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Sample records for inhibits cellular synthesis

  1. Anticancer agent CHS-828 inhibits cellular synthesis of NAD

    DEFF Research Database (Denmark)

    Olesen, U.H.; Christensen, M.K.; Bjorkling, F.

    2008-01-01

    Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NAD......, and thus in cancer cell survival. Here, we establish the cytotoxic mechanism of action of the small molecule inhibitor CHS-828 to result from impaired synthesis of NAD. Initially, we detected cross-resistance in cells between CHS-828 and a known inhibitor of Nampt, FK866, a compound of a structurally...... different class. We then showed that nicotinamide protects against CHS-828-mediated cytotoxicity. Finally, we observed that treatment with CHS-828 depletes cellular NAD levels in sensitive cancer cells. In conclusion, these results strongly suggest that, like FK866, CHS-828 kills cancer cells by depleting...

  2. Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

  3. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    International Nuclear Information System (INIS)

    Kultti, Anne; Pasonen-Seppaenen, Sanna; Jauhiainen, Marjo; Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I.

    2009-01-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  4. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    Energy Technology Data Exchange (ETDEWEB)

    Kultti, Anne, E-mail: anne.kultti@uku.fi [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Pasonen-Seppaenen, Sanna [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Jauhiainen, Marjo [Department of Pharmaceutical Chemistry, University of Kuopio, FIN-70211 Kuopio (Finland); Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I. [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland)

    2009-07-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  5. Andrographolide Suppresses MV4-11 Cell Proliferation through the Inhibition of FLT3 Signaling, Fatty Acid Synthesis and Cellular Iron Uptake

    Directory of Open Access Journals (Sweden)

    Xiao Chen

    2017-08-01

    Full Text Available Background: Andrographolide (ADR, the main active component of Andrographis paniculata, displays anticancer activity in various cancer cell lines, among which leukemia cell lines exhibit the highest sensitivity to ADR. In particular, ADR was also reported to have reduced drug resistance in multidrug resistant cell lines. However, the mechanism of action (MOA of ADR’s anticancer and anti-drug-resistance activities remain elusive. Methods: In this study, we used the MV4-11 cell line, a FLT3 positive acute myeloid leukemia (AML cell line that displays multidrug resistance, as our experimental system. We first evaluated the effect of ADR on MV4-11 cell proliferation. Then, a quantitative proteomics approach was applied to identify differentially expressed proteins in ADR-treated MV4-11 cells. Finally, cellular processes and signal pathways affected by ADR in MV4-11 cell were predicted with proteomic analysis and validated with in vitro assays. Results: ADR inhibits MV4-11 cell proliferation in a dose- and time-dependent manner. With a proteomic approach, we discovered that ADR inhibited fatty acid synthesis, cellular iron uptake and FLT3 signaling pathway in MV4-11 cells. Conclusions: ADR inhibits MV4-11 cell proliferation through inhibition of fatty acid synthesis, iron uptake and protein synthesis. Furthermore, ADR reduces drug resistance by blocking FLT3 signaling.

  6. Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: synthesis, phosphatase inhibition activity and cellular uptake studies.

    Science.gov (United States)

    Collins, Juliet; Cilibrizzi, Agostino; Fedorova, Marina; Whyte, Gillian; Mak, Lok Hang; Guterman, Inna; Leatherbarrow, Robin; Woscholski, Rudiger; Vilar, Ramon

    2016-04-28

    Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligands. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration than the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.

  7. Ketoconazole inhibits the cellular uptake of anandamide via inhibition of FAAH at pharmacologically relevant concentrations.

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    Emmelie Björklund

    Full Text Available The antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA.The effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC50 values of 17 and 18 µM, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC50 value (for the inhibitable component of 34 µM.The present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer.

  8. Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

    International Nuclear Information System (INIS)

    Minor, P.D.; Dimmock, N.J.

    1977-01-01

    Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

  9. Inhibition of cellular oxidation by fluoride

    Energy Technology Data Exchange (ETDEWEB)

    Borei, H

    1945-01-01

    An attempt has been made to investigate the inhibition by fluoride of the oxidative processes which occur in the cell by way of the cytochrome oxidase-cytochrome system. Some chemical and physical properties of the fluoride ion are discussed, together with certain quantitative methods for the determination of fluoride. An exhaustive review of the literature concerning the effect of fluoride on enzymic processes has been compiled. The experiments have shown that the point of the attack by fluoride is to be found among the cytochromes. The inhibitory mechanism appears to be such that the haemoprotein is prevented from taking part in the preceding and succeeding links in the reaction chain. The blocking action leaves the prosthetic group of the haemoprotein completely unchanged. The experimental results indicate the formation of a fluorophosphoprotein complex, analogous to that found in the case of enolase. Magnesium may possibly play a part in this process.

  10. TNF-α inhibits trophoblast integration into endothelial cellular networks.

    Science.gov (United States)

    Xu, B; Nakhla, S; Makris, A; Hennessy, A

    2011-03-01

    Preeclampsia has been linked to shallow trophoblast invasion and failure of uterine spiral artery transformation. Interaction between trophoblast cells and maternal uterine endothelium is critically important for this remodelling. The aim of our study was to investigate the effect of TNF-α on the interactions of trophoblast-derived JEG-3 cells into capillary-like cellular networks. We have employed an in vitro trophoblast-endothelial cell co-culture model to quantify trophoblast integration into endothelial cellular networks and to investigate the effects of TNF-α. Controlled co-cultures were also treated with anti-TNF-α antibody (5 μg/ml) to specifically block the effect of TNF-α. The invasion was evaluated by performing quantitative PCR (Q-PCR) to analyse gene expression of matrix metalloproteinases-2 (MMP-2), MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1, integrins (α(1)β(1) and α(6)β(4)), plasminogen activator inhibitor (PAI)-1, E-cadherin and VE-cadherin. JEG-3 cell integration into endothelial networks was significantly inhibited by exogenous TNF-α. The inhibition was observed in the range of 0.2-5 ng/ml, to a maximum 56% inhibition at the highest concentration. This inhibition was reversed by anti-TNF-α antibody. Q-PCR analysis showed that mRNA expression of integrins α(1)β(1) and MMP-2 was significantly decreased. VE-cadherin mRNA expression was significantly up-regulated (32-80%, p integration into maternal endothelial cellular networks, and this process involves the inhibition of MMP-2 and a failure of integrins switch from α(6)β(4) to α(1)β(1.) These molecular correlations reflect the changes identified in human preeclampsia. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Histone gene expression remains coupled to DNA synthesis during in vitro cellular senescence

    International Nuclear Information System (INIS)

    Zambetti, G.; Stein, G.; Stein, J.; Dell'Orco, R.

    1987-01-01

    Despite a decrease in the extent to which confluent monolayers of late compared to early passage CF3 human diploid fibroblasts can be stimulated to proliferate, the time course of DNA synthesis onset is similar regardless of the in vitro age of the cells. A parallel and stoichiometric relationship is maintained between the rate of DNA synthesis and the cellular levels of histone mRNA independent of the age of the cell cultures. Furthermore, DNA synthesis and cellular histone mRNA levels decline in a coordinate manner after inhibition of DNA replication by hydroxyurea treatment. These results indicate that while the proliferative activity of human diploid fibroblasts decreases with passage in culture, those cells that retain the ability to proliferate continue to exhibit a tight coupling of DNA replication and histone gene expression

  12. Radiation metagenesis and inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Dubinina, L.G.; Sergievskaya, S.P.; Kurashova, Z.I.; Dubinin, N.P.

    1983-01-01

    The study of modification of radiation mutagenesis and inhibition of the DNA synthesis by means of 1-β-D arabinofuranosylcytosine (ara-C) is carried out. It is shown that ara-C-acting on chromosomes in the G 1 phase and G 2 phase does not cause mutations in the C capillaris cells. The modification by means of ara-C radiation effect in the G 1 phase and G 2 phase correlates with duration and time of administering ara-C before and after irradiation. A new form of ara-C DNA synthesis inhibitor interaction with mutation processes has been found out. Protective effect of the DNA synthesis inhibitor (ara-C) from mutageneous radiation effect is stressed. Sensibilization of the radiation mutagenesis during cell treafment by the DNA synthesis inhibitor (ara-C) is shown. It is pointed out that emergence of sensibilization or protective effect, i. e. antimutagenesis phenomenon depends on conditions under which the synthesis inhibitor acted in G 1 and G 2 phases

  13. Chlorpromazine inhibits tumour necrosis factor synthesis and cytotoxicity in vitro.

    Science.gov (United States)

    Zinetti, M; Galli, G; Demitri, M T; Fantuzzi, G; Minto, M; Ghezzi, P; Alzani, R; Cozzi, E; Fratelli, M

    1995-11-01

    Chlorpromazine (CPZ) has been previously shown to protect against endotoxin [lipopolysaccharide (LPS)] lethality and inhibit the release of tumour necrosis factor in vivo. We investigated at the cellular level whether this was due to direct inhibition of tumour necrosis factor-alpha (TNF-alpha) synthesis, using LPS-stimulated THP-1 human monocytic leukemia cells. We also studied the effect of CPZ on human TNF-alpha action by assessing TNF-alpha cytotoxicity on mouse fibrosarcoma L929 cells. CPZ (1-100 microM) inhibited TNF-alpha production in THP-1 cells in a dose dependent manner by a maximum of 80%. This effect was comparable to that of two well-known inhibitory drugs, dexamethasone and cyclicAMP. Inhibition was also evident at the mRNA level. On the other hand CPZ (10-25 microM) also inhibited TNF-alpha activity: in fact it reduced the cytotoxicity of TNF-alpha on L929 cells (EC50 was increased four times) and could provide protection even as a post-treatment. CPZ inhibited TNF-induced apoptosis in L929 cells, as detected by analysis of nuclear morphology. However, since we showed that apoptosis was very limited, and was not the main mode of cell death in our conditions, this could not explain the overall protection. Since CPZ did not interfere with either the oligomerization state of TNF-alpha or its receptor binding, our data suggest that it reduced cytotoxicity by inhibiting some steps in the TNF-alpha signalling pathways.

  14. Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis.

    Science.gov (United States)

    Ulvatne, Hilde; Samuelsen, Ørjan; Haukland, Hanne H; Krämer, Manuela; Vorland, Lars H

    2004-08-15

    Most antimicrobial peptides have an amphipathic, cationic structure, and an effect on the cytoplasmic membrane of susceptible bacteria has been postulated as the main mode of action. Other mechanisms have been reported, including inhibition of cellular functions by binding to DNA, RNA and proteins, and the inhibition of DNA and/or protein synthesis. Lactoferricin B (Lfcin B), a cationic peptide derived from bovine lactoferrin, exerts slow inhibitory and bactericidal activity and does not lyse susceptible bacteria, indicating a possible intracellular target. In the present study incorporation of radioactive precursors into DNA, RNA and proteins was used to demonstrate effects of Lfcin B on macromolecular synthesis in bacteria. In Escherichia coli UC 6782, Lfcin B induces an initial increase in protein and RNA synthesis and a decrease in DNA synthesis. After 10 min, the DNA-synthesis increases while protein and RNA-synthesis decreases significantly. In Bacillus subtilis, however, all synthesis of macromolecules is inhibited for at least 20 min. After 20 min RNA-synthesis increases. The results presented here show that Lfcin B at concentrations not sufficient to kill bacterial cells inhibits incorporation of radioactive precursors into macromolecules in both Gram-positive and Gram-negative bacteria.

  15. Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

    Science.gov (United States)

    Fillingame, R H; Jorstad, C M; Morris, D R

    1975-01-01

    There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication. PMID:1060087

  16. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    International Nuclear Information System (INIS)

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site

  17. Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

    Science.gov (United States)

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

    2004-08-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

  18. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis

    Directory of Open Access Journals (Sweden)

    Li Q

    2013-07-01

    Full Text Available Qingli Li,1,2 Mark J Lambrechts,1 Qiuyang Zhang,1 Sen Liu,1 Dongxia Ge,1 Rutie Yin,2 Mingrong Xi,2 Zongbing You1 1Departments of Structural and Cellular Biology and Orthopaedic Surgery, Tulane Cancer Center and Louisiana Cancer Research Consortium, Tulane Center for Stem Cell Research and Regenerative Medicine, and Tulane Center for Aging, Tulane University Health Sciences Center, New Orleans, LA, USA; 2Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu, People’s Republic of China Abstract: Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA, are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy. Keywords: serine hydroxymethyltransferase, prostate cancer, apoptosis

  19. Cellular mechanisms for presynaptic inhibition of sensory afferents

    DEFF Research Database (Denmark)

    Perrier, Jean-Francois Marie; delgado-lezama, rodolfo; Christensen, Rasmus Kordt

    It is well established that presynaptic inhibition of primary afferents involves the activation of GABAA receptors located on presynaptic terminals. However, the source of GABA remains unknown. In an integrated preparation of the spinal cord of the adult turtle, we evoked dorsal root potentials...

  20. Inhibition of poliovirus RNA synthesis by brefeldin A.

    OpenAIRE

    Maynell, L A; Kirkegaard, K; Klymkowsky, M W

    1992-01-01

    Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infect...

  1. Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis.

    Directory of Open Access Journals (Sweden)

    Robert W Tilghman

    Full Text Available Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa, cells on soft substrates (150-300 Pa exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins and glycolysis (e.g., phosphofructokinase-1, whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical

  2. Physiological effects in bovine lymphocytes of inhibiting polyamine synthesis with ethylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Igarashi, K; Morris, D R

    1984-11-01

    Previous results have suggested that ethylglyoxal bis(guanylhydrazone) is a more specific inhibitor of polyamine biosynthesis than the widely used methylglyoxal bis(guanylhydrazone). The physiological effects on mitogenically activated lymphocytes of polyamine depletion with ethylglyoxal bis(guanylhydrazone) were examined. In the presence of ethylglyoxal bis(guanylhydrazone) and the ornithine decarboxylase inhibitor alpha-difluoromethylornithine, the cellular contents of putrescine, spermidine, and spermine were decreased by 75 to 90, 65 to 80, and 40 to 60%, respectively, compared with control cultures. Inhibition of DNA synthesis in these polyamine-deficient cells was always greater than that of protein synthesis. Upon addition of spermidine to the deficient cells, the cellular spermidine content was restored within 4 hr, but the complete recovery of macromolecular synthesis took 10 to 20 hr. Thymidine kinase and DNA polymerase alpha activities in polyamine-deficient cells were lower than those in normal cells, whereas RNA polymerase II and leucyl transfer RNA synthase activities were nearly equal to those in normal cells. These results and studies with 2-dimensional gel electrophoresis raise the possibility that polyamines may regulate the synthesis of specific proteins. Decreased synthesis of replication proteins in polyamine-deficient cells may be one reason for the reduced synthesis of DNA.

  3. Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

    International Nuclear Information System (INIS)

    Bennett, W.M.; Mela-Riker, L.M.; Houghton, D.C.; Gilbert, D.N.; Buss, W.C.

    1988-01-01

    Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. [3H]leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis

  4. Salmonella infection inhibits intestinal biotin transport: cellular and molecular mechanisms.

    Science.gov (United States)

    Ghosal, Abhisek; Jellbauer, Stefan; Kapadia, Rubina; Raffatellu, Manuela; Said, Hamid M

    2015-07-15

    Infection with the nontyphoidal Salmonella is a common cause of food-borne disease that leads to acute gastroenteritis/diarrhea. Severe/prolonged cases of Salmonella infection could also impact host nutritional status, but little is known about its effect on intestinal absorption of vitamins, including biotin. We examined the effect of Salmonella enterica serovar Typhimurium (S. typhimurium) infection on intestinal biotin uptake using in vivo (streptomycin-pretreated mice) and in vitro [mouse (YAMC) and human (NCM460) colonic epithelial cells, and human intestinal epithelial Caco-2 cells] models. The results showed that infecting mice with wild-type S. typhimurium, but not with its nonpathogenic isogenic invA spiB mutant, leads to a significant inhibition in jejunal/colonic biotin uptake and in level of expression of the biotin transporter, sodium-dependent multivitamin transporter. In contrast, infecting YAMC, NCM460, and Caco-2 cells with S. typhimurium did not affect biotin uptake. These findings suggest that the effect of S. typhimurium infection is indirect and is likely mediated by proinflammatory cytokines, the levels of which were markedly induced in the intestine of S. typhimurium-infected mice. Consistent with this hypothesis, exposure of NCM460 cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition of biotin uptake, sodium-dependent multivitamin transporter expression, and activity of the SLC5A6 promoter. The latter effects appear to be mediated, at least in part, via the NF-κB signaling pathway. These results demonstrate that S. typhimurium infection inhibits intestinal biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines.

  5. Metallofullerenol Inhibits Cellular Iron Uptake by Inducing Transferrin Tetramerization.

    Science.gov (United States)

    Li, Jinxia; Xing, Xueqing; Sun, Baoyun; Zhao, Yuliang; Wu, Zhonghua

    2017-10-18

    Herein, A549 tumor cell proliferation was confirmed to be positively dependent on the concentration of Fe 3+ or transferrin (Tf). Gd@C 82 (OH) 22 or C 60 (OH) 22 effectively inhibited the iron uptake and the subsequent proliferation of A549 cells. The conformational changes of Tf mixed with FeCl 3 , GdCl 3 , C 60 (OH) 22 or Gd@C 82 (OH) 22 were obtained by SAXS. The results demonstrate that Tf homodimers can be decomposed into monomers in the presence of FeCl 3 , GdCl 3 or C 60 (OH) 22 , but associated into tetramers in the presence of Gd@C 82 (OH) 22 . The larger change of SAXS shapes between Tf+C 60 (OH) 22 and Tf+FeCl 3 implies that C 60 (OH) 22 is bound to Tf, blocking the iron-binding site. The larger deviation of the SAXS shape from a possible crystal structure of Tf tetramer implies that Gd@C 82 (OH) 22 is bound to the Tf tetramer, thus disturbing iron transport. This study well explains the inhibition mechanism of Gd@C 82 (OH) 22 and C 60 (OH) 22 on the iron uptake and the proliferation of A549 tumor cells and highlights the specific interactions of a nanomedicine with the target biomolecules in cancer therapy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

    International Nuclear Information System (INIS)

    Dreslor, S.L.; Frattini, M.G.

    1987-01-01

    Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N 2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

  7. Cellular transformation by radiation: induction, promotion, and inhibition

    International Nuclear Information System (INIS)

    Borek, C.

    1981-01-01

    Radiation oncogenesis induced in utero in hamsters is expressed at a lower frequency than that induced in vitro. Quantitative studies carried out on hamster embryo cells indicate that neutrons are more effective in their carcinogenic potential than x-rays but also more toxic, that splitting the dose of x-rays at low doses leads to enhanced transformation, but that at high doses protracted radiation has a sparing effect. At all dose ranges survival was increased by protracting the radiation dose, thus suggesting that different repair processes must be involved for survival and transformation. In our qualitative studies, once cells are transformed by radiation, they exhibit a wide range of structural and functional phenotypic changes, some of which are membrane-associated and are expressed within days after induction. Our current studies on nutritional and hormonal influences on radiation transformation indicate the following: Pyrolysate products from broiled protein foods act in synergism with radiation to produce transformation, whereas vitamin A analogs are powerful, preventive agents. Retinoids inhibit both x-ray-induced transformation and its promotion by TPA; these modifications (enhancement by TPA, inhibition by retinoids) are not reflected in sister chromatid exchanges, but are reflected in the level of membrane associated enzymes Na/K ATPase. Whereas retinoids modify late events (expression, promotion), we find that thyroid hormone plays a crucial role in the early phases of radiation and chemically induced transformation. Our recent success in transforming human skin fibroblasts will enable quantitative and qualitative studies of radiation carcinogenesis in a system relevant to man

  8. Design, synthesis and cellular metabolism study of 4'-selenonucleosides.

    Science.gov (United States)

    Yu, Jinha; Sahu, Pramod K; Kim, Gyudong; Qu, Shuhao; Choi, Yoojin; Song, Jayoung; Lee, Sang Kook; Noh, Minsoo; Park, Sunghyouk; Jeong, Lak Shin

    2015-01-01

    4'-seleno-homonucleosides were synthesized as next-generation nucleosides, and their cellular phosphorylation was studied to confirm the hypothesis that bulky selenium atom can sterically hinder the approach of cellular nucleoside kinase to the 5'-OH for phosphorylation. 4'-seleno-homonucleosides (n = 2), with one-carbon homologation, were synthesized through a tandem seleno-Michael addition-SN2 ring cyclization. LC-MS analysis demonstrated that they were phosphorylated by cellular nucleoside kinases, resulting in anticancer activity. The bulky selenium atom played a key role in deciding the phosphorylation by cellular nucleoside kinases. [Formula: see text].

  9. Extracellular cystatin SN and cathepsin B prevent cellular senescence by inhibiting abnormal glycogen accumulation.

    Science.gov (United States)

    Oh, Sang-Seok; Park, Soojong; Lee, Ki-Won; Madhi, Hamadi; Park, Sae Gwang; Lee, Hee Gu; Cho, Yong-Yeon; Yoo, Jiyun; Dong Kim, Kwang

    2017-04-06

    Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated β-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3β phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.

  10. Inhibition of acetylcholine synthesis in vitro

    International Nuclear Information System (INIS)

    O-Neill, J.J.; Capacio, B.; Doukas, P.H.; Leech, R.; Ricciardi, F.; Sterling, G.H.

    1986-01-01

    In order to better understand diseases that stem from deficiencies in cholinergic activity, reproducible in vitro and in vivo models displaying cholinergic hypofunction are desirable. This necessitates the availability of specific inhibitors. This paper examines the design, synthesis and evaluation of quinuclidinyl compounds with structural features previously reported, but with certain key differences. Structure activity studies with in vitro assay systems are presented. In a few studies, choline was held constant and acetyl-CoA concentration was varied, but with a constant amount of ( 14 C) - acetyl CoA. Acetylcholine synthesis and CO 2 production from labelled glucose were measured in cerebral cortex slices from male rats after decapitation. The nanomoles of ACh and CO 2 produced from ( 14 C) -glucose were calculated from glucose specific activity. Results are presented

  11. Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

    Science.gov (United States)

    Fineran, Paul; Lloyd-Evans, Emyr; Lack, Nathan A; Platt, Nick; Davis, Lianne C; Morgan, Anthony J; Höglinger, Doris; Tatituri, Raju Venkata V; Clark, Simon; Williams, Ian M; Tynan, Patricia; Al Eisa, Nada; Nazarova, Evgeniya; Williams, Ann; Galione, Antony; Ory, Daniel S; Besra, Gurdyal S; Russell, David G; Brenner, Michael B; Sim, Edith; Platt, Frances M

    2016-11-18

    Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis , achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria

  12. Inhibition of Estradiol Synthesis Impairs Fear Extinction in Male Rats

    Science.gov (United States)

    Graham, Bronwyn M.; Milad, Mohammed R.

    2014-01-01

    Emerging research has demonstrated that the sex hormone estradiol regulates fear extinction in female rodents and women. Estradiol may also regulate fear extinction in males, given its role in synaptic plasticity in both sexes. Here we report that inhibition of estradiol synthesis during extinction training, via the aromatase inhibitor fadrozole,…

  13. Inhibiting Glycosphingolipid Synthesis Ameliorates Hepatic Steatosis in Obese Mice

    NARCIS (Netherlands)

    Zhao, Hongmei; Przybylska, Malgorzata; Wu, I.-Huan; Zhang, Jinhua; Maniatis, Panagiotis; Pacheco, Joshua; Piepenhagen, Peter; Copeland, Diane; Arbeeny, Cynthia; Shayman, James A.; Aerts, Johannes M.; Jiang, Canwen; Cheng, Seng H.; Yew, Nelson S.

    2009-01-01

    Steatosis in the liver is a common feature of obesity and type 2 diabetes and the precursor to the development of nonalcoholic steatohepatitis (NASH), cirrhosis, and liver failure. It has been shown previously that inhibiting glycosphingolipid (GSL) synthesis increases insulin sensitivity and lowers

  14. Inhibition of chloroplast protein synthesis following light chilling of tomato

    International Nuclear Information System (INIS)

    Kent, J.; Ort, D.

