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Sample records for phorbol ester-resistant human

  1. Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

    SciTech Connect

    Lee, Yong J.; Galoforo, S.S.; Berns, C.M.

    1997-08-01

    We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatmentmore » with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.« less

  2. CD4 down-modulation by ganglioside and phorbol ester inhibits human herpesvirus 7 infection.

    PubMed

    Yasukawa, M; Inoue, Y; Sada, E; Yakushijin, Y; Furukawa, M; Fujita, S

    1995-09-01

    Recently, data demonstrating that CD4 is an essential component of the receptor for human herpesvirus 7 (HHV-7) as well as for human immunodeficiency virus have been accumulating. Since gangliosides and phorbol esters are known to induce selective down-modulation of cell surface CD4 expression, it might be expected that treatment with these agents would interfere with HHV-7 infection of CD4+ T cells. The present study, undertaken to verify this possibility, demonstrated that addition of monosialoganglioside-GM1 or 12-O-tetradecanoylphorbol 13-acetate effectively induced disappearance of CD4 from the cell surface and also reduced HHV-7 infectivity, as judged by the CPE on virus-infected cells and studies of indirect immunofluorescence, TCID50 and semi-quantitative PCR of the HHV-7 genome. Taken together with previous studies, the present data strongly suggest that the CD4 molecule is a critical component of the receptor for HHV-7.

  3. Scavenging effect of berbamine on active oxygen radicals in phorbol ester-stimulated human polymorphonuclear leukocytes.

    PubMed

    Ju, H S; Li, X J; Zhao, B L; Han, Z W; Xin, W J

    1990-06-01

    The scavenging effect of berbamine (Ber) on active oxygen radicals was studied, using a spin-trapping technique and a chemiluminescence (CL) method in phorbol myristate acetate (PMA) stimulated polymorphonuclear leukocytes (PMN) and in four cell-free superoxide (O2-.) or hydroxyl radical (OH.) generating systems. Ber (0.1 to 0.3 mM) effectively reduced active oxygen radicals in PMN stimulated with PMA, but had no obvious effect on oxygen consumption during the respiratory burst of PMN, measured with spin probe oxymetry. Ber (0.3 mM) prominently inhibited the CL response of PMA-stimulated PMN. The agent remarkably quenched O2-. in xanthine/xanthine oxidase and irradiation riboflavin systems and OH. in the Fenton reaction. Its scavenging action on O2-. was stronger than that of Vitamin E in the xanthine/xanthine oxidase system but the same as Vitamin E in the riboflavin system, and its action on OH. was similar to that of Vitamin E.

  4. Phorbol ester and mitogens stimulate human fibroblast secretions of plasmin-activatable plasminogen activator and protease nexin, an antiactivator/antiplasmin

    PubMed Central

    1983-01-01

    Tumor-promoting phorbol esters have been reported to greatly increase plasminogen activator (PA) activity produced in numerous cell types. Many of these studies have employed a widely used fibrinolysis assay for PA activity that involves large-scale dilution of cell lysates or conditioned medium (CM) into buffer containing plasminogen and the plasmin substrate 125I-fibrin. This assay indicates that phorbol ester and the mitogens epidermal growth factor (EGF) and thrombin all stimulate secretion of PA activity in our human foreskin fibroblast cultures. However, these effects are not observed in a modified fibrinolysis assay employing undiluted conditioned culture medium unless the medium is first treated at pH 3, which inactivates the secreted protease inhibitor, protease nexin (PN). Moreover, a direct assay for plasminogen activator activity based on cleavage of 125I- plasminogen indicates that conditioned culture medium contains little if any active plasminogen activator either before or after treatment of the cultures with phorbol ester or EGF. Phorbol ester and mitogens do stimulate secretion of (a) an inactive PA that can be activated by plasmin and (b) PN, which inhibits both the activated form of the PA and plasmin. Secretions of the inactive PA and PN are further correlated in that release of both is stimulated most by phorbol ester, somewhat less by EGF, and least by thrombin. Significantly, these effects are not accompanied by increases in total protein secretion. We propose that fibroblasts secrete PA in an inactive form in the presence of PN to confine PA activity to an as yet undefined location or event. PMID:6224800

  5. beta. -Endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

    SciTech Connect

    Diamant, M.; Henricks, P.A.J.; Nijkamp, F.P.

    1989-01-01

    In the present study, the immunomodulatory effect of {beta}-endorphin ({beta}-E) and shorter pro-opiomelancortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity and their non-opioid derivatives were tested. With the exception of {alpha}-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10{sup {minus}17}M and higher than 10{sup {minus}8}M were without effect. {beta}-E andmore » dT{beta}E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations. {gamma}-E and dT{gamma}E proved to be less potent inhibitors, reaching maximal effect at higher concentrations. DE{gamma}E exerted an even less pronounced but still significant suppressive effect at the concentration of 10{sup {minus}10}M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone.« less

  6. Failure of matrix metalloproteinase-9 dimer induction by phorbol 12-myristate 13-acetate in normal human cell lines.

    PubMed

    Waheed Roomi, Mohd; Kalinovsky, Tatiana; Rath, Matthias; Niedzwiecki, Aleksandra

    2015-06-01

    Increasing experimental and clinical data has identified an association between increased levels of matrix metalloproteinase (MMP)-9 and shortened patient survival, cancer progression and metastasis. MMP-9 has a significant role in tumor cell invasion and metastasis, as it digests the basement membrane and components of the extracellular matrix. MMP-9 is secreted in either a monomeric or dimeric form. Although limited evidence exists concerning MMP-9 dimers, certain studies have demonstrated that the dimer is associated with aggressive tumor progression. This is believed to be due to the fact that cellular migration depends upon the MMP-9 dimer, and not the monomer. Our previous study revealed that cancer cell MMP-9 dimer secretion patterns could be divided into different categories, and that high MMP-9 and MMP-9 dimer secretion levels were correlated with the most aggressive cancer cell lines. It has been established that signal transduction pathways and cytokines, including those activated by phorbol 12-myristate 13-acetate (PMA), regulate the expression of MMPs. The aim of the present study was to analyze the expression patterns of MMP-2, MMP-9 and MMP-9 dimer in normal human cells from a number of tissues treated with PMA. Muscle, epithelial and connective tissues were selected for use in the present study, since adenosarcomas, carcinomas and sarcomas are derived from these tissue types, respectively. The cell lines were first cultured in 24-well tissue culture plates containing recommended media that was supplemented with 10% fetal bovine serum and antibiotics. When at confluency, the cells were washed and fresh medium was added. In addition, a parallel set of cultures was treated with PMA. Subsequent to a 24-h incubation period, the media were collected and analyzed using gelatinase zymography for the expression of MMP-2 and MMP-9 monomer and dimer forms. The results revealed that the cellular expression of MMP-2 and MMP-9 was dependent upon the primary

  7. Phorbol ester up-regulates capacities for nuclear translocation and phosphorylation of 5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes.

    PubMed

    Werz, O; Klemm, J; Samuelsson, B; Rådmark, O

    2001-04-15

    The leukotrienes are inflammatory mediators derived from arachidonic acid. It was demonstrated that the priming of leukocytes with phorbol-12-myristate-13-acetate (PMA) leads to the increased formation of 5-lipoxygenase (5-LO) products in parallel with the increased association of 5-LO with the nucleus and the activation of kinases that can phosphorylate 5-LO in vitro. Stimulation of the monocytic cell line Mono Mac 6 with calcium ionophore gave low 5-LO product formation and no detectable redistribution of 5-LO. However, after priming of Mono Mac 6 cells with phorbol esters, ionophore led to the association of 45% to 75% of cellular 5-LO with the nuclear membrane, to 5-LO kinase activation, to enhanced release of arachidonate, and to substantial leukotriene synthesis. Similar results were obtained for human polymorphonuclear leukocytes stimulated with low-dose ionophore. In addition, for each cell type, PMA priming up-regulated leukotriene biosynthesis in the presence of exogenous arachidonic acid. A protein kinase inhibitor, calphostin C, reduced the association of 5-LO with the nucleus and 5-LO kinase activity, and the formation of 5-LO products was inhibited. These results suggest that PMA up-regulates leukotriene biosynthesis not only by increasing the release of endogenous arachidonate, but also by increasing the capacity for 5-LO phosphorylation and for the translocation of 5-LO to the nucleus in leukocytes.

  8. System-wide analysis of the transcriptional network of human myelomonocytic leukemia cells predicts attractor structure and phorbol-ester-induced differentiation and dedifferentiation transitions

    NASA Astrophysics Data System (ADS)

    Sakata, Katsumi; Ohyanagi, Hajime; Sato, Shinji; Nobori, Hiroya; Hayashi, Akiko; Ishii, Hideshi; Daub, Carsten O.; Kawai, Jun; Suzuki, Harukazu; Saito, Toshiyuki

    2015-02-01

    We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.

  9. Galangin and Kaempferol Suppress Phorbol-12-Myristate-13-Acetate-Induced Matrix Metalloproteinase-9 Expression in Human Fibrosarcoma HT-1080 Cells

    PubMed Central

    Choi, Yu Jung; Lee, Young Hun; Lee, Seung-Taek

    2015-01-01

    Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9. PMID:25518925

  10. Galangin and kaempferol suppress phorbol-12-myristate-13-acetate-induced matrix metalloproteinase-9 expression in human fibrosarcoma HT-1080 cells.

    PubMed

    Choi, Yu Jung; Lee, Young Hun; Lee, Seung-Taek

    2015-01-01

    Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.

  11. Signal transduction of phorbol 12-myristate 13-acetate (PMA)-induced growth inhibition of human monocytic leukemia THP-1 cells is reactive oxygen dependent.

    PubMed

    Traore, Kassim; Trush, Michael A; George, Matthew; Spannhake, Ernst Wm; Anderson, Winston; Asseffa, Amha

    2005-08-01

    Human monocytic THP-1 cells can be induced to differentiate to macrophages when treated with phorbol 12-myristate 13-acetate (PMA). It is understood that before initiating cell differentiation, PMA treatment must first induce an inhibition of cell growth. Since the initial biochemical and molecular events that are associated with this growth inhibition have not been characterized, the present study was carried out to elucidate the molecular mechanisms associated with the PMA-induced growth arrest of THP-1 cells. Our results indicate that PMA inhibits THP-1 cells at G1-phase of the cell cycle, via a complex mechanism associated with the modulation of the expression of several cell cycle regulators, initiated by the cellular generation of reactive oxygen species (ROS). Both p21WAF1/CIP1 mRNA and protein were upregulated 24 h post PMA treatment as demonstrated by ribonuclease protection assay and Western blotting, respectively. Because these cells lack functional p53, this effect was independent of p53 activity. Electrophoretic mobility shift assay showed that the PMA-induced activation of the p21WAF1/CIP1 promoter was driven by the specific protein 1 (Sp1) transcription factor through Sp1-binding sites. Additionally, our study demonstrates that PMA-induces the upregulation of p21 through a protein kinase C (PKC)-mediated ROS-dependent signaling mechanism involving MAP kinase activation.

  12. Acute regulation of glucose transport after activation of human peripheral blood neutrophils by phorbol myristate acetate, fMLP, and granulocyte-macrophage colony-stimulating factor.

    PubMed

    Tan, A S; Ahmed, N; Berridge, M V

    1998-01-15

    Activation of human peripheral blood neutrophils by pathogens or by phorbol myristate acetate (PMA), fMLP, or myeloid growth factors generates a respiratory burst in which superoxide production plays an important role in killing invading microorganisms. Although the increased energy demands of activated neutrophils would be expected to be associated with increased glucose uptake and utilization, previous studies have shown that PMA inhibits 2-deoxyglucose (2-DOG) uptake. In this study, we show that PMA activation of neutrophils, isolated by methods not involving hypotonic lysis, increases the rate of 2-DOG uptake and results in a 1.6-fold to 2.1-fold increase in transporter affinity for glucose without changing Vmax. Increased transporter affinity in response to PMA was also observed with 3-O-methyglucose, which is not phosphorylated, and inclusion of glucose in the activation medium further increased respiratory burst activity. Increased 2-DOG uptake and increased transporter affinity for glucose were also observed with the peptide activator, fMLP, and with granulocyte-macrophage colony-stimulating factor (GM-CSF). The protein kinase C (PKC) inhibitor, calphostin C, and the tyrosine kinase inhibitor, genistein, inhibited both PMA- and fMLP-stimulated 2-DOG uptake. In contrast, genistein inhibited fMLP-induced superoxide production, but had little effect on the PMA-induced response, while staurosporine differentially inhibited PMA-induced superoxide production. These results show that neutrophil activation involves increased glucose transport and intrinsic activation of glucose transporter molecules. Both tyrosine kinases and PKC are implicated in the activation process.

  13. Butyrate Produced by Commensal Bacteria Potentiates Phorbol Esters Induced AP-1 Response in Human Intestinal Epithelial Cells

    PubMed Central

    Nepelska, Malgorzata; Cultrone, Antonietta; Béguet-Crespel, Fabienne; Le Roux, Karine; Doré, Joël; Arulampalam, Vermulugesan; Blottière, Hervé M.

    2012-01-01

    The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs) produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells. PMID:23300800

  14. Differential effects of phorbol 12-myristate 13-acetate and diacylglycerols on thromboxane A2-independent phospholipase A2 activation in collage-stimulated human platelets.

    PubMed

    Reddy, S; Rao, G H; Murthy, M

    1994-04-01

    We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC8 and OAG, and 1,3-DiC8 (a poor activator of PKC) on thromboxane A2 (TxA2)-independent phospholipase A2 (PLA2) activation in human platelets using collagen and A23187 as agonists. We measured PLA2 activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA2 synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC8, OAG, and 1,3-DiC8 increased TxA2-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC8, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC8) in priming TxA2-independent PLA2 activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA2-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligatory for priming PLA2 activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC8, OAG, and 1,3-DiC8 likely enhanced PLA2 activation via intracellular Ca2+ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA1, or nonspecific PLA2. Since both 1,2-DiC8 and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA1 may not

  15. Expression of interleukin-5 and granulocyte-macrophage colony-stimulating factor in human peripheral blood mononuclear cells after activation with phorbol myristate acetate.

    PubMed Central

    Staynov, D Z; Lee, T H

    1992-01-01

    The expression of interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied in peripheral blood mononuclear cells (PBMC) from atopic and non-atopic subjects after activation with phorbol 12-myristate 13-acetate (PMA). The levels of IL-5 and GM-CSF mRNA were monitored by quantitative polymerase chain reaction (PCR). IL-5 and GM-CSF mRNA was undetectable in quiescent cells. Following PMA stimulation, some atopic patients showed considerably higher levels of IL-5 and GM-CSF mRNA expression than the non-atopic subjects, and there was a significant correlation between the levels of these two cytokines. It was found that activation of IL-5 expression in PBMC requires protein synthesis as does activation of GM-CSF expression, and that PMA is only required during the first few hours of activation. The kinetics of activation indicated that the level of both mRNA increased over 15 hr and remained constant for another 20 hr. The accumulation of IL-5 mRNA lagged about 3 hr behind GM-CSF mRNA accumulation, suggesting that the expression of these two genes is regulated separately. However, GM-CSF expression was not required for IL-5 activation. Images Figure 1 Figure 4 Figure 5 PMID:1537597

  16. Co-ordinate expression of activin A and its type I receptor mRNAs during phorbol ester-induced differentiation of human K562 erythroleukemia cells.

    PubMed

    Hildén, K; Tuuri, T; Erämaa, M; Ritvos, O

    1999-07-20

    Activins were originally isolated based on their ability to stimulate follicle-stimulating hormone secretion but later they have been shown to regulate a number of different cellular functions such as nerve cell survival, mesoderm induction during early embryogenesis as well as hematopoiesis. We studied the regulation of activin A, a homodimer of betaA-subunits, mRNA and protein in K562 erythroleukemia cells, which are known to be induced toward the erythroid lineage in response to activin or TGF-beta or toward the megakaryocytic lineage by the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Here we show by Northern blot analysis as well as by Western and ligand blotting that TPA strongly promotes activin betaA-subunit mRNA and activin A protein expression in K562 cells in time- and concentration dependent manner. In contrast, neither activin A nor TGF-beta induced betaA-subunit mRNA expression during erythroid differentiation in K562 cells. Interestingly, whereas activin type II receptors are not regulated during K562 cell differentiation (Hilden et al. (1994) Blood 83, 2163-2170), we now show that the activin type I and IB receptor mRNAs are clearly induced by TPA but not by activin or TGF-beta. We also show that the inducing effect of TPA on expression of activin betaA-subunit mRNA is potentiated by the protein kinase A activator 8-bromo-cAMP. We conclude that activin A and its type I receptors appear to be co-ordinately up-regulated during megakaryocytic differentiation of K562 cells.

  17. Dual stimulus-dependent effect of Oenothera paradoxa extract on the respiratory burst in human leukocytes: suppressing for Escherichia coli and phorbol myristate acetate and stimulating for formyl-methionyl-leucyl-phenylalanine.

    PubMed

    Burzynska-Pedziwiatr, Izabela; Bukowiecka-Matusiak, Malgorzata; Wojcik, Marzena; Machala, Waldemar; Bienkiewicz, Malgorzata; Spolnik, Grzegorz; Danikiewicz, Witold; Wozniak, Lucyna Alicja

    2014-01-01

    Although a growing body of evidence suggests that plant polyphenols can modulate human immune responses, their simultaneous action on monocyte and neutrophil oxidative burst is currently poorly understood. Based on the hypothesis that various polyphenols contained in plant extracts might affect the oxidative burst of phagocytes, we evaluated the effects of ethanolic O. paradoxa extract polyphenols on monocyte and neutrophil oxidative burst in vitro activated by different stimuli, including opsonized bacteria E. coli, phorbol 12-myristate 13-acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (fMLP). Samples were analyzed by the dihydrorhodamine flow cytometry assay. Our results showed that the extract repressed significantly and dose-dependently reactive oxygen species production in both cell types stimulated with E. coli and PMA (P < 0.05) and its inhibitory efficiency was stimulus- and cell-type-dependent. Interestingly, there was significant stimulatory effect of the extract on bursting phagocytes induced by fMLP (P < 0.05). Additionally, several flavonoids and phenolic compounds as well as penta-galloyl-β-(D)-glucose (PGG), the representative of hydrolyzable tannins, were identified in the 60% extract by high-performance liquid chromatography (HPLC) coupled to electrospray ionization in negative ion mode. In summary, the ethanolic O. paradoxa extract, rich in flavonoids and phenolic compounds, exhibits dual stimulus-dependent effect on the respiratory burst in human leukocytes; hence, it might affect immune responses in humans.

  18. Effect of phorbol 12-myristate 13-acetate activated signaling pathways on 1α, 25 dihydroxyvitamin D3 regulated human 25-hydroxyvitamin D3 24-hydroxylase gene expression in differentiated Caco-2 cells

    PubMed Central

    Jiang, Yan; Fleet, James C.

    2012-01-01

    Phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, can modulate 1α, 25 dihydroxyvitamin D3 (1,25(OH)2D3)-induced expression of the 24-hydroxylase (CYP24A1) gene but this has not been studied in differentiated enterocytes, a primary 1,25(OH)2D3 target cell. We found that in differentiated Caco-2 cells, an established model of the mature absorptive epithelial cell, PMA significantly enhanced 1,25(OH)2D3-induced human CYP24A1 (hCYP24A1) mRNA accumulation and hCYP24A1 promoter-luciferase reporter gene activation by 150%. Reporter gene studies further identified the region between −298 to +74 bp in the hCYP24A1 promoter as critical for the PMA enhancing effect and chromatin immunoprecipitation (ChIP) analysis showed that PMA enhanced 1,25(OH)2D3-induced binding of vitamin D receptor to this region. PMA can activate PKC, ERK1/2 and p38 MAP kinases and inhibition of these signaling pathways reduced both 1,25(OH)2D3-induced hCYP24A1 gene transcription and the enhancing effect of PMA. The PMA enhancing effect on 1,25(OH)2D3 action was evident in a minimal promoter with three osteocalcin VDREs and was reduced after mutation of a putative vitamin D stimulatory site in the hCYP24A1 promoter. In contrast, mutation of a Ets binding site in the hCYP24A1 promoter had no impact on 1,25(OH)2D3 action or the PMA enhancing effect. These data suggest that in the differentiated enterocyte PMA-induced activation of several signaling pathways contribute to 1,25(OH)2D3-induced hCYP24A1 gene expression through multiple regulatory motifs within the proximal hCYP24A1 promoter. PMID:22174178

  19. Phorbol esters in seed oil of Jatropha curcas L. (saboodam in Thai) and their association with cancer prevention: from the initial investigation to the present topics.

    PubMed

    Fujiki, Hirota; Suttajit, Maitree; Rawangkan, Anchalee; Iida, Keisuke; Limtrakul, Pornngarm; Umsumarng, Sonthaya; Suganuma, Masami

    2017-08-01

    In 1988, we first reported the complete chemical structure of a new type of phorbol ester, abbreviated to DHPB, found in seed oil of Jatropha curcas L. (Saboodam in Thai) and its tumor-promoting activity on mouse skin. Although this seed oil contains toxic phorbol ester, it was planned to use it as a feasible renewable oil and the extracted seed cake as fertilizer. This utilization value opened a new science of Jatropha curcas. The main experimental results are cited from our publications, and the relevant literature screened from journals and PubMed. This paper begins with our original work on the structural elucidation of a new phorbol ester, 12-deoxy-16-hydroxyphorbol (DHPB): its tumor-promoting activity was compared with that of TPA. We think that it is timely to review the following research advances with Jatropha curcas, so numerous topics are classified as follows: (1) historical development of phorbol esters in seed oil; (2) toxicity of phorbol ester based on various bioassays; (3) degradation of phorbol ester; (4) a new pharmaceutical compound in seed; and (5) tumor promotion and progression with endogeneous tumor promoters in human carcinogenesis. The discovery of phorbol ester in seed oil raised awareness of the danger of public use of seed oil and seed cake in Thailand, and also indicated the necessity of discussing the concept of primary and tertiary cancer preventions. It is worthwhile to study the future benefits and cancer risks of globally distributed Jatropha curcas L.

  20. Occular and dermal toxicity of Jatropha curcas phorbol esters.

    PubMed

    Devappa, Rakshit K; Roach, Joy S; Makkar, Harinder P S; Becker, Klaus

    2013-08-01

    Jatropha curcas seeds are a promising feedstock for biodiesel production. However, Jatropha seed oil and other plant parts are toxic due to the presence of phorbol esters (PEs). The ever-increasing cultivation of toxic genotype of J. curcas runs the risk of increased human exposure to Jatropha products. In the present study, effects of J. curcas oil (from both toxic and nontoxic genotypes), purified PEs-rich extract and purified PEs (factors C1, C2, C(3mixture), (C4+C5)) on reconstituted human epithelium (RHE) and human corneal epithelium (HCE) were evaluated in vitro. The PEs were purified from toxic Jatropha oil. In both RHE and HCE, the topical application of PEs containing samples produced severe cellular alterations such as marked oedema, presence of less viable cell layers, necrosis and/or partial tissue disintegration in epithelium and increased inflammatory response (interleukin-1α and prostaglandin E2). When compared to toxic oil, histological alterations and inflammatory response were less evident (P<0.05) in nontoxic oil indicating the severity of toxicity was due to PEs. Conclusively, topical applications of Jatropha PEs are toxic towards RHE and HCE models, which represents dermal and occular toxicity respectively. Data obtained from this study would aid in the development of safety procedures for Jatropha biodiesel industries. It is advised to use protective gloves and glasses when handling PEs containing Jatropha products. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Phorbol esters inhibit ammoniagenesis and gluconeogenesis in proximal tubular segments.

    PubMed

    Chobanian, M C; Hammerman, M R

    1987-06-01

    To characterize the regulation of ammoniagenesis and gluconeogenesis in renal proximal tubule, ammonia and glucose productions were measured in suspensions of canine proximal tubular segments incubated with 10 mM L-glutamine. Productions were linear functions of time for 120 min and were decreased as extracellular pH was increased from 7.0 to 7.5 To ascertain whether activation of protein kinase c affects either process, we incubated segments with tumor-promoting phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA), or phorbol 12,13-dibutyrate, or with the inactive phorbol ester 4 alpha-phorbol. Ammoniagenesis and gluconeogenesis were inhibited by incubation with 10(-6) M of the two former compounds but not the latter compound. Inhibition of ammoniagenesis and gluconeogenesis occurred after incubation with as little as 10(-9) M phorbol 12,13-dibutyrate. Phorbol ester-induced inhibition was observed under conditions such that extracellular [Na+] was greater than intracellular [Na+], but not when extracellular [Na+] equaled intracellular [Na+], and was not observed in the presence of amiloride. Our findings are consistent with a role for protein kinase c in the control of ammoniagenesis and gluconeogenesis in proximal tubule. Such control could be mediated via stimulation of Na+-H+ exchange.

  2. Post-transcriptional regulation of apolipoprotein E expression in mouse macrophages by phorbol ester.

    PubMed Central

    Dory, L

    1993-01-01

    Phorbol ester-mediated differentiation of THP-1 cells (a human monocytic cell line) into mature macrophages is associated with a transcriptional induction of apolipoprotein E (apoE) expression [Auwerx, Deeb, Brunzell, Peng and Chait (1988) Biochemistry 27, 2651-2655]. Endotoxin, on the other hand, which may also act through activation of protein kinase C, is a potent inhibitor of apoE expression in mouse macrophages [Werb and Chin (1983) J. Biol. Chem. 258, 10642-10648]. The present experiments examine the effect of phorbol ester, an activator of protein kinase C, on the apoE expression in mouse thioglycollate-elicited peritoneal macrophages. Phorbol ester inhibits apoE expression in a specific, time- and dose-dependent manner. A 75% inhibition in the rate of apoE secretion, but not that of total protein, was observed following a 4.5 h incubation with 160 nM phorbol ester, although nearly full inhibition was obtained with 40 nM. The changes in apoE secretion were paralleled by similar changes in apoE synthesis, indicating synthesis as the primary site of action. The decreased rates of apoE synthesis are shown not to be due to increased apoE degradation. The profound inhibition of apoE synthesis was not accompanied by significant changes in apoE mRNA levels at any concentration of phorbol ester (up to 16 microM), or length of treatment (up to 24 h), suggesting a post-transcriptional locus of regulation of apoE expression. Although the early changes in apoE synthesis correlate with increased microsomal protein kinase C activity, the suppression of apoE expression persists even during conditions of nearly complete (> 95%) loss of protein kinase C activity, suggesting that the direct or indirect effect of protein kinase C on apoE expression is mediated by a stable phosphorylated protein, or that the observed effects are mediated through a protein kinase C species that is not readily downregulated by phorbol esters. The presented studies clearly demonstrate the

  3. Plasma application for detoxification of Jatropha phorbol esters

    NASA Astrophysics Data System (ADS)

    Kongmany, S.; Matsuura, H.; Furuta, M.; Okuda, S.; Imamura, K.; Maeda, Y.

    2013-06-01

    Atmospheric pressure non-thermal dielectric barrier discharge (DBD) plasma generated by helium gas at high voltage and input power of about 50 W was first applied to detoxification of Jatropha curcas phorbol esters (J. PEs) as well as standard phorbol ester (4β-12-O-tetradecanoyl phorbol-13-acetate, TPA) in water and methanol. Plasma irradiation on the solution sample was conducted for 15 min. In aqueous solution, only 16% of TPA was degraded and complete degradation of J. PEs was observed. On the contrary, complete degradation of both TPA and J. PEs in methanol was achieved by the same plasma irradiation condition. Hydroxyl radical (•OH) generated by plasma irradiation of the solution is expected as the main radical inducing the degradation of PEs.

  4. Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2

    SciTech Connect

    Jorgensen, Kjersti; Skrede, Martina; Cruciani, Veronique

    2005-04-01

    The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotidesmore » against PKC-{alpha}, or the PKC inhibitor Goe6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.« less

  5. Phorbol esters inhibit gluconeogenesis in canine renal proximal tubular segments.

    PubMed

    Rogers, S; Gavin, J R; Hammerman, M R

    1985-08-01

    Gluconeogenesis is a major metabolic function of the renal proximal tubular cell. To characterize the regulation of this process in proximal tubule, glucose production from gluconeogenic precursors was measured in proximal tubular segments prepared from dog kidney. Production of glucose was a linear function of time for up to 120 min of incubation at 37 degrees C under a variety of conditions. Lowering the pH of incubation media from 7.5 to 7.0 increased glucose synthesis. Production of glucose was inhibited by 3-mercaptopicolinate. Incubation of proximal tubular segments with insulin diminished synthesis of glucose. Incubation of segments with tumor-promoting phorbol esters, 12-O-tetradecanoylphorbol-13-acetate or phorbol 12,13-dibutyrate, resulted in decreased production of glucose. This effect was not observed following incubation with the inactive phorbol ester 4 alpha-phorbol. Changes in glucose synthesis could not be attributed to alterations in cell viability or in rates of glucose oxidation induced by experimental maneuvers. Our findings confirm the usefulness of proximal tubular segments for characterization of metabolic processes in this portion of the nephron. The experimental results are consistent with a role for protein kinase C in the control of gluconeogenesis in proximal tubule.

  6. Differential binding of cAMP-responsive-element (CRE)-binding protein-1 and activating transcription factor-2 to a CRE-like element in the human tissue-type plasminogen activator (t-PA) gene promoter correlates with opposite regulation of t-PA by phorbol ester in HT-1080 and HeLa cells.

    PubMed

    Costa, M; Medcalf, R L

    1996-05-01

    The human tissue-type plasminogen activator gene (t-PA) is induced by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in HeLa cells. Previous studies in transfected HeLa cells identified two cis-acting regulatory elements within the t-PA gene promoter responsible for both constitutive and PMA-inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide substitution (referred to in this report as t-PACRE) and another which bears similarity to the AP-2 recognition sequence. In HT-1080 fibrosarcoma cells, t-PA mRNA levels are expressed at higher constitutive levels and are suppressed by PMA. Nuclear run-on transcription experiments indicate that PMA-mediated suppression of t-PA in these cells is associated with a decrease in t-PA gene template activity. We designed experiments to determine whether nuclear t-PACRE or AP-2-like binding proteins were differentially expressed in HeLa and HT-1080 cells and, accordingly, if these could be correlated with the opposite effect of PMA on t-PA expression. Band shift analyses indicated that the migration profiles of HeLa and HT-1080 nuclear proteins interacting with the AP-2-like site were indistinguishable; however, those produced with the t-PACRE binding site were qualitatively and quantitatively distinct. The distribution of t-PACRE binding proteins in these cells was investigated in a supershift assay using specific antibodies against members of the fos/jun and CRE-binding protein (CREB)/activating transcription factor (ATF) families. In HT-1080 cells, CREB-1 was the most prominent t-PACRE-binding activity detected and was greatly increased in cells treated with PMA. In contrast, CREB-1 activity was absent in HeLa cells, but antibodies specific for ATF-2 produced a marked supershifted complex which was unaffected by PMA treatment. Since CREB-1 can repress transcription of other target genes (including c-jun) via association with identical cis-acting CRE

  7. Potential treatments to reduce phorbol esters levels in jatropha seed cake for improving the value added product.

    PubMed

    Sadubthummarak, Umapron; Parkpian, Preeda; Ruchirawat, Mathuros; Kongchum, Manoch; Delaune, R D

    2013-01-01

    Jatropha seed cake contains high amounts of protein and other nutrients, however it has a drawback due to toxic compounds. The aim of this study was to investigate the methods applied to detoxify the main toxin, phorbol esters in jatropha seed cake, to a safe and acceptable level by maintaining the nutritional values. Phorbol esters are tetracyclic diterpenoids-polycyclic compounds that are known as tumor promoters and hence exhibited the toxicity within a broad range of species. Mismanagement of the jatropha waste from jatropha oil industries would lead to contamination of the environment, affecting living organisms and human health through the food chain, so several methods were tested for reducing the toxicity of the seed cake. The results from this investigation showed that heat treatments at either 120°C or 220°C for 1 hour and then mixing with adsorbing bentonite (10%), nanoparticles of zinc oxide (100 μg/g) plus NaHCO3 at 4%, followed by a 4-week incubation period yielded the best final product. The remaining phorbol esters concentration (0.05-0.04 mg/g) from this treatment was less than that reported for the nontoxic jatropha varieties (0.11-0.27 mg/g). Nutritional values of the seed cake after treatment remained at the same levels found in the control group and these values were crude protein (20.47-21.40 + 0.17-0.25%), crude lipid (14.27-14.68 + 0.13-0.14%) and crude fiber (27.33-29.67 + 0.58%). A cytotoxicity test conducted using L929 and normal human dermal fibroblast cell lines confirmed that most of the toxic compounds, especially phorbol esters, were shown as completely eliminated. The results suggested that the detoxification of phorbol esters residues in the jatropha seed cake was possible while it also retained nutritional values. Therefore, the methods to detoxify phorbol esters are necessary to minimize the toxicity of jatropha seed cake. Further, it is essential to reduce the possible environmental impacts that may be generated

  8. Degradation of phorbol 12,13-diacetate in aqueous solution by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Kongmany, Santi; Furuta, Masakazu; Matsuura, Hiroto; Okuda, Shuichi; Imamura, Kiyoshi; Maeda, Yasuaki

    2014-12-01

    Phorbol esters (PEs) are highly toxic compounds that cause skin irritation, inflammation, and tumor promotion upon contact with humans or animals. These compounds are naturally present in Jatropha curcas L. To promote the use of J. curcas seed oil in bio-diesel production industries and reduce environmental concerns, it is necessary to find methods of degrading PEs. In this study, the degradation of phorbol 12,13-diacetate (PDA), as a representative PE, in aqueous solution at a concentration of 10 mg/L by 60Co-γ-irradiation was investigated. The results demonstrate that PDA was effectively degraded by this treatment and the degradation efficiency increased with the absorbed dose within the range of 0.5-3 kGy. Complete degradation of PDA was achieved at a dose of 3 kGy. In the presence of radical scavengers (i.e., methanol, tert-butanol, 2-propanol), reactive species from water radiolysis were scavenged, and significant inhibition of PDA degradation was observed at absorbed doses less than 1 kGy. In the presence of nitrous oxide, the generation of hydroxyl radicals (rad OH) was promoted during gamma irradiation and PDA degradation was drastically enhanced.

  9. The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium.

    PubMed

    Starr, Tregei; Bauler, Timothy J; Malik-Kale, Preeti; Steele-Mortimer, Olivia

    2018-01-01

    THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. However, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary widely. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The results show that THP-1 macrophages differentiated in high concentrations of PMA rapidly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular bacteria similar to primary human macrophages.

  10. Rapid activation of dormant presynaptic terminals by phorbol esters

    PubMed Central

    Chang, Chun Yun; Jiang, Xiaoping; Moulder, Krista L.; Mennerick, Steven

    2010-01-01

    Presynaptic stimulation stochastically recruits transmission according to the release probability (Pr) of synapses. The majority of central synapses have relatively low Pr, which includes synapses that are completely quiescent presynaptically. The presence of presynaptically dormant versus active terminals presumably increases synaptic malleability when conditions demand synaptic strengthening or weakening, perhaps by triggering second messenger signals. However, whether modulator-mediated potentiation involves recruitment of transmission from dormant terminals remains unclear. Here, by combining electrophysiological and fluorescence imaging approaches, we uncovered rapid presynaptic awakening by select synaptic modulators. A phorbol ester phorbol 12,13-dibutyrate (PDBu, a diacylglycerol analog), but not forskolin (an adenylyl cyclase activator) or elevated extracellular calcium, recruited neurotransmission from presynaptically dormant synapses. This effect was not dependent on protein kinase C activation. After PDBu-induced awakening, these previously dormant terminals had a synaptic Pr spectrum similar to basally active synapses naive to PDBu treatment. Dormant terminals did not seem to have properties of nascent or immature synapses, judged by NR2B N-methyl-D-aspartate receptor (NMDAR) receptor subunit contribution after PDBu-stimulated awakening. Strikingly, synapses rendered inactive by prolonged depolarization, unlike basally dormant synapses, were not awakened by PDBu. These results suggest that the initial release competence of synapses can dictate the acute response to second messenger modulation, and the results suggest multiple pathways to presynaptic dormancy and awakening. PMID:20668189

  11. The Phorbol Ester Fraction from Jatropha curcas Seed Oil: Potential and Limits for Crop Protection against Insect Pests

    PubMed Central

    Ratnadass, Alain; Wink, Michael

    2012-01-01

    The physic nut shrub, Jatropha curcas (Euphorbiaceae), has been considered as a “miracle tree”, particularly as a source of alternate fuel. Various extracts of the plant have been reported to have insecticidal/acaricidal or molluscicidal/anthelminthic activities on vectors of medical or veterinary interest or on agricultural or non-agricultural pests. Among those extracts, the phorbol ester fraction from seed oil has been reported as a promising candidate for use as a plant-derived protectant of a variety of crops, from a range of pre-harvest and post-harvest insect pests. However, such extracts have not been widely used, despite the “boom” in the development of the crop in the tropics during recent years, and societal concerns about overuse of systemic chemical pesticides. There are many potential explanations to such a lack of use of Jatropha insecticidal extracts. On the one hand, the application of extracts potentially harmful to human health on stored food grain, might not be relevant. The problem of decomposition of phorbol esters and other compounds toxic to crop pests in the field needing further evaluation before such extracts can be widely used, may also be a partial explanation. High variability of phorbol ester content and hence of insecticidal activity among physic nut cultivars/ecotypes may be another. Phytotoxicity to crops may be further limitation. Apparent obstacles to a wider application of such extracts are the costs and problems involved with registration and legal approval. On the other hand, more studies should be conducted on molluscicidal activity on slugs and land snails which are major pests of crops, particularly in conservation agriculture systems. Further evaluation of toxicity to natural enemies of insect pests and studies on other beneficial insects such as pollinators are also needed. PMID:23203190

  12. Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells

    SciTech Connect

    Jeng, A.Y.; Lichti, U.; Strickland, J.E.

    1985-11-01

    Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents. Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, the authors have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter. Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerolsmore » in cells prelabeled with (TH)arachidonic acid. Treatment with phospholipase C led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment. Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate. Treatment with phospholipase C yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding.« less

  13. Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

    SciTech Connect

    Emoto, T.; Kasai, K.; Hiraiwa, M.

    1988-01-01

    In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E/sub 1/ or E/sub 2/ (PGE/sub 1/ and PGE/sub 2/) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10/supmore » -9/ M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.« less

  14. Phorbol ester and spontaneous activity in SHR aorta

    SciTech Connect

    Moisey, D.M.; Cox, R.H.

    1986-03-01

    Thoracic aortas (TA) were excised from 6-week old SHR and WKY. 2mm rings were mounted isometrically at optimum preload. Spontaneous rhythmical activity developed in TA from SHR and had a frequency of 3-4/min with varying periods of quiescence between bursts of activity. The spontaneous activity often produced an increase in tension development which was associated with increased frequency of oscillations. Verapamil (10/sup -7/ M) or Ca/sup + +/-free solution added during the contractile phase resulted in an immediate loss of tension and spontaneous activity. Addition of ouabain (10/sup -4/ M) during the contractile phase of spontaneous activity, increased the frequencymore » of oscillations which appeared to fuse into a tetanus. Spontaneous rhythmical activity was infrequently observed in TA from WKY. However, addition of phorbol 12-myristate-13 acetate (TPA), frequently induced spontaneous rhythmic oscillations associated with tension development in TA from WKY. TPA contracted the SHR TA and increased the frequency of oscillations. SHR TA were more sensitive to TPA than WKY. This study demonstrates (1) spontaneous rhythmical activity, independent of agonist stimulation in TA from 6-week old SHR and (2) TPA induced spontaneous oscillatory activity. The mechanism underlying the spontaneous oscillatory activity may involve membrane coupling events and Na-pump difference between SHR and WKY.« less

  15. Isolation, stability and bioactivity of Jatropha curcas phorbol esters.

    PubMed

    Roach, Joy S; Devappa, Rakshit K; Makkar, Harinder P S; Becker, Klaus

    2012-04-01

    Jatropha curcas seed oil, which can be utilized for biodiesel production upon transesterification, is also rich in phorbol esters (PEs). In this study, PEs from J. curcas oil (Jatropha factors C₁ and C₂ (purified to homogeneity), Jatropha factors C₃ and (C₄+C₅) (obtained as mixtures) and PE-rich extract (containing all the above stated Jatropha factors) were investigated. The concentrations of Jatropha PEs were expressed equivalent to Jatropha factor C₁. In the snail (Physa fontinalis) bioassay, the order of potency (EC₅₀, μg/L) was: PE-rich extract

  16. Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment

    PubMed Central

    Zhou, Zhuo Long; Ma, Jing; Tong, Ming-Hui; Chan, Barbara Pui; Wong, Alice Sze Tsai; Ngan, Alfonso Hing Wan

    2016-01-01

    The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell–protein or cell–cell contact was also demonstrated. PMID:27994457

  17. Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment.

    PubMed

    Zhou, Zhuo Long; Ma, Jing; Tong, Ming-Hui; Chan, Barbara Pui; Wong, Alice Sze Tsai; Ngan, Alfonso Hing Wan

    The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell-protein or cell-cell contact was also demonstrated.

  18. Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

    SciTech Connect

    Onali, P.; Olianas, M.C.

    1987-03-23

    In rat striatal synaptosomes, 4..beta..-phorbol 12-myristate 13-acetate (PMA) and 4 ..beta..-phorbol 12,13-dibutyrate (PDBu), two activators of Ca/sup 2 +/-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C) tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 ..mu..M PMA and 1 ..mu..M PDBu. 4 ..beta..-Phorbol and 4 ..beta..-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 ..mu..M. PMA did not change the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C)DOPA. Addition of 1 mM EGTA to a Ca/sup 2 +/-freemore » incubation medium failed to affect PMA stimulation. KCl (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KCl addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis. 37 references, 3 figures, 3 tables.« less

  19. Phorbol ester induces apoptosis in HL-60 promyelocytic leukemia cells but not in HL-60 PET mutant.

    PubMed

    Macfarlane, D E; O'Donnell, P S

    1993-11-01

    One of the factors regulating the population size of a clone of proliferating cells is the induction of a physiological suicide mechanism known as apoptosis. We studied apoptosis in the HL-60 human promyelocytic leukemia cell line which differentiates when exposed to phorbol ester (S-cell), and in the PET-cell mutant of HL-60 which is defective in its response to phorbol ester. Exposing S-cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) (3 nM and above) induced morphological changes characteristic of apoptosis (visualized by light microscopy), and induced fragmentation of chromatin DNA to oligonucleosomal lengths. These changes were obvious in 48 h. In contrast, 1000 nM TPA for five days did not induce apoptosis in the PET-cell. DNA fragmentation was induced in both cell lines by A23187 (0.25 microM) and etoposide (7 microM). Novobiocin (600 and 900 microM) induced DNA fragmentation in S-cells, but higher concentrations inhibited fragmentation. Novobiocin is believed to induce DNA fragmentation by a direct action on DNA. In the case of PET-cells, novobiocin did not induce DNA fragmentation at any concentration, and prior treatment of PET-cells with novobiocin (300-1200 microM for 30 min) inhibited DNA fragmentation induced by A23187. Novobiocin inhibited cell growth equally in S-cell and PET-cells. It is concluded that the promyelocytes have the capacity to undergo apoptosis in response to agents which activate protein kinase C, and that the PET-cell has a mutation which disables both protein-kinase C-induced and novobiocin-induced DNA fragmentation, leaving intact the ability of novobiocin to protect DNA from calcium-entry-initiated fragmentation. The elucidation of the lesion responsible for the PET phenotype is likely to increase our understanding of this important pathway for regulating cellular proliferation and how it bears on leukemogenesis and chemotherapy.

  20. Chronic Li+ attenuates agonist- and phorbol ester-mediated Na+/H+ antiporter activity in HL-60 cells.

    PubMed

    Bitran, J A; Potter, W Z; Manji, H K; Gusovsky, F

    1990-04-25

    The effects of Li+ on signal transduction in dibutyryl cAMP-differentiated HL-60 cells were studied. Upon differentiation, these human promyelocytic leukemia cells express a chemotactic formyl peptide receptor, which is coupled through a guanine nucleotide-binding protein to phospholipase C. Stimulation with fMet-Leu-Phe results in changes in intracellular pH which are thought to be mediated by protein kinase C regulation of Na+/H+ antiporter function. Acute LiCl treatment (10 mM) was without any effect on Na+/H+ activity. However, pretreatment of HL-60 cells with 1 or 10 mM LiCl for at least 5 days resulted in a marked attenuation of fMet-Leu-Phe effects on Na+/H+ activity. In undifferentiated HL-60 cells, which lack fMet-Leu-Phe receptors, intracellular acidification induced by the proton ionophore nigericin generates an alkalinization response. Chronic (but not acute) Li+ treatment also resulted in an inhibition of the nigericin-mediated response. Furthermore, stimulation of the Na+/H+ antiporter by the phorbol ester, phorbol-12-myristate-13-acetate, was also markedly attenuated by chronic LiCl treatment, suggesting an impairment of protein kinase C activity. In contrast, fMet-Leu-Phe-induced increases in intracellular Ca2+ and phospho-inositide breakdown were unchanged in cells treated with Li+ for 5 days. These results indicate that chronic but not acute Li+ treatment alters intracellular pH regulation possibly at a site distal to the fMet-Leu-Phe receptor.

  1. Carbamylcholine and phorbol esters desensitize muscarinic receptors by different mechanisms in rat pancreatic acini.

    PubMed

    Blanchard, L M; Paquette, B; Larose, L; Morisset, J

    1990-01-01

    Pretreatment of rat pancreatic acini with phorbol 12-myristate, 13-acetate (PMA), a protein kinase C (PK-C) activator, caused the desensitization of carbamylcholine (CBC)-induced amylase release in a concentration- and time-dependent fashion. The less potent phorbol-12, 13-dibutyrate (PDBu) also provoked a desensitization, but the inactive 4-alpha-phorbol-12,13-didecanoate had no effect. PMA or PDBu also significantly reduced subsequent amylase release induced by caerulein or secretin in contrast to CBC, which only reduced amylase release induced by CBC or secretin. Preincubation of acini with PMA did not lead to a decrease in PMA or A23187-stimulated amylase release. A 3 h resting period did not restore the desensitization induced by PMA or PDBu. Pretreatment with PMA did not cause changes in muscarinic receptor high- and low-affinity populations as observed with CBC pretreatment. The PK-C inhibitor H-7 completely prevented the desensitization induced by PDBu but not that induced by CBC. TMB-8, another PK-C inhibitor, also completely prevented the desensitization induced by PDBu but only partially that induced by CBC. These results suggest that phorbol esters can induce desensitization of muscarinic receptor-stimulated amylase release by a different mechanism than that involved in muscarinic agonist-induced desensitization.

  2. Low- and high-affinity phorbol ester and diglyceride interactions with protein kinase C: 1-O-alkyl-2-acyl-sn-glycerol enhances phorbol ester- and diacylglycerol-induced activity but alone does not induce activity.

    PubMed

    Slater, S J; Seiz, J L; Stagliano, B A; Cook, A C; Milano, S K; Ho, C; Stubbs, C D

    2001-05-22

    Phorbol ester-induced conventional protein kinase C (PKCalpha, -betaIota/IotaIota, and -gamma) isozyme activities are potentiated by 1,2-diacyl-sn-glycerol. This has been attributed to a "cooperative" interaction of the two activators with two discrete sites termed the low- and high-affinity phorbol ester binding sites, respectively [Slater, S. J., Milano, S. K., Stagliano, B. A., Gergich, K. J., Ho, C., Mazurek, A., Taddeo, F. J., Kelly, M. B., Yeager, M. D., and Stubbs, C. D. (1999) Biochemistry 38, 3804-3815]. Here, we report that the 1-O-alkyl ether diglyceride, 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), like its 1,2-diacyl counterpart, 1-oleoyl-2-acetyl-sn-glycerol (OAG), also potentiated PKCalpha, -betaI/II, and -gamma activities induced by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). Similar to OAG, HAG was found to bind to the low-affinity phorbol ester binding site and to enhance high-affinity phorbol ester binding, and to decrease the level of Ca(2+) required for phorbol ester-induced activity, while being without effect on the Ca(2+) dependence of membrane association. Thus, similar to OAG, HAG may also potentiate phorbol ester-induced activity by interacting with the low-affinity phorbol ester binding site, leading to a reduced level of Ca(2+) required for the activating conformational change. However, HAG was found not to behave like a 1,2-diacyl-sn-glycerol in that alone it did not induce PKC activity, and also in that it enhanced OAG-induced activity. The results reveal HAG to be a member of a new class of "nonactivating" compounds that modulate PKC activity by interacting with the low-affinity phorbol ester binding site.

  3. Degradation of Jatropha curcas phorbol esters derived from Jatropha oil cake and their tumor-promoting activity.

    PubMed

    Nakao, Motoyuki; Hasegawa, Go; Yasuhara, Tadashi; Ishihara, Yoko

    2015-04-01

    Large amount of oil cake is generated during biodiesel production from Jatropha seeds. Although Jatropha oil cake is rich in plant nutrients, presence of toxic phorbol esters restricts the usage of oil cake as a fertilizer. The objective of this study is to evaluate the components and tumor promoting activity of phorbol esters in Jatropha oil cake-supplemented soil and plants grown in the treated soil. Contents and their biological activity of Jatropha phorbol esters in soil and plants were sequentially analyzed by high-performance liquid chromatography (HPLC) and in vitro cell transformation assay, respectively. Disappearance of Jatropha phorbol-ester-specific peaks were followed with HPLC during incubation of Jatropha oil cake with soil for five weeks. Along with the degradation of Jatropha phorbol ester in soil, tumor-promoting activity in the sample was also attenuated and ultimately disappeared. Jatropha phorbol esters and tumor promoting activity were not detected from mustard spinach grown in the Jatropha oil cake-supplemented soil. In addition, the esterase KM109 degrades DHPB (see definition below; Jatropha phorbol ester) and reduced its tumor-promoting activity. From these data, we conclude: (1) components and tumor promoting activity of Jatropha phorbol esters in the oil cake disappeared completely by incubation with soil for five-week, (2) Jatropha phorbol esters did not transfer into plants grown in the Jatropha oil cake-supplemented soil, and (3) DHPB can be degraded by esterase from soil bacterium. These observations are useful for utilization of Jatropha oil cake as a fertilizer. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding.

    PubMed

    Ikin, Annat F; Causevic, Mirsada; Pedrini, Steve; Benson, Lyndsey S; Buxbaum, Joseph D; Suzuki, Toshiharu; Lovestone, Simon; Higashiyama, Shigeki; Mustelin, Tomas; Burgoyne, Robert D; Gandy, Sam

    2007-12-09

    Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as alpha-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Rossner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPalpha. Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.

  5. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

    PubMed Central

    Ikin, Annat F; Causevic, Mirsada; Pedrini, Steve; Benson, Lyndsey S; Buxbaum, Joseph D; Suzuki, Toshiharu; Lovestone, Simon; Higashiyama, Shigeki; Mustelin, Tomas; Burgoyne, Robert D; Gandy, Sam

    2007-01-01

    Background Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as α-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Results Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Roßner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPα. Conclusion Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP. PMID:18067682

  6. Hydrocortisone inhibits phorbol ester stimulated release of histamine and arachidonic acid from rat mast cells.

    PubMed

    Heiman, A S; Crews, F T

    1985-07-31

    Purified rat peritoneal mast cells were incubated overnight with or without hydrocortisone (3 X 10(-6) M) and then stimulated with anti-IgE, somatostatin or a phorbol ester-ionophore combination, i.e., 12-O-tetradecanoyl-phorbol-13-acetate and A23187. The release of both histamine and [1-14C]arachidonic acid and its metabolites was determined. Hydrocortisone treatment markedly inhibited both anti-IgE and TPA-A23187 stimulated release, but not release stimulated by somatostatin. These results suggest that anti-inflammatory steroids may alter histamine release through an action involving the activation of the phosphatidylserine-calcium dependent protein kinase or its substrates.

  7. Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

    PubMed

    Pany, Satyabrata; Ghosh, Anamitra; You, Youngki; Nguyen, Nga; Das, Joydip

    2017-11-01

    Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca 2+ -sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca 2+ -sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1 H567K , a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis.

    PubMed

    Li, Chunhong; Ng, Ailing; Xie, Lifen; Mao, Huizhu; Qiu, Chengxiang; Srinivasan, Ramachandran; Yin, Zhongchao; Hong, Yan

    2016-01-01

    Casbene is a precursor to phorbol esters and down-regulating casbene synthase effectively reduces phorbol ester biosynthesis. Seed-specific reduction of phorbol ester (PE) helps develop Jatropha seed cake for animal nutrition. Phorbol esters (PEs) are diterpenoids present in some Euphorbiaceae family members like Jatropha curcas L. (Jatropha), a tropical shrub yielding high-quality oil suitable as feedstock for biodiesel and bio jet fuel. Jatropha seed contains up to 40 % of oil and can produce oil together with cake containing high-quality proteins. However, skin-irritating and cancer-promoting PEs make Jatropha cake meal unsuitable for animal nutrition and also raise some safety and environmental concerns on its planting and processing. Two casbene synthase gene (JcCASA163 and JcCASD168) homologues were cloned from Jatropha genome and both genes were highly expressed during seed development. In vitro functional analysis proved casbene synthase activity of JcCASA163 in converting geranylgeranyl diphosphate into casbene which has been speculated to be the precursor to PEs. A seed-specific promoter driving inverted repeats for RNAi interference targeting at either JcCASA163 or both genes could effectively down-regulate casbene synthase gene expression with concurrent marked reduction of PE level (by as much as 85 %) in seeds with no pleiotropic effects observed. Such engineered low PE in seed was heritable and co-segregated with the transgene. Our work implicated casbene synthase in Jatropha PE biosynthesis and provided evidence for casbene being the precursor for PEs. The success in reducing seed PE content through down-regulation of casbene synthase demonstrates the feasibility of intercepting PE biosynthesis in Jatropha seed to help address safety concerns on Jatropha plantation and seed processing and facilitate use of its seed protein for animal nutrition.

  9. Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region.

    PubMed Central

    Chakravarty, R; Rong, X H; Rice, R H

    1990-01-01

    The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation. Images Fig. 1. Fig. 3. PMID:1977383

  10. Insulin reverses the growth retardation effect of phorbol ester in chicken embryos during organogenesis.

    PubMed

    Girbau, M; Bassas, L; Roth, J; de Pablo, F

    1989-01-01

    The tumor promoting phorbol esters can affect early embryonic development by causing interference with the normal pathways of cellular growth and differentiation. The present study was designed to: a) define a time in organogenesis when a vertebrate embryo model, the chicken, was sensitive to the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA), and b) attempt a rescue of the embryos disturbed by TPA with simultaneous addition of insulin. In embryos treated at days 2 and 3 of development, TPA caused dose-dependent mortality. Survivors were biochemically retarded as indicated by their decreased weight, protein, DNA, RNA, total creatine kinase, triglycerides, phospholipids and cholesterol contents. When intermediate doses of TPA (50 ng/embryo) were applied together with insulin (100 ng/embryo), the embryonic growth disturbance was largely antagonized. These data, generated with an in vivo whole embryo, These data, generated with an in vivo whole embryo, support the strong link between the mode of action of insulin and signal transduction mechanisms typical of phorbol esters.

  11. Differential role of protein kinase C in desensitization of muscarinic receptor induced by phorbol esters and receptor agonists

    SciTech Connect

    Lai, Wi Sheung.

    1989-01-01

    PKC, a phorbol ester receptor, copurified with specific binding sites of ({sup 3}H)phorbol-12,13,-dibutyrate (({sup 3}H)PDBu). The specific binding of ({sup 3}H)PDBu to intact cells was saturable to a single class of binding sites. The PKC and phorbol ester receptors in N1E-115 cells can be down regulated by prolonged phorbol ester incubation. Phorbol 12-myristate 13-acetate (PMA) suppressed muscarinic receptor-mediated cyclic GMP response in a time-dependent and a concentration-dependent fashion and the suppressive effect of PMA could be attenuated by a protein kinase inhibitor, H-7, as well as by down-regulation of the PKC through long-term incubation with PDBu. Exposure of the cellsmore » to the muscarinic agonist carbamylcholine also desensitized subsequent CBC-mediated cyclic GMP response. However, pretreatment with carbamylcholine did not desensitize histamine-induced cyclic GMP formation while treatment with PMA suppressed this histamine-mediated response. Preincubation of the cells with CBC, but not with phorbol ester, resulted in down-regulation of muscarinic receptors. The loss of muscarinic receptors induced by agonist even occurred when the phosphoinositide hydrolysis response was suppressed.« less

  12. Decrease of epidermal histidase activity by tumor-promoting phorbol esters.

    PubMed

    Colburn, N H; Lau, S; Head, R

    1975-11-01

    The potent skin tumor promoter (12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulates epidermal macromolecular synthesis as well as proliferation, but little is known of specific functional aberrations produced by TPA. This report presents results of a study on the effects of TPA on epidermal histidase (L-histidine ammonia lyase), an enzyme found in normal epidermis but not in dermis or in mouse squamous cell carcinomas. Histidase activity was assayed on postmitochondrial supernatants obtained from hairless mouse epidermis after removal by keratotome. Topical TPA treatment at doses active in tumor promotion (1.7 to 17.0 nmoles/application) produced dose-dependent decreases in epidermal histidase specific activity at 19 hr posttreatment. The onset of the decrease occurred at 12 hr with recovery to control level specific activity by 5 days, showing kinetics similar to those obtained for stimulation of DNA synthesis. This decrease in histidase could not be attributed to a general inhibition of soluble protein synthesis or to the appearance of an inhibitor of histidase activity. The strong promoter TPA produced a greater histidase decrease than did the moderate promoter and mitogen 12,13-didecanoyl phorbol at equimolar dose, while phorbol, a nonpromoter and nonmitogen, produced no effects on histidase. The relationship of this histidase depression to tumor promotion and not initiation is further indicated by the finding that (a) Tween 60, a structurally unrelated tumor promotor, also produced a decrease in histidase; and (b) the tumor initiator urethan and an initiating dose of 9,10-dimethybenz(a)anthracene showed no effects on histadase activity.

