocytes, which are major components of the immune system. A significant reduction in their numbers can compromise the immune system, causing a higher risk for the development of infections which could jeopardize the success of the mission. Although there are no specific countermeasures utilized for the ARS resulting from exposure to space radiation(s), granulocyte colony-stimulating factor (G-CSF) has been proposed as a countermeasure for the low number of neutrophils caused by SPE radiation, but so far no countermeasure exists for a reduced number of circulating lymphocytes. The present study demonstrates that orally administered fructose significantly increases the number of peripheral lymphocytes reduced by exposure of mice to 2 Gy of gamma- or SPE-like proton radiation, making it a potential countermeasure for this biological end-point. PMID:25360417

  1. A Compact Laboratory Spectro-Goniometer (CLabSpeG) to Assess the BRDF of Materials. Presentation, Calibration and Implementation on Fagus sylvatica L. Leaves

    PubMed Central

    Biliouris, Dimitrios; Verstraeten, Willem W.; Dutré, Phillip; van Aardt, Jan A.N.; Muys, Bart; Coppin, Pol

    2007-01-01

    The design and calibration of a new hyperspectral Compact Laboratory Spectro-Goniometer (CLabSpeG) is presented. CLabSpeG effectively measures the bidirectional reflectance Factor (BRF) of a sample, using a halogen light source and an Analytical Spectral Devices (ASD) spectroradiometer. The apparatus collects 4356 reflectance data readings covering the spectrum from 350 nm to 2500 nm by independent positioning of the sensor, sample holder, and light source. It has an azimuth and zenith resolution of 30 and 15 degrees, respectively. CLabSpeG is used to collect BRF data and extract Bidirectional Reflectance Distribution Function (BRDF) data of non-isotropic vegetation elements such as bark, soil, and leaves. Accurate calibration has ensured robust geometric accuracy of the apparatus, correction for the conicality of the light source, while sufficient radiometric stability and repeatability between measurements are obtained. The bidirectional reflectance data collection is automated and remotely controlled and takes approximately two and half hours for a BRF measurement cycle over a full hemisphere with 125 cm radius and 2.4 minutes for a single BRF acquisition. A specific protocol for vegetative leaf collection and measurement was established in order to investigate the possibility to extract BRDF values from Fagus sylvatica L. leaves under laboratory conditions. Drying leaf effects induce a reflectance change during the BRF measurements due to the laboratory illumination source. Therefore, the full hemisphere could not be covered with one leaf. Instead 12 BRF measurements per leaf were acquired covering all azimuth positions for a single light source zenith position. Data are collected in radiance format and reflectance is calculated by dividing the leaf cycle measurement with a radiance cycle of a Spectralon reference panel, multiplied by a Spectralon reflectance correction factor and a factor to correct for the conical effect of the light source. BRF results of

  2. Orally Administered Fructose Increases the Numbers of Peripheral Lymphocytes Reduced by Exposure of Mice to Gamma or SPE-like Proton Radiation

    PubMed Central

    Romero-Weaver, A.L.; Ni, J.; Lin, L.; Kennedy, A.R.

    2014-01-01

    Exposure of the whole body or a major portion of the body to ionizing radiation can result in Acute Radiation Sickness (ARS), which can cause symptoms that range from mild to severe, and include death. One of the syndromes that can occur during ARS is the hematopoietic syndrome, which is characterized by a reduction in bone marrow cells as well as the number of circulating blood cells. Doses capable of causing this syndrome can result from conventional radiation therapy and accidental exposure to ionizing radiation. It is of concern that this syndrome could also occur during space exploration class missions in which astronauts could be exposed to significant doses of solar particle event (SPE) radiation. Of particular concern is the reduction of lymphocytes and granulocytes, which are major components of the immune system. A significant reduction in their numbers can compromise the immune system, causing a higher risk for the development of infections which could jeopardize the success of the mission. Although there are no specific countermeasures utilized for the ARS resulting from exposure to space radiation(s), granulocyte colony-stimulating factor (G-CSF) has been proposed as a countermeasure for the low number of neutrophils caused by SPE radiation, but so far no countermeasure exists for a reduced number of circulating lymphocytes. The present study demonstrates that orally administered fructose significantly increases the number of peripheral lymphocytes reduced by exposure of mice to 2 Gy of gamma- or SPE-like proton radiation, making it a potential countermeasure for this biological end-point. PMID:25360417

  3. Bamboo charcoal as adsorbent for SPE coupled with monolithic column-HPLC for rapid determination of 16 polycyclic aromatic hydrocarbons in water samples.

    PubMed

    Ma, Jiping; Li, Mo; Li, Jinhua; Rui, Cuijie; Xin, Yanping; Xue, Qinzhao; Chen, Lingxin

    2011-10-01

    The coupling of solid-phase extraction (SPE) using bamboo charcoal (BC) as an adsorbent with a monolithic column-high performance liquid chromatography (MC-HPLC) method was developed for the high-efficiency enrichment and rapid determination of 16 polycyclic aromatic hydrocarbons (PAHs) in water. Key influence factors, such as the type and the volume of the elution solvent, and the flow rate and the volume of the sample loading, were optimized to obtain a high SPE recovery and extraction efficiency. BC as an SPE adsorbent presented a high extraction efficiency due to its large specific surface area and high adsorption capacity; MC as an HPLC column accelerated the separation within 8 min because of its high porosity, fast mass transfer, and low-pressure resistance. The calibration curves for the PAHs extracted were linear in the range of 0.2-15 µg/L, with the correlation coefficients (r(2)) between 0.9970-0.9999. This method attained good precisions (relative standard deviation, RSD) from 3.5 to 10.9% for the standard PAHs I aqueous solutions at 5 µg/L; the method recoveries ranged in 52.6-121.6% for real spiked river water samples with 0.4 and 4 µg/L. The limits of detection (LODs, S/N = 3) of the method were determined from 11 and 87 ng/L. The developed method was demonstrated to be applicable for the rapid and sensitive determination of 16 PAHs in real environmental water samples. PMID:22586244

  4. Analysis of apoptosis in Caenorhabditis elegans.

    PubMed

    Lant, Benjamin; Derry, W Brent

    2014-05-01

    The nematode worm Caenorhabditis elegans has provided researchers with a wealth of information on the molecular mechanisms controlling programmed cell death (apoptosis). Its genetic tractability, optical clarity, and relatively short lifespan are key advantages for rapid assessment of apoptosis in vivo. The use of forward and reverse genetics methodology, coupled with in vivo imaging, has provided deep insights into how a multicellular organism orchestrates the self-destruction of specific cells during development and in response to exogenous stresses. Strains of C. elegans carrying mutations in the core elements of the apoptotic pathway, or in tissue-specific regulators of apoptosis, can be used for genetic analyses to reveal conserved mechanisms by which apoptosis is regulated in the somatic and reproductive (germline) tissue. Here we present an introduction to the study of apoptosis in C. elegans, including current techniques for visualization, analysis, and screening. PMID:24786497

  5. Induction of apoptosis by Shiga toxins

    PubMed Central

    Tesh, Vernon L

    2010-01-01

    Shiga toxins comprise a family of structurally and functionally related protein toxins expressed by Shigella dysenteriae serotype 1 and multiple serotypes of Escherichia coli. While the capacity of Shiga toxins to inhibit protein synthesis by catalytic inactivation of eukaryotic ribosomes has been well described, it is also apparent that Shiga toxins trigger apoptosis in many cell types. This review presents evidence that Shiga toxins induce apoptosis of epithelial, endothelial, leukocytic, lymphoid and neuronal cells. Apoptotic signaling pathways activated by the toxins are reviewed with an emphasis on signaling mechanisms that are shared among different cell types. Data suggesting that Shiga toxins induce apoptosis through the endoplasmic reticulum stress response and clinical evidence demonstrating apoptosis in humans infected with Shiga toxin-producing bacteria are briefly discussed. The potential for use of Shiga toxins to induce apoptosis in cancer cells is briefly reviewed. PMID:20210553

  6. Apoptosis inducers in chronic lymphocytic leukemia

    PubMed Central

    Billard, Christian

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by a typical defect in apoptosis and is still an incurable disease. Numerous apoptosis inducers have been described. These synthetic compounds and natural products (mainly derived from plants) display antileukemic properties in vitro and in vivo and some have even been tested in the clinic in CLL. They act through several different mechanisms. Most of them involve proteins of the Bcl-2 family, which are the key regulators in triggering the mitochondrial pathway of caspase-dependent apoptosis. Thus, the Mcl-1/Noxa axis appeared as a target. Here I overview natural and synthetic apoptosis inducers and their mechanisms of action in CLL cells. Opportunities for developing novel, apoptosis-based therapeutics are presented. PMID:24525395

  7. Profiling of impurities in p-methoxymethamphetamine (PMMA) by means of SPE/TLC method. Examination of the influence of experimental conditions according to 2(4) factorial.

    PubMed

    Kochana, J; Wilamowski, J; Parczewski, A

    2003-07-01

    In the present paper profiling of impurities in p-methoxymethamphetamine (PMMA) by means of SPE/TLC is reported. PMMA was synthesised by Leuckart procedure. The influence of experimental conditions on the profile quality was investigated. The experiments were carried out according to a 2(4) factorial. The proposed characteristics of the profile quality (optimisation criterions) are based on a matrix presentation of TLC patterns. They take into account, simultaneously, the number of spots revealed, differences between R(f) values and intensity of fluorescence. PMID:12850419

  8. Lymphocyte apoptosis in murine Pneumocystis pneumonia

    PubMed Central

    Shi, Xin; LeCapitaine, Nicole J; Rudner, Xiaowen L; Ruan, Sanbao; Shellito, Judd E

    2009-01-01

    Background Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. Methods Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. Results In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. Conclusion Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins. PMID:19558669

  9. Effects of ginseng saponins isolated from red ginseng on ultraviolet B-induced skin aging in hairless mice.

    PubMed

    Kim, Young Gon; Sumiyoshi, Maho; Sakanaka, Masahiro; Kimura, Yoshiyuki

    2009-01-01

    It is well-known that chronic ultraviolet B (UVB) exposure at low-dose causes skin photoaging including increases in skin thickness and wrinkle formation and reduction in skin elasticity. This study examined the effects of total saponins and ginsenoside Rb(1) isolated from Red Ginseng roots on skin thickness, elasticity, and wrinkle formation caused by long-term, low-dose UVB irradiation in hairless mice. The topical application of total ginseng saponins (10 pg or 100 ng/mouse) and ginsenoside Rb(1) (100 fg, 10 pg, or 1 ng/mouse) significantly inhibited increases in skin thickness and wrinkle formation and the reduction in skin elasticity induced by long-term UVB irradiation. Furthermore, we examined the histological effects of total saponins and ginsenoside Rb(1) in the skin of UVB-irradiated hairless mice. The increases in apoptotic, Ki-67-, and 8-hydroxy-2'-deoxyguanosine-positive cells induced by UVB exposure were prevented by the topical application of total saponins and ginsenoside Rb(1). Furthermore, total saponins and ginsenoside Rb(1) prevented the disruption of collagen fibers induced by the long-term UVB irradiation. Ginsenoside Rb(1) (100 fg, 10 pg, and 1 ng/ml) increased the Bcl-2 expression level in UVB-treated human keratinocytes. The protective effect of ginsenoside Rb(1) on UVB-mediated apoptosis may be due to the up-regulation of Bcl-2 expression. These results suggest that the protective effect of ginsenoside Rb(1) on skin photoaging induced by chronic UVB exposure may be due to the increase in collagen synthesis and/or the inhibition of matrix metalloproteinase expression in dermal fibroblasts. PMID:19041641

  10. Honey and Apoptosis in Human Gastric Mucosa

    PubMed Central

    Ghaffari, Aida; Somi, Mohammad H; Safaiyan, Abdolrasoul; Modaresi, Jabiz; Ostadrahimi, Alireza

    2012-01-01

    Background: Gastric cancer is the fourth most common malignancy in the world. Honey is a complex mixture of special biological active constituents. Honey possesses antioxidant and antitumor properties. Nutritional studies have indicated that consumption of honey modulates the risk of developing gastric cancer. On the other hand, apoptosis has been reported to play a decisive role in precancerous changes. Our chief study was conducted to assess the relationship between consumption of honey and apoptosis in human gastric mucosa. Method: This cross-sectional study was conducted on 98 subjects over 18 years old, referred to two hospitals in Tabriz, Iran. Subjects were undergone an upper gastrointestinal endoscopy, 62 subjects were finally enrolled. Honey consumption was assessed by a Food Frequency Questionnaire (FFQ) and apoptosis was detected by TUNEL technique. We tested polynomial curve to find the best fit between honey consumption and apoptosis. Results: A positive relation between honey consumption and apoptosis was found (P=0.024). Our results indicated that the final and the best fit curve was: apoptosis = 1.714+1.648(honey amount) - 0.533(honey amount)2 +1.833×10-5(honey amount)7. Conclusion: Honey consumption had positive effects on gastric cancer by inducing apoptosis in gastric mucosa. PMID:24688918

  11. Chemical Explosion Experiments to Improve Nuclear Test Monitoring [Developing a New Paradigm for Nuclear Test Monitoring with the Source Physics Experiments (SPE)

    SciTech Connect

    Snelson, Catherine M.; Abbott, Robert E.; Broome, Scott T.; Mellors, Robert J.; Patton, Howard J.; Sussman, Aviva J.; Townsend, Margaret J.; Walter, William R.

    2013-07-02

    A series of chemical explosions, called the Source Physics Experiments (SPE), is being conducted under the auspices of the U.S. Department of Energy’s National Nuclear Security Administration (NNSA) to develop a new more physics-based paradigm for nuclear test monitoring. Currently, monitoring relies on semi-empirical models to discriminate explosions from earthquakes and to estimate key parameters such as yield. While these models have been highly successful monitoring established test sites, there is concern that future tests could occur in media and at scale depths of burial outside of our empirical experience. This is highlighted by North Korean tests, which exhibit poor performance of a reliable discriminant, mb:Ms (Selby et al., 2012), possibly due to source emplacement and differences in seismic responses for nascent and established test sites. The goal of SPE is to replace these semi-empirical relationships with numerical techniques grounded in a physical basis and thus applicable to any geologic setting or depth.

  12. A fast method for screening and/or quantitation of tetrahydrocannabinol and metabolites in urine by automated SPE/LC/MS/MS.

    PubMed

    Jagerdeo, Eshwar; Montgomery, Madeline A; Karas, Roman P; Sibum, Martin

    2010-09-01

    Marijuana is one of the most commonly used illicit substances. The high usage of this substance results in it being commonly encountered in clinical samples throughout the USA and Europe. Due to its wide availability and use, marijuana is also commonly encountered in forensic toxicology laboratories. The proposed method utilized an automated solid phase extraction (SPE) coupled to liquid chromatography/mass spectrometry (LC/MS). The automated SPE procedure was developed using Hysphere C8-EC sorbent, and the high performance liquid chromatography (HPLC) separation was performed using an Xterra MS C(18) column with a total runtime of 10 min. The standard curves linearity generally fell between 6 and 500 ng/mL. The limits of detection ranged from 2 to 4 ng/mL, and the limits of quantitation ranged from 8 to 12 ng/mL. The bias and imprecision were determined using a simple analysis of variance (single factor). The results demonstrate bias as <11% and percent imprecision as <12% for all components at four quality control levels. This method has been in use for over 2 years and has been applied to numerous forensic samples. When compared to other published methods, it exceeds others in its simplicity and speed of analysis. This method takes advantage of robotics and automation for a total analysis time of 10 min, including sample preparation, separation, and detection. PMID:20582401

  13. Elevated urinary levels of carcinogenic N-nitrosamines in patients with urinary tract infections measured by isotope dilution online SPE LC-MS/MS.

    PubMed

    Hu, Chiung-Wen; Shih, Ying-Ming; Liu, Hung-Hsin; Chiang, Yi-Chen; Chen, Chih-Ming; Chao, Mu-Rong

    2016-06-01

    N-nitrosamines (NAms) are well-documented for their carcinogenic potential. Human exposure to NAms may arise from the daily environment and endogenous formation via the reaction of secondary amines with nitrites or from bacteria infection. We describe the use of isotope dilution online solid-phase extraction (SPE) LC-MS/MS to quantify nine NAms in human urine. This method was validated and further applied to healthy subjects and patients with urinary tract infection (UTI). N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPYR) and N-nitrosomorpholine (NMOR) were analyzed with an APCI source, while N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPIP), N-nitrosodi-n-propylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitrosodiphenylamine (NDPhA) were quantified with an ESI source, due to their effect on the sensitivity and chromatography. NDMA was the most abundant N-nitrosamine, while NDPhA was firstly identified in human. UTI patients had three to twelve-fold higher concentrations for NDMA, NPIP, NDEA, NMOR and NDBA in urine than healthy subjects, and the NAms were significantly decreased after antibiotics treatment. NDMA concentrations were also significantly correlated with the pH value, leukocyte esterase activity or nitrite in urines of UTI patients. Our findings by online SPE LC-MS/MS method evidenced that UTI patients experienced various NAms exposures, especially the potent carcinogen NDMA, which was likely induced by bacteria infection. PMID:26937867

  14. Bioanalytical method development for quantification of ulifloxacin, fenbufen and felbinac in rat plasma by solid-phase extraction (SPE) and HPLC with PDA detection.

    PubMed

    Ferrone, Vincenzo; Carlucci, Maura; Palumbo, Paola; Carlucci, Giuseppe

    2016-05-10

    A procedure based on solid-phase extraction (SPE) followed by high performance liquid chromatography (HPLC) with PDA detection has been developed for the analysis of multiple drugs in rat plasma. The analytes evaluated were ulifloxacin, fenbufen and felbinac. Eight different solid phase extraction cartridges were tested to evaluate their applicability for the isolation of drugs from rat plasma. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by HPLC using a Kinetex C18 EVO column and acetonitrile-10mM ammonium acetate-methanol as the mobile phase under gradient elution conditions. SPE combined with HPLC-PDA allowed the determination of drugs over a linear range of 0.05-15 μg/mL for ulifloxacin while 0.5-50 μg/mL for felbinac and fenbufen, with limit of detection at 0.05 for ulifloxacin and 0.5 for felbinac and fenbufen. Bond Elut Plexa sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 93.54% with relative standard deviation (RSD) <10%. The method was applied with good accuracy and precision in the determination of ulifloxacin, fenbufen and felbinac in rat plasma obtained from rats treated with selected drugs. This method permits its application to pharmacokinetic and pharmacodynamic studies of these analytes and will facilitate detailed investigations on the interactions between new fluoroquinolones and fenbufen. PMID:26898973

  15. Inactivation of the group A Streptococcus regulator srv results in chromosome wide reduction of transcript levels, and changes in extracellular levels of Sic and SpeB.

    PubMed

    Reid, Sean D; Chaussee, Michael S; Doern, Christopher D; Chaussee, Michelle A; Montgomery, Alison G; Sturdevant, Daniel E; Musser, James M

    2006-11-01

    Group A Streptococcus is characterized by the ability to cause a diverse number of human infections including pharyngitis, necrotizing fasciitis, toxic shock syndrome, and acute rheumatic fever, yet the regulation of streptococcal genes involved in disease processes and survival in the host is not completely understood. Genome scale analysis has revealed a complex regulatory network including 13 two-component regulatory systems and more than 100 additional putative regulators, the majority of which remain uncharacterized. Among these is the streptococcal regulator of virulence, Srv, the first Group A Streptococcus member of the Crp/Fnr family of transcriptional regulators. Previous work demonstrated that the loss of srv resulted in a significant decrease in Group A Streptococcus virulence. To begin to define the gene products influenced by Srv, we combined microarray and two-dimensional gel electrophoresis analysis. Loss of srv results in a chromosome wide reduction of gene transcription and changes in the production of the extracellular virulence factors Sic (streptococcal inhibitor of complement) and SpeB (cysteine proteinase). Sic levels are reduced in the srv mutant, whereas the extracellular concentration and activity of SpeB is increased. These data link Srv to the increasingly complex GAS regulatory network. PMID:16999824

  16. Rapid determination of memantine in human plasma by using nanoring carboxyl-functionalized paramagnetic molecularly imprinted polymer d-μ-SPE and UFLC-MS/MS.

    PubMed

    Qiu, Hai-Wen; Xia, Lei; Gong, Li-Min; Ruan, Lie-Min; Zhao, Yong-Gang

    2015-06-01

    A novel, simple, and sensitive method based on the use of dispersive micro-solid-phase extraction (d-μ-SPE) procedure combined with ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) for the determination of memantine (ME) was developed and validated over the linearity range 0.05-10.0 µg/L with 100 μL of human plasma using memantine-D6 (ME-D6) as the internal standard. The novel nanoring carboxyl-functionalized paramagnetic molecularly imprinted polymer (NR-CF-Mag-MIP) was synthesized by ultrasound-assisted suspension polymerization, using ME as a template molecule, methacrylic acid as a functional monomer, and divinylbenzene as a cross-linking agent. The NR-CF-Mag-MIP was used as the d-μ-SPE sorbent to extract ME from human plasma samples. The obtained results demonstrated the higher extraction capacity of NR-CF-Mag-MIP with recoveries between 97.6 and 101%. The limits of quantification (LOQs) for ME was 0.015 µg/L. Validation results on linearity, specificity, accuracy, precision, and stability, as well as on application to the analysis of samples taken up to 480 h after oral administration of 20 mg (two 10 mg capsules) of ME in healthy volunteers demonstrated the applicability to bioequivalence studies. PMID:25209851

  17. On-line SPE-UHPLC method using fused core columns for extraction and separation of nine illegal dyes in chilli-containing spices.