    1989-01-01

    In the present study we looked at the effects of a high light chill on the pulsed incorporation of 35 S methionine into total, stromal, and thylakoid proteins of lightly abraded leaflets of 18-21 day old tomato (Lycopersicon esculentum Mill ca. Floramerica) seedlings. Based on gel fluorographic patterns of marker proteins that are indicative of the net rates of chloroplast and cytoplasmic protein synthesis, there appears to be a nearly complete cessation of chloroplastic protein synthesis. No labeling is observed for either the stromal large subunit of Rubisco or the thylakoid-bound alpha and beta subunits of the coupling factor. One notable exception, however, appears to be the 32 kd, D1 protein. Its net synthetic rate remains high despite the inhibition of other chloroplastically synthesized proteins. The small subunit of Rubicso, LHCP-II, as well as several other proteins of known cytoplasmic origin, were still synthesized, albeit, at lower than control rates. Light chilling of chill-insensitive spinach produced a similar, but less dramatic differential behavior between chloroplastic and cytoplasmic protein synthesis. It appears, in chilling-sensitive plants, that chloroplast protein synthesis exhibits a greater sensitivity to low temperature inhibition than does cytoplasmic protein synthesis and that recovery of chloroplast protein synthesis may play an important role in recovery of photosynthetic activity following chilling

  15. Inhibition of nitric oxide synthesis enhances leukocyte rolling and adhesion in human microvasculature

    Directory of Open Access Journals (Sweden)

    Hossain Mokarram

    2012-07-01

    Full Text Available Abstract Background Nitric oxide (NO is a multifunctional signaling molecule that regulates important cellular events in inflammation including leukocyte recruitment. Previous studies have shown that pharmacological inhibition of NO synthesis induces leukocyte recruitment in various in vitro and animal models. However, it is not known whether NO modulation has similar effects on leukocyte-endothelial cell interactions within the human microvasculature. The present study explored the effect of systemic L-NAME treatment on leukocyte recruitment in the SCID-hu mouse model. Methods Human skin xenografts were transplanted in SCID mice to study human leukocyte dynamics in human vasculature. Early events of human leukocyte recruitment in human vasculature were studied using intravital microscopy. NO synthesis was pharmacologically inhibited using NG-nitro-L-arginine methyl ester (L-NAME. Immunohistochemical analysis was performed to elucidate E-selectin expression in human xenograft skin. Human neutrophil-endothelial cell interactions were also studied in an in vitro flow chamber assay system. P- and E-selectin expression on cultured human umbilical vein endothelial cells (HUVECs was measured using ELISA. Platelet-activating factor (PAF synthesis was detected using a TLC-based assay. Results L-NAME treatment significantly enhanced the rolling and adhesion of human leukocytes to the human vasculature. Functional blocking of P- and E-selectins significantly inhibited rolling but not adhesion induced by inhibition of NO synthesis. Systemic L-NAME treatment enhanced E-selectin expression in human xenograft skin. L-NAME treatment significantly enhanced P- and E-selectin expression on HUVECs. L-NAME treatment did not significantly modify neutrophil rolling or adhesion to HUVECs indicating that L-NAME−induced subtle P- and E-selectin expression was insufficient to elicit dynamic neutrophil-HUVEC interactions in vitro. Moreover, synthesis of endothelial

  16. Many Putative Endocrine Disruptors Inhibit Prostaglandin Synthesis

    DEFF Research Database (Denmark)

    Kristensen, David M.; Skalkam, Maria L.; Audouze, Karine Marie Laure

    2011-01-01

    Background: Prostaglandins (PGs) play key roles in development and maintenance of homeostasis of the adult body. Despite these important roles, it remains unclear whether the PG pathway is a target for endocrine disruption. However, several known endocrine disrupting compounds (EDCs) share a high...... suggest a hitherto unknown mode of action by EDCs through inhibition of the PG pathway and suggest new avenues to investigate effects of EDCs on reproductive and immunological disorders that have become increasingly common in recent decades....

  17. Novel Antimicrobial Peptides That Inhibit Gram Positive Bacterial Exotoxin Synthesis

    Science.gov (United States)

    Merriman, Joseph A.; Nemeth, Kimberly A.; Schlievert, Patrick M.

    2014-01-01

    Gram-positive bacteria, such as Staphylococcus aureus, cause serious human illnesses through combinations of surface virulence factors and secretion of exotoxins. Our prior studies using the protein synthesis inhibitor clindamycin and signal transduction inhibitors glycerol monolaurate and α-globin and β-globin chains of hemoglobin indicate that their abilities to inhibit exotoxin production by S. aureus are separable from abilities to inhibit growth of the organism. Additionally, our previous studies suggest that inhibition of exotoxin production, in absence of ability to kill S. aureus and normal flora lactobacilli, will prevent colonization by pathogenic S. aureus, while not interfering with lactobacilli colonization. These disparate activities may be important in development of novel anti-infective agents that do not alter normal flora. We initiated studies to explore the exotoxin-synthesis-inhibition activity of hemoglobin peptides further to develop potential agents to prevent S. aureus infections. We tested synthesized α-globin chain peptides, synthetic variants of α-globin chain peptides, and two human defensins for ability to inhibit exotoxin production without significantly inhibiting S. aureus growth. All of these peptides were weakly or not inhibitory to bacterial growth. However, the peptides were inhibitory to exotoxin production with increasing activity dependent on increasing numbers of positively-charged amino acids. Additionally, the peptides could be immobilized on agarose beads or have amino acid sequences scrambled and still retain exotoxin-synthesis-inhibition. The peptides are not toxic to human vaginal epithelial cells and do not inhibit growth of normal flora L. crispatus. These peptides may interfere with plasma membrane signal transduction in S. aureus due to their positive charges. PMID:24748386

  18. Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

    Science.gov (United States)

    Pal, Sandip; Dey, Subrata Kumar; Saha, Chabita

    2014-01-01

    Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×106 M−1 and 1.66×106 M−1, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC50 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition. PMID:25025898

  19. Interleukin-1 inhibits the synthesis of collagen by fibroblasts.

    Science.gov (United States)

    Bhatnagar, R; Penfornis, H; Mauviel, A; Loyau, G; Saklatvala, J; Pujol, J P

    1986-10-01

    Human dermal fibroblasts, exposed to human or porcine Interleukin-1, responded by an inhibition of collagen synthesis in a dose dependent manner. Incubation with Il-1 for more than 8 h was required to see an appreciable effect. The phenomenon was not dependent on the presence of serum in the culture medium. Since a stimulation of prostaglandin E2 secretion was also observed in presence of Il-1, we investigated the eventual role of arachidonic acid metabolites in the phenomenon. Inhibitors interfering with arachidonate metabolism, namely indomethacin, acetyl salicylic acid, BW 755 C and NDGA had no influence on the inhibition of collagen synthesis caused by Il-1. These data suggest that both cyclooxygenase and lipoxygenase derived metabolites of arachidonic acid are unlikely to play a role in the mechanism.

  20. The cellular origin of the hepatic cholesterol synthesis (1961)

    International Nuclear Information System (INIS)

    Chevallier, F.

    1961-01-01

    If rats are sacrificed within minutes after an injection of acetate 1 - 14 C the specific radioactivities of sterols precipitable with digitonine, extracted from liver parenchyma cells and from Kupffer cells are very close to each other, whatever the duration of the experiment may be. It follows that cholesterol synthesis probably occurs in both types of cells. A validation of this conclusion requires that the validity of certain assumptions be established. (authors) [fr

  1. 3-bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes.

    Science.gov (United States)

    Ehrke, Eric; Arend, Christian; Dringen, Ralf

    2015-07-01

    The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes. © 2014 Wiley Periodicals, Inc.

  2. Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells

    OpenAIRE

    Kim, Dongkyun; Kim, Song-Ja; Kang, Shin-Sung; Jin, Eun-Jung

    2009-01-01

    Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of ...

  3. Biomimetic synthesis of cellular SiC based ceramics from plant ...

    Indian Academy of Sciences (India)

    Unknown

    SiC based materials so derived can be used in structural applications and in designing high temperature filters and catalyst supports. Keywords. Biomimetic synthesis; carbonaceous biopreform; biomorphic Si–SiC ceramic composites; porous cellular SiC ceramics. 1. Introduction. In recent years, there has been tremendous ...

  4. Expression of PML tumor suppressor in A 431 cells reduces cellular growth by inhibiting the epidermal growth factor receptor expression

    International Nuclear Information System (INIS)

    Vallian, S.; Chang, K.S.

    2004-01-01

    Our previous studies showed that the promyelocytic leukemia, PML, protein functions as a cellular and growth suppressor. Transient expression of PML was also found to repress the activity of the epidermal growth factor receptor gene promoter. In this study we have examined the effects of PML on A431 cells, which express a high level of + protein. The PML gene was introduced into the cells using the adenovirus-mediated gene transfer system. Western blot analysis on the extracts from the cells expressing PML showed a significant repression in the expression of the epidermal growth factor receptor protein. The cells were examined for growth and DNA synthesis. The data showed a marked reduction in both growth and DNA synthesis rate in the cells expressing PML compared with the control cells. Furthermore, in comparison with the controls, the cells expressing PML were found to be more in G1 phase, fewer in S and about the same number in the G2/M phase. This data clearly demonstrated that the repression of epidermal growth factor receptor expression in A 431 cells by PML was associated with inhibition of cell growth and alteration of the cell cycle distribution, suggesting a novel mechanism for the known growth inhibitory effects of PML

  5. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

    1990-01-01

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  6. Inhibition of ribosomal RNA synthesis in yeast by ionizing radiations

    Energy Technology Data Exchange (ETDEWEB)

    Weber, K; Kiefer, J [Giessen Univ. (Germany, F.R.). Strahlenzentrum

    1984-12-01

    Synthesis of ribosomal RNA(r-RNA) was measured for 1 h after exposure of Saccharomyces cerevisiae to ..gamma..-rays, X-rays or ..cap alpha.. particles. ..gamma..- or X-ray induced transcription inhibition was always found to decrease exponentially with dose. D/sub 0/ values of 2150 or 1950 Gy were determined in wild-type cells, corresponding to a mean energy of about 60 eV per r-RNA gene. The finding of differential sensitivities of the two high molecular-weight r-RNA species which are cotranscribed from r-DNA is compatible with the existence of a transcription terminating mechanism. Cells from a mutant strain (rad-9), radiation sensitive to colony forming ability, showed an approximately equal sensitivity for transcription inhibition compared to the wild-type (D/sub 0/ (2095) = 2400 Gy). Inactivation of r-RNA synthesis in cells exposed to ..cap alpha..-particles at room-temperature showed a decreased sensitivity with higher particle fluences ('resistant tail'). This phenomenon was drastically reduced if the temperature during irradiation was lowered to 4/sup 0/C and completely abolished when dried cells were used. An inactivation cross-section for ..cap alpha..-particle induced transcription inhibition of about 0.02 ..mu..m/sup 2/ can be derived from the experimental data.

  7. Inhibition of rotavirus replication by downregulation of fatty acid synthesis.

    Science.gov (United States)

    Gaunt, Eleanor R; Cheung, Winsome; Richards, James E; Lever, Andrew; Desselberger, Ulrich

    2013-06-01

    Recently the recruitment of lipid droplets (LDs) to sites of rotavirus (RV) replication was reported. LDs are polymorphic organelles that store triacylglycerols, cholesterol and cholesterol esters. The neutral fats are derived from palmitoyl-CoA, synthesized via the fatty acid biosynthetic pathway. RV-infected cells were treated with chemical inhibitors of the fatty acid biosynthetic pathway, and the effects on viral replication kinetics were assessed. Treatment with compound C75, an inhibitor of the fatty acid synthase enzyme complex (FASN), reduced RV infectivity 3.2-fold (P = 0.07) and modestly reduced viral RNA synthesis (1.2-fold). Acting earlier in the fatty acid synthesis pathway, TOFA [5-(Tetradecyloxy)-2-furoic acid] inhibits the enzyme acetyl-CoA carboxylase 1 (ACC1). TOFA reduced the infectivity of progeny RV 31-fold and viral RNA production 6-fold. The effect of TOFA on RV infectivity and RNA replication was dose-dependent, and infectivity was reduced by administering TOFA up to 4 h post-infection. Co-treatment of RV-infected cells with C75 and TOFA synergistically reduced viral infectivity. Knockdown by siRNA of FASN and ACC1 produced findings similar to those observed by inhibiting these proteins with the chemical compounds. Inhibition of fatty acid synthesis using a range of approaches uniformly had a more marked impact on viral infectivity than on viral RNA yield, inferring a role for LDs in virus assembly and/or egress. Specific inhibitors of fatty acid metabolism may help pinpoint the critical structural and biochemical features of LDs that are essential for RV replication, and facilitate the development of antiviral therapies.

  8. Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI-Induced Cellular and DNA Toxicity

    Directory of Open Access Journals (Sweden)

    Gunjan Guha

    2011-01-01

    Full Text Available Hexavalent chromium Cr(VI is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae, commonly known as Henna. The extracts showed significant (P < .05 potential in scavenging free radicals (DPPH• and ABTS•+ and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05 positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI-induced oxidative cell damage.

  9. Effects of nicotine on cellular proliferation, cell cycle phase distribution, and macromolecular synthesis in human promyelocytic HL-60 leukaemia cells

    International Nuclear Information System (INIS)

    Konno, S.; Wu, J.M.; Chiao, J.W.

    1986-01-01

    Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in the human promyelocytic HL-60 leukemia cells, with 4 mM nicotine resulting in a 50% inhibition of cellular proliferation after 48-50h. Accompanying the anticellular effect of nicotine is a significant change in the cell cycle distribution of HL-60 cells. For example, treatment with 4 mM nicotine for 20h causes an increase in the proportion of G1-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes partial cell arrest in the G-1 phase which may in part account for its effects on cell growth. To determine whether nicotine changes the cellular uptake/transport to macromolecular precursors, HL-60 cells were treated with 216 mM nicotine for 30h, at the end of which time cells were labelled with ( 3 H)thymidine, ( 3 H)uridine, ( 14 C)lysine and( 35 S)methionine, the trichloroacetic acid soluble and insoluble radioactivities from each of the labelling conditions were determined. These studies show that nicotine mainly affects the ''de novo synthesis'' of proteins. (author)

  10. Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Ganges, M.B.; Robbins, J.H.; Jiang, H.; Hauser, C.; Tarone, R.E.

    1988-01-01

    The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radioresistant DNA synthesis. (author). 27 refs.; 1 fig.; 1 tab

  11. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

  12. Control of DNA synthesis in inhibited and activated Agrostemma githago seeds

    Energy Technology Data Exchange (ETDEWEB)

    Hecker, M [Sektion Biologie, FG Algemeine Botanik und Pflanzenphysiologie, Universitaet Greifswald (German Democratic Republic)

    1975-01-01

    The relationships between DNA synthesis and germination capacity of Agrostemma seeds had been studied. Protein synthesis and RNA synthesis were activated at the very beginning of imbibition, whereas DNA synthesis started in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30 degC), or aged seeds with a low germination capacity were characterized by a significantly reduced protein synthesis. DNA synthesis was also reduced. The inhibition of the protein synthesis of Agrostemma embryos fed with cycloheximide or actinomycin D caused a depression of DNA synthesis. The results indicated that the initiation of DNA synthesis of imbibing Agrostemma seeds depended on the synthesis of special proteins. Abscisic acid inhibited the growth as well as DNA synthesis of isolated Agrostemma embryos. Nitomycin inhibited germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also showed a reduced incorporation of /sup 3/H-thymidine by DNA. It is suggested that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, was involved in the mechanism of ripening of the Agrostemma seeds.

  13. Control of DNA synthesis in inhibited and activated Agrostemma githago seeds

    International Nuclear Information System (INIS)

    Hecker, M.

    1975-01-01

    The relationships between DNA synthesis and germination capacity of Agrostemma seeds had been studied. Protein synthesis and RNA synthesis were activated at the very beginning of imbibition, whereas DNA synthesis started in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30 degC), or aged seeds with a low germination capacity were characterized by a significantly reduced protein synthesis. DNA synthesis was also reduced. The inhibition of the protein synthesis of Agrostemma embryos fed with cycloheximide or actinomycin D caused a depression of DNA synthesis. The results indicated that the initiation of DNA synthesis of imbibing Agrostemma seeds depended on the synthesis of special proteins. Abscisic acid inhibited the growth as well as DNA synthesis of isolated Agrostemma embryos. Nitomycin inhibited germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also showed a reduced incorporation of 3 H-thymidine by DNA. It is suggested that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, was involved in the mechanism of ripening of the Agrostemma seeds. (author)

  14. Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Morris, D R; Jorstad, C M; Seyfried, C E

    1977-09-01

    The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

  15. Resveratrol Inhibition of Cellular Respiration: New Paradigm for an Old Mechanism

    Directory of Open Access Journals (Sweden)

    Luis Alberto Madrigal-Perez

    2016-03-01

    Full Text Available Resveratrol (3,4′,5-trihydroxy-trans-stilbene, RSV has emerged as an important molecule in the biomedical area. This is due to its antioxidant and health benefits exerted in mammals. Nonetheless, early studies have also demonstrated its toxic properties toward plant-pathogenic fungi of this phytochemical. Both effects appear to be opposed and caused by different molecular mechanisms. However, the inhibition of cellular respiration is a hypothesis that might explain both toxic and beneficial properties of resveratrol, since this phytochemical: (1 decreases the production of energy of plant-pathogenic organisms, which prevents their proliferation; (2 increases adenosine monophosphate/adenosine diphosphate (AMP/ADP ratio that can lead to AMP protein kinase (AMPK activation, which is related to its health effects, and (3 increases the reactive oxygen species generation by the inhibition of electron transport. This pro-oxidant effect induces expression of antioxidant enzymes as a mechanism to counteract oxidative stress. In this review, evidence is discussed that supports the hypothesis that cellular respiration is the main target of resveratrol.

  16. A Compact Synchronous Cellular Model of Nonlinear Calcium Dynamics: Simulation and FPGA Synthesis Results.

    Science.gov (United States)

    Soleimani, Hamid; Drakakis, Emmanuel M

    2017-06-01

    Recent studies have demonstrated that calcium is a widespread intracellular ion that controls a wide range of temporal dynamics in the mammalian body. The simulation and validation of such studies using experimental data would benefit from a fast large scale simulation and modelling tool. This paper presents a compact and fully reconfigurable cellular calcium model capable of mimicking Hopf bifurcation phenomenon and various nonlinear responses of the biological calcium dynamics. The proposed cellular model is synthesized on a digital platform for a single unit and a network model. Hardware synthesis, physical implementation on FPGA, and theoretical analysis confirm that the proposed cellular model can mimic the biological calcium behaviors with considerably low hardware overhead. The approach has the potential to speed up large-scale simulations of slow intracellular dynamics by sharing more cellular units in real-time. To this end, various networks constructed by pipelining 10 k to 40 k cellular calcium units are compared with an equivalent simulation run on a standard PC workstation. Results show that the cellular hardware model is, on average, 83 times faster than the CPU version.

  17. Arctigenin preferentially induces tumor cell death under glucose deprivation by inhibiting cellular energy metabolism.

    Science.gov (United States)

    Gu, Yuan; Qi, Chunting; Sun, Xiaoxiao; Ma, Xiuquan; Zhang, Haohao; Hu, Lihong; Yuan, Junying; Yu, Qiang

    2012-08-15

    Selectively eradicating cancer cells with minimum adverse effects on normal cells is a major challenge in the development of anticancer therapy. We hypothesize that nutrient-limiting conditions frequently encountered by cancer cells in poorly vascularized solid tumors might provide an opportunity for developing selective therapy. In this study, we investigated the function and molecular mechanisms of a natural compound, arctigenin, in regulating tumor cell growth. We demonstrated that arctigenin selectively promoted glucose-starved A549 tumor cells to undergo necrosis by inhibiting mitochondrial respiration. In doing so, arctigenin elevated cellular level of reactive oxygen species (ROS) and blocked cellular energy metabolism in the glucose-starved tumor cells. We also demonstrated that cellular ROS generation was caused by intracellular ATP depletion and played an essential role in the arctigenin-induced tumor cell death under the glucose-limiting condition. Furthermore, we combined arctigenin with the glucose analogue 2-deoxyglucose (2DG) and examined their effects on tumor cell growth. Interestingly, this combination displayed preferential cell-death inducing activity against tumor cells compared to normal cells. Hence, we propose that the combination of arctigenin and 2DG may represent a promising new cancer therapy with minimal normal tissue toxicity. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  18. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    Science.gov (United States)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  19. Madecassoside Inhibits Melanin Synthesis by Blocking Ultraviolet-Induced Inflammation

    Directory of Open Access Journals (Sweden)

    Eunsun Jung

    2013-12-01

    Full Text Available Madecassoside (MA, a pentacyclic triterpene isolated from Centella asitica (L., is used as a therapeutic agent in wound healing and also as an anti-inflammatory and anti-aging agent. However, the involvement of MA in skin-pigmentation has not been reported. This study was conducted to investigate the effects of MA on ultraviolet (UV-induced melanogenesis and mechanisms in a co-culture system of keratinocytes and melanocytes. MA significantly inhibited UVR-induced melanin synthesis and melanosome transfer in the co-culture system. These effects were further demonstrated by the MA-induced inhibition of protease-activated receptor-2 expression and its signaling pathway, cyclooxygenase-2, prostaglandin E2 and prostaglandin F2 alpha in keratinocytes. The clinical efficacy of MA was confirmed on artificially tanned human skin. MA significantly reduced UV-induced melanin index at 8 weeks after topical application. Overall, the study demonstrated significant benefits of MA use in the inhibition of hyperpigmentation caused by UV irradiation.

  20. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis.

    Science.gov (United States)

    Li, Qingli; Lambrechts, Mark J; Zhang, Qiuyang; Liu, Sen; Ge, Dongxia; Yin, Rutie; Xi, Mingrong; You, Zongbing

    2013-01-01

    Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose) polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy.

  1. The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

    Directory of Open Access Journals (Sweden)

    Ramón A. Lorca

    2011-01-01

    Full Text Available Although the physiological function of the cellular prion protein (PrPC remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+.

  2. Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

    Directory of Open Access Journals (Sweden)

    Richa Tyagi

    2015-02-01

    Full Text Available Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK. Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

  3. Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

    Science.gov (United States)

    Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

    2017-09-15

    Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Cockayne's syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation

    International Nuclear Information System (INIS)

    Lehmann, A.R.; Thompson, A.F.; Harcourt, S.A.

    1993-01-01

    Cockayne's syndrome (CS) is a rare autosomal recessive disorder with dwarfism, mental retardation, and otherwise clinically heterogeneous features. In cultured CS fibroblasts, the failure of RNA synthesis to recover to normal rates after UV-C irradiation provides a useful and relatively simple diagnostic test. We have measured post-UV-C RNA synthesis in 52 patients for whom a clinical diagnosis of CS was considered a possibility. Twenty-nine patients showed the defect characteristic of CS cells, and 23 had a normal response. We have attempted to correlate the cellular diagnosis with the different clinical features of the disorder. Clinical details of the patients were obtained from referring clinicians in the form of a questionnaire. Our results show that, apart from the cardinal features of dwarfism and mental retardation, sun sensitivity correlated best with a positive cellular diagnosis. Pigmentary retinopathy, gait defects, and dental caries were also good positive indicators, although several patients with a positive cellular diagnosis did not have these features. (Author)

  5. Fluorochemicals used in food packaging inhibit male sex hormone synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Rosenmai, A.K., E-mail: akjro@food.dtu.dk [Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, DK-2860 Søborg (Denmark); Nielsen, F.K. [Section of Toxicology, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen (Denmark); Pedersen, M. [Division of Food Chemistry, National Food Institute, Technical University of Denmark, DK-2860 Søborg (Denmark); Hadrup, N. [Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, DK-2860 Søborg (Denmark); Trier, X. [Division of Food Chemistry, National Food Institute, Technical University of Denmark, DK-2860 Søborg (Denmark); Christensen, J.H. [Department of Basic Sciences and Environment, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C. (Denmark); Vinggaard, A.M. [Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, DK-2860 Søborg (Denmark)

    2013-01-01

    Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS. The aim of this study was to elucidate the effects of six fluorochemicals on sex hormone synthesis and androgen receptor (AR) activation in vitro. Four PAPS and two metabolites, perfluorooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (8:2 FTOH) were tested. Hormone profiles, including eight steroid hormones, generally showed that 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH led to decreases in androgens (testosterone, dehydroepiandrosterone, and androstenedione) in the H295R steroidogenesis assay. Decreases were observed for progesterone and 17-OH-progesterone as well. These observations indicated that a step prior to progestagen and androgen synthesis had been affected. Gene expression analysis of StAR, Bzrp, CYP11A, CYP17, CYP21 and CYP19 mRNA showed a decrease in Bzrp mRNA levels for 8:2 monoPAPS and 8:2 FTOH indicating interference with cholesterol transport to the inner mitochondria. Cortisol, estrone and 17β-estradiol levels were in several cases increased with exposure. In accordance with these data CYP19 gene expression increased with 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH exposures indicating that this is a contributing factor to the decreased androgen and the increased estrogen levels. Overall, these results demonstrate that fluorochemicals present in food packaging materials and their metabolites can affect steroidogenesis through decreased Bzrp and increased CYP19 gene expression leading to lower androgen and higher estrogen levels. -- Highlights: ► Fluorochemicals found in 57% of paper and board food packaging were tested. ► Collectively six fluorochemicals were tested for antiandrogenic potential in vitro. ► Three out of six tested fluorochemicals inhibited

  6. Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock

    Directory of Open Access Journals (Sweden)

    Reut Shalgi

    2014-06-01

    Full Text Available During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from ∼1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes.