  13. Pleiotropic Effects of Chronic Phorbol Ester Treatment to Improve Glucose Transport in Insulin-Resistant Cardiomyocytes.

    PubMed

    Viglino, Christelle; Khoramdin, Bahareh; Praplan, Guillaume; Montessuit, Christophe

    2017-12-01

    Stimulation of glucose transport is an important determinant of myocardial susceptibility to ischemia and reperfusion. Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA). Deactivation of the Focal Adhesion Kinase (FAK) by FFA contributes to glucose transport impairment, and could be corrected by chronic treatment with the phorbol ester TPA. However, TPA must have effects in addition to FAK reactivation to restore stimulated glucose transport. Chronic treatment with TPA improved basal and stimulated glucose transport in FFA-exposed, but not in control cardiomyocytes. Chronic FFA exposure induced the activation of PKCδ and PKCϵ. TPA markedly downregulated the expression of PKCα, PKCδ, and PKCϵ, suggesting that PKCδ or PKCϵ activation could contribute to inhibition of glucose transport by FFA. Rottlerin, a specific PKCδ inhibitor, improved glucose transport in FFA-exposed cardiomyocytes; and PKCδ was reduced in the particulate fraction of FFA + TPA-exposed cardiomyocytes. TPA also activated Protein Kinase D 1(PKD1) in FFA-exposed cardiomyocytes, as assessed by autophosphorylation of PKD1 on Y916. Pharmaceutical inhibition of PKD1 only partially prevented the improvement of glucose transport by TPA. Chronic TPA treatment also increased basal and stimulated glycolysis and favored accumulation of lipid droplets in FFA-exposed cardiomyocytes. In conclusion, basal and stimulated glucose transport in cardiomyocytes is reduced by chronic FFA exposure, but restored by concomitant treatment with a phorbol ester. The mechanism of action of phorbol esters may involve downregulation of PKCδ, activation of PKD1 and a general switch from fatty acid to glucose metabolism. J. Cell. Biochem. 9999: 4716-4727, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. Induction of rat hepatic zinc thionein by phorbol ester-mediated protein kinase C pathway

    SciTech Connect

    Garrett, S.H.; Funk, A.E.; Brady, F.O.

    1986-05-01

    Metallothionein (MT) exists in rat liver mainly as a zinc protein. The levels of this protein fluctuate in response to a variety of internal and external stimuli. Among these inducers of MT are metals, glucocorticoids, catecholamines, and polypeptide hormones. Metals and glucocorticoids are primary inducers of MT, while the others operate either via adenylate cyclase/cAMP/cAMP-dependent protein kinase, or via phospholipase C/inositol 1,4,5-triphosphate, diacylglycerol/Ca/sup 2 +/-dependent protein kinase, protein kinase C. The authors have examined the role of the protein kinase C pathway in the induction of MT by using a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), to activate it. In vivomore » TPA is a good inducer of Zn/sub 7/-MT with an ED/sub 0.5/ of 26.5 nmoles/kg b.w. Maximal levels reached were about 7..mu..g Zn in MT/g liver, an induction increase of 8 to 10-fold. An inactive compound, 4..beta..-phorbol, and the vehicle (DMSO) did not stimulate the synthesis of Zn/sub 7/-MT. This induction by TPA requires de novo protein synthesis, as demonstrated by a cycloheximide/(/sup 35/S)-cysteine experiment. TPA stimulated Zn incorporation by 8.6-fold and (/sup 35/S)-cysteine incorporation by 4.8-fold during an 11h induction. These increases were blocked 100% by treatment with cycloheximide at -1 and +5h. These experiments have been repeated in cultured hepatocytes, using (/sup 35/S)-cysteine incorporation, slab SDS-PAGE, and autoradiography to quantitate MT levels.« less

  15. Phorbol myristate acetate stimulates RNA and casein synthesis in cultured mouse mammary gland tissues

    SciTech Connect

    Rillema, J.A.; Waters, S.B.

    1986-05-01

    Prolactin and phorbol myristate acetate (TPA) stimulate the rate of (/sup 3/H)uridine incorporation in cultured mouse mammary gland explants in a similar fashion. Both the time-courses and magnitude of responses were the same; in addition, maximum stimulatory concentrations of TPA and prolactin elicited a nonadditive response when tested together. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, abolished both the TPA and prolactin effects on (/sup 3/H)uridine incorporation. TPA also effected an enhanced rate of (/sup 3/H)leucine incorporation into a casein-rich phosphoprotein fraction, but only if the explants were also cultured with spermidine.

  16. Modulation of luteinizing hormone-stimulated inositol phosphate accumulation by phorbol esters in bovine luteal cells.

    PubMed

    Davis, J S

    1992-08-01

    The present studies were conducted to evaluate the effects of protein kinase C activators on the inositol phospholipid-phospholipase C second messenger system in isolated bovine luteal cells. This report describes the effects of phorbol esters on inositol phosphate accumulation in LH- and prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea of early pregnancy were dispersed with collagenase and luteal cells were prelabelled for 3 h with [3H]inositol. Inositol phosphates produced in response to LH or PGF2 alpha were analyzed by ion exchange column chromatography. The tumor promoter and protein kinase C activator 12-O-tetradecanolyphorbol 13-acetate (TPA) had no effect on basal levels of inositol phosphates but inhibited LH-stimulated accumulation of inositol mono-, bis-, and trisphosphates by 72%, 68%, and 65%, respectively. TPA reduced the response to maximally effective concentrations of LH and tripled the concentrations of LH required to evoke half-maximal accumulation of inositol mono-, bis-, trisphosphates. The inhibitory effects of TPA were rapid (5 min) whether added before or after treatment with LH. Treatment with TPA also reduced (58%) the initial phase of intracellular calcium mobilization in LH-treated cells. The inhibitory effects of TPA were not associated with acute reductions in [3H]inositol incorporation, [3H]inositol phospholipid levels, cAMP levels, or progesterone accumulation in control or LH-stimulated luteal cells. The effects of phorbol esters were concentration dependent and specific for active tumor promoters with 10-50 nM TPA producing maximal inhibitory effects. A synthetic diacylglycerol, 1-oleyl-2-acetylglycerol, mimicked the inhibitory effects of TPA. In contrast, pretreatment with a physiological activator of protein kinase C, PGF2 alpha, had no effect on LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA could not be explained by a generalized inhibition of phospholipase C or G

  17. Selective binding of phorbol esters and diacylglycerol by individual C1 domains of the PKD family.

    PubMed

    Chen, Jun; Deng, Fan; Li, Jun; Wang, Q Jane

    2008-04-15

    The PKD (protein kinase D) family are novel DAG (diacylglycerol) receptors. The twin C1 domains of PKD, designated C1a and C1b, have been shown to bind DAG or phorbol esters. However, their ligand-binding activities and selectivities have not been fully characterized. Here, binding activities of isolated C1a, C1b and intact C1a-C1b domains to DAG and phorbol esters were analysed. The isolated C1b domains of PKD isoforms bind [(3)H]PDBu ([20-(3)H]phorbol 12, 13-dibutyrate) with similar high affinities, while they exhibit weaker affinities towards a synthetic DAG analogue, DOG (1,2-dioctanoyl-sn-glycerol), as compared to the control. Mutating a conserved lysine residue at position 22 to tryptophan in C1b of PKD3 fully restores its affinity to DOG, indicating that this residue accounts for its weaker affinity to DOG. In contrast, the non-consensus residues in the isolated C1a domain of PKD mainly contribute to maintaining the protein's structural fold, since converting these residues in C1a of PKD3 to those in PKD1 or PKD2 drastically reduces the maximal number of active receptors, while only minimally impacting ligand-binding activities. Moreover, ligand-binding activities of C1a and C1b are sensitive to the structural context in an intact C1a-C1b domain and exhibit unique patterns of ligand selectivity. C1a and C1b in the intact C1a-C1b of PKD1 are opposite in selectivity for PDBu and DOG. In contrast, C1a of PKD3 exhibits 48-fold higher affinity to DOG as compared to C1b, although both domains bind PDBu with equivalent affinities. Accordingly, mutating C1a of a full-length PKD3-GFP greatly reduces DOG-induced plasma membrane translocation, but does not affect that induced by PMA. In summary, individual C1 domains of PKD isoforms differ in ligand-binding activity and selectivity, implying isoform-selective regulation of PKD by phorbol esters and DAG.

  18. Luminol-dependent photoemission from single neutrophil stimulated by phorbol ester and calcium ionophore--role of degranulation and myeloperoxidase

    SciTech Connect

    Suematsu, M.; Oshio, C.; Miura, S.

    1988-08-30

    Luminol-dependent photonic burst from phorbol ester-treated single neutrophil was visually investigated by using an ultrasensitive photonic image intensifier microscope. Neutrophils stimulated by phorbol myristate acetate (0.1 microgram/ml) alone produced a negligible level of photonic activities in the presence of luminol (10 micrograms/ml). The additional application of 0.1 microM Ca2+ ionophore A23187 induced explosive changes of photonic burst corresponding to the distribution of neutrophils, and these photonic activities were gradually spread to extracellular space. Sodium azide, which prevents myeloperoxidase activity, inhibited Ca2+ ionophore-induced photonic burst from phorbol ester-treated neutrophil. These findings suggest a prerequisite role of degranulation and myeloperoxidase release inmore » luminol-dependent photoemission from stimulated neutrophils.« less

  19. Active Oxygen Metabolites and Thromboxane in Phorbol Myristate Acetate Toxicity to the Isolated, Perfused Rat Lung.

    NASA Astrophysics Data System (ADS)

    Carpenter, Laurie Jean

    When administered intravenously or intratracheally to rats, rabbits and sheep, phorbol myristate acetate (PMA) produces changes in lung morphology and function are similar to those seen in humans with the adult respiratory distress syndrome (ARDS). Therefore, it is thought that information about the mechanism of ARDS development can be gained from experiments using PMA-treated animals. Currently, the mechanisms by which PMA causes pneumotoxicity are unknown. Results from other studies in rabbits and in isolated, perfused rabbit lungs suggest that PMA-induced lung injury is mediated by active oxygen species from neutrophils (PMN), whereas studies in sheep and rats suggest that PMN are not required for the toxic response. The role of PMN, active oxygen metabolites and thromboxane (TxA_2) in PMA-induced injury to isolated, perfused rat lungs (IPLs) was examined in this thesis. To determine whether PMN were required for PMA to produce toxicity to the IPL, lungs were perfused for 30 min with buffer containing various concentrations of PMA (in the presence or absence of PMN). When concentrations >=q57 ng/ml were added to medium devoid of added PMN, perfusion pressure and lung weight increased. When a concentration of PMA (14-28 ng/ml) that did not by itself cause lungs to accumulate fluid was added to the perfusion medium containing PMN (1 x 10 ^8), perfusion pressure increased, and lungs accumulated fluid. These results indicate that high concentrations of PMA produce lung injury which is independent of PMN, whereas injury induced by lower concentrations is PMN-dependent. To examine whether active oxygen species were involved in mediating lung injury induced by PMA and PMN, lungs were coperfused with the oxygen radical scavengers SOD and/or catalase. Coperfusion with either or both of these enzymes totally protected lungs against injury caused by PMN and PMA. These results suggest that active oxygen species (the hydroxyl radical in particular), mediate lung injury in

  20. Protein kinase Calpha contains two activator binding sites that bind phorbol esters and diacylglycerols with opposite affinities.

    PubMed

    Slater, S J; Ho, C; Kelly, M B; Larkin, J D; Taddeo, F J; Yeager, M D; Stubbs, C D

    1996-03-01

    Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160-17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC alpha. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKCalpha contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively.

  1. Down-modulation of receptors for phorbol ester tumor promoter in primary epidermal cells

    SciTech Connect

    Solanki, V.; Slaga, T.J.

    1982-01-01

    The specific (20-/sup 3/H)phorbol 12,13-dibutyrate ((/sup 3/H)PDBu) binding to intact epidermal cells displayed the phenomenon of down-modulation, i.e., the specific binding of (/sup 3/H)PDBu to its receptors on primary epidermal cells reached a maximum within 1 h and steadily declined thereafter. The apparent down-modulation of radiolabel resulted from a partial loss in the total number of receptors; the affinity of receptors for the ligand was essentially unchanged. A number of agents such as chloroquine, methylamine, or arginine which are known to prevent clustering, down-modulation, and/or internalization of several hormone receptors did not affect the down-modulation of phorbol ester receptors. Furthermore,more » cycloheximide had no effect either on down-modulation or on the binding capacity of cells. The surface binding capacity of down-modulated cells following a 90-min incubation with unlabeled ligand was almost returned to normal within 1 h. The effect of the antidepressant drug chlorpromazine, which is known to interact with calmodulin, on (/sup 3/H)PDBu binding was also investigated. Our data indicate that the effect of chlorpromazine on (/sup 3/H)PDBu binding is probably unrelated to its calmodulin-binding activity.« less

  2. [Effect of local anesthetics and phorbol ester on intracellular pH and rate of development of sea urchin embryos].

    PubMed

    Bozhkova, V P; Sharova, L V; Petriaevskaia, V B; Kozlov, D A; Khrust, Iu R

    1988-01-01

    The unfertilized eggs of sand-dollars (Scaphechinus mirabilis) were treated with local anesthetics (novocaine, lidocaine, trimecaine) or phorbol ester for 10 min. The treatment with the local anesthetics increased pH of the cytoplasm in the unfertilized eggs (pHc) and changed the value of the pHc increment in response to fertilization, as compared with the control eggs. In the treated embryos, the onset of immigration of the primary mesenchyme cells was delayed but the hatching was accelerated. The phorbol ester treatment rapidly accelerated development from gastrulation on and practically did not affect the preceding stages.

  3. Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α1A-adrenergic receptors.

    PubMed

    Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Romero-Ávila, M Teresa; Alfonzo-Méndez, Marco A; Pupo, André S; García-Sáinz, J Adolfo

    2017-12-01

    In LNCaP cells that stably express α 1A -adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α 1A -Adrenergic receptor interaction with β-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α 1A -adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α 1A -AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Phorbol esters seed content and distribution in Latin American provenances of Jatropha curcas L.: potential for biopesticide, food and feed.

    PubMed

    Bueso, Francisco; Sosa, Italo; Chun, Roldan; Pineda, Renan

    2016-01-01

    Jatropha curcas L. (Jatropha) is believed to have originated from Mexico and Central America. So far, characterization efforts have focused on Asia, Africa and Mexico. Non-toxic, low phorbol ester (PE) varieties have been found only in Mexico. Differences in PE content in seeds and its structural components, crude oil and cake from Jatropha provenances cultivated in Central and South America were evaluated. Seeds were dehulled, and kernels were separated into tegmen, cotyledons and embryo for PE quantitation by RP-HPLC. Crude oil and cake PE content was also measured. No phenotypic departures in seed size and structure were observed among Jatropha cultivated in Central and South America compared to provenances from Mexico, Asia and Africa. Cotyledons comprised 96.2-97.5 %, tegmen 1.6-2.4 % and embryo represented 0.9-1.4 % of dehulled kernel. Total PE content of all nine provenances categorized them as toxic. Significant differences in kernel PE content were observed among provenances from Mexico, Central and South America (P < 0.01), being Mexican the highest (7.6 mg/g) and Cabo Verde the lowest (2.57 mg/g). All accessions had >95 % of PEs concentrated in cotyledons, 0.5-3 % in the tegmen and 0.5-1 % in the embryo. Over 60 % of total PE in dehulled kernels accumulated in the crude oil, while 35-40 % remained in the cake after extraction. Low phenotypic variability in seed physical, structural traits and PE content was observed among provenances from Latin America. Very high-PE provenances with potential as biopesticide were found in Central America. No PE-free, edible Jatropha was found among provenances currently cultivated in Central America and Brazil that could be used for human consumption and feedstock. Furthermore, dehulled kernel structural parts as well as its crude oil and cake contained toxic PE levels.

  5. Inhibition of membrane lipid-independent protein kinase Calpha activity by phorbol esters, diacylglycerols, and bryostatin-1.

    PubMed

    Slater, S J; Taddeo, F J; Mazurek, A; Stagliano, B A; Milano, S K; Kelly, M B; Ho, C; Stubbs, C D

    1998-09-04

    The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKCalpha, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKCalpha also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKCalpha were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this "non-membrane" PKC activity was inhibited by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCalpha. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4alpha-TPA (4alpha-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4alpha-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4alpha-TPA also

  6. Screening for toxic phorbol esters in jerky pet treat products using LC–MS

    PubMed Central

    Nishshanka, Upul; Jayasuriya, Hiranthi; Chattopadhaya, Chaitali; Kijak, Philip J.; Chu, Pak-Sin; Reimschuessel, Renate; Tkachenko, Andriy; Ceric, Olgica; De Alwis, Hemakanthi G.

    2016-01-01

    Since 2007, the U.S. FDA’s Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats. Jerky used in pet treats contains glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Because some biodiesel is produced using oil from Jatropha curcas, a plant that contains toxic compounds including phorbol esters, CVM developed a liquid chromatography-mass spectrometry (LC–MS) screening method to evaluate investigational jerky samples for the presence of these toxins. Results indicated that the samples analyzed with the new method did not contain Jatropha toxins at or above the lowest concentration tested. PMID:27038400

  7. Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil.

    PubMed

    Pelletier, Guillaume; Padhi, Bhaja K; Hawari, Jalal; Sunahara, Geoffrey I; Poon, Raymond

    2015-06-01

    New health safety concerns may arise from the increasing production and use of Jatropha oil, a biodiesel feedstock that also contains toxic, pro-inflammatory, and co-carcinogenic phorbol esters. Based on the exceptional sensitivity of Madin-Darby canine kidney (MDCK) cells to the model phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a robust bioassay was developed to quantify the biological activity of Jatropha phorbol esters directly in oil, without sample extraction. We first verified that the characteristic response of MDCK cells to TPA was also observed following direct exposure to phorbol esters in Jatropha oil. We further confirmed that similarly to TPA, Jatropha oil's phorbol esters can activate protein kinase C (PKC). We then assessed the transcriptional response of MDCK cells to Jatropha oil exposure by measuring the expression of cyclooxygenase-2 (COX-2), a gene involved in inflammatory processes which is strongly upregulated following PKC activation. Based on the parameterization of a TPA dose-response curve, the transcriptional response of MDCK cells to Jatropha oil exposure was expressed in term of TPA toxic equivalent (TEQ), a convenient metric to report the inflammatory potential of complex mixtures. The sensitive bioassay described in this manuscript may prove useful for risk assessment, as it provides a quantitative method and a convenient metric to report the inflammatory potential of phorbol esters in Jatropha oil. This bioassay may also be adapted for the detection of bioactive phorbol esters in other matrices.

  8. Curcumin does not alter the phorbol ester effect on cell-cell transfer of lucifer yellow CH.

    PubMed

    Pásti, G; Kertai, P; Adány, R

    1995-05-01

    Curcumin, the dietary pigment responsible for the yellow color of curry, has been reported to be a potent inhibitor of tumor promotion in mouse epidermis. Since most tumor promoters inhibit cell-cell communication, we have examined the effect of curcumin on the reduction of gap junctional intercellular communication induced by the phorbol ester phorbol-12,13-dibutyrate (PDBu) in BALB/c 3T3 cells. Treatment of cells with 50 microM curcumin slightly inhibited the dye coupling evaluated by intercellular transfer of a fluorescent dye Lucifer Yellow CH; however, lower concentrations of curcumin did not affect the level of intercellular communication. Addition of 200 nM PDBu caused a rapid reduction of dye coupling, which was not altered by either pretreatment or simultaneous curcumin addition.

  9. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity*

    PubMed Central

    Czikora, Agnes; Lundberg, Daniel J.; Abramovitz, Adelle; Lewin, Nancy E.; Kedei, Noemi; Peach, Megan L.; Zhou, Xiaoling; Merritt, Raymond C.; Craft, Elizabeth A.; Braun, Derek C.; Blumberg, Peter M.

    2016-01-01

    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [3H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn7, Ser8, Ala19, and Ile21 with the corresponding residues from RasGRP1/3 (Thr7, Tyr8, Gly19, and Leu21, respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [3H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

  10. Daphnane and Phorbol Diterpenes, Anti-neuroinflammatory Compounds with Nurr1 Activation from the Roots and Stems of Daphne genkwa.

    PubMed

    Han, Baek-Soo; Minh, Nguyen Van; Choi, Ha-Young; Byun, Jung-Su; Kim, Won-Gon

    2017-01-01

    The methanol extract of the roots and stems of Daphne genkwa and its constituents yuanhuacin (1) and genkwanine N were previously reported to have Nurr1 activating effects and neuroprotective effects in an animal model of Parkinson's disease (PD). In this study, four more daphnane-type diterpenes (acutilonine F (2), wikstroemia factor M 1 (3), yuanhuadine (5), and yuanhuatine (6)) and two phorbol-type diterpenes (prostratin Q (4) and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (7)) were isolated as Nurr1 activating compounds from the D. genkwa extract. Consistent with their higher Nurr1 activating activity, compounds 1, 4, 5, and 7 exhibited higher inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial BV-2 cells with an IC 50 (µM) of 1-2, which was 15-30 times more potent than that of minocycline (29.9 µM), a well-known anti-neuroinflammatory agent. Additionally, these diterpenes reduced expression and transcription of LPS-induced pro-inflammatory cytokines in BV-2 cells. Thus, the daphnane-type and phorbol-type diterpenes had anti-neuroinflammatory activity with Nurr1 activation and could be responsible for the anti-PD effect of the roots and stems of D. genkwa.

  11. Rapid isolation and purification of phorbol esters from Jatropha curcas by high-speed countercurrent chromatography.

    PubMed

    Hua, Wan; Hu, Huiling; Chen, Fang; Tang, Lin; Peng, Tong; Wang, Zhanguo

    2015-03-18

    In this work, a high-speed countercurrent chromatography (HSCCC) method was established for the preparation of phorbol esters (PEs) from Jatropha curcas. n-Hexane-ethyl acetate-methanol-water (1.5:1.5:1.2:0.5, v/v) was selected as the optimum two-phase solvent system to separate and purify jatropha factor C1 (JC1) with a purity of 85.2%, as determined by HPLC, and to obtain a mixture containing four or five PEs. Subsequently, continuous semipreparative HPLC was applied to further purify JC1 (99.8% as determined by HPLC). In addition, UPLC-PDA and UPLC-MS were established and successfully used to evaluate the isolated JC1 and PE-rich crude extract. The purity of JC1 was only 87.8% by UPLC-UV. A peak (a compound highly similar to JC1) was indentified as the isomer of JC1 by comparing the characteristic UV absorption and MS spectra. Meanwhile, this strategy was also applied to analyze the PE-rich crude extract from J. curcas. It is interesting that there may be more than 15 PEs according to the same quasi-molecular ion peaks, highly similar sequence-specific fragment ions, and similar UV absorption spectrum.

  12. Potentiation of phorbol ester-induced coronary vasoconstriction in dogs following endothelium disruption

    SciTech Connect

    Roberts, R.B.; Ku, D.D.

    1986-03-05

    In the present study, the effect of phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA), activation of protein kinase C on coronary vascular reactivity was studied in isolated dog coronary arteries. Addition of TPA (10-100 nM) produced a slow, time- and dose-dependent contraction reaching a maximum at approx 2-3 hrs and was essentially irreversible upon washing. Disruption of the endothelium(EC) greatly accelerated the development as well as increase the magnitude of TPA contraction (50-100%). Prior treatment of vessels with phentolamine (1..mu..M), cyproheptadine (1..mu..H) and ibuprofen (1..mu..g/ml) did not alter the TPA contraction. Furthermore, in contrast to previously reported calcium-dependence of TPA contraction inmore » other vessels, complete removal of extracellular calcium (Ca/sub 0/) or addition of 1..mu..M nimodipine after TPA(30nM) resulted in only 32 +/- 4% and 25 +/- 3% reversal of TPA contraction, respectively. Addition of amiloride (10..mu..M to 1mM), however, resulted in a dose-dependent reversal of TPA contraction. The results of the present study indicate that a similar activation of protein kinase C by TPA leads to potent coronary vasoconstriction, which is not completely dependent on Ca/sub 0/. More importantly, these results further support their hypothesis that EC also functions as an inhibitory barrier to prevent circulating vasoconstrictors from exerting their deleterious constrictory effects.« less

  13. Effects of PMA (PHORBOL-12-MYRISTATE-13-ACETATE) on the Developing Rodent Brain.

    PubMed

    Dzietko, Mark; Hahnemann, Maria; Polley, Oliver; Sifringer, Marco; Felderhoff-Mueser, Ursula; Bührer, Christoph

    2015-01-01

    Perinatal infections have a negative impact on brain development. However, the underlying mechanisms leading to neurological impairment are not completely understood and reliable models of inflammation are urgently needed. Using phorbol-myristate-acetate as an activator of inflammation, we investigated the effect on the developing rodent brain. Neonatal rats and mice deficient in IL-18 or IRAK-4 were exposed to PMA. Brains were assessed for regulation of pro- and anti-inflammatory cytokines and cell death 24 hrs, 7 and 14 days after treatment. PMA induced an inflammatory response and caused widespread neurodegeneration in the brains of 3- and 7-day-old rats. In contrast, 14-day-old rats were resistant to the neurotoxic effect of PMA. Histological evaluation at the age of 14 and 21 days revealed a destruction of the cortical microstructure with decreased numerical density of neuronal cells. Mice deficient in IL-18 or IRAK-4 were protected against PMA induced brain injury. PMA treatment during a vulnerable period can alter brain development. IL-18 and IRAK-4 appear to be important for the development of PMA induced injury.

  14. Protection against apoptosis in chicken bursa and thymus cells by phorbol ester in vitro

    SciTech Connect

    Asakawa, J.; Thorbecke, G.J.

    1991-03-15

    Programmed suicide or apoptosis, due to activation of endogenous nucleases, occurs in immature CD4{sup {minus}}85{sup {minus}} mammalian thymus cells. Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study the authors have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hrs with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 basemore » pairs, indicative of apoptosis, was observed with both bursa and thymus cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristic acetate during culture and, more effectively, by PMA plus ionomycin, but not by ionomycin alone or by anti-{mu}. PMA did not affect the patterns of DNA fragmentation seen with spleen cells. Addition of the protein kinase C inhibitor staurosporin inhibited the preventive effect of PMA on apoptosis. PMA also greatly promoted the survival of bursa cells in culture, as assayed by percentage cell death and by {sup 3}H-thymidine incorporation. It is concluded that bursa and thymus cells from the chicken exhibit apoptosis. The data further suggest that protein kinase C activation protects apoptosis in cultured bursa cells.« less

  15. Silver nanoparticles impede phorbol myristate acetate-induced monocyte-macrophage differentiation and autophagy

    NASA Astrophysics Data System (ADS)

    Xu, Yingying; Wang, Liming; Bai, Ru; Zhang, Tianlu; Chen, Chunying

    2015-09-01

    Monocytes/macrophages are important constituents of the innate immune system. Monocyte-macrophage differentiation is not only crucial for innate immune responses, but is also related to some cardiovascular diseases. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials because of their broad-spectrum antimicrobial properties. However, the effect of AgNPs on the functions of blood monocytes is scarcely reported. Here, we report the impedance effect of AgNPs on THP-1 monocyte differentiation, and that this effect was mediated by autophagy blockade and lysosomal impairment. Firstly, AgNPs inhibit phorbol 12-myristate 13-acetate (PMA)-induced monocyte differentiation by down-regulating both expression of surface marker CD11b and response to lipopolysaccharide (LPS) stimulation. Secondly, autophagy is activated during PMA-induced THP-1 monocyte differentiation, and the autophagy inhibitor chloroquine (CQ) can inhibit this process. Thirdly, AgNPs block the degradation of the autophagy substrate p62 and induce autophagosome accumulation, which demonstrates the blockade of autophagic flux. Fourthly, lysosomal impairments including alkalization and decrease of lysosomal membrane stability were observed in AgNP-treated THP-1 cells. In conclusion, we demonstrate that the impedance of monocyte-macrophage differentiation by AgNPs is mediated by autophagy blockade and lysosomal dysfunction. Our results suggest that crosstalk exists in different biological effects induced by AgNPs.

  16. Effect of Dark Chocolate Extracts on Phorbol 12-Myristate 13-Acetate-Induced Oxidative Burst in Leukocytes Isolated by Normo-Weight and Overweight/Obese Subjects.

    PubMed

    Ioannone, Francesca; Sacchetti, Giampiero; Serafini, Mauro

    2017-01-01

    Oxidative and inflammatory stress represents a major risk factor for cardiovascular disease (CVD) in overweight and obese subjects. Between the different plant foods, chocolate has been shown to decrease CVD risk due to its antioxidant and anti-inflammatory properties. However, as we recently showed in epidemiological studies, meta-analyses, and human trials, dietary antioxidants resulted more effective in subjects characterized by an ongoing oxidative stress, than in healthy people. Aim of this work was to investigate the effect of different concentrations of chocolate phenolic extract (CPE) on in vitro free radical production, stimulated by phorbol 12-myristate 13-acetate (PMA), in leukocytes extracted from blood of normo-weight and overweight/obese subjects. Neutrophils from overweight/obese group had a significantly higher free radical production compared to the normo-weight group. In neutrophils, the lowest CPE concentration significantly reduced free radical production in overweight/obese group only, and higher CPE concentrations were effective in both groups. In monocytes, the CPE concentration that was significantly effective in reducing free radical production was lower in overweight/obese subjects than in normo-weight subjects. Chocolate polyphenol extracts inhibit oxidative burst in human neutrophils and monocytes with a higher efficiency in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese. Results of this study provide further evidence about a differential role of dietary antioxidant strictly related to the "stress" condition of the subjects.

  17. Effect of Dark Chocolate Extracts on Phorbol 12-Myristate 13-Acetate-Induced Oxidative Burst in Leukocytes Isolated by Normo-Weight and Overweight/Obese Subjects

    PubMed Central

    Ioannone, Francesca; Sacchetti, Giampiero; Serafini, Mauro

    2017-01-01

    Oxidative and inflammatory stress represents a major risk factor for cardiovascular disease (CVD) in overweight and obese subjects. Between the different plant foods, chocolate has been shown to decrease CVD risk due to its antioxidant and anti-inflammatory properties. However, as we recently showed in epidemiological studies, meta-analyses, and human trials, dietary antioxidants resulted more effective in subjects characterized by an ongoing oxidative stress, than in healthy people. Aim of this work was to investigate the effect of different concentrations of chocolate phenolic extract (CPE) on in vitro free radical production, stimulated by phorbol 12-myristate 13-acetate (PMA), in leukocytes extracted from blood of normo-weight and overweight/obese subjects. Neutrophils from overweight/obese group had a significantly higher free radical production compared to the normo-weight group. In neutrophils, the lowest CPE concentration significantly reduced free radical production in overweight/obese group only, and higher CPE concentrations were effective in both groups. In monocytes, the CPE concentration that was significantly effective in reducing free radical production was lower in overweight/obese subjects than in normo-weight subjects. Chocolate polyphenol extracts inhibit oxidative burst in human neutrophils and monocytes with a higher efficiency in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese. Results of this study provide further evidence about a differential role of dietary antioxidant strictly related to the “stress” condition of the subjects. PMID:28649567

  18. Mapping of QTLs for Seed Phorbol Esters, a Toxic Chemical in Jatropha curcas (L.)

    PubMed Central

    Amkul, Kitiya; Laosatit, Kularb; Shim, Sangrea; Lee, Suk-Ha; Tanya, Patcharin; Srinives, Peerasak

    2017-01-01

    Jatropha (Jatropha curcas L.) is an oil-bearing plant that has potential to be cultivated as a biodiesel crop. The seed cake after oil extraction has 40–50% protein that can be used in animal feeds. A major limitation in utilizing the cake is the presence of phorbol esters (PE), a heat-tolerant toxic chemical. To identify the quantitative trait loci (QTLs) for PE, we constructed a genetic linkage map from an F2 population of 95 individuals from a cross “Chai Nat” × “M10” using 143 simple sequence repeat (SSR) markers. M10 is low in seed PE while Chai Nat is high. Seeds from each F2 individual were quantified for PE content by high performance liquid chromatography. A single marker analysis revealed five markers from linkage group 3 (LG3) and nine markers from LG8 associated with seed PE. Inclusive composite interval mapping identified two QTLs, each on LG3 (qPE3.1) and LG8 (qPE8.1) responsible for the PE. qPE3.1 and qPE8.1 accounted for 14.10%, and 15.49% of total variation in seed PE, respectively. Alelle(s) from M10 at qPE3.1 increased seed PE, while at qPE8.1 decreased seed PE. qPE3.1 is a new loci for PE, while qPE8.1 is the same locus with that reported recently for PE. PMID:28820491

  19. The effects of phorbol ester activation and reactive oxygen species scavengers on the macrophage-mediated foreign body reaction to polyurethanes.

    PubMed

    McBane, Joanne E; Matheson, Loren A; Santerre, J Paul; Labow, Rosalind S

    2009-12-15

    Phorbol myristate acetate, a protein kinase C activator, inhibited monocyte-derived macrophage (MDM)-mediated degradation of aliphatic (HDI) polycarbonate-based polyurethanes but not degradation of the aromatic polycarbonate-based polyurethane (MDI). The objectives of this study were to determine if reactive oxygen species are involved in the phorbol myristate acetate effect on esterase activity and MDM-mediated polycarbonate-based polyurethane degradation and to find a good marker of material-initiated activation of MDM. The phorbol myristate acetate-dependent effects of the material chemistry on cell activation and degradation were evaluated by adding reactive oxygen species scavengers, catalase plus superoxide dismutase to MDM and assaying possible markers of MDM activation: esterase activity, acid phosphatase activity, and high molecular weight group box 1 protein (HMGB1). All treatments reduced the esterase activity in MDM on HDI but not in MDM on MDI. Acid phosphatase was inhibited in MDM to varying degrees on all surfaces by phorbol myristate acetate or catalase plus superoxide dismutase either alone or together. Secretion of HMGB1 from MDM on HDI431 was higher than MDI; however only secretion from MDM on HDI was inhibited by phorbol myristate acetate. In MDM on HDI, catalase plus superoxide dismutase reduced intracellular HMGB1 levels +/- phorbol myristate acetate; whereas, catalase, superoxide dismutase plus phorbol myristate acetate increased intracellular HMGB1 in MDM on MDI, suggesting that esterase and HMGB1 are more specific markers of activation than acid phosphatase. Manipulation of signaling pathways may provide insight surrounding the mechanism of activation for oxidative and/or hydrolytic degradative pathways in the MDM response to material surface chemistry.

  20. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus.

    PubMed

    Lund, Maria E; To, Joyce; O'Brien, Bronwyn A; Donnelly, Sheila

    2016-03-01

    The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/macrophages. This is because, following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics, in many respects, primary human macrophages. Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to inflammatory stimuli, as well as the development and screening of potential therapeutics, there is currently no standardised protocol for the reliable differentiation of THP-1 monocytes to a macrophage phenotype using PMA. Consequently, reports using THP-1 cells have demonstrated significant phenotypic and functional differences between resultant THP-1 macrophage populations, which are largely attributable to the varying PMA differentiation methods used. Thus, to guarantee consistency and reproducibility between studies, and to ensure the relevance of THP-1 cells as an appropriate model for primary human macrophages, it is crucial to develop a standardised protocol for the differentiation of THP-1 macrophages. Accordingly, we compared the function and phenotype of THP-1 macrophages generated using the range of published PMA differentiation protocols, specifically in response to the pro-inflammatory stimulus, lipopolysaccharide (LPS). Our results demonstrated that the function of the resultant THP-1 macrophage populations, as determined by tumour necrosis factor (TNF) secretion in response to LPS stimulation, varied significantly, and was dependent upon the concentration of PMA used to stimulate the differentiation of monocytes, and the period of rest following PMA exposure. These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  1. Comparison of effects of phorbol esters and glucose on protein kinase C activation and insulin secretion in pancreatic islets.

    PubMed Central

    Easom, R A; Hughes, J H; Landt, M; Wolf, B A; Turk, J; McDaniel, M L

    1989-01-01

    The tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces insulin secretion from isolated pancreatic islets, and this suggests a potential role for protein kinase C in the regulation of stimulus-secretion coupling in islets. In the present study, the hypothesis that the insulinotropic effect of TPA is mediated by activation of protein kinase C in pancreatic islets has been examined. TPA induced a gradual translocation of protein kinase C from the cytosol to a membrane-associated state which correlated with the gradual onset of insulin secretion. The pharmacologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not mimic this effect. TPA also induced a rapid time-dependent decline of total protein kinase C activity in islets and the appearance of a Ca2+- and phospholipid-independent protein kinase activity. Insulin secretion induced by TPA was completely suppressed (IC50 approximately 10 nM) by staurosporine, a potent protein kinase C inhibitor. Staurosporine also inhibited islet cytosolic protein kinase C activity at similar concentrations (IC50 approximately 2 nM). In addition, staurosporine partially (approximately 60%) inhibited glucose-induced insulin secretion at concentrations (IC50 approximately 10 nM) similar to those required to inhibit TPA-induced insulin secretion, suggesting that staurosporine may act at a step common to both mechanisms, possibly the activation of protein kinase C. However, stimulatory concentrations of glucose did not induce down-regulation of translocation of protein kinase C, and the inhibition of glucose-induced insulin release by staurosporine was incomplete. Significant questions therefore remain unresolved as to the possible involvement of protein kinase C in glucose-induced insulin secretion. PMID:2690823

  2. A phorbol ester-insensitive AP-1 motif mediates the stimulatory effect of insulin on rat malic enzyme gene transcription.

    PubMed

    Streeper, R S; Chapman, S C; Ayala, J E; Svitek, C A; Goldman, J K; Cave, A; O'Brien, R M

    1998-11-01

    In liver, insulin stimulates the transcription of the gene encoding the cytosolic form of malic enzyme (ME) and modulates protein binding to two putative insulin response sequences (IRSs) in the ME promoter. One of these IRSs resembles that identified in the phosphoenolpyruvate carboxykinase (PEPCK) gene, whereas the other resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the functional significance of these changes in protein binding, a series of truncated ME-chloramphenicol acetyl-transferase (CAT) fusion genes were transiently transfected into rat H4IIE hepatoma cells. Deletion of the PEPCK-like IRS motif had no effect on the stimulation of CAT expression by insulin. Instead, the stimulatory effect of insulin was mediated through an AP-1 motif and an Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME AP-1 motif and the AP-1 motif identified in the collagenase-1 gene promoter were able to confer a stimulatory effect of insulin on the expression of a heterologous fusion gene, but surprisingly only the latter was able to confer a stimulatory effect of phorbol esters. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatment has no additional effect. The collagenase and ME AP-1 motifs were both shown to bind mainly Jun D and Fra-2, with similar affinities. However, the results of a proteolytic clipping bandshift assay suggest that these proteins bind the collagenase and ME AP-1 motifs in distinct conformations, which potentially explain the differences in phorbol ester signaling through these elements.

  3. Niacinamide mitigated the acute lung injury induced by phorbol myristate acetate in isolated rat's lungs

    PubMed Central

    2012-01-01

    Background Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes. Methods The rat's lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 μg/g), PMA 4 μg/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (Kfc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined. Results PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent. Conclusions Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA

  4. Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

    PubMed Central

    Wang, J F; Komarov, P; Sies, H; de Groot, H

    1991-01-01

    Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2. PMID:1718262

  5. Molecular Basis for Failure of “Atypical” C1 Domain of Vav1 to Bind Diacylglycerol/Phorbol Ester*

    PubMed Central

    Geczy, Tamas; Peach, Megan L.; El Kazzouli, Saïd; Sigano, Dina M.; Kang, Ji-Hye; Valle, Christopher J.; Selezneva, Julia; Woo, Wonhee; Kedei, Noemi; Lewin, Nancy E.; Garfield, Susan H.; Lim, Langston; Mannan, Poonam; Marquez, Victor E.; Blumberg, Peter M.

    2012-01-01

    C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu9, Glu10, Thr11, Thr24, and Tyr26) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1. PMID:22351766

  6. Protein phosphorylation in electrically permeabilized islets of Langerhans. Effects of Ca2+, cyclic AMP, a phorbol ester and noradrenaline.

    PubMed Central

    Jones, P M; Salmon, D M; Howell, S L

    1988-01-01

    The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases. Images Fig. 1. Fig. 4. PMID:2845950

  7. The effect of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) on whole blood oxidative response as assessed by luminol-amplified chemiluminescence in dairy cows

    USDA-ARS?s Scientific Manuscript database

    The differences between lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) on whole blood oxidative response using luminol-amplified chemiluminescence (CL) are currently unknown in cattle. Luminol-dependent CL measures the amount of reactive oxygen species released from leukocytes a...

  8. Antioxidant and antiradical activities of Manihot esculenta Crantz (Euphorbiaceae) leaves and other selected tropical green vegetables investigated on lipoperoxidation and phorbol-12-myristate-13-acetate (PMA) activated monocytes.

    PubMed

    Tsumbu, Cesar N; Deby-Dupont, Ginette; Tits, Monique; Angenot, Luc; Franck, Thierry; Serteyn, Didier; Mouithys-Mickalad, Ange

    2011-09-01

    Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N'-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in "inflammation like" conditions was studied by fluorescence technique using 2',7'-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration.

  9. Antioxidant and Antiradical Activities of Manihot esculenta Crantz (Euphorbiaceae) Leaves and Other Selected Tropical Green Vegetables Investigated on Lipoperoxidation and Phorbol-12-myristate-13-acetate (PMA) Activated Monocytes

    PubMed Central

    Tsumbu, Cesar N.; Deby-Dupont, Ginette; Tits, Monique; Angenot, Luc; Franck, Thierry; Serteyn, Didier; Mouithys-Mickalad, Ange

    2011-01-01

    Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N′-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in “inflammation like” conditions was studied by fluorescence technique using 2′,7′-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration. PMID:22254126

  10. Influence of phorbol esters, and diacylglycerol kinase and lipase inhibitors on noradrenaline release and phosphoinositide hydrolysis in chromaffin cells.

    PubMed Central

    Jones, J. A.; Owen, P. J.; Boarder, M. R.

    1990-01-01

    1. We have investigated the modification of catecholamine efflux and inositol phosphate formation in cultured adrenal chromaffin cells by tetradecanoyl phorbol acetate (TPA) and inhibitors of diacylglycerol kinase (R 59,022) and diacylglycerol lipase (RG 80267), the two principal pathways of diacylglycerol metabolism. 2. TPA (1 nM to 1 microM) elicited a slow, calcium-dependent, sustained release of noradrenaline, which was partially blocked by the dihydropyridine calcium channel blocker (-)-202,791 and potentiated by the channel enhancer (+)-202,791. 3. R 59,022 enhanced noradrenaline efflux at 30 and 50 microM, while the lipase inhibitor RG 80267 failed to elicit release. 4. Neither R 59,022 nor RG 80267 affected bradykinin- or histamine-stimulated release, but both drugs substantially attenuated nicotine- and high K(+)-stimulated release. 5. Pretreatment for 10 min with TPA (but not the relatively inactive 4-methoxy TPA) or the non-phorbol protein kinase C stimulator mezerein potently inhibited bradykinin- and histamine-stimulated accumulation of total [3H]-inositol phosphate; inhibition of [3H]-inositol phosphate formation was also seen with 24 h TPA treatment. 6. Neither R 59,022 nor RG 80267, separately or together, affected bradykinin-stimulated [3H]-inositol phosphate formation. 7. Thus while the mechanism exists for inhibition of formation of inositol phosphates by stimulation of protein kinase C, these studies failed to show that this mechanism is activated by agonists acting on phospholipase C linked receptors. PMID:1963797

  11. Detoxification of Toxic Phorbol Esters from Malaysian Jatropha curcas Linn. Kernel by Trichoderma spp. and Endophytic Fungi

    PubMed Central

    Najjar, Azhar; Abdullah, Norhani; Saad, Wan Zuhainis; Ahmad, Syahida; Oskoueian, Ehsan; Abas, Faridah; Gherbawy, Youssuf

    2014-01-01

    The presence of phorbol esters (PEs) with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum JQ350879.1, T. harzianum JQ517493.1, Paecilomyces sinensis JQ350881.1, Cladosporium cladosporioides JQ517491.1, Fusarium chlamydosporum JQ350882.1, F. chlamydosporum JQ517492.1 and F. chlamydosporum JQ350880.1) was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS) and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p < 0.05) removed PEs with percentage losses of 96.9%–99.7%, while F. chlamydosporum strains showed percentage losses of 88.9%–92.2%. All fungal strains could utilize the PEs-rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%–96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs. PMID:24504029

  12. Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin.

    PubMed

    Wiejak, J; Surmacz, L; Wyroba, E

    2001-01-01

    The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LY uptake (575 ng LY/mg protein/hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/mg protein/hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin-FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in "mixing" endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.

  13. Effect of phorbol ester application and other mitogenic treatments on 3',5'-cyclic-nucleotide phosphodiesterase activity in mouse epidermis in vivo.

    PubMed

    Marks, F; Fürstenberger, G

    1980-11-01

    The effects of phorbol ester application and of other mitogenic treatments on the activity of 3',5'-cyclic-nucleotide phosphodiesterase were investigated in dorsal mouse epidermis in vivo. Local treatment with either the weak tumor promoter phorbol 12,13-dibenzoate or the strong promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the activity of the high affinity enzyme (Km = 4 microM). The enzymic changes began within the first hour after application, and lasted for about 5 days. maximal stimulations of approximately 300--400% were reached after 3--6 h with TPA application, whereas with phorbol dibenzoate the maximum could only be reached after 1--2 days. TPA stimulation of the enzyme depended on doses within the range of 0.2 to 20 nmol and could be completely prevented by cycloheximide, but not by 5-azacytidine, actinomycin D, 5,8,11,14-eicosatetraynoic acid or indomethacin. No evidence could be found for cAMP participation in enzyme induction. An increase in enzyme activity could also be observed after other mitogenic treatments such as local application of the weakly promoting phorbol esters C14:4-phorbol acetate ("Ti8") and 4.O-methyl-TPA, or of the non-promoting divalent cation ionophore A 23187, as well as after treatment with a depilatory cream. Skin massage or removal of the horny layer, which also stimulate mitosis, did not evoke a significant increase in enzyme activity. No apparent correlation exists between the hyperplasiogenic and tumor-promoting effectiveness of a manipulation and its effect on epidermal 3',5'-cyclic-nucleotide phosphodiesterase.

  14. Evidence for discrete diacylglycerol and phorbol ester activator sites on protein kinase C. Differences in effects of 1-alkanol inhibition, activation by phosphatidylethanolamine and calcium chelation.

    PubMed

    Slater, S J; Kelly, M B; Taddeo, F J; Rubin, E; Stubbs, C D

    1994-06-24

    Stimulation of protein kinase C (PKC) activity is achieved in vivo by diacylglycerol but can also be obtained with tumor-promoting phorbol esters. Evidence is presented indicating that these two classes of activator may interact at different regions of the enzyme. The activity of a calcium-dependent PKC isoform (PKC-I) preparation was determined using 1,2-dioleoylglycerol (DOG) together with the phorbol ester 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA). The resulting PKC activity was in excess of that attained with either activator alone, each being at a maximum concentration for activation. A similar result was obtained with purified PKC-alpha and -epsilon isoforms, indicating that the additive effect was not due to sites being on distinct enzyme molecules. Support for two dissimilar activator sites came from the observation that the inactive phorbol ester 4 alpha-TPA competed for TPA but not for DOG in PKC activation. Other differences were observed between TPA- and DOG-activated PKC. It was found that 1-butanol inhibited DOG-activated PKC-I, while being without effect on stimulation by TPA. Also, the inclusion of phosphatidylethanolamine in the lipid vesicles led to a potentiation of PKC-I activity which was greater when activation was achieved by DOG compared to TPA. Further, the calcium- and DOG-dependent active conformational change of PKC was fully reversible upon calcium chelation, while that stimulated by TPA was only partially reversible. These experiments taken together suggest that diacylglycerols and phorbol esters bind with different affinities and at different sites on PKC, and induce distinct activated conformational forms of the enzyme.

  15. Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study.

    PubMed

    Thangsunan, Patcharapong; Tateing, Suriya; Hannongbua, Supa; Suree, Nuttee

    2016-07-01

    Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.

  16. Studies on nitric oxide free radicals generated from polymorphonuclear leukocytes (PMN) stimulated by phorbol myristate acetate (PMA).

    PubMed

    Zhao, B L; Wang, J C; Hou, J W; Xin, W J

    1996-05-01

    The interaction of NO and O2- free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O2- by DMPO and addition of NG-monomethyl-arginine (NGMA) would significantly increase the trapped O2- by DMPO. It was proved that the formation of ONOO- by the reaction of NO and O2- was the main reason for the decrease of trapped O2- in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemiluminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO- synthesized were also studied in model systems.

  17. Degradation rates of phorbol esters in Jatropha curcas L. oil and pressed seeds under different storage conditions.

    PubMed

    Phasukarratchai, Naphatsarnan; Damrongsiri, Seelawut; Tongcumpou, Chantra

    2017-03-01

    Phorbol esters (PEs), found in Jatropha curcas crude oil (JCO) and J. curcas pressed seeds (JPS), are known as bioactive compounds in agricultural and pharmaceutical applications. The degradation rates of PEs in JCO and JPS under various conditions is important for the utilisation of PEs. Thus the objective of this study was to determine the PE degradation rates in JCO and JPS under different storage conditions. PE degradation rates were found to be first-order reactions. The slowest degradation rate was at 0.9 × 10 -3 d -1 for both JCO and JPS unexposed to light at 4 °C. Light intensity (1097 lx and 4690 lx, representing diffused sunlight and fluorescent lighting, respectively) and temperature (25 to 35 °C) were the significant degradation factors. Light exposure led to 280% to 380% higher degradation rates in JCO than in JPS due to light penetration through the transparent oil. Dried and sterilised JPS showed an 80% to 90% lower PE degradation rate than untreated JPS under all storage conditions since biodegradation was assembly limited. The PEs were unstable under the studied conditions, especially when exposed to light and room temperature. To protect against PE degradation, a material should be stored in a light-protected container and below 4 °C. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  18. Effects of phorbol myristate acetate and sivelestat on the lung injury caused by fat embolism in isolated lungs.

    PubMed

    Lin, Chia-Chih; Liu, Pei-Hsin; Kao, Shang Jyh; Chen, Hsing I

    2012-01-05

    Fat embolism syndrome (FES) associated with acute lung injury (ALI) is a clinical condition following long bone fracture. We have reported 14 victims due to ALI with FES. Our laboratory has developed an animal model that produced fat emboli (FE). The major purpose of this study was to test whether neutrophil activation with phorbol myristate acetate (PMA) and inhibition with sivelestat (SVT) exert protection on the lung. The lungs of Sprague-Dawley rats were isolated and perfused. FE was produced by addition of corn oil micelles into the lung perfusate. PMA and SVT were given simultaneously with FE. Parameters such as lung weight/body weight ratio, LW gain, exhaled nitric oxide (NO), protein concentration in bronchoalveolar lavage relating to ALI were measured. The neutrophil elastase (NE), myeloperoxidase, malondialdehyde and phopholipase A₂ activity were determined. We also measured the nitrate/nitrite, methyl guanidine (MG), and cytokines. Pulmonary arterial pressure and microvascular permeability were assessed. Lung pathology was examined and scored. The inducible and endothelial NO synthase (iNOS and eNOS) were detected. FE caused ALI and increased biochemical factors. The challenge also resulted in pulmonary hypertension and increased microvascular permeability. The NE appeared to be the first to reach its peak at 1 hr, followed by other factors. Coadministration with PMA exacerbated the FE-induced changes, while SVT attenuated the effects of FE. The FE-induced lung changes were enhanced by PMA, while SVT had the opposite effect. Sivelestat, a neutrophil inhibitor may be a therapeutic choice for patients with acute respiratory distress syndrome (ARDS) following fat embolism.

  19. Influence of alpha 1-adrenergic receptor stimulation and phorbol esters on hepatic Na+/K+-ATPase activity.

    PubMed

    Smart, J L; Deth, R C

    1988-01-01

    The effects of the alpha 1-adrenergic agonist phenylephrine (PE) and the phorbol ester 4 beta-phorbolmyristate-acetate (PMA) on sodium pump function were studied in the rat liver. In order to distinguish between direct and indirect influences, ouabain-sensitive 86Rb uptake by intact liver slices was compared with ouabain-sensitive Na+/K+-ATPase activity in plasma membranes isolated from PE and PMA-perfused livers. At a buffer Ca2+ level of 2.5 mmol/l, PE (10 mumol/l) caused an initial stimulation of both 86Rb uptake and Na+/K+-ATPase activity followed at 5 min by a decrease in both activities. Both actions were blocked by the alpha 1-antagonist prazosin. The decrease in ouabain-sensitive Na+/K+-ATPase was paralleled by an increase in Mg2+ ATPase activity. At a Ca2+ level of 1.5 mmol/l, PE stimulation of 86Rb uptake and Na+/K+-ATPase was sustained, and the inhibitory component was not expressed. PMA (4 mumol/l) reduced 86Rb uptake and Na+/K+-ATPase and similar to PE, this inhibition was paralleled by an increase of Mg2+-ATPase activity. 4 alpha-PMA, which does not activate protein kinase C, failed to influence 86Rb uptake or Na+/K+-ATPase. These results demonstrate that PE and PMA effects on ouabain-sensitive 86Rb uptake are preserved in isolated membranes, indicating a direct influence on the Na+/K+-ATPase. A role for protein kinase C in modulating sodium pump activity is suggested.

  20. 4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4

    PubMed Central

    Alexander, R; Kerby, A; Aubdool, AA; Power, AR; Grover, S; Gentry, C; Grant, AD

    2013-01-01

    Background and Purpose The Ca2+-permeable cation channel TRPV4 is activated by mechanical disturbance of the cell membrane and is implicated in mechanical hyperalgesia. Nerve growth factor (NGF) is increased during inflammation and causes mechanical hyperalgesia. 4α-phorbol 12,13-didecanoate (4αPDD) has been described as a selective TRPV4 agonist. We investigated NGF-induced hyperalgesia in TRPV4 wild-type (+/+) and knockout (–/–) mice, and the increases in [Ca2+]i produced by 4αPDD in cultured mouse dorsal root ganglia neurons following exposure to NGF. Experimental Approach Withdrawal thresholds to heat, von Frey hairs and pressure were measured in mice before and after systemic administration of NGF. Changes in intracellular Ca2+ concentration were measured by ratiometric imaging with Fura-2 in cultured DRG and trigeminal ganglia (TG) neurons during perfusion of TRPV4 agonists. Key Results Administration of NGF caused a significant sensitization to heat and von Frey stimuli in TRPV4 +/+ and –/– mice, but only TRPV4 +/+ mice showed sensitization to noxious pressure. 4αPDD stimulated a dose-dependent increase in [Ca2+]i in neurons from +/+ and –/– mice, with the proportion of responding neurons and magnitude of increase unaffected by the genotype. In contrast, the selective TRPV4 agonist GSK1016790A failed to stimulate an increase in intracellular Ca2+ in cultured neurons. Responses to 4αPDD were unaffected by pretreatment with NGF. Conclusions and Implications TRPV4 contributes to mechanosensation in vivo, but there is little evidence for functional TRPV4 in cultured DRG and TG neurons. We conclude that 4αPDD activates these neurons independently of TRPV4, so it is not appropriate to refer to 4αPDD as a selective TRPV4 agonist. PMID:22928864

  1. Phorbol esters enhance 1α,25 dihydroxyvitamin D3-regulated 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) gene expression through ERK-mediated phosphorylation of specific protein 3 (Sp3) in Caco-2 cells

    PubMed Central

    Jiang, Yan; Fleet, James C.

    2012-01-01

    Phorbol 12-myristate 13-acetate (PMA) increased 1,25(OH)2D3-induced human 25 hydroxyvitamin D-24 hydroxylase (hCYP24A1) gene expression and vitamin D receptor (VDR) binding to the hCYP24A1 promoter. It did not alter transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression, VDR binding to the TRPV6 promoter, or VDR binding to a crude chromatin preparation. PMA activated Extracellular signal-Regulated Kinases (ERK) 1/2 and p38 mitogen activated protein kinases (MAPK) and inhibiting these kinases reduced 1,25(OH)2D3-induced and PMA-enhanced hCYP24A1 promoter activity. Mithramycin A inhibits Specific Protein (Sp) family member binding to DNA and reduced 1,25(OH)2D3-induced and PMA-enhanced hCYP24A1 promoter activity. Sp1 or Sp3 siRNA knockdown reduced 1,25(OH)2D3-regulated hCYP24A1 promoter activity but only Sp3 siRNA reduced PMA-enhanced hCYP24A1 promoter activity. PMA increased MAPK-dependent Sp3 phosphorylation, Sp3-VDR interactions, and Sp3 binding to the hCYP24A1 promoter. These data suggest that MAPK signaling contributes to 1,25(OH)2D3-induced and PMA-enhanced CYP24A1 gene transcription by modulating Sp3 function. PMID:22871965

  2. Changes in the migratory properties of neural crest and early crest-derived cells in vivo following treatment with a phorbol ester drug.

    PubMed

    Sears, R; Ciment, G

    1988-11-01

    In previous work, we found that the phorbol ester drug 12-O-tetradecanoyl phorbol acetate (TPA) reversed the developmental restriction of melanogenesis that normally occurs in neural crest-derived Schwann cell precursors around embryonic Day 5 of quail development. That is, TPA treatment of dorsal root ganglia (DRG) from 7-day quail embryos caused Schwann cell precursors to regain the ability to give rise to melanocytes. In this paper, we examine other long-term effects of TPA on the differentiative and migratory properties of neural crest and crest-derived DRG cells, using heterospecific grafting methods. We report that TPA treatment in culture increased the extent of cell migration following grafting into host embryos, including some ectopic migration into the central nervous system and other locations. TPA did not, however, seem to change the fate of these crest-derived cells, except that some DRG cells underwent pigmentation, as had been observed previously. Interestingly, graft cells associated with peripheral nerves were found to be exclusively unpigmented, whereas graft cells found in all other locations, including the central nervous system, were both pigmented and unpigmented. This suggests that peripheral nerves may act in a fashion antagonistic to the effects of TPA. These findings are consistent with the notion that TPA treatment causes early crest-derived cells to regain developmental properties lost with developmental age.