    PubMed

    Khalikova, Maria A; Satínský, Dalibor; Smidrkalová, Tereza; Solich, Petr

    2014-12-01

    The presented work describes the development of a simple, fast and effective on-line SPE-UHPLC-UV/vis method using fused core particle columns for extraction, separation and quantitative analysis of the nine illegal dyes, most frequently found in chilli-containing spices. The red dyes Sudan I-IV, Sudan Red 7B, Sudan Red G, Sudan Orange G, Para Red, and Methyl Red were separated and analyzed in less than 9 min without labor-consuming pretreatment procedure. The chromatographic separation was performed on Ascentis Express RP-Amide column with gradient elution using mixture of acetonitrile and water, as a mobile phase at a flow rate of 1.0 mL min(-1) and 55°C of temperature. As SPE sorbent for cleanup and pre-concentration of illegal dyes short guard fused core column Ascentis Express F5 was used. The applicability of proposed method was proven for three different chilli-containing commercial samples. Recoveries for all compounds were between 90% and 108% and relative standard deviation ranged from 1% to 4% for within- and from 2% to 6% for between-day. Limits of detection showed lower values than required by European Union regulations and were in the range of 3.3-10.3 µg L(-1) for standard solutions, 5.6-235.6 µg kg(-1) for chilli-containing spices. PMID:25159432

  18. Inactivation of the group A Streptococcus regulator srv results in chromosome wide reduction of transcript levels, and changes in extracellular levels of Sic and SpeB

    PubMed Central

    Reid, Sean D.; Chaussee, Michael S.; Doern, Christopher D.; Chaussee, Michelle A.; Montgomery, Alison G.; Sturdevant, Daniel E.; Musser, James M.

    2009-01-01

    Group A Streptococcus is characterized by the ability to cause a diverse number of human infections including pharyngitis, necrotizing fasciitis, toxic shock syndrome, and acute rheumatic fever, yet the regulation of streptococcal genes involved in disease processes and survival in the host is not completely understood. Genome scale analysis has revealed a complex regulatory network including 13 two-component regulatory systems and more than 100 additional putative regulators, the majority of which remain uncharacterized. Among these is the streptococcal regulator of virulence, Srv, the first Group A Streptococcus member of the Crp/Fnr family of transcriptional regulators. Previous work demonstrated that the loss of srv resulted in a significant decrease in Group A Streptococcus virulence. To begin to define the gene products influenced by Srv, we combined microarray and two-dimensional gel electrophoresis analysis. Loss of srv results in a chromosome wide reduction of gene transcription and changes in the production of the extracellular virulence factors Sic (streptococcal inhibitor of complement) and SpeB (cysteine proteinase). Sic levels are reduced in the srv mutant, whereas the extracellular concentration and activity of SpeB is increased. These data link Srv to the increasingly complex GAS regulatory network. PMID:16999824

  19. Simultaneous determination of individual isothiocyanates in plant samples by HPLC-DAD-MS following SPE and derivatization with N-acetyl-l-cysteine.

    PubMed

    Pilipczuk, Tadeusz; Kusznierewicz, Barbara; Chmiel, Tomasz; Przychodzeń, Witold; Bartoszek, Agnieszka

    2017-01-01

    The procedure for the isothiocyanates (ITCs) determination that involves derivatization with N-acetyl-l-cysteine (NAC) and separation by HPLC was developed. Prior to derivatization, plant ITCs were isolated and purified using solid-phase extraction (SPE). The optimum conditions of derivatization are: 500μL of isopropanolic eluate obtained by SPE combined with 500μL of derivatizing reagent (0.2M NAC and 0.2M NaHCO3 in water) and reaction time of 1h at 50°C. The formed dithiocarbamates are directly analyzed by HPLC coupled with diode array detector and mass spectrometer if required. The method was validated for nine common natural ITCs. Calibration curves were linear (R(2)⩾0.991) within a wide range of concentrations and limits of detection were below 4.9nmol/mL. The recoveries were in the range of 83.3-103.7%, with relative standard deviations <5.4%. The developed method has been successfully applied to determine ITCs in broccoli, white cabbage, garden cress, radish, horseradish and papaya. PMID:27507514

  20. The SpeX Prism Library: 1000+ low-resolution, near-infrared spectra of ultracool M, L, T and Y dwarfs

    NASA Astrophysics Data System (ADS)

    Burgasser, Adam J.

    The SpeX Prism Library (SPL) is a uniform compilation of low-resolution (λ/Δλ ≈ 75-120), near-infrared (0.8--2.5 μm) spectra spanning a decade of observations with the IRTF SpeX spectrograph. Primarily containing ultracool M, L, T and Y dwarfs, this spectral library has been used in over 100 publications to date, facilitating a broad range of science on low mass stars, exoplanets, high redshift sources and instrument/survey design. I summarize the contents of the SPL and highlight a few of the key scientific results that have made use of this resource, as well as applications in education, outreach and art. I also outline the future plans of the SPL, which include a reanalysis of early data, better integration and dissemination of source and spectral metadata, conversion to Virtual Observatory formats, development of a Python software package for community analysis, and a design for a node-based visual programming platform that can facilitate citizen science and project-based learning in stellar spectroscopy. http://www.browndwarfs.org/spexprism

  1. Assessment of constituents in Allium by multivariate data analysis, high-resolution α-glucosidase inhibition assay and HPLC-SPE-NMR.

    PubMed

    Schmidt, Jeppe S; Nyberg, Nils T; Staerk, Dan

    2014-10-15

    Bulbs and leaves of 35 Allium species and cultivars bought or collected in 2010-2012 were investigated with multivariate data analysis, high-resolution α-glucosidase inhibition assays and HPLC-HRMS-SPE-NMR with the aim of exploring the potential of Allium as a future functional food for management of type 2 diabetes. It was found that 30 out of 106 crude extracts showed more than 80% inhibition of the α-glucosidase enzyme at a concentration of 40mg/mL (dry sample) or 0.4g/mL (fresh sample). High-resolution α-glucosidase biochromatograms of these extracts allowed fast identification of three analytes with α-glucosidase inhibitory activity, and subsequent HPLC-HRMS-SPE-NMR experiments allowed identification of these as N-p-coumaroyloctopamine, N-p-coumaroyltyramine, and quercetin. The distribution of these three compounds was mapped for all samples by HPLC-ESI-HRMS. Unsupervised principal component analysis of samples from 2012 indicated that a major difference between fresh material and dried material is the increased amount of quercetin, a known α-glucosidase inhibitor. PMID:24837940

  2. Chemical Explosion Experiments to Improve Nuclear Test Monitoring [Developing a New Paradigm for Nuclear Test Monitoring with the Source Physics Experiments (SPE)

    DOE PAGESBeta

    Snelson, Catherine M.; Abbott, Robert E.; Broome, Scott T.; Mellors, Robert J.; Patton, Howard J.; Sussman, Aviva J.; Townsend, Margaret J.; Walter, William R.

    2013-07-02

    A series of chemical explosions, called the Source Physics Experiments (SPE), is being conducted under the auspices of the U.S. Department of Energy’s National Nuclear Security Administration (NNSA) to develop a new more physics-based paradigm for nuclear test monitoring. Currently, monitoring relies on semi-empirical models to discriminate explosions from earthquakes and to estimate key parameters such as yield. While these models have been highly successful monitoring established test sites, there is concern that future tests could occur in media and at scale depths of burial outside of our empirical experience. This is highlighted by North Korean tests, which exhibit poormore » performance of a reliable discriminant, mb:Ms (Selby et al., 2012), possibly due to source emplacement and differences in seismic responses for nascent and established test sites. The goal of SPE is to replace these semi-empirical relationships with numerical techniques grounded in a physical basis and thus applicable to any geologic setting or depth.« less

  3. Simultaneous determination of isoflavones and resveratrols for adulteration detection of soybean and peanut oils by mixed-mode SPE LC-MS/MS.

    PubMed

    Zhao, Xin; Ma, Fei; Li, Peiwu; Li, Guangming; Zhang, Liangxiao; Zhang, Qi; Zhang, Wen; Wang, Xiupin

    2015-06-01

    To ensure authenticity of vegetable oils, isoflavones (genistein, genistin, daidzein and daidzin) and resveratrols (cis-resveratrol and trans-resveratrol) were selected as the putative markers for adulteration of soybean and peanut oils. Firstly, mixed mode solid-phase extraction coupled with liquid chromatography tandem mass spectrometry (mixed-mode SPE LC-MS/MS) method was developed to analyze isoflavones and resveratrols in vegetable oils. The concentration of marker compounds in vegetable oils were 0.08-1.47mgkg(-1) for daidzein, ND-78.9μgkg(-1) for daidzin, 0.40-5.89mgkg(-1) for genistein, 1.2-114.9μgkg(-1) for genistin, 3.1-85.0μgkg(-1) for trans-resveratrol and 1.9-51.0μgkg(-1) for cis-resveratrol, which are compatible with the raw materials for oil press. Additionally, the applicability of this method has been successfully tested in thirteen vegetable oils from the market. Mixed-mode SPE LC-MS/MS method can simultaneously detect isoflavones and resveratrols in vegetable oils and assess adulteration and quality of soybean and peanut oils. PMID:25624257

  4. The Role of Mitochondria in Apoptosis*

    PubMed Central

    Wang, Chunxin; Youle, Richard J.

    2016-01-01

    Mitochondria play key roles in activating apoptosis in mammalian cells. Bcl-2 family members regulate the release of proteins from the space between the mitochondrial inner and outer membrane that, once in the cytosol, activate caspase proteases that dismantle cells and signal efficient phagocytosis of cell corpses. Here we review the extensive literature on proteins released from the intermembrane space and consider genetic evidence for and against their roles in apoptosis activation. We also compare and contrast apoptosis pathways in Caenorhabditis elegans, Drosophila melanogaster, and mammals that indicate major mysteries remaining to be solved. PMID:19659442

  5. [The comeback of mitochondria in Drosophila apoptosis].

    PubMed

    Clavier, Amandine; Rincheval-Arnold, Aurore; Mignotte, Bernard; Guénal, Isabelle

    2016-05-01

    The role of the mitochondrion in mammalian cell apoptosis has been established since the mid-1990s. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, notably because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and apoptosis in Drosophila cell death occurs at the mitochondrial level. Numerous proteins that appear key for Drosophila apoptosis regulation constitutively or transiently bind to mitochondria. They participate in the cell death process at different levels such as degradation of an IAP caspase inhibitor, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. The aim of this review is to take stock of these events that might have their counterpart in humans. PMID:27225920

  6. Autophagy and apoptosis in liver injury

    PubMed Central

    Wang, Kewei

    2015-01-01

    Apoptosis is a primary characteristic in the pathogenesis of liver disease. Hepatic apoptosis is regulated by autophagic activity. However, mechanisms mediating their interaction remain to be determined. Basal level of autophagy ensures the physiological turnover of old and damaged organelles. Autophagy also is an adaptive response under stressful conditions. Autophagy can control cell fate through different cross-talk signals. A complex interplay between hepatic autophagy and apoptosis determines the degree of hepatic apoptosis and the progression of liver disease as demonstrated by pre-clinical models and clinical trials. This review summarizes recent advances on roles of autophagy that plays in pathophysiology of liver. The autophagic pathway can be a novel therapeutic target for liver disease. PMID:25927598

  7. Noninvasive real-time imaging of apoptosis.

    PubMed

    Laxman, Bharathi; Hall, Daniel E; Bhojani, Mahaveer Swaroop; Hamstra, Daniel A; Chenevert, Thomas L; Ross, Brian D; Rehemtulla, Alnawaz

    2002-12-24

    Strict coordination of proliferation and programmed cell death (apoptosis) is essential for normal physiology. An imbalance in these two opposing processes results in various diseases including AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemiareperfusion injury, cancer, autoimmune disease, among others. Objective and quantitative noninvasive imaging of apoptosis would be a significant advance for rapid and dynamic screening as well as validation of experimental therapeutic agents. Here, we report the development of a recombinant luciferase reporter molecule that when expressed in mammalian cells has attenuated levels of reporter activity. In cells undergoing apoptosis, a caspase-3-specific cleavage of the recombinant product occurs, resulting in the restoration of luciferase activity that can be detected in living animals with bioluminescence imaging. The ability to image apoptosis noninvasively and dynamically over time provides an opportunity for high-throughput screening of proapoptotic and antiapoptotic compounds and for target validation in vivo in both cell lines and transgenic animals. PMID:12475931

  8. Autophagy and apoptosis in liver injury.

    PubMed

    Wang, Kewei

    2015-01-01

    Apoptosis is a primary characteristic in the pathogenesis of liver disease. Hepatic apoptosis is regulated by autophagic activity. However, mechanisms mediating their interaction remain to be determined. Basal level of autophagy ensures the physiological turnover of old and damaged organelles. Autophagy also is an adaptive response under stressful conditions. Autophagy can control cell fate through different cross-talk signals. A complex interplay between hepatic autophagy and apoptosis determines the degree of hepatic apoptosis and the progression of liver disease as demonstrated by pre-clinical models and clinical trials. This review summarizes recent advances on roles of autophagy that plays in pathophysiology of liver. The autophagic pathway can be a novel therapeutic target for liver disease. PMID:25927598

  9. An AIF orthologue regulates apoptosis in yeast

    PubMed Central

    Wissing, Silke; Ludovico, Paula; Herker, Eva; Büttner, Sabrina; Engelhardt, Silvia M.; Decker, Thorsten; Link, Alexander; Proksch, Astrid; Rodrigues, Fernando; Corte-Real, Manuela; Fröhlich, Kai-Uwe; Manns, Joachim; Candé, Céline; Sigrist, Stephan J.; Kroemer, Guido; Madeo, Frank

    2004-01-01

    Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1). PMID:15381687

  10. Regulation of apoptosis by peroxisome proliferators.

    PubMed

    Roberts, Ruth A; Michel, Cecile; Coyle, Beth; Freathy, Caroline; Cain, Kelvin; Boitier, Eric

    2004-04-01

    Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis

  11. EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

    PubMed Central

    Allhorn, Maria; Olsén, Arne; Collin, Mattias

    2008-01-01

    Background The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. Results We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. Conclusion We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself. PMID:18182097

  12. Human mature erythroblasts are resistant to apoptosis.

    PubMed

    Hristoskova, Sashka; Holzgreve, Wolfgang; Hahn, Sinuhe; Rusterholz, Corinne

    2007-03-10

    Apoptosis plays an important role in red blood cell development, notably by regulating the fate of early erythroid progenitors. We show here that, by contrast, mature erythroblasts are resistant to apoptosis. Treatment of these cells with several apoptosis-inducing agents failed to trigger caspase activation and oligonucleosomal DNA fragmentation. Interestingly, we find that cytochrome c levels are dramatically reduced even though the cells contain mitochondria. Supplementation of cytosolic extracts from mature erythroblasts with cytochrome c, however, did not rescue caspase activation. This was not due to the presence of inhibitors of apoptosis, as these proteins were also missing in these cells. We also show that cytochrome c depletion is a normal event during erythroblast differentiation, which follows transient, developmentally induced caspase activation and correlates with the loss of response to cytokine withdrawal or drug-induced apoptosis. Our data therefore suggest that erythroblasts acquire resistance to apoptosis during maturation through the developmentally induced depletion of cytochrome c and other crucial regulators of the apoptotic machinery. PMID:17289021

  13. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  14. Ultrastructural definition of apoptosis in heart failure.

    PubMed

    Arbustini, Eloisa; Brega, Agnese; Narula, Jagat

    2008-06-01

    Cardiac myocytes die through apoptosis, oncosis, and autophagy. Apoptosis affects single cells and is morphologically characterized by nuclear fragmentation with generation of apoptotic bodies that can be seen either within dying cells or free in the interstitial spaces. Dead myocytes are removed by macrophages through phagocytosis without triggering inflammation. The circulating markers of myocyte necrosis are not increased by apoptosis. The morphologic changes of the induction and early execution phases are seen at electron microscopy while late fragmentation is visible on both light and electron microscopy. Immunoelectron microscopy provides combined functional and structural information showing cytochrome c immuno-labelling release from mitochondria, TUNEL labelling of apoptotic nuclei, annexin V translocation in the outer plasma cell layer. Oncosis is characterized by specific morphologic features that may coexist with apoptosis, especially in ischemic myocardium. Autophagy is a defense process that is associated with significant myocardial damage and necrosis when removal of the lysosomal content is impaired. Morphological features of apoptosis, oncosis, and autophagocytosis may coexist at the same time. Although dead myocytes showing characteristics of autophagy and apoptosis are rarely observed in human decompensated hearts, autophagic vacuoles, and early apoptotic changes may be seen more often in morphologically viable myocytes. Such features may occur in failing hearts of both ischemic and non-ischemic etiology. The shared mode of cardiac myocyte death in failing human hearts of different etiologies suggests that preservation of myocyte integrity may be possible by similar therapeutic strategies. PMID:18172761

  15. Ellagic acid plays a protective role against UV-B-induced oxidative stress by up-regulating antioxidant components in human dermal fibroblasts

    PubMed Central

    Baek, Beomyeol; Lee, Su Hee; Lim, Hye-Won

    2016-01-01

    Ellagic acid (EA), an antioxidant polyphenolic constituent of plant origin, has been reported to possess diverse pharmacological properties, including anti-inflammatory, anti-tumor and immunomodulatory activities. This work aimed to clarify the skin anti-photoaging properties of EA in human dermal fibroblasts. The skin anti-photoaging activity was evaluated by analyzing the reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP-2), total glutathione (GSH) and superoxide dismutase (SOD) activity levels as well as cell viability in dermal fibroblasts under UV-B irradiation. When fibroblasts were exposed to EA prior to UV-B irradiation, EA suppressed UV-B-induced ROS and proMMP-2 elevation. However, EA restored total GSH and SOD activity levels diminished in fibroblasts under UV-B irradiation. EA had an up-regulating activity on the UV-B-reduced Nrf2 levels in fibroblasts. EA, at the concentrations used, was unable to interfere with cell viabilities in both non-irradiated and irradiated fibroblasts. In human dermal fibroblasts, EA plays a defensive role against UV-B-induced oxidative stress possibly through an Nrf2-dependent pathway, indicating that this compound has potential skin antiphotoaging properties. PMID:27162481

  16. UV-B Induced Generation of Reactive Oxygen Species Promotes Formation of BFA-Induced Compartments in Cells of Arabidopsis Root Apices

    PubMed Central

    Yokawa, Ken; Kagenishi, Tomoko; Baluška, František

    2016-01-01

    UV-B radiation is an important part of the electromagnetic spectrum emitted by the sun. For much of the period of biological evolution organisms have been exposed to UV radiation, and have developed diverse mechanisms to cope with this potential stress factor. Roots are usually shielded from exposure to UV by the surrounding soil, but may nevertheless be exposed to high energy radiation on the soil surface. Due to their high sensitivity to UV-B radiation, plant roots need to respond rapidly in order to minimize exposure on the surface. In addition to root gravitropism, effective light perception by roots has recently been discovered to be essential for triggering negative root phototropism in Arabidopsis. However, it is not fully understood how UV-B affects root growth and phototropism. Here, we report that UV-B induces rapid generation of reactive oxygen species which in turn promotes the formation of BFA-induced compartments in the Arabidopsis root apex. During unilateral UV-B irradiation of roots changes in auxin concentration on the illuminated side have been recorded. In conclusion, UV-B-induced and ROS-mediated stimulation of vesicle recycling promotes root growth and induces negative phototropism. PMID:26793199

  17. UV-B Induced Generation of Reactive Oxygen Species Promotes Formation of BFA-Induced Compartments in Cells of Arabidopsis Root Apices.

    PubMed

    Yokawa, Ken; Kagenishi, Tomoko; Baluška, František

    2015-01-01

    UV-B radiation is an important part of the electromagnetic spectrum emitted by the sun. For much of the period of biological evolution organisms have been exposed to UV radiation, and have developed diverse mechanisms to cope with this potential stress factor. Roots are usually shielded from exposure to UV by the surrounding soil, but may nevertheless be exposed to high energy radiation on the soil surface. Due to their high sensitivity to UV-B radiation, plant roots need to respond rapidly in order to minimize exposure on the surface. In addition to root gravitropism, effective light perception by roots has recently been discovered to be essential for triggering negative root phototropism in Arabidopsis. However, it is not fully understood how UV-B affects root growth and phototropism. Here, we report that UV-B induces rapid generation of reactive oxygen species which in turn promotes the formation of BFA-induced compartments in the Arabidopsis root apex. During unilateral UV-B irradiation of roots changes in auxin concentration on the illuminated side have been recorded. In conclusion, UV-B-induced and ROS-mediated stimulation of vesicle recycling promotes root growth and induces negative phototropism. PMID:26793199

  18. Morphologic criteria and detection of apoptosis.

    PubMed

    Saraste, A

    1999-05-01

    Apoptosis is an organized, energy dependent process, which leads to cell death. Its definition is based on distinct morphological features [10] and demonstration of internucleosomal DNA degradation [27], executed by selectively activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include chromatic margination, nuclear condensation and fragmentation, and condensation of the cell with preservation of organelles. The process is followed by fragmentation of the cell into membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis characteristically occurs in insolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell death, a prototype of which is cell death due to ischemia (oncosis), is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles and rupture of the plasma membrane [15]. Groups of necrotic cells and inflammation are found in tissues [10, 15]. The significance of apoptosis has mostly been studied using the TUNEL assay that detects DNA strand breaks in tissue sections and allows quantification of apoptotic cells by light microscopy [6]. Common experience seems to be that the TUNEL assay is prone to false positive or negative findings. This has been explained by the dependence of the staining kinetics on the reagent concentration [17], fixation of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To obtain reliable and reproducible results, TUNEL assay should be carefully standardized by using tissue sections treated with DNAse (positive control of apoptosis). Quantification of apoptosis should include enough microscopic fields and identification of the cell type undergoing apoptosis

  19. Fully automated trace level determination of parent and alkylated PAHs in environmental waters by online SPE-LC-APPI-MS/MS.