  7. Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

    Science.gov (United States)

    Kim, Dong Kyun; Kim, Song Ja; Kang, Shin Sung; Jin, Eun Jung

    2009-09-30

    Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.

  8. Inhibition of HIV by Legalon-SIL is independent of its effect on cellular metabolism

    Energy Technology Data Exchange (ETDEWEB)

    McClure, Janela [Department of Laboratory Medicine, University of Washington, Seattle, WA (United States); Margineantu, Daciana H. [Department of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Sweet, Ian R. [Department of Medicine (Division of Metabolism, Endocrinology, and Nutrition), University of Washington, Seattle, WA (United States); Polyak, Stephen J., E-mail: polyak@uw.edu [Department of Laboratory Medicine, University of Washington, Seattle, WA (United States); Department of Global Health, University of Washington, Seattle, WA (United States)

    2014-01-20

    In this report, we further characterized the effects of silibinin (SbN), derived from milk thistle extract, and Legalon-SIL (SIL), a water-soluble derivative of SbN, on T cell metabolism and HIV infection. We assessed the effects of SbN and SIL on peripheral blood mononuclear cells (PBMC) and CEM-T4 cells in terms of cellular growth, ATP content, metabolism, and HIV infection. SIL and SbN caused a rapid and reversible (upon removal) decrease in cellular ATP levels, which was associated with suppression of mitochondrial respiration and glycolysis. SbN, but not SIL inhibited glucose uptake. Exposure of T cells to SIL (but not SbN or metabolic inhibitors) during virus adsorption blocked HIV infection. Thus, both SbN and SIL rapidly perturb T cell metabolism in vitro, which may account for its anti-inflammatory and anti-proliferative effects that arise with prolonged exposure of cells. However, the metabolic effects are not involved in SIL's unique ability to block HIV entry. - Highlights: • Silibinin (SbN) and Legalon-SIL (SIL) are cytoprotective mixtures of natural products. • SbN and SIL reduce T cell oxidative phosphorylation and glycolysis in vitro. • SIL but not SbN blocks entry of multiple HIV isolates into T cells in vitro. • SIL's suppression of HIV appears independent of its effects on T cell metabolism. • Metabolic effects of SIL and SbN may be relevant in inflammatory diseases.

  9. Inhibition of HIV by Legalon-SIL is independent of its effect on cellular metabolism

    International Nuclear Information System (INIS)

    McClure, Janela; Margineantu, Daciana H.; Sweet, Ian R.; Polyak, Stephen J.

    2014-01-01

    In this report, we further characterized the effects of silibinin (SbN), derived from milk thistle extract, and Legalon-SIL (SIL), a water-soluble derivative of SbN, on T cell metabolism and HIV infection. We assessed the effects of SbN and SIL on peripheral blood mononuclear cells (PBMC) and CEM-T4 cells in terms of cellular growth, ATP content, metabolism, and HIV infection. SIL and SbN caused a rapid and reversible (upon removal) decrease in cellular ATP levels, which was associated with suppression of mitochondrial respiration and glycolysis. SbN, but not SIL inhibited glucose uptake. Exposure of T cells to SIL (but not SbN or metabolic inhibitors) during virus adsorption blocked HIV infection. Thus, both SbN and SIL rapidly perturb T cell metabolism in vitro, which may account for its anti-inflammatory and anti-proliferative effects that arise with prolonged exposure of cells. However, the metabolic effects are not involved in SIL's unique ability to block HIV entry. - Highlights: • Silibinin (SbN) and Legalon-SIL (SIL) are cytoprotective mixtures of natural products. • SbN and SIL reduce T cell oxidative phosphorylation and glycolysis in vitro. • SIL but not SbN blocks entry of multiple HIV isolates into T cells in vitro. • SIL's suppression of HIV appears independent of its effects on T cell metabolism. • Metabolic effects of SIL and SbN may be relevant in inflammatory diseases

  10. Curcumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications.

    Science.gov (United States)

    Balasubramanyam, M; Koteswari, A Adaikala; Kumar, R Sampath; Monickaraj, S Finny; Maheswari, J Uma; Mohan, V

    2003-12-01

    There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric, Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca2+ influx suggest that a rise in cytosolic Ca2+ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca2+ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.

  11. Dextran Sulfate Sodium Inhibits Alanine Synthesis in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Carolyn M. Slupsky

    2011-04-01

    Full Text Available To understand and characterize the pathogenic mechanisms of inflammatory bowel disease, dextran sulfate sodium (DSS has been used to induce acute and chronic colitis in animal models by causing intestinal epithelium damage. The mechanism of action of DSS in producing this outcome is not well understood. In an effort to understand how DSS might impact epithelial cell metabolism, we studied the intestinal epithelial cell line Caco-2 incubated with 1% DSS over 56 hours using 1H NMR spectroscopy. We observed no difference in cell viability as compared to control cultures, and an approximately 1.5-fold increase in IL-6 production upon incubation with 1% DSS. The effect on Caco-2 cell metabolism as measured through changes in the concentration of metabolites in the cell supernatant included a three-fold decrease in the concentration of alanine. Given that the concentrations of other amino acids in the cell culture supernatant were not different between treated and control cultures over 56 hours suggest that DSS inhibits alanine synthesis, specifically alanine aminotransferase, without affecting other key metabolic pathways. The importance of alanine aminotransferase in inflammatory bowel disease is discussed.

  12. ROCK inhibition as a therapy for spinal muscular atrophy: understanding the repercussions on multiple cellular targets

    Science.gov (United States)

    Coque, Emmanuelle; Raoul, Cédric; Bowerman, Mélissa

    2014-01-01

    Spinal muscular atrophy (SMA) is the most common genetic disease causing infant death, due to an extended loss of motoneurons. This neuromuscular disorder results from deletions and/or mutations within the Survival Motor Neuron 1 (SMN1) gene, leading to a pathological decreased expression of functional full-length SMN protein. Emerging studies suggest that the small GTPase RhoA and its major downstream effector Rho kinase (ROCK), which both play an instrumental role in cytoskeleton organization, contribute to the pathology of motoneuron diseases. Indeed, an enhanced activation of RhoA and ROCK has been reported in the spinal cord of an SMA mouse model. Moreover, the treatment of SMA mice with ROCK inhibitors leads to an increased lifespan as well as improved skeletal muscle and neuromuscular junction pathology, without preventing motoneuron degeneration. Although motoneurons are the primary target in SMA, an increasing number of reports show that other cell types inside and outside the central nervous system contribute to SMA pathogenesis. As administration of ROCK inhibitors to SMA mice was systemic, the improvement in survival and phenotype could therefore be attributed to specific effects on motoneurons and/or on other non-neuronal cell types. In the present review, we will present the various roles of the RhoA/ROCK pathway in several SMA cellular targets including neurons, myoblasts, glial cells, cardiomyocytes and pancreatic cells as well as discuss how ROCK inhibition may ameliorate their health and function. It is most likely a concerted influence of ROCK modulation on all these cell types that ultimately lead to the observed benefits of pharmacological ROCK inhibition in SMA mice. PMID:25221469

  13. Fisetin inhibits cellular proliferation and induces mitochondria-dependent apoptosis in human gastric cancer cells.

    Science.gov (United States)

    Sabarwal, Akash; Agarwal, Rajesh; Singh, Rana P

    2017-02-01

    The anticancer effects of fisetin, a dietary agent, are largely unknown against human gastric cancer. Herein, we investigated the mechanisms of fisetin-induced inhibition of growth and survival of human gastric carcinoma AGS and SNU-1 cells. Fisetin (25-100 μM) caused significant decrease in the levels of G1 phase cyclins and CDKs, and increased the levels of p53 and its S15 phosphorylation in gastric cancer cells. We also observed that growth suppression and death of non-neoplastic human intestinal FHs74int cells were minimally affected by fisetin. Fisetin strongly increased apoptotic cells and showed mitochondrial membrane depolarization in gastric cancer cells. DNA damage was observed as early as 3 h after fisetin treatment which was accompanied with gamma-H2A.X(S139) phosphorylation and cleavage of PARP. Fisetin-induced apoptosis was observed to be independent of p53. DCFDA and MitoSOX analyses showed an increase in mitochondrial ROS generation in time- and dose-dependent fashion. It also increased cellular nitrite and superoxide generation. Pre-treatment with N-acetyl cysteine (NAC) inhibited ROS generation and also caused protection from fisetin-induced DNA damage. The formation of comets were observed in only fisetin treated cells which was blocked by NAC pre-treatment. Further investigation of the source of ROS, using mitochondrial respiratory chain (MRC) complex inhibitors, suggested that fisetin caused ROS generation specifically through complex I. Collectively, these results for the first time demonstrated that fisetin possesses anticancer potential through ROS production most likely via MRC complex I leading to apoptosis in human gastric carcinoma cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. ROCK inhibition as a therapy for spinal muscular atrophy: understanding the repercussions on multiple cellular targets

    Directory of Open Access Journals (Sweden)

    Emmanuelle eCoque

    2014-08-01

    Full Text Available Spinal muscular atrophy (SMA is the most common genetic disease causing infant death, due to an extended loss of motoneurons. This neuromuscular disorder results from deletions and/or mutations within the surviving motor neuron 1 (SMN1 gene, leading to a pathological decreased expression of functional full-length SMN protein. Emerging studies suggest that the small GTPase RhoA and its major downstream effector Rho kinase (ROCK, which both play an instrumental role in cytoskeleton organization, contribute to the pathology of motoneuron diseases. Indeed, an enhanced activation of RhoA and ROCK has been reported in the spinal cord of an SMA mouse model. Moreover, the treatment of SMA mice with ROCK inhibitors leads to an increased lifespan as well as improved skeletal muscle and neuromuscular junction pathology, without preventing motoneuron degeneration. Although motoneurons are the primary target in SMA, an increasing number of reports show that other cell types inside and outside the central nervous system contribute to SMA pathogenesis. As administration of ROCK inhibitors to SMA mice was systemic, the improvement in survival and phenotype could therefore be attributed to specific effects on motoneurons and/or on other non-neuronal cell types. In the present review, we will present the various roles of the RhoA/ROCK pathway in several SMA cellular targets including neurons, myocytes, glial cells, cardiomyocytes and pancreatic cells as well as discuss how ROCK inhibition may ameliorate their health and function. It is most likely a concerted influence of ROCK modulation on all these cell types that ultimately lead to the observed benefits of pharmacological ROCK inhibition in SMA mice.

  15. Melatonin Promotes Apoptosis of Oxaliplatin-resistant Colorectal Cancer Cells Through Inhibition of Cellular Prion Protein.

    Science.gov (United States)

    Lee, Jun Hee; Yoon, Yeo Min; Han, Yong-Seok; Yun, Chul Won; Lee, Sang Hun

    2018-04-01

    Drug resistance restricts the efficacy of chemotherapy in colorectal cancer. However, the detailed molecular mechanism of drug resistance in colorectal cancer cells remains unclear. The level of cellular prion protein (PrP C ) in oxaliplatin-resistant colorectal cancer (SNU-C5/Oxal-R) cells was assessed. PrP C level in SNU-C5/Oxal-R cells was significantly increased compared to that in wild-type (SNU-C5) cells. Superoxide dismutase and catalase activities were higher in SNU-C5/Oxal-R cells than in SNU-C5 cells. Treatment of SNU-C5/Oxal-R cells with oxaliplatin and melatonin reduced PrP C expression, while suppressing antioxidant enzyme activity and increasing superoxide anion generation. In SNU-C5/Oxal-R cells, endoplasmic reticulum stress and apoptosis were significantly increased following co-treatment with oxaliplatin and melatonin compared to treatment with oxaliplatin alone. Co-treatment with oxaliplatin and melatonin increased endoplasmic reticulum stress in and apoptosis of SNU-C5/Oxal-R cells through inhibition of PrP C , suggesting that PrP C could be a key molecule in oxaliplatin resistance of colorectal cancer cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  16. Multiple Molecular and Cellular Mechanisms of Action of Lycopene in Cancer Inhibition

    Directory of Open Access Journals (Sweden)

    Cristina Trejo-Solís

    2013-01-01

    Full Text Available Epidemiological studies suggest that including fruits, vegetables, and whole grains in regular dietary intake might prevent and reverse cellular carcinogenesis, reducing the incidence of primary tumours. Bioactive components present in food can simultaneously modulate more than one carcinogenic process, including cancer metabolism, hormonal balance, transcriptional activity, cell-cycle control, apoptosis, inflammation, angiogenesis and metastasis. Some studies have shown an inverse correlation between a diet rich in fruits, vegetables, and carotenoids and a low incidence of different types of cancer. Lycopene, the predominant carotenoid found in tomatoes, exhibits a high antioxidant capacity and has been shown to prevent cancer, as evidenced by clinical trials and studies in cell culture and animal models. In vitro studies have shown that lycopene treatment can selectively arrest cell growth and induce apoptosis in cancer cells without affecting normal cells. In vivo studies have revealed that lycopene treatment inhibits tumour growth in the liver, lung, prostate, breast, and colon. Clinical studies have shown that lycopene protects against prostate cancer. One of the main challenges in cancer prevention is the integration of new molecular findings into clinical practice. Thus, the identification of molecular biomarkers associated with lycopene levels is essential for improving our understanding of the mechanisms underlying its antineoplastic activity.

  17. Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf

    2012-01-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis...

  18. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

    Directory of Open Access Journals (Sweden)

    Anna Lundin

    2014-05-01

    Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  19. Dietary administration of scallion extract effectively inhibits colorectal tumor growth: cellular and molecular mechanisms in mice.

    Directory of Open Access Journals (Sweden)

    Palanisamy Arulselvan

    Full Text Available Colorectal cancer is a common malignancy and a leading cause of cancer death worldwide. Diet is known to play an important role in the etiology of colon cancer and dietary chemoprevention is receiving increasing attention for prevention and/or alternative treatment of colon cancers. Allium fistulosum L., commonly known as scallion, is popularly used as a spice or vegetable worldwide, and as a traditional medicine in Asian cultures for treating a variety of diseases. In this study we evaluated the possible beneficial effects of dietary scallion on chemoprevention of colon cancer using a mouse model of colon carcinoma (CT-26 cells subcutaneously inoculated into BALB/c mice. Tumor lysates were subjected to western blotting for analysis of key inflammatory markers, ELISA for analysis of cytokines, and immunohistochemistry for analysis of inflammatory markers. Metabolite profiles of scallion extracts were analyzed by LC-MS/MS. Scallion extracts, particularly hot-water extract, orally fed to mice at 50 mg (dry weight/kg body weight resulted in significant suppression of tumor growth and enhanced the survival rate of test mice. At the molecular level, scallion extracts inhibited the key inflammatory markers COX-2 and iNOS, and suppressed the expression of various cellular markers known to be involved in tumor apoptosis (apoptosis index, proliferation (cyclin D1 and c-Myc, angiogenesis (VEGF and HIF-1α, and tumor invasion (MMP-9 and ICAM-1 when compared with vehicle control-treated mice. Our findings may warrant further investigation of the use of common scallion as a chemopreventive dietary agent to lower the risk of colon cancer.

  20. Standardized Kaempferia parviflora Extract Inhibits Intrinsic Aging Process in Human Dermal Fibroblasts and Hairless Mice by Inhibiting Cellular Senescence and Mitochondrial Dysfunction

    Directory of Open Access Journals (Sweden)

    Ji-Eun Park

    2017-01-01

    Full Text Available Intrinsic skin aging is a complex biological phenomenon mainly caused by cellular senescence and mitochondrial dysfunction. This study evaluated the inhibitory effect of Kaempferia parviflora Wall ex. Baker ethanol extract (KPE on H2O2-stimulated cellular senescence and mitochondrial dysfunction both in vitro and in vivo. KPE significantly increased cell growth and suppressed senescence-associated β-galactosidase activation. KPE inhibited the expression of cell-cycle inhibitors (p53, p21, p16, and pRb and stimulated the expression of cell-cycle activators (E2F1 and E2F2. H2O2-induced hyperactivation of the phosphatidylinositol 3-kinase/protein kinase B (AKT signaling pathway was suppressed by KPE through regulated expression of forkhead box O3a (FoxO3a and mammalian target of rapamycin (mTOR. KPE attenuated inflammatory mediators (interleukin-6 (IL-6, IL-8, nuclear factor kappa B (NF-κB, and cyclooxygenase-2 (COX-2 and increased the mRNA expression of PGC-1α, ERRα, NRF1, and Tfam, which modulate mitochondrial biogenesis and function. Consequently, reduced ATP levels and increased ROS level were also reversed by KPE treatment. In hairless mice, KPE inhibited wrinkle formation, skin atrophy, and loss of elasticity by increasing the collagen and elastic fibers. The results indicate that KPE prevents intrinsic aging process in hairless mice by inhibiting cellular senescence and mitochondrial dysfunction, suggesting its potential as a natural antiaging agent.

  1. Inhibition of fatty acid synthesis in isolated adipocytes by 5-(tetradecyloxy)-2-furoic acid.

    Science.gov (United States)

    Halvorson, D L; McCune, S A

    1984-11-01

    The compound 5-(tetradecyloxy)-2-furoic acid (TOFA), a hypolipidemic agent, inhibits fatty acid synthesis, lactate and pyruvate accumulation and CO2 release in isolated rat adipocytes. TOFA stimulates the accumulation of citrate. ATP levels are not lowered by TOFA. In comparison with the natural fatty acid, oleate, TOFA exhibited a much greater inhibitory effect on lipogenesis. TOFyl-CoA formation within intact adipocytes was demonstrated. Although not inhibited by TOFA, acetyl-CoA carboxylase is inhibited by TOFyl-CoA. It is proposed that many of the metabolic effects of TOFA in isolated adipocytes can be explained by TOFyl-CoA inhibition of acetyl-CoA carboxylase. TOFA inhibits glycolysis as a secondary event with the primary event of inhibition of fatty acid synthesis causing an accumulation of citrate which is an inhibitor of phosphofructokinase.

  2. Cellular Development Associated with Induced Mycotoxin Synthesis in the Filamentous Fungus Fusarium graminearum

    Science.gov (United States)

    Menke, Jon; Weber, Jakob; Broz, Karen; Kistler, H. Corby

    2013-01-01

    Several species of the filamentous fungus Fusarium colonize plants and produce toxic small molecules that contaminate agricultural products, rendering them unsuitable for consumption. Among the most destructive of these species is F. graminearum, which causes disease in wheat and barley and often infests the grain with harmful trichothecene mycotoxins. Synthesis of these secondary metabolites is induced during plant infection or in culture in response to chemical signals. Our results show that trichothecene biosynthesis involves a complex developmental process that includes dynamic changes in cell morphology and the biogenesis of novel subcellular structures. Two cytochrome P-450 oxygenases (Tri4p and Tri1p) involved in early and late steps in trichothecene biosynthesis were tagged with fluorescent proteins and shown to co-localize to vesicles we provisionally call “toxisomes.” Toxisomes, the inferred site of trichothecene biosynthesis, dynamically interact with motile vesicles containing a predicted major facilitator superfamily protein (Tri12p) previously implicated in trichothecene export and tolerance. The immediate isoprenoid precursor of trichothecenes is the primary metabolite farnesyl pyrophosphate. Changes occur in the cellular localization of the isoprenoid biosynthetic enzyme HMG CoA reductase when cultures non-induced for trichothecene biosynthesis are transferred to trichothecene biosynthesis inducing medium. Initially localized in the cellular endomembrane system, HMG CoA reductase, upon induction of trichothecene biosynthesis, increasingly is targeted to toxisomes. Metabolic pathways of primary and secondary metabolism thus may be coordinated and co-localized under conditions when trichothecene biosynthesis occurs. PMID:23667578

  3. Antidepressant stimulation of CDP-diacylglycerol synthesis does not require monoamine reuptake inhibition

    Directory of Open Access Journals (Sweden)

    Aboukhatwa Marwa A

    2010-01-01

    Full Text Available Abstract Background Recent studies demonstrate that diverse antidepressant agents increase the cellular production of the nucleolipid CDP-diacylglycerol and its synthetic derivative, phosphatidylinositol, in depression-relevant brain regions. Pharmacological blockade of downstream phosphatidylinositide signaling disrupted the behavioral antidepressant effects in rats. However, the nucleolipid responses were resistant to inhibition by serotonin receptor antagonists, even though antidepressant-facilitated inositol phosphate accumulation was blocked. Could the neurochemical effects be additional to the known effects of the drugs on monoamine transmitter transporters? To examine this question, we tested selected agents in serotonin-depleted brain tissues, in PC12 cells devoid of serotonin transporters, and on the enzymatic activity of brain CDP-diacylglycerol synthase - the enzyme that catalyzes the physiological synthesis of CDP-diacylglycerol. Results Imipramine, paroxetine, and maprotiline concentration-dependently increased the levels of CDP-diacylglycerol and phosphatidylinositides in PC12 cells. Rat forebrain tissues depleted of serotonin by pretreatment with p-chlorophenylalanine showed responses to imipramine or maprotiline that were comparable to respective responses from saline-injected controls. With fluoxetine, nucleolipid responses in the serotonin-depleted cortex or hippocampus were significantly reduced, but not abolished. Each drug significantly increased the enzymatic activity of CDP-diacylglycerol synthase following incubations with cortical or hippocampal brain tissues. Conclusion Antidepressants probably induce the activity of CDP-diacylglycerol synthase leading to increased production of CDP-diacylglycerol and facilitation of downstream phosphatidylinositol synthesis. Phosphatidylinositol-dependent signaling cascades exert diverse salutary effects in neural cells, including facilitation of BDNF signaling and neurogenesis. Hence

  4. Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives.

    Science.gov (United States)

    Sanchez-Moreno, M; Ortega, J E; Valero, A

    1989-12-01

    High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat (Capra hircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

  5. Atorvastatin inhibits insulin synthesis by inhibiting the Ras/Raf/ERK/CREB pathway in INS-1 cells

    Science.gov (United States)

    Sun, Hongxi; Li, Yu; Sun, Bei; Hou, Ningning; Yang, Juhong; Zheng, Miaoyan; Xu, Jie; Wang, Jingyu; Zhang, Yi; Zeng, Xianwei; Shan, Chunyan; Chang, Bai; Chen, Liming; Chang, Baocheng

    2016-01-01

    Abstract Backround: Type 2 diabetes has become a global epidemic disease. Atorvastatin has become a cornerstone in the prevention and treatment of atherosclerosis. However, increasing evidence showed that statins can dose-dependently increase the risk of diabetes mellitus. The mechanism is not clear. Objective: The Ras complex pathway (Ras/Raf/extracellular signal-regulated kinase [ERK]/cAMP response element-binding protein [CREB]) is the major pathway that regulates the gene transcription. Except for the inhibition of cholesterol synthesis by inhibiting the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-COA) reductase, statins can also downregulate the phosphorylation of a series of downstream substrates including the key proteins of the Ras complex pathway, therefore may inhibit the insulin syntheses in pancreatic beta cells. In our study, we investigated the inhibitory effect and the underlying mechanism of atorvastatin on insulin synthesis in rat islets. Methods: Islets were isolated from Wistar rats and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. The insulin content in the medium was measured by radioimmunoassay before and after the treatment of 50 μM atorvastatin. Effect of atorvastatin on the expression of insulin message Ribonucleic acid (mRNA) in pancreatic islet beta cells was also detected using quantitative real-time polymerase chain reaction. Western blotting was used to explore the possible role of the Ras complex pathway (Ras/Raf/ERK/CREB) in atorvastatin-inhibited insulin synthesis. The effects of atorvastatin on the binding of nuclear transcription factor p-CREB with CRE in INS-1 cells were examined via chromatin immunoprecipitation assay. Results: Compared with the control group, the insulin level decreased by 27.1% at 24 hours after atorvastatin treatment. Atorvastatin inhibited insulin synthesis by decreasing insulin mRNA expression of pancreatic islet beta cells. The activities of Ras, Raf-1, and p-CREB in the Ras complex

  6. 17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

    Science.gov (United States)

    Joshi, Sandeep S; Jiang, Shunlin; Unni, Emmanual; Goding, Stephen R; Fan, Tao; Antony, Paul A; Hornyak, Thomas J

    2018-01-01

    Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of

  7. 17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model

    Science.gov (United States)

    Unni, Emmanual; Goding, Stephen R.; Fan, Tao; Antony, Paul A.; Hornyak, Thomas J.