  3. Synergistic activation of protein kinase Calpha, -betaI, and -gamma isoforms induced by diacylglycerol and phorbol ester: roles of membrane association and activating conformational changes.

    PubMed

    Slater, S J; Milano, S K; Stagliano, B A; Gergich, K J; Ho, C; Mazurek, A; Taddeo, F J; Kelly, M B; Yeager, M D; Stubbs, C D

    1999-03-23

    Protein kinase Calpha (PKCalpha) has been shown to contain two discrete activator sites with differing binding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiated level of activity [Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D. , Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancement of activity was examined with respect to the Ca2+ dependences of membrane association and accompanying conformational changes that lead to activation. The association of PKCalpha with membranes containing 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1, 2-dioleoylglycerol (DAG), determined from tryptophan to dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca2+ levels (

  4. ASB16165, a phosphodiesterase 7A inhibitor, reduces cutaneous TNF-alpha level and ameliorates skin edema in phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation model in mice.

    PubMed

    Kadoshima-Yamaoka, Kumiko; Goto, Megumi; Murakawa, Masao; Yoshioka, Ryosuke; Tanaka, Yoshitaka; Inoue, Hidekazu; Murafuji, Hidenobu; Kanki, Satomi; Hayashi, Yasuhiro; Nagahira, Kazuhiro; Ogata, Atsuto; Nakatsuka, Takashi; Fukuda, Yoshiaki

    2009-06-24

    Possible role of phosphodiesterase 7A (PDE7A) in skin inflammation was examined using ASB16165, a specific inhibitor for PDE7A. Epicutaneous application of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ear resulted in induction of skin edema, and topical treatment with ASB16165 inhibited the induction of skin edema in a dose-dependent manner. The TPA challenge also increased the level of TNF-alpha at the application site, and the ASB16165 treatment reduced the TNF-alpha level in the skin. In addition, ASB16165 suppressed the production of TNF-alpha by human keratinocytes stimulated in vitro with TPA and calcium ionophore. Forskolin, an activator of adenylyl cyclase, as well as dibutyryl cAMP also showed inhibitory effect on the TNF-alpha production in the cells, suggesting involvement of cAMP in TNF-alpha generation. These results demonstrate that PDE7A might regulate TNF-alpha production in keratinocytes in a cAMP-dependent fashion. As immunostaining analysis revealed that PDE7A is expressed in the epidermis and TNF-alpha is known to contribute to the TPA-induced edema, it is possible that the inhibitory effect of ASB16165 on skin edema in mouse TPA-induced dermatitis model is mediated by suppression of TNF-alpha production. This is the first report suggesting the association of PDE7A with the function of keratinocytes. ASB16165 will be useful as an agent for skin inflammation in which TNF-alpha plays a pathogenic role (e.g. psoriasis).

  5. Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B

    SciTech Connect

    Maier, Jana V., E-mail: Jana.maier@kit.edu; Volz, Yvonne; Berger, Caroline

    2010-10-22

    Research highlights: {yields}Bag-1 depletion only marginally affects the action of the glucocorticoid receptor but strongly regulates the activity of NF-{kappa}B. {yields}Bag-1 depletion attenuates phosphorylation and degradation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B p65 and p50. {yields}Bag-1 interacts with I{kappa}B{alpha} and partially restores I{kappa}B{alpha} and NF-{kappa}B activation in Bag-1 depleted cells. -- Abstract: Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulatemore » the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-{kappa}B. Furthermore phosphorylation and degradation of the inhibitor protein I{kappa}B{alpha} and nuclear accumulation of p65 and p50 NF-{kappa}B proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored I{kappa}B{alpha} and NF-{kappa}B activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.« less

  6. Linkage mapping in the oilseed crop Jatropha curcas L. reveals a locus controlling the biosynthesis of phorbol esters which cause seed toxicity

    PubMed Central

    King, Andrew J; Montes, Luis R; Clarke, Jasper G; Affleck, Julie; Li, Yi; Witsenboer, Hanneke; van der Vossen, Edwin; van der Linde, Piet; Tripathi, Yogendra; Tavares, Evanilda; Shukla, Parul; Rajasekaran, Thirunavukkarasu; van Loo, Eibertus N; Graham, Ian A

    2013-01-01

    Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring ‘nontoxic’ provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F2 mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F2 plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker-assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties. PMID:23898859

  7. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    NASA Technical Reports Server (NTRS)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  8. Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

    PubMed Central

    Yamamoto, Kazuhiro; Seki, Takahiro; Yamamoto, Hikaru; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2014-01-01

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca2+ and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate), a type of phorbol ester. Wild-type (WT) γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in pathogenesis of

  9. CAP37 Activation of PKC Promotes Human Corneal Epithelial Cell Chemotaxis

    PubMed Central

    Griffith, Gina L.; Russell, Robert A.; Kasus-Jacobi, Anne; Thavathiru, Elangovan; Gonzalez, Melva L.; Logan, Sreemathi; Pereira, H. Anne

    2013-01-01

    Purpose. The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. Methods. Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 μM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr505 phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. Results. Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr505. Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. Conclusions . These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ. PMID:24008408

  10. A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation*

    PubMed Central

    Azevedo, Estefania P.; Rochael, Natalia C.; Guimarães-Costa, Anderson B.; de Souza-Vieira, Thiago S.; Ganilho, Juliana; Saraiva, Elvira M.; Palhano, Fernando L.; Foguel, Debora

    2015-01-01

    Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

  11. Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed.

    PubMed

    Kongmany, Santi; Hoa, Truong Thi; Hanh, Le Thi Ngoc; Imamura, Kiyoshi; Maeda, Yasuaki; Boi, Luu Van

    2016-12-01

    Phorbol esters (PEs) are well known as the main toxic compounds in Jatropha curcas Linnaeus (JCL), the seed oil of which has been considered as a major feedstock for the production of biodiesel. In the present study, we investigated a series of PEs extracted from JCL seed kernels with methanol (MeOH), and identified more than seven components contained in the PEs. The isolation of main five components of a series of PEs was revised using a semi-preparative reversed phase HPLC analysis of ODS-3 column. The five peaks of components were successfully isolated, and peaks of J2, J3, J5, and J7 were assigned to be Jatropha factors C 1 , C 2 , C 3, and C 4/5 , but J6 was a mixture of Jatropha factor C 6 and its isomer based on the data of UV and LC-MS/MS, and J2 was identified using 1 H NMR analysis. By characterization using LC-MS/MS analysis, all components of a series of PEs were elucidated to be the 12-deoxy-16-hydroxyphorbol esters composed of isomeric form of dicarboxylic groups with same m/z value of 380. Copyright © 2016. Published by Elsevier B.V.

  12. Involvement of the antioxidative property of morusin in blocking phorbol ester-induced malignant transformation of JB6 P+ mouse epidermal cells.

    PubMed

    Cheng, Pai-Shan; Hu, Chao-Chin; Wang, Chau-Jong; Lee, Yean-Jang; Chung, Wei-Chia; Tseng, Tsui-Hwa

    2017-02-25

    Chemoprevention has been acknowledged as an important and practical strategy for managing cancer. We have previously synthesized morusin, a prenylated flavonoid that exhibits anti-cancer progression activity. In the present study, we evaluated the anti-cancer promotion potential of morusin by using the mouse epidermal JB6 P + cell model. Extensive evidence shows that tumor promotion by phorbol esters is due to the stimulation of reactive oxygen species (ROS). Therefore, the effect of morusin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ROS production was assessed. Noncytotoxic concentrations of morusin were found to dose-dependently reduce TPA-induced ROS production. Moreover, morusin inhibited TPA-induced activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) activation, which can mediate cell proliferation and malignant transformation. Furthermore, morusin inhibited the TPA upregulation of cyclooxygenase 2 (COX-2), which may be regulated by AP-1 and NF-κB. In addition, noncytotoxic concentrations of morusin reduced the TPA-promoted cell growth of JB6 P + cells and inhibited TPA-induced malignant properties, such as cytoskeletal rearrangement and cell migration of JB6 P + cells. Similar to the effects of glutathione (GSH) pretreatment, morusin inhibited TPA-induced expression of N-cadeherin and vimentin, which are malignant cell surface proteins. Finally, morusin treatment dose-dependently suppressed the TPA-induced anchorage-independent cell transformation of JB6 P + cells. In conclusion, our results evidence that morusin possesses anti-cancer promotion potential because of its antioxidant property, which mediates multiple transformation-associated gene expression. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Involvement of phorbol-12-myristate-13-acetate-induced protein 1 in goniothalamin-induced TP53-dependent and -independent apoptosis in hepatocellular carcinoma-derived cells

    SciTech Connect

    Kuo, Kung-Kai; Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; Chen, Yi-Ling

    2011-10-01

    The objective was to investigate the upstream apoptotic mechanisms that were triggered by a styrylpyrone derivative, goniothalamin (GTN), in tumor protein p53 (TP53)-positive and -negative hepatocellular carcinoma (HCC)-derived cells. Effects of GTN were evaluated by the flow cytometry, alkaline comet assay, immunocytochemistry, small-hairpin RNA interference, mitochondria/cytosol fractionation, quantitative reverse transcription-polymerase chain reaction, immunoblotting analysis and caspase 3 activity assays in two HCC-derived cell lines. Results indicated that GTN triggered phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA)-mediated apoptosis via TP53-dependent and -independent pathways. In TP53-positive SK-Hep1 cells, GTN furthermore induced TP53 transcription-dependent and -independent apoptosis. After GTN treatment, accumulation ofmore » reactive oxygen species, formation of DNA double-strand breaks, transactivation of TP53 and/or PMAIP1 gene, translocation of TP53 and/or PMAIP1 proteins to mitochondria, release of cytochrome c from mitochondria, cleavage of caspases and induction of apoptosis in both cell lines were sustained. GTN might represent a novel class of anticancer drug that induces apoptosis in HCC-derived cells through PMAIP1 transactivation regardless of the status of TP53 gene. - Highlights: > Goniothalamin (GTN) induced apoptosis in hepatocellular carcinomas-derived cells. > The apoptosis induced by GTN is PMAIP1-dependent, regardless of TP53 status. > The apoptosis induced by GTN might be TP53 transcription-dependent or -independent. > GTN-induced apoptosis is mitochondria- and caspases-mediated.« less

  14. Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

    SciTech Connect

    Henriksen, E.J.; Rodnick, K.J.; Holloszy, J.O.

    1989-12-25

    It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin. In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20%more » as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle.« less

  15. Enhanced detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide in combination with pokeweed mitogen and phorbol myristate acetate.

    PubMed

    Muthusamy, Natarajan; Breidenbach, Heather; Andritsos, Leslie; Flynn, Joseph; Jones, Jeffrey; Ramanunni, Asha; Mo, Xiaokui; Jarjoura, David; Byrd, John C; Heerema, Nyla A

    2011-02-01

    Reproducible cytogenetic analysis in CLL has been limited by the inability to obtain reliable metaphase cells for analysis. CpG oligonucleotide and cytokine stimulation have been shown to improve metaphase analysis of CLL cytogenetic abnormalities, but is limited by variability in the cytokine receptor levels, stability and biological activity of the cytokine in culture conditions and high costs associated with these reagents. We report here use of a novel, stable CpG, GNKG168 along with pokeweed mitogen (PWM) and phorbol 12-myristate 13-acetate (PMA) for conventional cytogenetic assessment in CLL. We demonstrate that the combined use of GNKG168+PWM/PMA increased the sensitivity of detection of chromosomal abnormalities compared to PWM/PMA (n=207, odds ratio=2.2, p=0.0002) and GNKG168 (n=219, odds ratio=1.5, p=0.0452). Further, a significant increase in sensitivity to detect complexity ≥3 with GNKG168+PWM/PMA compared to GNKG168 alone (odds ratio 8.0, p=0.0022) or PWM/PMA alone (odds ratio 9.6, p=0.0007) was observed. The trend toward detection of higher complexity was significantly greater with GNKG168+PWM/PMA compared to GNKG168 alone (p=0.0412). The increased sensitivity was mainly attributed to the addition of PWM/PMA with GNKG168 because GNKG168 alone showed no difference in sensitivity for detection of complex abnormalities (p=0.17). Comparison of fluorescence in situ hybridization (FISH) results with karyotypic results showed a high degree of consistency, although some complex karyotypes were present in cases with no adverse FISH abnormality. These studies provide evidence for potential use of GNKG168 in combination with PWM and PMA in karyotypic analysis of CLL patient samples to better identify chromosomal abnormalities for risk stratification.

  16. Enhanced Detection of Chromosomal Abnormalities in Chronic Lymphocytic Leukemia by Conventional Cytogenetics Using CpG Oligonucleotide in Combination with Pokeweed Mitogen and Phorbol Myristate Acetate

    PubMed Central

    Muthusamy, Natarajan; Breidenbach, Heather; Andritsos, Leslie; Flynn, Joseph; Jones, Jeffrey; Ramanunni, Asha; Mo, Xiaokui; Jarjoura, David; Byrd, John C.; Heerema, Nyla A.

    2011-01-01

    Reproducible cytogenetic analysis in CLL has been limited by the inability to obtain reliable metaphase cells for analysis. CpG oligonucleotide and cytokine stimulation have been shown to improve metaphase analysis of CLL cytogenetic abnormalities, but is limited by variability in the cytokine receptor levels, stability and biological activity of the cytokine in culture conditions and high costs associated with these reagents. We report here use of a novel, stable CpG, GNKG168 along with pokeweed mitogen (PWM) and phorbol 12-myristate 13-acetate (PMA) for conventional cytogenetic assessment in CLL. We demonstrate that the combined use of GNKG168+PWM/PMA increased the sensitivity of detection of chromosomal abnormalities compared to PWM/PMA (n=207, odds ratio=2.2, p=0.0002) and GNKG168 (n=219, odds ratio=1.5, p=0.0452). Further, a significant increase in sensitivity to detect complexity ≥3 with GNKG168+PWM/PMA compared to GNKG168 alone (odds ratio 8.0, p=0.0022) or PWM/PMA alone (odds ratio 9.6, p=0.0007) was observed. The trend toward detection of higher complexity was significantly greater with GNKG168+PWM/PMA compared to GNKG168 alone (p=0.0412). The increased sensitivity was mainly attributed to the addition of PWM/PMA with GNKG168 because GNKG168 alone showed no difference in sensitivity for detection of complex abnormalities (p=0.17). Comparison of fluorescence in situ hybridization (FISH) results with karyotypic results showed a high degree of consistency, although some complex karyotypes were present in cases with no adverse FISH abnormality. These studies provide evidence for potential use of GNKG168 in combination with PWM and PMA in karyotypic analysis of CLL patient samples to better identify chromosomal abnormalities for risk stratification. PMID:21494579

  17. Transplacental arsenic plus postnatal 12-O-teradecanoyl phorbol-13-acetate exposures associated with hepatocarcinogenesis induce similar aberrant gene expression patterns in male and female mouse liver

    SciTech Connect

    Liu Jie; Xie Yaxiong; Merrick, B. Alex

    2006-06-15

    Our prior work shows that in utero arsenic exposure alone is a complete transplacental carcinogen, producing hepatocellular carcinoma in adult male offspring but not in females. In a follow-up study to potentially promote arsenic-initiated tumors, mice were exposed to arsenic (85 ppm) from gestation day 8 to 18 and then exposed to 12-O-teradecanoyl phorbol-13-acetate (TPA), a well-known tumor promoter after weaning. The dermal application of TPA (2 {mu}g/0.1 ml acetone, twice/week for 21 weeks) after transplacental arsenic did not further increase arsenic-induced liver tumor formation in adult males but significantly increased liver tumor formation in adult females. Thus, for comparison,more » liver tumors and normal liver samples taken from adult male and female mice at necropsy were analyzed for aberrant gene/protein expression by microarray, real-time RT-PCR and Western blot analysis. Arsenic/TPA treatment resulted in increased expression of {alpha}-fetoprotein, k-ras, c-myc, estrogen receptor-{alpha}, cyclin D1, cdk2na, plasminogen activator inhibitor-1, cytokeratin-8, cytokeratin-18, glutathione S-transferases and insulin-like growth factor binding proteins in liver and liver tumors from both male and female mice. Arsenic/TPA also decreased the expression of BRCA1, betaine-homocysteine methyltransferase, CYP7B1, CYP2F2 and insulin-like growth factor-1 in normal and cancerous livers. Alterations in these gene products were associated with arsenic/TPA-induced liver tumors, regardless of sex. Thus, transplacental arsenic plus postnatal TPA exposure induced similar aberrant gene expression patterns in male and female mouse liver, which are persistent and potentially important to the mechanism of arsenic initiation of hepatocarcinogenesis.« less

  18. Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo

    SciTech Connect

    Kundu, Joydeb Kumar; Liu, Lijia; Shin, Jun-Wan

    2013-09-06

    Highlights: •Thymoquinone inhibits phorbol ester-induced COX-2 expression in mouse skin. •Thymoquinone attenuates phosphorylation of IκBα and DNA binding of NF-κB in mouse skin. •Thymoquinone inhibits phosphorylation of p38 MAP kinase, JNK and Akt in mouse skin. •Thymoquinone induces the expression of cytoprotective proteins in mouse skin. -- Abstract: Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2more » (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.« less

  19. Effect of silybin on phorbol myristate actetate-induced protein kinase C translocation, NADPH oxidase activity and apoptosis in human neutrophils.

    PubMed

    Varga, Zs; Ujhelyi, L; Kiss, A; Balla, J; Czompa, A; Antus, S

    2004-02-01

    Mechanism of the action of silybin (1) and its derivatives (2-4), possessing different lipid solubility in PMA-stimulated neutrophils was evaluated. Silybin (1) inhibited the calcium, phosphatidylserine- and diacylglycerol-dependent protein kinase C translocation and the NADPH oxidase activity in PMA-stimulated neutrophils and resulted in decreased apoptosis. Furthermore, silybin (1) inhibited xanthine oxidase activity and hem-mediated oxidative degradation of low-density lipoprotein, as well. Its derivatives (2-4), possessing different lipid-solubility, affected all the studied parameters. The lipid solubility of silybin (1) was enhanced by methylation (5'7'4''trimethylsilybin: 2), whereas a decrease in lipid-solubility by acetylation of compound 2 (5',7,'4"-trimethylsilybin-acetate: 3) or all the hydroxyl groups of silybin (peracetyl-silybin: 4) attenuated the antioxidant capacity by decreasing the inhibition in PKC translocation and NADPH oxidase activation. All the derivatives of silybin (2-4) showed no inhibition in cell free systems; e.g. did not alter the xanthine oxidase activity and the hem-mediated oxidative degradation of LDL. In conclusion, the antioxidant activity of (1) might be due to its ability to inhibit PKC translocation and NADPH oxidase activation in PMA-stimulated neutrophils. The increase of lipid solubility of silybin (1) supports its penetration through cell membrane and enhances its inhibitory effects. This structural modification of (1) might have pharmacological consequences.

  20. Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

    SciTech Connect

    Xun, C.Q.; Ensor, C.M.; Tai, H.H.

    1991-06-28

    Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

  1. MDP(Lysyl)GDP, a nontoxic muramyl dipeptide derivative, inhibits cytokine production by activated macrophages and protects mice from phorbol ester- and oxazolone-induced inflammation.

    PubMed

    Zunic, M; Bahr, G M; Mudde, G C; Meingassner, J G; Lam, C

    1998-07-01

    High levels of pro-inflammatory cytokines and nitric oxide are proposed to orchestrate pathophysiologic mechanism(s) associated with various inflammatory dermatoses. This study examines whether a water soluble 3-O-[N-acetylmuramyl-L-lysyl-D-iso]-2-di-on-glycine [MDP(Lysyl)GDP], a nontoxic and nonpyrogenic derivative of muramyl dipeptide (MDP), can inhibit the in vitro production of inflammatory mediators by lipopolysaccharide- or interferon-gamma-activated macrophages, and whether such an inhibitory effect can translate into in vivo protection of mice from irritant and allergic contact dermatitis. Thioglycollate-elicited peritoneal macrophages cultured in medium alone or in medium supplemented with MDP(Lysyl)GDP (1-100 microg per ml) expressed neither mRNA transcripts for inducible nitric oxide synthase, interleukin-1beta, and tumor necrosis factor-alpha, nor cytokine proteins and nitric oxide activity. Incubation of the cells with either lipopolysaccharide or interferon-gamma for 6 h resulted in a significant induction of inducible nitric oxide synthase, interleukin-1beta, and tumor necrosis factor-alpha mRNA, and the accumulation of high levels of monokines and nitrites in cultures by 24 h. Co-incubation of the macrophages with lipopolysaccharide or interferon-gamma and MDP(Lysyl)GDP (1-100 microg per ml) resulted in a concentration-dependent suppression of the steady-state mRNA transcripts for inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1beta, induced by lipopolysaccharide, but not by interferon-gamma. In mouse models of phorbol ester- and oxazolone-induced ear inflammation, topical application of MDP(Lysyl)GDP significantly suppressed ear swelling in a dose-dependent manner. Likewise, oral treatment with MDP(Lysyl)GDP at days -3, -2, and -1 before elicitation with oxazolone also significantly inhibited ear inflammation. Taken together, our findings suggest that MDP(Lysyl)GDP has the potential to be a therapeutic application in

  2. The Croton megalobotrys Müll Arg. traditional medicine in HIV/AIDS management: Documentation of patient use, in vitro activation of latent HIV-1 provirus, and isolation of active phorbol esters.

    PubMed

    Tietjen, Ian; Ngwenya, Barbara N; Fotso, Ghislain; Williams, David E; Simonambango, Sundana; Ngadjui, Bonaventure T; Andersen, Raymond J; Brockman, Mark A; Brumme, Zabrina L; Andrae-Marobela, Kerstin

    2018-01-30

    Current HIV therapies do not act on latent cellular HIV reservoirs; hence they are not curative. While experimental latency reversal agents (LRAs) can promote HIV expression in these cells, thereby exposing them to immune recognition, existing LRAs exhibit limited clinical efficacy and high toxicity. We previously described a traditional 3-step medicinal plant regimen used for HIV/AIDS management in Northern Botswana that inhibits HIV replication in vitro. Here we describe use of one component of the regimen that additionally contains novel phorbol esters possessing HIV latency-reversal properties. We sought to document experiences of traditional medicine users, assess the ability of traditional medicine components to reverse HIV latency in vitro, and identify pure compounds that conferred these activities. Experiences of two HIV-positive traditional medicine users (patients) were documented using qualitative interview techniques. Latency reversal activity was assessed using a cell-based model (J-Lat, clone 9.2). Crude plant extracts were fractionated by open column chromatography and reverse-phase HPLC. Compound structures were elucidated using NMR spectroscopy and mass spectrometry. Patients using the 3-step regimen reported improved health over several years despite no reported use of standard HIV therapies. Crude extracts from Croton megalobotrys Müll Arg. ("Mukungulu"), the third component of the 3-step regimen, induced HIV expression in J-lat cells to levels comparable to the known LRA prostratin. Co-incubation with known LRAs and pharmacological inhibitors indicated that the active agent(s) in C. megalobotrys were likely to be protein kinase C (PKC) activator(s). Consistent with these results, two novel phorbol esters (Namushen 1 and 2) were isolated as abundant components of C. megalobotrys and were sufficient to confer HIV latency reversal in vitro. We have identified novel LRAs of the phorbol ester class from a medicinal plant used in HIV/AIDS management

  3. Activity of artichoke leaf extract on reactive oxygen species in human leukocytes.

    PubMed

    Pérez-García, F; Adzet, T; Cañigueral, S

    2000-11-01

    Artichoke leaf extract was studied in human leukocytes for activity against oxidative stress using flow cytometry and dichlorofluorescin diacetate as a fluorescence probe. It produces a concentration-dependent inhibition of oxidative stress when cells are stimulated with agents that generate reactive oxygen species (ROS): hydrogen peroxide, phorbol-12-myristate-13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Cynarin, caffeic acid, chlorogenic acid, and luteolin, constituents of artichoke leaf extract, also show a concentration-dependent inhibitory activity in the above models, contributing to the antioxidant activity of the extract in human neutrophils.

  4. The stimulation of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils by phorbol myristate acetate, opsonized zymosan and IgG2-containing soluble immune complexes.

    PubMed Central

    Baxter, M A; Leslie, R G; Reeves, W G

    1983-01-01

    The kinetics of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils were determined following in vitro stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ) and soluble immune complexes of guinea-pig IgG2 (SIC). Superoxide production was recorded as chemiluminescence (CL) arising from the reductive cleavage of lucigenin. With PMA, both macrophages and neutrophils displayed a two-phase response consisting of a rapid initial burst of CL, which preceded ligand ingestion, followed by a plateau in the CL response which persisted for more than 30 min. By contrast, OZ induced a slow progressive increase in CL in both phagocytes which was consistent with the development of an oxidative burst concomitant with ingestion. The phagocytes differed in their responses to SIC, the macrophages displaying CL kinetics similar to those observed with PMA, whereas the neutrophils responded in the manner observed with OZ. The relationship between disparity in the patterns of macrophage and neutrophil CL responses to SIC and differences in their expression of Fc receptors for IgG2 (Coupland & Leslie, 1983) is discussed. PMID:6299935

  5. Phorbol esters induce death in MCF-7 breast cancer cells with altered expression of protein kinase C isoforms. Role for p53-independent induction of gadd-45 in initiating death.

    PubMed Central

    de Vente, J E; Kukoly, C A; Bryant, W O; Posekany, K J; Chen, J; Fletcher, D J; Parker, P J; Pettit, G J; Lozano, G; Cook, P P

    1995-01-01

    Protein kinase C (PKC) modulates growth, differentiation and apoptosis in a cell-specific fashion. Overexpression of PKC-alpha in MCF-7 breast cancer cells (MCF-7-PKC-alpha cell) leads to expression of a more transformed phenotype. The response of MCF-7 and MCF-7-PKC-alpha cells to phorbol esters (TPA) was examined. TPA-treated MCF-7 cells demonstrated a modest cytostatic response associated with a G1 arrest that was accompanied by Cip1 expression and retinoblastoma hypophosphorylation. While p53 was detected in MCF-7 cells, evidence for TPA-induced stimulation of p53 transcriptional activity was not evident. In contrast, TPA treatment induced death of MCF-7-PKC-alpha cells. Bryostatin 1, another PKC activator, exerted modest cytostatic effects on MCF-7 cells while producing a cytotoxic response at low doses in MCF-7-PKC-alpha cells that waned at higher concentrations. TPA-treated MCF-7-PKC-alpha cells accumulated in G2/M, did not express p53, displayed decreased Cip1 expression, and demonstrated a reduction in retinoblastoma hypophosphorylation. TPA-treated MCF-7-PKC-alpha cells expressed gadd-45 which occurred before the onset of apoptosis. Thus, alterations in the PKC pathway can modulate the decision of a breast cancer cell to undergo death or differentiation. In addition, these data show that PKC activation can induce expression of gadd45 in a p53-independent fashion. Images PMID:7560079

  6. Inhibitory effects of curcumin and capsaicin on phorbol ester-induced activation of eukaryotic transcription factors, NF-kappaB and AP-1.

    PubMed

    Surh, Y J; Han, S S; Keum, Y S; Seo, H J; Lee, S S

    2000-01-01

    Recently, considerable attention has been focused on identifying dietary and medicinal phytochemicals that can inhibit, retard or reverse the multi-stage carcinogenesis. Spices and herbs contain phenolic substances with potent antioxidative and chemopreventive properties. Curcumin, a yellow colouring agent from turmeric and capsaicin, a pungent principle of red pepper exhibit profound anticarcinogenic and antimutagenic activities. Two well-defined eukaryotic transcription factors, nuclear factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) have been implicated in pathogenesis of many human diseases including cancer. These transcription factors are known to be activated by a wide array of external stimuli, such as tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor, reactive oxygen species, bacterial lipopolysaccharide, and ultraviolet. In the present study, we found that topical application of TPA onto dorsal skin of female ICR mice resulted in marked activation of epidermal NF-kappaB and AP-1. Curcumin and capsaicin, when topically applied prior to TPA, significantly attenuated TPA-induced activation of each transcription factor in mouse skin. Likewise, both compounds inhibited NF-kappaB and AP-1 activation in cultured human promyelocytic leukemia (HL-60) cells stimulated with TPA. Based on these findings, it is likely that curcumin and capsaicin exert anti-tumor promotional effects through suppression of the tumor promoter-induced activation of transcription factors, NF-kappaB and AP-1.

  7. Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6

    PubMed Central

    Schmitt, J; Noble, A; Otsuka, M; Berry, P; Maitland, N J; Rumsby, M G

    2014-01-01

    Background: Malignancy alters cellular complex lipid metabolism and membrane lipid composition and turnover. Here, we investigated whether tumorigenesis in cancer-derived prostate epithelial cell lines influences protein kinase C-linked turnover of ethanolamine phosphoglycerides (EtnPGs) and alters the pattern of ethanolamine (Etn) metabolites released to the medium. Methods: Prostate epithelial cell lines P4E6, LNCaP and PC3 were models of prostate cancer (PCa). PNT2C2 and PNT1A were models of benign prostate epithelia. Cellular EtnPGs were labelled with [1-3H]-Etn hydrochloride. PKC was activated with phorbol ester (TPA) and inhibited with Ro31-8220 and GF109203X. D609 was used to inhibit PLD (phospholipase D). [3H]-labelled Etn metabolites were resolved by ion-exchange chromatography. Sodium oleate and mastoparan were tested as activators of PLD2. Phospholipase D activity was measured by a transphosphatidylation reaction. Cells were treated with ionomycin to raise intracellular Ca2+ levels. Results: Unstimulated cell lines release mainly Etn and glycerylphosphorylEtn (GPEtn) to the medium. Phorbol ester treatment over 3h increased Etn metabolite release from the metastatic PC3 cell line and the benign cell lines PNT2C2 and PNT1A but not from the tumour-derived cell lines P4E6 and LNCaP; this effect was blocked by Ro31-8220 and GF109203X as well as by D609, which inhibited PLD in a transphosphatidylation reaction. Only metastatic PC3 cells specifically upregulated Etn release in response to TPA treatment. Oleate and mastoparan increased GPEtn release from all cell lines at the expense of Etn. Ionomycin stimulated GPEtn release from benign PNT2C2 cells but not from cancer-derived cell lines P4E6 or PC3. Ethanolamine did not stimulate the proliferation of LNCaP or PC3 cell lines but decreased the uptake of choline (Cho). Conclusions: Only the metastatic basal PC3 cell line specifically increased the release of Etn on TPA treatment most probably by PKC activation of

  8. Dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppressed the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor.

    PubMed

    Lee, Hyun Jae; Park, Jin Sung; Yoon, Yong Pill; Shin, Ye Jin; Lee, Sang Kook; Kim, Yeong Shik; Hong, Jang-Hee; Son, Kun Ho; Lee, Choong Jae

    2015-05-15

    The root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine. We investigated whether dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis (Lour.) Merr. suppress the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor. Confluent NCI-H292 cells were pretreated with dioscin or methylprotodioscin for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. (1) Dioscin and methylprotodioscin suppressed the expression of MUC5AC mucin gene induced by EGF or PMA; (2) dioscin suppressed the production of MUC5AC mucin induced by either EGF at 10(-5) M (p < 0.05) and 10(-6) M (p < 0.05) or PMA at 10(-4) M (p < 0.05), 10(-5) M (p < 0.05) and 10(-6) M (p < 0.05); (3) methylprotodioscin also suppressed the production of MUC5AC mucin induced by either EGF at 10(-4) M (p < 0.05) or PMA at 10(-4) M (p < 0.05). These results suggest that dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppress the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Asparagus cochinchinensis as remedy for diverse inflammatory pulmonary diseases. Copyright © 2015 Elsevier GmbH. All rights reserved.

  9. Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

    PubMed

    Yoshimura, T; Mayumi, M; Yorifuji, T; Kim, K M; Heike, T; Miyanomae, T; Shinomiya, K; Mikawa, H

    1987-09-01

    A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

  10. Suppressed PHA Activation of T Lymphocytes in Simulated Microgravity is Restored by Direct Activation of Protein Kinase C with Phorbol Ester

    NASA Technical Reports Server (NTRS)

    Cooper, David; Pellis, Neal R.

    1997-01-01

    Various aspects of spaceflight, including microgravity, cosmic radiation, and physiological stress, may perturb immune function. We sought to understand the impact of microgravity alone on the cellular mechanisms critical to immunity. We utilized clinostatic RWV bioreactors that simulate aspects of microgravity to analyze the response of human PBMC to polyclonal activation. PHA responsiveness in the RWV was almost completely diminished. IL-2 and IFN-gamma secretion was reduced whereas IL- 1 beta and IL-6 secretion was increased, suggesting that monocytes may not be as adversely affected by simulated microgravity as T cells. Activation marker expression (CD25, CD69, CD71) was significantly reduced in RWV cultures. Furthermore, addition of exogenous IL-2 to these cultures did not restore proliferation. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness. However, PHA activation in Teflon culture bags that limit cell-substratum interactions did not suppress PHA activation. Furthermore, increasing cell density and, therefore, cell-cell interactions in the RWV cultures did not help restore PHA activation. However, placing PBMC within small collagen beads did partially restore PHA responsiveness. Activation of both PBMC and purified T cells with PMA and ionomycin was unaffected by RWV culture, indicating that signaling mechanisms downstream of PKC activation and calcium flux are not sensitive to simulated microgravity. Furthermore, submitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus, our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

  11. NOX5 in Human Spermatozoa

    PubMed Central

    Musset, Boris; Clark, Robert A.; DeCoursey, Thomas E.; Petheo, Gabor L.; Geiszt, Miklos; Chen, Yumin; Cornell, John E.; Eddy, Carlton A.; Brzyski, Robert G.; El Jamali, Amina

    2012-01-01

    Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H2O2 exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca2+ chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H2O2-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the HV1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and HV1. H2O2 treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility. PMID:22291013

  12. Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

    SciTech Connect

    Scherer, N.M.; Nathanson, N.M.

    1990-09-11

    Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA{sup {minus}}). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA{sup +} or PKA{sup {minus}} cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA{sup {minus}} cells. These data indicate that the basal activity of PKAmore » may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA{sup +} and PKA{sup {minus}} cells was accompanied by desensitization of functional responses. Exposure of PKA{sup +} cells to 10{sup {minus}7} M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 {plus minus} 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA{sup +} or PKA{sup {minus}} cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA{sup {minus}} cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA{sup +} or PKA{sup {minus}} cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%.« less

  13. Snapshot Profiling of the Antileishmanial Potency of Lead Compounds and Drug Candidates against Intracellular Leishmania donovani Amastigotes, with a Focus on Human-Derived Host Cells.

    PubMed

    Koniordou, Markela; Patterson, Stephen; Wyllie, Susan; Seifert, Karin

    2017-03-01

    This study characterized the in vitro potencies of antileishmanial agents against intracellular Leishmania donovani amastigotes in primary human macrophages, obtained with or without CD14-positive monocyte enrichment, phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells, and mouse peritoneal exudate macrophages (PEMs). Host cell-dependent potency was confirmed for pentavalent and trivalent antimony. Fexinidazole was inactive against intracellular amastigotes across the host cell panel. Fexinidazole sulfone, ( R )-PA-824, ( S )-PA-824, and VL-2098 displayed similar potency in all of the host cells tested. Copyright © 2017 American Society for Microbiology.

  14. Expression of the human c-fms proto-oncogene product (colony-stimulating factor-1 receptor) on peripheral blood mononuclear cells and choriocarcinoma cell lines.

    PubMed Central

    Rettenmier, C W; Sacca, R; Furman, W L; Roussel, M F; Holt, J T; Nienhuis, A W; Stanley, E R; Sherr, C J

    1986-01-01

    The c-fms gene product is related, and possibly identical, to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. Using antisera to a recombinant v-fms--coded polypeptide, glycoproteins encoded by the human c-fms locus were detected in mononuclear cells from normal peripheral blood and in promyelocytic HL-60 cells 24 h after induction of monocytic differentiation with phorbol ester. The 150-kD human c-fms--coded glycoprotein was expressed at the cell surface, was active as a tyrosine-specific protein kinase in vitro, and shared primary structural features with the product of the feline retroviral v-fms oncogene. A biochemically indistinguishable glycoprotein was detected in human choriocarcinoma cell lines. Like peripheral blood mononuclear cells and phorbol ester-treated HL-60 cells, the choriocarcinoma cells expressed high affinity binding sites for human CSF-1. In addition to serving as a lineage specific growth factor in hematopoiesis, CSF-1 may play a role in normal trophoblast development. Images PMID:3011859

  15. The production of collagenase by adherent mononuclear cells cultured from human peripheral blood.

    PubMed

    Louie, J S; Weiss, J; Ryhänen, L; Nies, K M; Rantala-Ryhänen, S; Uitto, J

    1984-12-01

    Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque centrifugation, and the cells adherent to plastic substrata were cultured in serum-free media supplemented with lactalbumin hydrolysate. These cell cultures, which consisted predominantly of monocyte-macrophages as judged by nonspecific esterase staining, accumulated collagenase in the medium. This collagenase resembled other vertebrate collagenases in that it cleaved native triple-helical type I collagen at a locus 3/4-length away from the amino-terminal end of the molecule. The collagenase activity was inhibited by Na2EDTA, dithiothreitol, and fetal calf serum, while the addition of Ca++ or N-ethylmaleimide enhanced the enzyme activity. The accumulation of collagenase in the culture media was markedly enhanced by the incubation of cells with concanavalin A or phorbol myristic acetate. In the presence of cycloheximide, the levels of collagenase activity were markedly reduced, suggesting that active protein synthesis was required to express the enzyme activity. In additional experiments, monocytes were further purified by counterflow centrifugation-elutriation. The collagenase production was markedly increased in cultures enriched in monocyte-macrophages and devoid of polymorphonuclear leukocytes. The accumulation of collagenase in monocyte cultures incubated for 48 hours in the presence of concanavalin A or phorbol myristic acetate was of the same order of magnitude as in parallel cultures containing the same number of polymorphonuclear leukocytes purified by Ficoll-Hypaque centrifugation and Plasmagel sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Anti-inflammatory effects of zinc in PMA-treated human gingival fibroblast cells

    PubMed Central

    Kim, Sangwoo; Jeon, Sangmi; Hui, Zheng; Kim, Young; Im, Yeonggwan; Lim, Wonbong; Kim, Changsu; Choi, Hongran; Kim, Okjoon

    2015-01-01

    Objectives: Abnormal cellular immune response has been considered to be responsible for oral lesions in recurrent aphthous stomatitis. Zinc has been known to be an essential nutrient metal that is necessary for a broad range of biological activities including antioxidant, immune mediator, and anti-inflammatory drugs in oral mucosal disease. The objective of this study was to investigate the effects of zinc in a phorbol-12-myristate-13-acetate (PMA)-treated inflammatory model on human gingival fibroblast cells (hGFs). Study Design: Cells were pre-treated with zinc chloride, followed by PMA in hGFs. The effects were assessed on cell viability, cyclooxygenease-1,2(COX-1/2) protein expression, PGE2 release, ROS production and cytokine release, Results: The effects were assessed on cell viability, COX1/2 protein expression, PGE2 release, ROS production, cytokine release. The results showed that, in the presence of PMA, zinc treatment leads to reduce the production of ROS, which results in decrease of COX-2 expression and PGE2 release. Conclusions: Thus, we suggest that zinc treatment leads to the mitigation of oral inflammation and may prove to be an alternative treatment for recurrent aphthous stomatitis. Key words:Zinc, inflammatory response, cytokines, phorbol-12-myristate-13-acetate, gingival fibroblasts cells. PMID:25662537

  17. Interleukin 1 production by human polymorphonuclear neutrophils.

    PubMed

    Tiku, K; Tiku, M L; Skosey, J L

    1986-05-15

    The purpose of this study was to determine whether human polymorphonuclear neutrophils (PMN), which share a common cell lineage with macrophages, could produce factors such as IL 1. Other properties which these two cell types share are their phagocytic nature and the common receptor and antigens on their cell surfaces. IL 1, in many of its physical, biochemical, and functional characteristics, is found to resemble endogenous pyrogen (EP). PMN have been cited as a possible cell source of EP, but there have also been reports in which the capacity of PMN to produce EP has been questioned. This study shows that normal human PMN can be stimulated by particulate agents such as zymosan and soluble agents such as phorbol myristic acetate to produce a factor(s) which induces proliferation of mouse thymocytes, i.e., PMN IL 1. This PMN IL 1 was released from PMN in a dose- and time-dependent fashion. PMN IL 1 was nondialyzable, was heat-labile, and was inactivated at pH below 5 and above 8. PMN IL 1 stimulated the proliferation of normal human synovial fibroblasts and caused release of a neutral protease (plasminogen activator) from synovial cells. The synovial and thymocyte-proliferating capacity of PMN IL 1 was not affected by the protease inhibitor aprotinin or by soybean trypsin inhibitor. Gel filtration studies estimate the m.w. of PMN IL 1 to be approximately 13,000 to 17,000.

  18. Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

    NASA Astrophysics Data System (ADS)

    Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda

    2004-06-01

    Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

  19. Functional activity of enucleated human polymorphonuclear leukocytes

    PubMed Central

    1983-01-01

    Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells. PMID:6309859

  20. Human chorionic gonadotropin induces human macrophages to form intracytoplasmic vacuoles mimicking Hofbauer cells in human chorionic villi.

    PubMed

    Yamaguchi, Munekage; Ohba, Takashi; Tashiro, Hironori; Yamada, Gen; Katabuchi, Hidetaka

    2013-01-01

    The most characteristic morphological feature of macrophages in the stroma of placental villi, known as Hofbauer cells, is their highly vacuolated appearance. They also show positive immunostaining for human chorionic gonadotropin (hCG) and express messenger ribonucleic acid of the luteinizing hormone/chorionic gonadotropin receptor with a deletion of exon 9 (LH/CG-R Δ9). Maternal hCG enters fetal plasma through the mesenchyme of the placental villi and promotes male sexual differentiation in early pregnancy; therefore, excess hCG may induce aberrant genital differentiation and hCG must be adjusted at the fetomaternal interface. We hypothesized that hCG is regulated by Hofbauer cells and that their peculiar vacuoles are involved in a cell-specific function. To assess the morphological modification and expression of LH/CG-R Δ9 in human macrophages after hCG exposure, the present study examined phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells, a human monocyte-macrophage cell line. hCG induced transient vacuole formation in PMA-treated THP-1 cells, morphologically mimicking Hofbauer cells. Immunocytochemistry showed that PMA-treated THP-1 cells incorporated hCG but not luteinizing hormone or follicle-stimulating hormone. Western blotting analyses demonstrated that PMA-treated THP-1 cells expressed an immunoreactive 60-kDa protein, designated as endogenous LH/CG-R Δ9. hCG induced a transient reduction in the LH/CG-R Δ9, which was synchronous with the appearance of cytoplasmic vacuoles. In conclusion, human macrophages regulating hCG via cytoplasmic LH/CG-R Δ9 mimic the morphological characteristics of Hofbauer cells. Their vacuoles may be associated with their cell-specific function to protect the fetus from exposure to excess maternal hCG during pregnancy. Copyright © 2012 S. Karger AG, Basel.

  1. Superoxide radical-scavenging effects from polymorphonuclear leukocytes and toxicity in human cell lines of newly synthesized organic selenium compounds.

    PubMed

    Tsukagoshi, Hiroyuki; Koketsu, Mamoru; Kato, Masahiko; Kurabayashi, Masahiko; Nishina, Atsuyoshi; Kimura, Hirokazu

    2007-12-01

    Synthetic organic selenium compounds such as 2-phenyl-1,2-benzisoselenazol-3(2H)-one may show glutathione peroxidase-like antioxidant activity. Recently, we synthesized new organic selenium compounds that are thought to be effective antioxidants. To study their possible applications as antioxidants, we evaluated two selenoureas, N,N-dimethylselenourea and 1-selenocarbamoylpyrrolidine, and two tertiary selenoamides, N-(phenylselenocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide, for their superoxide radical (O2-)-scavenging effects and toxicity. We measured (O2-)-scavenging effects in polymorphonuclear leukocytes (PMNs) with a specific, sensitive and real-time kinetic chemiluminescence method. Furthermore, the toxicity of these compounds was measured in some human cell lines and PMNs using the tetrazolium method. Hydrogen peroxide was measured by a scopoletin method. Finally, translocation of an NADPH oxidase component, p47 phagocyte oxidase, to the cell membrane was investigated by confocal laser scanning microscopy. N,N-Dimethylselenourea and 1-selenocarbamoylpyrrolidine effectively scavenged (O2-) released from 4beta-phorbol 12-myristate 13-acetate-stimulated PMNs, and the 50% inhibitory concentrations were 6.8 +/- 2.2 and 6.5 +/- 2.5 microm, respectively. N-(Phenylselenocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide also effectively scavenged (O2-) from PMNs, and the 50% inhibitory concentrations were 11.3 +/- 4.8 and 20.3 +/- 6.4 microm, respectively. Selenoureas showed very low toxicity in human cell lines and PMNs, even at high concentrations, whereas tertiary selenoamides were cytotoxic. These compounds did not produce significant amounts of hydrogen peroxide from 4beta-phorbol 12-myristate 13-acetate-stimulated PMNs. None of the compounds significantly affected the translocation of p47 phagocyte oxidase. Selenoureas acted as effective antioxidants and showed low toxicity in some human cells. Thus, these compounds might be new

  2. Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva.

    PubMed

    Xiao, Li; Okamura, Hisashi; Kumazawa, Yasuo

    2018-04-03

    Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 µg/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

  3. Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

    NASA Technical Reports Server (NTRS)

    Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

    1997-01-01

    A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

  4. Stimulation of bumetanide-sensitive Na+/K+/Cl- cotransport by different mitogens in synchronized human skin fibroblasts is essential for cell proliferation

    PubMed Central

    1991-01-01

    In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl- cotransport in the mitogenic signal of human skin fibroblast proliferation. The Na+/K+/Cl- cotransport was dramatically stimulated by either fetal calf serum, or by recombinant growth factors, added to quiescent G0/G1 human skin fibroblasts. The following mitogens, FGF, PDGF, alpha-thrombin, insulin-like growth factor-1, transforming growth factor-alpha, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13- acetate, all stimulated the Na+/K+/Cl- cotransport. In addition, all the above mitogens induced DNA synthesis in the synchronized human fibroblasts. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal, the effect of two specific inhibitors of the cotransport, furosemide and bumetanide, was tested on cell proliferation induced by the above recombinant growth factors. Bumetanide and furosemide inhibited synchronized cell proliferation as was measured by (a) cell exit from the G0/G1 phase measured by the use of flow cytometry, (b) cell entering the S-phase, determined by DNA synthesis, and (c) cell growth, measured by counting the cells. The inhibition by furosemide and bumetanide was reversible, removal of these compounds, completely released the cells from the block of DNA synthesis. In addition, the two drugs inhibited DNA synthesis only when added within the first 2-6 h of cell release. These results indicate that the effect of these drugs is specific, and is not due to an indirect toxic effect. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport plays a major role in the mitogenic signaling pathway of the human fibroblasts. PMID:2071675

  5. Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester.

    PubMed Central

    Sivanandam, Arun S; Mohan, Subburaman; Kita, Hirohito; Kapur, Sanjay; Chen, Shin-Tai; Linkhart, Thomas A; Bagi, Gyorgy; Baylink, David J; Qin, Xuezhong

    2004-01-01

    PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP-PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a approximately 470 kDa PAPP-A form (PAPP-A470) to a approximately 400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470

  6. Differential regulation of human RecQ family helicases in cell transformation and cell cycle.

    PubMed

    Kawabe, T; Tsuyama, N; Kitao, S; Nishikawa, K; Shimamoto, A; Shiratori, M; Matsumoto, T; Anno, K; Sato, T; Mitsui, Y; Seki, M; Enomoto, T; Goto, M; Ellis, N A; Ide, T; Furuichi, Y; Sugimoto, M

    2000-09-28

    Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated. In B cells this expression was stimulated within 5-40 h by the tumor promoting agent phorbol myristic acetate (PMA). Interestingly, RecQL5beta, an alternative splicing product of RecQL5 with a nuclear localization signal, is expressed in resting B cells without significant modulation of its synthesis by EBV or PMA, suggesting it has a role in resting cells. We also roughly determined the number of copies per cell for the five RecQ helicase in B cells. In addition, levels of the different RecQ helicases are modulated in different ways during the cell cycle of actively proliferating fibroblasts and umbilical endothelial cells. Our results support the view that the levels of WRN, BLM, RTS and RecQL1 are differentially up-regulated to guarantee genomic stability in cells that are transformed or actively proliferating.

  7. Identification of three related human GRO genes encoding cytokine functions

    SciTech Connect

    Haskill, S.; Peace, A.; Morris, J.

    1990-10-01

    The product of the human GRO gene is a cytokine with inflammatory and growth-regulatory properties; GRO is also called MGSA for melanoma growth-stimulatory activity. The authors have identified two additional genes, GRO{beta} and GRO{gamma}, that share 90{percent} and 86{percent} identity at the deduced amino acid level with the original GRO{alpha} isolate. One amino acid substitution of proline in GRO{alpha} by leucine in GRO{beta} and GRO{gamma} leads to a large predicted change in protein conformation. Significant differences also exist in the 3' untranslated region, including different numbers of ATTTA repeats associated with mRNA instability. A 122-base-pair region in the 3' regionmore » is conserved among the three GRO genes, and a part of it is also conserved in the Chinese hamster genome, suggesting a role in regulation. DNA hybridization with oligonucleotide probes and partial sequence analysis of the genomic clones confirm that the three forms are derived from related but different genes. Only one chromosomal locus has been identified, at 4q21, by using a GRO{alpha} cDNA clone that hybridized to all three genes. Expression studies reveal tissue-specific regulation as well as regulation by specific inducing agents, including interleukin 1, tumor necrosis factor, phorbol 12-myristate 13-acetate, and lipopolysaccharide.« less

  8. Hypochlorous acid regulates neutrophil extracellular trap release in humans

    PubMed Central

    Palmer, L J; Cooper, P R; Ling, M R; Wright, H J; Huissoon, A; Chapple, I L C

    2012-01-01

    Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine. PMID:22236002

  9. Hypochlorous acid regulates neutrophil extracellular trap release in humans.

    PubMed

    Palmer, L J; Cooper, P R; Ling, M R; Wright, H J; Huissoon, A; Chapple, I L C

    2012-02-01

    Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine. © 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.

  10. Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus

    PubMed Central

    Rubins, Kathleen H.; Hensley, Lisa E.; Relman, David A.; Brown, Patrick O.

    2011-01-01

    Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection. PMID:21267444

  11. Dioctanoylglycerol stimulates accumulation of [methyl-14C]choline and its incorporation into acetylcholine and phosphatidylcholine in a human cholinergic neuroblastoma cell line

    NASA Technical Reports Server (NTRS)

    Slack, B. E.; Richardson, U. I.; Nitsch, R. M.; Wurtman, R. J.

    1992-01-01

    Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [14C]choline uptake in cultured human neuroblastoma (LA-N-2) cells. As this effect has not, to our knowledge, been reported before, it was of interest to characterize it in more detail. In the presence of 500 microM dioctanoylglycerol the levels of [14C]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [14C]acetylcholine and long fatty acyl chain [14C]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [14C]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent Km for choline uptake to 56 +/- 9% of control (n = 4). The Vmax was not significantly altered. The stimulation of [14C]choline accumulation by dioctanoylglycerol was not dependent on protein kinase C activation; the effect was not mimicked by phorbol ester or by 1-oleoyl-2-acetylglycerol, and was not inhibited by the protein kinase C inhibitors H-7 or staurosporine, or by prolonged pretreatment with phorbol 12-myristate 13-acetate. The effect of dioctanoylglycerol was slightly (but not significantly) reduced by EGTA and strongly inhibited by the cell-permeant calcium chelator bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)ester. Although these results implicate elevated intracellular calcium in the response, dioctanoylglycerol did not increase phosphatidylinositol hydrolysis in LA-N-2 cells, and its effect was not inhibited by the diacylglycerol kinase inhibitor R 59 022 (which blocks the conversion of diacylglycerol to phosphatidic acid, a known stimulator of phosphatidylinositol hydrolysis).(ABSTRACT TRUNCATED AT 250 WORDS).

  12. Differential induction of apoptosis in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A

    SciTech Connect

    Kiguchi, Kaoru; Chubb, C.H.; Glesne, D.

    1994-09-01

    To investigate a possible relationship between apoptosis induction and protein phosphorylation in human breast carcinoma cells, the authors treated three such cell types, MB-231, MCF-7, and AU-565, wit okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, or phorbol 12 myristate 13-acetate, an activator of protein kinase C. They then examined these cells of the appearance of apoptosis markers. While OA caused multiplication arrest and cytotoxicity in all three cell lines, apoptosis was induced in MB-231 and MCF-7 cells but not in AU-565 cells. A similar cell-specific apoptosis induction was also observed after treatment with dinophysistoxin-1 (an activemore » OA analogue) and with calyculin A (a structurally unrelated protein phosphatase inhibitor) but not with analogues that either ar inactive or penetrate epithelial cells poorly. Phorbol 12-myristate 13-acetate also inhibited cell multiplication but was without effect in inducing apoptosis in these cells. Levels of the apoptosis-inhibitory protein BCL2 were examined in these cells, but they did to correlate with this differential susceptibility. They additionally treated the three cell types with 1-{beta}-D-arabinofuranosylcytosine and genistein to determine whether the AU-565 cell line would also be resistant to apoptosis induction by other chemical stimuli. Both of these agents led to the induction of apoptosis in all three cell lines. These results indicate that the AU-565 cells are specifically resistant to apoptosis induction by inhibitors of protein phosphatases 1 and 2A. This cell-specific resistance may thus allow one to identify cellular mediators of apoptosis by comparing protein phosphorylation patterns in these cells before and after treatment with OA or related inhibitors.« less

  13. Isolation of All CD44 Transcripts in Human Epidermis and Regulation of Their Expression by Various Agents.

    PubMed

    Teye, Kwesi; Numata, Sanae; Ishii, Norito; Krol, Rafal P; Tsuchisaka, Atsunari; Hamada, Takahiro; Koga, Hiroshi; Karashima, Tadashi; Ohata, Chika; Tsuruta, Daisuke; Saya, Hideyuki; Haftek, Marek; Hashimoto, Takashi

    2016-01-01

    CD44, a cell surface proteoglycan, is involved in many biological events. CD44 transcripts undergo complex alternative splicing, resulting in many functionally distinct isoforms. To date, however, the nature of these isoforms in human epidermis has not been adequately determined. In this study, we isolated all CD44 transcripts from normal human epidermis, and studied how their expressions are regulated. By RT-PCR, we found that a number of different CD44 transcripts were expressed in human epidermis, and we obtained all these transcripts from DNA bands in agarose and acrylamide gels by cloning. Detailed sequence analysis revealed 18 CD44 transcripts, 3 of which were novel. Next, we examined effects of 10 different agents on the expression of CD44 transcripts in cultured human keratinocytes, and found that several agents, particularly epidermal growth factor, hydrogen peroxide, phorbol 12-myristate 13-acetate, retinoic acid, calcium and fetal calf serum differently regulated their expressions in various patterns. Furthermore, normal and malignant keratinocytes were found to produce different CD44 transcripts upon serum stimulation and subsequent starvation, suggesting that specific CD44 isoforms are involved in tumorigenesis via different CD44-mediated biological pathways.

  14. Isolation of All CD44 Transcripts in Human Epidermis and Regulation of Their Expression by Various Agents

    PubMed Central

    Teye, Kwesi; Numata, Sanae; Ishii, Norito; Krol, Rafal P.; Tsuchisaka, Atsunari; Hamada, Takahiro; Koga, Hiroshi; Karashima, Tadashi; Ohata, Chika; Tsuruta, Daisuke; Saya, Hideyuki; Haftek, Marek; Hashimoto, Takashi

    2016-01-01

    CD44, a cell surface proteoglycan, is involved in many biological events. CD44 transcripts undergo complex alternative splicing, resulting in many functionally distinct isoforms. To date, however, the nature of these isoforms in human epidermis has not been adequately determined. In this study, we isolated all CD44 transcripts from normal human epidermis, and studied how their expressions are regulated. By RT-PCR, we found that a number of different CD44 transcripts were expressed in human epidermis, and we obtained all these transcripts from DNA bands in agarose and acrylamide gels by cloning. Detailed sequence analysis revealed 18 CD44 transcripts, 3 of which were novel. Next, we examined effects of 10 different agents on the expression of CD44 transcripts in cultured human keratinocytes, and found that several agents, particularly epidermal growth factor, hydrogen peroxide, phorbol 12-myristate 13-acetate, retinoic acid, calcium and fetal calf serum differently regulated their expressions in various patterns. Furthermore, normal and malignant keratinocytes were found to produce different CD44 transcripts upon serum stimulation and subsequent starvation, suggesting that specific CD44 isoforms are involved in tumorigenesis via different CD44-mediated biological pathways. PMID:27505250

  15. Antifungal activity against Cryptococcus neoformans strains and genotoxicity assessment in human leukocyte cells of Euphorbia tirucalli L.

    PubMed

    de Oliveira, Luís Flávio Souza; Fuentefria, Alexandre Meneghello; Klein, Fernanda da Silva; Machado, Michel Mansur

    2014-01-01

    In the last times, focus on plant research has increased all over the world. Euphorbia tirucalli L., a plant known popularly as Aveloz, and originally used in Africa, has been drawing attention for its use in the United States and Latin America, both for use as an ornamental plant and as a medicinal plant. E. tirucalli L. is a member of the family Euphorbiaceae and contains many diterpenoids and triterpenoids, in particular phorbol esters, apparently the main constituent of this plant, which are assumed to be responsible for their activities in vivo and in vitro. The in vitro antifungal activities of Euphorbia tirucalli (L.) against opportunistic yeasts were studied using microbroth dilution assay. The results showed that aqueous extract and latex preparation were effective against ten clinical strains of Cryptococcus neoformans in vitro (Latex and extract MIC range of 3.2 - > 411 μg/mL). Aiming the safe use in humans, the genotoxic effects of E. tirucalli were evaluated in human leukocytes cells. Our data show that both aqueous extract and latex preparation have no genotoxic effect in human leukocytes cells in vitro. Although the results cannot be extrapolated by itself for use in vivo, they suggest a good perspective for a therapeutic application in future. In conclusion, our results show that the aqueous extract and latex preparation from E. tirucalli L. are antifungal agents effectives against several strains of C. neoformans and do not provoke DNA damage in human leukocyte cells, considering the concentrations tested.