    PubMed

    Ramirez, Cesar E; Wang, Chengtao; Gardinali, Piero R

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds that enter the environment from natural and anthropogenic sources, often used as markers to determine the extent, fate, and potential effects on natural resources after a crude oil accidental release. Gas chromatography-mass spectrometry (GC-MS) after liquid-liquid extraction (LLE+GC-MS) has been extensively used to isolate and quantify both parent and alkylated PAHs. However, it requires labor-intensive extraction and cleanup steps and generates large amounts of toxic solvent waste. Therefore, there is a clear need for greener, faster techniques with enough reproducibility and sensitivity to quantify many PAHs in large numbers of water samples in a short period of time. This study combines online solid-phase extraction followed by liquid chromatography (LC) separation with dopant-assisted atmospheric pressure photoionization (APPI) and tandem MS detection, to provide a one-step protocol that detects PAHs at low nanograms per liter with almost no sample preparation and with a significantly lower consumption of toxic halogenated solvents. Water samples were amended with methanol, fortified with isotopically labeled PAHs, and loaded onto an online SPE column, using a large-volume sample loop with an auxiliary LC pump for sample preconcentration and salt removal. The loaded SPE column was connected to an UPLC pump and analytes were backflushed to a Thermo Hypersil Green PAH analytical column where a 20-min gradient separation was performed at a variable flow rate. Detection was performed by a triple-quadrupole MS equipped with a gas-phase dopant delivery system, using 1.50 mL of chlorobenzene dopant per run. In contrast, LLE+GC-MS typically use 150 mL of organic solvents per sample, and methylene chloride is preferred because of its low boiling point. However, this solvent has a higher environmental persistence than chlorobenzene and is considered a carcinogen. The automated system is capable of

  20. MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics

    SciTech Connect

    Luo, Quanzhou; Page, Jason S.; Tang, Keqi; Smith, Richard D.

    2007-01-01

    Silica-based monolithic narrow bore capillary columns (25 cm x 10 µm i.d.) with an integrated nanoESI emitter has been developed to provide high quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated nanoESI emitter adds no dead volume to the LC separation, allowing stable electrospray performance to be obtained at flow rates of ~10 nL/min. In an initial application we identified 5510 unique peptides covering 1443 distinct Shewanella oneidensis proteins from a 300 ng tryptic digest sample in a single 4-h LC-MS/MS analysis using a linear ion trap MS (LTQ). We found the use of an integrated monolithic ESI emitter provided enhanced resistance to clogging and good run-to-run reproducibility.

  1. Development, validation, and uncertainty measurement of multi-residue analysis of organochlorine and organophosphorus pesticides using pressurized liquid extraction and dispersive-SPE techniques.

    PubMed

    Sanyal, Doyeli; Rani, Anita; Alam, Samsul; Gujral, Seema; Gupta, Ruchi

    2011-11-01

    Simple and efficient multi-residue analytical methods were developed and validated for the determination of 13 organochlorine and 17 organophosphorous pesticides from soil, spinach and eggplant. Techniques namely accelerated solvent extraction and dispersive SPE were used for sample preparations. The recovery studies were carried out by spiking the samples at three concentration levels (1 limit of quantification (LOQ), 5 LOQ, and 10 LOQ). The methods were subjected to a thorough validation procedure. The mean recovery for soil, spinach and eggplant were in the range of 70-120% with median CV (%) below 10%. The total uncertainty was evaluated taking four main independent sources viz., weighing, purity of the standard, GC calibration curve and repeatability under consideration. The expanded uncertainty was well below 10% for most of the pesticides and the rest fell in the range of 10-20%. PMID:21210211

  2. Trauma patients’ elevated Tumor Necrosis Related Apoptosis Inducing Ligand (TRAIL) contributes to increased T cell apoptosis

    PubMed Central

    Bandyopadhyay, Gautam; Bankey, Paul E.; Miller-Graziano, Carol L.

    2012-01-01

    Immunosuppression resulting from excessive post-trauma apoptosis of hyperactivated Tcells is controversial. TRAIL mediated Tcell apoptosis decreases highly activated Tcells’ responses. Caspase-10, a particular TRAIL target, was increased in trauma patients’ Tcells with concomitantly elevated plasma TRAIL levels. These patients’ Tcells developed anergy, implicating increased TRAIL-mediated Tcell apoptosis in post-trauma Tcell anergy. Control Tcells cultured with patients’ sera containing high TRAIL levels increased their Caspase-10 activity and apoptosis. Stimulated primary Tcells are TRAIL apoptosis resistant. Increased plasma Thrombospondin-1 and Tcell expression of CD47, a Thrombospondin-1 receptor, preceded patients’ Tcell anergy. CD47 triggering of Tcells increased their sensitivity to TRAIL-induced apoptosis. Augmentation of Tcell TRAIL-induced apoptosis was secondary to CD47 triggered activation of the Src homology-containing phosphatase-1(SHP-1) and was partially blocked by a SHP-1 inhibitor. We suggest that combined post-trauma CD47 triggering, SHP-1 mediated NFκB suppression, and elevated TRAIL levels increase patients’ CD47 expressing Tcell apoptosis, thus contributing to subsequent Tcell anergy. PMID:22926077

  3. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    PubMed Central

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  4. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis.

    PubMed

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  5. Solamargine triggers hepatoma cell death through apoptosis

    PubMed Central

    XIE, XIAODONG; ZHU, HAITAO; YANG, HUIJIAN; HUANG, WENSI; WU, YINGYING; WANG, YING; LUO, YANLING; WANG, DONGQING; SHAO, GENBAO

    2015-01-01

    Solamargine (SM), a steroidal alkaloid glycoside extracted from the traditional Chinese herb Solanum incanum, has been evidenced to inhibit the growth and induce apoptosis in a number of human cancer cell lines. In the present study, the anticancer effect of SM and underlying molecular mechanism of SM-induced apoptosis were investigated on the human hepatocellular carcinoma cells, SMMC7721 and HepG2. The proliferation effects of SM on the SMMC7721 and HepG2 cell lines were evaluated using MTT and colony formation assays. In addition, the percentage of apoptosis was measured using an Annexin V/propidium iodide staining method and the cell cycle distribution mediated by SM was analyzed using flow cytometry. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-9, proliferating cell nuclear antigen (pcna) and Ki67 proteins were examined to further demonstrate the proliferate and apoptosis effects of SM on the hepatoma cells. The results indicated that SM effectively inhibited hepatoma cell proliferation and promoted apoptosis. SM resulted in cell cycle arrest at the G2/M phase in the two cell lines. In addition, SM downregulated the levels of proliferation-associated (Ki67 and pcna) and anti-apoptotic (Bcl-2) proteins, and promoted the activity of apoptosis-associated proteins (Bax, caspase-3 and caspase-9). Therefore, the activation of the Bcl-2/Bax and caspase signaling pathways may be involved in the SM-induced apoptosis of hepatoma cells. PMID:26170994

  6. Training-induced apoptosis in skeletal muscle.

    PubMed

    Boffi, F M; Cittar, J; Balskus, G; Muriel, M; Desmaras, E

    2002-09-01

    Apoptosis or programmed cell death is a genetically controlled response of cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include Ca2+i and oxygen free radicals/oxidative stress, which are also implicated in the pathogenesis of exercise-induced myopathies. To examine training-induced apoptosis, Thoroughbred horses were subjected to 3 months training programme on a treadmill. At the end of the training programme venous blood samples were taken for a creatine kinase (CK) assay. In addition, muscle biopsy samples were obtained for a membrane lipid peroxidation measurement by malondialdehyde (MDA) assay and for apoptosis detection. Apoptosis was studied by visualising the apoptotic myocytes on the paraffin sections by the modified TUNEL method. DNA laddering was evaluated by subjecting the DNA obtained from the biopsies to 1.5% agarose gel electrophoresis. There was a significant increase (P<0.05) of protein-bound MDA, and a nonsignificant trend (P = 0.14) for the control group to have higher levels of CK compared to the trained group. Under light microscopy, percentage of the TUNEL positive cells was higher (P<0.001) in the training group. This result was corroborated with the findings of DNA fragmentation by gel electrophoresis, which showed higher ladders of DNA band at the same group. In conclusion, these results clearly demonstrate that there is training-induced apoptosis in skeletal muscle. It is probable that apoptosis allows the work/recovery/rebound/supercompensation cycle, when unaccustomed muscle cells activate programmed cell death and are replaced by new and stronger cells, which is the mechanism for training-induced increases in fitness. PMID:12405700

  7. Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti(4+)-SPE enrichment for mass spectrometric analysis.

    PubMed

    Zhang, Ying; Peng, Ye; Bin, Zhichao; Wang, Huijie; Lu, Haojie

    2016-08-31

    N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti(4+)-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti(4+)-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified. PMID:27506354

  8. Outcompeting GC for the detection of legacy chlorinated pesticides: online-SPE UPLC APCI/MSMS detection of endosulfans at part per trillion levels.

    PubMed

    Quinete, Natalia; Wang, Jian; Fernandez, Adolfo; Castro, Joffre; Gardinali, Piero R

    2013-07-01

    Endosulfan, the last remaining organochlorine pesticide, has been the subject of a number of international regulations and restriction/banning action plans worldwide. Occurrence of endosulfan residues in South Florida environments has been widely described in the literature for more than two decades. This work describes a selective, sensitive, and fast online solid-phase extraction (SPE) method coupled with liquid chromatography separation and tandem mass spectrometry (LC-MS/MS) for the determination of endosulfan isomers and endosulfan sulfate in water samples at low part per trillion levels with very little sample preparation. A negative atmospheric pressure chemical ionization source was carefully optimized to produce reproducible spectra of the target compounds with no adduct ion formation. Selected reaction monitoring transitions were monitored and quantitation was performed against a per-deuterated internal standard β-endosulfan (d4). The automated online SPE clean-up was performed using only 20 mL of untreated water sample prior to LC-MS/MS analysis. The method was capable of separating and quantifying endosulfan within a 24-min run using acetonitrile and water as mobile phases and presenting statistically calculated method detection limits of 3, 10, and 7 ng/L for endosulfan sulfate, α-endosulfan, and β-endosulfan, respectively. In addition, a QuEChERS method was successfully developed and applied for endosulfan determination in sediments/soils, floating and submerged algal mats, and small fish. Minimal matrix effects were observed in all matrices analyzed and recoveries for all analytes ranged from 50-144 %. The developed methodology was applied to monitor the occurrence and to assess the potential transport of endosulfan in the Loveland Slough watershed, an area adjacent to Everglades National Park showing long-term contamination with endosulfans. PMID:23386002

  9. Evaluation of Apoptosis in Immunotoxicity Testing

    PubMed Central

    Nagarkatti, Mitzi; Rieder, Sadiye Amcaoglu; Vakharia, Dilip; Nagarkatti, Prakash S.

    2014-01-01

    Immunotoxicity testing is important in determining the toxic effects of chemical substances, medicinal products, airborne pollutants, cosmetics, medical devices, and food additives. The immune system of the host is a direct target of these toxicants, and the adverse effects include serious health complications such as susceptibility to infections, cancer, allergic reactions, and autoimmune diseases. One way to investigate the harmful effects of different chemicals is to study apoptosis in immune cell populations. Apoptosis is defined as the programmed cell death, and in general, this process helps in development and maintains homeostasis. However, in the case of an insult by a toxicant, apoptosis of the immune cells can lead to immunosuppression resulting in the development of cancer and the inability to fight infections. Apoptosis is characterized by cell shrinkage, nuclear condensation, changes in cell membrane and mitochondria, DNA fragmentation into 200 base oligomers, and protein degradation by caspases. Various methods are employed in order to investigate apoptosis. These methods include direct measurement of apoptotic cells with flow cytometry and in situ labeling, as well as RNA, DNA, and protein assays that are indicative of apoptotic molecules. PMID:19967519

  10. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    NASA Astrophysics Data System (ADS)

    Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng

    2013-02-01

    Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (ΔΨm). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ΔΨm. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.

  11. Targeting the Apoptosis Pathway in Hematologic Malignancies

    PubMed Central

    Zaman, Shadia; Wang, Rui; Gandhi, Varsha

    2014-01-01

    Apoptosis is a cell death program that is well-orchestrated for normal tissue homeostasis and for removal of damaged, old, or infected cells. It is regulated by intrinsic and extrinsic pathways. The intrinsic pathway responds to signals such as ultraviolet radiation or DNA damage and activates “executioner” caspases through a mitochondria-dependent pathway. The extrinsic pathway is activated by death signals induced, for example, by an infection that activates the immune system or receptor-mediated pathways. The extrinsic pathway signals also cascade down to executioner caspases that cleave target proteins and lead to cell death. Strict control of cellular apoptosis is important for the hematopoietic system as it has a high turnover rate. However, the apoptosis program is often deregulated in hematologic malignancies leading to the accumulation of malignant cells. Therefore, apoptosis pathways have been identified for development of anticancer therapeutics. We review here the proteins that have been targeted for anticancer drug development in hematologic malignancies. These include BCL-2 family proteins, death ligands and receptors, inhibitor of apoptosis family proteins, and caspases. Except for caspase activators, drugs that target each of these classes of proteins have advanced into clinical trials. PMID:24295132

  12. NMR exposure sensitizes tumor cells to apoptosis.

    PubMed

    Ghibelli, L; Cerella, C; Cordisco, S; Clavarino, G; Marazzi, S; De Nicola, M; Nuccitelli, S; D'Alessio, M; Magrini, A; Bergamaschi, A; Guerrisi, V; Porfiri, L M

    2006-03-01

    NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3-66 mT) static magnetic fields deeply affect apoptosis in a Ca2+ dependent fashion (Fanelli et al., 1999 FASEBJ., 13;95-102). The rationale of the present study is to examine whether exposure to the static magnetic fields of NMR can affect apoptosis induced on reporter tumor cells of haematopoietic origin. The impressive result was the strong increase (1.8-2.5 fold) of damage-induced apoptosis by NMR. This potentiation is due to cytosolic Ca2+ overload consequent to NMR-promoted Ca2+ influx, since it is prevented by intracellular (BAPTA-AM) and extracellular (EGTA) Ca2+ chelation or by inhibition of plasma membrane L-type Ca2+ channels. Three-days follow up of treated cultures shows that NMR decrease long term cell survival, thus increasing the efficiency of cytocidal treatments. Importantly, mononuclear white blood cells are not sensitised to apoptosis by NMR, showing that NMR may increase the differential cytotoxicity of antitumor drugs on tumor vs normal cells. This strong, differential potentiating effect of NMR on tumor cell apoptosis may have important implications, being in fact a possible adjuvant for antitumor therapies. PMID:16528477

  13. Lysosomal destabilization in p53-induced apoptosis

    PubMed Central

    Yuan, Xi-Ming; Li, Wei; Dalen, Helge; Lotem, Joseph; Kama, Rachel; Sachs, Leo; Brunk, Ulf T.

    2002-01-01

    The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders. PMID:11959917

  14. Determination of analytes in medical herbs extracts by SPE coupled with two-dimensional planar chromatography in combination with diode array scanning densitometry and HPLC-diode array detector.

    PubMed

    Tuzimski, Tomasz

    2011-01-01

    The purpose of this study is to demonstrate an application of 2-D high-performance planar chromatography-diode array detector (DAD) and HPLC-DAD after solid-phase extraction (SPE) for identification and quantitative analysis of pesticides (isoproturon, aziprotryne, hexazinone, flufenoxuron, methabenzthiazuron, procymidone, and α-cypermethrin) in Melissa officinalis L. (Labiatae) samples. The procedure described for the determination of compounds is inexpensive and can be applied to routine analysis of analytes in medical herbs' samples after preliminary cleanup and concentration by SPE. Average recoveries on C18 SPE cartridges of pesticides eluted with 5 mL tetrahydrofuran by the proposed HPLC-DAD method, before and after 2-D-high-performance planar chromatography separation of analytes from M. officinalis L. samples spiked with pesticide at a concentration level of 10 μg/g in plant material are presented. Method validation parameters for the quantification of pesticides by the proposed HPLC-DAD after SPE method are also presented. PMID:21171173

  15. The NFκB-inducing kinase is essential for the developmental programming of skin-resident and IL-17-producing γδ T cells.

    PubMed

    Mair, Florian; Joller, Stefanie; Hoeppli, Romy; Onder, Lucas; Hahn, Matthias; Ludewig, Burkhard; Waisman, Ari; Becher, Burkhard

    2015-01-01

    γδ T cells contribute to first line immune defense, particularly through their ability for rapid production of proinflammatory cytokines. The cytokine profile of γδ T cells is hard-wired already during thymic development. Yet, the molecular pathways underlying this phenomenon are incompletely understood. Here we show that signaling via the NFκB-inducing kinase (NIK) is essential for the formation of a fully functional γδ T cell compartment. In the absence of NIK, development of Vγ5(+) dendritic epidermal T cells (DETCs) was halted in the embryonic thymus, and impaired NIK function caused a selective loss of IL-17 expression by γδ T cells. Using a novel conditional mutant of NIK, we could show in vivo that NIK signaling in thymic epithelial cells is essential for the thymic hardwiring of γδ T cell cytokine production. PMID:26637788

  16. Apoptosis in Drosophila: which role for mitochondria?

    PubMed

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human. PMID:26679112

  17. Lipid metabolism, apoptosis and cancer therapy.

    PubMed

    Huang, Chunfa; Freter, Carl

    2015-01-01

    Lipid metabolism is regulated by multiple signaling pathways, and generates a variety of bioactive lipid molecules. These bioactive lipid molecules known as signaling molecules, such as fatty acid, eicosanoids, diacylglycerol, phosphatidic acid, lysophophatidic acid, ceramide, sphingosine, sphingosine-1-phosphate, phosphatidylinositol-3 phosphate, and cholesterol, are involved in the activation or regulation of different signaling pathways. Lipid metabolism participates in the regulation of many cellular processes such as cell growth, proliferation, differentiation, survival, apoptosis, inflammation, motility, membrane homeostasis, chemotherapy response, and drug resistance. Bioactive lipid molecules promote apoptosis via the intrinsic pathway by modulating mitochondrial membrane permeability and activating different enzymes including caspases. In this review, we discuss recent data in the fields of lipid metabolism, lipid-mediated apoptosis, and cancer therapy. In conclusion, understanding the underlying molecular mechanism of lipid metabolism and the function of different lipid molecules could provide the basis for cancer cell death rationale, discover novel and potential targets, and develop new anticancer drugs for cancer therapy. PMID:25561239

  18. Control of apoptosis by Drosophila DCAF12.

    PubMed

    Hwangbo, Dae-Sung; Biteau, Benoit; Rath, Sneha; Kim, Jihyun; Jasper, Heinrich

    2016-05-01

    Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. Here, we report the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. We find that DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD. PMID:26972874

  19. Downregulation of Reactive Oxygen Species in Apoptosis

    PubMed Central

    Jeong, Chul-Ho; Joo, Sang Hoon

    2016-01-01

    Generation of reactive oxygen species (ROS) by diverse anti-cancer drugs or phytochemicals has been closely related with the induction of apoptosis in cancers. Also, the downregulation of ROS by these chemicals has been found to block initiation of carcinogenesis. Therefore, modulation of ROS by phytochemicals emerges as a crucial mechanism to regulate apoptosis in cancer prevention or therapy. This review summarizes the current understanding of the selected chemical compounds and related cellular components that modulate ROS during apoptotic process. Metformin, quercetin, curcumin, vitamin C, and other compounds have been shown to downregulate ROS in the cellular apoptotic process, and some of them even induce apoptosis in cancer cells. The cellular components mediating the downregulation of ROS include nuclear factor erythroid 2-related factor 2 antioxidant signaling pathway, thioredoxin, catalase, glutathione, heme oxygenase-1, and uncoupling proteins. The present review provides information on the relationship between these compounds and the cellular components in modulating ROS in apoptotic cancer cells. PMID:27051644

  20. Shikonin causes cell-cycle arrest and induces apoptosis by regulating the EGFR–NF-κB signalling pathway in human epidermoid carcinoma A431 cells

    PubMed Central

    Tian, Rong; Li, You; Gao, Mei

    2015-01-01

    Shikonin, a naphthoquinone pigment isolated from the Chinese herbal Zicao, has been shown to exhibit antioxidant and anticancer effects. In the present study, we investigated the antiproliferative and pro-apoptotic effects of shikonin on A431 cells and explored the underlying molecular mechanisms. In the present study, our results showed that shikonin significantly inhibited the growth of A431 cells in a concentration- and time-dependent manner, and caused cell cycle arrest by upregulation of p21 and p27, and downregulation of cyclins and cyclin-dependent kinases. In addition, shikonin evidently induced apoptosis due to decreasing Bcl-2 expression, increasing Bax expression, activating caspase and inactivating NF-κB, while pretreatment with a pan-caspase inhibitor Z-Asp-CH2-DCB abrogated shikonin-induced apoptosis. Moreover, EGF could significantly increase the NF-κB DNA-binding activity and reversed the shikonin-induced inactivation of NF-κB. As anticipated AG1478 (EGFR inhibitor) and Bay11-7082 (NF-κB inhibitor) blocked EGF-reversed the inactivation of NF-κB induced by shikonin. Our data also showed that EGF could evidently reverse the shikonin-induced decreases in cell viability and increases in apoptosis. Then, the NF-κB inhibitors such as Bay11-7082, SN50, Helenalin and the EGFR inhibitor AG1478 and its downstream inhibitor such as PI3K inhibitor LY294002 and STAT3 inhibitor Stattic dramatically blocked EGF-reversed decreases in cell viability and increases in apoptosis induced by shikonin. Collectively, our findings indicated that shikonin inhibited cell growth and caused cell cycle arrest of the A431 cells through the regulation of apoptosis. Moreover, these effects were mediated at least partially by suppressing the activation of the EGFR–NF-κB signaling pathways. PMID:25720435

  1. Hydrogen peroxide, nitric oxide and UV RESISTANCE LOCUS8 interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts

    PubMed Central

    Wu, Qi; Su, Nana; Zhang, Xiaoyan; Liu, Yuanyuan; Cui, Jin; Liang, Yongchao

    2016-01-01

    The cross talk among hydrogen peroxide (H2O2), nitric oxide (NO) and UV RESISTANCE LOCUS8 (UVR8) in UV-B-induced anthocyanin accumulation in the hypocotyls of radish sprouts was investigated. The results showed that UV-B irradiation significantly increased the anthocyanin accumulation and the expression of UVR8, and a similar trend appeared in radish sprouts subjected to cadmium, chilling and salt stresses regardless of light source. However, these responses disappeared under dark exposure. These results suggest that abiotic stress-induced anthocyanin accumulation and UVR8 expression were light-dependent. Moreover, abiotic stresses all enhanced the production of H2O2 and exogenous H2O2 addition significantly increased the anthocyanin concentration and UVR8 transcription, while these increases were severely inhibited by addition of dimethylthiourea (DMTU, a chemical trap for H2O2). It seems to suggest that H2O2 played an important role in the anthocyanin biosynthesis. Furthermore, addition of 0.5 mM sodium nitroprusside (SNP, a NO-releasing compound) substantially induced the anthocyanin accumulation, and H2O2-induced anthocyanin accumulation and UVR8 expression were significantly suppressed by co-treatment with 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO, a NO scavenger), which was parallel with the expression of anthocyanin biosynthesis-related transcription factors and structural genes. All these results demonstrate that both H2O2 and NO are involved in UV-B-induced anthocyanin accumulation, and there is a crosstalk between them as well as a classical UVR8 pathway. PMID:27404993

  2. Hydrogen peroxide, nitric oxide and UV RESISTANCE LOCUS8 interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts.