    2018-01-01

    Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of

  8. DNA-synthesis inhibition and repair DNA-synthesis in CHO Ade- C cells: An alternative approach to genotoxicity testing

    International Nuclear Information System (INIS)

    Slamenova, D.; Papsova, E.; Gabelova, A.; Dusinska, M.; Collins, A.; Wsolova, L.

    1997-01-01

    We describe an alternative assay to determine genotoxicity. Its main feature is that it combines two measures in a single experiment; the inhibition of replicative DNA synthesis together with the stimulation of DNA repair. We show that, in tests of four different genotoxic agents, the assay gives results that are entirely consistent with what is known about the mode of action of these agents. In addition, we have demonstrated that chemical carcinogens requiring metabolic activation can be examined using a standard procedure of incubation with a microsomal activating fraction. We consider the combined assay for DNA synthesis inhibition and repair synthesis to be a useful way for the rapid pre-screening of chemicals suspected of genotoxic activity on the level of mammalian cells. (author)

  9. Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light

    International Nuclear Information System (INIS)

    Painter, R.B.

    1985-01-01

    The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion. (orig.)

  10. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  11. Ceruloplasmin Oxidation, a Feature of Parkinson's Disease CSF, Inhibits Ferroxidase Activity and Promotes Cellular Iron Retention

    KAUST Repository

    Olivieri, S.; Conti, A.; Iannaccone, S.; Cannistraci, C. V.; Campanella, A.; Barbariga, M.; Codazzi, F.; Pelizzoni, I.; Magnani, G.; Pesca, M.; Franciotta, D.; Cappa, S. F.; Alessio, M.

    2011-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by oxidative stress and CNS iron deposition. Ceruloplasmin is an extracellular ferroxidase that regulates cellular iron loading and export, and hence protects tissues from oxidative

  12. Heavy ion effects on yeast: Inhibition of ribosomal RNA synthesis

    International Nuclear Information System (INIS)

    Weber, K.J.; Schneider, E.; Kiefer, J.; Kraft, G.

    1990-01-01

    Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated

  13. Nitric oxide inhibits glycogen synthesis in isolated rat hepatocytes

    NARCIS (Netherlands)

    Sprangers, F.; Sauerwein, H. P.; Romijn, J. A.; van Woerkom, G. M.; Meijer, A. J.

    1998-01-01

    There is increasing evidence for the existence of intrahepatic regulation of glucose metabolism by Kupffer cell products. Nitric oxide (NO) is known to inhibit gluconeogenic flux through pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, NO may also influence glucose metabolism at

  14. Regulation of apoptosis, induced by phosphatidylcholine synthesis inhibition

    NARCIS (Netherlands)

    Sanden, Michiel Henrik Marie van der

    2004-01-01

    PC is the most abundant phospholipid in cellular membranes of mammalian tissues. In addition to its structural role in membranes and lipoproteins, PC functions as a major source of intracellular signalling molecules. All eukaryotic cell types and tissues display unique and stable profiles of PC and

  15. Cellulose synthesis inhibition, cell expansion, and patterns of cell wall deposition in Nitella internodes

    International Nuclear Information System (INIS)

    Richmond, P.A.; Metraux, J.P.

    1984-01-01

    The authors have investigated the pattern of wall deposition and maturation and correlated it with cell expansion and cellulose biosynthesis. The herbicide 2,6-dichlorobenzonitrile (DCB) was found to be a potent inhibitor of cellulose synthesis, but not of cell expansion in Nitella internodal cells. Although cellulose synthesis is inhibited during DCB treatment, matrix substances continue to be synthesized and deposited. The inhibition of cellulose microfibril deposition can be demonstrated by various techniques. These results demonstrate that matrix deposition is by apposition, not by intussusception, and that the previously deposited wall moves progressively outward while stretching and thinning as a result of cell expansion

  16. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    International Nuclear Information System (INIS)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-01-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

  17. Novel vanillin derivatives: Synthesis, anti-oxidant, DNA and cellular protection properties.

    Science.gov (United States)

    Scipioni, Matteo; Kay, Graeme; Megson, Ian; Kong Thoo Lin, Paul

    2018-01-01

    Antioxidants have been the subject of intense research interest mainly due to their beneficial properties associated with human health and wellbeing. Phenolic molecules, such as naturally occurring Resveratrol and Vanillin, are well known for their anti-oxidant properties, providing a starting point for the development of new antioxidants. Here we report, for the first time, the synthesis of a number of new vanillin through the reductive amination reaction between vanillin and a selection of amines. All the compounds synthesised, exhibited strong antioxidant properties in DPPH, FRAP and ORAC assays, with compounds 1b and 2c being the most active. The latter also demonstrated the ability to protect plasmid DNA from oxidative damage in the presence of the radical initiator AAPH. At cellular level, neuroblastoma SH-SY5Y cells were protected from oxidative damage (H 2 O 2 , 400 μM) with both 1b and 2c. The presence of a tertiary amino group, along with the number of vanillin moieties in the molecule contribute for the antioxidant activity. Furthermore, the delocalization of the electron pair of the nitrogen and the presence of an electron donating substituent to enhance the antioxidant properties of this new class of compounds. In our opinion, vanillin derivatives 1b and 2c described in this work can provide a viable platform for the development of antioxidant based therapeutics. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake

    International Nuclear Information System (INIS)

    Parab, Harshala J; Huang, Jing-Hong; Liu, Ru-Shi; Lai, Tsung-Ching; Jan, Yi-Hua; Wang, Jui-Ling; Hsiao, Michael; Chen, Chung-Hsuan; Hwu, Yeu-Kuang; Tsai, Din Ping; Chuang, Shih-Yi; Pang, Jong-Hwei S

    2011-01-01

    The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

  19. Synthesis of hydroxyeicosatetraenoic acids (HETE's) by adrenal glomerulosa cells and incorporation into cellular lipids

    International Nuclear Information System (INIS)

    Campbell, W.B.; Richards, C.F.; Brady, M.T.; Falck, J.R.

    1986-01-01

    The role of lipoxygenase metabolites of arachidonic acid (AA) in the regulation of aldosterone secretion was studied in isolated rat adrenal glomerulosa cells. Cells were incubated with 14 C-AA in the presence of angiotensin (AII). The media was extracted, metabolites isolated by HPLC, and structures of the metabolites determined by UV absorbance and mass spectrometry. The major products were 12- and 15-HETE with lesser amounts of 11- and 5-HETE. When adrenal cells were incubated with 15-, 12- or 5-HPETE or their respective HETE's (0.03-300nM), there was no significant change in basal or AII-stimulated aldosterone release. Cells were incubated with [ 3 H]-AA, -5-HETE, -15-HETE, -12-HETE or -LTB. The cellular lipids were extracted and analyzed by TLC. AA was incorporated into phospholipids (22%), cholesterol esters (50%) and triglycerides (21%). Neither the HETE's or LTB 4 were incorporated into phospholipids. 5-HETE was taken up into di- and mono-glycerides. The rates of incorporation of AA and 5-HETE were similar (+ 1/2 = 10 min). The incorporation of 5-HETE into glycerol esters did not modify the release of aldosterone by the cells. Thus, while adrenal cells synthesize HETE's, these eicosanoids do not appear to alter the synthesis of aldosterone

  20. Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake

    Energy Technology Data Exchange (ETDEWEB)

    Parab, Harshala J; Huang, Jing-Hong; Liu, Ru-Shi [Department of Chemistry, National Taiwan University, Taipei 106, Taiwan (China); Lai, Tsung-Ching; Jan, Yi-Hua; Wang, Jui-Ling; Hsiao, Michael; Chen, Chung-Hsuan [Genomics Research Center, Academia Sinica, Taipei 115, Taiwan (China); Hwu, Yeu-Kuang [Institute of Physics, Academia Sinica, Taipei 115, Taiwan (China); Tsai, Din Ping [Department of Physics, National Taiwan University, Taipei 106, Taiwan (China); Chuang, Shih-Yi; Pang, Jong-Hwei S, E-mail: rsliu@ntu.edu.tw, E-mail: mhsiao@gate.sinica.edu.tw [Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan (China)

    2011-09-30

    The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

  1. Cellular ATP synthesis mediated by type III sodium-dependent phosphate transporter Pit-1 is critical to chondrogenesis.

    Science.gov (United States)

    Sugita, Atsushi; Kawai, Shinji; Hayashibara, Tetsuyuki; Amano, Atsuo; Ooshima, Takashi; Michigami, Toshimi; Yoshikawa, Hideki; Yoneda, Toshiyuki

    2011-01-28

    Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent.

  2. Protein Synthesis Inhibition Blocks Consolidation of an Acrobatic Motor Skill

    Science.gov (United States)

    Kaelin-Lang, Alain; Dichgans, Johannes; Schulz, Jorg B.; Luft, Andreas R.; Buitrago, Manuel M.

    2004-01-01

    To investigate whether motor skill learning depends on de novo protein synthesis, adult rats were trained in an acrobatic locomotor task (accelerating rotarod) for 7 d. Animals were systemically injected with cycloheximide (CHX, 0.5 mg/kg, i.p.) 1 h before sessions 1 and 2 or sessions 2 and 3. Control rats received vehicle injections before…

  3. Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: An indication of cell cycle defects

    International Nuclear Information System (INIS)

    Hannan, Mohammed A.; Kunhi, Mohammed; Einspenner, Michael; Khan, Bashir A.; Al-Sedairy, Sultan

    1994-01-01

    In the present study, both post-irradiation DNA synthesis and G 2 phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by 3 H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G 2 phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G 2 phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G 2 accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cycle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G 2 phase accumulation developed elsewhere in characterizing AT heterozygote-like cell cycle anomaly in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle

  4. Influence of Macromolecular Biosynthesis on Cellular Autolysis in Streptococcus faecalis

    Science.gov (United States)

    Sayare, Mitchel; Daneo-Moore, Lolita; Shockman, Gerald D.

    1972-01-01

    The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 μg/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 μg/ml), tetracycline (0.5 μg/ml), puromycin (25 μg/ml), or 5-azacytidine (5 μg/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 μg/ml) and rifampin (0.01 μg/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis. PMID:4116754

  5. Inhibitors of poly (ADP-ribose) synthesis inhibit the two types of repair of potentially lethal damage

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi; Elkind, M.M.

    1994-01-01

    The purpose of this study was to examine whether 3-amino-benzamide (3ABA), an inhibitor of poly (ADP-ribose) synthesis, inhibits the two types of potentially lethal damage (PLD) repair, termed slow and fast. The fast-type PLD repair was measured by the decrease in survival of V79 Chinese hamster cells by postirradiation treatment with 3ABA. The slow-type PLD repair was measured by the increase in survival by posttreatment with conditioned medium (CM), which became conditioned by growing a crowed culture of cells and supports the slow-type PLD repair. Up to 1 mM 3-ABA inhibited the slow type repair; at doses of 2 mM and above, it inhibited the fast type of PLD repair. There are quantitative differences in cellular effects of 3ABA dependent on concentration. Poly (ADP-ribose) appears to play an important role in the PLD repairs and has little effect on the repair of sublethal damage. 10 refs., 2 figs

  6. A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

  7. Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

    Directory of Open Access Journals (Sweden)

    Mikael S Lindström

    Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

  8. Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

    Science.gov (United States)

    Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

    2012-10-26

    The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

  9. Stochastic cellular automata model of neurosphere growth: Roles of proliferative potential, contact inhibition, cell death, and phagocytosis.

    Science.gov (United States)

    Sipahi, Rifat; Zupanc, Günther K H

    2018-05-14

    Neural stem and progenitor cells isolated from the central nervous system form, under specific culture conditions, clonal cell clusters known as neurospheres. The neurosphere assay has proven to be a powerful in vitro system to study the behavior of such cells and the development of their progeny. However, the theory of neurosphere growth has remained poorly understood. To overcome this limitation, we have, in the present paper, developed a cellular automata model, with which we examined the effects of proliferative potential, contact inhibition, cell death, and clearance of dead cells on growth rate, final size, and composition of neurospheres. Simulations based on this model indicated that the proliferative potential of the founder cell and its progenitors has a major influence on neurosphere size. On the other hand, contact inhibition of proliferation limits the final size, and reduces the growth rate, of neurospheres. The effect of this inhibition is particularly dramatic when a stem cell becomes encapsulated by differentiated or other non-proliferating cells, thereby suppressing any further mitotic division - despite the existing proliferative potential of the stem cell. Conversely, clearance of dead cells through phagocytosis is predicted to accelerate growth by reducing contact inhibition. A surprising prediction derived from our model is that cell death, while resulting in a decrease in growth rate and final size of neurospheres, increases the degree of differentiation of neurosphere cells. It is likely that the cellular automata model developed as part of the present investigation is applicable to the study of tissue growth in a wide range of systems. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Inhibition of acetaminophen oxidation by cimetidine and the effects on glutathione and activated sulphate synthesis rates

    DEFF Research Database (Denmark)

    Dalhoff, K; Poulsen, H E

    1993-01-01

    inhibition of cytochrome P-450 drug oxidation by cimetidine in isolated rat hepatocytes. The synthesis rates of glutathione and PAPS were determined simultaneously by an established method based on trapping of radioactivity (35S) in the prelabelled glutathione and PAPS pools. Preincubation of the hepatocytes...

  11. Inhibition of prostaglandin synthesis after metabolism of menadione by cultured porcine endothelial cells

    International Nuclear Information System (INIS)

    Barchowsky, A.; Tabrizi, K.; Kent, R.S.; Whorton, A.R.

    1989-01-01

    We have examined the effects of menadione on porcine aortic endothelial cell prostaglandin synthesis. Addition of 1-20 microM menadione caused a dose- and time-dependent inhibition of stimulated prostaglandin synthesis with an IC50 of 5 microM at 15 min. Concentrations greater than 100 microM menadione were necessary to increase 51 Cr release from prelabeled cells. Recovery of enzyme inactivated by menadione required a 6-h incubation in 1% serum. In a microsomal preparation, menadione was shown to have no direct effect on conversion of arachidonic acid to prostaglandins. In intact cells menadione caused only a 40% inhibition of the conversion of PGH2 to prostacyclin. Enzymes involved in the incorporation and the release of arachidonic acid were not affected by menadione (20 microM, 15 min). Menadione undergoes oxidation/reduction reactions in intact cells leading to partial reduction of oxygen-forming, reactive oxygen species. In our cells menadione was found to increase KCN-resistant oxygen consumption. Further, an increased accumulation of H 2 O 2 was observed with a time course consistent with menadione-induced inhibition of prostaglandin synthesis. We conclude that menadione at sublethal doses caused inhibition of prostaglandin synthesis. The mechanism involves inactivation of PGH2 synthase by a reactive species resulting from metabolism of menadione by endothelial cells

  12. Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

    Science.gov (United States)

    Kleihues, P.; Magee, P. N.

    1973-01-01

    1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2–3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[14C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA. PMID:4774397

  13. Ebselen inhibits iron-induced tau phosphorylation by attenuating DMT1 up-regulation and cellular iron uptake.

    Science.gov (United States)

    Xie, Ling; Zheng, Wei; Xin, Na; Xie, Jing-Wei; Wang, Tao; Wang, Zhan-You

    2012-08-01

    Dysregulation of iron homeostasis is involved in the pathological process of Alzheimer's disease (AD). We have recently reported that divalent metal transporter 1 (DMT1) is upregulated in an AD transgenic mouse brain, and that silencing of DMT1, which reduces cellular iron influx, results in inhibition of amyloidogenesis in vitro, suggesting a potential target of DMT1 for AD therapy. In the present study, we tested the hypothesis that inhibition of DMT1 with ebselen, a DMT1 transport inhibitor, could affect tau phosphorylation. Human neuroblastoma SH-SY5Y cells were pre-treated with ebselen and then treated with ferrous sulfate (dissolved in ascorbic acid), and the effects of ebselen on tau phosphorylation and the relative signaling pathways were examined. Our results showed that ebselen decreased iron influx, reduced iron-induced ROS production, inhibited the activities of cyclin-dependent kinase 5 and glycogen synthase kinase 3β, and ultimately attenuated the levels of tau phosphorylation at the sites of Thr205, Ser396 and Thr231. The present study indicates that the neuroprotective effect of ebselen on AD is not only related to its antioxidant activity as reported previously, but is also associated with a reduction in tau phosphorylation by inhibition of DMT1. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Inhibition of hydrogenase synthesis by DNA gyrase inhibitors in Bradyrhizobium japonicum

    International Nuclear Information System (INIS)

    Novak, P.D.; Maier, R.J.

    1987-01-01

    Derepression of an uptake hydrogenase in Bradyrhizobium japonicum is dependent on a microaerophilic environment. Addition of DNA gyrase inhibitors during derepression of hydrogenase specifically prevented expression of the hydrogenase enzyme. Antibodies to individual hydrogenase subunits failed to detect the protein after derepression in the presence of inhibitors, although there was no general inhibition of protein synthesis. The general pattern of proteins synthesized from 14 C-labeled amino acids during derepression was no significantly different whether proteins were labeled in the presence or in the absence of gyrase inhibitors. In contrast, if transcription or translation was inhibited by addition of inhibitors of those functions, virtually no proteins were labeled during derepression. This indicated that most of the 14 C-labeled proteins were synthesized de novo during derepression, synthesis of most proteins was unaffected by gyrase inhibitors, and the dependence of hydrogenase synthesis on gyrase activity was a specific one

  15. Adiponectin inhibits insulin function in primary trophoblasts by PPARα-mediated ceramide synthesis.

    Science.gov (United States)

    Aye, Irving L M H; Gao, Xiaoli; Weintraub, Susan T; Jansson, Thomas; Powell, Theresa L

    2014-04-01

    Maternal adiponectin (ADN) levels are inversely correlated with birth weight, and ADN infusion in pregnant mice down-regulates placental nutrient transporters and decreases fetal growth. In contrast to the insulin-sensitizing effects in adipose tissue and muscle, ADN inhibits insulin signaling in the placenta. However, the molecular mechanisms involved are unknown. We hypothesized that ADN inhibits insulin signaling and insulin-stimulated amino acid transport in primary human trophoblasts by peroxisome proliferator-activated receptor-α (PPARα)-mediated ceramide synthesis. Primary human term trophoblast cells were treated with ADN and/or insulin. ADN increased the phosphorylation of p38 MAPK and PPARα. ADN inhibited insulin signaling and insulin-stimulated amino acid transport. This effect was dependent on PPARα, because activation of PPARα with an agonist (GW7647) inhibited insulin signaling and function, whereas PPARα-small interfering RNA reversed the effects of ADN on the insulin response. ADN increased ceramide synthase expression and stimulated ceramide production. C2-ceramide inhibited insulin signaling and function, whereas inhibition of ceramide synthase (with Fumonisin B1) reversed the effects of ADN on insulin signaling and amino acid transport. These findings are consistent with the model that maternal ADN limits fetal growth mediated by activation of placental PPARα and ceramide synthesis, which inhibits placental insulin signaling and amino acid transport, resulting in reduced fetal nutrient availability.

  16. MMS 1001 inhibits melanin synthesis via ERK activation.

    Science.gov (United States)

    Lee, Hyun-E; Song, Jiho; Kim, Su Yeon; Park, Kyoung-Chan; Min, Kyung Hoon; Kim, Dong-Seok

    2013-03-01

    Melanin plays major a role in pigmentation of hair, eyes, and skin in mammals. In this study, the inhibitory effects of MMS 1001 on alpha-MSH-stimulated melanogenesis were investigated in B16F10 melanoma cells. MMS 1001 did not show cytotoxic effects up to 10 microM. Melanin content and intracellular tyrosinase activity were inhibited by MMS 1001 treatment in a dose-dependent manner. In Western blot analysis, MITF expression was decreased by MMS 1001. In addition, tyrosinase expressions were also reduced after MMS 1001 treatment. Further results showed that the phosphorylation of ERK was induced by MMS 1001. Moreover, a specific MEK inhibitor, PD98059, abrogated the inhibitory effects of MMS 1001 on melanin production and tyrosinase expression. These results indicate that the hypopigmentary effects of MMS 1001 resulted from the inhibition of MITF and tyrosinase expression via phosphorylation of ERK. Thus, MMS 1001 could be developed as a new effective skin-whitening agent.

  17. Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

    Science.gov (United States)

    Liu, Long; Tian, Jiao; Nan, Hao; Tian, Mengmeng; Li, Yuan; Xu, Xiaodong; Huang, Baicheng; Zhou, Enmin; Hiscox, Julian A; Chen, Hongying

    2016-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of

  18. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    International Nuclear Information System (INIS)

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of [3H]thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity

  19. Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA

    International Nuclear Information System (INIS)

    Bodell, W.J.; Cleaver, J.E.; Roti Roti, J.L.

    1984-01-01

    The authors have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 0 C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 0 C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus, the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating steps(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 0 C with[ 3 H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 0 C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures. 23 references, 3 figures, 2 tables

  20. Perilipin 1 Mediates Lipid Metabolism Homeostasis and Inhibits Inflammatory Cytokine Synthesis in Bovine Adipocytes

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    Shiqi Zhang

    2018-03-01

    Full Text Available Dairy cows with ketosis displayed lipid metabolic disorder and high inflammatory levels. Adipose tissue is an active lipid metabolism and endocrine tissue and is closely related to lipid metabolism homeostasis and inflammation. Perilipin 1 (PLIN1, an adipocyte-specific lipid-coated protein, may be involved in the above physiological function. The aim of this study is to investigate the role of PLIN1 in lipid metabolism regulation and inflammatory factor synthesis in cow adipocytes. The results showed that PLIN1 overexpression upregulated the expression of fatty acid and triglyceride (TAG synthesis molecule sterol regulator element-binding protein-1c (SREBP-1c and its target genes, diacylglycerol acyltransferase (DGAT 1, and DGAT2, but inhibited the expression of lipolysis enzymes hormone-sensitive lipase (HSL and CGI-58 for adipose triglyceride lipase (ATGL, thus augmenting the fatty acids and TAG synthesis and inhibiting lipolysis. Importantly, PLIN1 overexpression inhibited the activation of the NF-κB inflammatory pathway and decreased the expression and content of tumor necrosis factor alpha (TNF-α, interleukin 1 beta (IL-1β, and interleukin 6 (IL-6 induced by lipopolysaccharide. Conversely, PLIN1 silencing inhibited TAG synthesis, promoted lipolysis, and overinduced the activation of the NF-κB inflammatory pathway in cow adipocytes. In ketotic cows, the expression of PLIN1 was markedly decreased, whereas lipid mobilization, NF-κB pathway, and downstream inflammatory cytokines were overinduced in adipose tissue. Taken together, these results indicate that PLIN1 can maintain lipid metabolism homeostasis and inhibit the NF-κB inflammatory pathway in adipocytes. However, low levels of PLIN1 reduced the inhibitory effect on fat mobilization, NF-κB pathway, and inflammatory cytokine synthesis in ketotic cows.