  16. Menadione modifies superoxide anion (O/sub 2/) generation in human neutrophils and HL-60 cells

    SciTech Connect

    Shakarjian, M.P.; Carchman, R.A.

    1986-03-05

    Menadione, a derivative of the natural vitamins K, has antineoplastic and antiinflammatory properties. Its profound biochemical effects upon resting and stimulated neutrophils make these cells a likely target of its purported therapeutic actions. They found superoxide dismutase-inhibitable O/sub 2/ production, a major mechanism of leukocyte-mediated cell damage, to be enhanced in resting neutrophils by menadione, as a function of cell number, time and concentration. Conversely, in phorbol myristate acetate (PMA)-stimulated neutrophils, menadione significantly reduced the rate and magnitude of O/sub 2/ generation. The release of O/sub 2/ induced by formylated peptide (fmlp) and calcium ionophore A23187, as well as PMAmore » were inhibited in a concentration dependent manner by menadione. Furthermore, inhibition was enhanced by incubation of resting neutrophils with menadione for 30 min prior to addition of stimulants. Also examined were the effects of menadione on human promyelocytic leukemia(HL-60) cells induced into granulocytic differentiation with dimethylsulfoxide. As in human neutrophils, menadione stimulated O/sub 2/ release in resting HL-60 cells and inhibited this response in PMA-treated cells, indicating the HL-60 cell line as suitable for the study of menadione's actions. Future experiments will elucidate the nature and loci of interaction of menadione with neutrophil activation mechanisms.« less

  17. Delta9 tetrahydrocannabinol and cannabidiol alter cytokine production by human immune cells.

    PubMed

    Srivastava, M D; Srivastava, B I; Brouhard, B

    1998-11-01

    Marijuana, a widely abused drug in the US, and its derivatives (cannabinoids) have been used in AIDS and cancer patients for treatment of intractable nausea and cachexia. Yet, objective investigations of the effect of cannabinoids on the human immune system are few. We investigated the effect of delta9 tetrahydrocannabinol (THC) and cannabidiol (CBD) on cytokine production in vitro by human leukemic T, B, eosinophilic and CD8+ NK cell lines as models. THC decreased constitutive production of IL-8, MIP-1alpha, MIP-1beta, and RANTES and phorbol ester stimulated production of TNF-alpha, GM-CSF and IFN-gamma by NK cells. It inhibited MIP-1beta in HTLV-1 positive B-cells but tripled IL-8, MIP-1alpha and MIP-1beta in B-cells and MIP-1beta in eosinophilic cells but doubled IL-8. Both cannabinoids strongly inhibited IL-10 production by HUT-78 T-cells. Results indicate that THC and nonpsychotropic CBD have complex lineage and derivative specific effects on cytokines consistent with previous animal studies. These effects while of potential benefits in some inflammatory/autoimmune diseases may worsen HIV infection, tumorigenesis and allergic inflammation in the lung.

  18. Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes.

    PubMed

    Li, Ye-Tao; Du, Xin-Ling

    2016-06-01

    Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

  19. Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase.

    PubMed

    Ellis, J A; Cross, A R; Jones, O T

    1989-09-01

    A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH----FAD----cytochrome b-245----O2.

  20. Nucleic acid binding of arylamines during the respiratory burst of human granulocytes

    SciTech Connect

    Corbett, M.D.; Corbett, B.R.

    Following stimulation with phorbol myristate acetate, human granulocytes were found to incorporate a series of arylamines into cellular nucleic acid. No such binding occurred if the granulocytes were not induced to undergo the respiratory burst. The relative amount of covalent binding to cellular DNA and RNA was found to depend strongly on the chemical structure of the arylamine. 2-Aminofluorene gave the highest ratio of DNA/RNA binding, while 4-nitroaniline showed a very low ratio of DNA/RNA binding. 4-Nitroaniline may bind only to RNA, since the degree of binding to DNA was at the level of detectability. Two other substrates, 4-chloroaniline andmore » 4-methylaniline, gave intermediary ratios of DNA/RNA binding. Studies on the possible role of the granulocyte enzyme myeloperoxidase in the activation and binding of these arylamines were conducted in vitro and also through the use of azide, an inhibitor of myeloperoxidase activity in cells. The results indicate that myeloperoxidase probably plays only a limited role in causing the covalent binding of arylamines to nucleic acid in human granulocytes. It is probably that other reactive oxygen species, which are not dependent upon myeloperoxidase for their production, are necessary for the bioactivation of some arylamines, especially for substrates such as 4-nitroaniline. A free-radical mechanism for arylamine bioactivation, and its potential role in arylamine toxicity, was presented in the context of the current scientific literature.« less

  1. 8-Mercaptoguanosine overcomes unresponsiveness of human neonatal B cells to polysaccharide antigens.

    PubMed

    Rijkers, G T; Dollekamp, I; Zegers, B J

    1988-10-01

    We have shown previously that pneumococcal polysaccharides behave as human T cell-independent type 2 Ag. When cultured in vitro with type 4 pneumococcal polysaccharides (PS4), human neonatal B cells do not or only marginally respond. Limiting dilution analysis of neonatal B cells polyclonally activated by a combination of phorbol esters, calcium ionophore, and T cells and T cell factors, however, showed that Ag-reactive B cells are present in cord blood. The frequency of anti-PS4 reactive B cells in cord blood is comparable with that of adult peripheral blood. In order to obtain more insight into the activation requirements of these PS4-reactive neonatal B cells, 8-mercaptoguanosine (8MGuo) was added to in vitro cultures. Addition of 0.5 to 1.0 mM 8MGuo resulted in a 3- to 10-fold amplification of the anti-PS4 response. the effect of 8 MGuo was most prominent when added 3 days after initiation of the culture. Based on kinetic studies, we propose that in vitro activated cells are target for 8MGuo. These data further indicate that neonatal unresponsiveness to polysaccharide Ag is not due to the physical absence of Ag-reactive B cells but more likely to be the consequence of different activation requirements as compared with adult B cells.

  2. Apolipoprotein A-I stimulates human placental lactogen release by activation of MAP kinase.

    PubMed

    Kanda, Y; Richards, R G; Handwerger, S

    1998-08-25

    Apolipoprotein A-I (apo A-I) stimulates human placental lactogen (hPL) release via protein kinase C (PKC)-dependent pathways. Since PKC has been shown to activate the MAP kinase cascade in other cell types, we examined the effect of two inhibitors of the MAP kinase cascade on apo A-I-induced hPL secretion and the effect of apo A-I on MAP kinase activity in human trophoblast cells. Apigenin (10 microM) and PD98059 (100 microM) inhibited apo A-I-induced hPL release by 94 and 73%, respectively. Moreover, apo A-I activated MAP kinase in a time- and dose-dependent manner. Activation of PKC by phorbol myristate acetate (PMA) stimulated MAP kinase activity, and down-regulation of PKC completely prevented apo A-I-stimulation of MAP kinase activity. Taken together, these results strongly suggest that activation of MAP kinase is involved in the intracellular mechanism of apo A-I-induced hPL release.

  3. Procyanidin trimer C1 derived from Theobroma cacao reactivates latent human immunodeficiency virus type 1 provirus.

    PubMed

    Hori, Takanori; Barnor, Jacob; Huu, Tung Nguyen; Morinaga, Osamu; Hamano, Akiko; Ndzinu, Jerry; Frimpong, Angela; Minta-Asare, Keren; Amoa-Bosompem, Mildred; Brandful, James; Odoom, John; Bonney, Joseph; Tuffour, Isaac; Owusu, Baffour-Awuah; Ofosuhene, Mark; Atchoglo, Philip; Sakyiamah, Maxwell; Adegle, Richard; Appiah-Opong, Regina; Ampofo, William; Koram, Kwadwo; Nyarko, Alexander; Okine, Laud; Edoh, Dominic; Appiah, Alfred; Uto, Takuhiro; Yoshinaka, Yoshiyuki; Uota, Shin; Shoyama, Yukihiro; Yamaoka, Shoji

    2015-04-03

    Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replication-competent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation largely depends on the NF-κB and MAPK signaling pathways because either overexpression of a super-repressor form of IκBα or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1 reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation, NF-κB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a potential compound for reactivation of latent HIV-1 reservoirs. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Effect of tannic acid, resveratrol and its derivatives, on oxidative damage and apoptosis in human neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Ignatowicz, Ewa; Krajka-Kuźniak, Violetta; Baer-Dubowska, Wanda

    2015-10-01

    In this study we compared the antioxidant and DNA protective activity of tannic acid and stilbene derivatives, resveratrol, 3,5,4(')-trimethoxystilbene (TMS) and pterostilbene in human neutrophils stimulated to oxidative burst by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in relation to apoptosis induction. All polyphenols within the concentration range 1-100 μM reduced the intracellular ROS and H2O2 production in the TPA-stimulated cells. Tannic acid was the most effective polyphenol in protection against DNA damage induced by TPA. In the resting neutrophils resveratrol and to lesser extent other polyphenols increased DNA damage and increased the level of p53. Pretreatment of the TPA-stimulated cells with tannic acid or stilbenes led to the induction of apoptosis. The most significant effect was observed as a result of treatment with TMS and resveratrol. These compounds appeared the most effective inducers of p53 in the TPA-challenged neutrophils, what may suggest that pro-apoptotic activity of these stilbenes might be related to p53 activation. Overall, the results of our present study demonstrate that tannic acid and stilbenes modulate the ROS production, ultimately leading to cell apoptosis in human neutrophils stimulated to oxidative burst. In resting neutrophils they exhibit pro-oxidant activity, which is accompanied by p53 induction. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. More Human than Human.

    PubMed

    Lawrence, David

    2017-07-01

    Within the literature surrounding nonhuman animals on the one hand and cognitively disabled humans on the other, there is much discussion of where beings that do not satisfy the criteria for personhood fit in our moral deliberations. In the future, we may face a different but related problem: that we might create (or cause the creation of) beings that not only satisfy but exceed these criteria. The question becomes whether these are minimal criteria, or hierarchical, such that those who fulfill them to greater degree should be afforded greater consideration. This article questions the validity and necessity of drawing divisions among beings that satisfy the minimum requirements for personhood; considering how future beings-intelligent androids, synthezoids, even alternate-substrate sentiences-might fit alongside the "baseline" human. I ask whether these alternate beings ought to be considered different to us, and why this may or may not matter in terms of a notion of "human community." The film Blade Runner, concerned in large part with humanity and its key synthezoid antagonist Roy Batty, forms a framing touchstone for my discussion. Batty is stronger, faster, more resilient, and more intelligent than Homo sapiens. His exploits, far beyond the capability of normal humans, are contrasted with his frailty and transient lifespan, his aesthetic appreciation of the sights he has seen, and his burgeoning empathy. Not for nothing does his creator within the mythos term him "more human than human."

  6. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    SciTech Connect

    Murison, G.; Chubb, C.B.H.; Maeda, S.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. Themore » THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.« less

  7. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    SciTech Connect

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site,more » to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.« less

  8. Human Development, Human Evolution.

    ERIC Educational Resources Information Center

    Smillie, David

    One of the truly remarkable events in human evolution is the unprecedented increase in the size of the brain of "Homo" over a brief span of 2 million years. It would appear that some significant selective pressure or opportunity presented itself to this branch of the hominid line and caused a rapid increase in the brain, introducing a…

  9. Humanizing the Humanities.

    ERIC Educational Resources Information Center

    Peters, Dennis

    1983-01-01

    Reviews some of the steps taken at Shoreline Community College to develop cooperative programs involving vocational and academic faculty, including the creation of a Humanities Advisory Council. Briefly describes some of the cooperative programs, e.g., symposia on critical issues in higher education, guest lectures, and high school outreach. (AYC)

  10. Defining MC1R Regulation in Human Melanocytes by Its Agonist α-Melanocortin and Antagonists Agouti Signaling Protein and β-Defensin 3

    PubMed Central

    Swope, Viki; Jameson, Joshua; McFarland, Kevin; Supp, Dorothy; Miller, William; McGraw, Dennis; Patel, Mira A.; Nix, Matthew A.; Milhauser, Glenn; Babcock, George; Abdel-Malek, Zalfa A.

    2012-01-01

    The melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor, plays an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human beta defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-MSH)-induced increase in the activities of adenylate cyclase, and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-o-tetradecanoyl phorbol 13-acetate, which activates PKC, increased, while exposure to ultraviolet radiation (UV) reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, while continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G-protein-coupled receptor kinases (GRK) 2, 3, 5, and 6, and β-arrestin 1. Therefore, in addition to MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV. PMID:22572817

  11. Regulation of hormone-induced Ca sup 2+ mobilization in the human platelets

    SciTech Connect

    Crouch, M.F.; Lapetina, E.G.

    1990-03-01

    {alpha}-Thrombin, {gamma}-thrombin, and platelet-activating factor each stimulated the mobilization of intracellular Ca{sup 2+} stores in aspirin-treated human platelets. This was followed by desensitization of the receptors, as shown by the return of the Ca{sup 2+} level to basal values and by the fact that a subsequent addition of a second different agonist, but not the same agonist, could again elicit a response. Epinephrine, acting on {alpha}{sub 2}-adrenergic receptors, was by itself ineffective at mobilizing Ca{sup 2+} stores. However, when added after the thrombin-induced response, epinephrine could evoke a considerable release of Ca{sup 2+} from cellular stores. This appeared to bemore » due to epinephrine recoupling thrombin receptors to phospholipase C. In support of this, epinephrine was able to induce the formation of inositol triphosphate when added after the response to thrombin had also become desensitized. Alone, epinephrine was without effect. Pre-activation of protein kinase C with the phorbol ester abolished these effects of epinephrine, suggesting that epinephrine was working by activating a protein which could be inactivated by phosphorylation. The current work is to characterize this protein that may be a member of the G{sub i}, GTP-binding protein family.« less

  12. Inhibition by fenoterol of human eosinophil functions including β2-adrenoceptor-independent actions

    PubMed Central

    TACHIBANA, A; KATO, M; KIMURA, H; FUJIU, T; SUZUKI, M; MORIKAWA, A

    2002-01-01

    Agonists at β2 adrenoceptors are used widely as bronchodilators in treating bronchial asthma. These agents also may have important anti-inflammatory effects on eosinophils in asthma. We examined whether widely prescribed β2-adrenoceptor agonists differ in ability to suppress stimulus-induced eosinophil effector functions such as superoxide anion (O2−) generation and degranulation. To examine involvement of cellular adhesion in such responses, we also investigated effects of β2 agonists on cellular adhesion and on CD11b expression by human eosinophils. O2− was measured using chemiluminescence. Eosinophil degranulation and adhesion were assessed by a radioimmunoassay for eosinophil protein X (EPX). CD11b expression was measured by flow cytometry. Fenoterol inhibited platelet-activating factor (PAF)-induced O2− generation by eosinophils significantly more than salbutamol or procaterol. Fenoterol partially inhibited PAF-induced degranulation by eosinophils similarly to salbutamol or procaterol. Fenoterol inhibited phorbol myristate acetate (PMA)-induced O2− generation and degranulation by eosinophils, while salbutamol or procaterol did not. Fenoterol inhibition of PMA-induced O2− generation was not reversed by ICI-118551, a selective β2-adrenoceptor antagonist. Fenoterol, but not salbutamol or procaterol, significantly inhibited PAF-induced eosinophil adhesion. Fenoterol inhibited O2− generation and degranulation more effectively than salbutamol or procaterol; these effects may include a component involving cellular adhesion. Inhibition also might include a component not mediated via β2 adrenoceptors. PMID:12452831

  13. Parallel secretion of pancreastatin and somatostatin from human pancreastatin producing cell line (QGP-1N).

    PubMed

    Funakoshi, A; Tateishi, K; Kitayama, N; Jimi, A; Matsuoka, Y; Kono, A

    1993-05-01

    In this investigation we studied pancreastatin (PST) secretion from a human PST producing cell line (QGP-1N) in response to various secretagogues. Immunocytochemical study revealed the immunoreactivity of PST and somatostatin (SMT) in the same cells of a monolayer culture. Ki-ras DNA point mutation on codon 12 was found. Carbachol stimulated secretion of PST and SMT and intracellular Ca2+ mobilization in the range of 10(-6)-10(-4) M. The secretion and Ca2+ mobilization were inhibited by atropine, a muscarinic receptor antagonist. Phorbol ester and calcium ionophore (A23187) stimulated secretion of PST and SMT. The removal of extracellular calcium suppressed both secretions throughout stimulation with 10(-5) M carbachol. Fluoride, a well-known activator of guanine nucleotide binding (G) protein, stimulated intracellular Ca2+ mobilization and secretion of PST and SMT in a dose-dependent manner in the range of 5-40 mM. Also, 10(-5) M carbachol and 20 mM fluoride stimulated inositol 1,4,5-triphosphate production. However, cholecystokinin and gastrin-releasing peptide did not stimulate Ca2+ mobilization or secretion of the two peptides. These results suggest that secretion of PST and SMT from QGP-1N cells is regulated mainly by acetylcholine in a parallel fashion through muscarinic receptors coupled to the activation of polyphosphoinositide breakdown by a G-protein and that increases in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  14. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    PubMed

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  15. Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome and associated flavoprotein.

    PubMed

    Borregaard, N; Tauber, A I

    1984-01-10

    Human neutrophils were fractionated by nitrogen cavitation and Percoll density centrifugation, and the subcellular localization of FAD-flavoprotein, b-cytochrome, NADH-cytochrome b5 reductase, and NADPH-dependent cytochrome c reductase were determined in normal cells, cells from two patients with chronic granulomatous disease (CGD), and normal cells that had been stimulated with phorbol myristate acetate. In normal cells, a FAD-flavoprotein is found in a 1:2 molar ratio, with cytochrome b in the fractions containing the specific granules. Triton X-114 phase distribution indicates that the b-cytochrome but not the b-cytochrome-associated flavoprotein is an integral membrane protein. 80% of this flavoprotein, as well as all the b-cytochrome, was absent in these fractions from 2 CGD patients, although these patients had normal quantities of FAD in the fractions containing plasma membranes and cytosol. During stimulation the b-cytochrome-associated flavoprotein of the granules translocates with the b-cytochrome to the plasma membrane where NADPH oxidase is localized. Definition of the role of these NADPH oxidase constituents may provide a molecular description of the normal neutrophil respiratory burst and the molecular defect(s) in CGD.

  16. The beetroot component betanin modulates ROS production, DNA damage and apoptosis in human polymorphonuclear neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Kostrzewa, Artur; Łuczak, Michał; Jagodziński, Paweł P; Baer-Dubowska, Wanda

    2012-06-01

    The aim of this study was to evaluate the effect of betanin, one of the beetroot major components, on ROS production, DNA damage and apoptosis in human resting and stimulated with phorbol 12-myristate13-acetate polymorphonuclear neutrophils, one of the key elements of the inflammatory response. Incubation of neutrophils with betanin in the concentration range 2-500 µM resulted in significant inhibition of ROS production (by 15-46%, depending on the ROS detection assay). The antioxidant capacity of betanin was most prominently expressed in the chemiluminescence measurements. This compound decreased also the percentage of DNA in comet tails in stimulated neutrophils, but only at the 24 h time point. In resting neutrophils an increased level of DNA in comet tails was observed. Betanin did not affect the activity of caspase-3, in resting neutrophils, but significantly enhanced the enzyme activity in stimulated neutrophils. The western blot analysis showed, however, an increased level of caspase-3 cleavage products as a result of betanin treatment both in resting and stimulated neutrophils. The results indicate that betanin may be responsible for the effect of beetroot products on neutrophil oxidative metabolism and its consequences, DNA damage and apoptosis. The dose and time dependent effects on these processes require further studies. Copyright © 2011 John Wiley & Sons, Ltd.

  17. α-Tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells.

    PubMed

    Huang, Huarong; Chen, Shaohua; Van Doren, Jeremiah; Li, Dongli; Farichon, Chelsea; He, Yan; Zhang, Qiuyan; Zhang, Kun; Conney, Allan H; Goodin, Susan; Du, Zhiyun; Zheng, Xi

    2015-06-01

    α‑Tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α‑tomatine on human myeloid leukemia HL‑60 cells were investigated. Treatment of HL‑60 cells with α‑tomatine resulted in growth inhibition and apoptosis in a concentration‑dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL‑60 cells. Growth inhibition and apoptosis induced by α‑tomatine in HL‑60 cells was partially abrogated by addition of cholesterol indicating that interactions between α‑tomatine and cell membrane‑associated cholesterol may be important in mediating the effect of α‑tomatine. Activation of nuclear factor‑κB by the phorbol ester, 12‑O‑tetradecanoylphorbol‑13‑acetate failed to prevent apoptosis in HL‑60 cells treated with α‑tomatine. In animal experiments, it was found that treatment of mice with α‑tomatine inhibited the growth of HL‑60 xenografts in vivo. Results from the present study indicated that α‑tomatine may have useful anti‑leukemia activities.

  18. α-tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells

    PubMed Central

    HUANG, HUARONG; CHEN, SHAOHUA; VAN DOREN, JEREMIAH; LI, DONGLI; FARICHON, CHELSEA; HE, YAN; ZHANG, QIUYAN; ZHANG, KUN; CONNEY, ALLAN H; GOODIN, SUSAN; DU, ZHIYUN; ZHENG, XI

    2015-01-01

    α-tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α-tomatine on human myeloid leukemia HL-60 cells were investigated. Treatment of HL-60 cells with α-tomatine resulted in growth inhibition and apoptosis in a concentration-dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL-60 cells. Growth inhibition and apoptosis induced by α-tomatine in HL-60 cells was partially abrogated by addition of cholesterol indicating that interactions between α-tomatine and cell membrane-associated cholesterol may be important in mediating the effect of α-tomatine. Activation of nuclear factor-κB by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate failed to prevent apoptosis in HL-60 cells treated with α-tomatine. In animal experiments, it was found that treatment of mice with α-tomatine inhibited the growth of HL-60 xenografts in vivo. Results from the present study indicated that α-tomatine may have useful anti-leukemia activities. PMID:25625536

  19. Arbutin and decrease of potentially toxic substances generated in human blood neutrophils

    PubMed Central

    Pečivová, Jana; Nosál', Radomír; Sviteková, Klára

    2014-01-01

    Neutrophils, highly motile phagocytic cells, constitute the first line of host defense and simultaneously they are considered to be central cells of chronic inflammation. In combination with standard therapeutic procedures, natural substances are gaining interest as an option for enhancing the effectiveness of treatment of inflammatory diseases. We investigated the effect of arbutin and carvedilol and of their combination on 4β-phorbol-12β-myristate-13α-acetate- stimulated functions of human isolated neutrophils. Cells were preincubated with the drugs tested and subsequently stimulated. Superoxide (with or without blood platelets, in the rate close to physiological conditions [1:50]) and HOCl generation, elastase and myeloperoxidase release were determined spectrophotometrically and phospholipase D activation spectrofluorometrically. The combined effect of arbutin and carvedilol was found to be more effective than the effect of each compound alone. Our study provided evidence supporting the potential beneficial effect of arbutin alone or in combination with carvedilol in diminishing tissue damage by decreasing phospholipase D, myeloperoxidase and elastase activity and by attenuating the generation of superoxide and the subsequently derived reactive oxygen species. The presented data indicate the ability of arbutin to suppress the onset and progression of inflammation. PMID:26109900

  20. Effects of flavonoids on human lymphocyte proliferative responses

    SciTech Connect

    Mookerjee, B.K.; Lee, T.P.; Logue, G.P.

    1986-01-01

    Flavonoids reversibly inhibit lymphocyte proliferative responses to phytomitogens, soluble antigens and phorbol esters by blocking an early event or events that follow stimulation. Quercetin and tangeretin inhibit thymidine transport in stimulated lymphocytes. These flavonoids reversibly inhibit antigen processing by monocytes and inhibit the expression of class II histocompatibility (DR) antigens in PBM cells.

  1. Heterogeneous expression of apolipoprotein-E by human macrophages

    PubMed Central

    Tedla, Nicodemus; Glaros, Elias N; Brunk, Ulf T; Jessup, Wendy; Garner, Brett

    2004-01-01

    Apolipoprotein-E (apoE) is expressed at high levels by macrophages. In addition to its role in lipid transport, macrophage-derived apoE plays an important role in immunoregulation. Previous studies have identified macrophage subpopulations that differ substantially in their ability to synthesize specific cytokines and enzymes, however, potential heterogeneous macrophage apoE expression has not been studied. Here we examined apoE expression in human THP-1 macrophages and monocyte-derived macrophages (MDM). Using immunocytochemistry and flow cytometry methods we reveal a striking heterogeneity in macrophage apoE expression in both cell types. In phorbol-ester-differentiated THP-1 macrophages, 5% of the cells over-expressed apoE at levels more than 50-fold higher than the rest of the population. ApoE over-expressing THP-1 macrophages contained condensed/fragmented nuclei and increased levels of activated caspase-3 indicating induction of apoptosis. In MDM, 3–5% of the cells also highly over-expressed apoE, up to 50-fold higher than the rest of the population; however, this was not associated with obvious nuclear alterations. The apoE over-expressing MDM were larger, more granular, and more autofluorescent than the majority of cells and they contained numerous vesicle-like structures that appeared to be coated by apoE. Flow cytometry experiments indicated that the apoE over-expressing subpopulation of MDM were positive for CD14, CD11b/Mac-1 and CD68. These observations suggest that specific macrophage subpopulations may be important for apoE-mediated immunoregulation and clearly indicate that subpopulation heterogeneity should be taken into account when investigating macrophage apoE expression. PMID:15500620

  2. Sulfite-Mediated Oxidation of Myeloperoxidase to a Free Radical: Immuno-Spin Trapping Detection in Human Neutrophils

    PubMed Central

    Ranguelova, Kalina; Rice, Annette B.; Lardinois, Olivier M.; Triquigneaux, Mathilde; Steinckwich, Natacha; Deterding, Leesa J.; Garantziotis, Stavros; Mason, Ronald P.

    2013-01-01

    Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of reactive sulfur trioxide (•SO3−), peroxymonosulfate (−O3SOO•) and sulfate (SO4•−) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of the present study is to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage possibly leading to (bi)sulfite-exacerbated allergic reactions. PMID:23376232

  3. Heparanase is preferentially expressed in human psoriatic lesions and induces development of psoriasiform skin inflammation in mice

    PubMed Central

    Hermano, Esther; Rubinstein, Ariel M.; Vlodavsky, Israel; Elkin, Michael

    2014-01-01

    Heparanase is the sole mammalian endoglycosidase that selectively degrades heparan sulfate, the key polysaccharide associated with the cell surface and extracellular matrix of a wide range of tissues. Extensively studied for its capacity to promote cancer progression, heparanase enzyme was recently implicated as an important determinant in several inflammatory disorders as well. Applying immunohistochemical staining, we detected preferential expression of heparanase by epidermal keratinocytes in human psoriatic lesions. To investigate the role of the enzyme in the pathogenesis of psoriasis, we utilized heparanase transgenic mice in a model of 12-O-tetradecanoyl phorbol 12-myristate 13-acetate (TPA)-induced cutaneous inflammation. We report that over-expression of the enzyme promotes development of mouse skin lesions that strongly recapitulate the human disease in terms of histomorphological appearance and molecular/cellular characteristics. Importantly, heparanase of epidermal origin appears to facilitate abnormal activation of skin-infiltrating macrophages, thus generating psoriasis-like inflammation conditions, characterized by induction of STAT3, enhanced NF-κB signaling, elevated expression of TNFα and increased vascularization. Taken together, our results reveal, for the first time, involvement of heparanase in the pathogenesis of psoriasis and highlight a role for the enzyme in facilitating abnormal interactions between immune and epithelial cell subsets of the affected skin. Heparanase inhibitors (currently under clinical testing in malignant diseases) could hence turn highly beneficial in psoriatic patients as well. PMID:24169805

  4. Permissiveness of human monocytes and monocyte-derived macrophages to infection by promastigotes of Leishmania (Viannia) panamensis.

    PubMed

    Bosque, F; Milon, G; Valderrama, L; Saravia, N G

    1998-12-01

    During natural infections, Leishmania is in contact with a variety of mononuclear phagocytic cells in different tissues, including resident macrophages and monocytes mobilized to the site of infection from the bone marrow and blood circulation. Because the functional capabilities of fully differentiated macrophages and blood monocytes differ, the outcome of infection by Leishmania may depend upon the stage of differentiation of the host cells. To address this question, we evaluated Leishmania panamensis infection of (1) the human promonocytic/histiocytic cell line U-937 before and after induction of differentiation by phorbol myristate acetate; (2) fresh human peripheral blood monocytes; and (3) macrophages derived from monocytes by differentiation in vitro. Based on the percentage of cells infected and the number of parasites per cell, macrophages derived from monocytes or by induction of differentiation of U-937 cells were significantly more permissive to infection by stationary-phase L. (Viannia) panamensis promastigotes than monocytes. Increasing time and maturation in culture prior to exposure to infective promastigotes was associated with the increased permissiveness of differentiated macrophages to infection (P<0.05). The percentage of cells infected and number of amastigotes per cell increased with time postinfection for both monocytes and macrophages but remained significantly greater for macrophages. The increased expression of CD68, CD16, and lysozyme, and decreased expression of peroxidase by macrophages cultured for 5 days in vitro compared with fresh monocytes, whether adherent or in suspension, supported the distinct maturation status of these cells.

  5. Assessment of Antioxidant Activity of Spray Dried Extracts of Psidium guajava Leaves by DPPH and Chemiluminescence Inhibition in Human Neutrophils

    PubMed Central

    Fernandes, M. R. V.; Azzolini, A. E. C. S.; Martinez, M. L. L.; Souza, C. R. F.; Lucisano-Valim, Y. M.; Oliveira, W. P.

    2014-01-01

    This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β-cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristate acetate (PMA) and the DPPH radical scavenging (DPPH∗ method). In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH• method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells. PMID:24822200

  6. Assessment of antioxidant activity of spray dried extracts of Psidium guajava leaves by DPPH and chemiluminescence inhibition in human neutrophils.

    PubMed

    Fernandes, M R V; Azzolini, A E C S; Martinez, M L L; Souza, C R F; Lucisano-Valim, Y M; Oliveira, W P

    2014-01-01

    This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β -cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristate acetate (PMA) and the DPPH radical scavenging (DPPH∗ method). In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH(•) method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells.

  7. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

    PubMed Central

    Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth J.; Siller, Richard; Srsen, Vlastimil; Morgan, Katie; Bryans, Anna; Kozlowska, Ada; Koulovasilopoulos, Andreas; Underwood, Ian; Smith, Stewart; del-Pozo, Jorge; Moss, Sharon; Thompson, Alexandra Inés; Henderson, Neil C.; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre-Olivier; Nelson, Leonard J.

    2017-01-01

    Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization. PMID:28134251

  8. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.

    PubMed

    Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth J; Siller, Richard; Srsen, Vlastimil; Morgan, Katie; Bryans, Anna; Kozlowska, Ada; Koulovasilopoulos, Andreas; Underwood, Ian; Smith, Stewart; Del-Pozo, Jorge; Moss, Sharon; Thompson, Alexandra Inés; Henderson, Neil C; Hayes, Peter C; Plevris, John N; Bagnaninchi, Pierre-Olivier; Nelson, Leonard J

    2017-01-30

    Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

  9. [Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils].

    PubMed

    Mikhal'chik, E V; Smolina, N V; Astamirova, T C; Gorudko, I V; Grigor'eva, D V; Ivanov, V A; Sokolov, A V; Kostevich, V A; Cherenkevich, S N; Panasenko, O M

    2013-01-01

    It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase--4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC50 = 20 microM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a "vicious circle" of neutrophil activation at the inflammatory site.

  10. Kafirin from Sorghum bicolor inhibition of inflammation in THP-1 human macrophages is associated with reduction of intracellular reactive oxygen species.

    PubMed

    Sullivan, Andrew C; Pangloli, Philipus; Dia, Vermont P

    2018-01-01

    Aberrant inflammation as a result of activation of the transmembrane protein Toll-like receptor 4 belonging to pattern recognition receptor and subsequent phosphorylation of signaling proteins facilitated by reactive oxygen species has been linked to a myriad of diseases. Sorghum is a drought-resistant cereal with health promoting properties associated with its biologically active substances such as kafirin. Kafirin is an alcohol soluble protein and accounts for as much as 70% of the total proteins in sorghum. The objective was to determine the effect of kafirin on lipopolysaccharide (LPS)-induced inflammation in THP-1 human macrophages. THP-1 human monocytic leukemia cells were differentiated into macrophages by phorbol-12-myristate 13-acetate followed by treatment of LPS with or without 50 μg/mL or 100 μg/mL concentrations of kafirin. Kafirin at 100 μg/mL reduced the production of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α by 28.3%, 74.0%, and 81.4%, respectively. Kafirin reduced production of intracellular reactive oxygen species is associated with reduced phosphorylation of extracellular regulated kinase1/2 and c-JUN N-terminal kinase and nuclear translocation of p65 and c-JUN transcription factors. Our results showed for the first time the anti-inflammatory property of kafirin purified from sorghum in LPS-induced THP-1 human macrophages. Published by Elsevier Ltd.

  11. Human niche, human behaviour, human nature.

    PubMed

    Fuentes, Agustin

    2017-10-06

    The concept of a 'human nature' or 'human natures' retains a central role in theorizing about the human experience. In Homo sapiens it is clear that we have a suite of capacities generated via our evolutionary past, and present, and a flexible capacity to create and sustain particular kinds of cultures and to be shaped by them. Regardless of whether we label these capacities 'human natures' or not, humans occupy a distinctive niche and an evolutionary approach to examining it is critical. At present we are faced with a few different narratives as to exactly what such an evolutionary approach entails. There is a need for a robust and dynamic theoretical toolkit in order to develop a richer, and more nuanced, understanding of the cognitively sophisticated genus Homo and the diverse sorts of niches humans constructed and occupied across the Pleistocene, Holocene, and into the Anthropocene. Here I review current evolutionary approaches to 'human nature', arguing that we benefit from re-framing our investigations via the concept of the human niche and in the context of the extended evolutionary synthesis (EES). While not a replacement of standard evolutionary approaches, this is an expansion and enhancement of our toolkit. I offer brief examples from human evolution in support of these assertions.

  12. Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

    PubMed Central

    Singh, Jagmohan

    2012-01-01

    Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca2+, KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and Gö-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K+ depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and Gö-6850 (each 10−5 M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs′ SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS. PMID:22790596

  13. Inhibition of Human Neutrophil Responses by Essential Oil of Artemisia kotuchovii and Its Constituents

    PubMed Central

    Schepetkin, Igor A.; Kushnarenko, Svetlana V.; Özek, Gulmira; Kirpotina, Liliya N.; Utegenova, Gulzhakhan A.; Kotukhov, Yuriy A.; Danilova, Alevtina N.; Özek, Temel; Başer, K. Hüsnü Can; Quinn, Mark T.

    2015-01-01

    Essential oils were obtained by hydrodistillation of the flowers+leaves and stems of Artemisia kotuchovii Kupr. (AKEOf+l and AKEOstm, respectively) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The primary components of the oils were estragole, (E)- and (Z)-β-ocimenes, methyl eugenol, limonene, spathulenol, β-pinene, myrcene, and (E)-methyl cinnamate. Seventy four constituents were present at concentrations from 0.1 to 1.0%, and 34 compounds were identified in trace (<0.1%) amounts in one or both plant components. Screening of the essential oils for biological activity showed that AKEOstm, but not AKEOf+l, inhibited N-formyl-Met-Leu-Phe (fMLF)-stimulated Ca2+ flux and chemotaxis and phorbol-12-myristate-13-acetate (PMA)-induced reactive oxygen species (ROS) production in human neutrophils. Selected pure constituents, representing >96% of the AKEOstm composition, were also tested in human neutrophils and HL-60 cells transfected with N-formyl peptide receptor 1 (FPR1). We found that one component, 6-methyl-3,5-heptadien-2-one (MHDO), inhibited fMLF- and interleukin 8 (IL-8)-stimulated Ca2+ flux, fMLF-induced chemotaxis, and PMA-induced ROS production in human neutrophils. MHDO also inhibited fMLF-induced Ca2+ flux in FPR1-HL60 cells. These results suggest that MHDO may be effective in modulating some innate immune responses, possibly by an inhibition of neutrophil migration and ROS production. PMID:25959257

  14. Inhibition of Human Neutrophil Responses by the Essential Oil of Artemisia kotuchovii and Its Constituents.

    PubMed

    Schepetkin, Igor A; Kushnarenko, Svetlana V; Özek, Gulmira; Kirpotina, Liliya N; Utegenova, Gulzhakhan A; Kotukhov, Yuriy A; Danilova, Alevtina N; Özek, Temel; Başer, K Hüsnü Can; Quinn, Mark T

    2015-05-27

    Essential oils were obtained by hydrodistillation of the flowers+leaves and stems of Artemisia kotuchovii Kupr. (AKEO(f+l) and AKEO(stm), respectively) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The primary components of the oils were estragole, (E)- and (Z)-β-ocimenes, methyleugenol, limonene, spathulenol, β-pinene, myrcene, and (E)-methyl cinnamate. Seventy-four constituents were present at concentrations from 0.1 to 1.0%, and 34 compounds were identified in trace (<0.1%) amounts in one or both plant components. Screening of the essential oils for biological activity showed that AKEO(stm), but not AKEOf+l, inhibited N-formyl-Met-Leu-Phe (fMLF)-stimulated Ca(2+) flux and chemotaxis and phorbol-12-myristate-13-acetate (PMA)-induced reactive oxygen species (ROS) production in human neutrophils. Selected pure constituents, representing >96% of the AKEO(stm) composition, were also tested in human neutrophils and HL-60 cells transfected with N-formyl peptide receptor 1 (FPR1). One component, 6-methyl-3,5-heptadien-2-one (MHDO), inhibited fMLF- and interleukin 8 (IL-8)-stimulated Ca(2+) flux, fMLF-induced chemotaxis, and PMA-induced ROS production in human neutrophils. MHDO also inhibited fMLF-induced Ca(2+) flux in FPR1-HL60 cells. These results suggest that MHDO may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration and ROS production.

  15. Dipyridamole decreases inflammatory metalloproteinase-9 expression and release by human monocytes.

    PubMed

    Massaro, Marika; Scoditti, Egeria; Carluccio, Maria Annunziata; Pellegrino, Mariangela; Calabriso, Nadia; Storelli, Carlo; Martines, Giuseppe; De Caterina, Raffaele

    2013-02-01

    Matrix metalloproteinase (MMP)-9 plays an important role in stroke by accelerating matrix degradation, disrupting the blood-brain barrier and increasing infarct size. Dipyridamole is an antiplatelet agent with recognised benefits in ischaemic stroke prevention. In addition to its antiplatelet properties, recent studies have reported that dipyridamole also features anti-inflammatory and anti-oxidant properties. We therefore investigated whether dipyridamole can ameliorate the proinflammatory profile of human monocytes, a source of MMP-9 in stroke, in terms of regulation of MMP-9 activity and expression, and explored underlying mechanisms. Human peripheral blood mononuclear cells (PBMC) and U937 cells were treated with increasing concentrations of dipyridamole (up to 10 µg/ml) for 60 minutes before stimulation with tumour necrosis factor (TNF)-α or phorbol myristate acetate (PMA). Exposure of PBMC and U937 to dipyridamole reduced TNF-α- and PMA-induced MMP-9 activity and protein release as well as MMP-9 mRNA, without significantly affecting the release of TIMP-1. This inhibitory effect was independent of dipyridamole-induced cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) increase. Correspondingly, dipyridamole also significantly inhibited TNF-α-induced nuclear factor (NF)-κB activation and nuclear translocation of the p65 NF-κB subunit through a mechanism involving the inhibition of IkBα degradation and p38 MAPK activation. In conclusion, dipyridamole, at therapeutically achievable concentrations, reduces the expression and release of MMP-9 through a mechanism involving p38 MAPK and NF-κB inhibition. These results indicate that dipyridamole exerts anti-inflammatory properties in human monocytes that may favourably contribute to its actions in the secondary prevention of stroke, independent of its antiplatelet properties.

  16. In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.

    PubMed

    Noubouossie, Denis F; Whelihan, Matthew F; Yu, Yuan-Bin; Sparkenbaugh, Erica; Pawlinski, Rafal; Monroe, Dougald M; Key, Nigel S

    2017-02-23

    NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins. © 2017 by The American Society of Hematology.

  17. High D-glucose reduces SLC29A1 promoter activity and adenosine transport involving specific protein 1 in human umbilical vein endothelium.

    PubMed

    Puebla, Carlos; Farías, Marcelo; González, Marcelo; Vecchiola, Andrea; Aguayo, Claudio; Krause, Bernardo; Pastor-Anglada, Marçal; Casanello, Paola; Sobrevia, Luis

    2008-06-01

    High D-glucose reduces human equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen-activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D-glucose-reduced hENT1-adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D-glucose (25 mM) reduced hENT1-adenosine transport and pGL3-hENT1(-1114) construct SLC29A1 reporter activity compared with normal D-glucose (5 mM). High D-glucose also reduced pGL3-hENT1(-1114) reporter activity compared with cells transfected with pGL3-hENT1(-795) construct. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D-glucose effects. Insulin (1 nM) and phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator), but not 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 100 nM, PMA less active analogue) reduced hENT1-adenosine transport. L-NAME and PD-98059 blocked insulin effects. L-NAME, PD-98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D-glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L-NAME, PD-98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D-glucose, an effect reversed by L-NAME and further reduced by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor) in high D-glucose. Thus, reduced hENT1-mediated adenosine transport in high D-glucose may result from increased Sp1 binding to SLC29A1 promoter down-regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia-associated endothelial

  18. TPA enhances growth hormone (GH) secretion effect of GH-releasing hormone (GHRH) by human gsp-positive pituitary somatotrophinomas.

    PubMed

    Lei, T; Bai, X; Hu, W; Xue, D; Jiang, X

    1999-01-01

    In recent years, one of the most exciting advances in the researches of pituitary adenomas is the discovery that 30%-40% of human pituitary somatotrophinomas carry somatic mutations of the gene for the alpha-subunit of the stimulatory GTP-binding protein, Gs (Gs alpha). These mutations, termed gsp oncogenes, may play an important role in the tumorigenesis of pituitary adenomas. Of 10 somatotrophinomas examined, 3 (30%) were proved to be gsp positive, as determined by sequence analysis of DNA generated by the polymerase chain reaction (PCR). GHRH exerted a significant stimulatory effect on GH secretion in 2 of 3 gsp-positive and 4 of 7 gsp-negative tumors. Moreover, phorbol ester, 1, 2-tetradecanoylphorbol-13-acetate (TPA), enhanced stimulation of lated the GH secretion effect exerted by GHRH in gsp-positive somatotrophinomas, whereas this effect was not observed in gsp-negative tumors. This result suggests that the protein kinase C signal system as well as adenylyl cyclase-cAMP-protein kinase A intracellular signal transduction system plays a pivotal role in GH secretory control of GHRH, which may work together via a cross-talk mechanism.

  19. Identification and characterization of human herpesvirus 8 open reading frame K9 viral interferon regulatory factor by a monoclonal antibody.

    PubMed

    Inagi, R; Okuno, T; Ito, M; Chen, J; Mori, Y; Haque, M; Zou, P; Yagi, H; Kiniwa, S; Saida, T; Ueyama, Y; Hayashi, K; Yamanishi, K

    1999-01-01

    To identify and characterize an open reading frame (ORF) in association with its mRNA and a coding protein recognized by anti-human herpesvirus 8 (HHV-8) monoclonal antibody B291. The antigen detected by B291 was characterized by immunofluorescence method, Western blot analysis, laser confocal microscopy, and immunohistology cDNA of the protein was identified by immunoscreening the HHV-8 cDNA library with B291 and sequenced. Structure and kinetics of the mRNA expression was investigated by 5',3' rapid amplification of cDNA ends (RACE) and Northern blot analysis. Viral interferon regulatory factor (vIRF; approximately 50 kd) was a protein recognized by B291. It was detected in 12-O-tetradecanoyl phorbol-13 acetate (TPA)-induced, but not uninduced, HHV-8-infected cell lines (BCBL-1 and BCP-1) and the tumor cells obtained from a SCID mouse injected with uninduced BCBL-1. The expression of vIRF was predominantly in the nucleus and was partially diminished by phosphonoformic acid (PFA) treatment, whereas the mRNA (approximately 2.4 kb) was accumulated. vIRF is an early protein expressed in the nucleus and may be involved in Kaposi's sarcoma tumor formation.

  20. Effect of clotrimazole on cytosolic Ca(2+) rise and viability in HA59T human hepatoma cells.

    PubMed

    Horng, Chi-Ting; Chiang, Ni-Na; Chen, I-Li; Liang, Wei-Zhe; Chen, I-Shu; Kuo, Daih-Huang; Shieh, Po-Chuen; Jan, Chung-Ren

    2013-04-01

    Abstract Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca(2+) homeostasis. This study examined the effect of clotrimazole on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in HA59T human hepatoma cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Clotrimazole induced [Ca(2+)](i) rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca(2+). Clotrimazole-evoked Ca(2+) entry was suppressed by store-operated channel inhibitors (nifedipine, econazole and SK&F96365) and protein kinase C modulators (GF109203X and phorbol, 12-myristate, 13-acetate). In Ca(2+)-free medium, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone abolished clotrimazole-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished clotrimazole-induced [Ca(2+)](i) rise. At 10-40 µM, clotrimazole inhibited cell viability, which was not reversed by chelating cytosolic Ca(2+). Clotrimazole at 10 and 30 µM also induced apoptosis. Collectively, in HA59T cells, clotrimazole-induced [Ca(2+)](i) rises by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. Clotrimazole also caused apoptosis.

  1. The diet-derived sulforaphane inhibits matrix metalloproteinase-9-activated human brain microvascular endothelial cell migration and tubulogenesis.

    PubMed

    Annabi, Borhane; Rojas-Sutterlin, Shanti; Laroche, Mathieu; Lachambre, Marie-Paule; Moumdjian, Robert; Béliveau, Richard

    2008-06-01

    Human brain microvascular endothelial cells (HBMECs) play an essential role as structural and functional components of the blood-brain barrier (BBB). While disruption of the BBB by the brain tumor-secreted matrix metalloproteinase-9 (MMP-9) favors tumor invasion, the role and regulation of MMP-9 secretion by HBMEC themselves in response to carcinogens or brain tumor-derived growth factors has received little attention. Our study delineates a unique brain endothelial phenotype in that MMP-9 secretion is increased upon phorbol 12-myristate 13-acetate (PMA) treatment of HBMEC. Sulforaphane (SFN), an isothiocyanate present in broccoli which exhibits chemopreventive properties, selectively inhibited the secretion of MMP-9 but not that of MMP-2. The decrease in MMP-9 gene expression correlated with a decrease in the expression of the mRNA stabilizing factor HuR protein triggered by SFN. PMA-induced HBMEC migration was also antagonized by SFN. Silencing of the MMP-9 gene inhibited PMA-induced MMP-9 secretion, cell migration, and in vitro tubulogenesis on Matrigel. While SFN inhibited the chemoattractive abilities of brain tumor-derived growth factors, it failed to inhibit PMA-induced tubulogenesis. Our data are indicative of a selective role for SFN to inhibit MMP-9-activated, but not basal, HBMEC migration, and tubulogenesis whose actions could add to SFN's antitumor properties.

  2. Inhibition of neutrophil elastase and metalloprotease-9 of human adenocarcinoma gastric cells by chamomile (Matricaria recutita L.) infusion.

    PubMed

    Bulgari, Michela; Sangiovanni, Enrico; Colombo, Elisa; Maschi, Omar; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

    2012-12-01

    This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. Copyright © 2012 John Wiley & Sons, Ltd.

  3. Extra- and intracellular formation of reactive oxygen species by human neutrophils in the presence of pheniramine, chlorpheniramine and brompheniramine.

    PubMed

    Jancinová, Viera; Drábiková, Katarína; Nosál', Radomír; Holománová, Dagmar

    2006-12-01

    Allergic inflammation was found to be accompanied by activation of neutrophils. Since this resulted in increased formation of reactive oxygen species, the antioxidative activity of antiallergic drugs was considered to decrease the risk of tissue damage. However, if the drug-induced inhibition of radical formation occurred intracellularly, it could disturb regulation of neutrophil functions and decrease bactericidal activity of these cells. Separate analysis of extra- and intracellular activity of antioxidative drugs is thus of particular importance. To differentiate the effects of three antiallergic H1-antihistamine drugs (pheniramine, chlor- and brompheniramine) on radical formation outside and inside human neutrophils. Formation of reactive oxygen species was determined in vitro using the chemiluminescence method. The chemiluminescence signal, initiated by phorbol-12-myristate-13-acetate, was enhanced either with isoluminol (extracellular) or with luminol in the presence of extracellular scavengers, superoxide dismutase and catalase (intracellular). The antihistamines tested displayed dual activity - they inhibited the extracellular and potentiated the intracellular chemiluminescence. Chlor- and brompheniramine were found to be more effective than pheniramine. Compared to other H1-antihistamines (such as dithiaden or loratadine, active both extra- and intracellularly), the observed inhibition caused by the pheniramines tested was unique since it occurred selectively outside neutrophils. This might indicate the ability of these drugs to minimise toxic effects of extracellular radicals without affecting intracellular oxidant production involved in regulation of neutrophil functions and in microbial killing.

  4. The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight

    NASA Technical Reports Server (NTRS)

    Hatton, J. P.; Gaubert, F.; Lewis, M. L.; Darsel, Y.; Ohlmann, P.; Cazenave, J. P.; Schmitt, D.

    1999-01-01

    Protein kinase C (PKC) is a family of serine/threonine kinases that play an important role in mediating intracellular signal transduction in eukaryotes. U937 cells were exposed to microgravity during a space shuttle flight and stimulated with a radiolabeled phorbol ester ([3H]PDBu) to both specifically label and activate translocation of PKC from the cytosol to the particulate fraction of the cell. Although significant translocation of PKC occurred at all g levels, the kinetics of translocation in flight were significantly different from those on the ground. In addition, the total quantity of [3H]PDBu binding PKC was increased in flight compared to cells at 1 g on the ground, whereas the quantity in hypergravity (1.4 g) was decreased with respect to 1 g. Similarly, in purified human peripheral blood T cells the quantity of PKCdelta varied in inverse proportion to the g level for some experimental treatments. In addition to these novel findings, the results confirm earlier studies which showed that PKC is sensitive to changes in gravitational acceleration. The mechanisms of cellular gravisensitivity are poorly understood but the demonstrated sensitivity of PKC to this stimulus provides us with a useful means of measuring the effect of altered gravity levels on early cell activation events.

  5. Human Augmentics: augmenting human evolution.

    PubMed

    Kenyon, Robert V; Leigh, Jason

    2011-01-01

    Human Augmentics (HA) refers to technologies for expanding the capabilities, and characteristics of humans. One can think of Human Augmentics as the driving force in the non-biological evolution of humans. HA devices will provide technology to compensate for human biological limitations either natural or acquired. The strengths of HA lie in its applicability to all humans. Its interoperability enables the formation of ecosystems whereby augmented humans can draw from other realms such as "the Cloud" and other augmented humans for strength. The exponential growth in new technologies portends such a system but must be designed for interaction through the use of open-standards and open-APIs for system development. We discuss the conditions needed for HA to flourish with an emphasis on devices that provide non-biological rehabilitation.

  6. Human Parvoviruses

    PubMed Central

    Söderlund-Venermo, Maria; Young, Neal S.

    2016-01-01

    SUMMARY Parvovirus B19 (B19V) and human bocavirus 1 (HBoV1), members of the large Parvoviridae family, are human pathogens responsible for a variety of diseases. For B19V in particular, host features determine disease manifestations. These viruses are prevalent worldwide and are culturable in vitro, and serological and molecular assays are available but require careful interpretation of results. Additional human parvoviruses, including HBoV2 to -4, human parvovirus 4 (PARV4), and human bufavirus (BuV) are also reviewed. The full spectrum of parvovirus disease in humans has yet to be established. Candidate recombinant B19V vaccines have been developed but may not be commercially feasible. We review relevant features of the molecular and cellular biology of these viruses, and the human immune response that they elicit, which have allowed a deep understanding of pathophysiology. PMID:27806994

  7. Image-Based Phenotypic Screening with Human Primary T Cells Using One-Dimensional Imaging Cytometry with Self-Tuning Statistical-Gating Algorithms.

    PubMed

    Wang, Steve S; Ehrlich, Daniel J

    2017-09-01

    The parallel microfluidic cytometer (PMC) is an imaging flow cytometer that operates on statistical analysis of low-pixel-count, one-dimensional (1D) line scans. It is highly efficient in data collection and operates on suspension cells. In this article, we present a supervised automated pipeline for the PMC that minimizes operator intervention by incorporating multivariate logistic regression for data scoring. We test the self-tuning statistical algorithms in a human primary T-cell activation assay in flow using nuclear factor of activated T cells (NFAT) translocation as a readout and readily achieve an average Z' of 0.55 and strictly standardized mean difference of 13 with standard phorbol myristate acetate/ionomycin induction. To implement the tests, we routinely load 4 µL samples and can readout 3000 to 9000 independent conditions from 15 mL of primary human blood (buffy coat fraction). We conclude that the new technology will support primary-cell protein-localization assays and "on-the-fly" data scoring at a sample throughput of more than 100,000 wells per day and that it is, in principle, consistent with a primary pharmaceutical screen.

  8. Identification by differential display of a mRNA specifically induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in T84 human colon carcinoma cells.

    PubMed

    Cafferata, E G; Gonzalez-Guerrico, A M; Pivetta, O H; Santa-Coloma, T A

    1996-07-01

    12-o-Tetradecanoylphorbol-13-Acetate (TPA) down-regulates the expression of the gene responsible for cystic fibrosis (CFTR). To understand the mechanism by which TPA down-regulates CFTR, we decided to study genes specifically induced by this phorbol ester in T84 human colon carcinoma cells, which highly express CFTR, using differential display. Several strategies that allowed us to overcome false-positive reactions in differential displays are described. We have detected different cDNAs obtained from mRNAs specifically induced by TPA. A cDNA fragment corresponding to a mRNA of approximately 2.2 kb was sequenced. Part of this sequence has been reported by others in GenBank and corresponds to a cDNA from a human lung library. The function is unknown and does not have any significant homology with other sequences. It is expressed after two hrs in T84 cells treated with TPA, reaching a maximum response by four hrs. The dose-response curve shows increased mRNA levels starting at 10 ng/ml of TPA and reaching a maximum by 50 ng/ml TPA (10-fold stimulation over control). This mRNA shows a rapid and large response to TPA and it might be involved in the differentiation of T84 cells induced by TPA.

  9. Identification of 1,8-cineole, borneol, camphor, and thujone as anti-inflammatory compounds in a Salvia officinalis L. infusion using human gingival fibroblasts.

    PubMed

    Ehrnhöfer-Ressler, Miriam M; Fricke, Kristina; Pignitter, Marc; Walker, Joel M; Walker, Jessica; Rychlik, Michael; Somoza, Veronika

    2013-04-10

    Drinking or gargling Salvia officinalis L. infusion (sage infusion) is thought to soothe a sore throat, tonsillitis, and inflamed, red gums, although structure-based scientific evidence for the key anti-inflammatory compounds in sage infusion is scarce. Human gingival fibroblasts (HGF-1) were treated with sage infusion (SI) or SI fractions containing either its volatile components and water (aqueous distillate, AD) or its dry matter (DM) for six hours. SI, AD, and DM reduced a mean phorbol-12-myristate-13-acetate/ionomycin (PMA/I)-stimulated release of the pro-inflammatory interleukins IL-6 and IL-8 by more than 50% (p < 0.05). Cellular uptake experiments and subsequent GC-MS analysis using stable-isotope-labeled internal standards revealed the presence of 1,8-cineole, borneol, camphor, and α-/β-thujone in SI-treated cells; LC-MS analysis demonstrated the presence of rosmarinic acid. A significant, more than 50% mean inhibition of PMA/I-induced IL-6 and IL-8 release was demonstrated for the volatile compounds 1,8-cineole, borneol, camphor, and thujone, but not for the nonvolatile rosmarinic acid when applied in concentrations representative of sage infusion. Therefore, the volatile compounds were found to be more effective than rosmarinic acid. 1,8-Cineole, borneol, camphor, and α-/β-thujone chiefly contribute to the anti-inflammatory activity of sage infusion in human gingival fibroblasts.

  10. Palmitic acid anilide-induced respiratory burst in human polymorphonuclear leukocytes is inhibited by a protein kinase C inhibitor, Ro 31-8220.

    PubMed

    Heiskanen, K M; Savolainen, K M

    1997-01-01

    Human polymorphonuclear leukocytes (PMNL) were exposed to palmitic acid anilide, an impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981, and to the corresponding fatty acid, palmitic acid. The effects of these compounds were studied on the production of reactive oxygen metabolites (ROM) and changes in the levels of free intracellular calcium. Palmitic acid anilide induced the production of reactive oxygen metabolites in PMNL. Interestingly, the palmitic acid anilide-induced respiratory burst was completely blocked by a protein kinase C inhibitor, Ro 31-8220. Moreover, palmitic acid anilide additively amplified the production of ROM caused by a chemotactic peptide, formyl-Methionyl-Leucyl-Phenylalanine (FMLP). In contrast, palmitic acid anilide did not have any effect on the production of ROM induced by a tumor promoter, phorbol myristate acetate (PMA). Palmitic acid, in turn, did not markedly induce the production of ROM nor did it amplify the agonist-induced respiratory burst. Neither of the compounds, alone or in combination with FMLP, affected the levels of intracellular calcium in PMNL. These results indicate that the aniline moiety in palmitic acid modifies its effects on the activation of human PMNL, and the subsequent oxidative burst. The present results also suggest that palmitic acid anilide may activate PMNL through a protein kinase C-dependent mechanism.