    PubMed

    Wu, Qi; Su, Nana; Zhang, Xiaoyan; Liu, Yuanyuan; Cui, Jin; Liang, Yongchao

    2016-01-01

    The cross talk among hydrogen peroxide (H2O2), nitric oxide (NO) and UV RESISTANCE LOCUS8 (UVR8) in UV-B-induced anthocyanin accumulation in the hypocotyls of radish sprouts was investigated. The results showed that UV-B irradiation significantly increased the anthocyanin accumulation and the expression of UVR8, and a similar trend appeared in radish sprouts subjected to cadmium, chilling and salt stresses regardless of light source. However, these responses disappeared under dark exposure. These results suggest that abiotic stress-induced anthocyanin accumulation and UVR8 expression were light-dependent. Moreover, abiotic stresses all enhanced the production of H2O2 and exogenous H2O2 addition significantly increased the anthocyanin concentration and UVR8 transcription, while these increases were severely inhibited by addition of dimethylthiourea (DMTU, a chemical trap for H2O2). It seems to suggest that H2O2 played an important role in the anthocyanin biosynthesis. Furthermore, addition of 0.5 mM sodium nitroprusside (SNP, a NO-releasing compound) substantially induced the anthocyanin accumulation, and H2O2-induced anthocyanin accumulation and UVR8 expression were significantly suppressed by co-treatment with 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO, a NO scavenger), which was parallel with the expression of anthocyanin biosynthesis-related transcription factors and structural genes. All these results demonstrate that both H2O2 and NO are involved in UV-B-induced anthocyanin accumulation, and there is a crosstalk between them as well as a classical UVR8 pathway. PMID:27404993

  3. Flavonoids Derived from Abelmoschus esculentus Attenuates UV-B Induced Cell Damage in Human Dermal Fibroblasts Through Nrf2-ARE Pathway

    PubMed Central

    Patwardhan, Juilee; Bhatt, Purvi

    2016-01-01

    Background: Ultraviolet-B (UV-B) radiation is a smaller fraction of the total radiation reaching the Earth but leads to extensive damage to the deoxyribonucleic acid (DNA) and other biomolecules through formation of free radicals altering redox homeostasis of the cell. Abelmoschus esculentus (okra) has been known in Ayurveda as antidiabetic, hypolipidemic, demulscent, antispasmodic, diuretic, purgative, etc. Objective: The aim of this study is to evaluate the protective effect of flavonoids from A. esculentus against UV-B-induced cell damage in human dermal fibroblasts. Materials and Methods: UV-B protective activity of ethyl acetate (EA) fraction of okra was studied against UV-B-induced cytotoxicity, antioxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway. Results: Flavonoid-rich EA fraction depicted a significant antioxidant potential also showing presence of rutin. Pretreatment of cells with EA fraction (10–30 μg/ml) prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Conclusion: Our study demonstrated for the 1st time that EA fraction of okra may reduce oxidative stress through Nrf2-ARE pathway as well as through endogenous enzymatic antioxidant system. These results suggested that flavonoids from okra may be considered as potential UV-B protective agents and may also be formulated into herbal sunscreen for topical application. SUMMARY Flavonoid-enriched ethyl acetate (EA) fraction from A. esculentus protected against ultraviolet-B (UV-B)-induced oxidative DNA damageEA fraction prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, and intracellular reactive oxygen species

  4. Apoptosis induced by dioscin in Hela cells.

    PubMed

    Cai, Jing; Liu, Mingjie; Wang, Zhao; Ju, Yong

    2002-02-01

    Dioscin, a saponin extracted from the root of Polygonatum Zanlanscianense Pamp, markedly inhibited proliferation of Hela cells. The results indicated that Hela cells underwent apoptosis in dose- and time-dependent manners when treated with Dioscin. Caspase-3, -8 and -9 activities were also detected. The low enzymatic activity of caspase-8 and high activity of caspase-9 showed that the mitochondrial pathway was activated in apoptosis. The reduced expression of the survival protein Bcl-2 also confirmed this result. These studies may be significant in finding a new drug to treat human cervical cancer. PMID:11853164

  5. Effects of ophiopogonin B on the proliferation and apoptosis of SGC-7901 human gastric cancer cells

    PubMed Central

    ZHANG, WEIYUE; ZHANG, QIAOYAN; JIANG, YIPING; LI, FENG; XIN, HAILIANG

    2016-01-01

    Ophiopogonin B (OP-B) is a bioactive component of Radix Ophiopogon japonicus, which is often used in traditional Chinese medicine to treat cancer. The present study aimed to investigate the antitumor activity of OP-B in gastric cancer. Cell Counting kit-8, flow cytometry with Annexin V-fluorescein isothiocyanate, Hoechst staining, mitochondrial membrane potential (MMP) detection, and reactive oxygen species (ROS) assay were used to detect the biological function of SGC-7901 gastric cancer cells. The results demonstrated that high concentrations of OP-B (5, 10 and 20 μmol/l) exerted potent antiproliferative effects on SGC-7901 cells in a dose-dependent manner. Furthermore, apoptotic rates were increased and cell morphology was altered following treatment with OP-B. In addition, OP-B-induced apoptosis of SGC-7901 cells was associated with loss of MMP and increased ROS generation. Western blotting indicated that treatment with OP-B increased the protein expression levels of caspase-3 and B-cell lymphoma 2 (Bcl-2)-associated X protein, whereas the expression levels of Bcl-2 and the phosphorylation levels of extracellular signal-regulated kinases 1/2 and c-Jun N-terminal kinases 1/2 were decreased. These results suggest that OP-B may be considered a potential inhibitor of gastric cancer progression, and may be used as an alternative compound for its treatment. PMID:27121658

  6. Apoptosis after reperfused myocardial infarction: Role of angiotensin II

    PubMed Central

    Jugdutt, Bodh I

    2004-01-01

    Angiotensin II (Ang II) plays a significant role in apoptosis after myocardial infarction (MI) and reperfused MI. Cumulative evidence suggests that Ang II is a major contributor to cardiomyocyte (CM) apoptosis and left ventricular (LV) dysfunction after acute reperfused MI and that apoptosis mediates a major portion of early LV dysfunction. Importantly, blockade of the Ang II type 1 receptor (AT1R) limits CM apoptosis and LV dysfunction after acute reperfused MI. Ang II type 2 receptor activation during AT1R blockade contributes to these beneficial effects. The role of Ang II and apoptosis in chronic LV remodelling, healing and post-MI heart failure is more complex and involves effects on the CMs, fibroblasts and vascular cells. The long-term effects of agents targeting apoptosis after reperfused MI, including AT1R blockade, on apoptosis in different cell types, windows of enhanced apoptosis and the appropriate timing of therapy need to be considered. PMID:19641712

  7. A novel method for detection of apoptosis

    SciTech Connect

    Zagariya, Alexander M.

    2012-04-15

    There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II*). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

  8. Farnesol-induced apoptosis in Candida albicans.

    PubMed

    Shirtliff, Mark E; Krom, Bastiaan P; Meijering, Roelien A M; Peters, Brian M; Zhu, Jingsong; Scheper, Mark A; Harris, Megan L; Jabra-Rizk, Mary Ann

    2009-06-01

    Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival. PMID:19364863

  9. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY

    EPA Science Inventory

    Apoptosis, a form of programmed cell death, occurs in the nervous system throughout development, but with a preponderance of cell death occurring during the prenatal and perinatal periods. Aberrant periods of increased or decreased cell death, induced by toxicants in air, water,...

  10. Fluorescence spectroscopy to assess apoptosis in myocardium

    NASA Astrophysics Data System (ADS)

    Ranji, Mahsa; Matsubara, Muneaki; Grosso, Michael A.; Jaggard, Dwight L.; Chance, Britton; Gorman, Robert C.; Gorman, Joseph H., III

    2007-02-01

    Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction. 1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.

  11. Techniques to Distinguish Apoptosis from Necroptosis.

    PubMed

    Feoktistova, Maria; Wallberg, Fredrik; Tenev, Tencho; Geserick, Peter; Leverkus, Martin; Meier, Pascal

    2016-04-01

    The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis. PMID:27037077

  12. Ceramide in apoptosis: a revisited role.

    PubMed

    Levade, Thierry; Malagarie-Cazenave, Sophie; Gouazé, Valérie; Ségui, Bruno; Tardy, Claudine; Betito, Susan; Andrieu-Abadie, Nathalie; Cuvillier, Olivier

    2002-08-01

    The sphingolipid ceramide has recently emerged as a new transducer or modulator of apoptotic cell death. This function, however, has recently been challenged. Here, in the light of recent observations, the role of ceramide in apoptosis signaling is discussed. PMID:12374195

  13. Effect of hyaluronic acid on chondrocyte apoptosis

    PubMed Central

    Barreto, Ronald Bispo; Sadigursky, David; de Rezende, Marcia Uchoa; Hernandez, Arnaldo José

    2015-01-01

    OBJECTIVE: To determine the percentage of apoptotic cells in a contusion model of osteoarthritis (OA) and to assess whether intra-articular injection of high doses of hyaluronic acid (HA) immediately after trauma reduces chondrocyte apoptosis. METHODS: Forty knees from adult rabbits were impacted thrice with a 1 kg block released through a 1 meter tall cylinder (29.4 Joules). Subsequently, 2 mL of HA was injected in one knee and 2 mL saline in the contra-lateral knee. Medication were administered twice a week for 30 days, when animals were sacrificed. Specimens were prepared for optical microscopy exam and terminal deoxynucleotidyl transferase end labeling assay (TUNEL). RESULTS: The apoptosis rate in the contusion model was 68.01% (± 19.73%), a higher rate than previously described. HA significantly reduced the rate of apoptosis to 53.52% (± 18.09) (p <0.001). CONCLUSION: Intra-articular HA administration started immediately after trauma reduces impact-induced chondrocyte apoptosis rates in rabbits. Level of Evidence I, Experimental Study. PMID:27069407

  14. Sodium orthovanadate inhibits p53-mediated apoptosis.

    PubMed

    Morita, Akinori; Yamamoto, Shinichi; Wang, Bing; Tanaka, Kaoru; Suzuki, Norio; Aoki, Shin; Ito, Azusa; Nanao, Tomohisa; Ohya, Soichiro; Yoshino, Minako; Zhu, Jin; Enomoto, Atsushi; Matsumoto, Yoshihisa; Funatsu, Osamu; Hosoi, Yoshio; Ikekita, Masahiko

    2010-01-01

    Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. PMID:20048077

  15. High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS.

    PubMed

    Ghassabian, Sussan; Moosavi, Seyed Mojtaba; Valero, Yarmarly Guerra; Shekar, Kiran; Fraser, John F; Smith, Maree T

    2012-08-15

    A rapid LC-MS/MS assay method for simultaneous quantification of morphine, fentanyl, midazolam and their major metabolites: morphine-3-β-D-glucuronide (M3G), morphine-6-β-D-glucuronide (M6G), norfentanyl, 1'-hydroxymidazolam (1-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) in samples of human plasma has been developed and validated. Robotic on-line solid phase extraction (SPE) instrumentation was used to elute the eight analytes of interest from polymeric SPE cartridges to which had been added aliquots (150 μL) of human plasma and aliquots (150 μL) of a mixture of two internal standards, viz. morphine-d3 (200 ng/mL) and 1'-hydroxymidazolam-d5 (50 ng/mL) in 50 mM ammonium acetate buffer (pH 9.25). Cartridges were washed using 10% methanol in ammonium acetate buffer, pH 9.25 (1 mL, 2 mL/min) before elution with mobile phase comprising 0.1% formic acid in water (A) and acetonitrile (B) with a flow rate of 0.6 mL/min using an 11.5 min run time. The analytes were separated on a C18 X-Terra® analytical column. The linear concentration ranges were 0.5-100 ng/mL for fentanyl, norfentanyl and midazolam; 1-200 ng/mL for 4-hydroxymidazolam, 2.5-500 ng/mL for 1'-hydroxymidazolam and 3.5-700 ng/mL for morphine, M3G, and M6G. The method showed acceptable within-run and between-run precision (relative standard deviation (RSD) and accuracy <20%) for quality control (QC) samples spiked at concentrations of 80% and 50% of the ULOQ, 3 times higher than the LLOQ, and also at the LLOQ. Furthermore, analytes were stable in samples (after mixing with internal standard) for at least 48 h in the autosampler (except for 4-hydroxymidazolam which decreased by 22% after 24 h), 5 h at room temperature and after three cycles of freeze and thaw. No autosampler carry-over was observed and the absolute recovery (the area ratio of analyte in plasma relative to that in ammonium acetate buffer 50 mM, pH 9.25) was in the range 40% (midazolam) to 110% (morphine). The assay was applied successfully to

  16. An on-line SPE-HPLC method for effective sample preconcentration and determination of fenoxycarb and cis, trans-permethrin in surface waters.

    PubMed

    Šatínský, Dalibor; Naibrtová, Linda; Fernández-Ramos, Carolina; Solich, Petr

    2015-09-01

    A new on-line SPE-HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl-Hexyl (100×4.6 mm), particle size 2.7 µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0 mL min(-1), column temperature 45°C. Large volume sample injection (1500 µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6 mm); fused-core particle size 2.7 µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5 mL min(-1) through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225 nm was used and the method reached the limits of detection (LOD) at ng mL(-1) levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8 min. PMID:26003701

  17. Yield and depth Estimation of Selected NTS Nuclear and SPE Chemical Explosions Using Source Equalization by modeling Local and Regional Seismograms (Invited)

    NASA Astrophysics Data System (ADS)

    Saikia, C. K.; Roman-nieves, J. I.; Woods, M. T.

    2013-12-01

    Source parameters of nuclear and chemical explosions are often estimated by matching either the corner frequency and spectral level of a single event or the spectral ratio when spectra from two events are available with known source parameters for one. In this study, we propose an alternative method in which waveforms from two or more events can be simultaneously equalized by setting the differential of the processed seismograms at one station from any two individual events to zero. The method involves convolving the equivalent Mueller-Murphy displacement source time function (MMDSTF) of one event with the seismogram of the second event and vice-versa, and then computing their difference seismogram. MMDSTF is computed at the elastic radius including both near and far-field terms. For this method to yield accurate source parameters, an inherent assumption is that green's functions for the any paired events from the source to a receiver are same. In the frequency limit of the seismic data, this is a reasonable assumption and is concluded based on the comparison of green's functions computed for flat-earth models at various source depths ranging from 100m to 1Km. Frequency domain analysis of the initial P wave is, however, sensitive to the depth phase interaction, and if tracked meticulously can help estimating the event depth. We applied this method to the local waveforms recorded from the three SPE shots and precisely determined their yields. These high-frequency seismograms exhibit significant lateral path effects in spectrogram analysis and 3D numerical computations, but the source equalization technique is independent of any variation as long as their instrument characteristics are well preserved. We are currently estimating the uncertainty in the derived source parameters assuming the yields of the SPE shots as unknown. We also collected regional waveforms from 95 NTS explosions at regional stations ALQ, ANMO, CMB, COR, JAS LON, PAS, PFO and RSSD. We are

  18. PECAM-1, apoptosis and CD34+ precursors.

    PubMed

    Zocchi, Maria R; Poggi, A

    2004-11-01

    Apoptosis is a physiological process that controls tissue homeostasis, in combination with survival signals delivered by distinct receptors that bind hormones, growth factors or extracellular matrix components. The extrinsic pathway of apoptosis is due to the triggering of death receptors and the activation of the caspase cascade; the intrinsic pathway is due to withdrawal of growth factors and mainly related to mitochondrial metabolism. The choice between survival or apoptosis, which is the result of such different integrated environmental signals, is crucial for the maintainance of bone marrow reservoir of hematopoietic precursors (HPC). CD34+ HPC can receive multiple survival signals during homing and maturation, due to different interactions with adhesion molecules expressed on endothelial and bone marrow stromal cells, proteins of the extracellular matrix and chemokines or growth factors. Among them, the signal delivered via platelet endothelial cell adhesion molecule-1 (PECAM-1) seems to contribute to the resistance of this cell population to starvation, and it is related to the maintainance of mitochondrial metabolism. Indeed, this molecule, originally described as an adhesion receptor belonging to the immunoglobulin superfamily, capable of homophilic and heterophilic interactions, turned out to be a signalling molecule, containing an immunoreceptor tyrosine-based inhibitory motifs (ITIM) within its cytoplasmic domain. In particular, it has been shown that PECAM-1 binds to different kinases and phosphatases, including the phosphatidylinositide-3-kinase that phosphorylates Akt, which, in turn can upregulate transcription and function of antiapoptotic proteins, such as Bcl-2 and Bcl-x or A1, responsible for the rescue from mitochondrial apoptosis. The possible role of PECAM-1 engagement in the prevention of starvation-induced apoptosis of HPC precursors and in the maintainance of their survival is discussed. PMID:15512808

  19. Erythrocyte ion channels in regulation of apoptosis.

    PubMed

    Lang, Florian; Birka, Christina; Myssina, Svetlana; Lang, Karl S; Lang, Philipp A; Tanneur, Valerie; Duranton, Christophe; Wieder, Thomas; Huber, Stephan M

    2004-01-01

    Erythrocytes lack mitochondria and nuclei, key organelles in the regulation of apoptosis. Until recently, erythrocytes were thus not considered subject to this type of cell death. However, exposure of erythrocytes to the Ca2+ ionophore ionomycin was shown to induce cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the cell surface, all typical features of apoptosis. Further studies revealed the participation of ion channels in the regulation of erythrocyte "apoptosis." Osmotic shock, oxidative stress and energy depletion all activate a Ca2(+)-permeable non-selective cation channel in the erythrocyte cell membrane. The subsequent increase of Ca2+ concentration stimulates a scramblase leading to breakdown of cell membrane phosphatidylserine asymmetry and activates Ca2+ sensitive K+ (Gardos) channels leading to KCl loss and (further) cell shrinkage. Phosphatidylserine exposure and cell shrinkage are blunted in the nominal absence of extracellular Ca2+, in the presence of the cation channel inhibitors amiloride or ethylisopropylamiloride, at increased extracellular K+ or in the presence of the Gardos channel inhibitors clotrimazole or charybdotoxin. Thus, increase of cytosolic Ca2+ and cellular loss of K+ participate in the triggering of erythrocyte scramblase. Nevertheless, phosphatidylserine exposure is not completely abrogated in the nominal absence of Ca2+, pointing to additional Ca2(+)-independent pathways. One of those is activation of sphingomyelinase with subsequent formation of ceramide which in turn leads to stimulation of erythrocyte scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Erythropoietin inhibits the non-selective cation channel and thus

  20. Synthesis, Anticancer Activity, Effect on Cell Cycle Profile, and Apoptosis-Inducing Ability of Novel Hexahydrocyclooctathieno[2,3-d]pyrimidine Derivatives.

    PubMed

    Kassab, Asmaa Elsayed; Gedawy, Ehab Mohamed; El-Malah, Afaf Ali; Abdelghany, Tamer Mohamed; Abdel-Bakky, Mohamed Sadek

    2016-01-01

    A novel series of hexahydrocyclooctathieno[2,3-d]pyrimidines was synthesized. Investigation of the anticancer activity of these derivatives revealed that compounds 2a and b showed broad-spectrum anticancer activity in nanomolar to micromolar concentrations. In particular, compound 2b showed a concentration required for 50% inhibition of cell growth (GI50) value of less than 1 µM against 20 cancer cell lines. Compounds 2a and b induced G2/M- and S-phase cell cycle arrest in human colon adenocarcinoma (HCT116) and human breast adenocarcinoma (MCF7) cell lines with a concomitant increase in the pre-G cell population in a time-dependent manner. Furthermore, compound 2b increased the nuclear expression of the proapoptotic protein cleaved caspase-3, indicating that apoptosis has an important role, at least in part, in the cancer cell death induced by the new compounds. PMID:27150481

  1. Intrinsic apoptosis and NF-κB signaling are potential molecular targets for chemoprevention by black tea polyphenols in HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo.