  1. Perilipin 1 Mediates Lipid Metabolism Homeostasis and Inhibits Inflammatory Cytokine Synthesis in Bovine Adipocytes.

    Science.gov (United States)

    Zhang, Shiqi; Liu, Guowen; Xu, Chuang; Liu, Lei; Zhang, Qiang; Xu, Qiushi; Jia, Hongdou; Li, Xiaobing; Li, Xinwei

    2018-01-01

    Dairy cows with ketosis displayed lipid metabolic disorder and high inflammatory levels. Adipose tissue is an active lipid metabolism and endocrine tissue and is closely related to lipid metabolism homeostasis and inflammation. Perilipin 1 (PLIN1), an adipocyte-specific lipid-coated protein, may be involved in the above physiological function. The aim of this study is to investigate the role of PLIN1 in lipid metabolism regulation and inflammatory factor synthesis in cow adipocytes. The results showed that PLIN1 overexpression upregulated the expression of fatty acid and triglyceride (TAG) synthesis molecule sterol regulator element-binding protein-1c (SREBP-1c) and its target genes, diacylglycerol acyltransferase (DGAT) 1, and DGAT2, but inhibited the expression of lipolysis enzymes hormone-sensitive lipase (HSL) and CGI-58 for adipose triglyceride lipase (ATGL), thus augmenting the fatty acids and TAG synthesis and inhibiting lipolysis. Importantly, PLIN1 overexpression inhibited the activation of the NF-κB inflammatory pathway and decreased the expression and content of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) induced by lipopolysaccharide. Conversely, PLIN1 silencing inhibited TAG synthesis, promoted lipolysis, and overinduced the activation of the NF-κB inflammatory pathway in cow adipocytes. In ketotic cows, the expression of PLIN1 was markedly decreased, whereas lipid mobilization, NF-κB pathway, and downstream inflammatory cytokines were overinduced in adipose tissue. Taken together, these results indicate that PLIN1 can maintain lipid metabolism homeostasis and inhibit the NF-κB inflammatory pathway in adipocytes. However, low levels of PLIN1 reduced the inhibitory effect on fat mobilization, NF-κB pathway, and inflammatory cytokine synthesis in ketotic cows.

  2. Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro.

    Science.gov (United States)

    Lambrecht, Jennifer A; Browne, Beth Ann; Downs, Diana M

    2010-11-05

    The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

  3. Injury-induced inhibition of small intestinal protein and nucleic acid synthesis

    International Nuclear Information System (INIS)

    Carter, E.A.; Hatz, R.A.; Yarmush, M.L.; Tompkins, R.G.

    1990-01-01

    Small intestinal mucosal weight and nutrient absorption are significantly diminished early after cutaneous thermal injuries. Because these intestinal properties are highly dependent on rates of nucleic acid and protein synthesis, in vivo incorporation of thymidine, uridine, and leucine into small intestinal deoxyribonucleic acid, ribonucleic acid, and proteins were measured. Deoxyribonucleic acid synthesis was markedly decreased with the lowest thymidine incorporation in the jejunum (p less than 0.01); these findings were confirmed by autoradiographic identification of radiolabeled nuclei in the intestinal crypts. Protein synthesis was decreased by 6 h postinjury (p less than 0.01) but had returned to normal by 48 h. Consistent with a decreased rate of protein synthesis, ribonucleic acid synthesis was also decreased 18 h postinjury (p less than 0.01). These decreased deoxyribonucleic acid, ribonucleic acid, and protein synthesis rates are not likely a result of ischemia because in other studies of this injury model, intestinal blood flow was not significantly changed by the burn injury. Potentially, factors initiating the acute inflammatory reaction may directly inhibit nucleic acid and protein synthesis and lead to alterations in nutrient absorption and intestinal barrier function after injury

  4. Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles

    International Nuclear Information System (INIS)

    Teimoori, Salma; Seesuay, Watee; Jittavisutthikul, Surasak; Chaisri, Urai; Sookrung, Nitat; Densumite, Jaslan; Saelim, Nawannaporn; Chulanetra, Monrat; Maneewatch, Santi; Chaicumpa, Wanpen

    2016-01-01

    A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials. - Highlights: • Cell penetrable human scFvs (transbodies) to Ebolavirus (EBOV) VP40 were produced. • The transbodies inhibited egress of EBOV-like particles (VLPs) from human hepatocytes. • They interacted with VP40 CTD basic residues important for plasma membrane binding. • And hence interfere with viral matrix assembly and viral progeny budding. • This is the first report on human antibodies that target intracellular EBOV VP40.

  5. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

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    Nan Wang

    2017-05-01

    Full Text Available Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1, a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8, and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2. Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs. Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP, cysteinyl aspartate specific proteases-3 (caspase-3, cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

  6. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis

    OpenAIRE

    Li, Qingli; Lambrechts, Mark J; Zhang, Qiuyang; Liu, Sen; Ge, Dongxia; Yin, Rutie; Xi, Mingrong; You, Zongbing

    2013-01-01

    Qingli Li,1,2 Mark J Lambrechts,1 Qiuyang Zhang,1 Sen Liu,1 Dongxia Ge,1 Rutie Yin,2 Mingrong Xi,2 Zongbing You1 1Departments of Structural and Cellular Biology and Orthopaedic Surgery, Tulane Cancer Center and Louisiana Cancer Research Consortium, Tulane Center for Stem Cell Research and Regenerative Medicine, and Tulane Center for Aging, Tulane University Health Sciences Center, New Orleans, LA, USA; 2Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan Un...

  7. Diethyl 2-(Phenylcarbamoylphenyl Phosphorothioates: Synthesis, Antimycobacterial Activity and Cholinesterase Inhibition

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    Jarmila Vinšová

    2014-05-01

    Full Text Available A new series of 27 diethyl 2-(phenylcarbamoylphenyl phosphorothioates (thiophosphates was synthesized, characterized by NMR, IR and CHN analyses and evaluated against Mycobacterium tuberculosis H37Rv, Mycobacterium avium and two strains of Mycobacterium kansasii. The best activity against M. tuberculosis was found for O-{4-bromo-2-[(3,4-dichlorophenylcarbamoyl]phenyl} O,O-diethyl phosphorothioate (minimum inhibitory concentration of 4 µM. The highest activity against nontuberculous mycobacteria was exhibited by O-(5-chloro-2-{[4-(trifluoromethylphenyl]carbamoyl}-phenyl O,O-diethyl phosphorothioate with MIC values from 16 µM. Prepared thiophosphates were also evaluated against acetylcholinesterase from electric eel and butyrylcholinesterase from equine serum. Their inhibitory activity was compared to that of the known cholinesterases inhibitors galanthamine and rivastigmine. All tested compounds showed a higher (for AChE inhibition and comparable (for BChE inhibition activity to that of rivastigmine, with IC50s within the 8.04 to 20.2 µM range.

  8. Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

    International Nuclear Information System (INIS)

    Ulrich, F.

    1988-01-01

    When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [ 3 H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others,suggest that deoxyribose damages DNA

  9. Inhibition of human arterial smooth muscle (HASM) cell proliferation and collagen synthesis by protamine

    International Nuclear Information System (INIS)

    Drucker, D.E.; Graham, M.F.; Diegelmann, R.F.; Greenfield, L.J.

    1986-01-01

    Atherosclerotic plaques result from vascular smooth muscle cell proliferation and collagen deposition. The authors have been studying factors which modulate HASM cell proliferation and collagen synthesis. HASM cells were isolated from the media of normal human thoracic and infrarenal aortas and grown in vitro. Cell numbers were determined by direct counting and collagen synthesis was measured by incorporation of 3 H-proline into collagenase-digestible protein. In this study, protamine (200 μg/ml) was tested and found to cause a 55% reduction of HASM cell proliferation which was reversible when the cells were returned to control medium or when heparin (100 μg/ml) was added with protamine. Protamine caused a constant 33% decrease in non-collagen protein (NCP) synthesis per cell. In contrast, collagen synthesis was inhibited in dose dependent fashion (88% reduction at 200 μg/ml). Protamine blocks HASM cell proliferation and specifically inhibits collagen production. The exact mechanism of this inhibition is unclear but may be related to a transcriptional event since protamine has a high affinity for DNA

  10. Aqueous fraction from Cuscuta japonica seed suppresses melanin synthesis through inhibition of the p38 mitogen-activated protein kinase signaling pathway in B16F10 cells.

    Science.gov (United States)

    Jang, Ji Yeon; Kim, Ha Neui; Kim, Yu Ri; Choi, Yung Hyun; Kim, Byung Woo; Shin, Hwa Kyoung; Choi, Byung Tae

    2012-05-07

    Semen cuscutae has been used traditionally to treat pimples and alleviate freckles and melasma in Korea. The present study aimed to investigate the inhibitory effect of Cuscuta japonica Choisy seeds on alpha-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis. The aqueous fraction from Semen cuscutae (AFSC) was used to determine anti-melanogenic effects by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay and Western blot analysis for melanin synthesis-related signaling proteins in B16F10 mouse melanoma cells. AFSC markedly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related proteins (TRPs). Moreover, AFSC significantly decreased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK) signaling through the down-regulation of α-MSH-induced cAMP. Furthermore, we confirmed that the specific inhibitor of p38 MAPK (SB203580)-mediated suppressed melanin synthesis and tyrosinase activity was further attenuated by AFSC. AFSC also further decreased SB203580-mediated suppression of MITF and TRP expression. These results indicate that AFSC inhibits p38 MAPK phosphorylation with suppressed cAMP levels and subsequently down-regulate MITF and TRP expression, which results in a marked reduction of melanin synthesis and tyrosinase activity in α-MSH-stimulated B16F10 cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  11. Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats.

    Science.gov (United States)

    Schubring-Giese, Maximilian; Leemburg, Susan; Luft, Andreas Rüdiger; Hosp, Jonas Aurel

    2016-01-01

    Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session.

  12. Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

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    Moina Hasni Ebou

    Full Text Available Diabetes is a major complication of chronic Glucocorticoids (GCs treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1 and 2 (Tph2, leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

  13. Neomycin inhibits PDGF-induced IP3 formation and DNA synthesis but not PDGF-stimulated uptake of inorganic phosphate in C3H/10T1/2 fibroblasts.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Holmsen, H

    1990-09-01

    Porcine PDGF was found to increase [3H]inositol trisphosphate, [3H]thymidine incorporation and 32P-labelling of polyphosphoinositides in C3H/10T1/2 Cl 8 fibroblasts. These responses to PDGF stimulation were all inhibited by 5 mM neomycin, a polycationic aminoglycoside formerly known to inhibit polyphosphoinositide turnover. PDGF also markedly increased the cellular uptake of inorganic [32P]Pi. This response of PDGF was not inhibited by neomycin (5 mM). Thus, neomycin inhibited PDGF-induced IP3 formation, 32P-labelling of polyphosphoinositides and DNA synthesis, but not cellular uptake of inorganic phosphate. These effects of neomycin suggest a bifurcation of the initial part of the PDGF-induced signal transduction, separating at the receptor level or before phospholipase C activation.

  14. MECHANISM OF ACTION OF ANTIBIOTICS WHICH INHIBIT SYNTHESIS OF BACTERIAL CELL WALL

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    Indira Mujezinović

    2013-03-01

    Full Text Available Bacterial cell possess a cell wall, which is a main difference from mammalian cells. Its basic function is to provide the strength of bacteria, keeps its shape and provides an unusually high internal osmotic pressure. Synthesis of (construction of bacterial cell wall occurs in at least three phases. All of these three phases can be influence by a variety of antibiotics in way to inhibit its synthesis. The most important drugs that act in this manner are ß-lactam antibiotics (penicillins, cephalosporins, cephamycins and other ß-lactams. They interfere with the synthesis of the bacterial cell wall peptidoglycan. After attachment to penicillin binding proteins (PBP on bacteria, they inhibit the transpeptidation enzyme that cross-links the peptide chain attached to the backbone of the peptidoglycan. The final bactericidal event is the inactivation of an inhibitor of autolytic enzymes in the cell wall, wich leads to lysis of the bacteria. Vancomycin inhibits the release of the building block unit from the carrier, thus preventing its addition to the growing end of the peptidoglycan. Cycloserine, which is a structural analogue of D-alanine, prevents the addition of the two terminal alanine residue to the initial tripeptide side-chain on N-acetylmuramic acid by competitive inhibition. Bacitracin interferes with the regeneration of the lipid carrier by blocking its dephosphorylation. Key words: bacterial cell wall, paptidoglycan, antibiotics, ß-lactams

  15. Inhibition of DNA synthesis and radiosensitization effects of thalidomide on esophageal carcinoma TE1 cells

    International Nuclear Information System (INIS)

    Yu Jingping; Sun Suping; Sun Zhiqiang; Sun Meiling; Liu Fenju

    2010-01-01

    Objective: To explore the radiosensitization effect of thalidomide combined with X-ray on esophageal carcinoma TE1 cells. Methods: Cell scratch assay was used to detect the inhibition ability of different concentration of Thalidomide on cell invasion and metastasis. H 3 -TdR incorporation assay was used to investigate the inhibition of DNA synthesis in TE1 cells by treated with Thalidomide singly or combination with X-rays. The colony formation assay was used to analyze the radiosensitization of Thalidomide effect on TE1 cells. Results: Thalidomide had obvious inhibition effect on TE1 cell metastasis, DNA synthesis and colony formation, which were correlated with drug concentration. The values D 0 , D q and SF 2 in TE1 cells were gradually decreased with thalidomide concentration increased. When the concentration of thalidomide was 100μg/ml, the SER D 0 and SER D 0 and SER D q were (1.4±0.2) and (1.5±0.1), respectively, While the concentration of thalidomide was 150 μg/ml, the SER D 0 and SER D q were (1.5±0.2) and (1.8±0.2), respectively. Conclusions: Thalidomide could inhibit TE1 cell invasion, metastasis, DNA synthesis, and significantly enhance the radiosensitizing effect on esophageal carcinoma TE1 cells. (authors)

  16. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

    Directory of Open Access Journals (Sweden)

    Hardy Michele E

    2008-01-01

    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  17. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    International Nuclear Information System (INIS)

    Polatnick, J.; Wool, S.H.

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [ 3 H] uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity. (Author)

  18. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    Energy Technology Data Exchange (ETDEWEB)

    Polatnick, J.; Wool, S.H. (United States Department of Agriculture, Science and Education, Greenport, New York (USA). Agricultural Research, Plum Island Animal Disease Center)

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated (/sup 3/H) uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity.

  19. DCB-3503, a tylophorine analog, inhibits protein synthesis through a novel mechanism.

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    Ying Wang

    Full Text Available BACKGROUND: DCB-3503, a tylophorine analog, inhibits the growth of PANC-1 (human pancreatic ductal cancer cell line and HepG2 (human hepatocellular cancer cell line tumor xenografts in nude mice. The inhibition of growth leads to cancer cell differentiation instead of cell death. However, the mechanisms of action of tylophorine analogs is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that DCB-3503 suppresses the expression of pro-oncogenic or pro-survival proteins with short half-lives, including cyclin D1, survivin, beta-catenin, p53, and p21, without decreasing their mRNA levels. Proteasome inhibitor reversed the inhibitory effect of DCB-3503 on expression of these proteins. DCB-3503 inhibited the incorporation of radiolabeled amino acid and thymidine, and to a much lesser degree of uridine, in a panel of cell lines. The mechanism of inhibition of protein synthesis is different from that of cycloheximide (CHX as assayed in cell culture and HeLa in vitro translation system. Furthermore, in contrast to rapamycin, DCB-3503 does not affect protein synthesis through the mTOR pathway. DCB-3503 treatment shifts the sedimentation profiles of ribosomes and mRNAs towards the polysomal fractions while diminishing monosome abundance, indicative of the inhibition of the elongation step of protein synthesis. Preferential down regulation of several studied proteins under these conditions is likely due to the relative short half-lives of these proteins. CONCLUSION/SIGNIFICANCE: The inhibitory effect of DCB-3503 on translation is apparently distinct from any of the current anticancer compounds targeting protein synthesis. Translation inhibitors with novel mechanism could complement current chemotherapeutic agents for the treatment of human cancers and suppress the occurrence of drug resistance.

  20. Indomethacin Inhibits Cancer Cell Migration via Attenuation of Cellular Calcium Mobilization

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    Ke-Li Tsai

    2013-06-01

    Full Text Available Non-steroidal anti-inflammatory drugs (NSAIDs were shown to reduce the risk of colorectal cancer recurrence and are widely used to modulate inflammatory responses. Indomethacin is an NSAID. Herein, we reported that indomethacin can suppress cancer cell migration through its influence on the focal complexes formation. Furthermore, endothelial growth factor (EGF-mediated Ca2+ influx was attenuated by indomethacin in a dose dependent manner. Our results identified a new mechanism of action for indomethacin: inhibition of calcium influx that is a key determinant of cancer cell migration.

  1. Cockayne's syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, A.R.; Thompson, A.F.; Harcourt, S.A. (Medical Research Council, Brighton (United Kingdom). Cell Mutation Unit); Stefanini, Miria (Consiglio Nazionale delle Ricerche, Pavia (Italy). Ist. di Genetica Biochimica ed Evoluzionistica); Norris, P.G. (Addenbrooke' s Hospital, Cambridge (United Kingdom))

    1993-08-01

    Cockayne's syndrome (CS) is a rare autosomal recessive disorder with dwarfism, mental retardation, and otherwise clinically heterogeneous features. In cultured CS fibroblasts, the failure of RNA synthesis to recover to normal rates after UV-C irradiation provides a useful and relatively simple diagnostic test. We have measured post-UV-C RNA synthesis in 52 patients for whom a clinical diagnosis of CS was considered a possibility. Twenty-nine patients showed the defect characteristic of CS cells, and 23 had a normal response. We have attempted to correlate the cellular diagnosis with the different clinical features of the disorder. Clinical details of the patients were obtained from referring clinicians in the form of a questionnaire. Our results show that, apart from the cardinal features of dwarfism and mental retardation, sun sensitivity correlated best with a positive cellular diagnosis. Pigmentary retinopathy, gait defects, and dental caries were also good positive indicators, although several patients with a positive cellular diagnosis did not have these features. (Author).

  2. Inhibition of prostaglandin synthesis after metabolism of menadione by cultured porcine endothelial cells.

    OpenAIRE

    Barchowsky, A; Tabrizi, K; Kent, R S; Whorton, A R

    1989-01-01

    We have examined the effects of menadione on porcine aortic endothelial cell prostaglandin synthesis. Addition of 1-20 microM menadione caused a dose- and time-dependent inhibition of stimulated prostaglandin synthesis with an IC50 of 5 microM at 15 min. Concentrations greater than 100 microM menadione were necessary to increase 51Cr release from prelabeled cells. Recovery of enzyme inactivated by menadione required a 6-h incubation in 1% serum. In a microsomal preparation, menadione was show...

  3. Inhibition of ATP synthesis by fenbufen and its conjugated metabolites in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-01-01

    in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen...... and fenbufen show any protective effect on fenbufen mediated inhibition of oxidative phosphorylation. Inclusion of NADPH in mitochondrial preparations with fenbufen did not modulate the inhibitory effects, suggesting no role of CYP mediated oxidative metabolites on the ATP synthesis in isolated mitochondria...

  4. Fluorochemicals used in food packaging inhibit male sex hormone synthesis

    DEFF Research Database (Denmark)

    Rosenmai, Anna Kjerstine; Nielsen, F. K.; Pedersen, Mikael

    2013-01-01

    Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS. The....... Overall, these results demonstrate that fluorochemicals present in food packaging materials and their metabolites can affect steroidogenesis through decreased Bzrp and increased CYP19 gene expression leading to lower androgen and higher estrogen levels.......Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS....... The aim of this study was to elucidate the effects of six fluorochemicals on sex hormone synthesis and androgen receptor (AR) activation in vitro. Four PAPS and two metabolites, perfluorooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (8:2 FTOH) were tested. Hormone profiles, including eight steroid...

  5. Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation.

    Science.gov (United States)

    Huang, Hsueh-Meei; Zhang, Hui; Xu, Hui; Gibson, Gary E

    2003-01-20

    Mitochondrial dysfunction occurs in many neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. alpha-Keto-beta-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca(2+)](i) by 23% (Pcalcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca(2+) abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.

  6. Ceruloplasmin Oxidation, a Feature of Parkinson's Disease CSF, Inhibits Ferroxidase Activity and Promotes Cellular Iron Retention

    KAUST Repository

    Olivieri, S.

    2011-12-14

    Parkinson\\'s disease is a neurodegenerative disorder characterized by oxidative stress and CNS iron deposition. Ceruloplasmin is an extracellular ferroxidase that regulates cellular iron loading and export, and hence protects tissues from oxidative damage. Using two-dimensional electrophoresis, we investigated ceruloplasmin patterns in the CSF of human Parkinson\\'s disease patients. Parkinson\\'s disease ceruloplasmin profiles proved more acidic than those found in healthy controls and in other human neurological diseases (peripheral neuropathies, amyotrophic lateral sclerosis, and Alzheimer\\'s disease); degrees of acidity correlated with patients\\' pathological grading. Applying an unsupervised pattern recognition procedure to the two-dimensional electrophoresis images, we identified representative pathological clusters. In vitro oxidation of CSF in two-dimensional electrophoresis generated a ceruloplasmin shift resembling that observed in Parkinson\\'s disease and co-occurred with an increase in protein carbonylation. Likewise, increased protein carbonylation was observed in Parkinson\\'s disease CSF, and the same modification was directly identified in these samples on ceruloplasmin. These results indicate that ceruloplasmin oxidation contributes to pattern modification in Parkinson\\'s disease. From the functional point of view, ceruloplasmin oxidation caused a decrease in ferroxidase activity, which in turn promotes intracellular iron retention in neuronal cell lines as well as in primary neurons, which are more sensitive to iron accumulation. Accordingly, the presence of oxidized ceruloplasmin in Parkinson\\'s disease CSF might be used as a marker for oxidative damage and might provide new insights into the underlying pathological mechanisms.

  7. Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

    Science.gov (United States)

    Emadi Baygi, Modjtaba; Soheili, Zahra Soheila; Schmitz, Ingo; Sameie, Shahram; Schulz, Wolfgang A

    2010-12-01

    The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.

  8. TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Rafael Zúñiga

    2018-03-01

    Full Text Available TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.

  9. Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants

    International Nuclear Information System (INIS)

    Dairou, Julien; Petit, Emile; Ragunathan, Nilusha; Baeza-Squiban, Armelle; Marano, Francelyne; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2009-01-01

    Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and β-naphthylamine (β-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H 2 O 2 or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

  10. Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

    Science.gov (United States)

    Jürets, Alexander; Le Bras, Marie; Staffler, Günther; Stein, Gesine; Leitner, Lukas; Neuhofer, Angelika; Tardelli, Matteo; Turkof, Edvin; Zeyda, Maximilian; Stulnig, Thomas M

    2016-01-01

    Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.

  11. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    Directory of Open Access Journals (Sweden)

    Charalampos Rallis

    2014-01-01

    Target of rapamycin complex 1 (TORC1, which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.

  12. Interleukin-6 promotes the migration and cellular senescence and inhibits apoptosis of human intrahepatic biliary epithelial cells.

    Science.gov (United States)

    Li, Ran; Dong, Juan; Bu, Xiu-Qin; Huang, Yong; Yang, Jing-Yu; Dong, Xuan; Liu, Jie

    2018-02-01

    Biliary epithelial cells (BEC) are closely related to some immune regulatory bile duct diseases. However, the complexity and polymorphism of the morphology and function of bile duct cells have hindered further investigation. Therefore, the aim of this study is to investigate how interleukin-6 (IL-6) affects the migration, cellular senescence, and apoptosis of human intrahepatic biliary epithelial cells (HIBECs). The HIBECs were stimulated by different concentrations of IL-6 (0, 5, 10, 15, and 20 ng/mL, respectively). Transwell assay was performed in order to measure the migration abilities, positive β-Galactosidase staining for the cellular senescence of HIBECs, MTT assay for changes of proliferation after IL-6 treatment and flow cytometry for cell cycle and apoptosis. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were conducted in order to detect the mRNA and protein expressions of epithelial-mesenchymal transition (EMT) markers in HIBECs. In comparison to the 0 ng/mL group, in the 5, 10, 15, and 20 ng/mL groups, a significant increase in the number of migratory HIBECs, proliferation, along with mRNA and protein expressions of EMT markers was observed. While the mRNA and protein expressions of epithelial markers, the number of β-galactosidase positive staining cells, as well as apoptosis rate of HIBECs dramatic decreased. Further, the aforementioned changes were significantly more evident in the 15 and 20 ng/mL groups in comparison to the 5 and 10 ng/mL groups. IL-6 may stimulate EMT, enhance the migration and proliferation, and inhibit apoptosis of HIBECs, thus delaying cellular senescence. © 2017 Wiley Periodicals, Inc.