  11. Expression of the Peptide Antibiotic Human β-Defensin 1 in Cultured Gingival Epithelial Cells and Gingival Tissue

    PubMed Central

    Krisanaprakornkit, Suttichai; Weinberg, Aaron; Perez, Christopher N.; Dale, Beverly A.

    1998-01-01

    Human β-defensin-1 (hBD-1) is a member of the family of small cationic antimicrobial peptides that have been identified in several mucosal epithelia. Because human gingival epithelium is a site that is constantly challenged by oral microorganisms, we examined the expression of hBD-1 in human gingival epithelial and fibroblast cell cultures and tissue samples. Cell cultures were challenged with cell wall extracts of Porphyromonas gingivalis or Fusobacterium nucleatum, Escherichia coli lipopolysaccharide, tumor necrosis factor alpha, or phorbol myristate acetate. hBD-1 mRNA was detected in unstimulated and stimulated cultures by reverse transcription (RT)-PCR using several primer sets specific for hBD-1. Gingival epithelial cells, but not gingival fibroblasts, expressed a product of the predicted size for hBD-1 mRNA. The sequence of the PCR product was identical to that of hBD-1. hBD-1 mRNA expression was not significantly modulated by any of the stimulants tested. Human gingival tissues from noninflamed and inflamed sites were also analyzed by RT-PCR. hBD-1 mRNA was expressed in all tissue samples. The relative expression of hBD-1 mRNA was similar in noninflamed and inflamed tissues obtained from each of four patients undergoing treatment for periodontitis. However, the relative expression of hBD-1 mRNA varied in gingival biopsies obtained from 15 different normal individuals, and the relative hBD-1 expression was unrelated to interleukin-8 expression. Our findings show the constitutive expression of hBD-1 mRNA in cultured epithelial cells and gingival tissues but not gingival fibroblasts. These findings suggest that expression of hBD-1 may play a role as part of the innate host defenses in maintaining normal gingival health. PMID:9712771

  12. Integrin engagement mediates the human polymorphonuclear leukocyte response to a fungal pathogen-associated molecular pattern.

    PubMed

    Lavigne, Liz M; O'Brien, Xian M; Kim, Minsoo; Janowski, Jessie W; Albina, Jorge E; Reichner, Jonathan S

    2007-06-01

    Extravasation of leukocytes from peripheral blood is required for an effective inflammatory response at sites of tissue infection. Integrins help mediate extravasation and navigate the leukocyte to the infectious source. A novel role for integrins in regulating the effector response to a cell wall component of fungal pathogens is the subject of the current study. Although phagocytosis is useful for clearance of unicellular fungi, the immune response against large, noningestible hyphae is not well-understood. Fungal beta-glucan, a pathogen-associated molecular pattern, activates production of superoxide anion in leukocytes without the need for phagocytosis. To model polymorphonuclear leukocyte (PMN) recognition of fungi under conditions in which phagocytosis cannot occur, beta-glucan was covalently immobilized onto tissue culture plastic. Plasma membrane-associated respiratory burst was measured by reduction of ferricytochrome C. Results show that the human PMN oxidative burst response to immobilized beta-glucan is suppressed by addition of beta(1) integrin ligands to the beta-glucan matrix. Suppression was dose dependent and steric hindrance was ruled out. beta(1) integrin ligands did not affect respiratory burst to ingestible beta-glucan-containing particles, phorbol esters or live yeast hyphae. Furthermore, in the absence of matrix, Ab activation of VLA3 or VLA5, but not other beta(1) integrins, also prevented beta-glucan-induced respiratory burst. beta(1)-induced suppression was blocked and burst response restored by treating neutrophils with either the cell-binding fragment of soluble human Fn, cyclic RGD peptide, or Ab specific to VLA3 or VLA5. Together these findings extend the functional role of beta(1) integrins to include modulating PMN respiratory burst to a pathogen-associated molecular pattern.

  13. Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes

    SciTech Connect

    Ramos, C.L.; Pou, S.; Britigan, B.E.

    1992-04-25

    Using the electron spin resonance/spin trapping system, 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol, hydroxyl radical was detected as the alpha-hydroxyethyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH3)OH, from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. 4-POBN-CH(CH3)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by treatment with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators did not appreciably alter 4-POBN-CH(CH3)OH, which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-catalyzed Haber-Weiss reaction. Kinetic differences between transition metal-dependent and -independentmore » mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH3)-OH accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyte-derived macrophages, which lack myeloperoxidase, required the addition of supplemental iron. The addition of purified myeloperoxidase to an enzymatic superoxide generating system resulted in the detection of hydroxyl radical that was dependent upon the presence of chloride and was inhibited by superoxide dismutase, catalase, and azide. These findings implicated the reaction of hypochlorous acid and superoxide to produce hydroxyl radical. 4-POBN-CH(CH3)OH was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. These results support the ability of human neutrophils and monocytes to generate hydroxyl radical through a myeloperoxidase-dependent mechanism.« less

  14. Exposure to 60-Hz magnetic fields and proliferation of human astrocytoma cells in vitro.

    PubMed

    Wei, M; Guizzetti, M; Yost, M; Costa, L G

    2000-02-01

    Epidemiological studies have suggested that exposure to electric and magnetic fields (EMF) may be associated with an increased incidence of brain tumors, most notably astrocytomas. However, potential cellular or molecular mechanisms involved in these effects of EMF are not known. In this study we investigated whether exposure to 60-Hz sinusoidal magnetic fields (0.3-1.2 G for 3-72 h) would cause proliferation of human astrocytoma cells. Sixty-Hertz magnetic fields (MF) caused a time- and dose-dependent increase in proliferation of astrocytoma cells, measured by (3)H-thymidine incorporation and by flow cytometry, and strongly potentiated the effect of two agonists (the muscarinic agonist carbachol and the phorbol ester PMA). However, MF had no effect on DNA synthesis of rat cortical astrocytes, i.e., of similar, nontransformed cells. To determine the amount of heating induced by MF, temperatures were also recorded in the medium. Both 1.2 G MF and a sham exposure caused a 0.7 degrees C temperature increase in the medium; however, (3)H-thymidine incorporation induced by sham exposure was significantly less than that caused by MF. GF 109203X, a rather specific protein kinase C (PKC) inhibitor, and down-regulation of PKC inhibited the effect of MF on basal and on agonist-stimulated (3)H-thymidine incorporation. These data indicate that MF can increase the proliferation of human astrocytoma cells and strongly potentiate the effects of two agonists. These findings may provide a biological basis for the observed epidemiological associations between MF exposure and brain tumors. Copyright 2000 Academic Press.

  15. Echinacea purpurea (L.) Moench modulates human T-cell cytokine response☆

    PubMed Central

    Fonseca, Fabiana N.; Papanicolaou, Genovefa; Lin, Hong; Lau, Clara B.S.; Kennelly, Edward J.; Cassileth, Barrie R.; Cunningham-Rundles, Susanna

    2014-01-01

    The study objective was to evaluate the composition of a neutral and weakly acidic water-soluble extract from Echinacea purpurea (L.) Moench (EchNWA) previously shown to modify murine influenza infection, and to assess immunomodulatory effects on human T-cells. EchNWA extract from fresh aerial parts was extracted with water, ethanolic precipitation, and size-exclusion chromatography. The chemical profile of EchNWA was characterized by chromatography (size-exclusion, HPLC, GC–MS), and small molecule finger-print analysis performed by HPLC–PDA. Jurkat T-cells at high and low cell density were pretreated or not with doses of EchNWA, followed by activation with phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). Interleukin-2 (IL-2) and interferon gamma (IFNg) cytokine secretions were measured by multi-cytokine luminex technology. Results showed that EchNWA contains 80% polysaccharides, predominantly a 10 kDa entity; phenolic compounds, cynarin, cichoric and caftaric acids, but no detectable alkylamides. Cytokine production required stimulation and was lower after PMA+I activation in high-density compared to low-density conditions. EchNWA mediated a strong dose-dependent enhancement of high-density T-cell production of IL-2 and IFNg response to PMA+I. EchNWA alone did not stimulate T-cells. EchNWA enhanced mean fluorescence intensity of IL-2 in Jurkat T-cells activated by PMA+1 or ionomycin alone. Conversely EchNWA mediated modest but significant suppression of IFNg response and reduced the percentage of CD25+ T-cells under low-density conditions. Conclusions are that EchNWA polysaccharides, but not phenolic compounds have dose-related adjuvant effects on human T-cell cytokine responses characterized by enhancing and suppressive effects that are regulated by T-cell density. PMID:24434371

  16. Echinacea purpurea (L.) Moench modulates human T-cell cytokine response.

    PubMed

    Fonseca, Fabiana N; Papanicolaou, Genovefa; Lin, Hong; Lau, Clara B S; Kennelly, Edward J; Cassileth, Barrie R; Cunningham-Rundles, Susanna

    2014-03-01

    The study objective was to evaluate the composition of a neutral and weakly acidic water-soluble extract from Echinacea purpurea (L.) Moench (EchNWA) previously shown to modify murine influenza infection, and to assess immunomodulatory effects on human T-cells. EchNWA extract from fresh aerial parts was extracted with water, ethanolic precipitation, and size-exclusion chromatography. The chemical profile of EchNWA was characterized by chromatography (size-exclusion, HPLC, GC-MS), and small molecule fingerprint analysis performed by HPLC-PDA. Jurkat T-cells at high and low cell density were pretreated or not with doses of EchNWA, followed by activation with phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). Interleukin-2 (IL-2) and interferon gamma (IFNg) cytokine secretions were measured by multi-cytokine luminex technology. Results showed that EchNWA contains 80% polysaccharides, predominantly a 10kDa entity; phenolic compounds, cynarin, cichoric and caftaric acids, but no detectable alkylamides. Cytokine production required stimulation and was lower after PMA+I activation in high-density compared to low-density conditions. EchNWA mediated a strong dose-dependent enhancement of high-density T-cell production of IL-2 and IFNg response to PMA+I. EchNWA alone did not stimulate T-cells. EchNWA enhanced mean fluorescence intensity of IL-2 in Jurkat T-cells activated by PMA+1 or ionomycin alone. Conversely EchNWA mediated modest but significant suppression of IFNg response and reduced the percentage of CD25+ T-cells under low-density conditions. Conclusions are that EchNWA polysaccharides, but not phenolic compounds have dose-related adjuvant effects on human T-cell cytokine responses characterized by enhancing and suppressive effects that are regulated by T-cell density. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Secretion of the human T cell leukemia virus type I transactivator protein tax.

    PubMed

    Alefantis, Timothy; Mostoller, Kate; Jain, Pooja; Harhaj, Edward; Grant, Christian; Wigdahl, Brian

    2005-04-29

    Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.

  18. Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

    PubMed

    Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

    1996-05-01

    The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

  19. Characterization of Treefoil Peptide Genes in Iron-Ion or X-Irradiated Human Cells

    NASA Technical Reports Server (NTRS)

    Balcer-Kubiczek, E. K.; Harrison, G. H.; Xu, J. F.; Zhou, X. F.

    1999-01-01

    The gastrointestinal (GI) tract is especially sensitive to ionizing radiation, probably because of its high rate of cell turn over. Most of the data in the literature concerns the histological/anatomical description of damage rather than functional studies. In fact, previous reports in humans have shown that, at doses of 2 Gy or more, functional abnormalities appear indicating that in radiation sensitive tissues the effects of radiation are not limited to cell death. GI functions are controlled in particular by GI peptides. One hypothesis is that ionizing radiation may modulate the synthesis and release of these peptides and consequently may contribute largely to abnormalities in GI function. However, no previous studies have been concerned with GI-specific gene expression in irradiated GI tissues. The family of human trefoil peptides comprises three members thus far, all of which are expressed in specific regions of the GI tract. In addition, two trefoil peptides, pS2 (TFFI) and HITF (TFF2) are expressed in breast tissue. Their exact function in GI and breast tissues is unclear but mucosal integrity, repair, mucin secretion and responsiveness to hormones have been shown. We recently isolated and characterized pS2 as a novel p53- and estrogen receptor-independent gene whose MRNA expression in several cells lines was found to be delayed 4 to 7 days after irradiation with X-rays, fission neutrons or 1 GeV/n Fe-ions. The aim of the present study was to determine whether pS2 and HITF have a similar induction kinetics in irradiated gastric and breast cell lines, and whether they have the phorbol ester (TPA) responsive element (TRE).

  20. Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia

    PubMed Central

    Jia, Hong Peng; Look, Dwight C.; Tan, Ping; Shi, Lei; Hickey, Melissa; Gakhar, Lokesh; Chappell, Mark C.; Wohlford-Lenane, Christine; McCray, Paul B.

    2009-01-01

    Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1β and TNFα acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase “recognition motif.” Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface. PMID:19411314

  1. Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function.

    PubMed

    Wright, Lesley; Bubb, William; Davidson, John; Santangelo, Rosemary; Krockenberger, Mark; Himmelreich, Uwe; Sorrell, Tania

    2002-11-01

    Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4-5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites-acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and gamma-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas.

  2. IN VITRO EFFECTS OF A COMMERCIAL HERBAL MEDICINE USED AS AFRICAN TRADITIONAL MEDICINE ON HUMAN NEUTROPHILS.

    PubMed

    Mothibe, Mmamosheledi Elsie; Kahler-Venter, Christinah; Osuch, Elżbieta

    2017-01-01

    Commercial herbal medicines (CHMs) being marketed as immune boosters or tonics, have gained widespread popularity. The many herbal mixtures sold have not been tested for efficacy and safety, despite their modern packaging and presentations. It is imperative that these herbal mixtures be investigated for their effects on human neutrophils. The selected herbal mixture (HM), Stametta ™ Body healing liquid, is common in retail outlets in Pretoria, South Africa (SA) and is used as an immune booster or intended to strengthen the body. Isolated neutrophils as well as those in whole blood phagocytes were obtained from blood samples collected from consenting healthy adult volunteers. The neutrophils were incubated with the HM at different strengths, and taken through a luminol-enhanced luminescence assay, using activators- phorbol myristate acetate and N-formyl-methionyl-leucyl-phenylalanine. The HM had variable stimulatory and inhibitory effects on the luminescence activity of healthy isolated and non-isolated human neutrophils. The effects, ranging from weak to potent were either directly or inversely related to the concentration of the HM and were mediated through a direct protein kinase C activating mechanism and an indirect formyl peptide receptor-linked mechanism. The findings have shown the immunomodulatory potential of Stametta ™. The in vitro inhibitory and stimulatory effects on neutrophils which are furthermore time-based, suggest variable effects on the immune system, which may be beneficial as well as risky. The effects at different concentrations highlight the importance of appropriate dosing. It would therefore be prudent to caution users of this commercial herbal medicine accordingly.

  3. In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL-2 production in human T-cells.

    PubMed

    Midgett, Kristin; Peden-Adams, Margie M; Gilkeson, Gary S; Kamen, Diane L

    2015-05-01

    Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)-2 production in the human Jurkat T-cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD-3/anti CD-28, or anti CD-3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml(-1) PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml(-1) PFOA. Jurkat cells stimulated with PHA/PMA or anti CD-3 exhibited decreased IL-2 production beginning at 50 µg PFOS ml(-1) and 5 µg PFOS ml(-1) respectively, but stimulation with anti-CD3/anti-CD28 resulted in no changes compared with the control. Addition of the PPAR-alpha antagonist GW6471 to PFOS-dosed cells stimulated with PHA/PMA resulted in decreases in IL-2 production starting at 50 µg PFOS ml(-1), which suggests PFOS affected T-cell IL-2 production via PPAR-alpha-independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL-2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL-2 production, but PFOS suppressed IL-2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL-2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. Copyright © 2014 John Wiley & Sons, Ltd.

  4. In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL-2 production in human T-cells

    PubMed Central

    Midgett, Kristin; Peden-Adams, Margie M.; Gilkeson, Gary S.; Kamen, Diane L.

    2014-01-01

    Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)-2 production in the human Jurkat T-cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD-3/anti CD-28, or anti CD-3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 μg ml−1 PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μg ml−1 PFOA. Jurkat cells stimulated with PHA/PMA or anti CD-3 exhibited decreased IL-2 production beginning at 50 μg PFOS ml−1 and 5 μg PFOS ml−1 respectively, but stimulation with anti-CD3/anti-CD28 resulted in no changes compared with the control. Addition of the PPAR-alpha antagonist GW6471 to PFOS-dosed cells stimulated with PHA/PMA resulted in decreases in IL-2 production starting at 50 μg PFOS ml−1, which suggests PFOS affected T-cell IL-2 production via PPAR-alpha-independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL-2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL-2 production, but PFOS suppressed IL-2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL-2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. PMID:25056757

  5. The effects of N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA) on the changes in free intracellular calcium and the production of reactive oxygen metabolites in human leukocytes.

    PubMed

    Heiskanen, K M; Naarala, J; Savolainen, K M

    1995-06-26

    Human leukocytes were exposed to N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA), a postulated impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981. Changes induced by 5-VTPA alone and together with a chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), a tumor promoter, phorbol myristate acetate (PMA), or a synthetic diacylglycerol, dioctanoyl-s,n-glycerol (DiC8) in free intracellular calcium levels ([Ca2+]i) and in the induction of oxidative burst were measured. 5-VTPA elevated dose-dependently [Ca2+]i and induced the production of reactive oxygen metabolites in leukocytes. 5-VTPA also amplified FMLP-induced increase in [Ca2+]i, but was without an effect on FMLP-induced oxidative burst. On the contrary, 5-VTPA amplified dose-dependently PMA- and DiC8-induced respiratory burst. The present results indicate that 5-VTPA may interfere with transmembrane signalling in human leukocytes. 5-VTPA may elevate [Ca2+]i by acting directly on the membrane, or by acting through Ca(2+)-mobilizing receptors. Moreover, 5-VTPA also clearly amplified responses produced through protein kinase C stimulation. Thus, 5-VTPA may act on human leukocytes by affecting Ca(2+)-metabolism and the activity of protein kinase C.

  6. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

    1997-01-01

    Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

  7. Human natural killer cells exhibit negative regulatory function by ectopic expression of hFoxp3 gene.

    PubMed

    Wang, Fei; Jiang, Wen; Zhang, Cai; Tian, Zhigang; Zhang, Jian

    2013-06-15

    Foxp3 is a key marker of CD4CD25 regulatory T cells and appears highly specific for regulatory T cells. Human dendritic cells transfected with foxp3 gene also exhibit immunosuppressive functions. We want to understand whether natural killer (NK) cells could be endowed with regulatory properties by transduction of Foxp3 gene. A recombinant vector (pRV.GFP Foxp3) or control vector (pRV.GFP WWRR) was transferred into NKL/NK-92 cells by an electroporation method. The hFoxp3 gene-modified NK cells were characterized with regard to their proliferation, cytokine production, and cytotoxicity and their regulatory effects on activated human peripheral blood mononuclear cells (hPBMCs) in vitro and trans vivo delayed-type hypersensitivity assay. We found that the ectopic expression of hFoxp3 in human NK cells resulted in the high production of the immunosuppressive cytokine, interleukin (IL)-10. Luciferase reporter assay showed that the expression of IL-10 is directly regulated by Foxp3. We observed that NKL.Foxp3 cells inhibited the proliferation and activation of phorbol-12-myristate-13-acetate/ionomycin-stimulated hPBMCs; furthermore, NKL.Foxp3 cells significantly suppressed the delayed-type hypersensitivity response, which was induced by anti-CD3 monoclonal antibody-activated hPBMCs. NKL.Foxp3 cell-mediated negative regulatory function was dependent on IL-10 production. Our findings indicated that NK cells acquired IL-10 phenotype by transduction with foxp3 gene and provided evidence that Foxp3 could exert regulatory function not only in regulatory T cells but also in NK cells. These results suggested that Foxp3 gene-modified NK cells might be potential usefulness on graft-versus-host disease or some autoimmune diseases.

  8. Extracellular Cl− regulates human SO42−/anion exchanger SLC26A1 by altering pH-sensitivity of anion transport

    PubMed Central

    Wu, Meng; Heneghan, John F.; Vandorpe, David H.; Escobar, Laura I.; Wu, Bai-Lin; Alper, Seth L.

    2016-01-01

    Genetic deficiency of the SLC26A1 anion exchanger in mice is known to be associated with hyposulfatemia and hyperoxaluria with nephrolithiasis, but many aspects of human SLC26A1 function remain to be explored. We report here the functional characterization of human SLC26A1, a DIDS-sensitive, electroneutral sodium-independent anion exchanger transporting sulfate, oxalate, bicarbonate, thiosulfate and (with divergent properties) chloride. Human SLC26A1-mediated anion exchange differs from that of its rodent orthologs in its stimulation by alkaline pHo and inhibition by acidic pHo but not pHi, and in its failure to transport glyoxylate. SLC26A1-mediated transport of sulfate and oxalate is highly dependent on allosteric activation by extracellular chloride or nonsubstrate anions. Extracellular chloride stimulates apparent Vmax of human SLC26A1-mediated sulfate uptake by conferring a two-log decrease in sensitivity to inhibition by extracellular protons, without changing transporter affinity for extracellular sulfate. In contrast to SLC26A1-mediated sulfate transport, SLC26A1-associated chloride transport is activated by acid pHo, shows reduced sensitivity to DIDS, and exhibits cation-dependence of its DIDS-insensitive component. Human SLC26A1 resembles SLC26 paralogs in its inhibition by phorbol ester activation of PKC, which differs in its undiminished polypeptide abundance at or near the oocyte surface. Mutation of SLC26A1 residues corresponding to candidate anion binding site-associated residues in avian SLC26A5/prestin altered anion transport in patterns resembling those of prestin. However, rare SLC26A1 polymorphic variants from a patient with renal Fanconi Syndrome and from a patient with nephrolithiasis/calcinosis exhibited no loss-of-function phenotypes consistent with disease pathogenesis. PMID:27125215

  9. Extracellular Cl(-) regulates human SO4 (2-)/anion exchanger SLC26A1 by altering pH sensitivity of anion transport.

    PubMed

    Wu, Meng; Heneghan, John F; Vandorpe, David H; Escobar, Laura I; Wu, Bai-Lin; Alper, Seth L

    2016-08-01

    Genetic deficiency of the SLC26A1 anion exchanger in mice is known to be associated with hyposulfatemia and hyperoxaluria with nephrolithiasis, but many aspects of human SLC26A1 function remain to be explored. We report here the functional characterization of human SLC26A1, a 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS)-sensitive, electroneutral sodium-independent anion exchanger transporting sulfate, oxalate, bicarbonate, thiosulfate, and (with divergent properties) chloride. Human SLC26A1-mediated anion exchange differs from that of its rodent orthologs in its stimulation by alkaline pHo and inhibition by acidic pHo but not pHi and in its failure to transport glyoxylate. SLC26A1-mediated transport of sulfate and oxalate is highly dependent on allosteric activation by extracellular chloride or non-substrate anions. Extracellular chloride stimulates apparent V max of human SLC26A1-mediated sulfate uptake by conferring a 2-log decrease in sensitivity to inhibition by extracellular protons, without changing transporter affinity for extracellular sulfate. In contrast to SLC26A1-mediated sulfate transport, SLC26A1-associated chloride transport is activated by acid pHo, shows reduced sensitivity to DIDS, and exhibits cation dependence of its DIDS-insensitive component. Human SLC26A1 resembles SLC26 paralogs in its inhibition by phorbol ester activation of protein kinase C (PKC), which differs in its undiminished polypeptide abundance at or near the oocyte surface. Mutation of SLC26A1 residues corresponding to candidate anion binding site-associated residues in avian SLC26A5/prestin altered anion transport in patterns resembling those of prestin. However, rare SLC26A1 polymorphic variants from a patient with renal Fanconi Syndrome and from a patient with nephrolithiasis/calcinosis exhibited no loss-of-function phenotypes consistent with disease pathogenesis.

  10. Human tolerances.

    DOT National Transportation Integrated Search

    1962-04-01

    The ultimate limitations in flight performance and in future civil air carrier equipment are the limitations imposed by what may be termed human tolerances. This is particularly applicable to the matter of the supersonic transport. The discussi...

  11. Human expunction

    NASA Astrophysics Data System (ADS)

    Klee, Robert

    2017-10-01

    Thomas Nagel in `The Absurd' (Nagel 1971) mentions the future expunction of the human species as a `metaphor' for our ability to see our lives from the outside, which he claims is one source of our sense of life's absurdity. I argue that the future expunction (not to be confused with extinction) of everything human - indeed of everything biological in a terran sense - is not a mere metaphor but a physical certainty under the laws of nature. The causal processes by which human expunction will take place are presented in some empirical detail, so that philosophers cannot dismiss it as merely speculative. I also argue that appeals to anthropic principles or to forms of mystical cosmology are of no plausible avail in the face of human expunction under the laws of physics.

  12. Human Health

    MedlinePlus

    ... Water Supply Agriculture Indigenous Peoples Ecosystems and Biodiversity Oceans Responses Regions Northeast Southeast Midwest Great Plains Southwest ... Peoples Widespread Impacts Ecosystems and Biodiversity Human Health Oceans Infrastructure Responses Northeast Northwest Southeast Alaska Midwest Hawai' ...

  13. Synergistic induction of gene expression during the differentiation into mature macrophage in human myeloblastic leukemia cells treated with TPA and KH1060.

    PubMed

    Shimizu, Takahisa; Kuromi, Aya; Takeda, Ken

    2009-06-01

    Treatment of human myeloblastic leukemia ML-1 cells with the phorbol ester TPA in combination with the vitamin D(3) analogue KH1060 will induce a synergistic differentiation to mature macrophage with multinuclei. To investigate the mechanism underlying this differentiation and the synergistic effect, a cDNA microarray and Northern blot analysis were used to examine gene expression profiles of ML-1 cells treated with TPA and/or KH1060. Results show that KH1060 enhanced several TPA-induced gene expressions and that TPA enhanced several KH1060-induced gene expressions. Studies with inhibitors of signaling molecules suggested that PKC and MAPK pathways play an important role in the differentiation induced by TPA and KH1060, and that they are associated with the synergistic induction of genes. The results of this study indicate the possibility that the expression of various genes are induced synergistically by cross-talk between TPA and KH1060 signals. It is likely that the synergistic effect on gene expression leads to the synergistic induction of differentiation by both reagents.

  14. Profile of CYP19A1 mRNA expression and aromatase activity during syncytialization of primary human villous trophoblast cells at term.

    PubMed

    Hudon Thibeault, Andrée-Anne; Vaillancourt, Cathy; Sanderson, J Thomas

    2018-05-01

    Estrogen production by the human villous trophoblast is dependent on the biosynthetic enzyme aromatase (CYP19; CYP19A1) and is crucial for successful placental development and pregnancy outcome. Using villous cytotrophoblast cells (vCTs) freshly isolated from normal term placenta, we characterized the promoter-specific expression of CYP19A1 mRNA (derived from promoters I.1, I.4, I.8 or total transcript) and aromatase activity during villous trophoblast syncytialization. CYP19A1 mRNA levels and aromatase activity in vCTs reached a maximum after about 48 h of culture. The cAMP inducer forskolin (10 μM) and protein kinase C stimulant phorbol myristate acetate (1 μM) increased CYP19A1 mRNA levels by 1.8- and 1.6-fold, respectively, as well as inducing aromatase catalytic activity. Dexamethasone (100 nM) and vascular endothelial growth factor (5 ng/mL) decreased CYP19A1 mRNA levels, while having no effect on aromatase activity. Our results emphasize the importance of not solely studying CYP19A1 regulation and function at the mRNA level but also considering posttranslational mechanisms that alter the final catalytic activity of aromatase. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Inhibitory effects of kaempferol on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9.

    PubMed

    Li, Chenglin; Zhao, Yuanwei; Yang, Dan; Yu, Yanyan; Guo, Hao; Zhao, Ziming; Zhang, Bei; Yin, Xiaoxing

    2015-02-01

    Matrix metalloproteinases (MMPs) have been regarded as major critical molecules assisting tumor cells during metastasis, for excessive ECM (ECM) degradation, and cancer cell invasion. In the present study, in vitro and in vivo assays were employed to examine the inhibitory effects of kaempferol, a natural polyphenol of flavonoid family, on tumor metastasis. Data showed that kaempferol could inhibit adhesion, migration, and invasion of MDA-MB-231 human breast carcinoma cells. Moreover, kaempferol led to the reduced activity and expression of MMP-2 and MMP-9, which were detected by gelatin zymography, real-time PCR, and western blot analysis, respectively. Further elucidation of the mechanism revealed that kaempferol treatment inhibited the activation of transcription factor activator protein-1 (AP-1) and MAPK signaling pathway. Moreover, kaempferol repressed phorbol-12-myristate-13-acetate (PMA)-induced MMP-9 expression and activity through suppressing the translocation of protein kinase Cδ (PKCδ) and MAPK signaling pathway. Our results also indicated that kaempferol could block the lung metastasis of B16F10 murine melanoma cells as well as the expression of MMP-9 in vivo. Taken together, these results demonstrated that kaempferol could inhibit cancer cell invasion through blocking the PKCδ/MAPK/AP-1 cascade and subsequent MMP-9 expression and its activity. Therefore, kaempferol might act as a therapeutic potential candidate for cancer metastasis.

  16. All-Trans Retinoic Acid Inhibits Human Colorectal Cancer Cells RKO Migration via Downregulating Myosin Light Chain Kinase Expression through MAPK Signaling Pathway.

    PubMed

    Zuo, Li; Yang, Xiaoping; Lu, Man; Hu, Ruolei; Zhu, Huaqing; Zhang, Sumei; Zhou, Qing; Chen, Feihu; Gui, Shuyu; Wang, Yuan

    2016-10-01

    All-trans-retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells. However, the underlying mechanism is unclear. Here, we demonstrate that ATRA inhibited colorectal cancer cells RKO (human colon adenocarcinoma cell) migration by downregulating cell movement and increasing cell adhesion. ATRA inhibited the expression and activation of myosin light chain kinase (MLCK) in RKO cells, while the expression level of MLC phosphatase (MLCP) had no change in RKO cells treated with or without ATRA. The expression and activity of MLC was also inhibited in RKO cells exposed to ATRA. Intriguingly, ATRA increased the expression of occludin messenger RNA (mRNA) and protein and its localization on cell membrane. However, ATRA did not change the expression of zonula occludens 1 (ZO-1), but increased the accumulation of ZO-1 on RKO cells membrane. ML-7, an inhibitor of MLCK, significantly inhibited RKO cell migration. Furthermore, knockdown of endogenous MLCK expression inhibited RKO migration. Mechanistically, we showed that MAPK-specific inhibitor PD98059 enhanced the inhibitory effect of ATRA on RKO migration. In contrast, phorbol 12-myristate 13-acetate (PMA) attenuated the effects of ATRA in RKO cells. Moreover, knocking down endogenous extracellular signal-regulated kinase (ERK) expression inhibited MLCK expression in the RKO cells. In conclusion, ATRA inhibits RKO migration by reducing MLCK expression via extracellular signal-regulated kinase 1/Mitogen-activated protein kinase (ERK1/MAPK) signaling pathway.

  17. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts.

    PubMed

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-06-16

    Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

  18. Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella.

    PubMed

    Renwick, Julie; Reeves, Emer P; Wientjes, Frans B; Kavanagh, Kevin

    2007-01-01

    Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA) activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels. Primarily these results correlate translocation of hemocyte 47 and 67kDa proteins with PMA induced oxidase activity. Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens.

  19. Flavonoids targeting of IκB phosphorylation abrogates carcinogen-induced MMP-9 and COX-2 expression in human brain endothelial cells

    PubMed Central

    Tahanian, Elizabeth; Sanchez, Luis Arguello; Shiao, Tze Chieh; Roy, René; Annabi, Borhane

    2011-01-01

    Brain endothelial cells play an essential role as structural and functional components of the blood–brain barrier (BBB). Increased BBB breakdown and brain injury are associated with neuroinflammation and are thought to trigger mechanisms involving matrix metalloproteinase upregulation. Emerging evidence also indicates that cyclooxygenase (COX) inhibition limits BBB disruption, but the mechanisms linking metalloproteinase to COX remain unknown. In this study, we sought to investigate the nuclear factor-kappa B (NF-κB) signaling pathway, a common pathway in both the regulation of matrix metalloproteinase-9 (MMP-9) and COX-2 expression, and the inhibitory properties of several chemopreventive flavonoids. Human brain microvascular endothelial cells were treated with a combination of phorbol 12-myristate 13-acetate (PMA), a carcinogen documented to increase MMP-9 and COX-2 through NF-κB, and several naturally occurring flavonoids. Among the molecules tested, we found that fisetin, apigenin, and luteolin specifically and dose-dependently antagonized PMA-induced COX-2 and MMP-9 gene and protein expressions as assessed by qRT-PCR, immunoblotting, and zymography respectively. We further demonstrate that flavonoids impact on IκK-mediated phosphorylation activity as demonstrated by the inhibition of PMA-induced IκB phosphorylation levels. Our results suggest that BBB disruption during neuroinflammation could be pharmacologically reduced by a specific class of flavonoids acting as NF-κB signal transduction inhibitors. PMID:21625419

  20. Human immunodeficiency virus infection of helper T cell clones. Early proliferative defects despite intact antigen-specific recognition and interleukin 4 secretion.

    PubMed Central

    Laurence, J; Friedman, S M; Chartash, E K; Crow, M K; Posnett, D N

    1989-01-01

    HIV selectively inhibited the proliferative response of clonal CD4+ T lymphocytes to alloantigen while other alloantigen-dependent responses were unperturbed. Specifically, impaired blastogenesis could be dissociated from alloantigen-specific induction of the B cell activation molecule CD23, IL-4 release, and inositol lipid hydrolysis. In addition, membrane expression of pertinent T cell receptor molecules, including CD2, CD3, and T cell antigen receptor (Ti), remained intact. Using two MHC class II-specific human CD4+ helper T cell clones, the proliferative defect was shown to be an early consequence of HIV infection, occurring within 4 d of viral inoculation and preceding increases in mature virion production. It was generalizable to three distinct methods of T cell activation, all independent of antigen-presenting cells: anti-CD3 mediated cross-linking of the CD3/Ti complex; anti-CD2 and phorbol 12-myristic 13-acetate (PMA); and anti-CD28 plus PMA. These abnormalities were not mitigated by addition of exogenous IL-2, even though expression of the IL-2 receptor (CD25) was unaltered. These studies define a selective blockade in T cell function early after HIV exposure that could serve as a model for certain in vivo manifestations of AIDS. PMID:2470786

  1. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts

    PubMed Central

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-01-01

    Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements. PMID:27322248

  2. Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties.

    PubMed

    Nguyen, Su Duy; Maaninka, Katariina; Lappalainen, Jani; Nurmi, Katariina; Metso, Jari; Öörni, Katariina; Navab, Mohamad; Fogelman, Alan M; Jauhiainen, Matti; Lee-Rueckert, Miriam; Kovanen, Petri T

    2016-02-01

    Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. The findings identify C-terminal cleavage of apoA-I by human mast

  3. Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties

    PubMed Central

    Nguyen, Su Duy; Maaninka, Katariina; Lappalainen, Jani; Nurmi, Katariina; Metso, Jari; Öörni, Katariina; Navab, Mohamad; Fogelman, Alan M.; Jauhiainen, Matti; Lee-Rueckert, Miriam

    2016-01-01

    Objective— Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. Approach and Results— Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α–activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB–dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)– and M-CSF (macrophage colony-stimulating factor)–differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)–activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. Conclusions— The

  4. Anti-inflammatory, membrane-stabilizing interactions of salmeterol with human neutrophils in vitro.

    PubMed

    Anderson, R; Feldman, C; Theron, A J; Ramafi, G; Cole, P J; Wilson, R

    1996-04-01

    1. We have investigated the effects of salmeterol (0.3-50 microM) on several pro-inflammatory activities of human neutrophils in vitro. 2. Oxidant production by FMLP- and calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (0.3-3 microM) of salmeterol, while the responses of phorbol myristate acetate- and opsonised zymosan-stimulated cells were affected only by higher concentrations (3-50 microM) of the drug. At these concentrations salmeterol is not cytotoxic, nor does it act as a scavenger of superoxide. 3. These anti-oxidative interactions of salmeterol with neutrophils were insensitive to propranolol but could be eliminated by washing the cells, or by pretreatment with low concentrations (1-2 microM) of the pro-oxidative, membrane-destabilizing phospholipids, lysophosphatidylcholine (LPC), platelet activating factor (PAF) and lysoPAF (LPAF). 4. At concentrations of 6.25-50 microM salmeterol interfered with several other activities of stimulated neutrophils, including intracellular calcium fluxes, phospholipase A2 activity and synthesis of PAF. 5. In an assay of membrane-stabilizing activity, salmeterol (25 and 50 microM) neutralized the haemolytic action of LPC, PAF and LPAF. 6. Of the other commonly used beta 2-adrenoceptor agonists, fenoterol, and formoterol, but not salbutamol, caused moderate inhibition of neutrophil oxidant generation by a superoxide-scavenging mechanism. However, unlike salmeterol, these agents possessed only weak membrane stabilizing properties. 7. We conclude that salmeterol antagonizes the pro-inflammatory, pro-oxidative activity of several bioactive lipids implicated in the pathogenesis of bronchial asthma, by a mechanism related to the membrane-stabilizing, rather than to the beta 2-agonist properties of this agent.

  5. CFTR Expression in Human Neutrophils and the Phagolysosomal Chlorination Defect in Cystic Fibrosis

    PubMed Central

    Painter, Richard G.; Valentine, Vincent G.; Lanson, Nicholas A.; Leidal, Kevin; Zhang, Qiang; Lombard, Gisele; Thompson, Connie; Viswanathan, Anand; Nauseef, William M.; Wang, Guangdi; Wang, Guoshun

    2010-01-01

    Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC/MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically pre-labeled with 13C-L-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides the evidence to suggest that CFTR channel expression in neutrophils and its dysfunction affects neutrophil chlorination of phagocytosed bacteria. PMID:16922501

  6. Intracellular trafficking and fate of chimeric adenovirus 5/F35 in human B lymphocytes.

    PubMed

    Samson, Mélanie; Jung, Daniel

    2011-09-01

    Investigation of the molecular processes that control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte-associated pathogenesis. Adenovirus-mediated gene transfer provides a powerful tool for investigating these processes. However, we observed variation in transgene expression among normal human peripheral blood B lymphocytes from different donors and at distinct stages of differentiation. It is recognized that efficient gene transfer is highly dependent on the intracellular route by which the viruses travel within the host cell. Thus, we aimed to examine this aspect in the present study. We analyzed the binding, uptake, intracellular trafficking and fate of CY3-labelled Ad5/F35 vectors in lymphoid cell lines and primary B cells. Furthermore, we decreased protein synthesis levels and rapid endocytosis in a plasma cell line exhibiting a high level of protein synthesis activity and activated transcription and endocytosis in primary B cells, which are less active than plasma cells. Major differences in intracellular trafficking pattern between B cells and plasma cell line U266 were identified that explain the observed divergence in transgene expression efficiency. Importantly, modification of the transcriptional or translational activity of U266 cells reverted the Ad5/F35 endocytic trafficking to that seen in B cells, with a loss of transgene expression, whereas activation of B cells with phorbol 12-myristate 13-acetate had the opposite effects. Taken together, these results suggest that Ad5/F35 is more efficiently transduced in cells with a strong transcriptional activity as a result of differences in intracellular trafficking. This finding extends our current knowledge of the mechanisms of adenovirus-mediated gene transfer. Copyright © 2011 John Wiley & Sons, Ltd.

  7. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  8. Mitochondrial dysfunction as the cause of the failure to precondition the diabetic human myocardium.

    PubMed

    Hassouna, Ashraf; Loubani, Mahmoud; Matata, Bashir M; Fowler, Alan; Standen, Nicholas B; Galiñanes, Manuel

    2006-02-01

    We have shown previously that human diabetic myocardium cannot be preconditioned. Here, we have investigated the basis of this cardioprotective deficit. Right atrial sections from four patient groups-non-diabetic, insulin-dependent diabetes mellitus (IDDM), non-insulin-dependent diabetes mellitus (NIDDM) receiving glibenclamide, and NIDDM receiving metformin-were subjected to one of the following protocols: aerobic control, simulated ischemia/reoxygenation, ischemic preconditioning before ischemia, and pharmacological preconditioning with alpha 1 agonist phenylephrine, adenosine, the mito-K(ATP) channel opener diazoxide, the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA), or the p38 mitogen-activated protein kinase (p38MAPK) activator anisomycin. Cellular damage was assessed using creatine kinase leakage and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. In mitochondrial preparations from non-diabetic and diabetic myocardium, mitochondrial membrane potential (Psi(m)) was assessed using JC-1 dye, and production of reactive oxygen species was determined. Preconditioning with ischemia, phenylephrine, adenosine, or diazoxide failed to protect diabetic myocardium. However, activation of PKC or p38MAPK was still protective. In isolated non-diabetic mitochondria, diazoxide partially depolarized Psi(m), an effect not seen in diabetic mitochondria. Furthermore, diazoxide increased superoxide production in non-diabetic but not in diabetic mitochondria. Our results show that the cardioprotective deficit in diabetic myocardium arises upstream of PKC and p38MAPK. We suggest that mitochondrial dysfunction in diabetic myocardium, possibly dysfunctional mito-K(ATP) channels, leads to impaired depolarization and superoxide production, and that this causes the inability to respond to preconditioning.

  9. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  10. Differential Surface Expression of ADAM10 and ADAM17 on Human T Lymphocytes and Tumor Cells

    PubMed Central

    Kabelitz, Dieter; Janssen, Ottmar

    2013-01-01

    A disintegrin and metalloproteases (ADAMs) have been implicated in many processes controlling organismic development and integrity. Important substrates of ADAM proteases include growth factors, cytokines and their receptors and adhesion proteins. The inducible but irreversible cleavage of their substrates alters cell-cell communication and signaling. The crucial role of ADAM proteases (e.g. ADAM10 and 17) for mammalian development became evident from respective knockout mice, that displayed pre- or perinatal lethality with severe defects in many organs and tissues. Although many substrates for these two ADAM proteases were identified over the last decade, the regulation of their surface appearance, their enzymatic activity and their substrate specificity are still not well understood. We therefore analyzed the constitutive and inducible surface expression of ADAM10 and ADAM17 on a variety of human T cell and tumor cell lines. We demonstrate that ADAM10 is constitutively present at comparably high levels on the majority of the tested cell types. Stimulation with phorbol ester and calcium ionophore does not significantly alter the amount of surface ADAM10, except for a slight down-regulation from T cell blasts. Using FasL shedding as a readout for ADAM10 activity, we show that PKC activation and calcium mobilization are both prerequisite for activation of ADAM10 resulting in a production of soluble FasL. In contrast to ADAM10, the close relative ADAM17 is detected at only low levels on unstimulated cells. ADAM17 surface expression on T cell blasts is rapidly induced by stimulation. Since this inducible mobilization of ADAM17 is sensitive to inhibitors of actin filament formation, we propose that ADAM17 but not ADAM10 is prestored in a subcellular compartment that is transported to the cell surface in an activation- and actin-dependent manner. PMID:24130797

  11. Role of ATM in bystander signaling between human monocytes and lung adenocarcinoma cells.

    PubMed

    Ghosh, Somnath; Ghosh, Anu; Krishna, Malini

    2015-12-01

    The response of a cell or tissue to ionizing radiation is mediated by direct damage to cellular components and indirect damage mediated by radiolysis of water. Radiation affects both irradiated cells and the surrounding cells and tissues. The radiation-induced bystander effect is defined by the presence of biological effects in cells that were not themselves in the field of irradiation. To establish the contribution of the bystander effect in the survival of the neighboring cells, lung carcinoma A549 cells were exposed to gamma-irradiation, 2Gy. The medium from the irradiated cells was transferred to non-irradiated A549 cells. Irradiated A549 cells as well as non-irradiated A549 cells cultured in the presence of medium from irradiated cells showed decrease in survival and increase in γ-H2AX and p-ATM foci, indicating a bystander effect. Bystander signaling was also observed between different cell types. Phorbol-12-myristate-13-acetate (PMA)-stimulated and gamma-irradiated U937 (human monocyte) cells induced a bystander response in non-irradiated A549 (lung carcinoma) cells as shown by decreased survival and increased γ-H2AX and p-ATM foci. Non-stimulated and/or irradiated U937 cells did not induce such effects in non-irradiated A549 cells. Since ATM protein was activated in irradiated cells as well as bystander cells, it was of interest to understand its role in bystander effect. Suppression of ATM with siRNA in A549 cells completely inhibited bystander effect in bystander A549 cells. On the other hand suppression of ATM with siRNA in PMA stimulated U937 cells caused only a partial inhibition of bystander effect in bystander A549 cells. These results indicate that apart from ATM, some additional factor may be involved in bystander effect between different cell types. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

    PubMed Central

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-01-01

    HIV type 1 (HIV-1) infects CD4+ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages. PMID:26056317

  13. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

    PubMed

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-06-23

    HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

  14. Regulation of muscarinic acetylcholine receptors in the 1321N1 human astrocytoma cell line

    SciTech Connect

    Hoover, R.K.

    1989-01-01

    The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of 1321N1 human astrocytoma cells was studied. Binding was studied in both intact cells and cell lysates. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand ({sup 3}H)QNB or the hydrophilic radioligand ({sup 3}H)NMS. In contrast, full agonists exhibited markedly lower apparent affinities in intact cells with ({sup 3}H)QNB than with ({sup 3}H)NMS. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptormore » subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs ({sup 3}H)QNB were markedly higher. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. In other studies, the effects of the protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) on muscarinic receptor downregulation and internalization in 1321N1 cells were determined. PMA alone did not induce muscarinic receptor downregulation but instead decreased both the rate and final extent of downregulation induced by the agonist carbachol. The specificity of other protein kinase C activators for inhibiting carbachol-induced downregulation indicated involvement of protein kinase C. Furthermore, the protein kinase C inhibitor staurosporine prevented the inhibitory effect of PMA on downregulation. However, staurosporine did not inhibit agonist-induced downregulation.« less

  15. Nucleosome repositioning during differentiation of a human myeloid leukemia cell line

    PubMed Central

    Mallm, Jan-Philipp; Olins, Ada L.

    2017-01-01

    ABSTRACT Cell differentiation is associated with changes in chromatin organization and gene expression. In this study, we examine chromatin structure following differentiation of the human myeloid leukemia cell line (HL-60/S4) into granulocytes with retinoic acid (RA) or into macrophage with phorbol ester (TPA). We performed ChIP-seq of histone H3 and its modifications, analyzing changes in nucleosome occupancy, nucleosome repeat length, eu-/heterochromatin redistribution and properties of epichromatin (surface chromatin adjacent to the nuclear envelope). Nucleosome positions changed genome-wide, exhibiting a specific class of alterations involving nucleosome loss in extended (∼1kb) regions, pronounced in enhancers and promoters. Genes that lost nucleosomes at their promoters showed a tendency to be upregulated. On the other hand, nucleosome gain did not show simple effects on transcript levels. The average genome-wide nucleosome repeat length (NRL) did not change significantly with differentiation. However, we detected an approximate 10 bp NRL decrease around the haematopoietic transcription factor (TF) PU.1 and the architectural protein CTCF, suggesting an effect on NRL proximal to TF binding sites. Nucleosome occupancy changed in regions associated with active promoters in differentiated cells, compared with untreated HL-60/S4 cells. Epichromatin regions revealed an increased GC content and high nucleosome density compared with surrounding chromatin. Epichromatin showed depletion of major histone modifications and revealed enrichment with PML body-associated genes. In general, chromatin changes during HL-60/S4 differentiation appeared to be more localized to regulatory regions, compared with genome-wide changes among diverse cell types studied elsewhere. PMID:28406749

  16. Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes.

    PubMed Central

    Rook, G A; Steele, J; Fraher, L; Barker, S; Karmali, R; O'Riordan, J; Stanford, J

    1986-01-01

    Previous studies have shown that recombinant interferon gamma (IFN-gamma), crude T cell supernatants, or appropriate T-cell lines can cause total inhibition of the growth of M. tuberculosis inside murine peritoneal macrophages. In similar experiments with human monocytes much smaller effects are seen. This could be due to the relative immaturity of these cells. Because dihydroxy vitamin D3 (1,25-(OH)2 D3) can cause phenotypic differentiation of immature leukemic lines into macrophage-like cells, we have explored the possibility that exposure to cholecalciferol metabolites in vitro might increase the ability of monocytes to control proliferation of M. tuberculosis, or cause monocytes to mature into cells able to respond appropriately to IFN-gamma. Incubation of monocytes with three cholecalciferol metabolites induced anti-tuberculosis activity to an extent that correlated with their binding affinities to the intracellular receptor protein for the derivatives. 1,25-(OH)2 D3 also primed monocytes for phorbol myristate acetate-triggered reduction of nitroblue tetrazolium. The effects were additive rather than synergistic with those of IFN-gamma. Monocytes incubated with IFN-gamma developed 25-OH D3 1-hydroxylase activity, detected by conversion of tritiated 25-(OH) D3 to a more polar metabolite which coeluted with 1,25-(OH)2 D3 on straight and reverse-phase HPLC. The latter is a more active form in vivo. These findings help to explain claims for the efficacy of vitamin D in the treatment of some forms of tuberculosis, and also the occasional finding of raised serum calcium, and disturbed vitamin D metabolism in these patients. PMID:3002968

  17. Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

    PubMed Central

    Grodzki, Ana Cristina G.; Giulivi, Cecilia; Lein, Pamela J.

    2013-01-01

    The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O2 and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O2) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O2 versus 5% O2 indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O2 significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O2 decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology. PMID:23355903

  18. Ion transport regulated by protease-activated receptor 2 in human airway Calu-3 epithelia

    PubMed Central

    Sato, Shinji; Ito, Yasushi; Kondo, Masashi; Ohashi, Takamasa; Ito, Satoru; Nakayama, Shinsuke; Shimokata, Kaoru; Kume, Hiroaki

    2005-01-01

    We examined the mechanisms underlying anion secretion mediated by protease-activated receptor 2 (PAR2) and its role in the regulation of ion transport, using polarized human airway Calu-3 cells. PAR2 stimulation by trypsin and a PAR2-activating peptide (PAR2AP), especially from the basolateral aspect, caused transient Cl− secretion due to cytosolic Ca2+ mobilization. Antagonists of PI-PLC (U73122, ET-18-OCH3) and inositol 1,4,5-triphosphate (xestospongin C (Xest C)) were without effect on the PAR2AP-mediated Cl− secretion, whereas it was attenuated by D609 (a PC-PLC inhibitor) and phorbol 12-myristate 13 acetate (PMA, a PKC activator). Even 30 min after removal of PAR2AP after a 10-min-exposure, cells were still poorly responsive to PAR2 stimulation, but the reduced responsiveness was upregulated by a PKC inhibitor, GF109203X (GFX). Pretreatment with PAR2AP did not affect responses to anion secretagogues, such as isoproterenol, forskolin, thapsigargin, 1-ethyl-2-benzimdazolinone, and adenosine, but ATP-induced responses were significantly reduced. Nystatin permeabilization studies revealed that the presence of PAR2AP prevented ATP-induced increments in basolateral membrane K+ conductance without affecting apical membrane Cl− conductance. ATP-elicited Ca2+ mobilization, which was sensitive to D609 and PMA, was inhibited by the pretreatment with PAR2AP, and this inhibition was blunted by the presence of GFX. Collectively, stimulation of PAR2 generates a brief response of Cl− secretion through PC-PLC-mediated pathway, followed by not only auto-desensitization of PAR2 itself but also cross-desensitization of a PC-PLC-coupled purinoceptor. The two types of desensitization seem likely to have PKC-mediated downregulation of PC-PLC in common. PMID:16025139

  19. Human Monkeypox

    PubMed Central

    McCollum, Andrea M.; Damon, Inger K.

    2018-01-01

    Human monkeypox is a zoonotic Orthopoxvirus with a presentation similar to smallpox. Clinical differentiation of the disease from smallpox and varicella is difficult. Laboratory diagnostics are principal components to identification and surveillance of disease, and new tests are needed for a more precise and rapid diagnosis. The majority of human infections occur in Central Africa, where surveillance in rural areas with poor infrastructure is difficult but can be accomplished with evidence-guided tools and educational materials to inform public health workers of important principles. Contemporary epidemiological studies are needed now that populations do not receive routine smallpox vaccination. New therapeutics and vaccines offer hope for the treatment and prevention of monkeypox; however, more research must be done before they are ready to be deployed in an endemic setting. There is a need for more research in the epidemiology, ecology, and biology of the virus in endemic areas to better understand and prevent human infections. PMID:19025497

  20. Human monkeypox.

    PubMed

    McCollum, Andrea M; Damon, Inger K

    2014-01-01

    Human monkeypox is a zoonotic Orthopoxvirus with a presentation similar to smallpox. Clinical differentiation of the disease from smallpox and varicella is difficult. Laboratory diagnostics are principal components to identification and surveillance of disease, and new tests are needed for a more precise and rapid diagnosis. The majority of human infections occur in Central Africa, where surveillance in rural areas with poor infrastructure is difficult but can be accomplished with evidence-guided tools and educational materials to inform public health workers of important principles. Contemporary epidemiological studies are needed now that populations do not receive routine smallpox vaccination. New therapeutics and vaccines offer hope for the treatment and prevention of monkeypox; however, more research must be done before they are ready to be deployed in an endemic setting. There is a need for more research in the epidemiology, ecology, and biology of the virus in endemic areas to better understand and prevent human infections.

  1. Transcriptional regulation of the human NAD(P)H:quinone oxidoreductase (NQO1) gene by monofunctional inducers.

    PubMed

    Wang, B; Williamson, G

    1996-06-03

    The upstream region of the human NAD(P)H:quinone oxidoreductase (NQO1) gene contains a functional antioxidant responsive element (ARE) and an overlapping 12-O-tetradecanoyl-phorbol-13-acetate responsive element (TRE), with the sequence TGACTCAGCA. We show that the ARE (TGACNNNGCA) is required for induction by redox cycling phenolics (p-benzoquinone, catechol and hydroquinone), which are monofunctional inducers and induce NQO1 without the requirement for activation by cytochrome P-450. The TRE (TGACTCA) is involved only in basal expression. A plasmid containing overlapping ARE-TRE (TGACTCAGCA) sequences (-587 to -379) from the NAD(P)H:quinone oxidoreductase gene was transiently transfected into Hep G2 cells. In the absence of inducers, basal expression was 4-fold higher than in F9 cells (which lack AP-1 activity). Using subcloned oligonucleotides containing the ARE-TRE sequence (-473 to -440), the ARE sequence alone (TCA changed to GAC) and the TRE sequence alone (GC changed to TA), the basal level of expression was in the order: TRE > TRE-ARE > ARE in Hep G2 cells. Using F9 cells, basal expression was detected using the combination ARE-TRE sequence or the ARE, but not the TRE alone, p-Benzoquinone, catechol and hydroquinone, but not resorcinol, induced gene expression in both Hep G2 and F9 cells via the ARE-TRE and ARE sequences, but the TRE sequence did not contribute to this induction. We therefore conclude that induction of human NAD(P)H:quinone oxidoreductase by monofunctional inducers is via the ARE and not the TRE, and that the induction is mediated by proteins other than Fos and Jun.

  2. Mometasone Furoate Suppresses PMA-Induced MUC-5AC and MUC-2 Production in Human Airway Epithelial Cells

    PubMed Central

    Koontongkaew, Sittichai; Monthanapisut, Paopanga; Pattanacharoenchai, Napaporn

    2017-01-01

    Background Mucus hypersecretion from airway epithelium is a characteristic feature of airway inflammatory diseases. Tumor necrosis factor α (TNF-α) regulates mucin synthesis. Glucocorticoids including mometasone fuorate (MF) have been used to attenuate airway inflammation. However, effects of MF on mucin production have not been reported. Methods Effects of MF and budesonide (BUD) on the phorbol-12-myristate-13-acetate (PMA)–induction of mucin and TNF-α in human airway epithelial cells (NCI-H292) were investigated in the present study. Confluent NCI-H292 cells were pretreated with PMA (200 nM) for 2 hours. Subsequently, the cells were stimulated with MF (1–500 ng/mL) or BUD (21.5 ng/mL) for 8 hours. Dexamethasone (1 µg/mL) was used as the positive control. Real-time polymerase chain reaction was used to determine MUC2 and MUC5AC mRNA levels. The level of total mucin, MUC2, MUC5AC, and TNF-α in culture supernatants were measured using enzyme-linked immunosorbent assay. Results MF and BUD significantly suppressed MUC2 and MUC5AC gene expression in PMA-stimulated NCI-H292 cells. The inhibitory effects of the two steroid drugs were also observed in the production of total mucin, MUC2 and MUC5AC proteins, and TNF-α. Conclusion Our findings demonstrated that MF and BUD attenuated mucin and TNF-α production in PMA-induced human airway epithelial cells. PMID:28119748

  3. Monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

    SciTech Connect

    Zwadlo, G.; Schlegel, R.; Sorg, C.

    1986-07-15

    A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at M/sub r/ 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-..gamma.. (IFN-..gamma..), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) Ylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphotyces, and all tested humanmore » cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG ranulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr.« less

  4. Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects.

    PubMed

    Stepensky, Polina; Keller, Baerbel; Buchta, Mary; Kienzler, Anne-Kathrin; Elpeleg, Orly; Somech, Raz; Cohen, Sivan; Shachar, Idit; Miosge, Lisa A; Schlesier, Michael; Fuchs, Ilka; Enders, Anselm; Eibel, Hermann; Grimbacher, Bodo; Warnatz, Klaus

    2013-02-01

    Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history. We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency. Molecular, immunologic, and functional assays were performed. The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells. Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response

  5. Aliphatic alcohols of illegally produced spirits can act synergistically on superoxide-anion production by human granulocytes.