    PubMed

    Murugan, R Senthil; Priyadarsini, R Vidya; Ramalingam, K; Hara, Y; Karunagaran, D; Nagini, S

    2010-11-01

    Antiproliferative and apoptosis inducing effects of black tea polyphenols (Polyphenon-B) on HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo were investigated. Viability of HepG2 cells was evaluated by the MTT assay, and apoptosis by AO-EB and DAPI staining, cell cycle analysis, and annexin V-PI assay. For the in vivo study, male Sprague-Dawley rats treated with dimethylaminoazobenzene (DAB) (0.06%) were used. The expression of Bcl-2 and NF-κB family members were analyzed by immunoblotting. Administration of Polyphenon-B induced dose-dependent inhibition of growth of HepG2 cells and reduced tumor incidence in DAB administered animals. HepG2 cells also exhibited morphological features characteristic of apoptotic cell death. In addition, administration of Polyphenon-B increased the expression of Bax, tBid, Smac/Diablo, cytochrome C, Apaf-1, caspases, and IκB with PARP cleavage, and decreased the expression of Bcl-2, Bcl-xL, pBad, NF-κB, p-IκB-α, IKKβ and Ub in both HepG2 cells and in DAB-treated animals. These results provide evidence that Polyphenon-B effectively inhibits proliferation and induces apoptosis both in vitro and in vivo by inhibiting NF-κB, and inducing intrinsic apoptosis by modulating the expression of a network of interrelated molecules eventually culminating in caspase-mediated cell death. PMID:20828598

  2. SpeX spectroscopy of unresolved very low mass binaries. II. Identification of 14 candidate binaries with late-M/early-L and T dwarf components

    SciTech Connect

    Bardalez Gagliuffi, Daniella C.; Burgasser, Adam J.; Nicholls, Christine P.; Gelino, Christopher R.; Looper, Dagny L.; Schmidt, Sarah J.; Cruz, Kelle; West, Andrew A.; Gizis, John E.; Metchev, Stanimir

    2014-10-20

    Multiplicity is a key statistic for understanding the formation of very low mass (VLM) stars and brown dwarfs. Currently, the separation distribution of VLM binaries remains poorly constrained at small separations (≤1 AU), leading to uncertainty in the overall binary fraction. We approach this problem by searching for late-M/early-L plus T dwarf spectral binaries whose combined light spectra exhibit distinct peculiarities, allowing for separation-independent identification. We define a set of spectral indices designed to identify these systems, and we use a spectral template fitting method to confirm and characterize spectral binary candidates from a library of 815 spectra from the SpeX Prism Spectral Libraries. We present 11 new binary candidates, confirm 3 previously reported candidates, and rule out 2 previously identified candidates, all with primary and secondary spectral types in the range M7-L7 and T1-T8, respectively. We find that subdwarfs and blue L dwarfs are the primary contaminants in our sample and propose a method for segregating these sources. If confirmed by follow-up observations, these systems may add to the growing list of tight separation binaries, whose orbital properties may yield further insight into brown dwarf formation scenarios.

  3. High-resolution bioactivity profiling combined with HPLC-HRMS-SPE-NMR: α-Glucosidase inhibitors and acetylated ellagic acid rhamnosides from Myrcia palustris DC. (Myrtaceae).

    PubMed

    Wubshet, Sileshi G; Moresco, Henrique H; Tahtah, Yousof; Brighente, Inês M C; Staerk, Dan

    2015-08-01

    Type 2 diabetes (T2D) is an endocrine metabolic disease with a worldwide prevalence of more than 8%, and an expected increase close to 50% in the next 15-20years. T2D is associated with severe and life-threatening complications like retinopathy, neuropathy, nephropathy, and cardiovascular diseases, and therefore improved drug leads or functional foods containing α-glucosidase inhibitors are needed for management of blood glucose. In this study, leaves of Myrcia palustris were investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR. This led to identification of casuarinin, myricetin 3-O-β-d-(6″-galloyl)galactopyranoside, kaempferol 3-O-β-d-galactopyranoside, myricetin, and quercetin as α-glucosidase inhibitors. In addition, four acetylated ellagic acid rhamnosides, i.e., 4-O-(2″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(2″,3″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(3″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, and 4-O-(2″,3″,4″-O-triacetyl-α-l-rhamnopyranosyl)ellagic acid were identified. PMID:25935545

  4. New method for the determination of carbamate and pyrethroid insecticides in water samples using on-line SPE fused core column chromatography.

    PubMed

    Fernández-Ramos, C; Satínský, D; Solich, P

    2014-11-01

    A new HPLC column-switching method using large volume sample injection and fused-core columns for on-line solid phase extraction have been developed for the determination of the following carbamates and pyrethroids: aldicarb, carbaryl, pirimicarb, carbofuran, kadethrin, flumethrin, fenpropathrin, fenoxycarb, tau-fluvalinate and fenvalerate, in surface water samples. Sudan I was used as internal standard. The proposed method was performed using 100 µl sample injection followed by an on-line solid phase extraction procedure and finally the compounds were identified and quantified by liquid chromatography with ultraviolet detection. The separation was carried out on C-18 reversed phase column based on fused-core particle technology. The influence of the injected sample volume, the variables affecting to SPE process and the conditions for the separation on an analytical column, were studied and optimized. The limits of detection ranged from 5.5 to 8.9 µg L(-1), and limits of quantification from 18.4 to 29.7 µg L(-1), while inter- and intra-day variability was under 15%. This new analytical procedure was satisfactorily applied for the determination of these organic pollutants in surface water samples located in Czech Republic. Concentration levels were found for some of these pollutants up to 26.11 µg L(-1) in the river Elbe and up to 34.53 µg L(-1) in the closed lakes samples. PMID:25127636

  5. Determination of perfluorinated sulfonate and perfluorinated acids in tissues of free-living European beaver (castor fiber L.) by d-SPE/ micro-UHPLC-MS/MS.

    PubMed

    Surma, Magdalena; Giżejewski, Zygmunt; Zieliński, Henryk

    2015-10-01

    Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the main representatives of an rising class of persistent organic pollutants (POPs), perfluorochemicals (PFCs). In this study, determination of selected PFCs concentration in liver, brain, tail, adipose and peritoneum tissues of free-living European beaver (Castor fiber L.) was addressed. Tissue samples, collected from beavers living in Masurian Lakeland (NE Poland), were analyzed by dispersive Solid Phase Extraction (d-SPE) with micro-UHPLC-MS/MS system. In a group of ten selected pefrluorinated compounds only two perfluorinated acids (PFOA and PFNA) and one perfluorinated sulfonate (PFOS) were quantified. PFOA was detected in all analysed tissue samples in both female and male beavers in a range from 0.55 to 0.98ngg(-1) ww whereas PFOS was identified in all analyzed female beaver tissues and only in liver, subcutaneous adipose and peritoneum tissues of male beavers at the concentration level from 0.86 to 5.08ngg(-1) ww. PFNA was only identified in female beaver tissues (liver, subcutaneous adipose and peritoneum) in a range from 1.50 to 6.61ngg(-1) ww. This study demonstrated the bioaccumulation of PFCs in tissue samples collected from beavers living in area known as green lungs of Poland. The results provided in this study indicate for the increasing risk of PFCs occurrence in the environment and the level of PFCs in tissue of free-living European beavers may serve as bioindicator of environmental pollution by these compounds. PMID:26143169

  6. Determination of parent and substituted polycyclic aromatic hydrocarbons in high-fat salmon using a modified QuEChERS extraction, dispersive SPE and GC-MS

    PubMed Central

    Forsberg, Norman D.; Wilson, Glenn R.; Anderson, Kim A.

    2011-01-01

    A fast and easy modified QuEChERS (quick, easy, cheap, rugged and safe) extraction method has been developed and validated for determination of 33 parent and substituted polycyclic aromatic hydrocarbons (PAHs) in high-fat smoked salmon that greatly enhances analyte recovery compared to traditional QuEChERS procedures. Sample processing includes extraction of PAHs into a solution of ethyl acetate, acetone and iso-octane followed by cleanup with dispersive SPE and analysis by GC-MS in SIM mode. Method performance was assessed in spike recovery experiments (500 ng/g wet weight) in three commercially available smoked salmon with 3 – 11% fat. Recoveries of some 2, 3 and 5-ring PAHs were improved 50 – 200% over traditional methods, while average recovery across all PAHs was improved 67%. Method precision was good with replicate extractions typically yielding relative standard deviations < 10% and detection limits were in the low ng/g range. With this method, a single analyst could extract and cleanup ≥ 60 samples for PAH analysis in an 8 hour work day. PMID:21732651

  7. Determination of urinary aromatic amines in smokers and nonsmokers using a MIPs-SPE coupled with LC-MS/MS method.

    PubMed

    Yu, Jingjing; Wang, Sheng; Zhao, Ge; Wang, Bing; Ding, Li; Zhang, Xiaobing; Xie, Jianping; Xie, Fuwei

    2014-05-01

    Urinary aromatic amines (AAs) could be used as biomarkers for human exposure to AAs in cigarette smoke. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of urinary AAs (i.e. 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP)) in smokers and nonsmokers. A molecularly imprinted polymers (MIPs) solid phase extraction (SPE) cartridge was applied to purify urine samples and no derivatization reaction was involved. Each analytes used respective stable isotope internal standards, which could well compensate matrix effect. Lower limit of detections (LODs) for four AAs were obtained and in the range of 1.5-5ngL(-1). Recovery ranged from 87.7±4.5% to 111.3±6.4% and precision were less than 9.9%. The method was applied to analyze urine samples of 40 smokers and 10 nonsmokers. The 24h urinary excretion amounts of total AAs were higher for smokers compared with nonsmokers. What's more, 1-NA, 3-ABP and 4-ABP excretion amounts showed significant differences (p<0.05) between smokers and nonsmokers. PMID:24735928

  8. A new SPE/GC-fid method for the determination of cholesterol oxidation products. Application to subcutaneous fat from Iberian dry-cured ham.

    PubMed

    Narváez-Rivas, Mónica; Pham, Alessandra J; Schilling, M Wes; León-Camacho, Manuel

    2014-05-01

    A new method for the isolation and analysis of cholesterol oxidation products (COPs) using solid phase extraction (SPE) and silica columns was developed using gas chromatography-flame ion detection (GC-FID). The method comprises of saponification and liquid-liquid extraction of the unsaponifiable fraction prior to the isolation and derivatization of the COPs to trimethylsilyl ethers. The COPs used in this study are cholestane-5α-6α-epoxide, cholestane-3β-5α-6β-triol, 25-hydroxycholesterol and 5-cholesten-3β-ol-7-one. In order to identify the COPs fraction a GC-ion-trap-mass spectrometry experiment were conducted using authentic standards to verify the presence of the COPs. The method was effective at rapidly separating the COPs (25 min run). Calibration curves were linear with the LODs and LOQs bellow 0.03 and 0.07 mgkg(-1) for all cases, respectively. This methodology gave a total recovery for every compound that was used in the study. Betulin was used as an internal standard to monitor the recovery. The method was validated with a standard mixture of COPs. The method has been applied to characterize the COP fraction of subcutaneous fat from Iberian dry-cured ham. Cholestane-5α-6α-epoxide, cholestane-3β-5α-6β-triol, 25-hydroxycholesterol and 5-cholesten-3β-ol-7-one have been identified for the first time in these samples. PMID:24720962

  9. Targeted analysis of omega-6-derived eicosanoids in human serum by SPE-LC-MS/MS for evaluation of coronary artery disease.

    PubMed

    Fernández Peralbo, María Auxiliadora; Priego-Capote, Feliciano; Galache-Osuna, José Gabriel; Luque de Castro, María Dolores

    2013-10-01

    A targeted approach has been applied to quantitative analysis of eicosanoids derived from omega-6 fatty acids in serum from individuals diagnosed with coronary artery disease (CAD). The target metabolites were series-2 prostaglandins, thromboxane B2, hydroxyeicosatetraenoic acids, and hydroxyoctadecadienoic acids. The method was based on SPELC-MS/MS in selected reaction monitoring mode for highly selective and sensitive determination of the target eicosanoids. The combination of SPE and LC-MS/MS involved the benefits from both direct analysis of serum without a step for protein precipitation and fully automation of the analysis. The method allowed comparison of omega-6-derived eicosanoids in serum from patients diagnosed with CAD and from control individuals. The effect of treatment with aspirin on the profile of the target compounds was evaluated through its incidence on the different pathways. Finally, the serum levels of the target metabolites in patients diagnosed with CAD were also statistically examined according to the severity of the coronary lesion stratified as stable angina, non-ST-elevation acute coronary syndrome, and acute myocardial infarction. PMID:24228265

  10. Novel magnetic SPE method based on carbon nanotubes filled with cobalt ferrite for the analysis of organochlorine pesticides in honey and tea.

    PubMed

    Du, Zhuo; Liu, Miao; Li, Gongke

    2013-10-01

    A novel magnetic SPE method based on magnetic cobalt ferrite filled carbon nanotubes (MFCNTs) coupled with GC with electron capture detection was developed to determine organochlorine pesticides (OCPs) in tea and honey samples. The MFCNTs were prepared through the capillarity of carbon nanotubes for drawing mixed cobalt and iron nitrates solution into their inner cavity followed by heating to 550°C under Ar to form the cobalt ferrite nanoparticles. SEM images provided visible evidence of the filled cobalt ferrite nanoparticles in the multiwalled nanotubes. X-ray photoelectron spectroscopy indicated no adhesion of cobalt ferrite nanoparticles and metal salts on the outer surface of the MFCNTs. Eight OCPs were extracted with the MFCNTs. The enrichment factors were in the range of 52-68 for eight OCPs. The LODs for the eight OCPs were in the range of 1.3-3.6 ng/L. The recoveries of the OCPs for honey and tea samples were 83.2-128.7 and 72.6-111.0%, respectively. The RSDs for these samples were below 6.8%. The new method is particularly suited to extract nonpolar and weakly polar analytes from a complex matrix and could potentially be extended to other target analytes. PMID:23926126

  11. Rapid and selective quantification of L-theanine in ready-to-drink teas from Chinese market using SPE and UPLC-UV.

    PubMed

    Chen, Guoqiang; Wang, Yun; Song, Weiqi; Zhao, Bo; Dou, Yuling

    2012-11-15

    An ultra performance liquid chromatography (UPLC) method combined with solid phase extraction (SPE) sample pre-treatment was developed and validated for the rapid quantification of L-theanine in ready-to-drink (RTD) teas. UPLC analysis of twenty-seven RTD teas from the Chinese market revealed that the L-theanine levels in various types of RTD teas were significantly different. RTD green teas were found to contain highest mean L-theanine level (37.85±20.54 mg/L), followed by jasmine teas (36.60±12.08 mg/L), Tieguanying teas (18.54±3.46 mg/L) black teas (16.89±6.56), Pu-erh teas (11.31±0.90 mg/L) and oolong teas (3.85±2.27 mg/L). The ratio of total polyphenols content to L-theanine content could be used as a featured parameter for differentiating RTD teas. L-theanine in RTD teas could be a reliable quality parameter that is complementary to total polyphenols. PMID:22868106

  12. SpeX SPECTROSCOPY OF UNRESOLVED VERY LOW MASS BINARIES. I. IDENTIFICATION OF 17 CANDIDATE BINARIES STRADDLING THE L DWARF/T DWARF TRANSITION

    SciTech Connect

    Burgasser, Adam J.; Cruz, Kelle L.; Cushing, Michael; Looper, Dagny L.; Gelino, Christopher R.; Kirkpatrick, J. Davy; Faherty, Jacqueline K.; Reid, I. Neill

    2010-02-20

    We report the identification of 17 candidate brown dwarf binaries whose components straddle the L dwarf/T dwarf transition. These sources were culled from a large near-infrared spectral sample of L and T dwarfs observed with the Infrared Telescope Facility SpeX spectrograph. Candidates were selected on the basis of spectral ratios which segregate known (resolved) L dwarf/T dwarf pairs from presumably single sources. Composite templates, constructed by combining 13,581 pairs of absolute flux-calibrated spectra, are shown to provide statistically superior fits to the spectra of our 17 candidates as compared to single templates. Ten of these candidates appear to have secondary components that are significantly brighter than their primaries over the 1.0-1.3 {mu}m band, indicative of rapid condensate depletion at the L dwarf/T dwarf transition. Our results support prior indications of enhanced multiplicity amongst early-type T dwarfs; 53% +- 7% of the T0-T4 dwarfs in our spectral sample are found to be either resolved or unresolved (candidate) pairs, although this is consistent with an intrinsic (volume complete) brown dwarf binary fraction of only 15%. If verified, this sample of spectral binaries more than doubles the number of known L dwarf/T dwarf transition pairs, enabling a broader exploration of this poorly understood phase of brown dwarf atmospheric evolution.

  13. High-Resolution α-Glucosidase Inhibition Profiling Combined with HPLC-HRMS-SPE-NMR for Identification of Antidiabetic Compounds in Eremanthus crotonoides (Asteraceae).

    PubMed

    Silva, Eder Lana E; Lobo, Jonathas Felipe Revoredo; Vinther, Joachim Møllesøe; Borges, Ricardo Moreira; Staerk, Dan

    2016-01-01

    α-Glucosidase inhibitors decrease the cleavage- and absorption rate of monosaccharides from complex dietary carbohydrates, and represent therefore an important class of drugs for management of type 2 diabetes. In this study, a defatted ethyl acetate extract of Eremanthus crotonoides leaves with an inhibitory concentration (IC50) of 34.5 μg/mL towards α-glucosidase was investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR. This led to identification of six α-glucosidase inhibitors, namely quercetin (16), trans-tiliroside (17), luteolin (19), quercetin-3-methyl ether (20), 3,5-di-O-caffeoylquinic acid n-butyl ester (26) and 4,5-di-O-caffeoylquinic acid n-butyl ester (29). In addition, nineteen other metabolites were identified. The most active compounds were the two regioisomeric di-O-caffeoylquinic acid derivatives 26 and 29, with IC50 values of 5.93 and 5.20 μM, respectively. This is the first report of the α-glucosidase inhibitory activity of compounds 20, 26, and 29, and the findings support the important role of Eremanthus species as novel sources of new drugs and/or herbal remedies for treatment of type 2 diabetes. PMID:27322221

  14. An Optimized High Throughput Clean-Up Method Using Mixed-Mode SPE Plate for the Analysis of Free Arachidonic Acid in Plasma by LC-MS/MS

    PubMed Central

    Wang, Wan; Qin, Suzi; Li, Linsen; Chen, Xiaohua; Wang, Qunjie; Wei, Junfu

    2015-01-01

    A high throughput sample preparation method was developed utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. Plasma was mixed with 3% aqueous ammonia and loaded into each well of 96-well plate. After washing with water and methanol sequentially, 3% of formic acid in acetonitrile was used to elute arachidonic acid. The collected fraction was injected onto a reversed phase column at 30°C with mobile phase of acetonitrile/water (70 : 30, v/v) and detected by LC-MS/MS coupled with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r2 = 0.9999). The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%. The limit of detection is 3 ng/mL. PMID:25873969

  15. High-resolution hyaluronidase inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of anti-necrosis constituents in Chinese plants used to treat snakebite.

    PubMed

    Liu, Yueqiu; Staerk, Dan; Nielsen, Mia N; Nyberg, Nils; Jäger, Anna K

    2015-11-01

    Inhibition of the necrotizing hyaluronidase, phospholipase A2 and protease enzymes in four snake venoms by crude water and ethanol extracts of 88 plant species used against snakebites in traditional Chinese medicine was measured. High-resolution hyaluronidase inhibition profiles were constructed for the 22 plants showing highest hyaluronidase inhibition, and the results were used to guide subsequent structural analysis towards specific hyaluronidase inhibitors. Structural analysis was performed by high-performance liquid chromatography, high-resolution mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR. This allowed identification of four non-tannin inhibitors, i.e., lansiumamide B (6) from Clausena excavata Burm.f., myricetin 3-O-β-D-glucopyranoside (7) from Androsace umbellata (Lour.) Merr., and vitexin (8) and 4',7-dihydroxy-5-methoxyflavone-8-C-β-D-glucopyranoside (9) from Oxalis corniculata L. Absolute configuration of 2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide (1) was determined using the Mosher method, which revealed two enantiomers, i.e., (2S,3R)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide and (2R,3S)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide with a ratio of 7:3. PMID:26386983

  16. Preparation of molecularly imprinted polymers using theanine as dummy template and its application as SPE sorbent for the determination of eighteen amino acids in tobacco.

    PubMed

    Zhu, Fengling; Wang, Jing; Zhu, Lijun; Tan, Lanlan; Feng, Guanglin; Liu, Shaomin; Dai, Ya; Wang, Hua

    2016-04-01

    In this paper, a novel dummy template molecularly imprinted polymer (DMIP) based on a vinyl-SiO2 microspheres surface for the simultaneous selective recognition and enrichment of 18 amino acids was prepared via a surface molecular imprinting technique using theanine as a dummy template. Compared to the imprinted polymers prepared using traditional polymerization techniques, the obtained DMIPs exhibited a regular spherical shape and were relatively monodisperse. The maximal sorption capacity (Qmax) of the resulting DMIPs for the 18 amino acids was up to 1444.3 mg g(-1). A kinetic binding study showed that the sorption capacity reached 85.40% of Qmax in 25 min and sorption equilibrium at 30 min. The imprint factors of the sorbents ranged from 2.86 to 6.9 for the 18 amino acids, which indicated that the DMIP sorbents have high selectivity. An HPLC-UV method for the simultaneous determination of 18 amino acids in tobacco and tobacco smoke was developed using the DMIPs as sorbents for solid phase extraction (SPE) in the sample pretreatment procedure. Under the optimum experimental conditions, the materials had enrichment factors of up to 200 for the amino acids, and the recoveries of the 18 amino acids in tobacco smoke were in the range from 79% to 104% with relative standard deviations of less than 7.4%. It indicated that the obtained DMIP sorbents could specifically recognize the amino acids from complicated samples. PMID:26838422

  17. Investigation of structural heterogeneity at the SPE site using combined P–wave travel times and Rg phase velocities

    SciTech Connect

    Rowe, Charlotte A.; Patton, Howard J.

    2015-10-01

    Here, we present analyses of the 2D seismic structure beneath Source Physics Experiments (SPE) geophone lines that extended radially at 100 m spacing from 100 to 2000 m from the source borehole. With seismic sources at only one end of the geophone lines, standard refraction profiling methods cannot resolve seismic velocity structures unambiguously. In previous work, we demonstrated overall agreement between body-wave refraction modeling and Rg dispersion curves for the least complex of the five lines. A more detailed inspection supports a 2D reinterpretation of the structure. We obtained Rg phase velocity measurements in both the time and frequency domains, then used iterative adjustment of the initial 1D body-wave model to predict Rg dispersion curves to fit the observed values. Our method applied to the most topographically severe of the geophone lines is supplemented with a 2D ray-tracing approach, whose application to P-wave arrivals supports the Rg analysis. In addition, midline sources will allow us to refine our characterization in future work.