  13. Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration1[W][OA

    Science.gov (United States)

    Vivancos, Pedro Diaz; Driscoll, Simon P.; Bulman, Christopher A.; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H.

    2011-01-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway. PMID:21757634

  14. Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration.

    Science.gov (United States)

    Vivancos, Pedro Diaz; Driscoll, Simon P; Bulman, Christopher A; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H

    2011-09-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.

  15. Synthesis of biocompatible nanoparticle drug complexes for inhibition of mycobacteria

    International Nuclear Information System (INIS)

    Bhave, Tejashree; Ghoderao, Prachi; Sanghavi, Sonali; Babrekar, Harshada; Bhoraskar, S V; Ganesan, V; Kulkarni, Anjali

    2013-01-01

    Tuberculosis (TB) is one of the most critical infectious diseases affecting the world today. Current TB treatment involves six months long daily administration of four oral doses of antibiotics. Due to severe side effects and the long treatment, a patient's adherence is low and this results in relapse of symptoms causing an alarming increase in the prevalence of multi-drug resistant (MDR) TB. Hence, it is imperative to develop a new drug delivery technology wherein these effects can be reduced. Rifampicin (RIF) is one of the widely used anti-tubercular drugs (ATD). The present study discusses the development of biocompatible nanoparticle–RIF complexes with superior inhibitory activity against both Mycobacterium smegmatis (M. smegmatis) and Mycobacterium tuberculosis (M. tuberculosis). Iron oxide nanoparticles (NPs) synthesized by gas phase condensation and NP-RIF complexes were tested against M. smegmatis SN2 strain as well as M. tuberculosis H37Rv laboratory strain. These complexes showed significantly better inhibition of M. smegmatis SN2 strain at a much lower effective concentration (27.5 μg ml −1 ) as compared to neat RIF (125 μg ml −1 ). Similarly M. tuberculosis H37Rv laboratory strain was susceptible to both nanoparticle–RIF complex and neat RIF at a minimum inhibitory concentration of 0.22 and 1 μg ml −1 , respectively. Further studies are underway to determine the efficacy of NPs–RIF complexes in clinical isolates of M. tuberculosis as well as MDR isolates. (paper)

  16. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J.

    1991-01-01

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [ 3 H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [ 3 H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [ 3 H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone

  17. Cardiac glycosides induce cell death in human cells by inhibiting general protein synthesis.

    Directory of Open Access Journals (Sweden)

    Andrea Perne

    2009-12-01

    Full Text Available Cardiac glycosides are Na(+/K(+-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive.Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+/K(+-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+/K(+-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels.The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans.

  18. Inhibition of fatty acid synthesis decreases very low density lipoprotein secretion in the hamster.

    Science.gov (United States)

    Arbeeny, C M; Meyers, D S; Bergquist, K E; Gregg, R E

    1992-06-01

    The hamster was developed as a model to study very low density lipoprotein (VLDL) metabolism, since, as is the case in humans, the hamster liver was found to synthesize apoB-100 and not apoB-48. The effect of inhibiting fatty acid synthesis on the hepatic secretion of VLDL triglyceride (TG) and apolipoprotein (apo) B-100 in this model was then investigated. In an in vivo study, hamsters were fed a chow diet containing 0.15% TOFA (5-tetradecyloxy-2-furancarboxylic acid), an inhibitor of acetyl-CoA carboxylase. After 6 days of treatment, plasma triglyceride and cholesterol levels were decreased by 30.2% and 11.6%, respectively. When the secretion of VLDL-TG by the liver was measured in vivo after injection of Triton WR 1339, TOFA treatment was found to decrease VLDL-TG secretion by 40%. In subsequent in vitro studies utilizing cultured primary hamster hepatocytes, incubation with 20 microM TOFA for 4 h resulted in 98% and 76% inhibition in fatty acid and triglyceride synthesis, respectively; VLDL-TG secretion was decreased by 90%. When hepatocytes were pulsed with [3H]leucine, incubation with TOFA resulted in a 50% decrease in the incorporation of radiolabel into secreted VLDL apoB-100. The results of this study indicate that inhibition of intracellular triglyceride synthesis decreases the secretion of VLDL-TG and apoB-100, and does not result in the secretion of a dense, triglyceride-depleted lipoprotein.

  19. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

    Science.gov (United States)

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-09-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods.

  20. Differential chlorate inhibition of Chaetomium globosum germination, hyphal growth, and perithecia synthesis.

    Science.gov (United States)

    Biles, Charles L; Wright, Desiree; Fuego, Marianni; Guinn, Angela; Cluck, Terry; Young, Jennifer; Martin, Markie; Biles, Josiah; Poudyal, Shubhra

    2012-12-01

    Chaetomium globosum Kunze:Fr is a dermatophytic, dematiaceous fungus that is ubiquitous in soils, grows readily on cellulolytic materials, and is commonly found on water-damaged building materials. Chlorate affects nitrogen metabolism in fungi and is used to study compatibility among anamorphic fungi by inducing nit mutants. The effect of chlorate toxicity on C. globosum was investigated by amending a modified malt extract agar (MEA), oat agar, and carboxymethyl cellulose agar (CMC) with various levels of potassium chlorate (KClO(3)). C. globosum perithecia production was almost completely inhibited (90-100 %) at low levels of KClO(3) (0.1 mM) in amended MEA. Inhibition of perithecia production was also observed on oat agar and CMC at 1 and 10 mM, respectively. However, hyphal growth in MEA was only inhibited 20 % by 0.1-100 mM KClO(3) concentrations. Hyphal growth was never completely inhibited at the highest levels tested (200 mM). Higher levels of KClO(3) were needed on gypsum board to inhibit perithecia synthesis. In additional experiments, KClO(3) did not inhibit C. globosum, Fusarium oxysporum, Aspergillus niger, Penicillum expansum, and airborne fungal spore germination. The various fungal spores were not inhibited by KClO(3) at 1-100 mM levels. These results suggest that C. globosum perithecia synthesis is more sensitive to chlorate toxicity than are hyphal growth and spore germination. This research provides basic information that furthers our understanding about perithecia formation and may help in developing control methods for fungal growth on building materials.

  1. Restoration of microRNA‑218 increases cellular chemosensitivity to cervical cancer by inhibiting cell‑cycle progression.

    Science.gov (United States)

    Dong, Ruofan; Qiu, Haifeng; Du, Guiqiang; Wang, Yuan; Yu, Jinjin; Mao, Caiping

    2014-12-01

    We previously reported frequent loss of microRNA‑218 (miR‑218) in human cervical cancer, which was associated with tumor progression and poor prognosis. In this study, we investigated whether restoration of the miR‑218 level is a valid strategy for the treatment of cervical cancer. The expression of miR‑218 in cervical cancer samples and cell lines was quantified by reverse transcription TaqMan quantitative (RT‑q)PCR. Overexpression of miR‑218 was achieved by both transient and stable transfection, using a miR‑218 mimic and a miR‑218‑expressing plasmid, respectively. Alterations in cellular proliferation and cell‑cycle progression were measured by the MTT assay and flow cytometry analysis. Nude mice bearing SiHa xenografts were used to investigate the functions of miR‑218 and carboplatin on tumor growth and weight. The expression of cycle‑related proteins was detected by western blotting and immunohistochemical staining. In vitro, miR‑218 significantly inhibited cellular growth in all four cell lines tested (P=0.021 for CaSki, P=0.009 for HeLa, P=0.016 for SiHa, and P=0.029 for C33A). Overexpression of miR‑218 induced G1 phase arrest and reduced expression of cyclin D1 and CDK4. In vivo, restoration of miR‑218 notably inhibited tumor growth and decreased tumor weight. In primary cultured samples, tumors with high levels of miR‑218 were more sensitive to carboplatin (R2=0.3319, P=0.0026); consistently, miR‑218 overexpression suppressed tumor growth, induced cell‑cycle arrest, and reduced the cyclin D1 level. Based on these and previous results, we conclude that restoration of the miR‑218 level inhibits the growth of cervical cancer cells both in vitro and in vivo; furthermore, overexpression of miR‑218 sensitizes cervical cancer cells to carboplatin. Our findings suggest a novel therapy for cervical cancer based on miR‑218, especially in patients with reduced levels of miR‑218.

  2. The E-domain region of mechano-growth factor inhibits cellular apoptosis and preserves cardiac function during myocardial infarction.

    Science.gov (United States)

    Mavrommatis, Evangelos; Shioura, Krystyna M; Los, Tamara; Goldspink, Paul H

    2013-09-01

    Insulin-like growth factor-1 (IGF-1) isoforms are expressed via alternative splicing. Expression of the minor isoform IGF-1Eb [also known as mechano-growth factor (MGF)] is responsive to cell stress. Since IGF-1 isoforms differ in their E-domain regions, we are interested in determining the biological function of the MGF E-domain. To do so, a synthetic peptide analog was used to gain mechanistic insight into the actions of the E-domain. Treatment of H9c2 cells indicated a rapid cellular uptake mechanism that did not involve IGF-1 receptor activation but resulted in a nuclear localization. Peptide treatment inhibited the intrinsic apoptotic pathway in H9c2 cells subjected to cell stress with sorbitol by preventing the collapse of the mitochondrial membrane potential and inhibition of caspase-3 activation. Therefore, we administered the peptide at the time of myocardial infarction (MI) in mice. At 2 weeks post-MI cardiac function, gene expression and cell death were assayed. A significant decline in both systolic and diastolic function was evident in untreated mice based on PV loop analysis. Delivery of the E-peptide ameliorated the decline in function and resulted in significant preservation of cardiac contractility. Associated with these changes were an inhibition of pathologic hypertrophy and significantly fewer apoptotic nuclei in the viable myocardium of E-peptide-treated mice post-MI. We conclude that administration of the MGF E-domain peptide may provide a means of modulating local tissue IGF-1 autocrine/paracrine actions to preserve cardiac function, prevent cell death, and pathologic remodeling in the heart.

  3. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    International Nuclear Information System (INIS)

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-01-01

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [ 3 H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [ 3 H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-κB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  4. Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

    Science.gov (United States)

    Chase, Amanda J.; Daijogo, Sarah

    2014-01-01

    ABSTRACT Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition. PMID:24371074

  5. Synthesis of heterocycles: Indolo (2,1-a) isoquinolines, renewables, and aptamer ligands for cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Beasley, Jonathan [Ames Laboratory (AMES), Ames, IA (United States)

    2013-01-01

    In this thesis, we explore both total syntheses and methodologies of several aromatic heterocyclic molecules. Extensions of the Kraus indole synthesis toward 2-substituted and 2,3-disubstituted indoles, as well as biologically attractive indolo[2,1-a]isoquinolines are described. Recent renewable efforts directed to commodity maleic acid and the first reported furan-based ionic liquids are described. Our total synthesis of mRNA aptamer ligand PDC-Gly, and its dye coupled forms, plus aminoglycoside dye coupled ligands used in molecular imaging, are described.

  6. Oleic Acid and Hydroxytyrosol Inhibit Cholesterol and Fatty Acid Synthesis in C6 Glioma Cells

    Directory of Open Access Journals (Sweden)

    Paola Priore

    2017-01-01

    Full Text Available Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives for a healthier brain. Here, we investigated the effects of oleic acid (OA and hydroxytyrosol (HTyr, two important extra virgin olive oil compounds, on lipid synthesis in C6 glioma cells. OA and HTyr inhibited both de novo fatty acid and cholesterol syntheses without affecting cell viability. The inhibitory effect of the individual compounds was more pronounced if OA and HTyr were administered in combination. A reduction of polar lipid biosynthesis was also detected, while triglyceride synthesis was marginally affected. To clarify the lipid-lowering mechanism of these compounds, their effects on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase-ACC and fatty acid synthase-FAS and cholesterologenesis (3-hydroxy-3-methylglutaryl-CoA reductase-HMGCR were investigated in situ by using digitonin-permeabilized C6 cells. ACC and HMGCR activities were especially reduced after 4 h of 25 μM OA and HTyr treatment. No change in FAS activity was observed. Inhibition of ACC and HMGCR activities is corroborated by the decrease of their mRNA abundance and protein level. Our results indicate a direct and rapid downregulatory effect of the two olive oil compounds on lipid synthesis in C6 cells.

  7. Inhibition of Fatty Acid Synthesis Induces Apoptosis of Human Pancreatic Cancer Cells.

    Science.gov (United States)

    Nishi, Koji; Suzuki, Kenta; Sawamoto, Junpei; Tokizawa, Yuma; Iwase, Yumiko; Yumita, Nagahiko; Ikeda, Toshihiko

    2016-09-01

    Cancer cells tend to have a high requirement for lipids, including fatty acids, cholesterol and triglyceride, because of their rapid proliferative rate compared to normal cells. In this study, we investigated the effects of inhibition of lipid synthesis on the proliferation and viability of human pancreatic cancer cells. Of the inhibitors of lipid synthesis that were tested, 5-(tetradecyloxy)-2-furoic acid (TOFA), which is an inhibitor of acetyl-CoA carboxylase, and the fatty acid synthase (FAS) inhibitors cerulenin and irgasan, significantly suppressed the proliferation of MiaPaCa-2 and AsPC-1 cells. Treatment of MiaPaCa-2 cells with these inhibitors significantly increased the number of apoptotic cells. In addition, TOFA increased caspase-3 activity and induced cleavage of poly (ADP-ribose) polymerase in MiaPaCa-2 cells. Moreover, addition of palmitate to MiaPaCa-2 cells treated with TOFA rescued cells from apoptotic cell death. These results suggest that TOFA induces apoptosis via depletion of fatty acids and that, among the various aspects of lipid metabolism, inhibition of fatty acid synthesis may be a notable target for the treatment of human pancreatic cancer cells. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  8. Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Ventura, A M; Ortega, J M; Schumacher, R I; Meneghini, R

    1987-01-01

    In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. Using metabolic inhibitors, it has been shown that (1) even the low repair rate exhibited by V79 cells is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of small replicons.

  9. Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine.

    Science.gov (United States)

    McFarlane, Matthew R; Liang, Guosheng; Engelking, Luke J

    2014-01-24

    Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

  10. Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine*

    Science.gov (United States)

    McFarlane, Matthew R.; Liang, Guosheng; Engelking, Luke J.

    2014-01-01

    Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes. PMID:24337570

  11. Ammonia inhibits long-term potentiation via neurosteroid synthesis in hippocampal pyramidal neurons.

    Science.gov (United States)

    Izumi, Y; Svrakic, N; O'Dell, K; Zorumski, C F

    2013-03-13

    Neurosteroids are a class of endogenous steroids synthesized in the brain that are believed to be involved in the pathogenesis of neuropsychiatric disorders and memory impairment. Ammonia impairs long-term potentiation (LTP), a synaptic model of learning, in the hippocampus, a brain region involved in memory acquisition. Although mechanisms underlying ammonia-mediated LTP inhibition are not fully understood, we previously found that the activation of N-methyl-d-aspartate receptors (NMDARs) is important. Based on this, we hypothesize that metabolic stressors, including hyperammonemia, promote untimely NMDAR activation and result in neural adaptations that include the synthesis of allopregnanolone (alloP) and other GABA-potentiating neurosteroids that dampen neuronal activity and impair LTP and memory formation. Using an antibody against 5α-reduced neurosteroids, we found that 100 μM ammonia acutely enhanced neurosteroid immunostaining in pyramidal neurons in the CA1 region of rat hippocampal slices. The enhanced staining was blocked by finasteride, a selective inhibitor of 5α-reductase, a key enzyme required for alloP synthesis. Finasteride also overcame LTP inhibition by 100 μM ammonia, as did picrotoxin, an inhibitor of GABA-A receptors. These results indicate that GABA-enhancing neurosteroids, synthesized locally within pyramidal neurons, contribute significantly to ammonia-mediated synaptic dysfunction. These results suggest that the manipulation of neurosteroid synthesis could provide a strategy to improve cognitive function in individuals with hyperammonemia. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

    Energy Technology Data Exchange (ETDEWEB)

    Gorrochategui, Eva; Pérez-Albaladejo, Elisabet [Department of Environmental Chemistry, IDAEA–CSIC, 08034 Barcelona, Catalonia (Spain); Casas, Josefina [Department of Biomedicinal Chemistry, IQAC–CSIC, 08034 Barcelona, Catalonia (Spain); Lacorte, Sílvia, E-mail: slbqam@cid.csic.es [Department of Environmental Chemistry, IDAEA–CSIC, 08034 Barcelona, Catalonia (Spain); Porte, Cinta, E-mail: cinta.porte@cid.csic.es [Department of Environmental Chemistry, IDAEA–CSIC, 08034 Barcelona, Catalonia (Spain)

    2014-06-01

    The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA > PFOS ≫ PFNA > PFOA > PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80 μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells. - Highlights: • Eight perfluorinated chemicals of different chain lengths have been selected. • Long chain ones – PFOS, PFDoA, PFNA, PFOA – were cytotoxic in placenta cells. • The uptake of long chain perfluorinated chemicals by cells was comparatively higher. • PFOS, PFOA and the short chain PFBS significantly inhibited aromatase activity. • A mixture of perfluorinated chemicals significantly altered placenta cell

  13. Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

    International Nuclear Information System (INIS)

    Gorrochategui, Eva; Pérez-Albaladejo, Elisabet; Casas, Josefina; Lacorte, Sílvia; Porte, Cinta

    2014-01-01

    The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA > PFOS ≫ PFNA > PFOA > PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80 μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells. - Highlights: • Eight perfluorinated chemicals of different chain lengths have been selected. • Long chain ones – PFOS, PFDoA, PFNA, PFOA – were cytotoxic in placenta cells. • The uptake of long chain perfluorinated chemicals by cells was comparatively higher. • PFOS, PFOA and the short chain PFBS significantly inhibited aromatase activity. • A mixture of perfluorinated chemicals significantly altered placenta cell

  14. Inhibition of local estrogen synthesis in the hippocampus impairs hippocampal memory consolidation in ovariectomized female mice.

    Science.gov (United States)

    Tuscher, Jennifer J; Szinte, Julia S; Starrett, Joseph R; Krentzel, Amanda A; Fortress, Ashley M; Remage-Healey, Luke; Frick, Karyn M

    2016-07-01

    The potent estrogen 17β-Estradiol (E2) plays a critical role in mediating hippocampal function, yet the precise mechanisms through which E2 enhances hippocampal memory remain unclear. In young adult female rodents, the beneficial effects of E2 on memory are generally attributed to ovarian-synthesized E2. However, E2 is also synthesized in the adult brain in numerous species, where it regulates synaptic plasticity and is synthesized in response to experiences such as exposure to females or conspecific song. Although de novo E2 synthesis has been demonstrated in rodent hippocampal cultures, little is known about the functional role of local E2 synthesis in mediating hippocampal memory function. Therefore, the present study examined the role of hippocampal E2 synthesis in hippocampal memory consolidation. Using bilateral dorsal hippocampal infusions of the aromatase inhibitor letrozole, we first found that blockade of dorsal hippocampal E2 synthesis impaired hippocampal memory consolidation. We next found that elevated levels of E2 in the dorsal hippocampus observed 30min after object training were blocked by dorsal hippocampal infusion of letrozole, suggesting that behavioral experience increases acute and local E2 synthesis. Finally, aromatase inhibition did not prevent exogenous E2 from enhancing hippocampal memory consolidation, indicating that hippocampal E2 synthesis is not necessary for exogenous E2 to enhance hippocampal memory. Combined, these data are consistent with the hypothesis that hippocampally-synthesized E2 is necessary for hippocampus-dependent memory consolidation in rodents. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Porphyrins with directly meso-attached disaccharide moieties: Synthesis, self-assembly and cellular study

    Czech Academy of Sciences Publication Activity Database

    Malachowska, M.; Sperduto, C.; Darmostuk, M.; Monti, D.; Venanzi, M.; Mancini, G.; D'Acunto, C.W.; Králová, Jarmila; Ruml, T.; Wimmer, Zdeněk; Drasar, P.

    2016-01-01

    Roč. 20, č. 7 (2016), s. 773-784 ISSN 1088-4246 Institutional support: RVO:61389030 ; RVO:68378050 Keywords : derivatives * aggregation * steroids * sucrose * porphyrinoids * carbohydrates * self-assembly * cellular localisation * liposomes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.043, year: 2016

  16. Green Tea Polyphenol Epigallocatechin-3-Gallate Enhance Glycogen Synthesis and Inhibit Lipogenesis in Hepatocytes

    Directory of Open Access Journals (Sweden)

    Jane J. Y. Kim

    2013-01-01

    Full Text Available The beneficial effects of green tea polyphenols (GTP against metabolic syndrome and type 2 diabetes by suppressing appetite and nutrient absorption have been well reported. However the direct effects and mechanisms of GTP on glucose and lipid metabolism remain to be elucidated. Since the liver is an important organ involved in glucose and lipid metabolism, we examined the effects and mechanisms of GTP on glycogen synthesis and lipogenesis in HepG2 cells. Concentrations of GTP containing 68% naturally occurring (−-epigallocatechin-3-gallate (EGCG were incubated in HepG2 cells with high glucose (30 mM under 100 nM of insulin stimulation for 24 h. GTP enhanced glycogen synthesis in a dose-dependent manner. 10 μM of EGCG significantly increased glycogen synthesis by 2fold (P<0.05 compared with insulin alone. Western blotting revealed that phosphorylation of Ser9 glycogen synthase kinase 3β and Ser641 glycogen synthase was significantly increased in GTP-treated HepG2 cells compared with nontreated cells. 10 μM of EGCG also significantly inhibited lipogenesis (P<0.01. We further demonstrated that this mechanism involves enhanced expression of phosphorylated AMP-activated protein kinase α and acetyl-CoA carboxylase in HepG2 cells. Our results showed that GTP is capable of enhancing insulin-mediated glucose and lipid metabolism by regulating enzymes involved in glycogen synthesis and lipogenesis.

  17. A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

    Science.gov (United States)

    Hoffmann, Julia; Schneider, Carola; Heinbockel, Lena; Brandenburg, Klaus; Reimer, Rudolph; Gabriel, Gülsah

    2014-04-01

    Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Alteration of cellular behavior and response to PI3K pathway inhibition by culture in 3D collagen gels.

    Directory of Open Access Journals (Sweden)

    Brian Fallica

    Full Text Available Most investigations into cancer cell drug response are performed with cells cultured on flat (2D tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D extracellular matrix (ECM is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.

  19. Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line.

    Science.gov (United States)

    Moeslinger, Thomas; Friedl, Roswitha; Spieckermann, Paul Gerhard

    2006-06-20

    Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of

  20. Synthesis of the oxysterol, 24(S, 25-epoxycholesterol, parallels cholesterol production and may protect against cellular accumulation of newly-synthesized cholesterol

    Directory of Open Access Journals (Sweden)

    Brown Andrew J

    2007-04-01

    Full Text Available Abstract Aim The effects of 24(S,25-epoxycholesterol (24,25EC on aspects of cholesterol homeostasis is well-documented. When added to cells, 24,25EC decreases cholesterol synthesis and up-regulates cholesterol efflux genes, including ABCA1. Synthesis of 24,25EC occurs in a shunt of the mevalonate pathway which also produces cholesterol. Therefore, 24,25EC synthesis should be subject to the same negative feedback regulation as cholesterol synthesis. To date, no role has been ascribed to 24,25EC in light of the fact that increased accumulation of cholesterol should decrease formation of this oxysterol through feedback inhibition. This leads to the intriguing paradox: why inhibit production of an apparently important regulator of cholesterol homeostasis when it is needed most? Methods We used a combination of pharmacological and genetic approaches in Chinese Hamster Ovary cell-lines to investigate this paradox. Endogenous synthesis of 24,25EC was manipulated using partial inhibition of the enzyme, Oxidosqualene Cyclase. Changes in cholesterol and 24,25EC synthesis were determined using metabolic labelling with [1-14C]-acetate, thin-layer chromatography and phosphorimaging. Transcriptional effects mediated via SREBP and LXR were analysed by luciferase reporter assays. Results We showed that cholesterol addition to cells lead to a rapid and preferential inhibition of 24,25EC synthesis. Addition of 24,25EC resulted in parallel inhibition of 24,25EC and cholesterol synthesis. Furthermore, we used a variety of approaches to examine the relationship between cholesterol and 24,25EC synthesis, including cell-lines with different rates of cholesterol synthesis, varying cholesterol synthetic rates by pre-treatment with a statin, or lipoprotein cholesterol loading of macrophages. In all cases, we showed that 24,25EC synthesis faithfully tracked cholesterol synthesis. Moreover, changes in 24,25EC synthesis exerted downstream effects, reducing SREBP

  1. Lobatamide C: total synthesis, stereochemical assignment, preparation of simplified analogues, and V-ATPase inhibition studies.