    PubMed

    Arnyas, Ervin M; Pál, László; Kovács, Csilla; Adány, Róza; McKee, Martin; Szűcs, Sándor

    2012-10-01

    Aliphatic alcohols present in illegally produced spirits in a large number of low and middle income countries have been implicated in the etiology of chronic liver disease and cirrhosis. Previous studies have confirmed that chronic alcoholism can lead to increased susceptibility to infectious diseases. Reduced superoxide-anion (O(2)·(-)) production by granulocytes could provide a mechanism by which antimicrobial defense is impaired in alcoholics. In vitro experiments have also demonstrated that ethanol can inhibit granulocyte O(2)·(-) generation. Aliphatic alcohols consumed as contaminants of illicit spirits may also influence O(2)·(-) production thereby contributing to a decrease in microbicidal activity. The aim of this study was to investigate this possibility. It measured the O(2)·(-) production by human granulocytes following treatment of the cells with aliphatic alcohol contaminants found in illicit spirits. Granulocytes were isolated from human buffy coats with centrifugal elutriation and then treated with individual aliphatic alcohols and their mixture. The O(2)·(-) production was stimulated with phorbol-12-13-dibutyrate and N-formyl-methionyl-leucyl-phenylalanine (FMLP) and measured by superoxide dismutase inhibitable reduction of ferricytochrome c. Aliphatic alcohols of illegally produced spirits inhibited the FMLP-induced O(2)·(-) production in a concentration dependent manner. They suppressed O(2)·(-) generation at 2.5-40 times lower concentrations when combined than when tested individually. Aliphatic alcohols found in illegally produced spirits can inhibit FMLP-induced O(2)·(-) production by granulocytes in a concentration-dependent manner. Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit O(2)·(-) formation in heavy episodic drinkers.

  6. Human vulnerability.

    PubMed

    De Siqueira, José Eduardo; Segre, Marco

    2008-01-01

    We consider essential when addressing human vulnerability, to perceive it under two diverse prisms, therefore, complementary: the individual and the collective. The first part will present the human being as the protagonist isolated from this circumstance and it will be performed in the first person, in a nearly colloquial manner. One's vulnerability, while a part of a social network, will be addressed in the second part of the text. It will not be difficult to realize how the individual on the condition of autonomous being or integrated element in a wide social network dies of the same illness, which seems to be a strong characteristic of post-modernity.

  7. [Human monkeypox].

    PubMed

    Chastel, C

    2009-03-01

    Unlike other recent viral emergences, which were in majority caused by RNA viruses, the monkeypox results from infection by a DNA virus, an orthopoxvirus closely related to both vaccine and smallpox viruses and whose two genomic variants are known. Unexpectedly isolated from captive Asiatic monkeys and first considered as an laboratory curiosity, this virus was recognised in 1970 as an human pathogen in tropical Africa. Here it was responsible for sporadic cases following intrusions (for hunting) into tropical rain forests or rare outbreak with human-to-human transmission as observed in 1996 in Democratic Republic of Congo. As monkeypox in humans is not distinguishable from smallpox (a disease globally eradicated in 1977) it was only subjected to vigilant epidemiological surveillance and not considered as a potential threat outside Africa. This point of view radically changed in 2003 when monkeypox was introduced in the USA by African wild rodents and spread to 11 different states of this country. Responsible for 82 infections in American children and adults, this outbreak led to realize the sanitary hazards resulting from international trade of exotic animals and scientific investigations increasing extensively our knowledge of this zoonosis.

  8. Nothing Human

    ERIC Educational Resources Information Center

    Wharram, C. C.

    2014-01-01

    In this essay C. C. Wharram argues that Terence's concept of translation as a form of "contamination" anticipates recent developments in philosophy, ecology, and translation studies. Placing these divergent fields of inquiry into dialogue enables us read Terence's well-known statement "I am a human being--I deem nothing…

  9. Human Trafficking

    ERIC Educational Resources Information Center

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  10. Humanizing Calculus

    ERIC Educational Resources Information Center

    Cirillo, Michelle

    2007-01-01

    In this article, the author explores the history and the mathematics used by Newton and Leibniz in their invention of calculus. The exploration of this topic is intended to show students that mathematics is a human invention. Suggestions are made to help teachers incorporate the mathematics and the history into their own lessons. (Contains 3…

  11. Two human osteoblast-like osteosarcoma cell lines show distinct expression and differential regulation of parathyroid hormone-related protein.

    PubMed

    Jemtland, R; Rian, E; Olstad, O K; Haug, E; Bruland, O S; Bucht, E; Gautvik, K M

    1999-06-01

    Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA 3' splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines. PTHrP(1-139) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detected in TPXM cells. Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA. In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 microM forskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells. The different responsiveness to agents activating PKA- and PKC-dependent pathways

  12. Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration.

    PubMed

    Gravel, Annie; Dubuc, Isabelle; Wallaschek, Nina; Gilbert-Girard, Shella; Collin, Vanessa; Hall-Sedlak, Ruth; Jerome, Keith R; Mori, Yasuko; Carbonneau, Julie; Boivin, Guy; Kaufer, Benedikt B; Flamand, Louis

    2017-07-15

    Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39 , U90 , and U100 , without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state. IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the

  13. The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells.

    PubMed

    Kifor, O; Diaz, R; Butters, R; Brown, E M

    1997-05-01

    The extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR) is a G protein-coupled receptor that activates phospholipase C (PLC). In the present studies, we assessed Ca2+(o)-dependent changes in the generation of inositol phosphates (IP), free arachidonic acid (AA), and phosphatidylbutanol (PtdBtOH) by PLC, phospholipase A2 (PLA2), and phospholipase D (PLD), respectively, in bovine parathyroid cells as well as in wild-type or CaR-transfected human embryonic kidney (HEK293) cells (HEK-WT and HEK-CaR, respectively). Elevated Ca2+(o) increased the formation of IPs in parathyroid cells as well in HEK-CaR but not in HEK-WT cells. High Ca2+(o) also elicited time- and dose-dependent increases in PtdBtOH in parathyroid cells and HEK-CaR but not in HEK-WT cells. Brief treatment of parathyroid and HEK-CaR cells with an activator of protein kinase C (PKC), phorbol 12-myristate,13-acetate (PMA), stimulated PLD activity at both low and high Ca2+(o). Moreover, high Ca2+(o)-stimulated PLD activity was abolished following down-regulation of PKC by overnight phorbol myristate acetate (PMA) pretreatment, suggesting that CaR-mediated activation of PLD depends largely upon stimulation of PKC. High Ca2+(o) likewise increased the release of free AA in parathyroid and HEK-CaR but not in HEK-WT cells. Mepacrine, a general PLA2 inhibitor, and AACOCF3, an inhibitor of cytosolic PLA2, reduced AA release in parathyroid cells at high Ca2+(o), suggesting a major role for PLA2 in high Ca2+(o)-elicited AA release. Pretreatment of parathyroid cells with PMA stimulated release of AA at low and high Ca2+(o), while a PKC inhibitor, chelerythrine, reduced AA release at high Ca2+(o) to the level observed with low Ca2+(o) alone. Thus, PKC contributes importantly to the high Ca2+(o)-evoked, CaR-mediated activation of not only PLD but also PLA2. Finally, high Ca2+(o)-stimulated production of IP, PtdBtOH, and AA all decreased substantially in parathyroid cells cultured for 4 days, in which expression of the Ca

  14. Human Rights in the Humanities

    ERIC Educational Resources Information Center

    Harpham, Geoffrey

    2012-01-01

    Human rights are rapidly entering the academic curriculum, with programs appearing all over the country--including at Duke, Harvard, Northeastern, and Stanford Universities; the Massachusetts Institute of Technology; the Universities of Chicago, of Connecticut, of California at Berkeley, and of Minnesota; and Trinity College. Most of these…

  15. Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

    PubMed

    Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

    2016-04-15

    The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation.

    PubMed

    Chien, Chih-Chiang; Wu, Ming-Shun; Shen, Shing-Chuan; Yang, Liang-Yo; Wu, Wen-Shin; Chen, Yen-Chou

    2013-04-01

    The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Human monoclonal antibodies targeting nonoverlapping epitopes on insulin-like growth factor II as a novel type of candidate cancer therapeutics.

    PubMed

    Chen, Weizao; Feng, Yang; Zhao, Qi; Zhu, Zhongyu; Dimitrov, Dimiter S

    2012-07-01

    Soluble ligands are important targets for therapy of cancers and other diseases. Therapeutic monoclonal antibodies (mAb) against such ligands block their interactions with corresponding receptors but do not enhance their removal from the circulation and can increase their half-lives because of the long half-lives of the antibodies. We have hypothesized that mAbs targeting two or more nonoverlapping epitopes on the same ligand could form oligomeric antibody-ligand complexes that can bind to cells expressing Fc gamma receptors (FcγRs) with high avidity leading to their fast and irreversible removal from the circulation. Insulin-like growth factor II (IGF-II) is an example of such ligands and an important target for human cancer therapy. We identified two mAbs, m610.27 and m630.3, which bound to nonoverlapping epitopes on IGF-II with nanomolar affinity, and generated a bispecific antibody, m660. m660 inhibited the interaction of human IGF-II (hIGF-II) with the human breast cancer cell line MCF-7, hIGF-II-mediated IGF receptor type I and insulin receptor phosphorylation, and cell growth. In the presence of hIGF-II, large complexes of m660 were formed that bound to FcγRII-expressing BJAB cells much more efficiently than the monospecific antibody-hIGF-II complexes and were presumably phagocytosed by phorbol 12-myristate 13-acetate-stimulated macrophage-like U937 cells. A mixture of m610.27 and m630.3 exhibited similar properties. To our knowledge, these mAbs are the first reported to target nonoverlapping epitopes on a cancer-related ligand and could represent a novel class of candidate therapeutics against cancers. This approach could also be used to irreversibly eliminate other disease-related soluble ligands. ©2012 AACR.

  18. Human genetics

    SciTech Connect

    Carlson, E.A.

    1984-01-01

    This text provides full and balanced coverage of the concepts requisite for a thorough understanding of human genetics. Applications to both the individual and society are integrated throughout the lively and personal narrative, and the essential principles of heredity are clearly presented to prepare students for informed participation in public controversies. High-interest, controversial topics, including recombinant DNA technology, oncogenes, embryo transfer, environmental mutagens and carcinogens, IQ testing, and eugenics encourage understanding of important social issues.

  19. Human Capital, (Human) Capabilities and Higher Education

    ERIC Educational Resources Information Center

    Le Grange, L.

    2011-01-01

    In this article I initiate a debate into the (de)merits of human capital theory and human capability theory and discuss implications of the debate for higher education. Human capital theory holds that economic growth depends on investment in education and that economic growth is the basis for improving the quality of human life. Human capable…

  20. Differentiation, early response gene expression, and apoptosis induction in human breast tumor cells by Okadaic Acid and related inhibitors of protein phosphatases 1 and 2A. Okadaic acid effects on human breast tumor cells

    SciTech Connect

    Kiguchi, K.; Giometti, C.; Chubb, C.H.

    1992-08-20

    Okadaic acid (OA), a tumor promoter and an inhibitor of protein phosphatases (PPH) 1 and 2A, was tested for its ability to induce events associated with differentiation and apoptosis induction in the human MCF-7, AU-565, and MB-231 breast tumor cells. Differentiation in these cells was characterized by inhibition of cell multiplication, reactivity with monoclonal antibodies to {alpha}-lactalbumin and {beta}-casein, and the appearance of large lipid droplets; apoptosis was characterized by the appearance of cells with segmented and fragmented nuclei. In the MCF-7 cell line, OA at nanomolar concentrations elicited within 5 min an increase in the phosphorylation of a setmore » of cellular proteins, within hours expression of the early response genes, junB, c-jun, and c-fos and within days manifestation of differentiation and apoptosis markers. Differentiation and apoptosis were also induced by dinophysistoxin-1 and calyculin A, two other tumor promoters and inhibitors of PPH 1 and 2A, but not by OA tetramethyl ether, an inactive OA derivative, or microcystin LR, a PPH 1 and 2A inhibitor that penetrates epithelial cells poorly. OA induced both differentiation and apoptosis in MB-231 cells and MCF-7, but only differentiation in AU-565 cells. Phorbol 12-myristate 13-acetate (PMA), a tumor promoter that is not an inhibitor of PPH 1 and 2A but rather an activator of protein kinase C, also induced within minutes the phosphorylation of proteins, within hours the expression of early response genes, and within days differentiation, but not apoptosis; yet PMA was able to attenuate apoptosis induced by the okadaic acid class of tumor promoters. These results indicate that OA and related agents can induce processes that result in tumor breast cell differentiation and apoptosis, and this induction is associated with their ability to inhibit PPH 1 and 2A. Yet apoptosis is not necessarily required for differentiation induction by these agents.« less

  1. Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

    SciTech Connect

    Wu, Yang-Chang; Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan; Sureshbabu, Munisamy

    2013-02-01

    Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changesmore » in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.« less

  2. Neurotoxicity of "ecstasy" and its metabolites in human dopaminergic differentiated SH-SY5Y cells.

    PubMed

    Ferreira, Patrícia Silva; Nogueira, Tiago Bernandes; Costa, Vera Marisa; Branco, Paula Sério; Ferreira, Luísa Maria; Fernandes, Eduarda; Bastos, Maria Lourdes; Meisel, Andreas; Carvalho, Félix; Capela, João Paulo

    2013-02-04

    "Ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) is a widely abused recreational drug, reported to produce neurotoxic effects, both in laboratory animals and in humans. MDMA metabolites can be major contributors for MDMA neurotoxicity. This work studied the neurotoxicity of MDMA and its catechol metabolites, α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) in human dopaminergic SH-SY5Y cells differentiated with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. Differentiation led to SH-SY5Y neurons with higher ability to accumulate dopamine and higher resistance towards dopamine neurotoxicity. MDMA catechol metabolites were neurotoxic to SH-SY5Y neurons, leading to caspase 3-independent cell death in a concentration- and time-dependent manner. MDMA did not show a concentration- and time-dependent death. Pre-treatment with the antioxidant and glutathione precursor, N-acetylcysteine (NAC), resulted in strong protection against the MDMA metabolites' neurotoxicity. Neither the superoxide radical scavenger, tiron, nor the inhibitor of the dopamine (DA) transporter, GBR 12909, prevented the metabolites' toxicity. Cells exposed to α-MeDA showed an increase in intracellular glutathione (GSH) levels, which, at the 48 h time-point, was not dependent in the activity increase of γ-glutamylcysteine synthetase (γ-GCS), revealing a possible transient effect. Importantly, pre-treatment with buthionine sulfoximine (BSO), an inhibitor of γ-GCS, prevented α-MeDA induced increase in GSH levels, but did not augment this metabolite cytotoxicity. Even so, BSO pre-treatment abolished NAC protective effects against α-MeDA neurotoxicity, which were, at least partially, due to GSH de novo synthesis. Inversely, pre-treatment of cells with BSO augmented N-Me-α-MeDA-induced neurotoxicity, but only slightly affected NAC neuroprotection. In conclusion, MDMA catechol metabolites promote differential toxic effects to differentiated dopaminergic human SH

  3. Protein kinase C activates non-capacitative calcium entry in human platelets

    PubMed Central

    Rosado, Juan A; Sage, Stewart O

    2000-01-01

    In many non-excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store-mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non-capacitative Ca2+ entry mechanism in human platelets. Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura-2. Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store-mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration-dependent manner. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro-31-8220. Unlike store-mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store-mediated Ca2+ entry were blocked by xestospongin C. These results indicate that the non-capacitative divalent cation entry pathway is regulated independently of store-mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. These results support the existence of a mechanism for receptor-evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G-protein-coupled receptors

  4. An in vitro method to investigate the microbicidal potential of human macrophages for use in psychosomatic research.

    PubMed

    Kuebler, Ulrike; Ehlert, Ulrike; Zuccarella, Claudia; Sakai, Miho; Stemmer, Andreas; Wirtz, Petra H

    2013-01-01

    Psychological states relate to changes in circulating immune cells, but associations with immune cells in peripheral tissues such as macrophages have hardly been investigated. Here, we aimed to implement and validate a method for measuring the microbicidal potential of ex vivo isolated human monocyte-derived macrophages (HMDMs) as an indicator of macrophage activation. The method was implemented and validated for two blood sampling procedures (short-term cannula insertion versus long-term catheter insertion) in 79 participants (34 women, 45 men) aged between 18 and 75 years. The method principle is based on the reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-dis-ulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) by superoxide anions, the first in a series of pathogen-killing reactive oxygen species produced by phorbol myristate acetate-activated HMDM. Cytochrome c reduction and current generation were measured as reference methods for validation purposes. We further evaluated whether depressive symptom severity (Beck Depression Inventory) and chronic stress (Chronic Stress Screening Scale) were associated with macrophage microbicidal potential. The assay induced superoxide anion responses by HMDM in all participants. Assay results depended on blood sampling procedure (cannula versus catheter insertion). Interassay variability as a measure for assay reliability was 10.92% or less. WST-1 reduction scores correlated strongly with results obtained by reference methods (cytochrome c: r = 0.57, p = .026; current generation: r values ≥ 0.47, p values <.033) and with psychological factors (depressive symptom severity: r = 0.35 [cannula insertion] versus r = -0.54 [catheter insertion]; chronic stress: r = 0.36 [cannula insertion]; p values ≤ .047). Our findings suggest that the implemented in vitro method investigates microbicidal potential of HMDM in a manner that is valid and sensitive to psychological measures.

  5. [Effect of pulsed electromagnetic fields (PEMF) on human periodontal ligament in vitro. Alterations of intracellular Ca2+].

    PubMed

    Satake, T; Yasu, N; Kakai, Y; Kawamura, T; Sato, T; Nakano, T; Amino, S; Ishiwata, Y; Saito, S

    1990-03-01

    The concept of orthodontic tooth movement is based on the hypothesis that teeth move as a result of the biological response of periodontal tissues to the mechanical forces applied. There is a widely held hypothesis that mechanical stress generates an electrical signal which sets in motion the subsequent events, as in bone exposed to mechanical forces electrical currents are produced affect bone growth and remodeling. This implies a transduction mechanism which translates the electrical signal into a biochemical message, recognizable by the cellular machine. This study is aimed at the identification of the message and the investigation of its control. In fact, the effect of Pulsed Electromagnetic Fields (PEMF) on the intracellular second messenger, cytoplasmic Ca2+ in Human Periodontal Ligament Fibroblasts (HPLF) was investigated. The resting intracellular ionized calcium concentration ([Ca+2]i) of HPLF cells was 232.7 +/- 25.0 nM, and with PEMF [Ca2+]i increased from 12 hrs to 499.0 +/- 115.5 nM up to 12 hrs, then reached to a steady level through 24 hrs. The PEMF were also found to decrease the responses towards epidermal growth factor (EGF) and serum, when the degree of response was based on the intracellular Ca2+ transient. These effects of PEMF were mimicked by 12-0-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C. Some reports have suggested that fibroblasts of the periodontal ligament contain high alkaline phosphatase (ALPase) activity as much as osteoblast. Since similar results concerning the [Ca2+]i were obtained in osteoblast (OB)-like cells, this experiment also supports the hypothesis that fibroblasts of periodontal ligament have osteoblastic features.

  6. Human lung tissue macrophages, but not alveolar macrophages, express matrix metalloproteinases after direct contact with activated T lymphocytes.

    PubMed

    Ferrari-Lacraz, S; Nicod, L P; Chicheportiche, R; Welgus, H G; Dayer, J M

    2001-04-01

    Human alveolar macrophages (AM) and lung tissue macrophages (LTM) have a distinct localization in the cellular environment. We studied their response to direct contact with activated T lymphocytes in terms of the production of interstitial collagenase (MMP-1), 92-kD gelatinase (MMP-9), and of TIMP-1, one of the counter-regulatory tissue inhibitors of metalloproteinases. Either AM obtained by bronchoalveolar lavage or LTM obtained by mincing and digestion of lung tissue were exposed for 48 h to plasma membranes of T lymphocytes previously activated with phorbol myristate acetate and phytohemagglutinin for 24 h. Membranes of activated T cells strongly induced the production of MMP-1, MMP-9, and TIMP-1 exclusively in LTM but not in AM, whereas membranes from unstimulated T cells failed to induce the release of MMPs. Both populations of mononuclear phagocytes spontaneously released only small amounts of MMPs and TIMP-1. Similar results were obtained when MMP and TIMP-1 expression was analyzed at pretranslational and biosynthetic levels, respectively. Blockade experiments with cytokine antagonists revealed the involvement of T-cell membrane-associated interleukin-1 and tumor necrosis factor-alpha in MMP production by LTM upon contact with T cells. These data suggest that the ability of lung macrophages to produce MMPs after direct contact with activated T cells is related to the difference in phenotype of mononuclear phagocytes and cell localization. In addition, these observations indicate that cell-cell contact represents an important biological mechanism in potentiating the inflammatory response of mononuclear phagocytes in the lungs.

  7. Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

    SciTech Connect

    Hara, T.; Jung, L.K.L.; FU, S.M.

    1986-03-01

    With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% ofmore » the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.« less

  8. Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells.

    PubMed

    Patil, Rajeshwari H; Naveen Kumar, M; Kiran Kumar, K M; Nagesh, Rashmi; Kavya, K; Babu, R L; Ramesh, Govindarajan T; Chidananda Sharma, S

    2018-03-01

    The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti-inflammatory effects of glucocorticoids. Activator protein-1 is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type-II A549 cells to examine the effect of dexamethasone on the phorbol ester (PMA)/Lipopolysaccharide (LPS) induced pro-inflammatory cytokines and AP-1 factors. A549 cells were treated with and without PMA or LPS or dexamethasone and the cell viability and nitric oxide production was measured by MTT assay and Griess reagent respectively. Expression of pro-inflammatory cytokines and AP-1 factors mRNA were measured using semi quantitative RT-PCR. The PMA/LPS treated cells show significant 2-3 fold increase in the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α), cyclo‑oxygenase-2 (COX-2) and specific AP-1 factors (c-Jun, c-Fos and Jun-D). Whereas, pretreatment of cells with dexamethasone significantly inhibited the LPS induced nitric oxide production and PMA/LPS induced mRNAs expression of above pro-inflammatory cytokines, COX-2 and AP-1 factors. Cells treated with dexamethasone alone at both the concentrations inhibit the mRNAs expression of IL-1β, IL-6 and TNF-α compared to control. Our study reveals that dexamethasone decreased the mRNAs expression of c-Jun and c-Fos available for AP-1 formation suggested that AP-1 is the probable key transcription factor involved in the anti-inflammatory activity of dexamethasone. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases which may be useful for treatment of lung inflammatory diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Functional expression of the human thiazide-sensitive NaCl cotransporter in Madin-Darby canine kidney cells.

    PubMed

    de Jong, Joke C; Willems, Peter H G M; van den Heuvel, Lambertus P W J; Knoers, Nine V A M; Bindels, René J M

    2003-10-01

    The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which is expressed on the apical membrane of epithelial cells lining the distal convoluted tubule, is responsible for the reabsorption of 5% to 10% of filtered Na(+) and Cl(-). To date, functional studies on the structural and regulatory requirements for localized trafficking and ion-transporting activity of NCC have been hampered by lack of a suitable cell system expressing this cotransporter. Reported here is the functional expression of human NCC (hNCC) in a polarized mammalian cell of renal origin-that is, the high-resistance Madin-Darby canine kidney (MDCK) cell. Western blot testing revealed that the cells predominantly expressed the complex glycosylated (approximately 140 kD) form of hNCC. hNCC was present primarily in the apical part of the cell. The functionality of hNCC was demonstrated by the gain of thiazide-sensitive Na(+) uptake and transepithelial transport activity. Na(+) uptake was significantly increased after short-term (15 min) treatment with forskolin, whereas cyclic guanosine monophosphate, wortmannin, phorbol 12-myriatate 13-acetate, and staurosporine were without effect. This indicates that hNCC activity is regulated through cyclic adenosine monophosphate, rather than via cyclic guanosine monophosphate, phospho-inositide 3-kinases or protein kinase C. Aldosterone did not alter Na(+) uptake in the short term (15 min) but significantly increased the transport activity in the long term (16 h). The latter effect of aldosterone was due to an effect on the cytomegalovirus promoter/enhancer driving the expression of hNCC. hNCC-MDCK cells are a good model for the study of the regulation of apical trafficking and ion-transporting activity of hNCC.

  10. Human Astroviruses

    PubMed Central

    Pintó, Rosa M.; Guix, Susana

    2014-01-01

    SUMMARY Human astroviruses (HAtVs) are positive-sense single-stranded RNA viruses that were discovered in 1975. Astroviruses infecting other species, particularly mammalian and avian, were identified and classified into the genera Mamastrovirus and Avastrovirus. Through next-generation sequencing, many new astroviruses infecting different species, including humans, have been described, and the Astroviridae family shows a high diversity and zoonotic potential. Three divergent groups of HAstVs are recognized: the classic (MAstV 1), HAstV-MLB (MAstV 6), and HAstV-VA/HMO (MAstV 8 and MAstV 9) groups. Classic HAstVs contain 8 serotypes and account for 2 to 9% of all acute nonbacterial gastroenteritis in children worldwide. Infections are usually self-limiting but can also spread systemically and cause severe infections in immunocompromised patients. The other groups have also been identified in children with gastroenteritis, but extraintestinal pathologies have been suggested for them as well. Classic HAstVs may be grown in cells, allowing the study of their cell cycle, which is similar to that of caliciviruses. The continuous emergence of new astroviruses with a potential zoonotic transmission highlights the need to gain insights on their biology in order to prevent future health threats. This review focuses on the basic virology, pathogenesis, host response, epidemiology, diagnostic assays, and prevention strategies for HAstVs. PMID:25278582

  11. Human Heredity: Genetic Mechanisms in Humans.

    ERIC Educational Resources Information Center

    Blank, C. E.

    1988-01-01

    Discussed are some of the uncertainties in human genetic mechanisms that are often presented as dogma in Biology textbooks. Presented is a brief historical background and illustrations involving chromosome abnormality in humans and linkage studies in humans. (CW)

  12. Human Neutrophils Convert the Sebum-derived Polyunsaturated Fatty Acid Sebaleic Acid to a Potent Granulocyte Chemoattractant*

    PubMed Central

    Cossette, Chantal; Patel, Pranav; Anumolu, Jaganmohan R.; Sivendran, Sashikala; Lee, Gue Jae; Gravel, Sylvie; Graham, François D.; Lesimple, Alain; Mamer, Orval A.; Rokach, Joshua; Powell, William S.

    2008-01-01

    Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP+, the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B4, indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of

  13. Preclinical Study of Novel Gene Silencer Pyrrole-Imidazole Polyamide Targeting Human TGF-β1 Promoter for Hypertrophic Scars in a Common Marmoset Primate Model

    PubMed Central

    Igarashi, Jun; Fukuda, Noboru; Inoue, Takashi; Nakai, Shigeki; Saito, Kosuke; Fujiwara, Kyoko; Matsuda, Hiroyuki; Ueno, Takahiro; Matsumoto, Yoshiaki; Watanabe, Takayoshi; Nagase, Hiroki; Bando, Toshikazu; Sugiyama, Hiroshi; Itoh, Toshio; Soma, Masayoshi

    2015-01-01

    We report a preclinical study of a pyrrole-imidazole (PI) polyamide that targets the human transforming growth factor (hTGF)-β1 gene as a novel transcriptional gene silencer in a common marmoset primate model. We designed and then synthesized PI polyamides to target the hTGF-β1 promoter. We examined effects of seven PI polyamides (GB1101-1107) on the expression of hTGF-β1 mRNA stimulated with phorbol 12-myristate 13-acetate (PMA) in human vascular smooth muscle cells. GB1101, GB1105 and GB1106 significantly inhibited hTGF-β1 mRNA expression. We examined GB1101 as a PI polyamide to hTGF-β1 for hypertrophic scars in marmosets in vivo. Injection of GB1101 completely inhibited hypertrophic scar formation at 35 days post-incision and inhibited cellular infiltration, TGF-β1 and vimentin staining, and epidermal thickness. Mismatch polyamide did not affect hypertrophic scarring or histological changes. Epidermis was significantly thinner with GB1101 than with water and mismatch PI polyamides. We developed the PI polyamides for practical ointment medicines for the treatment of hypertrophic scars. FITC-labeled GB1101 with solbase most efficiently distributed in the nuclei of epidermal keratinocytes, completely suppressed hypertropic scarring at 42 days after incision, and considerably inhibited epidermal thickness and vimentin-positive fibroblasts. PI polyamides targeting hTGF-β1 promoter with solbase ointment will be practical medicines for treating hypertrophic scars after surgical operations and skin burns. PMID:25938472

  14. Human Factors Planning Guidelines

    DOT National Transportation Integrated Search

    1996-01-01

    To ensure human factors considerations are fully incorporated in the system : development, the Integrated Product Team (IPT) or Program Manager initiates a : Human Factors Program (HFP) that addresses the human performance and human : resource parame...

  15. NATO Human View Architecture and Human Networks

    NASA Technical Reports Server (NTRS)

    Handley, Holly A. H.; Houston, Nancy P.

    2010-01-01

    The NATO Human View is a system architectural viewpoint that focuses on the human as part of a system. Its purpose is to capture the human requirements and to inform on how the human impacts the system design. The viewpoint contains seven static models that include different aspects of the human element, such as roles, tasks, constraints, training and metrics. It also includes a Human Dynamics component to perform simulations of the human system under design. One of the static models, termed Human Networks, focuses on the human-to-human communication patterns that occur as a result of ad hoc or deliberate team formation, especially teams distributed across space and time. Parameters of human teams that effect system performance can be captured in this model. Human centered aspects of networks, such as differences in operational tempo (sense of urgency), priorities (common goal), and team history (knowledge of the other team members), can be incorporated. The information captured in the Human Network static model can then be included in the Human Dynamics component so that the impact of distributed teams is represented in the simulation. As the NATO militaries transform to a more networked force, the Human View architecture is an important tool that can be used to make recommendations on the proper mix of technological innovations and human interactions.

  16. The Digital Humanities as a Humanities Project

    ERIC Educational Resources Information Center

    Svensson, Patrik

    2012-01-01

    This article argues that the digital humanities can be seen as a humanities project in a time of significant change in the academy. The background is a number of scholarly, educational and technical challenges, the multiple epistemic traditions linked to the digital humanities, the potential reach of the field across and outside the humanities,…

  17. Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

    PubMed Central

    Moffett, John; Kratz, Erica; Myers, Jason; Stachowiak, Ewa K.; Florkiewicz, Robert Z.; Stachowiak, Michal K.

    1998-01-01

    Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively. PMID:9693381

  18. Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells

    PubMed Central

    Kohler, Thomas P.; Scholz, Annemarie; Kiachludis, Delia; Hammerschmidt, Sven

    2016-01-01

    Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during

  19. Human Rhinoviruses

    PubMed Central

    Lamson, Daryl M.; St. George, Kirsten; Walsh, Thomas J.

    2013-01-01

    Human rhinoviruses (HRVs), first discovered in the 1950s, are responsible for more than one-half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work. Advances in molecular methods have enhanced our understanding of the genomic structure of HRV and have led to the characterization of three genetically distinct HRV groups, designated groups A, B, and C, within the genus Enterovirus and the family Picornaviridae. HRVs are traditionally associated with upper respiratory tract infection, otitis media, and sinusitis. In recent years, the increasing implementation of PCR assays for respiratory virus detection in clinical laboratories has facilitated the recognition of HRV as a lower respiratory tract pathogen, particularly in patients with asthma, infants, elderly patients, and immunocompromised hosts. Cultured isolates of HRV remain important for studies of viral characteristics and disease pathogenesis. Indeed, whether the clinical manifestations of HRV are related directly to viral pathogenicity or secondary to the host immune response is the subject of ongoing research. There are currently no approved antiviral therapies for HRVs, and treatment remains primarily supportive. This review provides a comprehensive, up-to-date assessment of the basic virology, pathogenesis, clinical epidemiology, and laboratory features of and treatment and prevention strategies for HRVs. PMID:23297263

  20. Chemical Carcinogen-Induced Changes in tRNA Metabolism in Human Cells

    DTIC Science & Technology

    1982-11-01

    promotion of neoplasia , 7-methylguanine, queuine, phorbol esters, tumor promoters. 20.1 ABSTRACT (Continue mn reverse aide It neeeem4 md Identif by belk...4ethylguanine) may be involved in inducing the 0-hypomodification of tRNA associated with neoplasia . This could 330 Vol. I10A, No. 1, 1982 BIOCHEMICAL AND...alterations in tRN’A metabolism associated with neoplasia . Potential interrelationships between the changes in tRNA modification and catabolism are

  1. 1998--Human Rights Year.

    ERIC Educational Resources Information Center

    Mock, Karen R.

    1998-01-01

    Promotes teaching human rights at every educational level in conjunction with the United Nations-declared Human Rights Year. Provides World Wide Web addresses for resources on human rights and the Human Rights Year, including the United Nations and Amnesty International. Summarizes UN-suggested guidelines for human rights commemoration…

  2. Sevoflurane modulates the release of reactive oxygen species, myeloperoxidase, and elastase in human whole blood: Effects of different stimuli on neutrophil response to volatile anesthetic in vitro.

    PubMed

    Minguet, Grégory; Franck, Thierry; Joris, Jean; Serteyn, Didier

    2017-12-01

    Volatile anesthetics have been shown to modulate polymorphonuclear neutrophil (PMN) functions. The aim of this study was to examine the impact of clinically relevant concentrations of sevoflurane (SEVO), a volatile anesthetic, on the release of reactive oxygen species (ROS), myeloperoxidase (MPO), and elastase (EL) from human activated PMNs. For this purpose, samples of whole blood were collected from healthy volunteers and exposed in vitro to 2.3% or 4.6% SEVO in air. To assess for a stimulus-dependent effect of the volatile anesthetic, PMNs were activated using different validated protocols. Artificial stimulation of neutrophils involved either a combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). In addition, a combination of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) was also tested as a natural activation mean of PMNs. The production of ROS by PMNs was assessed by L-012 chemiluminescence. Total MPO and EL released in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, degranulation of the active fraction of MPO was also measured by specific immunological extraction followed by enzymatic detection (SIEFED). Overall, SEVO enhanced the release of ROS, MPO, and EL following artificial stimulation of PMNs but the volatile anesthetic inhibited the degranulation of active MPO and EL after neutrophil exposure to LPS and TNF-α. This study highlighted that the effect of SEVO on activated PMNs is dependent on the conditions of cell stimulation. These properties should be taken into consideration in future studies investigating immunomodulatory effects of volatile anesthetics.

  3. The two calcium-binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils.

    PubMed

    Kerkhoff, C; Klempt, M; Kaever, V; Sorg, C

    1999-11-12

    Recently, we identified the two myeloid related protein-8 (MRP8) (S100A8) and MRP14 (S100A9) as fatty acid-binding proteins (Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U., and Sorg, C. (1997) FEBS Lett. 408, 81-84). Here we present data that the S100A8/A9 protein complex represents the exclusive arachidonic acid-binding proteins in human neutrophils. Binding and competition studies revealed evidence that (i) fatty acid binding was dependent on the calcium concentration; (ii) fatty acid binding was specific for the protein complex formed by S100A8 and S100A9, whereas the individual components were unable to bind fatty acids; (iii) exclusively polyunsaturated fatty acids were bound by S100A8/A9, whereas saturated (palmitic acid, stearic acid) and monounsaturated fatty acids (oleic acid) as well as arachidonic acid-derived eicosanoids (15-hydroxyeicosatetraenoic acid, prostaglandin E(2), thromboxane B(2), leukotriene B(4)) were poor competitors. Stimulation of neutrophil-like HL-60 cells with phorbol 12-myristate 13-acetate led to the secretion of S100A8/A9 protein complex, which carried the released arachidonic acid. When elevation of intracellular calcium level was induced by A23187, release of arachidonic acid occurred without secretion of S100A8/A9. In view of the unusual abundance in neutrophilic cytosol (approximately 40% of cytosolic protein) our findings assign an important role for S100A8/A9 as mediator between calcium signaling and arachidonic acid effects. Further investigations have to explore the exact function of the S100A8/A9-arachidonic acid complex both inside and outside of neutrophils.

  4. Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

    SciTech Connect

    Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata

    2008-06-01

    The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activitymore » was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 {mu}g/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 {mu}g/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1{beta} (83%) or TNF-{alpha} (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity.« less

  5. Bioassay-Guided Isolation of Iridoids and Phenylpropanoids from Aerial Parts of Lamium album and Their Anti-inflammatory Activity in Human Neutrophils.

    PubMed

    Czerwińska, Monika E; Świerczewska, Anita; Woźniak, Marta; Kiss, Anna K

    2017-08-01

    In traditional medicine, flowers and aerial parts of Lamium album are assigned by their anti-inflammatory, antiseptic, and mucolytic activities, and are used in chronic bronchitis and pharyngitis as well as skin, vaginal, and cervical inflammation.The aim of the present study was to compare effects of ethanolic extracts prepared from flowers and aerial parts of L. album on selected functions of human neutrophils, which are involved in an inflammatory response. In order to identify the compounds engaged in the anti-inflammatory activity of extracts, the bioassay-guided isolation of compounds was performed based on the inhibition of cytokine secretion by stimulated neutrophils.The extracts were phytochemically characterized with the HPLC-DAD-MS n method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase secretion by neutrophils was established spectrophotometrically. The levels of cytokine (interleukin 8, TNF- α ) production after extract treatment was measured by ELISA.The most abundant constituents of extracts were phenylpropanoids, iridoids, flavonoids, and phenolic acids. Both extracts at concentrations of 25 and 100 µg/mL significantly inhibited reactive oxygen species production, and myeloperoxidase and interleukin 8 secretion. The phenylethanoid glycosides, such as lamiusides A, B, and C as well as 6″-O- β -D-glucopyranosylmartynoside, were isolated and identified. The cells treated with 6″- O - β -D-glucopyranosylmartynoside and lamiuside B produced 29.47 ± 7.11 % and 64.67 ± 5.25 % of interleukin 8, respectively, compared to non-treated control cells.Our results support the traditional use of L. album and indicate it as a potential source of natural anti-inflammatory constituents, such as phenylpropanoids. Georg Thieme Verlag KG Stuttgart

  6. Inhibitory effect of known antioxidants and of press juice from herring (Clupea harengus) light muscle on the generation of free radicals in human monocytes.

    PubMed

    Gunnarsson, Gujón; Undeland, Ingrid; Sannaveerappa, Thippeswamy; Sandberg, Ann-Sofie; Lindgård, Ann; Mattsson-Hultén, Lillemor; Soussi, Bassam

    2006-10-18

    Reactive oxygen species (ROS) can cause oxidative stress, which has been linked to various diseases. It has been suggested that antioxidant-rich foods can reduce such oxidative stress. However, the lack of suitable model systems to screen for in vivo effects of food-derived antioxidants has prevented a clear consensus in this area. In this study, the aim was to use a single-cell model system (human monocyte) to evaluate whether certain pure antioxidants and complex muscle extracts (herring light muscle press juice, PJ) could prevent ROS formation under in vivo like conditions. ROS were excreted from the monocytes upon stimulation with phorbol myristate acetate and were then detected as isoluminol-enhanced chemiluminescence (CL). Adding 2000 units of catalase and 50 units of superoxide dismutase to the monocytes model lowered the CL response by 35 and 86%, respectively. Ascorbate (14.1 mM) lowered the response by 99%, alpha-tocoperhol (188 microM) by 37%, and Trolox (50 microM) by almost 100%. Crude herring PJ gave a dose-dependent reduction in the CL response. At 10, 100, and 1000 times dilution, the PJ reduced the CL signal by 93, 60.5, and 10.6%. PJ fractionated into low molecular weight (LMW) (<1000 Da) and high molecular weight (>3500 Da) fractions decreased the CL response by 52.9 and 71.4%, respectively, at a 100-fold dilution. Evaluation of the PJ samples in the oxygen radical absorbance capacity test indicated that proteins may be the primary radical scavenging compounds of PJ, whereas the ROS-preventing effect obtained from the LMW fraction may also be attributed to other mechanisms. Thus, this study proved that the monocyte assay can be a useful tool for studying whether food-derived antioxidants can limit ROS production under physiologically relevant conditions. It also showed that herring contains numerous aqueous compounds demonstrating antioxidative effects in the monocyte model system.

  7. Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

    PubMed

    Dunne, J; Lynch, S; O'Farrelly, C; Todryk, S; Hegarty, J E; Feighery, C; Doherty, D G

    2001-09-15

    IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.

  8. Inactivation of the antifungal and immunomodulatory properties of human cathelicidin LL-37 by aspartic proteases produced by the pathogenic yeast Candida albicans.

    PubMed

    Rapala-Kozik, Maria; Bochenska, Oliwia; Zawrotniak, Marcin; Wolak, Natalia; Trebacz, Grzegorz; Gogol, Mariusz; Ostrowska, Dominika; Aoki, Wataru; Ueda, Mitsuyoshi; Kozik, Andrzej

    2015-06-01

    Constant cross talk between Candida albicans yeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used by C. albicans to cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivate C. albicans at sites of candidal infection and that C. albicans uses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage-the LL-25 peptide-is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates that C. albicans can effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Inactivation of the Antifungal and Immunomodulatory Properties of Human Cathelicidin LL-37 by Aspartic Proteases Produced by the Pathogenic Yeast Candida albicans

    PubMed Central

    Bochenska, Oliwia; Zawrotniak, Marcin; Wolak, Natalia; Trebacz, Grzegorz; Gogol, Mariusz; Ostrowska, Dominika; Aoki, Wataru; Ueda, Mitsuyoshi; Kozik, Andrzej

    2015-01-01

    Constant cross talk between Candida albicans yeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used by C. albicans to cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivate C. albicans at sites of candidal infection and that C. albicans uses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage—the LL-25 peptide—is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates that C. albicans can effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate. PMID:25847962

  10. IVI human factors strategy

    DOT National Transportation Integrated Search

    1999-11-01

    This document focuses on human factors research that supports the Intelligent Vehicle Initiative (IVI). The status of the problem areas within categories used often by the human factors community to organize human factors process is discussed. A simi...

  11. Human Factors Job Aid

    DOT National Transportation Integrated Search

    1996-12-09

    The purpose of this Human Factors Job Aid is to serve as a desk reference for : human factors integration during system acquisition. The first chapter contains : an overview of the FAA human factors process in system acquisitions. The : remaining eig...

  12. Engineered human vaccines

    SciTech Connect

    Sandhu, J.S.

    1994-01-01

    The limitations of human vaccines in use at present and the design requirements for a new generation of human vaccines are discussed. The progress in engineering of human vaccines for bacteria, viruses, parasites, and cancer is reviewed, and the data from human studies with the engineered vaccines are discussed, especially for cancer and AIDS vaccines. The final section of the review deals with the possible future developments in the field of engineered human vaccines and the requirement for effective new human adjuvants.

  13. Human-machine interactions

    DOEpatents

    Forsythe, J Chris [Sandia Park, NM; Xavier, Patrick G [Albuquerque, NM; Abbott, Robert G [Albuquerque, NM; Brannon, Nathan G [Albuquerque, NM; Bernard, Michael L [Tijeras, NM; Speed, Ann E [Albuquerque, NM

    2009-04-28

    Digital technology utilizing a cognitive model based on human naturalistic decision-making processes, including pattern recognition and episodic memory, can reduce the dependency of human-machine interactions on the abilities of a human user and can enable a machine to more closely emulate human-like responses. Such a cognitive model can enable digital technology to use cognitive capacities fundamental to human-like communication and cooperation to interact with humans.

  14. 5'-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells.

    PubMed

    Garcia-Gil, M; Pesi, R; Perna, S; Allegrini, S; Giannecchini, M; Camici, M; Tozzi, M G

    2003-01-01

    5'-Aminoimidazole-4-carboxamide riboside (AICA riboside) has been previously shown to be toxic to two neuronal cell models [Neuroreport 11 (2000) 1827]. In this paper we demonstrate that AICA riboside promotes apoptosis in undifferentiated human neuroblastoma cells (SH-SY5Y), inducing a raise in caspase-3 activity. In order to exert its effect on viability, AICA riboside must enter the cells and be phosphorylated to the ribotide, since both a nucleoside transport inhibitor, and an inhibitor of adenosine kinase produce an enhancement of the viability of AICA riboside-treated cells. Short-term incubations (2 h) with AICA riboside result in five-fold increase in the activity of AMP-dependent protein kinase (AMPK). However, the activity of AMPK is not significantly affected at prolonged incubations (48 h), when the apoptotic effect of AICA riboside is evident. The results demonstrate that when the cell line is induced to differentiate both toward a cholinergic phenotype (with retinoic acid) or a noradrenergic phenotype (with phorbol esters), the toxic effect is significantly reduced, and in the case of the noradrenergic phenotype differentiation, the riboside is completely ineffective in promoting apoptosis. This reduction of effect correlates with an overexpression of Bcl-2 during differentiation. AICA riboside, derived from the hydrolysis of the ribotide, an intermediate of purine de novo synthesis, is absent in normal healthy cells; however it may accumulate in those individuals in which an inborn error of purine metabolism causes an increase in the rate of de novo synthesis and/or an overexpression of cytosolic 5'-nucleotidase, that appears to be the enzyme responsible for AICA ribotide hydrolysis. In fact, 5'-nucleotidase activity has been shown to increase in patients affected by Lesch-Nyhan syndrome in which both acceleration of de novo synthesis and accumulation of AICA ribotide has been described, and also in other neurological disorders of unknown etiology

  15. Pesticides and Human Health

    MedlinePlus

    ... Low-Risk Pesticides Organic Pesticide Ingredients Pesticide Incidents Human Exposure Pet Exposure Environmental Incident Illegal Pesticide Activity Problem With Labels or Containers Emergency Human Poisonings ...

  16. The mouse S100A15 ortholog parallels genomic organization, structure, gene expression, and protein-processing pattern of the human S100A7/A15 subfamily during epidermal maturation.

    PubMed

    Wolf, Ronald; Voscopoulos, Christopher J; FitzGerald, Peter C; Goldsmith, Paul; Cataisson, Christophe; Gunsior, Michele; Walz, Markus; Ruzicka, Thomas; Yuspa, Stuart H

    2006-07-01

    The calcium-binding proteins of the human S100A7/A15 (hS100A7/A15) subfamily are differentially expressed in normal and pathological epidermis. The hS100A7 (psoriasin) and S100A15 reside in a chromosomal cluster of highly similar paralogs. To exploit the power of mouse models for determining functions of gene products, the corresponding S100A7/A15 ortholog was cloned and examined in murine skin. The single mouse S100A15 (mS100A15) gene encodes a protein of 104 amino acids with a predicted molecular weight of 12,870 Da and two EF-hand calcium binding sites. Using gene-specific primers and specific antibodies, expression of mS100A15 in both skin and isolated keratinocytes is confined to differentiating granular and cornified epidermal cells. Immunoblotting of epidermal extracts revealed a series of high molecular weight bands that are also recognized by an antibody for transglutaminase-mediated protein crosslinks. mS100A15 expression is upregulated in cultured keratinocytes induced to differentiate by calcium or phorbol esters. Maximal induction occurs concordantly with expression of late differentiation markers. Induction is enhanced in keratinocytes overexpressing protein kinase Calpha and is dependent on activator protein-1 transcription factors. The regulation, expression pattern and crosslinking of mS100A15 are consistent with the characteristics of the human orthologs, providing a valid surrogate model to study changes in these proteins associated with cutaneous pathologies.

  17. Human Research Program Opportunities

    NASA Technical Reports Server (NTRS)

    Kundrot, Craig E.

    2014-01-01

    The goal of HRP is to provide human health and performance countermeasures, knowledge, technologies, and tools to enable safe, reliable, and productive human space exploration. The Human Research Program was designed to meet the needs of human space exploration, and understand and reduce the risk to crew health and performance in exploration missions.

  18. Upholding the Humanities.

    ERIC Educational Resources Information Center

    Cohen, Arthur M.

    Suggestions for improving the state of the humanities in two-year colleges are presented, based on the findings of a nationwide study of humanities faculty. Among the policy recommendations are: (1) administrators/faculty leaders should organize lay committees as advisors to humanities programs and should involve humanities instructors with such…

  19. Visualizing Humans by Computer.

    ERIC Educational Resources Information Center

    Magnenat-Thalmann, Nadia

    1992-01-01

    Presents an overview of the problems and techniques involved in visualizing humans in a three-dimensional scene. Topics discussed include human shape modeling, including shape creation and deformation; human motion control, including facial animation and interaction with synthetic actors; and human rendering and clothing, including textures and…

  20. Preference for human eyes in human infants.

    PubMed

    Dupierrix, Eve; de Boisferon, Anne Hillairet; Méary, David; Lee, Kang; Quinn, Paul C; Di Giorgio, Elisa; Simion, Francesca; Tomonaga, Masaki; Pascalis, Olivier

    2014-07-01

    Despite evidence supporting an early attraction to human faces, the nature of the face representation in neonates and its development during the first year after birth remain poorly understood. One suggestion is that an early preference for human faces reflects an attraction toward human eyes because human eyes are distinctive compared with other animals. In accord with this proposal, prior empirical studies have demonstrated the importance of the eye region in face processing in adults and infants. However, an attraction for the human eye has never been shown directly in infants. The current study aimed to investigate whether an attraction for human eyes would be present in newborns and older infants. With the use of a preferential looking time paradigm, newborns and 3-, 6-, 9-, and 12-month-olds were simultaneously presented with a pair of nonhuman primate faces (chimpanzees and Barbary macaques) that differed only by the eyes, thereby pairing a face with original nonhuman primate eyes with the same face in which the eyes were replaced by human eyes. Our results revealed that no preference was observed in newborns, but a preference for nonhuman primate faces with human eyes emerged from 3months of age and remained stable thereafter. The findings are discussed in terms of how a preference for human eyes may emerge during the first few months after birth. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Dopamine D1 stimulation of Na+,K+,Cl- cotransport in human NPE cells: effects of multiple hormones.

    PubMed

    Riese, K; Beyer, A T; Lui, G M; Crook, R B

    1998-07-01

    To determine the effects of dopamine on Na+,K+,Cl- cotransport in human ciliary nonpigmented epithelial (NPE) cells. The authors used 86Rb+ as a marker for K+ to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured fetal human NPE monolayers. Na+,K+,Cl- cotransport was stimulated 1.63-fold by 10 microM dopamine. Stimulation was dose dependent, with a maximum stimulation occurring at 10 microM dopamine and an EC50 of 0.5 microM. NaK-ATPase (measured as ouabain-sensitive, bumetanide-insensitive 86Rb+ uptake) and bumetanide-insensitive, ouabain-insensitive 86Rb+ uptake were not affected by dopamine. The D1-receptor-specific antagonist, SCH23390, inhibited stimulation by 10 microM dopamine more than 90% at 1 microM, with an IC50 of 4 nM, whereas the D2-receptor-specific antagonist, sulpiride, was over 250 times less effective. Similarly, a D1 agonist, SKF81297, was more potent than the D2 agonist bromocriptine in stimulation of Na+,K+,Cl- cotransport. The beta-adrenergic antagonists timolol and propranolol did not significantly inhibit stimulation of Na+,K+,Cl- cotransport by dopamine. Conversely, SCH23390, showed minimal inhibition of isoproterenol stimulation of Na+,K+,Cl- cotransport. Stimulation by maximally stimulating concentrations of isoproterenol and dopamine were not additive, but were similar to stimulation by 1 microM forskolin, suggesting that adenylyl cyclase may be close to maximally activated by either catecholamine. In vivo concentrations (stimulation approximately 25% over control) of dopamine, isoproterenol, and norepinephrine added together stimulated Na+,K+,Cl- cotransport 80% to 89% of stimulation by maximal concentrations of these drugs. The protein kinase A inhibitor N-[2-p-bromocinnamylaminoethyl]-5-isoquinolinesulfonamide (H-89) blocked dopamine stimulation of Na+,K+,Cl- cotransport by more than 75%, whereas phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, given with 10 microM dopamine inhibited Na

  2. ISS Payload Human Factors

    NASA Technical Reports Server (NTRS)

    Ellenberger, Richard; Duvall, Laura; Dory, Jonathan

    2016-01-01

    The ISS Payload Human Factors Implementation Team (HFIT) is the Payload Developer's resource for Human Factors. HFIT is the interface between Payload Developers and ISS Payload Human Factors requirements in SSP 57000. ? HFIT provides recommendations on how to meet the Human Factors requirements and guidelines early in the design process. HFIT coordinates with the Payload Developer and Astronaut Office to find low cost solutions to Human Factors challenges for hardware operability issues.

  3. Economics of human trafficking.

    PubMed

    Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V

    2010-01-01

    Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans.

  4. Indicators: Human Disturbance

    EPA Pesticide Factsheets

    Human disturbance is a measure of the vulnerability of aquatic resources to a variety of harmful human activities such as tree removal, road building, construction near shorelines/streambanks, and artificial hardening of lakeshores with retaining walls.

  5. Statement on Human Cloning

    MedlinePlus

    ... for the Advancement of Science Statement on Human Cloning Tweet The American Association for the Advancement of ... for this statement on human cloning. Ban Reproductive Cloning AAAS endorses a legally enforceable ban on efforts ...

  6. Telling the Human Story.

    ERIC Educational Resources Information Center

    Richardson, Miles

    1987-01-01

    Proposes that one of the fundamental human attributes is telling stories. Explores the debate on whether Neanderthals possessed language ability. Discusses the role of the "human story" in teaching anthropology. (DH)

  7. Human bites (image)

    MedlinePlus

    Human bites present a high risk of infection. Besides the bacteria which can cause infection, there is ... the wound extends below the skin. Anytime a human bite has broken the skin, seek medical attention.

  8. The Growing Human Population.

    ERIC Educational Resources Information Center

    Keyfitz, Nathan

    1989-01-01

    Discusses the issue of human population. Illustrates the projections of the growing human population in terms of developed and less developed countries. Describes the family planning programs in several countries. Lists three references for further reading. (YP)

  9. Human Papillomavirus (HPV) Vaccine

    MedlinePlus

    Why get vaccinated?HPV vaccine prevents infection with human papillomavirus (HPV) types that are associated with cause ... at http://www.cdc.gov/hpv. HPV Vaccine (Human Papillomavirus) Information Statement. U.S. Department of Health and ...

  10. Pathfinder: Humans in space

    NASA Technical Reports Server (NTRS)

    Anderson, John L.

    1988-01-01

    Viewgraphs are presented on the Pathfinder program. Information is given on human exploration of the solar system, technical requirements interfaces, program objectives, space suits, human performance, man-machine systems, space habitats, life support systems, and artificial gravity

  11. Human papillomavirus molecular biology.

    PubMed

    Harden, Mallory E; Munger, Karl

    Human papillomaviruses are small DNA viruses with a tropism for squamous epithelia. A unique aspect of human papillomavirus molecular biology involves dependence on the differentiation status of the host epithelial cell to complete the viral lifecycle. A small group of these viruses are the etiologic agents of several types of human cancers, including oral and anogenital tract carcinomas. This review focuses on the basic molecular biology of human papillomaviruses. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. HUMAN PAPILLOMAVIRUS MOLECULAR BIOLOGY

    PubMed Central

    Harden, Mallory E.; Munger, Karl

    2016-01-01

    Human papillomaviruses are small DNA viruses with a tropism for squamous epithelia. A unique aspect of human papillomavirus molecular biology involves dependence on the differentiation status of the host epithelial cell to complete the viral lifecycle. A small group of these viruses are the etiologic agents of several types of human cancers, including oral and anogenital tract carcinomas. This review focuses on the basic molecular biology of human papillomaviruses. PMID:28528688

  13. International Human Rights Kit.

    ERIC Educational Resources Information Center

    Woito, Robert, Ed.

    Designed for students, educators, and citizens interested in human rights, the booklet presents resources for learning about the facts, perspectives, and existing procedures and institutions to promote human rights. Chapter one explores the relationship between human rights and war. Chapter two presents a self-survey to help readers clarify…

  14. Human Rights Educational Resources.

    ERIC Educational Resources Information Center

    Flowers, Nancy

    1998-01-01

    Gives a variety of educational resources on human rights that include videos, resource notebooks, books, publications, and websites along with short descriptions of the materials. Provides the contact information for a list of human-rights organizations, such as the Center for Human Rights Education and the Franklin and Eleanor Roosevelt…

  15. Robotics of human movements.

    PubMed

    van der Smagt, Patrick; Grebenstein, Markus; Urbanek, Holger; Fligge, Nadine; Strohmayr, Michael; Stillfried, Georg; Parrish, Jonathon; Gustus, Agneta

    2009-01-01

    The construction of robotic systems that can move the way humans do, with respect to agility, stability and precision, is a necessary prerequisite for the successful integration of robotic systems in human environments. We explain human-centered views on robotics, based on the three basic ingredients (1) actuation; (2) sensing; and (3) control, and formulate detailed examples thereof.

  16. Intervention and Human Rights.

    ERIC Educational Resources Information Center

    Goldberg, Arthur J.

    1988-01-01

    Defends the right of nations to criticize human rights violations within other nations. Pointing out that the human rights protections cannot be left to domestic jurisdiction, Goldberg cites numerous treaties and declarations which make human rights protection a matter of international law. (GEA)

  17. Human Relations Ideabook.

    ERIC Educational Resources Information Center

    McRae, Jean A.

    Intended for local human relations committees and commissions, this ideabook may serve as a do-it-yourself kit, a springboard for human relations action, showing concrete action steps that can be taken. Ideas and suggestions given in the booklet may be used to provide a basis for organizing a human relations committee; awaken the educational…

  18. Production Of Human Antibodies

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  19. Human Systems Roadmap Review

    DTIC Science & Technology

    2016-02-09

    1 Distribution Statement A: Approved for Public Release Human Systems Roadmap Review Dr. John Tangney, SES Director, Human and Bioengineered...Right Job , Right Skills Our Forces Prepared for Global Challenges Effective, Natural Human-Machine Teaming Ensuring Warfighter Safety and... robotics & autonomous systems  Enhanced interaction & trust w/autonomous systems; increased SA for operators

  20. Human Rights Resource Catalogue.

    ERIC Educational Resources Information Center

    Zambrano, Elias, Comp.