  18. Pharmacokinetic study of six flavones in rat plasma and tissues after oral administration of 'JiangYaBiFeng' using SPE-HPLC-DAD.

    PubMed

    Zeng, Hua-jin; Yang, Ran; Guo, Cheng; Wang, Qing-wen; Qu, Ling-bo; Li, Jian-jun

    2011-12-01

    In this study, a high performance liquid chromatography (HPLC) coupled with diode array detection (DAD) for simultaneous determination of six flavones including baicalein, sophoricoside, rutin, baicalin, quercetin and genistein in rat plasma and tissues after oral administration of JiangYaBifeng (JYBF) tablets was developed. The investigated analytes in plasma and tissues were extracted and purified with liquid-liquid extraction and solid phase extraction (SPE). Chromatographic separation was accomplished on a DIONEX Acclaim C18 column (250mm×4.6mm, 5.0μm particle size) with a simple linear gradient elution. The calibration curves for all the flavones had good linearity in the measured range with R(2) higher than 0.9983. The relative errors (REs) of the intra- and inter-day accuracy at different flavones levels were all less than ±10%. The proposed method enables unambiguous identification and quantification of investigated flavones in vivo. This is the first report on determination of the major flavones in rat plasma and tissues after oral administration of JYBF tablets. The results provided a meaningful basis for evaluating the clinical application of this medicine. PMID:21831556

  19. Determination of parent and substituted polycyclic aromatic hydrocarbons in high-fat salmon using a modified QuEChERS extraction, dispersive SPE and GC-MS.

    PubMed

    Forsberg, Norman D; Wilson, Glenn R; Anderson, Kim A

    2011-08-10

    A fast and easy modified QuEChERS (quick, easy, cheap, rugged and safe) extraction method has been developed and validated for determination of 33 parent and substituted polycyclic aromatic hydrocarbons (PAHs) in high-fat smoked salmon that greatly enhances analyte recovery compared to traditional QuEChERS procedures. Sample processing includes extraction of PAHs into a solution of ethyl acetate, acetone and isooctane followed by cleanup with dispersive SPE and analysis by GC-MS in SIM mode. Method performance was assessed in spike recovery experiments (500 μg/g wet weight) in three commercially available smoked salmon with 3-11% fat. Recoveries of some 2-, 3- and 5-ring PAHs were improved 50-200% over traditional methods, while average recovery across all PAHs was improved 67%. Method precision was good with replicate extractions typically yielding relative standard deviations <10%, and detection limits were in the low ng/g range. With this method, a single analyst could extract and clean up ≥60 samples for PAH analysis in an 8 h work day. PMID:21732651

  20. Key regulators of apoptosis execution as biomarker candidates in melanoma

    PubMed Central

    Charles, Emilie M; Rehm, Markus

    2014-01-01

    Resistance to apoptosis is frequently detected in malignant melanoma, a skin cancer with rapidly growing incidence rates. Apoptosis resistance may develop with disease progression and may be associated with the poor responsiveness of metastatic melanoma to apoptosis-inducing treatments, such as genotoxic chemotherapy and radiotherapy. Likewise, the efficacy of novel treatment options (targeted kinase inhibitors and immunotherapeutics) that indirectly lead to cell death may depend on the susceptibility of melanoma to apoptosis. At its core, apoptosis execution is regulated by the interplay between a comparatively small number of pro- and anti-apoptotic proteins, and consequently numerous studies have investigated the potential of these players as biomarker candidates. Here, we provide a comprehensive overview of biomarker discovery studies focusing on key regulators of apoptosis execution, critically review the findings of these studies, and outline strategies that address current limitations and challenges in exploiting regulators of apoptosis execution as prognostic or predictive biomarkers in melanoma. PMID:27308353

  1. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    PubMed Central

    Musumeci, Giuseppe; Castrogiovanni, Paola; Trovato, Francesca Maria; Weinberg, Annelie Martina; Al-Wasiyah, Mohammad K.; Alqahtani, Mohammed H.; Mobasheri, Ali

    2015-01-01

    Cell death with morphological and molecular features of apoptosis has been detected in osteoarthritic (OA) cartilage, which suggests a key role for chondrocyte death/survival in the pathogenesis of OA. Identification of biomarkers of chondrocyte apoptosis may facilitate the development of novel therapies that may eliminate the cause or, at least, slow down the degenerative processes in OA. The aim of this review was to explore the molecular markers and signals that induce chondrocyte apoptosis in OA. A literature search was conducted in PubMed, Scopus, Web of Science and Google Scholar using the keywords chondrocyte death, apoptosis, osteoarthritis, autophagy and biomarker. Several molecules considered to be markers of chondrocyte apoptosis will be discussed in this brief review. Molecular markers and signalling pathways associated with chondroycte apoptosis may turn out to be therapeutic targets in OA and approaches aimed at neutralizing apoptosis-inducing molecules may at least delay the progression of cartilage degeneration in OA. PMID:26334269

  2. Rabies virus matrix protein induces apoptosis by targeting mitochondria.

    PubMed

    Zan, Jie; Liu, Juan; Zhou, Jian-Wei; Wang, Hai-Long; Mo, Kai-Kun; Yan, Yan; Xu, Yun-Bin; Liao, Min; Su, Shuo; Hu, Rong-Liang; Zhou, Ji-Yong

    2016-09-10

    Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination. PMID:27426727

  3. Dual high-resolution α-glucosidase and radical scavenging profiling combined with HPLC-HRMS-SPE-NMR for identification of minor and major constituents directly from the crude extract of Pueraria lobata.

    PubMed

    Liu, Bingrui; Kongstad, Kenneth T; Qinglei, Sun; Nyberg, Nils T; Jäger, Anna K; Staerk, Dan

    2015-02-27

    The crude methanol extract of Pueraria lobata was investigated by dual high-resolution α-glucosidase inhibition and radical scavenging profiling combined with hyphenated HPLC-HRMS-SPE-NMR. Direct analysis of the crude extract without preceding purification was facilitated by combining chromatograms from two analytical-scale HPLC separations of 120 and 600 μg on-column, respectively. High-resolution α-glucosidase and radical scavenging profiles were obtained after microfractionation of the eluate in 96-well microplates. This allowed full bioactivity profiling of individual peaks in the HPLC chromatogram of the crude methanol extract. Subsequent HPLC-HRMS-SPE-NMR analysis allowed identification of 21 known compounds in addition to two new compounds, i.e., 3'-methoxydaidzein 8-C-[α-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and 6″-O-malonyl-3'-methoxydaidzin, as well as an unstable compound tentatively identified as 3'-de-O-methylpuerariafuran. PMID:25679337

  4. Activin B induces human endometrial cancer cell adhesion, migration and invasion by up-regulating integrin β3 via SMAD2/3 signaling

    PubMed Central

    Xiong, Siyuan; Klausen, Christian; Cheng, Jung-Chien; Zhu, Hua; Leung, Peter C.K.

    2015-01-01

    Endometrial cancer is the fourth most common female cancer and the most common gynecological malignancy. Although it comprises only ~10% of all endometrial cancers, the serous histological subtype accounts for ~40% of deaths due to its aggressive behavior and propensity to metastasize. Histopathological studies suggest that elevated expression of activin/inhibin βB subunit is associated with reduced survival in non-endometrioid endometrial cancers (type II, mostly serous). However, little is known about the specific roles and mechanisms of activin (βB dimer) in serous endometrial cancer growth and progression. In the present study, we examined the biological functions of activin B in type II endometrial cancer cell lines, HEC-1B and KLE. Our results demonstrate that treatment with activin B increases cell migration, invasion and adhesion to vitronectin, but does not affect cell viability. Moreover, we show that activin B treatment increases integrin β3 mRNA and protein levels via SMAD2/3-SMAD4 signaling. Importantly, siRNA knockdown studies revealed that integrin β3 is required for basal and activin B-induced cell migration, invasion and adhesion. Our results suggest that activin B-SMAD2/3-integrin β3 signaling could contribute to poor patient survival by promoting the invasion and/or metastasis of type II endometrial cancers. PMID:26384307

  5. Apigenin, a bioactive flavonoid from Lycopodium clavatum, stimulates nucleotide excision repair genes to protect skin keratinocytes from ultraviolet B-induced reactive oxygen species and DNA damage.

    PubMed

    Das, Sreemanti; Das, Jayeeta; Paul, Avijit; Samadder, Asmita; Khuda-Bukhsh, Anisur Rahman

    2013-10-01

    In this study, we examined the antioxidative and the DNA protective potentials of apigenin, a flavonoid polyphenol isolated from Lycopodium clavatum, in both in-vitro (HaCaT skin keratinocytes) and in-vivo (mice) models against UV-B radiation. We used DAPI staining in UV-B-irradiated HaCaT skin keratinocytes pre-treated with and without apigenin to assess DNA damage. We also used a flow-cytometric analysis in mice exposed to UV-B radiation with or without topical application of apigenin to assess, through a comet assay, chromosomal aberrations and quanta from reactive oxygen species (ROS) generation. Data from the stability curves for the Gibb's free energy determined from a melting-temperature profile study indicated that apigenin increased the stability of calf thymus DNA. Immunofluorescence studies revealed that apigenin caused a reduction in the number of cyclobutane pyrimidine dimers (CPDs) after 24 h, the time at which the nucleotide excision repair (NER) genes were activated. Thus, apigenin accelerated reversal of UV-B-induced CPDs through up-regulation of NER genes, removal of cyclobutane rings, inhibition of ROS generation, and down-regulation of NF-κB and MAPK, thereby revealing the precise mechanism of DNA repair. PMID:24139463

  6. Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury

    PubMed Central

    Sakhatskyy, Pavlo; Newton, Julie; Shamirian, Paul; Hsiao, Vivian; Curren, Sean; Gabino Miranda, Gustavo Andres; Pedroza, Mesias; Blackburn, Michael R.; Rounds, Sharon

    2013-01-01

    Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases. PMID:23316066

  7. Investigation of epothilone B-induced cell death mechanisms in human epithelial cancer cells -in consideration of combined treatment with ionizing radiation.

    PubMed

    Baumgart, Tonja; Kriesen, Stephan; Neels, Oliver; Hildebrandt, Guido; Manda, Katrin

    2015-07-01

    Epothilone B was shown to have promising chemo- and radiosensitizing effects on cells, but the mechanisms underlying cell death remain ambiguous. The aim of the study was to examine selected cell death pathways on the basis of FaDu and A549 cells. Western blot analyses were used for investigation of specific apoptotic markers. Immunofluorescence imaging and flow cytometry were utilized for examination of cell death mechanisms. DNA-staining was used for studying influence of epothilone B on micronucleus rate. We showed that epothilone B can initiate cell death via apoptosis and mitotic catastrophe, but induction of cell death was cell type specific. PMID:25919223

  8. Autophagy and apoptosis dysfunction in neurodegenerative disorders.

    PubMed

    Ghavami, Saeid; Shojaei, Shahla; Yeganeh, Behzad; Ande, Sudharsana R; Jangamreddy, Jaganmohan R; Mehrpour, Maryam; Christoffersson, Jonas; Chaabane, Wiem; Moghadam, Adel Rezaei; Kashani, Hessam H; Hashemi, Mohammad; Owji, Ali A; Łos, Marek J

    2014-01-01

    Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimer's diseases, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders. PMID:24211851

  9. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  10. Control of apoptosis by asymmetric cell division.

    PubMed

    Hatzold, Julia; Conradt, Barbara

    2008-04-01

    Asymmetric cell division and apoptosis (programmed cell death) are two fundamental processes that are important for the development and function of multicellular organisms. We have found that the processes of asymmetric cell division and apoptosis can be functionally linked. Specifically, we show that asymmetric cell division in the nematode Caenorhabditis elegans is mediated by a pathway involving three genes, dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail, that directly control the enzymatic machinery responsible for apoptosis. Interestingly, the MIDA1-like protein GlsA of the alga Volvox carteri, as well as the Snail-related proteins Snail, Escargot, and Worniu of Drosophila melanogaster, have previously been implicated in asymmetric cell division. Therefore, C. elegans dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail may be components of a pathway involved in asymmetric cell division that is conserved throughout the plant and animal kingdoms. Furthermore, based on our results, we propose that this pathway directly controls the apoptotic fate in C. elegans, and possibly other animals as well. PMID:18399720

  11. Lithium protects ethanol-induced neuronal apoptosis

    SciTech Connect

    Zhong Jin . E-mail: jizhong@iupui.edu; Yang Xianlin; Yao Weiguo; Lee Weihua

    2006-12-01

    Lithium is widely used for the treatment of bipolar disorder. Recent studies have demonstrated its neuroprotective effect. Ethanol is a potent neurotoxin that is particularly harmful to the developing nervous system. In this study, we evaluated lithium's neuroprotection against ethanol-induced apoptosis. Transient exposure of infant mice to ethanol caused apoptotic cell death in brain, which was prevented significantly by administering a low dose of lithium 15 min later. In cultured cerebellar granule neurons, ethanol-induced apoptosis and activation of caspase-3/9, both of which were prevented by lithium. However, lithium's protection is not mediated by its commonly known inhibition of glycogen synthase3{beta}, because neither ethanol nor lithium has significant effects on the phosphorylation of Akt (ser473) or GSK3{beta} (ser9). In addition, the selective GSK-3{beta} inhibitor SB-415286 was unable to prevent ethanol-induced apoptosis. These data suggest lithium may be used as a potential preventive measure for ethanol-induced neurological deficits.

  12. Alcohol and Apoptosis: Friends or Foes?

    PubMed Central

    Rodriguez, Ana; Chawla, Karan; Umoh, Nsini A.; Cousins, Valerie M.; Ketegou, Assama; Reddy, Madhumati G.; AlRubaiee, Mustafa; Haddad, Georges E.; Burke, Mark W.

    2015-01-01

    Alcohol abuse causes 79,000 deaths stemming from severe organ damage in the United States every year. Clinical manifestations of long-term alcohol abuse on the cardiac muscle include defective contractility with the development of dilated cardiomyopathy and low-output heart failure; which has poor prognosis with less than 25% survival for more than three years. In contrast, low alcohol consumption has been associated with reduced risk of cardiovascular disease, however the mechanism of this phenomenon remains elusive. The aim of this study was to determine the significance of apoptosis as a mediating factor in cardiac function following chronic high alcohol versus low alcohol exposure. Adult rats were provided 5 mM (low alcohol), 100 mM (high alcohol) or pair-fed non-alcohol controls for 4–5 months. The hearts were dissected, sectioned and stained with cresyl violet or immunohistochemically for caspase-3, a putative marker for apoptosis. Cardiomyocytes were isolated to determine the effects of alcohol exposure on cell contraction and relaxation. High alcohol animals displayed a marked thinning of the left ventricular wall combined with elevated caspase-3 activity and decreased contractility. In contrast, low alcohol was associated with increased contractility and decreased apoptosis suggesting an overall protective mechanism induced by low levels of alcohol exposure. PMID:26610584

  13. Increased small intestinal apoptosis in coeliac disease.

    PubMed Central

    Moss, S F; Attia, L; Scholes, J V; Walters, J R; Holt, P R

    1996-01-01

    BACKGROUND: Coeliac disease (CD) mucosa is flattened despite epithelial hyperproliferation. AIMS: To establish mechanisms of cell loss in CD. PATIENTS: 14 controls, 17 active CD patients, and 16 maintained with gluten free diet. METHODS: Programmed cell death was examined in small intestinal biopsy specimens by staining fragmented DNA using terminal uridine deoxynucleotidyl nick end labelling (TUNEL), in comparison with haematoxylin and eosin stained adjacent sections. Double staining with anti-CD45 antibodies determined the origin of apoptotic cells. Apoptosis was graded from 1-3 (< 5, 5-20, > 20% respectively). Proliferating cells, immunostained by Ki-67 (MIB-1) antibody, were counted. RESULTS: Apoptotic cells were seen rarely by haematoxylin and eosin but more readily by TUNEL. In controls, 1.4 +/- 0.2% of epithelial cells were apoptotic (mean grade 1.1), mainly located in the upper villus. In active CD, frequent apoptotic cells were distributed throughout the crypt-villus unit (mean grade 2.4), decreasing after treatment to 1.1 (p < 0.001) even when still histologically abnormal. CD45 antibodies rarely stained apoptotic cells in active CD. The number of TUNEL positive cells correlated with proliferating cell number (p < 0.001). CONCLUSION: Enterocyte apoptosis is greatly increased in untreated CD, correlates with proliferation, and falls to normal with a gluten free diet, before histological improvement. Increased apoptosis may be responsible for villous atrophy in CD. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9038662

  14. Ion channels and apoptosis in cancer

    PubMed Central

    Bortner, Carl D.; Cidlowski, John A.

    2014-01-01

    Humans maintain a constant cell number throughout their lifespan. This equilibrium of cell number is accomplished when cell proliferation and cell death are kept balanced, achieving a steady-state cell number. Abnormalities in cell growth or cell death can lead to an overabundance of cells known as neoplasm or tumours. While the perception of cancer is often that of an uncontrollable rate of cell growth or increased proliferation, a decrease in cell death can also lead to tumour formation. Most cells when detached from their normal tissue die. However, cancer cells evade cell death, tipping the balance to an overabundance of cell number. Therefore, overcoming this resistance to cell death is a decisive factor in the treatment of cancer. Ion channels play a critical role in cancer in regards to cell proliferation, malignant angiogenesis, migration and metastasis. Additionally, ion channels are also known to be critical components of apoptosis. In this review, we discuss the modes of cell death focusing on the ability of cancer cells to evade apoptosis. Specifically, we focus on the role ion channels play in controlling and regulating life/death decisions and how they can be used to overcome resistance to apoptosis in the treatment of cancer. PMID:24493752

  15. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    PubMed Central

    Kuo, Chen-Tzu; Chen, Bing-Chang; Yu, Chung-Chi; Weng, Chih-Ming; Hsu, Ming-Jen; Chen, Chien-Chih; Chen, Mei-Chieh; Teng, Che-Ming; Pan, Shiow-Lin; Bien, Mauo-Ying; Shih, Chung-Hung; Lin, Chien-Huang

    2009-01-01

    In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis. PMID:19405983

  16. Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food--validation and proof-of-concept study with caper buds.

    PubMed

    Wiese, Stefanie; Wubshet, Sileshi G; Nielsen, John; Staerk, Dan

    2013-12-15

    This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N(1),N(5),N(10)-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N(1),N(10)-di-(E)-caffeoyl-N(5)-p-(E)-coumaroyl spermidine, N(1)-(E)-caffeoyl-N(5),N(10)-di-p-(E)-coumaroyl spermidine, N(10)-(E)-caffeoyl-N(1),N(5)-di-p-(E)-coumaroyl spermidine, and N(1),N(5),N(10)-tri-p-(E)-coumaroyl spermidine amides. PMID:23993578

  17. Gardenin B-induced cell death in human leukemia cells involves multiple caspases but is independent of the generation of reactive oxygen species.

    PubMed

    Cabrera, Javier; Saavedra, Ester; Del Rosario, Henoc; Perdomo, Juan; Loro, Juan F; Cifuente, Diego A; Tonn, Carlos E; García, Celina; Quintana, José; Estévez, Francisco

    2016-08-25

    Flavonoids have attracted great interest due to their possible anticancer activities. Here we investigated the antiproliferative activity of the flavonoids isolated from Baccharis scandens against human leukemia cell lines and found that the methoxyflavonoid gardenin B was the most cytotoxic compound against HL-60 and U-937 cells, showing IC50 values between 1.6 and 3.0 μM, but had no significant cytotoxic effects against quiescent or proliferating human peripheral blood mononuclear cells. These effects on viability were accompanied by the concentration- and time-dependent appearance of apoptosis as evidenced by DNA fragmentation, formation of apoptotic bodies and a sub-G1 ratio increase. Comparative studies with the best-studied bioflavonoid quercetin indicate that gardenin B is a more cytotoxic and more apoptotic inducer than quercetin. Cell death induced by gardenin B was associated with: (i) a significant induction of caspase-2, -3, -8 and -9 activities; (ii) cleavage of the initiator caspases (caspase-2, -8 and -9), of the executioner caspase-3, and of poly(ADP-ribose) polymerase; and (iii) a concentration-dependent reactive oxygen species generation. In conclusion, apoptosis induced by gardenin B is associated with activation of both the extrinsic and the intrinsic apoptotic pathways of cell death and occurs through a mechanism that is independent of the generation of reactive oxygen species. PMID:27423764

  18. Determination of six phthalic acid esters in orange juice packaged by PVC bottle using SPE and HPLC-UV: application to the migration study.

    PubMed

    Guo, Zhiyong; Wei, Danyi; Wang, Meili; Wang, Sui

    2010-10-01

    A high-performance liquid chromatographic assay is described for the determination of six phthalic acid esters (PAEs) in orange juice packaged in polyvinyl chloride (PVC) bottle. Samples were extracted by solid-phase extraction (SPE) cartridges and separated by a C₁₈ column. The calibration curves were all linear with a correlation coefficient r > 0.9900. The limits of detection for the assay ranged from 2.6 to 13.8 ng/mL. Expressed as the within- and between-day coefficient of variation (CV), precision was 1.4-13.4% and 1.9-13.3%, respectively, and relative errors were 7.6-12.8% and -9.0-14.2%, respectively. The recovery ranged from 76.8 to 112.3% with the CV from 0.3 to 11.3%. The proposed methodology was applied for studing the migration of the selected PAEs into orange juice packaged in PVC bottle. Di-ethyl phthalate (DEP) and di-(2-ethylhexyl) phthalate (DEHP) were detected in the orange juice without the other four PAEs. Concentrations would increase with the storage time and reach up to 0.385 μg/mL and 0.662 μg/mL, respectively, when the expiration date arrived. The level of DEHP was about 110 times higher than the limiting one in drink water (6 ppb) regulated by U.S. EPA. Results suggest that PVC plasticized by DEHP should not be used as the packaging material for orange juice. PMID:20875239

  19. WINGS-SPE II: A catalog of stellar ages and star formation histories, stellar masses and dust extinction values for local clusters galaxies

    NASA Astrophysics Data System (ADS)

    Fritz, J.; Poggianti, B. M.; Cava, A.; Valentinuzzi, T.; Moretti, A.; Bettoni, D.; Bressan, A.; Couch, W. J.; D'Onofrio, M.; Dressler, A.; Fasano, G.; Kjærgaard, P.; Moles, M.; Omizzolo, A.; Varela, J.