    Science.gov (United States)

    Shen, Ruichao; Lin, Cheng Ting; Bowman, Emma Jean; Bowman, Barry J; Porco, John A

    2003-07-02

    The total synthesis and stereochemical assignment of the potent antitumor macrolide lobatamide C, as well as synthesis of simplified lobatamide analogues, is reported. Cu(I)-mediated enamide formation methodology has been developed to prepare the highly unsaturated enamide side chain of the natural product and analogues. A key fragment coupling employs base-mediated esterification of a beta-hydroxy acid and a salicylate cyanomethyl ester. Three additional stereoisomers of lobatamide C have been prepared using related synthetic routes. The stereochemistry at C8, C11, and C15 of lobatamide C was assigned by comparison of stereoisomers and X-ray analysis of a crystalline derivative. Synthetic lobatamide C, stereoisomers, and simplified analogues have been evaluated for inhibition of bovine chromaffin granule membrane V-ATPase. The salicylate phenol, enamide NH, and ortho-substitution of the salicylate ester have been shown to be important for V-ATPase inhibitory activity.

  2. Kinetics of radiation-induced apoptosis in neonatal urogenital tissues with and without protein synthesis inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Gobe, G.C.; Harmon, B.; Schoch, E.; Allan, D.J. [Queensland Univ., St. Lucia, QLD (Australia). Dept. of Chemistry

    1996-12-31

    The difference in incidence of radiation-induced apoptosis between two neonatal urogenital tissues, kidney and testis, was analysed over a 24h period. Concurrent administration of cycloheximide (10mg/kg body weight), a protein synthesis inhibitor, with radiation treatment was used to determine whether new protein synthesis had a role in induction of apoptosis in this in vivo model. Many chemotherapeutic drugs act via protein synthesis inhibition, and we believe that the results of this latter analysis may provide information for the planning of concurrent radio and chemotherapy. Apoptosis was quantified using morphological parameters, and verified by DNA gel electrophoresis for the typical banding pattern, and by electron microscopy. The proliferative index in tissues was studied, using [6-{sup 3}H]-thymidine uptake ( 1h prior to euthanasia and collection of tissues) and autoradiography as indicators of cell proliferation (S-phase). Tissue was collected 2, 4, 6, 8, and 24h after radiation treatment. Expression of one of the apoptosis-associated genes, Bcl-2 (an apoptosis inhibitor/cell survival gene), was studied using immunohistochemistry. Apoptosis peaked at 4h in the testis and 6h in the kidney, emphasising the necessity of knowing tissue differences in radiation response if comparing changes at a particular time. A higher proportion (almost five fold) of the apoptotic cells died in S-phase in the kidney than the testis, over the 24h. Protein synthesis inhibition completely negated induction of apoptosis in both tissues. Necrosis was not identified at any time. Cycloheximide treatment greatly diminished Bcl-2 expression. The differences in response of the two tissues to irradiation relates to their innate cell (genetic) controls, which may be determined by their state of differentiation at time of treatment, or the tissue type. This in vivo study also suggests the model may be useful for analysis of other cancer therapies for example polychemotherapies or chemo

  3. Kinetics of radiation-induced apoptosis in neonatal urogenital tissues with and without protein synthesis inhibition

    International Nuclear Information System (INIS)

    Gobe, G.C.; Harmon, B.; Schoch, E.; Allan, D.J.

    1996-01-01

    The difference in incidence of radiation-induced apoptosis between two neonatal urogenital tissues, kidney and testis, was analysed over a 24h period. Concurrent administration of cycloheximide (10mg/kg body weight), a protein synthesis inhibitor, with radiation treatment was used to determine whether new protein synthesis had a role in induction of apoptosis in this in vivo model. Many chemotherapeutic drugs act via protein synthesis inhibition, and we believe that the results of this latter analysis may provide information for the planning of concurrent radio and chemotherapy. Apoptosis was quantified using morphological parameters, and verified by DNA gel electrophoresis for the typical banding pattern, and by electron microscopy. The proliferative index in tissues was studied, using [6- 3 H]-thymidine uptake ( 1h prior to euthanasia and collection of tissues) and autoradiography as indicators of cell proliferation (S-phase). Tissue was collected 2, 4, 6, 8, and 24h after radiation treatment. Expression of one of the apoptosis-associated genes, Bcl-2 (an apoptosis inhibitor/cell survival gene), was studied using immunohistochemistry. Apoptosis peaked at 4h in the testis and 6h in the kidney, emphasising the necessity of knowing tissue differences in radiation response if comparing changes at a particular time. A higher proportion (almost five fold) of the apoptotic cells died in S-phase in the kidney than the testis, over the 24h. Protein synthesis inhibition completely negated induction of apoptosis in both tissues. Necrosis was not identified at any time. Cycloheximide treatment greatly diminished Bcl-2 expression. The differences in response of the two tissues to irradiation relates to their innate cell (genetic) controls, which may be determined by their state of differentiation at time of treatment, or the tissue type. This in vivo study also suggests the model may be useful for analysis of other cancer therapies for example polychemotherapies or chemo

  4. Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade

    Science.gov (United States)

    Fiandalo, Michael V.; Stocking, John J.; Pop, Elena A.; Wilton, John H.; Mantione, Krystin M.; Li, Yun; Attwood, Kristopher M.; Azabdaftari, Gissou; Wu, Yue; Watt, David S.; Wilson, Elizabeth M.; Mohler, James L.

    2018-01-01

    Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert 5α-androstane-3α,17β-diol (DIOL) to dihydrotestosterone (DHT). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to DHT. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the 5α-reductase inhibitor, dutasteride, decreased DHT levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of DHT synthesis provides a therapeutic strategy to inhibit CRPC development and growth. PMID:29541409

  5. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Bag, Jnanankur, E-mail: jbag@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2011-05-13

    Highlights: {yields} Depletion of cellular PABP level arrests mRNA translation in HeLa cells. {yields} PABP knock down leads to apoptotic cell death. {yields} PABP depletion does not affect transcription. {yields} PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  6. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    International Nuclear Information System (INIS)

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-01-01

    Highlights: → Depletion of cellular PABP level arrests mRNA translation in HeLa cells. → PABP knock down leads to apoptotic cell death. → PABP depletion does not affect transcription. → PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  7. Cold injury, blood-brain barrier changes, and leukotriene synthesis: Inhibition by phenidone

    International Nuclear Information System (INIS)

    Robichaud, L.J.; Marcoux, F.W.

    1990-01-01

    Transcranial cold injury in rats and guinea pigs induced cerebral extravasation of albumin labeled with Evans blue dye or 125 I, respective indicators of the area and amount of blood-brain barrier (BBB) disruption. Radioimmunoassay of brain extracts showed that cold injury induced leukotriene (LT)C4 in rat and guinea pig brains 15 min after injury. In guinea pigs, the LT synthesis inhibitor phenidone (30 mg/kg, i.p.) completely blocked cold-induced LTC4 in brain. Phenidone (30 and 100 mg/kg) also inhibited cerebral tissue accumulation of 125 I-albumin and dye in rats and guinea pigs. Phenidone is reported to show antioxidant properties and selective lipoxygenase inhibition of arachidonic acid metabolism compared to cyclooxygenase inhibitors, meclofenamate sodium, and other nonsteroidal anti-inflammatory agents. Since several oxygen and hydroxyl radical scavengers and the cyclooxygenase inhibitor, meclofenamate sodium, did not inhibit protein extravasation, the findings support a role for LT as a mediator of cold-induced changes in BBB permeability in rats and guinea pigs and suggest that the inhibitory effects of phenidone on BBB permeability may be due to inhibition of LT production

  8. The assembly and properties of protobiological structures - The beginnings of cellular peptide synthesis

    Science.gov (United States)

    Fox, S. W.; Nakashima, T.

    1980-01-01

    New data indicate that lysine-rich proteinoids have the ability to catalyze the synthesis of peptide bonds from a variety of amino acids and ATP. This capacity is evident in aqueous solution, in suspension of phase-separated complexes of lysine-rich proteinoid with acidic proteinoids, and in suspension of phase-separated particles composed of lysine-rich proteinoids with polynucleotides. Since the proteinoid complexes can contain other catalytic activities, including ability to catalyze internucleotide bond formation, it is inferred that the first protocells on earth already had a number of biological types of activity.

  9. Evaluation of adamantane hydroxamates as botulinum neurotoxin inhibitors: synthesis, crystallography, modeling, kinetic and cellular based studies.

    Science.gov (United States)

    Šilhár, Peter; Silvaggi, Nicholas R; Pellett, Sabine; Čapková, Kateřina; Johnson, Eric A; Allen, Karen N; Janda, Kim D

    2013-03-01

    Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based and thus have a limited window of treatment opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a therapeutic strategy for the treatment of botulism as it may provide an effective post exposure remedy. Using a combination of crystallographic and modeling studies a series of hydroxamates derived from 1-adamantylacetohydroxamic acid (3a) were prepared. From this group of compounds, an improved potency of about 17-fold was observed for two derivatives. Detailed mechanistic studies on these structures revealed a competitive inhibition model, with a K(i)=27 nM, which makes these compounds some of the most potent small molecule, non-peptidic BoNT/A LC inhibitors reported to date. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

    Science.gov (United States)

    Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

    2017-12-09

    Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

  11. Inhibiting corticosterone synthesis during fear memory formation exacerbates cued fear extinction memory deficits within the single prolonged stress model.

    Science.gov (United States)

    Keller, Samantha M; Schreiber, William B; Stanfield, Briana R; Knox, Dayan

    2015-01-01

    Using the single prolonged stress (SPS) animal model of post-traumatic stress disorder (PTSD), previous studies suggest that enhanced glucocorticoid receptor (GR) expression leads to cued fear extinction retention deficits. However, it is unknown how the endogenous ligand of GRs, corticosterone (CORT), may contribute to extinction retention deficits in the SPS model. Given that CORT synthesis during fear learning is critical for fear memory consolidation and SPS enhances GR expression, CORT synthesis during fear memory formation could strengthen fear memory in SPS rats by enhancing GR activation during fear learning. In turn, this could lead to cued fear extinction retention deficits. We tested the hypothesis that CORT synthesis during fear learning leads to cued fear extinction retention deficits in SPS rats by administering the CORT synthesis inhibitor metyrapone to SPS and control rats prior to fear conditioning, and observed the effect this had on extinction memory. Inhibiting CORT synthesis during fear memory formation in control rats tended to decrease cued freezing, though this effect never reached statistical significance. Contrary to our hypothesis, inhibiting CORT synthesis during fear memory formation disrupted extinction retention in SPS rats. This finding suggests that even though SPS exposure leads to cued fear extinction memory deficits, CORT synthesis during fear memory formation enhances extinction retention in SPS rats. This suggests that stress-induced CORT synthesis in previously stressed rats can be beneficial. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. A Network of AOPs for reduced thyroid hormone synthesis derived from inhibition of Thyroperoxidase - A common Molecular Initiating Event Leading to Species-Specific Indices of Adversity.

    Science.gov (United States)

    This collection of 3 AOPs describe varying outcomes of adversity dependent upon species in response to inhibition of thyroperoxidase (TPO) during development. Chemical inhibition of TPO, the molecular-initiating event (MIE), results in decreased thyroid hormone (TH) synthesis, a...

  13. Total synthesis of [2-11C]thymidine from [11C]urea: A tracer of choice for measurement of cellular proliferation using PET

    International Nuclear Information System (INIS)

    Labar, D.; Vander Borght, T.

    1990-01-01

    In preliminary studies of cellular proliferation with [methyl- 11 C]thymidine, the labelled degradative products mask the progressive incorporation of the tracer into DNA. The authors have developed a procedure for the synthesis of [2- 11 C]thymidine to circumvent this difficulty, using a [ 11 C]urea precursor

  14. Estradiol inhibits hepatic stellate cell area and collagen synthesis in the chicken liver.

    Science.gov (United States)

    Nishimura, Shotaro; Teshima, Akifumi; Kawabata, Fuminori; Tabata, Shoji

    2017-11-01

    Hepatic stellate cells (HSCs) are the main collagen-producing cells in the liver. The HSC area and amount of collagen fibers are different between male and female chickens. This study was performed to confirm the effect of estradiol on collagen synthesis in the growing chicken liver. Blood estradiol levels in chicks were compared at 4 and 8 weeks of age, and the collagen fibril network in liver tissue was observed at 8 weeks by scanning electron microscopy. Intraperitoneal administrations of estradiol and tamoxifen to male and female chicks, respectively, were performed daily from 5 to 8 weeks of age. The areas of HSCs and collagen contents were measured in the liver tissue. The blood estradiol level was higher in females than in males, and the collagen fibril network was denser in males than in females at 8 weeks of age. Estradiol administration in males induced decreases in the HSC area and collagen content of the liver. Conversely, tamoxifen administration in females induced an increase in the HSC area but did not facilitate collagen synthesis. Based on these results, estradiol inhibits the area and collagen synthesis of HSCs in the growing chicken liver under normal physiological conditions. © 2017 Japanese Society of Animal Science.

  15. Synthesis of diethylenetriaminepentaacetic acid conjugated inulin and utility for cellular uptake of liposomes

    International Nuclear Information System (INIS)

    Essien, H.; Lai, J.Y.; Hwang, K.J.

    1988-01-01

    The synthesis, binding of radioactive cations, liposomal encapsulation, and biodistribution of the oxidized-inulin reaction product with ethylenediamine and diethylenetriaminepentaacetic acid (4) are described. The four-step synthesis of the inulin derivative proceeded in a good overall yield of 72%. The complex of the inulin derivative with either 67 Ga3+ or 111 In3+ was stable in vivo and did not readily distribute into tissues, being excreted primarily in urine after intravenous administration to mice. The liposome-entrapped inulin derivative can be loaded with radioactive heavy metal cations by mobile ionophores in high radiochemical yields of 80-91%. Following the intravenous administration of the liposomal encapsulation of the indium-111-labeled inulin derivative, the entrapped compound had a biodistribution characteristic of liposomes and allowed an estimation of the extent of the intracellular uptake of liposomes. The ability of the inulin derivative to chelate many different types of metals will allow the use of this probe for studying subtle differences in tissue distribution resulting from different drug targeting or delivery protocols in the same animal by multiple labeling techniques. Moreover, the chelate-conjugated inulin permits studies of the applications of drug delivery systems in primates or human subjects by noninvasive techniques such as gamma-scintigraphic or nuclear magnetic resonance imaging methods

  16. Block of glucocorticoid synthesis during re-activation inhibits extinction of an established fear memory.

    Science.gov (United States)

    Blundell, Jacqueline; Blaiss, Cory A; Lagace, Diane C; Eisch, Amelia J; Powell, Craig M

    2011-05-01

    The pharmacology of traumatic memory extinction has not been fully characterized despite its potential as a therapeutic target for established, acquired anxiety disorders, including post-traumatic stress disorder (PTSD). Here we examine the role of endogenous glucocorticoids in traumatic memory extinction. Male C57BL/6J mice were injected with corticosterone (10 mg/kg, i.p.) or metyrapone (50 mg/kg, s.c.) during re-activation of a contextual fear memory, and compared to vehicle groups (N=10-12 per group). To ensure that metyrapone was blocking corticosterone synthesis, we measured corticosterone levels following re-activation of a fear memory in metyrapone- and vehicle-treated animals. Corticosterone administration following extinction trials caused a long-lasting inhibition of the original fear memory trace. In contrast, blockade of corticosteroid synthesis with metyrapone prior to extinction trials enhanced retrieval and prevented extinction of context-dependent fear responses in mice. Further behavioral analysis suggested that the metyrapone enhancement of retrieval and prevention of extinction were not due to non-specific alterations in locomotor or anxiety-like behavior. In addition, the inhibition of extinction by metyrapone was rescued by exogenous administration of corticosterone following extinction trials. Finally, we confirmed that the rise in corticosterone during re-activation of a contextual fear memory was blocked by metyrapone. We demonstrate that extinction of a classical contextual fear memory is dependent on endogenous glucocorticoid synthesis during re-activation of a fear memory. Our data suggest that decreased glucocorticoids during fear memory re-activation may contribute to the inability to extinguish a fear memory, thus contributing to one of the core symptoms of PTSD. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Targeting (cellular) lysosomal acid ceramidase by B13: design, synthesis and evaluation of novel DMG-B13 ester prodrugs.

    Science.gov (United States)

    Bai, Aiping; Szulc, Zdzislaw M; Bielawski, Jacek; Pierce, Jason S; Rembiesa, Barbara; Terzieva, Silva; Mao, Cungui; Xu, Ruijuan; Wu, Bill; Clarke, Christopher J; Newcomb, Benjamin; Liu, Xiang; Norris, James; Hannun, Yusuf A; Bielawska, Alicja

    2014-12-15

    Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition.

    Science.gov (United States)

    Marques-Ramos, Ana; Candeias, Marco M; Menezes, Juliane; Lacerda, Rafaela; Willcocks, Margaret; Teixeira, Alexandre; Locker, Nicolas; Romão, Luísa

    2017-11-01

    The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions. © 2017 Marques-Ramos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Action of caffeine on x-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Tolmach, L.J.; Jones, R.W.; Busse, P.M.

    1977-01-01

    Treatment of HeLa S3 cells with 1 mM caffeine delays progression through G1 by 1.5 hours but causes no other detectable inhibition of cell progression; it sometimes results in a large stimulation of thymidine incorporation. When this concentration is applied to cells that have been irradiated with 1-krad doses of 220-kV x rays, there is a marked suppression of both the inhibition of DNA synthesis and G2 arrest induced by the radiation. Larger doses require higher concentrations of caffeine to suppress the inhibition of DNA synthesis. Delaying addition until the rate of synthesis is at its minimum (1.5 hours after irradiation with 1 krad) results in a slightly accelerated recovery of the rate. Treatment before or during irradiation is without effect on the inhibition. Removal of the caffeine as late as 6 hours after its addition at the time of irradiation results in a prompt inhibition in DNA synthesis that mimics that observed immediately after irradiation in the absence of caffeine. These findings raise the possibility that the depression in rate of DNA systhesis might not result from radiation damage introduced into the replicon initiation system, but rather may be an indirect consequence of damage residing elsewhere in the irradiated cell

  20. Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

    Science.gov (United States)

    Lee, Dae-Hee; Lee, Yong J

    2008-10-01

    Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

  1. Dose-dependent inhibition of BACE-1 by the monoterpenoid 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone in cellular and mouse models of Alzheimer's disease.

    Science.gov (United States)

    Videira, Rita; Castanheira, Pedro; Grãos, Mário; Resende, Rosa; Salgueiro, Lígia; Faro, Carlos; Cavaleiro, Carlos

    2014-06-27

    BACE-1 is an aspartic protease involved in the conversion of amyloid precursor protein (APP) to amyloid-β (Aβ) in vivo, which is one of the key steps in the development and progression of Alzheimer's disease. In a previous screening procedure for inhibitors of BACE-1 activity, the oil of Lavandula luisieri was identified as the most potent among several essential oils. The inhibitory effect of this essential oil on Aβ production was also demonstrated in a cellular assay. The composition of the volatile oil and the isolation of the compound responsible for the inhibitory activity were also reported. The present work focused on the characterization of the inhibition of BACE-1 by this active compound, a monoterpene necrodane ketone, 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1), with assessment of its Ki value and the type of inhibition. The dose-related effects of the compound were also evaluated using two different cell lines, with determinations of the respective EC50 values. The entire oil and the 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1) were tested on a triple transgenic mouse model of Alzheimer's disease. The overall results showed that compound 1 displayed a dose-dependent inhibition of BACE-1 in cellular and mouse models of Alzheimer's disease and is therefore capable of passing through cellular membranes and the blood-brain barrier.

  2. Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation.

    Directory of Open Access Journals (Sweden)

    Kuo-Feng Weng

    2009-09-01

    Full Text Available Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell-virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71 3C protease (3C(pro cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3' pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C(pro. CstF-64 was cleaved in vitro by 3C(pro but neither by mutant 3C(pro (in which the catalytic site was inactivated nor by another EV71 protease 2A(pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C(pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500. An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3'-end pre-mRNA processing and polyadenylation in 3C(pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C(pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

  3. Synthesis and biological investigation of PIM mimics carrying biotin or a fluorescent label for cellular imaging.

    Science.gov (United States)

    Front, Sophie; Bourigault, Marie-Laure; Rose, Stéphanie; Noria, Ségueni; Quesniaux, Valérie F J; Martin, Olivier R

    2013-01-16

    Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-inflammatory agents. Recently, heterocyclic analogues of PIMs in which the inositol is replaced by a piperidine (aza-PIM mimics) or a tetrahydropyran moiety (oxa-PIM mimics) have been prepared by short synthetic sequences and shown to retain the biological activity of the parent PIM structures. In this investigation, the aza-PIM analogue was used as a convenient scaffold to link biotin or a fluorescent label (tetramethyl-rhodamine) by way of an aminocaproyl spacer, with the goal of using these conjugates for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM-biotin conjugate binds to macrophage membranes with rapid kinetics.

  4. Synthesis, anticancer activity, and inhibition of tubulin polymerization by conformationally restricted analogues of lavendustin A.

    Science.gov (United States)

    Mu, Fanrong; Hamel, Ernest; Lee, Debbie J; Pryor, Donald E; Cushman, Mark

    2003-04-24

    Compounds in the lavendustin A series have been shown to inhibit both protein-tyrosine kinases (PTKs) and tubulin polymerization. Since certain lavendustin A derivatives can exist in conformations that resemble both the trans-stilbene structure of the PTK inhibitor piceatannol and the cis-stilbene structure of the tubulin polymerization inhibitor combretastatin A-4, the possibility exists that the ratio of the two types of activities of the lavendustins could be influenced through the synthesis of conformationally restricted analogues. Accordingly, the benzylaniline structure of a series of pharmacologically active lavendustin A fragments was replaced by either their cis- or their trans-stilbene relatives, and effects on both inhibition of tubulin polymerization and cytotoxicity in cancer cell cultures were monitored. Both dihydrostilbene and 1,2-diphenylalkyne congeners were also prepared and evaluated biologically. Surprisingly, conformational restriction of the bridge between the two aromatic rings of the lavendustins had no significant effect on biological activity. On the other hand, conversion of the three phenolic hydroxyl groups of the lavendustin A derivatives to their corresponding methyl ethers consistently abolished their ability to inhibit tubulin polymerization and usually decreased cytotoxicity in cancer cell cultures as well, indicating the importance of at least one of the phenolic hydroxyl groups. Further investigation suggested that the phenolic hydroxyl group in the salicylamide ring was required for activity, while the two phenol moieties in the hydroquinone ring could be methylated with retention of activity. Two of the lavendustin A derivatives displayed IC(50) values of 1.4 microM for inhibition of tubulin polymerization, which ranks them among the most potent of the known tubulin polymerization inhibitors.

  5. Carbamazepine (Tegretol) inhibits in vivo iodide uptake and hormone synthesis in rat thyroid glands

    International Nuclear Information System (INIS)

    Villa, S.M.; Alexander, N.M.