    This document provides information about 25 programs/brochures which focus on human rights topics. Specific topics include: (1) counselor preparation; (2) multicultural awareness; (3) abuse and neglect; (4) Acquired Immune Deficiency Syndrome; (5) self-awareness; (6) human rights awareness and human rights of students; (7) cultural diversity; (8)…

  1. Whose Human Rights?

    ERIC Educational Resources Information Center

    Rendel, Margherita

    During the last 50 years, principles, institutions, and policies of human rights have been developed worldwide. This book brings together European and international conventions on human rights, the rights of women, and the users and uses of education, and places them in their wider context. It examines issues in how human rights work, the ways in…

  2. Human Machine Learning Symbiosis

    ERIC Educational Resources Information Center

    Walsh, Kenneth R.; Hoque, Md Tamjidul; Williams, Kim H.

    2017-01-01

    Human Machine Learning Symbiosis is a cooperative system where both the human learner and the machine learner learn from each other to create an effective and efficient learning environment adapted to the needs of the human learner. Such a system can be used in online learning modules so that the modules adapt to each learner's learning state both…

  3. Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells.

    PubMed

    Sierra-Rivera, Crystel A; Franco-Molina, Moisés A; Mendoza-Gamboa, Edgar; Zapata-Benavides, Pablo; Santaolalla-Tapia, Jesús; Coronado-Cerda, Erika E; Tamez-Guerra, Reyes S; Rodríguez-Padilla, Cristina

    2016-12-01

    Differentiation induction therapy is an attractive approach in leukemia treatment due to the fact that in blast crisis stage, leukemic cells lose their differentiation capacity. Therefore, it has been proposed as a therapeutic strategy to induce terminal differentiation of leukemic blast cells into a specific lineage, leading to prevention of high proliferation rates. The aim of the present study was to demonstrate the potential of cell differentiation and death induced by bovine dialyzable leukocyte extract (bDLE) in the K562 cell line. For this purpose K562 and MOLT-3 human leukemic cell lines and primary human monocytes and murine peritoneal macrophages were exposed to bDLE, phorbol myristate acetate (PMA) and dimethyl sulfoxide for 96 h, and the viability, proliferation and cell cycle were evaluated. To determine the lineage that led to cell differentiation, Romanowsky staining was performed to observe the morphological changes following the treatments, and the expression of the surface markers cluster of differentiation (CD)14 + , CD68 + , CD163 + and CD42a + , as well as the phagocytic activity, and the production of nitric oxide (NO) (assessed by colorimetric assay), cytokines [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] and chemokines [chemokine (C-C motif) ligand (CCL)2, CCL5 and chemokine (C-X-C motif) ligand 8] in cell supernatants was assessed by flow cytometry. The results of the present study reveal that high doses of bDLE increase the cell death in K562 and MOLT-3 lines, without affecting the viability of human monocytes and murine peritoneal macrophages. Furthermore, low doses of bDLE induce differentiation in K562 cells towards a monocyte/macrophage lineage with an M2 phenotype, and induced moderately upregulated expression of CD42 + , a megakaryocytic marker. Cell cycle arrest in the S and G 2 /M phases was observed in bDLE-treated K562 cells, which demonstrated similar phagocytic activity, NO levels and cytokine and chemokine

  4. Selective induction of cyclo-oxygenase-2 activity in the permanent human endothelial cell line HUV-EC-C: biochemical and pharmacological characterization

    PubMed Central

    Miralpeix, Montserrat; Camacho, Mercedes; López-Belmonte, Jesús; Canalías, Francesca; Beleta, Jordi; Ma Palacios, José; Vila, Luis

    1997-01-01

    Cyclo-oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX-1 and COX-2 which are encoded by different genes. COX-1 is expressed constitutively and is known to be the site of action of aspirin and other non-steroidal anti-inflammatory drugs. COX-2 may be induced by a series of pro-inflammatory stimuli and its role in the development of inflammation has been claimed. Endothelial cells are an important physiological source of prostanoids and the selective induction of COX-2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines. The HUV-EC-C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12-myristate 13-acetate (PMA) and interleukin-1β (IL-1β). Both PMA and IL-1β produced dose- and time-dependent increases of the synthesis of the COX-derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2. The increase of COX activity by PMA or IL-1β correlated with an increase of the enzyme's apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected. Treatment of the cells with PMA induced a time-dependent increase in the expression of both COX-1 and COX-2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX-2 isoenzyme at the protein level. The enzymatic activity of the PMA-induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX-1 selective assay. Classical non-steroidal anti-inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three

  5. Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells

    PubMed Central

    Sierra-Rivera, Crystel A.; Franco-Molina, Moisés A.; Mendoza-Gamboa, Edgar; Zapata-Benavides, Pablo; Santaolalla-Tapia, Jesús; Coronado-Cerda, Erika E.; Tamez-Guerra, Reyes S.; Rodríguez-Padilla, Cristina

    2016-01-01

    Differentiation induction therapy is an attractive approach in leukemia treatment due to the fact that in blast crisis stage, leukemic cells lose their differentiation capacity. Therefore, it has been proposed as a therapeutic strategy to induce terminal differentiation of leukemic blast cells into a specific lineage, leading to prevention of high proliferation rates. The aim of the present study was to demonstrate the potential of cell differentiation and death induced by bovine dialyzable leukocyte extract (bDLE) in the K562 cell line. For this purpose K562 and MOLT-3 human leukemic cell lines and primary human monocytes and murine peritoneal macrophages were exposed to bDLE, phorbol myristate acetate (PMA) and dimethyl sulfoxide for 96 h, and the viability, proliferation and cell cycle were evaluated. To determine the lineage that led to cell differentiation, Romanowsky staining was performed to observe the morphological changes following the treatments, and the expression of the surface markers cluster of differentiation (CD)14+, CD68+, CD163+ and CD42a+, as well as the phagocytic activity, and the production of nitric oxide (NO) (assessed by colorimetric assay), cytokines [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] and chemokines [chemokine (C-C motif) ligand (CCL)2, CCL5 and chemokine (C-X-C motif) ligand 8] in cell supernatants was assessed by flow cytometry. The results of the present study reveal that high doses of bDLE increase the cell death in K562 and MOLT-3 lines, without affecting the viability of human monocytes and murine peritoneal macrophages. Furthermore, low doses of bDLE induce differentiation in K562 cells towards a monocyte/macrophage lineage with an M2 phenotype, and induced moderately upregulated expression of CD42+, a megakaryocytic marker. Cell cycle arrest in the S and G2/M phases was observed in bDLE-treated K562 cells, which demonstrated similar phagocytic activity, NO levels and cytokine and chemokine production

  6. Training Humans for the Human Domain

    DTIC Science & Technology

    2015-11-01

    TRAINING HUMANS FOR THE HUMAN DOMAIN Dr. Steve Tatham Mr. Keir Giles U.S. ARMY WAR COLLEGE ~~~ ~O.L STRATEGIC STUDIES INSTITUTE Report...ADDRESS(ES) U.S. Army War College ,Strategic Studies Institute,47 Ashburn Drive,Carlisle,PA,17013-5010 8. PERFORMING ORGANIZATION REPORT NUMBER 9...ABSTRACT unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 i The United States Army War College

  7. The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation.

    PubMed

    Bentham, Matthew; Mazaleyrat, Sabine; Harris, Mark

    2003-10-01

    The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.

  8. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  9. Human-technology Integration

    NASA Astrophysics Data System (ADS)

    Mullen, Katharine M.

    Human-technology integration is the replacement of human parts and extension of human capabilities with engineered devices and substrates. Its result is hybrid biological-artificial systems. We discuss here four categories of products furthering human-technology integration: wearable computers, pervasive computing environments, engineered tissues and organs, and prosthetics, and introduce examples of currently realized systems in each category. We then note that realization of a completely artificial sytem via the path of human-technology integration presents the prospect of empirical confirmation of an aware artificially embodied system.

  10. [Human factors in medicine].

    PubMed

    Lazarovici, M; Trentzsch, H; Prückner, S

    2017-01-01

    The concept of human factors is commonly used in the context of patient safety and medical errors, all too often ambiguously. In actual fact, the term comprises a wide range of meanings from human-machine interfaces through human performance and limitations up to the point of working process design; however, human factors prevail as a substantial cause of error in complex systems. This article presents the full range of the term human factors from the (emergency) medical perspective. Based on the so-called Swiss cheese model by Reason, we explain the different types of error, what promotes their emergence and on which level of the model error prevention can be initiated.

  11. Biological Races in Humans

    PubMed Central

    Templeton, Alan R.

    2013-01-01

    Races may exist in humans in a cultural sense, but biological concepts of race are needed to access their reality in a non-species-specific manner and to see if cultural categories correspond to biological categories within humans. Modern biological concepts of race can be implemented objectively with molecular genetic data through hypothesis-testing. Genetic data sets are used to see if biological races exist in humans and in our closest evolutionary relative, the chimpanzee. Using the two most commonly used biological concepts of race, chimpanzees are indeed subdivided into races but humans are not. Adaptive traits, such as skin color, have frequently been used to define races in humans, but such adaptive traits reflect the underlying environmental factor to which they are adaptive and not overall genetic differentiation, and different adaptive traits define discordant groups. There are no objective criteria for choosing one adaptive trait over another to define race. As a consequence, adaptive traits do not define races in humans. Much of the recent scientific literature on human evolution portrays human populations as separate branches on an evolutionary tree. A tree-like structure among humans has been falsified whenever tested, so this practice is scientifically indefensible. It is also socially irresponsible as these pictorial representations of human evolution have more impact on the general public than nuanced phrases in the text of a scientific paper. Humans have much genetic diversity, but the vast majority of this diversity reflects individual uniqueness and not race. PMID:23684745

  12. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    SciTech Connect

    Bencherif, M.; Lukas, R.J.

    1991-06-01

    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10more » microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.« less

  13. Neutrophil extracellular traps possess anti-human respiratory syncytial virus activity: possible interaction with the viral F protein.

    PubMed

    Souza, Priscila Silva Sampaio; Barbosa, Lia Vezenfard; Diniz, Larissa Figueiredo Alves; da Silva, Gabriel Soares; Lopes, Bruno Rafael Pereira; Souza, Pedro Miyadaira Ribeiro; de Araujo, Gabriela Campos; Pessoa, Diogo; de Oliveira, Juliana; Souza, Fátima Pereira; Toledo, Karina Alves

    2018-04-02

    Human respiratory syncytial virus (hRSV) is one of the main etiological agents of diseases of the lower respiratory tract, and is often responsible for the hospitalization of children and the elderly. To date, treatments are only palliative and there is no vaccine available. The airways of patients infected with hRSV exhibit intense neutrophil infiltration, which is responsible for the release of neutrophil extracellular traps (NETs). These are extracellular structures consisting of DNA associated with intracellular proteins, and are efficient in capturing and eliminating various microorganisms, including some viruses. hRSV induces the release of NETs into the lung tissue of infected individuals; however, the pathophysiological consequences of this event have not been elucidated. The objective of this study was to utilize in vitro and in silico assays to investigate the impact of NETs on hRSV infection. NETs, generated by neutrophils stimulated with phorbol myristate acetate (PMA), displayed long fragments of DNA and an electrophoretic profile suggestive of the presence of proteins that are classically associated with these structures (elastase, cathepsin G, myeloperoxidase, and histones). The presence of NETs (>2 µg/ml) in HEp-2 cell culture medium resulted in cellular cytotoxicity of less than 50%. Pre-incubation (1 h) of viral particles (multiplicity of infection (MOI) values of 0.1, 0.5, and 1.0) with NETs (2-32 µg/ml) resulted in cellular protection from virus-induced death of HEp-2 cells. Concurrently, there was a reduction in the formation of syncytia, which is related to decreased viral spread in infected tissue. Results from western blotting and molecular docking, suggest interactions between F protein of the hRSV viral envelope and BPI (bactericidal permeability-increasing protein), a microbicidal member of NETs. Interactions occurred at sites important for the neutralization and coordination of the hRSV infection/replication process. Our results

  14. Sulfatide-induced L-selectin activation generates intracellular oxygen radicals in human neutrophils: modulation by extracellular adenosine.

    PubMed

    Bengtsson, T; Grenegård, M; Olsson, A; Sjögren, F; Stendahl, O; Zalavary, S

    1996-08-28

    The sulfated form of galactocerebrosides (sulfatides) have recently been established as ligands for L-selectin. In this study we show that exposure of human neutrophils to sulfatides induces a transient generation of oxygen radicals, revealed by the luminol-enhanced chemiluminescence (CL) technique. The CL response was mainly located intracellularly, and was dependent on sulfation of the galactose ring, since non-sulfated galactocerebrosides had no effect. Sulfatides also dramatically amplified the CL response triggered by the chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP). This effect was primarily due to an increased (up to 10-fold) intracellular generation of oxygen metabolites. Removal or blocking of L-selectin with chymotrypsin and monoclonal antibodies, respectively, markedly reduced the effects of sulfatides. Furthermore, sulfatides amplified the CL response triggered by ionomycin, whereas the response induced by phorbol-12-myristate-13-acetate was slightly reduced. The tyrosine kinase inhibitor, genistein, markedly inhibited the oxygen radical production induced by sulfatides, and totally abolished the potentiating effects of sulfatides in fMLP- and ionomycin-stimulated neutrophils. Sulfatides also triggered a transient rise in the intracellular free calcium concentration, [Ca2+]i. Consequently, L-selectin activation through sulfatides appear to affect oxidase activity through a Ca(2+)-dependent pathway involving tyrosine phosphorylation. Adenosine is an anti-inflammatory agent predominately released from the vascular endothelium which might suppress an inappropriate activation of the oxidase during L-selectin-mediated rolling of neutrophils. Indeed, we found that adenosine inhibited the oxidative burst induced by sulfatides, mainly by attenuating the intracellular generation of oxygen radicals. However, 10-100 times higher concentration of exogenous adenosine was required to inhibit the CL response induced by sulfatides to the same extent

  15. Human rights and bioethics.

    PubMed

    Barilan, Y M; Brusa, M

    2008-05-01

    In the first part of this article we survey the concept of human rights from a philosophical perspective and especially in relation to the "right to healthcare". It is argued that regardless of meta-ethical debates on the nature of rights, the ethos and language of moral deliberation associated with human rights is indispensable to any ethics that places the victim and the sufferer in its centre. In the second part we discuss the rise of the "right to privacy", particularly in the USA, as an attempt to make the element of personal free will dominate over the element of basic human interest within the structure of rights and when different rights seem to conflict. We conclude by discussing the relationship of human rights with moral values beyond the realm of rights, mainly human dignity, free will, human rationality and response to basic human needs.

  16. Integrated Environmental Modelling: human decisions, human challenges

    USGS Publications Warehouse

    Glynn, Pierre D.

    2015-01-01

    Integrated Environmental Modelling (IEM) is an invaluable tool for understanding the complex, dynamic ecosystems that house our natural resources and control our environments. Human behaviour affects the ways in which the science of IEM is assembled and used for meaningful societal applications. In particular, human biases and heuristics reflect adaptation and experiential learning to issues with frequent, sharply distinguished, feedbacks. Unfortunately, human behaviour is not adapted to the more diffusely experienced problems that IEM typically seeks to address. Twelve biases are identified that affect IEM (and science in general). These biases are supported by personal observations and by the findings of behavioural scientists. A process for critical analysis is proposed that addresses some human challenges of IEM and solicits explicit description of (1) represented processes and information, (2) unrepresented processes and information, and (3) accounting for, and cognizance of, potential human biases. Several other suggestions are also made that generally complement maintaining attitudes of watchful humility, open-mindedness, honesty and transparent accountability. These suggestions include (1) creating a new area of study in the behavioural biogeosciences, (2) using structured processes for engaging the modelling and stakeholder communities in IEM, and (3) using ‘red teams’ to increase resilience of IEM constructs and use.

  17. Human research subjects as human research workers.

    PubMed

    Lynch, Holly Fernandez

    2014-01-01

    Biomedical research involving human subjects has traditionally been treated as a unique endeavor, presenting special risks and demanding special protections. But in several ways, the regulatory scheme governing human subjects research is counter-intuitively less protective than the labor and employment laws applicable to many workers. This Article relies on analogical and legal reasoning to demonstrate that this should not be the case; in a number of ways, human research subjects ought to be fundamentally recast as human research workers. Like other workers protected under worklaw, biomedical research subjects often have interests that diverge from those in positions of control but little bargaining power for change. Bearing these important similarities in mind, the question becomes whether there is any good reason to treat subjects and protected workers differently as a matter of law. With regard to unrestricted payment, eligibility for a minimum wage, compensation for injury, and rights to engage in concerted activity, the answer is no and human subjects regulations ought to be revised accordingly.

  18. Integrated Environmental Modelling: Human decisions, human challenges

    USGS Publications Warehouse

    Glynn, Pierre D.

    2015-01-01

    Integrated Environmental Modelling (IEM) is an invaluable tool for understanding the complex, dynamic ecosystems that house our natural resources and control our environments. Human behaviour affects the ways in which the science of IEM is assembled and used for meaningful societal applications. In particular, human biases and heuristics reflect adaptation and experiential learning to issues with frequent, sharply distinguished, feedbacks. Unfortunately, human behaviour is not adapted to the more diffusely experienced problems that IEM typically seeks to address. Twelve biases are identified that affect IEM (and science in general). These biases are supported by personal observations and by the findings of behavioural scientists. A process for critical analysis is proposed that addresses some human challenges of IEM and solicits explicit description of (1) represented processes and information, (2) unrepresented processes and information, and (3) accounting for, and cognizance of, potential human biases. Several other suggestions are also made that generally complement maintaining attitudes of watchful humility, open-mindedness, honesty and transparent accountability. These suggestions include (1) creating a new area of study in the behavioural biogeosciences, (2) using structured processes for engaging the modelling and stakeholder communities in IEM, and (3) using ‘red teams’ to increase resilience of IEM constructs and use.

  19. Sensitization to human milk.

    PubMed

    Schulmeister, U; Swoboda, I; Quirce, S; de la Hoz, B; Ollert, M; Pauli, G; Valenta, R; Spitzauer, S

    2008-01-01

    Allergy to milk is one of the earliest manifestations of IgE-mediated allergies and affects about 2.5% of newborn children. Several reports indicate that milk-allergic patients may be sensitized also to human milk proteins. To analyse the specificity and possible biological relevance of IgE reactivity to human milk antigens in milk-allergic patients. The specificity of IgE reactivity to cow's milk and human milk antigens was analysed with sera from milk-allergic children and adults by IgE immunoblotting. IgE cross-reactivity between milk antigens was studied by immunoblot inhibition experiments. That IgE reactivity to human milk antigens is not due to alloreactivity or due to the transmission of foreign antigens into mother's milk was demonstrated through the analysis of milk samples from genetically unrelated mothers before and after intake of dietary milk products. The biological relevance of IgE reactivity to human milk was confirmed by skin testing. Results IgE antibodies to human milk were found in more than 80% of the tested milk-allergic patients. Cross-reactive IgE-reactive human antigens such as alpha-lactalbumin and non-cross-reactive human milk antigens were identified. Immediate-type skin reactions could be elicited with human milk samples in patients with IgE reactivity to human milk. IgE reactivity to human milk in milk-allergic patients can be due to cross- sensitization and genuine sensitization to human milk and may cause allergic symptoms. IgE-mediated sensitization to human milk is common in milk-allergic patients and may require diagnostic testing and monitoring.

  20. Human organ markets and inherent human dignity.

    PubMed

    MacKellar, Calum

    2014-01-01

    It has been suggested that human organs should be bought and sold on a regulated market as any other material property belongingto an individual. This would have the advantage of both addressing the grave shortage of organs available for transplantation and respecting the freedom of individuals to choose to do whatever they want with their body parts. The old arguments against such a market in human organs are, therefore, being brought back into question. The article examines the different arguments both in favour and against the sale of human organs. It concludes that the body and any of its elements is a full expression of the whole person. As such, they cannot have a price if the individual is to retain his or her full inherent dignity and if society is to retain and protect this very important concept.

  1. Human Performance in Space

    NASA Technical Reports Server (NTRS)

    Jones, Patricia M.; Fiedler, Edna

    2010-01-01

    Human factors is a critical discipline for human spaceflight. Nearly every human factors research area is relevant to space exploration -- from the ergonomics of hand tools used by astronauts, to the displays and controls of a spacecraft cockpit or mission control workstation, to levels of automation designed into rovers on Mars, to organizational issues of communication between crew and ground. This chapter focuses more on the ways in which the space environment (especially altered gravity and the isolated and confined nature of long-duration spaceflight) affects crew performance, and thus has specific novel implications for human factors research and practice. We focus on four aspects of human performance: neurovestibular integration, motor control and musculo-skeletal effects, cognitive effects, and behavioral health. We also provide a sampler of recent human factors studies from NASA.

  2. The Human Cell Atlas.

    PubMed

    Regev, Aviv; Teichmann, Sarah A; Lander, Eric S; Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

    2017-12-05

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

  3. Robotics for Human Exploration

    NASA Technical Reports Server (NTRS)

    Fong, Terrence; Deans, Mathew; Bualat, Maria

    2013-01-01

    Robots can do a variety of work to increase the productivity of human explorers. Robots can perform tasks that are tedious, highly repetitive or long-duration. Robots can perform precursor tasks, such as reconnaissance, which help prepare for future human activity. Robots can work in support of astronauts, assisting or performing tasks in parallel. Robots can also perform "follow-up" work, completing tasks designated or started by humans. In this paper, we summarize the development and testing of robots designed to improve future human exploration of space.

  4. The psychology of humanness.

    PubMed

    Haslam, Nick; Loughnan, Steve; Holland, Elise

    2013-01-01

    This chapter explores the ways in which the concept of "humanness" illuminates a wide and fascinating variety of psychological phenomena. After introducing the concept--everyday understandings of what it is to be human--we present a model of the diverse ways in which humanness can be denied to people. According to this model people may be perceived as lacking uniquely human characteristics, and thus likened to animals, or as lacking human nature, and thus likened to inanimate objects. Both of these forms of dehumanization occur with varying degrees of subtlety, from the explicit uses of derogatory animal metaphors, to stereotypes that ascribe lesser humanness or simpler minds to particular groups, to nonconscious associations between certain humans and nonhumans. After reviewing research on dehumanization through the lens of our model we examine additional topics that the psychology of humanness clarifies, notably the perception of nonhuman animals and the objectification of women. Humanness emerges as a concept that runs an integrating thread through a variety of research literatures.

  5. [Effects of Mycobacterium tuberculosis antigen and anti-TCRγδ antibody on IL-17 production of human γδT cells in polarization culture].

    PubMed

    Chen, Li; Xia, Hui; Wang, Hongtao; Sheng, Lingling; Xu, Zhiqing; Sun, Yao; Tang, Jie; Li, Baiqing

    2015-02-01

    To investigate the effects of Mycobacterium tuberculosis heat-resistant antigen (MTB-HAg) and anti-TCR γδ monoclonal antibody (mAb) on the induction and differentiation of human IL-17-producing γδT cells under polarization culture conditions. From human peripheral blood mononuclear cells, the γδT cells were purified by magnetic-activated cell sorting (MACS) and then stimulated with MTB-HAg or anti-TCRγδ mAb and with or without anti-CD28 mAb, and cultured in the presence or absence of the cytokine cocktails (CK) (IL-1β, IL-6, TGF-β and IL-23) for 10 to 12 days. The polarized cultured γδT cells were collected and re-stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 6 hours, and the number of IL-17-producing cells was detected by ELISPOT. The number of IL-17-producing cells (per 10⁵ γδT cells) in anti-TCRγδ combined with CK group was significantly higher than that in the anti-TCRγδ group. Meanwhile, the IL-17-producing γδT cell number in anti-TCRγδ combined with anti-CD28 group was lower than that in the anti-TCRγδ combined with anti-CD28 and CK group. The IL-17-producing γδT cell number of anti-TCRγδ combined with anti-CD28 and CK group was significantly higher than that in anti-TCRγδ combined with CK group, and that in MTB-HAg combined with anti-CD28 and CK group was significantly lower than that in anti-TCRγδ combined with anti-CD28 and CK group, but higher than that in MTB-HAg combined with anti-CD28 group. The CK (IL-1β, IL-6, TGF-β and IL-23) required for the differentiation of Th17 cells also induced the differentiation of IL-17⁺ γδT cells after pre-activated by MTB-HAg or anti-TCRγδ antibody, while anti-TCRγδ mAb showed the stronger stimulatory effect than MTB-HAg. In addition, CD28 co-stimulation enhanced the differentiation of IL-17 from activated γδT cells.

  6. Developing Human Resources through Actualizing Human Potential

    ERIC Educational Resources Information Center

    Clarken, Rodney H.

    2012-01-01

    The key to human resource development is in actualizing individual and collective thinking, feeling and choosing potentials related to our minds, hearts and wills respectively. These capacities and faculties must be balanced and regulated according to the standards of truth, love and justice for individual, community and institutional development,…

  7. Health and Humanity: Humanities 401 Syllabus.

    ERIC Educational Resources Information Center

    Snowden, Fraser; Taylor, Maxine

    A syllabus for the "Health and Humanities" interdisciplinary course at Northwestern State University, Louisiana, is presented. An introduction suggests that with the proliferation of technological advances in the field of health care, there is a need for reconsideration of many moral, ethical, legal, and humanistic questions. Information…

  8. Environment and the Humanities.

    ERIC Educational Resources Information Center

    Allen, Rodney F., Ed.; And Others

    As a conference report, the booklet is primarily devoted to abstracts of papers presented at a Conference on Environment and Humanities held in Tallahassee, Florida, April 25-27, 1976. Dr. Huston Smith of Syracuse University, the main speaker, addressed the issue of "Humanities and Environmental Awareness." Other topics discussed…

  9. Human Sociobiology: Wilson's Fallacy.

    ERIC Educational Resources Information Center

    Lehrman, Nathaniel S.

    1981-01-01

    Presents an introduction to and a critique of E.O. Wilson's new science of sociobiology, which focuses on explaining the social behavior of species as diverse as ants, apes, and humans. Suggests that Wilson has gone beyond his data in claiming that complex human behaviors such as altruism are caused to any extent by genetic, as opposed to…

  10. Human-System Technology

    DTIC Science & Technology

    2005-11-10

    Computing, this multidisciplinary field exploits advances in cognitive research together with those in computer science and related areas to optimize the...deep understanding of human cognition, perception, and/or locomotion; the relevant areas of computer science ; and the nature of the human activity to be

  11. Humane Education Projects Handbook.

    ERIC Educational Resources Information Center

    Junior League of Ogden, UT.

    This handbook was developed to promote interest in humane education and to encourage the adoption of humane education projects. Although specifically designed to assist Junior Leagues in developing such projects, the content should prove valuable to animal welfare organizations, zoos, aquariums, nature centers, and other project-oriented groups…

  12. Humanism within Globalization

    ERIC Educational Resources Information Center

    Weber, Jennifer E.

    2014-01-01

    The complexity of adult learning connects it to almost all other facets of human endeavor. Consequently, the future of adult education depends, to a large extent on who participates and the quality of such participation. Quality participation, when teamed with environments committed to a concern for humanity, launches opportunities for varied…

  13. Human Powered Centrifuge

    NASA Technical Reports Server (NTRS)

    Mulenburg, Gerald M. (Inventor); Vernikos, Joan (Inventor)

    1997-01-01

    A human powered centrifuge has independently established turntable angular velocity and human power input. A control system allows excess input power to be stored as electric energy in a battery or dissipated as heat through a resistors. In a mechanical embodiment, the excess power is dissipated in a friction brake.

  14. HUMAN HEALTH RESEARCH STRATEGY

    EPA Science Inventory

    The mission of the U.S. Environmental Protection Agency (EPA) is to protect public health and safeguard the environment. Risk assessment is an integral part of this mission in that it identifies and characterizes environmentally related human health problems. The Human Health Re...

  15. Assessment of Human Factors

    NASA Technical Reports Server (NTRS)

    Mount, Frances; Foley, Tico

    1999-01-01

    Human Factors Engineering, often referred to as Ergonomics, is a science that applies a detailed understanding of human characteristics, capabilities, and limitations to the design, evaluation, and operation of environments, tools, and systems for work and daily living. Human Factors is the investigation, design, and evaluation of equipment, techniques, procedures, facilities, and human interfaces, and encompasses all aspects of human activity from manual labor to mental processing and leisure time enjoyments. In spaceflight applications, human factors engineering seeks to: (1) ensure that a task can be accomplished, (2) maintain productivity during spaceflight, and (3) ensure the habitability of the pressurized living areas. DSO 904 served as a vehicle for the verification and elucidation of human factors principles and tools in the microgravity environment. Over six flights, twelve topics were investigated. This study documented the strengths and limitations of human operators in a complex, multifaceted, and unique environment. By focusing on the man-machine interface in space flight activities, it was determined which designs allow astronauts to be optimally productive during valuable and costly space flights. Among the most promising areas of inquiry were procedures, tools, habitat, environmental conditions, tasking, work load, flexibility, and individual control over work.

  16. Fungi that Infect Humans.

    PubMed

    Köhler, Julia R; Hube, Bernhard; Puccia, Rosana; Casadevall, Arturo; Perfect, John R

    2017-06-01

    Fungi must meet four criteria to infect humans: growth at human body temperatures, circumvention or penetration of surface barriers, lysis and absorption of tissue, and resistance to immune defenses, including elevated body temperatures. Morphogenesis between small round, detachable cells and long, connected cells is the mechanism by which fungi solve problems of locomotion around or through host barriers. Secretion of lytic enzymes, and uptake systems for the released nutrients, are necessary if a fungus is to nutritionally utilize human tissue. Last, the potent human immune system evolved in the interaction with potential fungal pathogens, so few fungi meet all four conditions for a healthy human host. Paradoxically, the advances of modern medicine have made millions of people newly susceptible to fungal infections by disrupting immune defenses. This article explores how different members of four fungal phyla use different strategies to fulfill the four criteria to infect humans: the Entomophthorales, the Mucorales, the Ascomycota, and the Basidiomycota. Unique traits confer human pathogenic potential on various important members of these phyla: pathogenic Onygenales comprising thermal dimorphs such as Histoplasma and Coccidioides ; the Cryptococcus spp. that infect immunocompromised as well as healthy humans; and important pathogens of immunocompromised patients- Candida , Pneumocystis , and Aspergillus spp. Also discussed are agents of neglected tropical diseases important in global health such as mycetoma and paracoccidiomycosis and common pathogens rarely implicated in serious illness such as dermatophytes. Commensalism is considered, as well as parasitism, in shaping genomes and physiological systems of hosts and fungi during evolution.

  17. Quantification of human responses

    NASA Technical Reports Server (NTRS)

    Steinlage, R. C.; Gantner, T. E.; Lim, P. Y. W.

    1992-01-01

    Human perception is a complex phenomenon which is difficult to quantify with instruments. For this reason, large panels of people are often used to elicit and aggregate subjective judgments. Print quality, taste, smell, sound quality of a stereo system, softness, and grading Olympic divers and skaters are some examples of situations where subjective measurements or judgments are paramount. We usually express what is in our mind through language as a medium but languages are limited in available choices of vocabularies, and as a result, our verbalizations are only approximate expressions of what we really have in mind. For lack of better methods to quantify subjective judgments, it is customary to set up a numerical scale such as 1, 2, 3, 4, 5 or 1, 2, 3, ..., 9, 10 for characterizing human responses and subjective judgments with no valid justification except that these scales are easy to understand and convenient to use. But these numerical scales are arbitrary simplifications of the complex human mind; the human mind is not restricted to such simple numerical variations. In fact, human responses and subjective judgments are psychophysical phenomena that are fuzzy entities and therefore difficult to handle by conventional mathematics and probability theory. The fuzzy mathematical approach provides a more realistic insight into understanding and quantifying human responses. This paper presents a method for quantifying human responses and subjective judgments without assuming a pattern of linear or numerical variation for human responses. In particular, quantification and evaluation of linguistic judgments was investigated.

  18. Teaching Human Rights Law.

    ERIC Educational Resources Information Center

    Berman, Howard R.

    1985-01-01

    The international community has developed a system of human rights law relevant to many areas of legal encounter, which American law schools have been slow to incorporate into curricula. Teaching human rights law provides an opportunity for law schools to enrich the learning process and contribute creatively to the respect for rights in society.…

  19. Humanities in Engineering Education.

    ERIC Educational Resources Information Center

    Ruprecht, Robert

    1997-01-01

    States that engineers contribute tremendously to the changing face of the earth, and the ever more urgent call for languages, management, and law competencies for engineers is an expression of the need for a grounding in humanities. Discusses the role of humanities in engineering education in the context of world economics and the role of…

  20. Incorporating Human Interindividual Biotransformation ...

    EPA Pesticide Factsheets

    The protection of sensitive individuals within a population dictates that measures other than central tendencies be employed to estimate risk. The refinement of human health risk assessments for chemicals metabolized by the liver to reflect data on human variability can be accomplished through (1) the characterization of enzyme expression in large banks of human liver samples, (2) the employment of appropriate techniques for the quantification and extrapolation of metabolic rates derived in vitro, and (3) the judicious application of physiologically based pharmacokinetic (PBPK) modeling. While in vitro measurements of specific biochemical reactions from multiple human samples can yield qualitatively valuable data on human variance, such measures must be put into the perspective of the intact human to yield the most valuable predictions of metabolic differences among humans. For quantitative metabolism data to be the most valuable in risk assessment, they must be tied to human anatomy and physiology, and the impact of their variance evaluated under real exposure scenarios. For chemicals metabolized in the liver, the concentration of parent chemical in the liver represents the substrate concentration in the MichaelisMenten description of metabolism. Metabolic constants derived in vitro may be extrapolated to the intact liver, when appropriate conditions are met. Metabolic capacity Vmax; the maximal rate of the reaction) can be scaled directly to the concentration

  1. Human Granulocytic Anaplasmosis, Japan

    PubMed Central

    Gaowa; Wuritu; Kawamori, Fumihiko; Wu, Dongxing; Yoshikawa, Yuko; Chiya, Seizou; Fukunaga, Kazutoshi; Funato, Toyohiko; Shiojiri, Masaaki; Nakajima, Hideki; Hamauzu, Yoshiji; Takano, Ai; Kawabata, Hiroki; Ando, Shuji; Kishimoto, Toshio

    2013-01-01

    We retrospectively confirmed 2 cases of human Anaplasma phagocytophilum infection. Patient blood samples contained unique p44/msp2 for the pathogen, and antibodies bound to A. phagocytophilum antigens propagated in THP-1 rather than HL60 cells. Unless both cell lines are used for serodiagnosis of rickettsiosis-like infections, cases of human granulocytic anaplasmosis could go undetected. PMID:23460988

  2. Human granulocytic Anaplasmosis, Japan.

    PubMed

    Ohashi, Norio; Gaowa; Wuritu; Kawamori, Fumihiko; Wu, Dongxing; Yoshikawa, Yuko; Chiya, Seizou; Fukunaga, Kazutoshi; Funato, Toyohiko; Shiojiri, Masaaki; Nakajima, Hideki; Hamauzu, Yoshiji; Takano, Ai; Kawabata, Hiroki; Ando, Shuji; Kishimoto, Toshio

    2013-02-01

    We retrospectively confirmed 2 cases of human Anaplasma phagocytophilum infection. Patient blood samples contained unique p44/msp2 for the pathogen, and antibodies bound to A. phagocytophilum antigens propagated in THP-1 rather than HL60 cells. Unless both cell lines are used for serodiagnosis of rickettsiosis-like infections, cases of human granulocytic anaplasmosis could go undetected.

  3. Human Simulated Diving Experiments.

    ERIC Educational Resources Information Center

    Bruce, David S.; Speck, Dexter F.

    1979-01-01

    This report details several simulated divinq experiments on the human. These are suitable for undergraduate or graduate laboratories in human or environmental physiology. The experiment demonstrates that a diving reflex is precipitated by both facial cooling and apnea. (Author/RE)

  4. Manage "Human Capital" Strategically

    ERIC Educational Resources Information Center

    Odden, Allan

    2011-01-01

    To strategically manage human capital in education means restructuring the entire human resource system so that schools not only recruit and retain smart and capable individuals, but also manage them in ways that support the strategic directions of the organization. These management practices must be aligned with a district's education improvement…

  5. The Humanities' Value

    ERIC Educational Resources Information Center

    Harpham, Geoffrey Galt

    2009-01-01

    Why should society support the humanities when so many people are suffering from the effects of the economic crisis? What claim do the humanities, or scholarship generally, have on increasingly limited resources? Shouldn't such pursuits be considered luxuries at a time when people should be focusing on essentials? The alleviation of human…

  6. IMMUNOASSAY HUMAN EXPOSURE STUDIES

    EPA Science Inventory

    The Human Exposure Research Branch has developed several enzyme-linked immunosorbent assay (ELISA) methods to support human exposure assessment studies. Immunoassays to detect low levels (<10 ng/mL) of chlorpyrifos in food, track-in dirt and house dust have been applied to sam...

  7. Human Mind Maps

    ERIC Educational Resources Information Center

    Glass, Tom

    2016-01-01

    When students generate mind maps, or concept maps, the maps are usually on paper, computer screens, or a blackboard. Human Mind Maps require few resources and little preparation. The main requirements are space where students can move around and a little creativity and imagination. Mind maps can be used for a variety of purposes, and Human Mind…

  8. The great human expansion.

    PubMed

    Henn, Brenna M; Cavalli-Sforza, L L; Feldman, Marcus W

    2012-10-30

    Genetic and paleoanthropological evidence is in accord that today's human population is the result of a great demic (demographic and geographic) expansion that began approximately 45,000 to 60,000 y ago in Africa and rapidly resulted in human occupation of almost all of the Earth's habitable regions. Genomic data from contemporary humans suggest that this expansion was accompanied by a continuous loss of genetic diversity, a result of what is called the "serial founder effect." In addition to genomic data, the serial founder effect model is now supported by the genetics of human parasites, morphology, and linguistics. This particular population history gave rise to the two defining features of genetic variation in humans: genomes from the substructured populations of Africa retain an exceptional number of unique variants, and there is a dramatic reduction in genetic diversity within populations living outside of Africa. These two patterns are relevant for medical genetic studies mapping genotypes to phenotypes and for inferring the power of natural selection in human history. It should be appreciated that the initial expansion and subsequent serial founder effect were determined by demographic and sociocultural factors associated with hunter-gatherer populations. How do we reconcile this major demic expansion with the population stability that followed for thousands years until the inventions of agriculture? We review advances in understanding the genetic diversity within Africa and the great human expansion out of Africa and offer hypotheses that can help to establish a more synthetic view of modern human evolution.

  9. Portraits of Human Greatness.

    ERIC Educational Resources Information Center

    Saint Anselm's Coll., Manchester, NH.

    Examined is the Humanities Program at St. Anselm College, a two-year program of readings and lectures ordered chronologically from ancient to contemporary times--from the age of Classical Greek thought and the Old Testament to the twentieth century. The first year of the Humanities Program is organized in eight units on general modes of…

  10. Dogs catch human yawns.

    PubMed

    Joly-Mascheroni, Ramiro M; Senju, Atsushi; Shepherd, Alex J

    2008-10-23

    This study is the first to demonstrate that human yawns are possibly contagious to domestic dogs (Canis familiaris). Twenty-nine dogs observed a human yawning or making control mouth movements. Twenty-one dogs yawned when they observed a human yawning, but control mouth movements did not elicit yawning from any of them. The presence of contagious yawning in dogs suggests that this phenomenon is not specific to primate species and may indicate that dogs possess the capacity for a rudimentary form of empathy. Since yawning is known to modulate the levels of arousal, yawn contagion may help coordinate dog-human interaction and communication. Understanding the mechanism as well as the function of contagious yawning between humans and dogs requires more detailed investigation.

  11. Human Space Flight

    NASA Technical Reports Server (NTRS)

    Woolford, Barbara; Mount, Frances

    2004-01-01

    The first human space flight, in the early 1960s, was aimed primarily at determining whether humans could indeed survive and function in micro-gravity. Would eating and sleeping be possible? What mental and physical tasks could be performed? Subsequent programs increased the complexity of the tasks the crew performed. Table 1 summarizes the history of U.S. space flight, showing the projects, their dates, crew sizes, and mission durations. With over forty years of experience with human space flight, the emphasis now is on how to design space vehicles, habitats, and missions to produce the greatest returns to human knowledge. What are the roles of the humans in space flight in low earth orbit, on the moon, and in exploring Mars?

  12. Mars Human Exploration Objectives

    NASA Technical Reports Server (NTRS)

    Briggs, Geoff

    1998-01-01

    This paper reviews the objectives and other considerations of Human exploration of Mars. The objectives of human exploration of Mars are: (1) to learn how Mars is similar to, and different from, Earth; (2) to explore possible life, past and present; (3) to discover what Mars is like now from the perspective of Geoscience and geologic history; and (4) how did Mars form and how did its formation differ from Earth. Considerations of human Martian exploration involve: (1) having a capable base laboratory; (2) having long range transportation; (3) having operational autonomy of the crew, and the requirement of the crew to possess a range of new cognitive processes along with easy communications with terrestrial colleagues; and finally (4) creating the human habitat along with human factors which involve more than just survivability.

  13. Beliefs about Human Extinction

    SciTech Connect

    Tonn, Bruce Edward

    2009-11-01

    This paper presents the results of a web-based survey about futures issues. Among many questions, respondents were asked whether they believe humans will become extinct. Forty-five percent of the almost 600 respondents believe that humans will become extinct. Many of those holding this believe felt that humans could become extinct within 500-1000 years. Others estimated extinction 5000 or more years into the future. A logistic regression model was estimated to explore the bases for this belief. It was found that people who describe themselves a secular are more likely to hold this belief than people who describe themselves as beingmore » Protestant. Older respondents and those who believe that humans have little control over their future also hold this belief. In addition, people who are more apt to think about the future and are better able to imagine potential futures tend to also believe that humans will become extinct.« less

  14. Chemical Carcinogen-Induced Changes in tRNA Metabolism in Human Cells

    DTIC Science & Technology

    1984-11-20

    uptake Chart 7 by POD was not affected by concurrent treatment with 0.1 mM quercetin (Chart 7). Epidermal growth factor (100 nM) also had no affect on...Therefore, we examined the affect on queuine uptake of quercetin , a flavanoid inhibitor of protein kinase C which binds to a site separate from the...phorbol ester binding site (12). Quercetin did not relieve the POO-effected inhibition of rQT3 uptake.. Although some residual activity of membrane bound

  15. Digital Human Modeling

    NASA Technical Reports Server (NTRS)

    Dischinger, H. Charles, Jr.

    2017-01-01

    The development of models to represent human characteristics and behaviors in human factors is broad and general. The term "model" can refer to any metaphor to represent any aspect of the human; it is generally used in research to mean a mathematical tool for the simulation (often in software, which makes the simulation digital) of some aspect of human performance and for the prediction of future outcomes. This section is restricted to the application of human models in physical design, e.g., in human factors engineering. This design effort is typically human interface design, and the digital models used are anthropometric. That is, they are visual models that are the physical shape of humans and that have the capabilities and constraints of humans of a selected population. They are distinct from the avatars used in the entertainment industry (movies, video games, and the like) in precisely that regard: as models, they are created through the application of data on humans, and they are used to predict human response; body stresses workspaces. DHM enable iterative evaluation of a large number of concepts and support rapid analysis, as compared with use of physical mockups. They can be used to evaluate feasibility of escape of a suited astronaut from a damaged vehicle, before launch or after an abort (England, et al., 2012). Throughout most of human spaceflight, little attention has been paid to worksite design for ground workers. As a result of repeated damage to the Space Shuttle which adversely affected flight safety, DHM analyses of ground assembly and maintenance have been developed over the last five years for the design of new flight systems (Stambolian, 2012, Dischinger and Dunn Jackson, 2014). The intent of these analyses is to assure the design supports the work of the ground crew personnel and thereby protect the launch vehicle. They help the analyst address basic human factors engineering questions: can a worker reach the task site from the work platform

  16. [Medicine and human rights].

    PubMed

    Lavik, N J

    1994-01-10

    Medicine's task to promote health without inflicting harm is parallel to the two dimensions of human rights-"the positive rights", meaning the right of fulfillment of certain basic needs, and "the negative rights", meaning protection from harmful violations. A historical review shows that the practice of medicine has been an integral part of every civilization in human history, but that the ideas of human rights are a more recent invention of the human mind. In spite of old philosophical traditions about the unique value of the human being, the idea that all human beings are created equal and born with the same rights has only been truly recognized in recent centuries, and has been elaborated through conventions and declarations in national and international political institutions after World War II. At the crossroads between human rights and medicine, five points are high-lighted: the extensive prevalence of man-made disease, the relationship between medical diagnoses, ethics and law, the connection between healing and moral rehabilitation, the relationship between health, psychology and fair trials in society, and the role of physicians in conflicts of loyalty.

  17. Human Milk Banking.

    PubMed

    Haiden, Nadja; Ziegler, Ekhard E

    2016-01-01

    Human milk banks play an essential role by providing human milk to infants who would otherwise not be able to receive human milk. The largest group of recipients are premature infants who derive very substantial benefits from it. Human milk protects premature infants from necrotizing enterocolitis and from sepsis, two devastating medical conditions. Milk banks collect, screen, store, process, and distribute human milk. Donating women usually nurse their own infants and have a milk supply that exceeds their own infants' needs. Donor women are carefully selected and are screened for HIV-1, HIV-2, human T-cell leukemia virus 1 and 2, hepatitis B, hepatitis C, and syphilis. In the milk bank, handling, storing, processing, pooling, and bacterial screening follow standardized algorithms. Heat treatment of human milk diminishes anti-infective properties, cellular components, growth factors, and nutrients. However, the beneficial effects of donor milk remain significant and donor milk is still highly preferable in comparison to formula. © 2017 S. Karger AG, Basel.

  18. Human Space Exploration

    NASA Technical Reports Server (NTRS)

    Jeevarajan, Antony

    2014-01-01

    The Mars probe, launched by India a few months ago, is on its way to Mars. At this juncture, it is appropriate to talk about the opportunities presented to us for the Human Exploration of Mars. I am planning to highlight some of the challenges to take humans to Mars, descend, land, stay, ascend and return home safely. The logistics of carrying the necessary accessories to stay at Mars will be delivered in multiple stages using robotic missions. The primary ingredients for human survival is air, water, food and shelter and the necessity to recycle the primary ingredients will be articulated. Humans have to travel beyond the van Allen radiation belt under microgravity condition during this inter-planetary travel for about 6 months minimum one way. The deconditioning of human system under microgravity conditions and protection of humans from Galactic cosmic radiation during the travel should be taken into consideration. The multi-disciplinary effort to keep the humans safe and functional during this journey will be addressed.

  19. Human ehrlichiosis and anaplasmosis.

    PubMed

    Ismail, Nahed; Bloch, Karen C; McBride, Jere W

    2010-03-01

    Human ehrlichiosis and anaplasmosis are acute febrile tick-borne diseases caused by various members of the genera Ehrlichia and Anaplasma (Anaplasmataceae). Human monocytotropic ehrlichiosis has become one of the most prevalent life-threatening tick-borne disease in the United States. Ehrlichiosis and anaplasmosis are becoming more frequently diagnosed as the cause of human infections, as animal reservoirs and tick vectors have increased in number and humans have inhabited areas where reservoir and tick populations are high. Ehrlichia chaffeensis, the etiologic agent of human monocytotropic ehrlichiosis (HME), is an emerging zoonosis that causes clinical manifestations ranging from a mild febrile illness to a fulminant disease characterized by multiorgan system failure. Anaplasma phagocytophilum causes human granulocytotropic anaplasmosis (HGA), previously known as human granulocytotropic ehrlichiosis. This article reviews recent advances in the understanding of ehrlichial diseases related to microbiology, epidemiology, diagnosis, pathogenesis, immunity, and treatment of the 2 prevalent tick-borne diseases found in the United States, HME and HGA. 2010 Elsevier Inc. All rights reserved.

  20. Archaea on human skin.

    PubMed

    Probst, Alexander J; Auerbach, Anna K; Moissl-Eichinger, Christine

    2013-01-01

    The recent era of exploring the human microbiome has provided valuable information on microbial inhabitants, beneficials and pathogens. Screening efforts based on DNA sequencing identified thousands of bacterial lineages associated with human skin but provided only incomplete and crude information on Archaea. Here, we report for the first time the quantification and visualization of Archaea from human skin. Based on 16 S rRNA gene copies Archaea comprised up to 4.2% of the prokaryotic skin microbiome. Most of the gene signatures analyzed belonged to the Thaumarchaeota, a group of Archaea we also found in hospitals and clean room facilities. The metabolic potential for ammonia oxidation of the skin-associated Archaea was supported by the successful detection of thaumarchaeal amoA genes in human skin samples. However, the activity and possible interaction with human epithelial cells of these associated Archaea remains an open question. Nevertheless, in this study we provide evidence that Archaea are part of the human skin microbiome and discuss their potential for ammonia turnover on human skin.

  1. Archaea on Human Skin

    PubMed Central

    Probst, Alexander J.; Auerbach, Anna K.; Moissl-Eichinger, Christine

    2013-01-01

    The recent era of exploring the human microbiome has provided valuable information on microbial inhabitants, beneficials and pathogens. Screening efforts based on DNA sequencing identified thousands of bacterial lineages associated with human skin but provided only incomplete and crude information on Archaea. Here, we report for the first time the quantification and visualization of Archaea from human skin. Based on 16 S rRNA gene copies Archaea comprised up to 4.2% of the prokaryotic skin microbiome. Most of the gene signatures analyzed belonged to the Thaumarchaeota, a group of Archaea we also found in hospitals and clean room facilities. The metabolic potential for ammonia oxidation of the skin-associated Archaea was supported by the successful detection of thaumarchaeal amoA genes in human skin samples. However, the activity and possible interaction with human epithelial cells of these associated Archaea remains an open question. Nevertheless, in this study we provide evidence that Archaea are part of the human skin microbiome and discuss their potential for ammonia turnover on human skin. PMID:23776475

  2. Genetics of human hydrocephalus

    PubMed Central

    Williams, Michael A.; Rigamonti, Daniele

    2006-01-01

    Human hydrocephalus is a common medical condition that is characterized by abnormalities in the flow or resorption of cerebrospinal fluid (CSF), resulting in ventricular dilatation. Human hydrocephalus can be classified into two clinical forms, congenital and acquired. Hydrocephalus is one of the complex and multifactorial neurological disorders. A growing body of evidence indicates that genetic factors play a major role in the pathogenesis of hydrocephalus. An understanding of the genetic components and mechanism of this complex disorder may offer us significant insights into the molecular etiology of impaired brain development and an accumulation of the cerebrospinal fluid in cerebral compartments during the pathogenesis of hydrocephalus. Genetic studies in animal models have started to open the way for understanding the underlying pathology of hydrocephalus. At least 43 mutants/loci linked to hereditary hydrocephalus have been identified in animal models and humans. Up to date, 9 genes associated with hydrocephalus have been identified in animal models. In contrast, only one such gene has been identified in humans. Most of known hydrocephalus gene products are the important cytokines, growth factors or related molecules in the cellular signal pathways during early brain development. The current molecular genetic evidence from animal models indicate that in the early development stage, impaired and abnormal brain development caused by abnormal cellular signaling and functioning, all these cellular and developmental events would eventually lead to the congenital hydrocephalus. Owing to our very primitive knowledge of the genetics and molecular pathogenesis of human hydrocephalus, it is difficult to evaluate whether data gained from animal models can be extrapolated to humans. Initiation of a large population genetics study in humans will certainly provide invaluable information about the molecular and cellular etiology and the developmental mechanisms of human

  3. Oncogenic human papillomaviruses

    PubMed Central

    2017-01-01

    Human papillomaviruses (HPVs) are an ancient group of viruses with small, double-stranded DNA circular genomes. They are species-specific and have a strict tropism for mucosal and cutaneous stratified squamous epithelial surfaces of the host. A subset of these viruses has been demonstrated to be the causative agent of several human cancers. Here, we review the biology, natural history, evolution and cancer association of the oncogenic HPVs. This article is part of the themed issue ‘Human oncogenic viruses’. PMID:28893940

  4. Oncogenic human papillomaviruses.

    PubMed

    McBride, Alison A

    2017-10-19

    Human papillomaviruses (HPVs) are an ancient group of viruses with small, double-stranded DNA circular genomes. They are species-specific and have a strict tropism for mucosal and cutaneous stratified squamous epithelial surfaces of the host. A subset of these viruses has been demonstrated to be the causative agent of several human cancers. Here, we review the biology, natural history, evolution and cancer association of the oncogenic HPVs.This article is part of the themed issue 'Human oncogenic viruses'. © 2017 The Authors.

  5. Avian and human metapneumovirus.

    PubMed

    Broor, Shobha; Bharaj, Preeti

    2007-04-01

    Pneumovirus infection remains a significant problem for both human and veterinary medicine. Both avian pneumovirus (aMPV, Turkey rhinotracheitis virus) and human metapneumovirus (hMPV) are pathogens of birds and humans, which are associated with respiratory tract infections. Based on their different genomic organization and low level of nucleotide (nt) and amino acid (aa) identity with paramyxoviruses in the genus Pneumovirus, aMPV and hMPV have been classified into a new genus referred to as Metapneumovirus. The advancement of our understanding of pneumovirus biology and pathogenesis of pneumovirus disease in specific natural hosts can provide us with strategies for vaccine formulations and combined antiviral and immunomodulatory therapies.

  6. Human Genome Project

    SciTech Connect

    Block, S.; Cornwall, J.; Dally, W.

    1998-01-04

    The study reviews Department of Energy supported aspects of the United States Human Genome Project, the joint National Institutes of Health/Department of Energy program to characterize all human genetic material, to discover the set of human genes, and to render them accessible for further biological study. The study concentrates on issues of technology, quality assurance/control, and informatics relevant to current effort on the genome project and needs beyond it. Recommendations are presented on areas of the genome program that are of particular interest to and supported by the Department of Energy.

  7. Human exposure to aluminium.

    PubMed

    Exley, Christopher

    2013-10-01

    Human activities have circumvented the efficient geochemical cycling of aluminium within the lithosphere and therewith opened a door, which was previously only ajar, onto the biotic cycle to instigate and promote the accumulation of aluminium in biota and especially humans. Neither these relatively recent activities nor the entry of aluminium into the living cycle are showing any signs of abating and it is thus now imperative that we understand as fully as possible how humans are exposed to aluminium and the future consequences of a burgeoning exposure and body burden. The aluminium age is upon us and there is now an urgent need to understand how to live safely and effectively with aluminium.

  8. Aluminium in human sweat.

    PubMed

    Minshall, Clare; Nadal, Jodie; Exley, Christopher

    2014-01-01

    It is of burgeoning importance that the human body burden of aluminium is understood and is measured. There are surprisingly few data to describe human excretion of systemic aluminium and almost no reliable data which relate to aluminium in sweat. We have measured the aluminium content of sweat in 20 healthy volunteers following mild exercise. The concentration of aluminium ranged from 329 to 5329μg/L. These data equate to a daily excretion of between 234 and 7192μg aluminium and they strongly suggest that perspiration is the major route of excretion of systemic aluminium in humans. Copyright © 2013 Elsevier GmbH. All rights reserved.

  9. Human Computer Interaction

    NASA Astrophysics Data System (ADS)

    Bhagwani, Akhilesh; Sengar, Chitransh; Talwaniper, Jyotsna; Sharma, Shaan

    2012-08-01

    The paper basically deals with the study of HCI (Human computer interaction) or BCI(Brain-Computer-Interfaces) Technology that can be used for capturing brain signals and translating them into commands that allow humans to control (just by thinking) devices such as computers, robots, rehabilitation technology and virtual reality environments. The HCI is based as a direct communication pathway between the brain and an external device. BCIs are often aimed at assisting, augmenting, or repairing human cognitive or sensory-motor functions.The paper also deals with many advantages of BCI Technology along with some of its applications and some major drawbacks.

  10. Human Use Index

    EPA Pesticide Factsheets

    Human land uses may have major impacts on ecosystems, affecting biodiversity, habitat, air and water quality. The human use index (also known as U-index) is the percentage of human land use in an area, including agriculture, urban and suburban development, and mining. Low values indicate little disturbance of natural land cover. More information about these resources, including the variables used in this study, may be found here: https://edg.epa.gov/data/Public/ORD/NERL/ReVA/ReVA_Data.zip.

  11. Human Use Index (Future)

    EPA Pesticide Factsheets

    Human land uses may have major impacts on ecosystems, affecting biodiversity, habitat, air and water quality. The human use index (also known as U-index) is the percentage of human land use in an area, including agriculture, urban and suburban development, and mining. Low values indicate little disturbance of natural land cover. More information about these resources, including the variables used in this study, may be found here: https://edg.epa.gov/data/Public/ORD/NERL/ReVA/ReVA_Data.zip.

  12. The Concept of Being Human.

    ERIC Educational Resources Information Center

    Purcell, Royal

    This analysis of the relationship between humanism and humanitarianism outlines educational goals that should lead to a more humane world. Section 1, an outline of human life examines six substructures--human life, individuality, amenity, contact, actualization, and problems. A definition and examples of humanism in section 2 are elaborated into a…

  13. Aerospace Human Factors

    NASA Technical Reports Server (NTRS)

    Jordan, Kevin

    1999-01-01

    The following contains the final report on the activities related to the Cooperative Agreement between the human factors research group at NASA Ames Research Center and the Psychology Department at San Jose State University. The participating NASA Ames division has been, as the organization has changed, the Aerospace Human Factors Research Division (ASHFRD and Code FL), the Flight Management and Human Factors Research Division (Code AF), and the Human Factors Research and Technology Division (Code IH). The inclusive dates for the report are November 1, 1984 to January 31, 1999. Throughout the years, approximately 170 persons worked on the cooperative agreements in one capacity or another. The Cooperative Agreement provided for research personnel to collaborate with senior scientists in ongoing NASA ARC research. Finally, many post-MA/MS and post-doctoral personnel contributed to the projects. It is worth noting that 10 former cooperative agreement personnel were hired into civil service positions directly from the agreements.