    2011-02-01

    Context. The WIde-field Nearby Galaxy clusters Survey (wings) is a project whose primary goal is to study the galaxy populations in clusters in the local universe (z < 0.07) and of the influence of environment on their stellar populations. This survey has provided the astronomical community with a high quality set of photometric and spectroscopic data for 77 and 48 nearby galaxy clusters, respectively. Aims: In this paper we present the catalog containing the properties of galaxies observed by the wings SPEctroscopic (wings-spe) survey, which were derived using stellar populations synthesis modelling approach. We also check the consistency of our results with other data in the literature. Methods: Using a spectrophotometric model that reproduces the main features of observed spectra by summing the theoretical spectra of simple stellar populations of different ages, we derive the stellar masses, star formation histories, average age and dust attenuation of galaxies in our sample. Results: ~ 5300 spectra were analyzed with spectrophotometric techniques, and this allowed us to derive the star formation history, stellar masses and ages, and extinction for the wings spectroscopic sample that we present in this paper. Conclusions: The comparison with the total mass values of the same galaxies derived by other authors based on sdss data, confirms the reliability of the adopted methods and data. Based on observations taken at the Anglo Australian Telescope (3.9 m- AAT), and at the William Herschel Telescope (4.2 m- WHT).Full Table 2 is available in electronic form both at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/526/A45, and by querying the wings database at http://web.oapd.inaf.it/wings/new/index.html

  20. Validation and uncertainties evaluation of an isotope dilution-SPE-LC-MS/MS for the quantification of drug residues in surface waters.

    PubMed

    Brieudes, V; Lardy-Fontan, S; Lalere, B; Vaslin-Reimann, S; Budzinski, H

    2016-01-01

    The present work describes the development and validation of a reference method conducted at the French National Institute of Metrology (LNE) for the quantitative determination of psychoactive compounds in the dissolved fraction of surface waters. More specifically an isotope dilution-SPE-LC-MS/MS based method has been implemented for the characterization of a broad range of analytes belonging to different classes of psychotropic drugs such as benzodiazepines, antidepressants, stimulants, opiates and opioids, anticonvulsants, anti-dementia drugs, analgesics as well as the anti-inflammatory drug diclofenac in the low ng L(-1) range of concentration. Full validation of the method was performed following procedures described by the French standard NF T90-210. Limits of quantification between 0.14 and 3.54 ng L(-1) were obtained. Method recoveries from 71 to 123% were observed with standard deviation below 10% in intermediate precision conditions. Accuracy was determined for every compound: measurement errors were between -4 and +1% and standard deviations in intermediate precision conditions were included within a 1-9% interval. Finally, measurement uncertainties were evaluated following the Guide to the expression of uncertainty in measurement (GUM). Expanded uncertainties (k=2) ranged from 2% for carbamazepine, EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) and venlafaxine to 17% for diazepam. The validated method was implemented to Seine river surface waters demonstrating its fitness for purpose. All compounds were detected and 22 out of 25 analytes were quantified. More specifically, measured concentration ranged from 0.39 ng L(-1) for MDMA (3,4-methylene-dioxy-N-methylamphetamine) to 182 ng L(-1) for gabapentine. PMID:26695245

  1. Study of spatial and temporal distribution of antimicrobial in water and sediments from caging fish farms by on-line SPE-LC-MS/MS.

    PubMed

    Monteiro, Sérgio H; Francisco, Jeane G; Andrade, Graziela C R M; Botelho, Rafael G; Figueiredo, Leila A; Tornisielo, Valdemar L

    2016-09-01

    An on-line solid phase extraction liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of 12 antimicrobials in sediment and surface water was developed and validated. Furthermore, the spatial and temporal antimicrobials distributions in the sediment and in the water of four fish farms located in the hydroelectric dam of Ilha Solteira Reservoir in Brazil were investigated over four seasons in three sampling sites: at the fish cages, 100 and 1,000 m downstream far from the cages. The method was performed using an Agilent Zorbax 80 SB-C8 column (9.4 × 15 mm, 5 µm) as the loading column, and the Agilent Zorbax Eclipse Plus C18 column (3.0 × 100 mm, 3.5 µm) as a separation column within a run time of 13 min. The limits of quantification were less than 9 ng·L(-1) for the antibiotics in water and 16 µg·kg(-1) in sediment; the recovery ranged from 80 to 119%, with a variation coefficient less than 11%, and the repeatability was lower than 15%. Oxytetracycline was found in the water in all sample seasons. However, florfenicol was identified in April and October 2013 and January 2014, and tetracycline was present in July 2013. Regarding the sediment, oxytetracycline and tetracycline were found in all sampling periods, but chlortetracycline was only identified in January 2014. The spatial distribution of antimicrobials showed that the main pollution source came from the fish farms. This study demonstrated that the proposed method is reliable for the monitoring of antimicrobials in water and sediments and it showed contamination in both matrices from Ilha Solteira Reservoir. PMID:27249158

  2. Optimization and Simultaneous Determination of Alkyl Phenol Ethoxylates and Brominated Flame Retardants in Water after SPE and Heptafluorobutyric Anhydride Derivatization followed by GC/MS.

    PubMed

    Chokwe, Tlou B; Okonkwo, Jonathan O; Sibali, Linda L; Ncube, Esper J

    2012-10-01

    A gas chromatography-mass spectrometry (GC-MS) method was investigated for the simultaneous analysis of two types of endocrine disrupting compounds (EDCs), i.e., alkylphenol ethoxylates and brominated flame retardants (BFRs), by extraction and derivatization followed by GC-MS. Different solid phase extraction (SPE) cartridges (Cleanert PestiCarb, C18, Cleanert-SAX and Florosil), solvents (toluene, tetrahydrofuran, acetone, acetonitrile and ethyl acetate) and bases (NaHCO3, triethylamine and pyridine) were tested and the best chromatographic analysis was achieved by extraction with Strata-X (33 μm, Reverse Phase) cartridge and derivatization with heptafluorobutyric anhydride at 55 °C under Na2CO3 base in hexane. It was observed that APE together with lower substituted PBBs (PBB1, PBB10, PBB18 and PBB49), HBCD and TBBPA can be determined simultaneously under the same GC conditions. This simple and reliable analytical method was applied to determining trace amounts of these compounds from wastewater treatment plant samples. The recoveries of the target compounds from simulated water were above 60 %. The limit of detection ranged from 0.01 to 0.15 μg L(-1) and the limit of quantification ranged from 0.05 to 0.66 μg L(-1). There were no appreciable differences between filtered and unfiltered wastewater samples from Leeuwkil treatment plant although concentration of target analytes in filtered influent was slightly lower than the concentration of target analytes in unfiltered influent water. The concentrations of the target compounds from the wastewater treatment were determined from LOQ upwards. PMID:23864736

  3. Role for Serine Protease HtrA (DegP) of Streptococcus pyogenes in the Biogenesis of Virulence Factors SpeB and the Hemolysin Streptolysin S

    PubMed Central

    Lyon, William R.; Caparon, Michael G.

    2004-01-01

    The serine protease HtrA is involved in the folding and maturation of secreted proteins, as well as in the degradation of proteins that misfold during secretion. Depletion of HtrA has been shown to affect the sensitivity of many organisms to thermal and environmental stresses, as well as being essential for virulence in many pathogens. In the present study, we compared the behaviors of several different HtrA mutants of the gram-positive pathogen Streptococcus pyogenes (group A streptococcus). Consistent with prior reports, insertional inactivation of htrA, the gene that encodes HtrA, resulted in a mutant that grew poorly at 37°C. However, an identical phenotype was observed when a similar polar insertion was placed immediately downstream of htrA in the streptococcal chromosome, suggesting that the growth defect of the insertion mutant was not a direct result of insertional inactivation of htrA. This conclusion was supported by the observation that a nonpolar deletion mutation of htrA did not produce the growth defect. However, this mutation did affect the production of several secreted virulence factors whose biogenesis requires extensive processing. For the SpeB cysteine protease, the loss of HtrA was associated with a failure to proteolytically process the zymogen to an active protease. For the streptolysin S hemolysin, a dramatic increase in hemolytic activity resulted from the depletion of HtrA. Interestingly, HtrA-deficient mutants were not attenuated in a murine model of subcutaneous infection. These data add to the growing body of information that implies an important role for HtrA in the biogenesis of secreted proteins in gram-positive bacteria. PMID:14977969

  4. Role for serine protease HtrA (DegP) of Streptococcus pyogenes in the biogenesis of virulence factors SpeB and the hemolysin streptolysin S.

    PubMed

    Lyon, William R; Caparon, Michael G

    2004-03-01

    The serine protease HtrA is involved in the folding and maturation of secreted proteins, as well as in the degradation of proteins that misfold during secretion. Depletion of HtrA has been shown to affect the sensitivity of many organisms to thermal and environmental stresses, as well as being essential for virulence in many pathogens. In the present study, we compared the behaviors of several different HtrA mutants of the gram-positive pathogen Streptococcus pyogenes (group A streptococcus). Consistent with prior reports, insertional inactivation of htrA, the gene that encodes HtrA, resulted in a mutant that grew poorly at 37 degrees C. However, an identical phenotype was observed when a similar polar insertion was placed immediately downstream of htrA in the streptococcal chromosome, suggesting that the growth defect of the insertion mutant was not a direct result of insertional inactivation of htrA. This conclusion was supported by the observation that a nonpolar deletion mutation of htrA did not produce the growth defect. However, this mutation did affect the production of several secreted virulence factors whose biogenesis requires extensive processing. For the SpeB cysteine protease, the loss of HtrA was associated with a failure to proteolytically process the zymogen to an active protease. For the streptolysin S hemolysin, a dramatic increase in hemolytic activity resulted from the depletion of HtrA. Interestingly, HtrA-deficient mutants were not attenuated in a murine model of subcutaneous infection. These data add to the growing body of information that implies an important role for HtrA in the biogenesis of secreted proteins in gram-positive bacteria. PMID:14977969

  5. Change Detection via Cross-Borehole and VSP Seismic Surveys for the Source Physics Experiments (SPE) at the Nevada National Security Site (NNSS)

    NASA Astrophysics Data System (ADS)

    Knox, H. A.; Abbott, R. E.; Bonal, N. D.; Aldridge, D. F.; Preston, L. A.; Ober, C.

    2012-12-01

    In support of the Source Physics Experiment (SPE) at the Nevada National Security Site (NNSS), we have conducted two cross-borehole seismic experiments in the Climax Stock. The first experiment was conducted prior to the third shot in this multi-detonation program using two available boreholes and the shot hole, while the second experiment was conducted after the shot using four of the available boreholes. The first study focused on developing a well-characterized 2D pre-explosion Vp model including two VSPs and a seismic refraction survey, as well as quantifying baseline waveform similarity at reoccupied sites. This was accomplished by recording both "sparker" and accelerated weight drop sources on a hydrophone string and surface geophones. In total more than 18,500 unique source-receiver pairs were acquired during this testing. In the second experiment, we reacquired aproximately 8,800 source-receiver pairs and performed a cross-line survey allowing for a 3D post-explosion Vp model. The data acquired from the reoccupied sites was processed using cross-correlation methods and change detection methodologies, including comparison of the tomographic images. The survey design and subsequent processing provided an opportunity to investigate seismic wave propagation through damaged rock. We also performed full waveform forward modelling for a granitic body hosting a perched aquifer. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  6. Quantitative determination of opioids in whole blood using fully automated dried blood spot desorption coupled to on-line SPE-LC-MS/MS.

    PubMed

    Verplaetse, Ruth; Henion, Jack

    2016-01-01

    Opioids are well known, widely used painkillers. Increased stability of opioids in the dried blood spot (DBS) matrix compared to blood/plasma has been described. Other benefits provided by DBS techniques include point-of-care collection, less invasive micro sampling, more economical shipment, and convenient storage. Current methodology for analysis of micro whole blood samples for opioids is limited to the classical DBS workflow, including tedious manual punching of the DBS cards followed by extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. The goal of this study was to develop and validate a fully automated on-line sample preparation procedure for the analysis of DBS micro samples relevant to the detection of opioids in finger prick blood. To this end, automated flow-through elution of DBS cards was followed by on-line solid-phase extraction (SPE) and analysis by LC-MS/MS. Selective, sensitive, accurate, and reproducible quantitation of five representative opioids in human blood at sub-therapeutic, therapeutic, and toxic levels was achieved. The range of reliable response (R(2)  ≥0.997) was 1 to 500 ng/mL whole blood for morphine, codeine, oxycodone, hydrocodone; and 0.1 to 50 ng/mL for fentanyl. Inter-day, intra-day, and matrix inter-lot accuracy and precision was less than 15% (even at lower limits of quantitation (LLOQ) level). The method was successfully used to measure hydrocodone and its major metabolite norhydrocodone in incurred human samples. Our data support the enormous potential of DBS sampling and automated analysis for monitoring opioids as well as other pharmaceuticals in both anti-doping and pain management regimens. PMID:26607771

  7. 1H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations

    PubMed Central

    Agnolet, Sara; Jaroszewski, Jerzy W.; Verpoorte, Robert

    2010-01-01

    Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, 1H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that 1H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d6. Unexpected or unwanted extract constituents were also easily identified in the 1H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of 1H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that 1H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts. Electronic supplementary material The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users. PMID:20526353

  8. SpeB proteolysis with imaged capillary isoelectric focusing for the characterization of domain-specific charge heterogeneities of reference and biosimilar Rituximab.

    PubMed

    Zhang, Zichuan; Perrault, Ronel; Zhao, Yun; Ding, Julia

    2016-05-01

    The charge variations of therapeutic monoclonal antibody reveal important information of the post-translational modifications that may potentially impact the potency and safety of pharmaceutical products, especially during the evaluation of biosimilarity of therapeutic proteins. In this work, a novel SpeB-based proteolysis strategy coupling with imaged capillary isoelectric focusing was developed for the determination of domain-specific charge heterogeneities of innovator and generic Rituximab drug products from United States, European and Indian markets. It was observed that innovator Rituximab from the United States and Europe share highly similar peak distributions and charge heterogeneities with 26.2-26.6% Fc/2, 28.9-29.3% LC and 44.4-44.5% Fd peak areas detected, respectively, while multiple basic variations of Fc/2 and less acidic LC and Fd species were found from generic Rituximab from India with 20.9% Fc/2, 32.3% LC and 46.9% Fd peak areas detected. It was also demonstrated that structural changes caused by Carboxypeptidase B treatment and deamidation study at pH extremes could be sensitively captured with the established method, with the results further indicating that the generic product's basic variations of Fc/2 were un-cleaved Lysine residues, while the lack of certain acidic peaks on LC and Fd probably was due to the lower level of deamidation. This new strategy could become a useful tool to reveal domain-specific charge heterogeneities profiles of a variety of therapeutic monoclonal antibodies in regulated environments. PMID:27038651

  9. Carboxyl terminus of HSC70-interacting protein (CHIP) down-regulates NF-κB-inducing kinase (NIK) and suppresses NIK-induced liver injury.

    PubMed

    Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

    2015-05-01

    Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease. PMID:25792747

  10. The DR6 protein from human herpesvirus-6B induces p53-independent cell cycle arrest in G{sub 2}/M

    SciTech Connect

    Schleimann, Mariane H.; Hoberg, Søren; Solhøj Hansen, Aida; Bundgaard, Bettina; Witt, Christoffer T.; Kofod-Olsen, Emil; Höllsberg, Per

    2014-03-15

    HHV-6B infection inhibits cell proliferation in G{sub 2}/M, but no protein has so far been recognized to exert this function. Here we identify the protein product of direct repeat 6, DR6, as an inhibitor of G{sub 2}/M cell-cycle progression. Transfection of DR6 reduced the total number of cells compared with mock-transfected cells. Lentiviral transduction of DR6 inhibited host cell DNA synthesis in a p53-independent manner, and this inhibition was DR6 dose-dependent. A deletion of 66 amino acids from the N-terminal part of DR6 prevented efficient nuclear translocation and the ability to inhibit DNA synthesis. DR6-induced accumulation of cells in G{sub 2}/M was accompanied by an enhanced expression of cyclin B1 that accumulated predominantly in the cytoplasm. Pull-down of cyclin B1 brought down pCdk1 with the inactivating phosphorylation at Tyr15. Together, DR6 delays cell cycle with an accumulation of cells in G{sub 2}/M and thus might be involved in HHV-6B-induced cell-cycle arrest. - Highlights: • HHV-6B-encoded DR6 protein inhibits cell proliferation. • DR6 inhibits host cell DNA synthesis independent of p53. • DR6 delays the cell cycle in G{sub 2}/M. • An N-terminal sequence is necessary for DR6 function. • DR6 induces cytoplasmic accumulation of cyclin B1.

  11. Screening of UV-B-induced genes from apple peels by SSH: possible involvement of MdCOP1-mediated signaling cascade genes in anthocyanin accumulation.

    PubMed

    Peng, Ting; Saito, Takanori; Honda, Chikako; Ban, Yusuke; Kondo, Satoru; Liu, Ji-Hong; Hatsuyama, Yoshimichi; Moriguchi, Takaya

    2013-07-01

    Suppression subtractive hybridization (SSH) was employed to identify candidate genes involved in red coloration in apple peel with the ultraviolet (UV)-B-treated 'Mutsu'. After reverse Northern blotting verification, nearly 80 clones were successfully sequenced. Large portions of the expressed sequence tags (ESTs) are well characterized anthocyanin biosynthesis-related genes, such as chalcone synthase (11A5), flavonol synthase (12F3), anthocyanidin synthase (11H5) and UDP-glycosyl transferase (14A12) whose presence proved the success of SSH. Eight ESTs were selected for quantitative real-time polymerase chain reaction analysis and their expressions were all elevated in 'Induction', further confirming the reliability of the SSH library. One EST, 11F4 (CONSTITUTIVE PHOTOMORPHOGENIC 1: COP1) with putative function in light signal relay was further analyzed in 'Mutsu' and 'Tsugaru', along with MdHY5 (ELONGATED HYPOCOTYL 5: the downstream target of COP1), MdMYB22 (a possible flavonol-specific activator under the regulation of HY5, belonging to the SG7/PRODUCTION OF FLAVONOL GLYCOSIDES family) and MdMYBA. Results showed that MdCOP1, MdHY5, MdMYB22 and MdMYBA were all UV-B inducible genes and anthocyanin accumulation occurred after their increased expressions. Moreover, their expressions and anthocyanin content were enhanced under UV-B plus 17°C treatment. The presence of G box, a known consensus binding site of HY5, in the MdMYBA promoter region implicated that it could be regulated by MdHY5, which was verified by the result of the yeast one-hybrid analysis. Our data suggested that UV-B irradiation would induce the utmost upstream light signaling factor, MdCOP1, which activates MdHY5 signaling by binding to the promoter regions of MdMYBs, and finally leads to the red coloration of apple peels. PMID:23171407

  12. miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells.

    PubMed

    Amodio, Nicola; Bellizzi, Dina; Leotta, Marzia; Raimondi, Lavinia; Biamonte, Lavinia; D'Aquila, Patrizia; Di Martino, Maria Teresa; Calimeri, Teresa; Rossi, Marco; Lionetti, Marta; Leone, Emanuela; Passarino, Giuseppe; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2013-12-01

    Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells. PMID:24091729

  13. miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

    PubMed Central

    Amodio, Nicola; Bellizzi, Dina; Leotta, Marzia; Raimondi, Lavinia; Biamonte, Lavinia; D’Aquila, Patrizia; Di Martino, Maria Teresa; Calimeri, Teresa; Rossi, Marco; Lionetti, Marta; Leone, Emanuela; Passarino, Giuseppe; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2013-01-01

    Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells. PMID:24091729

  14. Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

    PubMed

    Bockstael, Olivier; Tenenbaum, Liliane; Dalkara, Deniz; Melas, Catherine; De Witte, Olivier; Levivier, Marc; Chtarto, Abdelwahed

    2014-01-01

    We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are

  15. Artesunate induces AIF-dependent apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  16. Apoptosis as a Mechanism for Liver Disease Progression

    PubMed Central

    Guicciardi, Maria Eugenia; Gores, Gregory J.

    2011-01-01

    Hepatocyte injury is ubiquitous in clinical practice, and the mode of cell death associated with this injury is often apoptosis, especially by death receptors. Information from experimental systems demonstrates that hepatocyte apoptosis is sufficient to cause liver hepatic fibrogenesis. The mechanisms linking hepatocyte apoptosis to hepatic fibrosis remain incompletely understood, but likely relate to engulfment of apoptotic bodies by professional phagocytic cells and stellate cells, and release of mediators by cells undergoing apoptosis. Inhibition of apoptosis with caspase inhibitors has demonstrated beneficial effects in murine models of hepatic fibrosis. Recent studies implicating Toll-like receptor 9 (TLR9) in liver injury and fibrosis are also of particular interest. Engulfment of apoptotic bodies is one mechanism by which the TLR9 ligand (CpG DNA motifs) could be delivered to this intracellular receptor. These concepts suggest therapy focused on interrupting the cellular mechanisms linking apoptosis to fibrosis would be useful in human liver diseases. PMID:20960379

  17. CD45 regulates apoptosis in peripheral T lymphocytes.

    PubMed

    Liu, Zhe; Dawes, Ritu; Petrova, Svetla; Beverley, Peter C L; Tchilian, Elma Z

    2006-06-01

    Programmed cell death (apoptosis) is a key mechanism for regulating lymphocyte numbers. Murine lymph node lymphocytes cultured in vitro without added stimuli show significant levels of apoptosis over 24 h, detectable by staining with Annexin V. CD4 and CD8 T lymphocytes from transgenic (Tg) mice expressing single CD45RABC or CD45RO isoforms show increased apoptosis and the extent of apoptosis is inversely correlated with the level of CD45 expression. CD45 Tg cells exhibit phosphatidyl serine translocation and DNA oligonucleosome formation, and can be partially rescued from apoptosis by culture in caspase inhibitors or common gamma-chain-binding cytokines. We conclude that CD45 is an important regulator of spontaneous apoptosis in T lymphocytes and this mechanism may contribute to the disease associations reported for individuals expressing CD45 variant alleles. PMID:16621865

  18. Participation of cyclin A in Myc-induced apoptosis.

    PubMed Central

    Hoang, A T; Cohen, K J; Barrett, J F; Bergstrom, D A; Dang, C V

    1994-01-01

    The involvement of c-Myc in cellular proliferation or apoptosis has been linked to differential cyclin gene expression. We observed that in both proliferating cells and cells undergoing apoptosis, cyclin A (but not B, C, D1, and E) mRNA level was elevated in unsynchronized Myc-overexpressing cells when compared with parental Rat1a fibroblasts. We further demonstrated that Zn(2+)-inducible cyclin A expression was sufficient to cause apoptosis. When Myc-induced apoptosis was blocked by coexpression of Bcl-2, the levels of cyclin C, D1, and E mRNAs were also elevated. Thus, while apoptosis induced by c-Myc is associated with an elevated cyclin A mRNA level, protection from apoptosis by coexpressed Bcl-2 is associated with a complementary increase in cyclin C, D1, and E mRNAs. Images PMID:8041712

  19. Platelets induce apoptosis via membrane-bound FasL

    PubMed Central

    Schleicher, Rebecca I.; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O’Reilly, Lorraine; Meuth, Sven G.; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank

    2015-01-01

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. PMID:26232171

  20. Bystander Macrophage Apoptosis after Mycobacterium tuberculosis H37Ra Infection▿

    PubMed Central

    Kelly, Deirdre M.; ten Bokum, Annemieke M. C.; O'Leary, Seonadh M.; O'Sullivan, Mary P.; Keane, Joseph

    2008-01-01

    Human macrophages infected with Mycobacterium tuberculosis may undergo apoptosis. Macrophage apoptosis contributes to the innate immune response against M. tuberculosis by containing and limiting the growth of mycobacteria and also by depriving the bacillus of its niche cell. Apoptosis of infected macrophages is well documented; however, bystander apoptosis of uninfected macrophages has not been described in the setting of M. tuberculosis. We observed that uninfected human macrophages underwent significant bystander apoptosis 48 and 96 h after they came into contact with macrophages infected with avirulent M. tuberculosis. The bystander apoptosis was significantly greater than the background apoptosis observed in uninfected control cells cultured for the same length of time. There was no evidence of the involvement of tumor necrosis factor alpha, Fas, tumor necrosis factor-related apoptosis-inducing ligand, transforming growth factor β, Toll-like receptor 2, or MyD88 in contact-mediated bystander apoptosis. This newly described phenomenon may further limit the spread of M. tuberculosis by eliminating the niche cells on which the bacillus relies. PMID:17954721

  1. Apoptosis in vascular cells induced by cold atmospheric plasma treatment

    NASA Astrophysics Data System (ADS)

    Sladek, Raymond; Stoffels, Eva

    2006-10-01

    Apoptosis is a natural mechanism of cellular self-destruction. It can be triggered by moderate, yet irreversible damage. Apoptosis plays a major role in tissue renewal. Artificial apoptosis induction will become a novel therapy that meets all requirements for tissue-saving surgery. Diseased tissues can disappear without inflammation and scarring. This is particularly important in treatment of blockages in body tracts (e.g. cardiovascular diseases). Artificial induction of apoptosis can be achieved by means of cold plasma treatment. In this work an atmospheric micro-plasma operated in helium/air has been used to induce apoptosis in vascular cells. Parametric studies of apoptosis induction have been conducted; the efficiency is almost 100%. The apoptotic factors are ROS/RNS (reactive oxygen and nitrogen species). Their densities in the plasma have been measured by mass spectrometry. For apoptosis induction, RNS seem to be more important than ROS, because of their relative abundance. Moreover, addition of a ROS scavenger (ascorbic acid) to the cell culture medium does not reduce the occurrence of apoptosis. Cold plasma is a very efficient tool for fundamental studies of apoptosis, and later, for controlled tissue removal in vivo.

  2. Apoptosis in the vasculature: mechanisms and functional importance

    PubMed Central

    Mallat, Ziad; Tedgui, Alain

    2000-01-01

    Apoptotic death has now been recognized in a number of common and threatening vascular diseases, including atherosclerosis. Interest in apoptosis research relates to the fact that apoptosis, in contrast to oncosis, is a highly regulated process of cell death which raises the hope for the development of specific therapeutic strategies to alter disease progression. This review summarizes the mechanisms involved in vascular endothelial and smooth muscle cell survival/apoptosis, and the potential roles of apoptotic death in atherosclerosis and restenosis. The potential effects of modulation of apoptosis in these diseases are also discussed. PMID:10882378

  3. Research Advances on Pathways of Nickel-Induced Apoptosis

    PubMed Central

    Guo, Hongrui; Chen, Lian; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Wu, Bangyuan

    2015-01-01

    High concentrations of nickel (Ni) are harmful to humans and animals. Ni targets a number of organs and produces multiple toxic effects. Apoptosis is important in Ni-induced toxicity of the kidneys, liver, nerves, and immune system. Apoptotic pathways mediated by reactive oxygen species (ROS), mitochondria, endoplasmic reticulum (ER), Fas, and c-Myc participate in Ni-induced cell apoptosis. However, the exact mechanism of apoptosis caused by Ni is still unclear. Understanding the mechanism of Ni-induced apoptosis may help in designing measures to prevent Ni toxicity. PMID:26703593

  4. Role of PUMA in methamphetamine-induced neuronal apoptosis.

    PubMed

    Chen, Chuanxiang; Qincao, Litao; Xu, Jingtao; Du, Sihao; Huang, Enping; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

    2016-01-01

    Exposure to methamphetamine (METH), a widely used illicit drug, has been shown to cause neuron apoptosis. p53 upregulated modulator of apoptosis (PUMA) is a key mediator in neuronal apoptosis. This study aimed to examine the effects of PUMA in METH-induced neuronal apoptosis. We determined PUMA protein expression in PC12 cells and SH-SY5Y cells after METH exposure using western blot. We also observed the effect of METH on neuronal apoptosis after silencing PUMA expression with siRNA using TUNEL staining and flow cytometry. Additionally, to investigate possible mechanisms of METH-induced PUMA-mediated neuronal apoptosis, we measured the protein expression of apoptotic markers, including cleaved caspase-3, cleaved PARP, Bax, B-cell leukemia/lymphoma-2 (Bcl-2) and cytochrome c (cyto c), after METH treatment with or without PUMA knockdown. Results showed that METH exposure induced cell apoptosis, increased PUMA protein levels, activated caspase-3 and PARP, elevated Bax and reduced Bcl-2 expression, as well as increased the release of cyto c from mitochondria to the cytoplasm in both PC12 and SH-SY5Y cells. All these effects were attenuated or reversed after silencing PUMA. A schematic depicting the role of PUMA in METH-induced mitochondrial apoptotic pathway was proposed. Our results suggest that PUMA plays an important role in METH-triggered apoptosis and it may be a potential target for ameliorating neuronal injury and apoptosis caused by METH. PMID:26524635

  5. Shifting the balance of mitochondrial apoptosis: therapeutic perspectives

    PubMed Central

    Fulda, Simone

    2012-01-01

    Signaling via the intrinsic (mitochondrial) pathway of apoptosis represents one of the critical signal transduction cascades that control the regulation of cell death. This pathway is typically altered in human cancers, thereby providing a suitable target for therapeutic intervention. Members of the Bcl-2 family of proteins as well as cell survival signaling cascades such as the PI3K/Akt/mTOR pathway are involved in the regulation of mitochondria-mediated apoptosis. Therefore, further insights into the molecular mechanisms that form the basis for the control of mitochondria-mediated apoptosis will likely open new perspectives to bypass evasion of apoptosis and treatment resistance in human cancers. PMID:23061040

  6. Mitosis and mitochondrial priming for apoptosis.

    PubMed

    Pedley, Robert; Gilmore, Andrew P

    2016-07-01

    Cell division is a period of danger for cells, as inaccurate segregation of chromosomes can lead to loss of cell viability or aneuploidy. In order to protect against these dangers, cells ultimately initiate mitochondrial apoptosis if they are unable to correctly exit mitosis. A number of important chemotherapeutics exploit this response to delayed mitotic exit, but despite this, the molecular mechanism of the apoptotic timer in mitosis has proved elusive. Some recent studies have now shed light on this, showing how passage through the cell cycle fine-tunes a cell's apoptotic sensitivity such that it can respond appropriately when errors arise. PMID:27016149

  7. Taurine induces the apoptosis of breast cancer cells by regulating apoptosis-related proteins of mitochondria.

    PubMed

    Zhang, Xiali; Lu, Hongfei; Wang, Yibing; Liu, Chunju; Zhu, Weifeng; Zheng, Shuangyan; Wan, Fusheng

    2015-01-01

    Taurine (Tau), the most abundant free amino acid in humans has numerous potential health benefits through its antioxidant and anti-inflammatory properties. However, limited studies have assessed its effect on tumors and the antitumor mechanism remains unknown. The present study investigated the cellular and molecular changes induced by Tau, leading to the induction of apoptosis in human breast cancer cell lines MCF-7 and MDA-MB-231. MCF-7 is p53 proficient (p53+/+) and MDA-MB-231 is a p53 null mutant (p53-/-). Cell proliferation and viability were assessed by MTT. Flow cytometry and hoechst33342 fluorescent staining were employed to detect apoptosis. Spectrophotometry was used to detect caspase-3 activity. Reverse transcription-polymerase chain reaction and western blot analysis were used to detect the levels of mRNA and proteins of p53-upregulated modulator of apoptosis (PUMA), Bax and Bcl-2. Finally, the affect of Tau on the growth of MDA-MB-231-cell-nude mice xenografts was examined. In the study, Tau inhibited growth and induced apoptosis of the two cell lines in a concentration- and time-dependent manner. Notably, the inhibitory effect of Tau on p53-/- cancer cells was clearly significant compared to the p53+/+ cancer cells. Further studies showed that Tau promoted apoptosis in human breast cancer cells and inhibited the growth of tumor in nude mice by inducing the expression of PUMA, which further up- and downregulated the expression of Bax and Bcl-2 protein, giving rise to increased activation of caspase-3. Collectively, these results indicate that Tau is a potent candidate for the chemotherapy of breast cancer through increasing the PUMA expression independent of p53 status. PMID:25395275

  8. An increase of granulosa cell apoptosis mediates aqueous neem (Azadirachta indica) leaf extract-induced oocyte apoptosis in rat

    PubMed Central

    Tripathi, Anima; Shrivastav, Tulsidas G; Chaube, Shail K

    2013-01-01

    Objective: Neem plant (Azadirachta indica) has been extensively used in Ayurvedic system of medicine for female fertility regulation for a long time, but its mechanism of action remains poorly understood. Hence, the present study was aimed to determine whether an increase of granulosa cell apoptosis is associated with aqueous neem leaf extract (NLE)-induced oocyte apoptosis. Materials and Methods: Sexually immature female rats of 20 days old were fed NLE (50 mg/day) for 10 days and then subjected to superovulation induction protocol. The morphological changes in cumulus oocyte complexes (COCs), rate of oocyte apoptosis, hydrogen peroxide (H2O2), total nitrite, and cytochrome c concentrations, inducible nitric oxide synthase (iNOS), cytochrome c, p53, Bcl2 and Bax expressions, deoxyribonucleic acid (DNA) fragmentation, and estradiol 17β level in granulosa cells collected from preovulatory COCs were analyzed. Results: Aqueous NLE increased H2O2 concentration and decreased catalase activity, increased iNOS expression and total nitrite concentration, increased p53, Bax, and p53 expressions but decreased Bcl2 expression, increased cytochrome c concentration and induced DNA fragmentation in granulosa cells. An increased granulosa cell apoptosis resulted in reduced estradiol 17β concentration and induced apoptosis in ovulated oocytes. Conclusion: We conclude that aqueous NLE-induced granulosa cell apoptosis through the mitochondria-mediated pathway, reduced estradiol 17β concentration and induced apoptosis in ovulated oocytes. Thus, granulosa cell apoptosis mediates NLE-induced oocyte apoptosis during female fertility regulation in rat. PMID:23776837

  9. Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells.

    PubMed

    Kato, Satsuki; Sugimura, Norihiko; Nakashima, Keisuke; Nishihara, Tatsuji; Kowashi, Yusuke

    2005-03-01

    It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans

  10. The genomic underpinnings of apoptosis in the silkworm, Bombyx mori

    PubMed Central

    2010-01-01

    Background Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes. Results From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis. Conclusions Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori. PMID:21040523

  11. FemtoSpeX: a versatile optical pump-soft X-ray probe facility with 100 fs X-ray pulses of variable polarization.

    PubMed

    Holldack, Karsten; Bahrdt, Johannes; Balzer, Andreas; Bovensiepen, Uwe; Brzhezinskaya, Maria; Erko, Alexei; Eschenlohr, Andrea; Follath, Rolf; Firsov, Alexander; Frentrup, Winfried; Le Guyader, Loïc; Kachel, Torsten; Kuske, Peter; Mitzner, Rolf; Müller, Roland; Pontius, Niko; Quast, Torsten; Radu, Ilie; Schmidt, Jan Simon; Schüssler-Langeheine, Christian; Sperling, Mike; Stamm, Christian; Trabant, Christoph; Föhlisch, Alexander

    2014-09-01

    Here the major upgrades of the femtoslicing facility at BESSY II (Khan et al., 2006) are reviewed, giving a tutorial on how elliptical-polarized ultrashort soft X-ray pulses from electron storage rings are generated at high repetition rates. Employing a 6 kHz femtosecond-laser system consisting of two amplifiers that are seeded by one Ti:Sa oscillator, the total average flux of photons of 100 fs duration (FWHM) has been increased by a factor of 120 to up to 10(6) photons s(-1) (0.1% bandwidth)(-1) on the sample in the range from 250 to 1400 eV. Thanks to a new beamline design, a factor of 20 enhanced flux and improvements of the stability together with the top-up mode of the accelerator have been achieved. The previously unavoidable problem of increased picosecond-background at higher repetition rates, caused by `halo' photons, has also been solved by hopping between different `camshaft' bunches in a dedicated fill pattern (`3+1 camshaft fill') of the storage ring. In addition to an increased X-ray performance at variable (linear and elliptical) polarization, the sample excitation in pump-probe experiments has been considerably extended using an optical parametric amplifier that supports the range from the near-UV to the far-IR regime. Dedicated endstations covering ultrafast magnetism experiments based on time-resolved X-ray circular dichroism have been either upgraded or, in the case of time-resolved resonant soft X-ray diffraction and reflection, newly constructed and adapted to femtoslicing requirements. Experiments at low temperatures down to 6 K and magnetic fields up to 0.5 T are supported. The FemtoSpeX facility is now operated as a 24 h user facility enabling a new class of experiments in ultrafast magnetism and in the field of transient phenomena and phase transitions in solids. PMID:25177998

  12. Mitochondria in human pluripotent stem cell apoptosis.

    PubMed

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients. PMID:26828436

  13. Evolution of the Animal Apoptosis Network

    PubMed Central

    Zmasek, Christian M.; Godzik, Adam

    2013-01-01

    The number of available eukaryotic genomes has expanded to the point where we can evaluate the complete evolutionary history of many cellular processes. Such analyses for the apoptosis regulatory networks suggest that this network already existed in the ancestor of the entire animal kingdom (Metazoa) in a form more complex than in some popular animal model organisms. This supports the growing realization that regulatory networks do not necessarily evolve from simple to complex and that the relative simplicity of these networks in nematodes and insects does not represent an ancestral state, but is the result of secondary simplifications. Network evolution is not a process of monotonous increase in complexity, but a dynamic process that includes lineage-specific gene losses and expansions, protein domain reshuffling, and emergence/reemergence of similar protein architectures by parallel evolution. Studying the evolution of such networks is a challenging yet interesting subject for research and investigation, and such studies on the apoptosis networks provide us with interesting hints of how these networks, critical in so many human diseases, have developed. PMID:23457257

  14. UV-B-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor and acceptor side components.

    PubMed

    Vass, I; Sass, L; Spetea, C; Bakou, A; Ghanotakis, D F; Petrouleas, V

    1996-07-01

    Inhibition of photosystem II electron transport by UV-B radiation has been studied in isolated spinach photosystem II membrane particles using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements. UV-B irradiation results in the rapid inhibition of oxygen evolution and the decline of variable chlorophyll fluorescence. These effects are accompanied by the loss of the multiline EPR signal arising from the S2 state of the water-oxidizing complex and the induction of Signal IIfast originating from stabilized Try-Z+. The EPR signals from the QA-Fe2+ acceptor complex, Tyr-D+, and the oxidized non-heme iron (Fe3+) are also decreased during the course of UV-B irradiation, but at a significantly slower rate than oxygen evolution and the multiline signal. The decrease of the Fe3+ signal at high g values (g = 8.06, g = 5.6) is accompanied by the induction of another EPR signal at g = 4.26 that arises most likely from the same Fe3+ ion in a modified ligand environment. UV-B irradiation also affects cytochrome b-559. The g = 2.94 EPR signal that arises from the dark- oxidized form is enhanced, whereas the light inducible g = 3.04 signal that arises from the photo-oxidizable population of cytochrome b-559 is diminished. UV-B irradiation also induces the degradation of the D1 reaction center protein. The rate of the D1 protein loss is slower than the inhibition of oxygen evolution and of the multiline signal but follows closely the loss of Signal IIslow, the QA-Fe2+ and the Fe3+ EPR signals, as well as the release of protein-bound manganese. It is concluded from the results that UV-B radiation affects photosystem II redox components at both the donor and acceptor side. The primary damage occurs at the water-oxidizing complex. Modification and/or inactivation of tyrosine-D, cytochrome b-559, and the QAFe2+ acceptor complex are subsequent events that coincide more closely with the UV-B-induced damage to the protein structure of the photosystem II reaction

  15. FAS Expression and Apoptosis in the Fish Parasite Ichthyophthirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish that survive an infection by Ichthyophthirius multifiliis (Ich) develop antibodies that immobilize and kill Ich theronts. Apoptosis of I. multifiliis theronts was caused by cutaneous antibody from channel catfish immune to Ich. The mechanism for cutaneous antibody to induce apoptosis of theron...

  16. ROS signaling during granzyme B-mediated apoptosis

    PubMed Central

    Martinvalet, Denis

    2015-01-01

    Reactive oxygen species (ROS) are involved in cell signaling, aging, and death and play a role in carcinogenesis. However, whether ROS are bystanders or active effectors of apoptosis was unclear until recently. New evidence shows that the killer lymphocyte protease granzyme B activates a conserved biochemical pathway centered on respiratory chain disruption to trigger mitocentric ROS-dependent apoptosis. PMID:27308474

  17. FAS EXPRESSION AND APOPTOSIS IN THE FISH PARASITE ICHTHYOPHTHIRIUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish that survive an infection by Ichthyophthirius multifiliis (Ich) develop antibodies that immobilize and kill Ich theronts. Apoptosis of I. multifiliis theronts was caused by cutaneous antibody from channel catfish immune to Ich. The mechanism for cutaneous antibody to induce apoptosis of theron...

  18. PRMT6 mediates CSE induced inflammation and apoptosis.

    PubMed

    Kang, Naixing; Chen, Ping; Chen, Yan; Zeng, Huihui; He, Xue; Zhu, Yingqun

    2015-01-01

    Cigarette smoke extract (CSE) induces apoptosis and inflammation, but the mechanism is unknown. Arginine methyltransferase (PRMT6) catalyzes the asymmetric di-methylation of histone H3 arginine 2 (H3R2me2a) to control global level transcription. We hypothesized that PRMT6 mediates CSE induced apoptosis and inflammation through H3R2me2a. The apoptosis after CSE treatment in human umbilical vein endothelial cells (HUVECs) was fully measured with real-time reverse transcription PCR, western blotting and Annexin-V staining. Meanwhile, the inflammation in HUVECs after CSE exposure was detected with real-time reverse transcription PCR, western blotting and ELISA. CSE treatment promoted apoptosis and inflammation in HUVECs, coinciding with the decreased protein abundance of PRMT6. Meanwhile, HUVECs transfected with PRMT6 expressing plasmid inhibited the CSE-induced apoptosis and inflammation. Also, the inhibition of PRMT6 promoted the apoptosis and inflammation in HUVECs induced by CSE. Notably, H3R2me2a was associated with the modulation of PRMT6 in CSE induced apoptosis and inflammation in HUVECs. In conclusion, PRMT6 mediates CSE induced apoptosis and inflammation through H3R2me2a in HUVECs. PMID:25481537

  19. Mitochondrion-mediated apoptosis induced by photofrin-PDT

    NASA Astrophysics Data System (ADS)

    Wu, Yunxia; Xing, Da

    2007-05-01

    Apoptosis is an important cellular event that plays a key role in pathogeny and therapy of many diseases. The mechanisms of the initiation and regulation of PDT-induced apoptosis are complex. Some PDT-associated apoptosis pathways involved plasma membrane death receptors, mitochondria, lysosomes and endoplasmic reticulum (ER). In order to determine the apoptosis pathway induced by Photofrin-PDT, we used flu