  14. The Human Hazard.

    ERIC Educational Resources Information Center

    Tickell, Crispin

    1995-01-01

    Examines the plight of environmental refugees and the adequacy of political responses to the situation. Discusses the consequences of accelerated environmental change, particularly the impact of global warming on human migration. (LZ)

  15. Human Spaceflight Safety Hearing

    NASA Image and Video Library

    2009-12-02

    Dr. Joseph R. Fragola, Vice President, Valador, Inc., testifies during a hearing before the House Subcommitte on Space and Aeronautics regarding Safety of Human Spaceflight on Capitol Hill, Wednesday, Dec. 2, 2009, in Washington. Photo Credit: (NASA/Bill Ingalls)

  16. Human Spaceflight Safety Hearing

    NASA Image and Video Library

    2009-12-02

    Constellation Program Manager Jeff Hanley testifies during a hearing before the House Subcommitte on Space and Aeronautics regarding Safety of Human Spaceflight on Capitol Hill, Wednesday, Dec. 2, 2009, in Washington. Photo Credit: (NASA/Bill Ingalls)

  17. Human Spaceflight Safety Hearing

    NASA Image and Video Library

    2009-12-02

    Bretton Alexander, President of the Commercial Spaceflight Federation, testifies during a hearing before the House Subcommitte on Space and Aeronautics regarding Safety of Human Spaceflight on Capitol Hill, Wednesday, Dec. 2, 2009, in Washington. Photo Credit: (NASA/Bill Ingalls)

  18. Human Spaceflight Safety Hearing

    NASA Image and Video Library

    2009-12-02

    Reitred astronaut Lt. Gen. Thomas Stafford testifies during a hearing before the House Subcommitte on Space and Aeronautics regarding Safety of Human Spaceflight on Capitol Hill, Wednesday, Dec. 2, 2009, in Washington. Photo Credit: (NASA/Bill Ingalls)

  19. Human Spaceflight Safety Hearing

    NASA Image and Video Library

    2009-12-02

    Jeff Hanley, Constellation Program Manager at NASA, testifies before a hearing before the House Subcommitte on Space and Aeronautics regarding Safety of Human Spaceflight on Capitol Hill, Wednesday, Dec. 2, 2009, in Washington. Photo Credit: (NASA/Bill Ingalls)

  20. Will Technology Humanize Us?

    ERIC Educational Resources Information Center

    Snider, Robert C.

    1972-01-01

    The author considers the question of whether technology will cause humanization or dehumanization in the schools. He concludes that we can not stop tecchnology; we can only give it direction and purpose. (Author/MS)

  1. Human Papillomavirus (HPV)

    MedlinePlus

    ... Prevention Listen Español Text Size Email Print Share HPV (Human Papillomavirus) Vaccine: What You Need to Know ( ... men get cancer and other diseases from HPV. HPV vaccine HPV vaccine is approved by FDA and ...

  2. Humanism vs. Behaviorism

    ERIC Educational Resources Information Center

    Hunter, Madeline

    1977-01-01

    Author argues that humanism and behaviorism are not necessarily exclusive of one another, and that principles of behaviorism, when thoughtfully applied, can lead to the achievement of humanistic goals. (RW)

  3. Bridging Humanism and Behaviorism.

    ERIC Educational Resources Information Center

    Chu, Lily

    1980-01-01

    Humanistic behaviorism may provide the necessary bridge between behaviorism and humanism. Perhaps the most humanistic approach to teaching is to learn how certain changes will help students and how these changes can be accomplished. (Author/MLF)

  4. Spaceflight Versus Human Spaceflight

    NASA Astrophysics Data System (ADS)

    Barr, Stephanie

    2013-09-01

    Spaceflight is challenging. Human spaceflight is far more challenging,.Those familiar with spaceflight recognize that human spaceflight is more than tacking an environmental control system on an existing spacecraft, that there are a number of serious technical challenges involved in sending people out into space and bringing them back home safely.The return trip, bringing the crew back to the surface of the earth safely, is more than just an additional task, it's the new imperative. Differences between manned and unmanned spaceflight are more than technical. The human element forces a change in philosophy, a mindset that will likely touch every aspect of flight from launch through mission and return. Seasoned space professionals used to the paradigms and priorities of unmanned flight need to be cognizant of these differences and some of the implications, perhaps most especially because mission success and human safety priorities are sometimes contradictory.

  5. Pushing Human Frontiers

    NASA Technical Reports Server (NTRS)

    Zubrin, Robert

    2005-01-01

    With human colonization of Mars, I think you will see a higher standard of civilization, just as America set a higher standard of civilization which then promulgated back into Europe. I think that if you want to maximize human potential, you need a higher standard of civilization, and that becomes an example that benefits everyone. Without an open frontier, closed world ideologies, such as the Malthus Theory, tend to come to the forefront. It is that there are limited resources; therefore, we are all in deadly competition with each other for the limited pot. The result is tyrannical and potentially genocidal regimes, and we've already seen this in the twentieth century. There s no truth in the Malthus Theory, because human beings are the creators of their resources. With every mouth comes a pair of hands and a brain. But if it seems to be true, you have a vector in this direction, and it is extremely unfortunate. It is only in a universe of infinite resources that all humans can be brothers and sisters. The fundamental question which affects humanity s sense of itself is whether the world is changeable or fixed. Are we the makers of our world or just its inhabitants? Some people have a view that they re living at the end of history within a world that s already defined, and there is no fundamental purpose to human life because there is nothing humans can do that matters. On the other hand, if humans understand their own role as the creators of their world, that s a much more healthy point of view. It raises the dignity of humans. Indeed, if we do establish a new branch of human civilization on Mars that grows in time and potency to the point where it cannot really settle Mars, but transforms Mars, and brings life to Mars, we will prove to everyone and for all time the precious and positive nature of the human species and every member of it.

  6. Human Exploration of Mars

    NASA Image and Video Library

    2016-10-22

    The scientific knowledge and technologies needed to make human exploration of Mars happen are within our reach. NASA 360 joins Dr. Jim Green, Director of NASA’s Planetary Science Division, as he discusses how NASA is preparing for human exploration of the Red Planet. This video was created from a live recording at the Viking 40th Anniversary Symposium in July 2016. To watch the original talk please visit: http://bit.ly/2bk1PGk

  7. Evolution and human sexuality.

    PubMed

    Gray, Peter B

    2013-12-01

    The aim of this review is to put core features of human sexuality in an evolutionary light. Toward that end, I address five topics concerning the evolution of human sexuality. First, I address theoretical foundations, including recent critiques and developments. While much traces back to Darwin and his view of sexual selection, more recent work helps refine the theoretical bases to sex differences and life history allocations to mating effort. Second, I consider central models attempting to specify the phylogenetic details regarding how hominin sexuality might have changed, with most of those models honing in on transitions from a possible chimpanzee-like ancestor to the slightly polygynous and long-term bonded sociosexual partnerships observed among most recently studied hunter-gatherers. Third, I address recent genetic and physiological data contributing to a refined understanding of human sexuality. As examples, the availability of rapidly increasing genomic information aids comparative approaches to discern signals of selection in sexuality-related phenotypes, and neuroendocrine studies of human responses to sexual stimuli provide insight into homologous and derived mechanisms. Fourth, I consider some of the most recent, large, and rigorous studies of human sexuality. These provide insights into sexual behavior across other national samples and on the Internet. Fifth, I discuss the relevance of a life course perspective to understanding the evolution of human sexuality. Most research on the evolution of human sexuality focuses on young adults. Yet humans are sexual beings from gestation to death, albeit in different ways across the life course, and in ways that can be theoretically couched within life history theory. Copyright © 2013 Wiley Periodicals, Inc.

  8. Human exploration mission studies

    NASA Technical Reports Server (NTRS)

    Cataldo, Robert L.

    1990-01-01

    This paper describes several case studies of human space exploration, considered by the NASA's Office of Exploration in 1988. Special attention is given to the mission scenarios, the critical technology required in these expeditions, and the extraterrestrial power requirements of significant system elements. The cases examined include a manned expedition to Phobos, the inner Martian moon; a human expedition to Mars; the Lunar Observatory; and a lunar outpost to early Mars evolution.

  9. Human-centered Computing

    NASA Astrophysics Data System (ADS)

    Sebe, Nicu

    Computing is at one of itsmost excitingmoments in history, playing an essential role in supporting many important human activities. The explosion in the availability of information in various media forms and through multiple sensors and devices means, on one hand, that the amount of data we can collect will continue to increase dramatically, and, on the other hand, that we need to develop new paradigms to search, organize, and integrate such information to support all human activities.

  10. Human ocular anatomy.

    PubMed

    Kels, Barry D; Grzybowski, Andrzej; Grant-Kels, Jane M

    2015-01-01

    We review the normal anatomy of the human globe, eyelids, and lacrimal system. This contribution explores both the form and function of numerous anatomic features of the human ocular system, which are vital to a comprehensive understanding of the pathophysiology of many oculocutaneous diseases. The review concludes with a reference glossary of selective ophthalmologic terms that are relevant to a thorough understanding of many oculocutaneous disease processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Mapping the human genome

    SciTech Connect

    Annas, G.C.; Elias, S.

    1992-01-01

    This article is a review of the book Mapping the Human Genome: Using Law and Ethics as Guides, edited by George C. Annas and Sherman Elias. The book is a collection of essays on the subject of using ethics and laws as guides to justify human gene mapping. It addresses specific issues such problems related to eugenics, patents, insurance as well as broad issues such as the societal definitions of normality.

  12. Human Germline Genome Editing.

    PubMed

    Ormond, Kelly E; Mortlock, Douglas P; Scholes, Derek T; Bombard, Yvonne; Brody, Lawrence C; Faucett, W Andrew; Garrison, Nanibaa' A; Hercher, Laura; Isasi, Rosario; Middleton, Anna; Musunuru, Kiran; Shriner, Daniel; Virani, Alice; Young, Caroline E

    2017-08-03

    With CRISPR/Cas9 and other genome-editing technologies, successful somatic and germline genome editing are becoming feasible. To respond, an American Society of Human Genetics (ASHG) workgroup developed this position statement, which was approved by the ASHG Board in March 2017. The workgroup included representatives from the UK Association of Genetic Nurses and Counsellors, Canadian Association of Genetic Counsellors, International Genetic Epidemiology Society, and US National Society of Genetic Counselors. These groups, as well as the American Society for Reproductive Medicine, Asia Pacific Society of Human Genetics, British Society for Genetic Medicine, Human Genetics Society of Australasia, Professional Society of Genetic Counselors in Asia, and Southern African Society for Human Genetics, endorsed the final statement. The statement includes the following positions. (1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy. (2) Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. There should be no prohibition on making public funds available to support this research. (3) Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input. Copyright © 2017 American Society of Human Genetics. All rights reserved.

  13. Nutritionally relevant concentrations of resveratrol and hydroxytyrosol mitigate oxidative burst of human granulocytes and monocytes and the production of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

    PubMed

    Bigagli, Elisabetta; Cinci, Lorenzo; Paccosi, Sara; Parenti, Astrid; D'Ambrosio, Mario; Luceri, Cristina

    2017-02-01

    The health benefits of bio-active phenolic compounds have been largely investigated in vitro at concentrations which exceed those reachable in vivo. We investigated and compared the anti-inflammatory effects of resveratrol, hydroxytyrosol and oleuropein at physiologically relevant concentrations by using in vitro models of inflammation. Human granulocytes and monocytes were stimulated with phorbol myristate acetate (PMA) and the ability of resveratrol, hydroxytyrosol and oleuropein to inhibit the oxidative burst and CD11b expression was measured. Nitric oxide (NO), prostaglandin E2 (PGE2) levels, COX-2, iNOS, TNFα, IL-1β and miR-146a expression and activation of the transcription factor Nrf2 were evaluated in macrophages RAW 264.7 stimulated with LPS (1μg/ml) for 18h, exposed to resveratrol, hydroxytyrosol and oleuropein (5 and 10μM). Synergistic effects were explored as well, together with the levels of PGE2, COX-2 and IL-1β expression in macrophages after 6h of LPS stimulation. PGE2 and COX-2 expression were also assessed on human monocytes. All the tested compounds inhibited granulocytes oxidative burst in a concentration dependent manner and CD11b expression was also significantly counteracted by resveratrol and hydroxytyrosol. The measurement of oxidative burst in human monocytes produced similar effects being resveratrol more active. Hydroxytyrosol and resveratrol inhibited the production of NO and PGE2 but did not reduce iNOS, TNFα or IL-1β gene expression in LPS-stimulated RAW 264.7 for 18h. Resveratrol slightly decreased COX-2 expression after 18h but not after 6h, but reduced PGE2 levels after 6h. Resveratrol and hydroxytyrosol 10μM induced NRf2 nuclear translocation and reduced miR-146a expression in LPS treated RAW 264.7. Overall, we reported an anti-inflammatory effect of resveratrol and hydroxytyrosol at low, nutritionally relevant concentrations, involving the inhibition of granulocytes and monocytes activation, the modulation of miR-146a

  14. Cytokines in human milk.

    PubMed

    Garofalo, Roberto

    2010-02-01

    Epidemiologic studies conducted in the past 30 years to investigate the protective functions of human milk strongly support the notion that breastfeeding prevents infantile infections, particularly those affecting the gastrointestinal and respiratory tracts. However, more recent clinical and experimental observations also suggest that human milk not only provides passive protection, but also can directly modulate the immunological development of the recipient infant. The study of this remarkable defense system in human milk has been difficult because of its biochemical complexity, the small concentration of certain bioactive components, the compartmentalization of some of these agents, the dynamic quantitative and qualitative changes of milk during lactation, and the lack of specific reagents to quantify these agents. However, a host of bioactive substances, including hormones, growth factors, and immunological factors such as cytokines, have been identified in human milk. Cytokines are pluripotent polypeptides that act in autocrine/paracrine fashions by binding to specific cellular receptors. They operate in networks and orchestrate the development and functions of immune system. Several different cytokines and chemokines have been discovered in human milk in the past years, and the list is growing very rapidly. This article will review the current knowledge about the increasingly complex network of chemoattractants, activators, and anti-inflammatory cytokines present in human milk and their potential role in compensating for the developmental delay of the neonate immune system. Copyright 2010. Published by Mosby, Inc.

  15. The Human Cell Atlas

    PubMed Central

    Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

    2017-01-01

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community. PMID:29206104

  16. Habitability and Human Factors Contributions to Human Space Flight

    NASA Technical Reports Server (NTRS)

    Sumaya, Jennifer Boyer

    2011-01-01

    This slide presentation reviews the work of the Habitability and Human Factors Branch in support of human space flight in two main areas: Applied support to major space programs, and Space research. The field of Human Factors applies knowledge of human characteristics for the design of safer, more effective, and more efficient systems. This work is in several areas of the human space program: (1) Human-System Integration (HSI), (2) Orion Crew Exploration Vehicle, (3) Extravehicular Activity (EVA), (4) Lunar Surface Systems, (5) International Space Station (ISS), and (6) Human Research Program (HRP). After detailing the work done in these areas, the facilities that are available for human factors work are shown.

  17. Human behavior and human performance: Psychomotor demands

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The results of several experiments are presented in abstract form. These studies are critical for the interpretation and acceptance of flight based science to be conducted by the Behavior and Performance project. Some representative titles are as follow: External audio for IBM/PC compatible computers; A comparative assessment of psychomotor performance (target prediction by humans and macaques); Response path (a dependent measure for computer maze solving and other tasks); Behavioral asymmetries of psychomotor performance in Rhesus monkey (a dissociation between hand preference and skill); Testing primates with joystick based automated apparatus; and Environmental enrichment and performance assessment for ground or flight based research with primates;

  18. Microtubule organization during human parthenogenesis.

    PubMed

    Terada, Yukihiro; Hasegawa, Hisataka; Ugajin, Tomohisa; Murakami, Takashi; Yaegashi, Nobuo; Okamura, Kunihiro

    2009-04-01

    In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.

  19. OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

    SciTech Connect

    Omar, Hany A.; Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514; Arafa, El-Shaimaa A.

    2013-11-01

    Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt–nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to studymore » the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt–NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt–NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9's clinical promise as a component of anticancer therapy. - Highlights: • The antiangiogenic activity of OSU-A9 in HUVECs was explored. • OSU-A9 inhibited HUVECs proliferation, migration, invasion and tube formation. • OSU-A9

  20. Effect of Atmospheric PM2.5 on Expression Levels of NF-κB Genes and Inflammatory Cytokines Regulated by NF-κB in Human Macrophage.

    PubMed

    Zhang, Yuezhu; Wang, Shuyue; Zhu, Jian; Li, Chunyan; Zhang, Tianrong; Liu, Hongbo; Xu, Qi; Ye, Xiaofang; Zhou, Liting; Ye, Lin

    2018-02-02

    Exposure to PM2.5 induces systemic inflammation, and the NF-κB signaling pathway plays an important role in the inflammation process. We aim to clarify whether the expression of NF-κB gene family affects inflammation caused by PM2.5. Human monocytic cells (THP-1) were induced to differentiate into macrophages using phorbol myristate acetate. The macrophages were then treated with 100, 200, and 400 μg/ml of PM2.5 for 12, 24, and 48 h, respectively. Then, we determined the survival rate of macrophages through the MTT assay. The TNF-α and CRP levels in the cell culture medium were measured through enzyme-linked immunosorbent assay. The NF-κB1, NF-κB2, RelA, RelB, and Rel mRNA levels in macrophages were measured with reverse transcriptase-polymerase chain reaction. As a consequence, the survival rate of macrophages decreased with increasing PM2.5 exposure time and dose. The TNF-α levels in PM2.5-treated groups were lower as compared with the control group and in contrast to the NF-κB mRNA levels at all exposure times. The TNF-α level in the 400-μg/ml group and the NF-κB1, NF-κB2, RelB, and Rel mRNA levels in all PM2.5-treated groups were found to be higher at 24 h than at 12 h. Furthermore, the TNF-α, CRP, and NF-κB2 mRNA levels in the group treated with 400 μg/ml PM2.5 were higher at 48 h that at 12 and 24 h. On the other hand, the NF-κB1, RelA, RelB, and Rel mRNA levels in all PM2.5-treated groups were lower as compared to levels of TNF-α, CRP, and NF-κB2 mRNA. The levels of NF-κB genes and inflammatory cytokines demonstrated different correlations at different exposure times. Therefore, we conclude that PM2.5 reduces the survival rate of macrophages. As macrophages are exposed to PM2.5, the NF-κB gene family expression is increased, which subsequently affects inflammatory factor levels.

  1. Spaceflight Human System Standards

    NASA Technical Reports Server (NTRS)

    Holubec, Keith; Tillman, Barry; Connolly, Jan

    2009-01-01

    NASA created a new approach for human system integration and human performance standards. NASA created two documents a standard and a reference handbook. The standard is titled NASA Space Flight Human-System Standard (SFHSS) and consists of two-volumes: Volume 1- Crew Health This volume covers standards needed to support astronaut health (medical care, nutrition, sleep, exercise, etc.) Volume 2 Human Factors, Habitability and Environmental Health This volume covers the standards for system design that will maintain astronaut performance (ie., environmental factors, design of facilities, layout of workstations, and lighting requirements). It includes classic human factors requirements. The new standards document is written in terms so that it is applicable to a broad range of present and future NASA systems. The document states that all new programs prepare system-specific requirements that will meet the general standards. For example, the new standard does not specify a design should accommodate specific percentiles of a defined population. Rather, NASA-STD-3001, Volume 2 states that all programs shall prepare program-specific requirements that define the user population and their size ranges. The design shall then accommodate the full size range of those users. The companion reference handbook, Human Integration Design Handbook (HIDH), was developed to capture the design consideration information from NASA-STD-3000, and adds spaceflight lessons learned, gaps in knowledge, example solutions, and suggests research to further mature specific disciplines. The HIDH serves two major purposes: HIDH is the reference document for writing human factors requirements for specific systems. HIDH contains design guidance information that helps insure that designers create systems which safely and effectively accommodate the capabilities and limitations of space flight crews.

  2. Human Milk Fortification.

    PubMed

    Simmer, Karen

    2015-01-01

    Human milk is the feed of choice for preterm infants. However, human milk does not provide enough nutrition, especially protein, for preterm infants to achieve target growth rates similar to those in utero (15-20 g/kg per day). Fortifiers for human milk, manufactured from bovine milk, are commercially available and routinely used for patients born <32 weeks' gestation prior to discharge home. Recent recommended dietary intakes (RDI) have been revised. Up to 4.2 g of protein and 135 kcal/kg per day is recommended for infants born very preterm. Additional supplements are needed to current commercial fortifiers to achieve these RDI and reduce the incidence of ex-uterine growth failure. A human milk fortifier that is manufactured from donor human milk is available in some developed countries and may confer some clinical benefits, including a reduction in necrotizing enterocolitis. Fortification can be added in a standardized protocol as per manufacturers' instructions. Human milk composition can be analyzed and fortification individualized to take into account the large variation from mother to mother. Alternatively, fortification can be increased in a stepwise manner based on assumed composition while monitoring blood urea levels for safety. The current aim is to prevent preterm infants dropping percentiles and falling below the 10th percentile at 36 weeks' corrected gestational age or discharge home. More data are required on how best to fortify human milk for preterm infants to achieve optimal growth, development and health outcomes in the long term. There is an urgent need for well-designed and informed randomized clinical trials in this vulnerable preterm population. © 2015 Nestec Ltd., Vevey/S. Karger AG, Basel.

  3. Meeting human needs

    NASA Technical Reports Server (NTRS)

    Nicogossian, Arnauld E.

    1992-01-01

    Manned space flight can be viewed as an interaction of three general elements: the human crewmember, spacecraft systems, and the environment. While the human crewmember is a crucial element in the system, certain physiological, psychological, environ- mental and spacecraft systems factors can compromise human performance in space. These factors include atmospheric pressure, physiology, uncertainties associated with space radiation, the potential for exposure to toxic materials in the closed environment, and spacecraft habitability. Health protection in space, for current and future missions, relies on a philosophy of risk reduction, which in the space program is achieved in four ways-through health maintenance, health care, design criteria, an selection and training. Emphasis is place upon prevention, through selection criteria and careful screening. Spacecraft health care systems must be absolutely reliable, and they will be automated and computerized to the maximum extent possible, but still designed with the human crewmember's capabilities in mind. The autonomy and technological sophistication of future missions will require a greater emphasis on high-level interaction between the human operator and automated systems, with effective allocation of tasks between humans and machines. Performance in space will include complex tasks during extravehicular activity (EVA) and on planetary surfaces, and knowledge of crewmembers' capability and limitations during such operations will be critical to mission success. Psychological support will become increasingly important on space missions, as crews spend long periods in remote and potentially hazardous environments. The success of future missions will depend on both individual psychological health and group cohesion and productivity, particularly as crew profiles become more heterogeneous. Thus, further human factors are needed in the area of small-group dynamics and performance.

  4. Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets.

    PubMed

    Pelech, S L; Samiei, M; Charest, D L; Howard, S L; Salari, H

    1991-05-15

    The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.

  5. Effect of Chimaerins, Novel Receptors for Phorbol Esters, on Breast Cancer Cell Proliferation and Cell Cycle Progression

    DTIC Science & Technology

    2006-07-01

    recently solved structure of 2- chimerin, we generated an AdV encoding for a 2-chimerin mutant locked in the constitutively active conformation ...and cell proliferation entirely depended on its Rac-GAP activity; (4) heregulin beta1 (HRG), an EGF- like growth factor and a mitogen for breast cancer...Caloca et al., 2003; Canagarajah et al., 2004). Accumulating evidence indicates that Rac plays a critical role in the control of actin cytoskeleton

  6. Reversal of a developmental restriction in neural crest-derived cells of avian embryos by a phorbol ester drug.

    PubMed

    Ciment, G; Glimelius, B; Nelson, D M; Weston, J A

    1986-12-01

    Neural crest cells and some of the crest-derived cells of dorsal root ganglia (DRG) of early avian embryos give rise to pigment cells when placed in culture. DRG from older embryos, however, fail to do so under comparable culture conditions. This age-dependent loss of melanogenic ability might be explained either by the death of a subpopulation of latent melanoblasts within early DRG, or the imposition of additional developmental restrictions in multipotent DRG cells. We show here that 12-O-tetradecanoylphorbol-13-acetate (TPA) causes some DRG cells to undergo pigmentation in cultures from older embryos, indicating that the loss of melanogenic ability in older embryos is not due to cell death. These pigment cells also display morphogenetic properties of normal melanocytes, including the ability to invade feather primordia. In addition to DRG, various other neural crest-derivatives contain cells similarly affected by TPA, including cells within sympathetic ganglia and peripheral nerves. We suggest that TPA reverses the developmental restriction of melanogenic ability that is normally imposed on neural crest-derived cells that migrate to various sites in avian embryos where melanogenesis does not normally occur.

  7. Why Geo-Humanities

    NASA Astrophysics Data System (ADS)

    Graells, Robert Casals i.; Sibilla, Anna; Bohle, Martin

    2016-04-01

    Anthropogenic global change is a composite process. It consists of societal processes (in the 'noosphere') and natural processes (in the 'bio-geosphere'). The 'noosphere' is the ensemble of social, cultural or political insights ('shared subjective mental concepts') of people. Understanding the composite of societal and natural processes ('human geo-biosphere intersections'), which shapes the features of anthropogenic global change, would benefit from a description that draws equally on natural sciences, social sciences and humanities. To that end it is suggested to develop a concept of 'geo-humanities': This essay presents some aspects of its scope, discussing "knowledge that is to manage", "intentions that are to shape", "choices that are to justify" and "complexity that is to handle". Managing knowledge: That people understand anthropogenic global change requires their insights into how 'human geosphere intersections' function. Insights are formed ('processed') in the noosphere by means of interactions between people. Understanding how 'human geosphere intersections' functions combines scientific, engineering and economic studies with studies of the dynamics of the noosphere. Shaping intentions: During the last century anthropogenic global change developed as the collateral outcome of humankind's accumulated actions. It is caused by the number of people, the patterns of their consumption of resources, and the alterations of their environments. Nowadays, anthropogenic global chance is either an intentional negligence or a conscious act. Justifying choices: Humanity has alternatives how to alter Earth at planetary scale consciously. For example, there is a choice to alter the geo-biosphere or to adjust the noosphere. Whatever the choice, it will depend on people's world-views, cultures and preferences. Thus beyond issues whether science and technology are 'sound' overarching societal issues are to tackle, such as: (i) how to appropriate and distribute natural

  8. The Human Serum Metabolome

    PubMed Central

    Psychogios, Nikolaos; Hau, David D.; Peng, Jun; Guo, An Chi; Mandal, Rupasri; Bouatra, Souhaila; Sinelnikov, Igor; Krishnamurthy, Ramanarayan; Eisner, Roman; Gautam, Bijaya; Young, Nelson; Xia, Jianguo; Knox, Craig; Dong, Edison; Huang, Paul; Hollander, Zsuzsanna; Pedersen, Theresa L.; Smith, Steven R.; Bamforth, Fiona; Greiner, Russ; McManus, Bruce; Newman, John W.; Goodfriend, Theodore; Wishart, David S.

    2011-01-01

    Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca. PMID:21359215

  9. Human hybrid hybridoma

    SciTech Connect

    Tiebout, R.F.; van Boxtel-Oosterhof, F.; Stricker, E.A.M.

    1987-11-15

    Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. The authors have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. They fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1kappa antibody directed against tetanus toxiod and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture mediummore » containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassay. The results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies. Bispecific antibodies activity was measured by means of two radioimmunoassays.« less

  10. Healthy human gut phageome

    PubMed Central

    Manrique, Pilar; Bolduc, Benjamin; Walk, Seth T.; van der Oost, John; de Vos, Willem M.; Young, Mark J.

    2016-01-01

    The role of bacteriophages in influencing the structure and function of the healthy human gut microbiome is unknown. With few exceptions, previous studies have found a high level of heterogeneity in bacteriophages from healthy individuals. To better estimate and identify the shared phageome of humans, we analyzed a deep DNA sequence dataset of active bacteriophages and available metagenomic datasets of the gut bacteriophage community from healthy individuals. We found 23 shared bacteriophages in more than one-half of 64 healthy individuals from around the world. These shared bacteriophages were found in a significantly smaller percentage of individuals with gastrointestinal/irritable bowel disease. A network analysis identified 44 bacteriophage groups of which 9 (20%) were shared in more than one-half of all 64 individuals. These results provide strong evidence of a healthy gut phageome (HGP) in humans. The bacteriophage community in the human gut is a mixture of three classes: a set of core bacteriophages shared among more than one-half of all people, a common set of bacteriophages found in 20–50% of individuals, and a set of bacteriophages that are either rarely shared or unique to a person. We propose that the core and common bacteriophage communities are globally distributed and comprise the HGP, which plays an important role in maintaining gut microbiome structure/function and thereby contributes significantly to human health. PMID:27573828

  11. Human herpesvirus 6.

    PubMed Central

    Braun, D K; Dominguez, G; Pellett, P E

    1997-01-01

    Human herpesvirus 6 variant A (HHV-6A) and human herpesvirus 6 variant B (HHV-6B) are two closely related yet distinct viruses. These visuses belong to the Roseolovirus genus of the betaherpesvirus subfamily; they are most closely related to human herpesvirus 7 and then to human cytomegalovirus. Over 95% of people older than 2 years of age are seropositive for either or both HHV-6 variants, and current serologic methods are incapable of discriminating infection with one variant from infection with the other. HHV-6A has not been etiologically linked to any human disease, but such an association will probably be found soon. HHV-6B is the etiologic agent of the common childhood illness exanthem subitum (roseola infantum or sixth disease) and related febrile illnesses. These viruses are frequently active and associated with illness in immunocompromised patients and may play a role in the etiology of Hodgkin's disease and other malignancies. HHV-6 is a commensal inhabitant of brains; various neurologic manifestations, including convulsions and encephalitis, can occur during primary HHV-6 infection or in immunocompromised patients. HHV-6 and distribution in the central nervous system are altered in patients with multiple sclerosis; the significance of this is under investigation. PMID:9227865

  12. Human Skin Fungal Diversity

    PubMed Central

    Findley, Keisha; Oh, Julia; Yang, Joy; Conlan, Sean; Deming, Clayton; Meyer, Jennifer A.; Schoenfeld, Deborah; Nomicos, Effie; Park, Morgan; Kong, Heidi H.; Segre, Julia A.

    2013-01-01

    Traditional culture-based methods have incompletely defined the etiology of common recalcitrant human fungal skin diseases including athlete’s foot and toenail infections. Skin protects humans from invasion by pathogenic microorganisms, while providing a home for diverse commensal microbiota1. Bacterial genomic sequence data have generated novel hypotheses about species and community structures underlying human disorders2,3,4. However, microbial diversity is not limited to bacteria; microorganisms such as fungi also play major roles in microbial community stability, human health and disease5. Genomic methodologies to identify fungal species and communities have been limited compared with tools available for bacteria6. Fungal evolution can be reconstructed with phylogenetic markers, including ribosomal RNA gene regions and other highly conserved genes7. Here, we sequenced and analyzed fungal communities of 14 skin sites in 10 healthy adults. Eleven core body and arm sites were dominated by Malassezia fungi, with species-level classifications revealing greater topographical resolution between sites. By contrast, three foot sites, plantar heel, toenail, and toeweb, exhibited tremendous fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that skin physiologic attributes and topography differentially shape these two microbial communities. These results provide a framework for future investigation of interactions between pathogenic and commensal fungal and bacterial communities in maintaining human health and contributing to disease pathogenesis. PMID:23698366

  13. Human occupancy detection

    NASA Astrophysics Data System (ADS)

    Brown, David A.

    1994-10-01

    In the area of security and surveillance technologies, the problem of the arrival in Canada of illegal and undesirable ship and truck cargo loads is steadily increasing. As the volumes of cargo arrivals increase so do the Immigration and Customs problems related to the determination of the validity of those cargo contents. Of special concern to Immigration Control Authorities around the world is the emerging and increasing trend of illegal smuggling of human beings hidden inside of shipping containers. Beginning in 1992, Immigration Control Authorities in Canada observed an escalation of alien people smuggling through the use of cargo shipping containers arriving in the Port of Montreal. This paper will present to the audience the recently completed Immigration Canada Human Occupancy Detection project by explaining the design, development and testing of human occupancy detectors. The devices are designed to electronically detect the presence of persons hiding inside of shipping containers, without the requirement of opening the container doors. The human occupancy detection concepts are based upon the presence of carbon dioxide or other human waste characteristics commonly found inside of shipping containers.

  14. Infants' Responses to Real Humans and Representations of Humans

    ERIC Educational Resources Information Center

    Heron, Michelle; Slaughter, Virginia

    2010-01-01

    Infants' responses to typical and scrambled human body shapes were assessed in relation to the realism of the human body stimuli presented. In four separate experiments, infants were familiarized to typical human bodies and then shown a series of scrambled human bodies on the test. Looking behaviour was assessed in response to a range of different…

  15. Cardiovascular Deconditioning in Humans: Human Studies Core

    NASA Technical Reports Server (NTRS)

    Williams, Gordon

    1999-01-01

    Major cardiovascular problems, secondary to cardiovascular deconditioning, may occur on extended space missions. While it is generally assumed that the microgravity state is the primary cause of cardiovascular deconditioning, sleep deprivation and disruption of diurnal rhythms may also play an important role. Factors that could be modified by either or both of these perturbations include: autonomic function and short-term cardiovascular reflexes, vasoreactivity, circadian rhythm of cardiovascular hormones (specifically the renin-angiotensin system) and renal sodium handling and hormonal influences on that process, venous compliance, cardiac mass, and cardiac conduction processes. The purpose of the Human Studies Core is to provide the infrastructure to conduct human experiments which will allow for the assessment of the likely role of such factors in the space travel associated cardiovascular deconditioning process and to develop appropriate countermeasures. The Core takes advantage of a newly-created Intensive Physiologic Monitoring (IPM) Unit at the Brigham and Women's Hospital, Boston, MA, to perform these studies. The Core includes two general experimental protocols. The first protocol involves a head down tilt bed-rest study to simulate microgravity. The second protocol includes the addition of a disruption of circadian rhythms to the simulated microgravity environment. Before and after each of these environmental manipulations, the subjects will undergo acute stressors simulating changes in volume and/or stress, which could occur in space and on return to Earth. The subjects are maintained in a rigidly controlled environment with fixed light/dark cycles, activity pattern, and dietary intake of nutrients, fluids, ions and calories.

  16. Is humanity suicidal?

    PubMed

    Wilson, E O

    1993-01-01

    The world's fauna and flora has entered a crisis unparalleled since the end of the Mesozoic Era, with the extinction rate of species now elevated to more than a thousand times that existing before the coming of humanity. Scientists and policy makers are ill-prepared to moderate this hemorrhaging, because so little is known of the biology of the Earth's millions of species and because so little effort has been directed toward conservation thus far. With the vanished species will go great potential wealth in scientific knowledge, new products, ecosystems services, and part of the natural world in which the human species originated. The need for new research and improved management is thus urgent. If it is not met, humanity will likely survive, but in a world biologically impoverished for all time.

  17. The human toxome project.

    PubMed

    Bouhifd, Mounir; Andersen, Melvin E; Baghdikian, Christina; Boekelheide, Kim; Crofton, Kevin M; Fornace, Albert J; Kleensang, Andre; Li, Henghong; Livi, Carolina; Maertens, Alexandra; McMullen, Patrick D; Rosenberg, Michael; Thomas, Russell; Vantangoli, Marguerite; Yager, James D; Zhao, Liang; Hartung, Thomas

    2015-01-01

    The Human Toxome Project, funded as an NIH Transformative Research grant 2011-2016, is focused on developing the concepts and the means for deducing, validating and sharing molecular pathways of toxicity (PoT). Using the test case of estrogenic endocrine disruption, the responses of MCF-7 human breast cancer cells are being phenotyped by transcriptomics and mass-spectroscopy-based metabolomics. The bioinformatics tools for PoT deduction represent a core deliverable. A number of challenges for quality and standardization of cell systems, omics technologies and bioinformatics are being addressed. In parallel, concepts for annotation, validation and sharing of PoT information, as well as their link to adverse outcomes, are being developed. A reasonably comprehensive public database of PoT, the Human Toxome Knowledge-base, could become a point of reference for toxicological research and regulatory test strategies.

  18. The Human Toxome Project

    PubMed Central

    Bouhifd, Mounir; Andersen, Melvin E.; Baghdikian, Christina; Boekelheide, Kim; Crofton, Kevin M.; Fornace, Albert J.; Kleensang, Andre; Li, Henghong; Livi, Carolina; Maertens, Alexandra; McMullen, Patrick D.; Rosenberg, Michael; Thomas, Russell; Vantangoli, Marguerite; Yager, James D.; Zhao, Liang; Hartung, Thomas

    2016-01-01

    Summary The Human Toxome Project, funded as an NIH Transformative Research grant 2011–2016, is focused on developing the concepts and the means for deducing, validating and sharing molecular pathways of toxicity (PoT). Using the test case of estrogenic endocrine disruption, the responses of MCF-7 human breast cancer cells are being phenotyped by transcriptomics and mass-spectrometry-based metabolomics. The bioinformatics tools for PoT deduction represent a core deliverable. A number of challenges for quality and standardization of cell systems, omics technologies and bioinformatics are being addressed. In parallel, concepts for annotation, validation and sharing of PoT information, as well as their link to adverse outcomes, are being developed. A reasonably comprehensive public database of PoT, the Human Toxome Knowledge-base, could become a point of reference for toxicological research and regulatory test strategies. PMID:25742299

  19. Teleoperator Human Factors Study

    NASA Technical Reports Server (NTRS)

    1986-01-01

    An investigation of the spectrum of space teleoperation activities likely in the 1985 to 1995 decade focused on the resolution of critical human engineering issues and characterization of the technology effect on performance of remote human operators. The study began with the identification and documentation of a set of representative reference teleoperator tasks. For each task, technology, development, and design options, issues, and alternatives that bear on human operator performance were defined and categorized. A literature survey identified existing studies of man/machine issues. For each teleoperations category, an assessment was made of the state of knowledge on a scale from adequate to void. The tests, experiments, and analyses necessary to provide the missing elements of knowledge were then defined. A limited set of tests were actually performed, including operator selection, baseline task definition, control mode study, lighting study, camera study, and preliminary time delay study.

  20. Human-Robot Interaction

    NASA Technical Reports Server (NTRS)

    Rochlis-Zumbado, Jennifer; Sandor, Aniko; Ezer, Neta

    2012-01-01

    Risk of Inadequate Design of Human and Automation/Robotic Integration (HARI) is a new Human Research Program (HRP) risk. HRI is a research area that seeks to understand the complex relationship among variables that affect the way humans and robots work together to accomplish goals. The DRP addresses three major HRI study areas that will provide appropriate information for navigation guidance to a teleoperator of a robot system, and contribute to the closure of currently identified HRP gaps: (1) Overlays -- Use of overlays for teleoperation to augment the information available on the video feed (2) Camera views -- Type and arrangement of camera views for better task performance and awareness of surroundings (3) Command modalities -- Development of gesture and voice command vocabularies

  1. Preparing for Human Exploration

    NASA Technical Reports Server (NTRS)

    Drake, Bret G.; Joosten, B. Kent

    1998-01-01

    NASA's Human Exploration and Development of Space (HEDS) Enterprise is defining architectures and requirements for human exploration that radically reduce the costs of such missions through the use of advanced technologies, commercial partnerships and innovative systems strategies. In addition, the HEDS Enterprise is collaborating with the Space Science Enterprise to acquire needed early knowledge about Mars and to demonstrate critical technologies via robotic missions. This paper provides an overview of the technological challenges facing NASA as it prepares for human exploration. Emphasis is placed on identifying the key technologies including those which will provide the most return in terms of reducing total mission cost and/or reducing potential risk to the mission crew. Top-level requirements are provided for those critical enabling technology options currently under consideration.

  2. Scientists and Human Rights

    NASA Astrophysics Data System (ADS)

    Makdisi, Yousef

    2012-02-01

    The American Physical Society has a long history of involvement in defense of human rights. The Committee on International Freedom of Scientists was formed in the mid seventies as a subcommittee within the Panel On Public Affairs ``to deal with matters of an international nature that endangers the abilities of scientists to function as scientists'' and by 1980 it was established as an independent committee. In this presentation I will describe some aspects of the early history and the impetus that led to such an advocacy, the methods employed then and how they evolved to the present CIFS responsibility ``for monitoring concerns regarding human rights for scientists throughout the world''. I will also describe the current approach and some sample cases the committee has pursued recently, the interaction with other human rights organizations, and touch upon some venues through which the community can engage to help in this noble cause.

  3. Abortion and human rights.

    PubMed

    Shaw, Dorothy

    2010-10-01

    Abortion has been a reality in women's lives since the beginning of recorded history, typically with a high risk of fatal consequences, until the last century when evolutions in the field of medicine, including techniques of safe abortion and effective methods of family planning, could have ended the need to seek unsafe abortion. The context of women's lives globally is an important but often ignored variable, increasingly recognised in evolving human rights especially related to gender and reproduction. International and regional human rights instruments are being invoked where national laws result in violations of human rights such as health and life. The individual right to conscientious objection must be respected and better understood, and is not absolute. Health professional organisations have a role to play in clarifying responsibilities consistent with national laws and respecting reproductive rights. Seeking common ground using evidence rather than polarised opinion can assist the future focus. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. Human immune system variation

    PubMed Central

    Brodin, Petter; Davis, Mark M.

    2017-01-01

    The human immune system is highly variable between individuals but relatively stable over time within a given person. Recent conceptual and technological advances have enabled systems immunology analyses, which reveal the composition of immune cells and proteins in populations of healthy individuals. The range of variation and some specific influences that shape an individual’s immune system is now becoming clearer. Human immune systems vary as a consequence of heritable and non-heritable influences, but symbiotic and pathogenic microbes and other non-heritable influences explain most of this variation. Understanding when and how such influences shape the human immune system is key for defining metrics of immunological health and understanding the risk of immune-mediated and infectious diseases. PMID:27916977

  5. The Human Centrifuge

    NASA Astrophysics Data System (ADS)

    van Loon, Jack J. W. A.

    2009-01-01

    Life on Earth has developed at unit gravity, 9.81 m/s2, which was a major factor especially when vertebrates emerged from water onto land in the late Devonian, some 375 million years ago. But how would nature have evolved on a larger planet? We are able to address this question simply in experiments using centrifuges. Based on these studies we have gained valuable insights in the physiological process in plants and animals. They adapt to a new steady state suitable for the high-g environments applied. Information on mammalian adaptations to hyper-g is interesting or may be even vital for human space exploration programs. It has been shown in long duration animal hypergravity studies, ranging from snails, rats to primates, that various structures like muscles, bones, neuro-vestibular, or the cardio-vascular system are affected. However, humans have never been exposed to a hyper-g environment for long durations. Centrifuge studies involving humans are mostly in the order of hours. The current work on human centrifuges are all focused on short arm systems to apply short periods of artificial gravity in support of long duration space missions in ISS or to Mars. In this paper we will address the possible usefulness of a large human centrifuge on Earth. In such a centrifuge a group of humans can be exposed to hypergravity for, in principle, an unlimited period of time like living on a larger planet. The input from a survey under scientists working in the field of gravitational physiology, but also other disciplines, will be discussed.

  6. Human push capability.

    PubMed

    Barnett, Ralph L; Liber, Theodore

    2006-02-22

    Use of unassisted human push capability arises from time to time in the areas of crowd and animal control, the security of locked doors, the integrity of railings, the removal of tree stumps and entrenched vehicles, the manoeuvering of furniture, and athletic pursuits such as US football or wrestling. Depending on the scenario, human push capability involves strength, weight, weight distribution, push angle, footwear/floor friction, and the friction between the upper body and the pushed object. Simple models are used to establish the relationships among these factors.

  7. Biodemography of human ageing

    PubMed Central

    Vaupel, James W.

    2014-01-01

    Human senescence has been delayed by a decade. This finding, documented in 1994 and bolstered since, is a fundamental discovery about the biology of human ageing, and one with profound implications for individuals, society and the economy. Remarkably, the rate of deterioration with age seems to be constant across individuals and over time: it seems that death is being delayed because people are reaching old age in better health. Research by demographers, epidemiologists and other biomedical researchers suggests that further progress is likely to be made in advancing the frontier of survival — and healthy survival — to even greater ages. PMID:20336136

  8. Ayahuasca and human destiny.

    PubMed

    McKenna, Dennis J

    2005-06-01

    In this essay, the author shares his personal reflections gleaned from a lifetime of research with ayahuasca, and speculates on the societal, political, planetary, and evolutionary implications of humanity's aeons-old symbiosis with this shamanic plant. The thesis is developed that at this critical historical juncture, ayahuasca has developed a strategy to broadcast its message to a wider world--a reflection of the urgent need to avert global ecological catastrophe. While ayahuasca has much to teach us, the critical question is, will humanity hear it, and heed it, in time?

  9. Human Factors Model

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Jack is an advanced human factors software package that provides a three dimensional model for predicting how a human will interact with a given system or environment. It can be used for a broad range of computer-aided design applications. Jack was developed by the computer Graphics Research Laboratory of the University of Pennsylvania with assistance from NASA's Johnson Space Center, Ames Research Center and the Army. It is the University's first commercial product. Jack is still used for academic purposes at the University of Pennsylvania. Commercial rights were given to Transom Technologies, Inc.

  10. We Are Human Beings

    PubMed Central

    McGee, Andrew

    2016-01-01

    In this paper, I examine Jeff McMahan’s arguments for his claim that we are not human organisms, and the arguments of Derek Parfit to the same effect in a recent paper. McMahan uses these arguments to derive conclusions concerning the moral status of embryos and permanent vegetative state (PVS) patients. My claim will be that neither thinker has successfully shown that we are not human beings, and therefore these arguments do not establish the ethical conclusions that McMahan has sought to draw from the arguments in respect of the moral status of embryos and PVS patients. PMID:26810918

  11. Human Care Trust (NGO).

    PubMed

    1993-01-01

    Human Care Trust, a nongovernmental organization, has established a team to detect HIV cases, create awareness of HIV/AIDS and serve mankind against HIV/AIDS disease. This program is devoted to serve humanity without any discrimination on the basis of religion, race, and creed. As this is a charitable organization, it totally depends on the donations and assistance from local and overseas sources. For more information, please contact: Qamar D.K. Mall, President, Post Box No 5116, Model Town, Lahore, Pakistan. full text

  12. Human MSH2 protein

    DOEpatents

    de la Chapelle, Albert; Vogelstein, Bert; Kinzler, Kenneth W.

    1997-01-01

    The human MSH2 gene, responsible for hereditary non-polyposis colorectal cancer, was identified by virtue of its homology to the MutS class of genes, which are involved in DNA mismatch repair. The sequence of cDNA clones of the human gene are provided, and the sequence of the gene can be used to demonstrate the existence of germ line mutations in hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in replication error.sup.+ (RER.sup.+) tumor cells.

  13. Human MSH2 protein

    DOEpatents

    Chapelle, A. de la; Vogelstein, B.; Kinzler, K.W.

    1997-01-07

    The human MSH2 gene, responsible for hereditary non-polyposis colorectal cancer, was identified by virtue of its homology to the MutS class of genes, which are involved in DNA mismatch repair. The sequence of cDNA clones of the human gene are provided, and the sequence of the gene can be used to demonstrate the existence of germ line mutations in hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in replication error{sup +} (RER{sup +}) tumor cells. 19 figs.

  14. Epigenetics and Human Disease

    PubMed Central

    Zoghbi, Huda Y.; Beaudet, Arthur L.

    2016-01-01

    SUMMARY Genetic causes for human disorders are being discovered at an unprecedented pace. A growing subclass of disease-causing mutations involves changes in the epigenome or in the abundance and activity of proteins that regulate chromatin structure. This article focuses on research that has uncovered human diseases that stem from such epigenetic deregulation. Disease may be caused by direct changes in epigenetic marks, such as DNA methylation, commonly found to affect imprinted gene regulation. Also described are disease-causing genetic mutations in epigenetic modifiers that either affect chromatin in trans or have a cis effect in altering chromatin configuration. PMID:26834142

  15. Disorders of Human Hemoglobin

    NASA Astrophysics Data System (ADS)

    Bank, Arthur; Mears, J. Gregory; Ramirez, Francesco

    1980-02-01

    Studies of the human hemoglobin system have provided new insights into the regulation of expression of a group of linked human genes, the γ -δ -β globin gene complex in man. In particular, the thalassemia syndromes and related disorders of man are inherited anemias that provide mutations for the study of the regulation of globin gene expression. New methods, including restriction enzyme analysis and cloning of cellular DNA, have made it feasible to define more precisely the structure and organization of the globin genes in cellular DNA. Deletions of specific globin gene fragments have already been found in certain of these disorders and have been applied in prenatal diagnosis.

  16. [Controversies about human cloning].

    PubMed

    Ziga, Jusuf

    2006-01-01

    Human cloning is one of the greatest scientific and research challenges that human kind faced so far. As much as we have reasons for excitement in front of achievements of the genetic engineering, also are great the fears about it. There is inevitable question: is the man aware where can cloning as an attack on a "natural laws for the life reproduction" can lead, or to say its impact on evolution diversities within kind that we belongs to. In this paper we have focus on finding the answer on the last question, with application of a multidisciplinary approach, which means not only scientific, but also ethical, as well as theology.

  17. Human Modeling For Ground Processing Human Factors Engineering Analysis

    NASA Technical Reports Server (NTRS)

    Tran, Donald; Stambolian, Damon; Henderson, Gena; Barth, Tim

    2011-01-01

    There have been many advancements and accomplishments over that last few years using human modeling for human factors engineering analysis for design of spacecraft and launch vehicles. The key methods used for this are motion capture and computer generated human models. The focus of this paper is to explain the different types of human modeling used currently and in the past at Kennedy Space Center (KSC) currently, and to explain the future plans for human modeling for future spacecraft designs.

  18. Human Modeling for Ground Processing Human Factors Engineering Analysis

    NASA Technical Reports Server (NTRS)

    Stambolian, Damon B.; Lawrence, Brad A.; Stelges, Katrine S.; Steady, Marie-Jeanne O.; Ridgwell, Lora C.; Mills, Robert E.; Henderson, Gena; Tran, Donald; Barth, Tim

    2011-01-01

    There have been many advancements and accomplishments over the last few years using human modeling for human factors engineering analysis for design of spacecraft. The key methods used for this are motion capture and computer generated human models. The focus of this paper is to explain the human modeling currently used at Kennedy Space Center (KSC), and to explain the future plans for human modeling for future spacecraft designs

  19. The Exploration of Mars by Humans: Why Mars? Why Humans?

    NASA Technical Reports Server (NTRS)

    Levine, Joel S.

    2011-01-01

    As we commemorate the 50th anniversary of Yuri Gagarin's historic flight in 1961, the first flight of a human in space, plans are underway for another historic human mission. Plans are being developed for a human mission to Mars. Once we reach Mars, the human species will become the first two-planet species. Both the Bush Administration (in 2004) and the Obama Administration (in 2010) proposed a human mission to Mars as a national goal of the United States.

  20. The Case for the Humanities.

    ERIC Educational Resources Information Center

    Cary, Michael S.

    1981-01-01

    Describes the current impoverishment of the humanities and the gulf separating the humanities from the sciences. Discusses the need for adequate humanities instruction at the elementary-secondary level. Suggests that humanities teachers rediscover the Italian Renaissance spirit to improve their teaching. (SB)

  1. Human Rights: The Essential Reference.

    ERIC Educational Resources Information Center

    Devine, Carol; Hansen, Carol Rae; Wilde, Ralph; Bronkhorst, Daan; Moritz, Frederic A.; Rolle, Baptiste; Sherman, Rebecca; Southard, Jo Lynn; Wilkinson, Robert; Poole, Hilary, Ed.

    This reference work documents the history of human rights theory, explains each article of the Universal Declaration of Human Rights, explores the contemporary human rights movement, and examines the major human rights issues facing the world today. This book is the first to combine historical and contemporary perspectives on these critical…

  2. Human Challenges in Exploration Missions

    NASA Technical Reports Server (NTRS)

    Lloyd, Charles W.

    2007-01-01

    This viewgraph presents an overview using pictures some of the history of human exploration of the new frontiers of Earth and then examines some of the challenges to human exploration of space. Particular attention is given to the environmental factors and to the social and human factors that effect humans in space environments.

  3. Making IBM's Computer, Watson, Human

    ERIC Educational Resources Information Center

    Rachlin, Howard

    2012-01-01

    This essay uses the recent victory of an IBM computer (Watson) in the TV game, "Jeopardy," to speculate on the abilities Watson would need, in addition to those it has, to be human. The essay's basic premise is that to be human is to behave as humans behave and to function in society as humans function. Alternatives to this premise are considered…

  4. Humanism: Tough or Tender?

    ERIC Educational Resources Information Center

    Cordell, Franklin D.; Luna, F. C.

    1978-01-01

    Describes the minimum conditions for creating the humanistic school. Humanism is based on principles that fall into three basic categories: a set of beliefs about self and the nature of social reality, a set of skills to be learned by teachers, administrators, and pupils, and a set of necessary conditions in the social system. (Author/RK)

  5. Neurobiology and the Humanities

    PubMed Central

    Zeki, Semir

    2014-01-01

    Can the arts and humanities contribute significantly to the study of the brain? Similar brain processes are involved in humanistic and scientific inference, and in this essay, I argue that conclusions reached by one are relevant to the other. PMID:25277451

  6. Humane Treatment of Animals.

    ERIC Educational Resources Information Center

    Dawson, Joan Smithey

    This booklet is designed to give teachers resource information about the humane treatment of and care for animals. The topics are presented as springboards for discussion and class activity. Topics include the care of dogs, cats, birds, horses, and fish; wildlife and ecological relationships; and careers with animals. Illustrations on some pages…

  7. Sarcocystis in Humans

    USDA-ARS?s Scientific Manuscript database

    Humans serve as definitive hosts for two known species of Sarcocystis, acquired from eating undercooked pork or beef, and resulting in gastrointestinal infection. Sporocysts excreted from these infected persons are each infectious for pigs and cattle, the intermediate hosts, resulting in the develop...

  8. Marketing Human Resource Development.

    ERIC Educational Resources Information Center

    Frank, Eric, Ed.

    1994-01-01

    Describes three human resource development activities: training, education, and development. Explains marketing from the practitioners's viewpoint in terms of customer orientation; external and internal marketing; and market analysis, research, strategy, and mix. Shows how to design, develop, and implement strategic marketing plans and identify…

  9. Predictors of human rotation.

    PubMed

    Stochl, Jan; Croudace, Tim

    2013-01-01

    Why some humans prefer to rotate clockwise rather than anticlockwise is not well understood. This study aims to identify the predictors of the preferred rotation direction in humans. The variables hypothesised to influence rotation preference include handedness, footedness, sex, brain hemisphere lateralisation, and the Coriolis effect (which results from geospatial location on the Earth). An online questionnaire allowed us to analyse data from 1526 respondents in 97 countries. Factor analysis showed that the direction of rotation should be studied separately for local and global movements. Handedness, footedness, and the item hypothesised to measure brain hemisphere lateralisation are predictors of rotation direction for both global and local movements. Sex is a predictor of the direction of global rotation movements but not local ones, and both sexes tend to rotate clockwise. Geospatial location does not predict the preferred direction of rotation. Our study confirms previous findings concerning the influence of handedness, footedness, and sex on human rotation; our study also provides new insight into the underlying structure of human rotation movements and excludes the Coriolis effect as a predictor of rotation.

  10. Humanism in Education

    ERIC Educational Resources Information Center

    Armstrong, Michael

    2015-01-01

    This is the text of Michael Armstrong's address to the Brian Simon Centenary conference, held at the Institute of Education on 26 March 2015. Michael Armstrong celebrates the humanism that underlay Brian's belief in a common system of education, democratic and non-selective, and finds its counterpart in the creative practice of school children.

  11. Fighting for Human Rights

    ERIC Educational Resources Information Center

    Ong, Bao

    2011-01-01

    Speak Truth To Power consists of 17 teacher-developed lessons based on the stories of rights advocates from all over the world. The lessons were created for sixth-through 12th-grade students, and have come to New York schools thanks to the Robert F. Kennedy Center for Justice and Human Rights and the New York State United Teachers union. Speak…

  12. Synthesizing Humanism and Behaviorism.

    ERIC Educational Resources Information Center

    Linder, Steven

    The old argument of humanism versus behaviorism is slowly being replaced by a synthesis of these two foundations of thought. This slow professional recognition of synthesis is due to three basic shortcomings in the professional community rather than extreme differences in ideological thought. They are: (1) psychological terminology being equated…

  13. Tackling Human Rights

    ERIC Educational Resources Information Center

    McLester, Susan

    2005-01-01

    In 2003, four high school students from the Tashkent International School in the capital city confronted the issue of their nation's human rights problems head on by researching the topic and publishing their findings on the Web. The site, "Uzbekistan: Opaque Reality," was created as an entry for the non-profit Global SchoolNet's Doors…

  14. Learning to Be Human

    ERIC Educational Resources Information Center

    Macmurray, John

    2012-01-01

    This article presents "Learning to be Human", which John Macmurray delivered on 5 May 1958 as the annual public lecture at Moray House College of Education, now part of Edinburgh University. The key themes of the paper are ones to which Macmurray returned again and again in both his educational and his philosophical writing for over 40 years and…

  15. Human Movement and Preference.

    ERIC Educational Resources Information Center

    Bechtel, Robert B.

    Why and how architecture and interior design can stimulate exploratory locomotion is discussed in terms of research and testing into human response. Two kinds of locomotion are defined, exploratory and habitual, with regard to locomotion and behavior responses to the architectural environment. Measuring responses with a hodometer is outlined with…

  16. Human Biology: Experimental.

    ERIC Educational Resources Information Center

    New York City Board of Education, Brooklyn, NY. Bureau of Curriculum Development.

    Education is a process of adapting to change, and the rate of change is especially rapid in science today. This curriculum in human biology is an alternative to the New York State courses in general and Regents biology, and it has been designed to focus on change from the standpoint of the urban student. It is designed to provide students with…

  17. Doctors and human rights.

    PubMed

    Forrest, D M

    1991-01-01

    This article describes the roles of Amnesty International's Medical Groups and the Medical Foundation for the Care of Victims of Torture in campaigning for and treating those suffering the physical and psychological effects of human rights abuse in peace and war.

  18. Human immunodeficiency virus.

    PubMed

    Skinner, Anita

    2016-11-23

    What was the nature of the CPD activity, practice-related feedback and/or event and/or experience in your practice? The CPD article discussed the importance of human immunodeficiency virus (HIV) testing and diagnosing the condition as early as possible, so that antiretroviral treatment can be initiated and patient outcomes improved.