    1987-01-01

    Decreased serum concentrations of T3 and T4 occur in patients treated with the anticonvulsant drug carbamazepine (CBZ), but with rare exception, these patients remain euthyroid. The mechanism that accounts for diminished hormone levels is unknown, and our objective was to study the direct effect of CBZ on iodide uptake and hormone synthesis in thyroid glands of CBZ-treated and pair-fed control rats. Chronic ingestion (per os) of CBZ in male rats reduced the four hour thyroid 131I-iodide uptake by approximately 60%. This inhibition occurred after the animals had received sufficient CBZ to attain plasma CBZ concentrations of 0.8 microgram/ml. Continued treatment with CBZ ranging from 560 to 800 mg/kg/day for 14 days did not result in further inhibition of iodide uptake even though the plasma CBZ concentrations had increased 6-20 fold. No inhibition of iodide uptake was apparent when the animals initially received CBZ ranging from 40 to 152 mg/kg body weight for 22 days when there were no detectable levels of plasma CBZ. Overall growth rates of CBZ-treated rats were slightly (6-10%) less than the pair-fed control animals. Plasma T4 concentrations were reduced by 18% (p less than 0.05) in the CBZ-fed animals, while T3 concentrations were diminished by 53% (p less than 0.01). CBZ appeared to alter thyroidal iodide transport because the thyroid:plasma iodide ratios were decreased by 26% in the drug-treated rats. The distribution of radioiodine in thyroidal iodoamino acids was essentially the same in both groups of rats but the absolute quantities of radioiodine were more than 2.5 times greater in the control rats. CBZ failed to inhibit peroxidase-catalyzed iodide and guaiacol oxidation in vitro

  6. Further in vitro studies on the biochemistry of the inhibition of nucleic acid and protein synthesis induced by arsenic

    Energy Technology Data Exchange (ETDEWEB)

    Baron, D; Kunick, I; Frischmuth, I; Petres, J

    1975-01-01

    Human lymphocytes, stimulated by PHA, and exposed to increasing Na/sub 2/ HAsO/sub 4/ concentrations, show an identical incorporation rate for /sup 14/C thymidine and /sup 14/C-TTP into the DNA. /sup 14/C uridine is incorporated 3-4% less at an As concentration of 1.0 ..mu..g/ml medium, above this however approximately 15% less into the RNA as compared to /sup 14/C-UTP. In free pyrimidine bases, the incorporation of labelled triphosphates into the DNA and RNA is significantly reduced above 1 ..mu..g and 10 ..mu..g Na/sub 2/HAsO/sub 4/. Corresponding to its approximately uniform distribution into DNA and RNA, the incorporation rate of /sup 14/C-ATP above 10 ..mu..g Na/sub 2/HAsO/sub 4//ml culture medium lies between that of /sup 14/C-UTP and /sup 14/C-TTP. The incorporation of /sup 14/C alanine and /sup 14/C leucine into cellular protein is not reduced below 10 ..mu..g Na/sub 2/HAsO/sub 4//ml medium. Compared to /sup 14/c-UTP, /sup 14/C-ATP and /sup 14/C-TTP it is increased by the factor of 1.8; 2.5 and 6.8 respectively. The inhibition of enzymes of the dark repair mechanisms and the synthesis of biopolymers together with their altered sequence and the involvement of long-lived messenger RNA serve as an explanation of the observed alterations of the lymphocyte metabolism, caused by arsenic.

  7. On the synthesis of a bio-inspired dual-cellular fluidic flexible matrix composite adaptive structure based on a non-dimensional dynamics model

    International Nuclear Information System (INIS)

    Li, Suyi; Wang, K W

    2013-01-01

    A recent study investigated the dynamic characteristics of an adaptive structure concept featuring dual fluidic flexible matrix composite (F 2 MC) cells inspired by the configuration of plant cells and cell walls. This novel bio-inspired system consists of two F 2 MC cells with different fiber angles connected through internal fluid circuits. It was discovered that the dual F 2 MC cellular structure can be characterized as a two degree of freedom damped mass–spring oscillator, and can be utilized as a vibration absorber or an enhanced actuator under different operation conditions. These results demonstrated that the concept is promising and further investigations are needed to develop methodologies for synthesizing future multi-cellular F 2 MC structural systems. While interesting, the previous study focused on specific case studies and analysis. That is, the outcome did not provide insight that could be generalized, or tools for synthesizing a multiple F 2 MC cellular structure. This paper attempts to address this important issue by developing a non-dimensional dynamic model, which reveals good physical insights as well as identifying crucial constitutive parameters for F 2 MC cellular design. Working with these parameters, rather than physical variables, can greatly simplify the mathematics involved in the study. A synthesis tool is then developed for the dual-cellular structure, and it is found that for each set of achievable target poles and zero, there exist multiple F 2 MC cellular designs, forming a design space. The presented physical insights and synthesis tool for the dual-cellular structure will be the building blocks for future investigation on cellular structures with a larger number of cells. (paper)

  8. Depletion of microglia and inhibition of exosome synthesis halt tau propagation

    Science.gov (United States)

    Asai, Hirohide; Ikezu, Seiko; Tsunoda, Satoshi; Medalla, Maria; Luebke, Jennifer; Haydar, Tarik; Wolozin, Benjamin; Butovsky, Oleg; Kügler, Sebastian; Ikezu, Tsuneya

    2015-01-01

    Accumulation of pathological tau protein is a major hallmark of Alzheimer’s disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target. PMID:26436904

  9. The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

    Science.gov (United States)

    Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; García, Noemí Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nuñez, Gabriel; McDonald, Christine

    2013-01-01

    BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD. PMID:22387394

  10. Dasatinib synergises with irinotecan to suppress hepatocellular carcinoma via inhibiting the protein synthesis of PLK1.

    Science.gov (United States)

    Xu, Li; Zhu, Yuanrun; Shao, Jinjin; Chen, Min; Yan, Hao; Li, Guanqun; Zhu, Yi; Xu, Zhifei; Yang, Bo; Luo, Peihua; He, Qiaojun

    2017-04-11

    Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumour and has poor prognosis. Currently, systematic chemotherapy is the only approach to prolong survival. Thus the development of new treatment regimens is urgently needed to improve the therapeutic efficacy. Our study intended to assess the combination of dasatinib and irinotecan against HCC and made an effort to develop a potential medical choice for advanced HCC patients. We used SRB colorimetric assay and clonogenic assay to assess antitumour effect in vitro and HCC xenograft model to assess antitumour effect in vivo. We applied flow cytometry and western blotting to explore the mechanism of the combined therapy. Knockdown and overexpression of PLK1 are also applied for validation. We confirmed that dasatinib has synergistic effect with irinotecan (or SN38) on HCC both in vitro and in vivo. The effect is due to arisen apoptosis rate of HCC cells that is accompanied by mitochondria dysfunction. The enhanced antitumour efficacy of SN38 could be explained by additional inhibition of PLK1, which is triggered by dasatinib. Unlike existed PLK1 inhibitors, dasatinib does not inhibit PLK1 activity in a direct way. Instead, we found that dasatinib reduces PLK1 level by interfering with its protein synthesis progress. We validated that this kind of downregulation of PLK1 level has a key role in the synergistic effect of the two agents. Dasatinib is able to reinforce the anti-HCC efficacy of irinotecan/SN38 by downregulation of PLK1 synthesis. The combination of the two agents might be a potential medical choice for HCC therapy.

  11. Quantitative study of myocardial microcirculation in arterial hypertension due to progressive inhibition of NO synthesis

    Directory of Open Access Journals (Sweden)

    Leila Maria Meirelles Pereira

    1999-11-01

    Full Text Available OBJECTIVE: To study the quantitative changes in intramyocardial blood vessels in rats in whom nitric oxide synthesis was inhibited. METHODS: Four groups of 10 rats were studied: control (C25 and C40 and L-NAME (L25 and L40. The animals L25 and L40 received L-NAME in the dosage of 50mg/kg/day for 25 and 40 days, respectively. On days 26 and 41 the animals in groups 25 and 40 were sacrificed. Analysis of the myocardium was performed using light microscopy and stereology. RESULTS: Arterial blood pressure and heart weight increased 74.5 and 57.8% after 25 days and 90.2 and 34.6% after 40 days, respectively. Comparing the L-NAME rats with the respective controls revealed that vessel volume density decreased 31.3% after 40 days, and the vessel length-density decreased 53.5% after 25 days and 25.7% after 40 days. The mean cross-sectional area of the vessels showed an important reduction of 154.6% after 25 days. The intramyocardial vessels decreased significantly in length- density in the L-NAME animals. The mean cross-sectional area of the vessels, which normally increases during heart growth between 25 and 40 days, showed a precocious increase by the 25th day in the L-NAME rats. This suggests an increase of the size of the heart, including blood vessels. CONCLUSION: The inhibition of the NO synthesis provokes rarefaction in the intramyocardial vessels that progresses with the time of administration of L-NAME.

  12. Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

    Science.gov (United States)

    Uluisik, Irem; Kaya, Alaattin; Fomenko, Dmitri E.; Karakaya, Huseyin C.; Carlson, Bradley A.; Gladyshev, Vadim N.; Koc, Ahmet

    2011-01-01

    Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance. PMID:22114689

  13. Boron stress activates the general amino acid control mechanism and inhibits protein synthesis.

    Directory of Open Access Journals (Sweden)

    Irem Uluisik

    Full Text Available Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.

  14. Circadian rhythm genes mediate fenvalerate-induced inhibition of testosterone synthesis in mouse Leydig cells.

    Science.gov (United States)

    Guo, Yichen; Shen, Ouxi; Han, Jingjing; Duan, Hongyu; Yang, Siyuan; Zhu, Zhenghong; Tong, Jian; Zhang, Jie

    2017-01-01

    Fenvalerate (Fen), a widely used pesticide, is known to impair male reproductive functions by mechanisms that remain to be elucidated. Recent studies indicated that circadian clock genes may play an important role in successful male reproduction. The aim of this study was to determine the effects of Fen on circadian clock genes involved in the biosynthesis of testosterone using TM3 cells derived from mouse Leydig cells. Data demonstrated that the circadian rhythm of testosterone synthesis in TM3 cells was disturbed following Fen treatment as evidenced by changes in the circadian rhythmicity of core clock genes (Bmal1, Rev-erbα, Rorα). Further, the observed altered rhythms were accompanied by increased intracellular Ca 2+ levels and modified steroidogenic acute regulatory (StAR) mRNA expression. Thus, data suggested that Fen inhibits testosterone synthesis via pathways involving intracellular Ca 2+ and clock genes (Bmal1, Rev-Erbα, Rorα) as well as StAR mRNA expression in TM3 cells.

  15. Inhibition of hyaluronan synthesis reduces versican and fibronectin levels in trabecular meshwork cells.

    Directory of Open Access Journals (Sweden)

    Kate E Keller

    Full Text Available Hyaluronan (HA is a major component of the extracellular matrix (ECM and is synthesized by three HA synthases (HAS. Similarities between the HAS2 knockout mouse and the hdf mutant mouse, which has a mutation in the versican gene, suggest that HA and versican expression may be linked. In this study, the relationship between HA synthesis and levels of versican, fibronectin and several other ECM components in trabecular meshwork cells from the anterior segment of the eye was investigated. HA synthesis was inhibited using 4-methylumbelliferone (4MU, or reduced by RNAi silencing of each individual HAS gene. Quantitative RT-PCR and immunoblotting demonstrated a reduction in mRNA and protein levels of versican and fibronectin. Hyaluronidase treatment also reduced versican and fibronectin levels. These effects could not be reversed by addition of excess glucose or glucosamine or exogenous HA to the culture medium. CD44, tenascin C and fibrillin-1 mRNA levels were reduced by 4MU treatment, but SPARC and CSPG6 mRNA levels were unaffected. Immunostaining of trabecular meshwork tissue after exposure to 4MU showed an altered localization pattern of HA-binding protein, versican and fibronectin. Reduction of versican by RNAi silencing did not affect HA concentration as assessed by ELISA. Together, these data imply that HA concentration affects synthesis of certain ECM components. Since precise regulation of the trabecular meshwork ECM composition and organization is required to maintain the aqueous humor outflow resistance and intraocular pressure homeostasis in the eye, coordinated coupling of HA levels and several of its ECM binding partners should facilitate this process.

  16. Glycosaminoglycan-functionalized poly-lactide-co-glycolide nanoparticles: synthesis, characterization, cytocompatibility, and cellular uptake

    Directory of Open Access Journals (Sweden)

    Lamichhane SP

    2015-01-01

    Full Text Available Surya P Lamichhane,1 Neha Arya,1,2 Nirdesh Ojha,3 Esther Kohler,1 V Prasad Shastri1,2,41Institute for Macromolecular Chemistry, University of Freiburg, Freiburg, 2Helmholtz Virtual Institute on “Multifunctional Biomaterials for Medicine”, 3Laboratory for Process Technology, Department of Microsystems Engineering, University of Freiburg, Freiburg, 4Centre for Biological Signaling Studies (BIOSS, University of Freiburg, Freiburg, GermanyAbstract: The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG production is altered in many diseases (or pathologies, NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA NPs in the range of 100–150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549 cells, human pulmonary microvascular endothelial cells (HPMEC, and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100–600 µg/mL by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%–65% in comparison to that of

  17. Glycosphingolipid synthesis inhibitor AMP-DNM lowers plasma cholesterol levels by promoting fecal cholesterol excretion without inhibiting cholesterol absorption

    NARCIS (Netherlands)

    Vrins, Carlos L. J.; Bietrix, Florence; Lombardo, Elisa; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Overkleeft, Herman S.; Aerts, Johannes M.

    2012-01-01

    Inhibition of glycosphingolipid synthesis with iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM) increases fecal neutral sterol output in mice. To investigate which pathways were involved in this increase, C57BI/6J mice were treated with AMP-DNM and/or ezetimibe. Fecal

  18. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

    DEFF Research Database (Denmark)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia

    2016-01-01

    to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during...... recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits...

  19. Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway [version 2; referees: 2 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Paul Fineran

    2017-06-01

    Full Text Available Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC, a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC.  Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed.  Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in

  20. Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Paul Fineran

    2016-11-01

    Full Text Available Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC, a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were

  1. Codon-usage-based inhibition of HIV protein synthesis by human schlafen 11.

    Science.gov (United States)

    Li, Manqing; Kao, Elaine; Gao, Xia; Sandig, Hilary; Limmer, Kirsten; Pavon-Eternod, Mariana; Jones, Thomas E; Landry, Sebastien; Pan, Tao; Weitzman, Matthew D; David, Michael

    2012-11-01

    In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination.

  2. Peripheral Injection of SB203580 Inhibits the Inflammatory-Dependent Synthesis of Proinflammatory Cytokines in the Hypothalamus

    Directory of Open Access Journals (Sweden)

    Andrzej P. Herman

    2014-01-01

    Full Text Available The study was designed to determine the effects of peripheral injection of SB203580 on the synthesis of interleukin- (IL- 1β, IL-6, and tumor necrosis factor (TNF α in the hypothalamus of ewes during prolonged inflammation. Inflammation was induced by the administration of lipopolysaccharide (LPS (400 ng/kg over 7 days. SB203580 is a selective ATP-competitive inhibitor of the p38 mitogen-activated protein kinase (MAPK, which is involved in the regulation of proinflammatory cytokines IL-1β, IL-6 and TNFα synthesis. Intravenous injection of SB203580 successfully inhibited (P<0.01 synthesis of IL-1β and reduced (P<0.01 the production of IL-6 in the hypothalamus. The p38 MAPK inhibitor decreased (P<0.01 gene expression of TNFα but its effect was not observed at the level of TNFα protein synthesis. SB203580 also reduced (P<0.01 LPS-stimulated IL-1 receptor type 1 gene expression. The conclusion that inhibition of p38 MAPK blocks LPS-induced proinflammatory cytokine synthesis seems to initiate new perspectives in the treatment of chronic inflammatory diseases also within the central nervous system. However, potential proinflammatory effects of SB203580 treatment suggest that all therapies using p38 MAPK inhibitors should be introduced very carefully with analysis of all expected and unexpected consequences of treatment.

  3. Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

    Science.gov (United States)

    Xie, Xiaolei; Le, Li; Fan, Yanxin; Lv, Lin; Zhang, Junjie

    2012-07-01

    Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

  4. Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

    Science.gov (United States)

    Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

    2013-09-01

    Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

  5. Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

    International Nuclear Information System (INIS)

    Geraci, Fabiana; Pinsino, Annalisa; Turturici, Guiseppina; Savona, Rosalia; Giudice, Giovanni; Sconzo, Gabriella

    2004-01-01

    Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20 h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages

  6. Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish.

    Science.gov (United States)

    Li, Jia-Min; Li, Ling-Yu; Qin, Xuan; Degrace, Pascal; Demizieux, Laurent; Limbu, Samwel M; Wang, Xin; Zhang, Mei-Ling; Li, Dong-Liang; Du, Zhen-Yu

    2018-01-01

    Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor [0.05% body weight (BW)/d] for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) β-oxidation capability, and other molecular and biochemical assays of lipid, glucose, and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin ( mtor ), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be

  7. Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish

    Directory of Open Access Journals (Sweden)

    Jia-Min Li

    2018-05-01

    Full Text Available Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor [0.05% body weight (BW/d] for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG concentrations, fatty acid (FA β-oxidation capability, and other molecular and biochemical assays of lipid, glucose, and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin (mtor, and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model

  8. Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-01-01

    Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects...... of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48 ± 2.74 μM)>celecoxib (IC50: 14.92 ± 6.40 μM)>valdecoxib (IC50: 161.4 ± 28.6 μM)>rofecoxib (IC50...... correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs...

  9. Cellular targets of the myeloperoxidase-derived oxidant hypothiocyanous acid (HOSCN) and its role in the inhibition of glycolysis in macrophages

    DEFF Research Database (Denmark)

    Love, D; Barrett, T.J.; White, M.Y.

    2016-01-01

    the cellular targets of HOSCN in macrophages (J774A.1). We report that multiple thiol-containing proteins involved in metabolism and glycolysis; fructose bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and creatine kinase, together with a number of chaperone......, antioxidant and structural proteins, were modified in a reversible manner in macrophages treated with HOSCN. The modification of the metabolic enzymes was associated with a decrease in basal glycolysis, glycolytic reserve, glycolytic capacity and lactate release, which was only partly reversible on further...... incubation in the absence of HOSCN. Inhibition of glycolysis preceded cell death and was seen in cells exposed to low concentrations (r25 mM) of HOSCN. The ability of HOSCN to inhibit glycolysis and perturb energy production is likely to contribute to the cell death seen in macrophages on further incubation...

  10. Inhibition of Hyaluronic Acid Synthesis Suppresses Angiogenesis in Developing Endometriotic Lesions.

    Directory of Open Access Journals (Sweden)

    Carla N Olivares

    Full Text Available The development and long-term survival of endometriotic lesions is crucially dependent on an adequate vascularization. Hyaluronic acid (HA through its receptor CD44 has been described to be involved in the process of angiogenesis.To study the effect of HA synthesis inhibition using non-toxic doses of 4-methylumbelliferone (4-MU on endometriosis-related angiogenesis.The cytotoxicity of different in vitro doses of 4-MU on endothelial cells was firstly tested by means of a lactate dehydrogenase assay. The anti-angiogenic action of non-cytotoxic doses of 4-MU was then assessed by a rat aortic ring assay. In addition, endometriotic lesions were induced in dorsal skinfold chambers of female BALB/c mice, which were daily treated with an intraperitoneal injection of 0.9% NaCl (vehicle group; n = 6, 20 mg/kg 4-MU (n = 8 or 80 mg/kg 4-MU (n = 7 throughout an observation period of 14 days. The effect of 4-MU on their vascularization, survival and growth were studied by intravital fluorescence microscopy, histology and immunohistochemistry.Non-cytotoxic doses of 4-MU effectively inhibited vascular sprout formation in the rat aortic ring assay. Endometriotic lesions in dorsal skinfold chambers of 4-MU-treated mice dose-dependently exhibited a significantly smaller vascularized area and lower functional microvessel density when compared to vehicle-treated controls. Histological analyses revealed a downregulation of HA expression in 4-MU-treated lesions. This was associated with a reduced density of CD31-positive microvessels within the lesions. In contrast, numbers of PCNA-positive proliferating and cleaved caspase-3-positive apoptotic cells did not differ between 4-MU-treated and control lesions.The present study demonstrates for the first time that targeting the synthesis of HA suppresses angiogenesis in developing endometriotic lesions. Further studies have to clarify now whether in the future this anti-angiogenic effect can be used beneficially for the

  11. C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis.

    Science.gov (United States)

    Li, Yanning; Zhong, Yan; Gong, Wenjian; Gao, Xuehan; Qi, Huanli; Liu, Kun; Qi, Jinsheng

    2018-07-01

    Activation of transforming growth factor β1 (TGFB1)/SMAD3 signaling may lead to additional synthesis of collagen type IV (COL4), which is a major contributor to extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can attenuate fibrosis to have unique beneficial effects in DN. However, whether and how C-peptide affects TGFB1/SMAD3-activated COL4 synthesis is unclear. In this study, pathological changes, expression of COL4 a1-a5 chains ( Col4a1-a5 ), COL4 distribution and protein and TGFB1 and SMAD3 protein were first assessed in a rat model of diabetes. Then, rat mesangial cells were treated with high glucose (HG) and/or C-peptide to investigate the underlying mechanism. Col4a1-a5 expression, COL4 protein and secretion, TGFB1 protein, SMAD3 nuclear translocation and binding of SMAD3 to its cognate sites in the promoters of Col4a1a2 , Col4a3a4 and Col4a5 were measured. It was found that C-peptide attenuated glomerular pathological changes and suppressed renal Col4a1 -a5 mRNA expression, COL4 protein content and TGFB1 protein content. C-peptide had a dose-dependent effect to inhibit Col4a1-a5 mRNA expression, COL4 protein content and secretion, in HG-stimulated mesangial cells. In addition, the HG-induced increase in TGFB1 protein content was significantly reduced by C-peptide. Although not apparently affecting SMAD3 nuclear translocation, C-peptide prevented SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters in HG-stimulated mesangial cells. In conclusion, C-peptide could prevent SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters, to inhibit COL4 generation. These results may provide a mechanism for the alleviation of fibrosis in DN by C-peptide. © 2018 Society for Endocrinology.

  12. Dissociation of histone and DNA synthesis in x-irradiated HeLa cells

    International Nuclear Information System (INIS)

    Bases, R.; Mendez, F.

    1971-01-01

    Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1,000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal

  13. Generalization of fear inhibition by disrupting hippocampal protein synthesis-dependent reconsolidation process.

    Science.gov (United States)

    Yang, Chih-Hao; Huang, Chiung-Chun; Hsu, Kuei-Sen

    2011-09-01

    Repetitive replay of fear memories may precipitate the occurrence of post-traumatic stress disorder and other anxiety disorders. Hence, the suppression of fear memory retrieval may help prevent and treat these disorders. The formation of fear memories is often linked to multiple environmental cues and these interconnected cues may act as reminders for the recall of traumatic experiences. However, as a convenience, a simple paradigm of one cue pairing with the aversive stimulus is usually used in studies of fear conditioning in animals. Here, we built a more complex fear conditioning model by presenting several environmental stimuli during fear conditioning and characterize the effectiveness of extinction training and the disruption of reconsolidation process on the expression of learned fear responses. We demonstrate that extinction training with a single-paired cue resulted in cue-specific attenuation of fear responses but responses to other cures were unchanged. The cue-specific nature of the extinction persisted despite training sessions combined with D-cycloserine treatment reveals a significant weakness in extinction-based treatment. In contrast, the inhibition of the dorsal hippocampus (DH) but not the basolateral amygdala (BLA)-dependent memory reconsolidation process using either protein synthesis inhibitors or genetic disruption of cAMP-response-element-binding protein-mediated transcription comprehensively disrupted the learned connections between fear responses and all paired environmental cues. These findings emphasize the distinct role of the DH and the BLA in the reconsolidation process of fear memories and further indicate that the disruption of memory reconsolidation process in the DH may result in generalization of fear inhibition.

  14. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Science.gov (United States)

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-09-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+)-K(+) ATPases and Na(+)-K(+)-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+) channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+)-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+)-activated basolateral K(+) channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+)-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  15. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Directory of Open Access Journals (Sweden)

    Pawin Pongkorpsakol

    2014-09-01

    Full Text Available Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84 cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+-K(+ ATPases and Na(+-K(+-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+ channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+-activated basolateral K(+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  16. Effects of exogenous fatty acids and inhibition of de novo fatty acid synthesis on disaturated phosphatidylcholine production by fetal lung cells and adult type II cells.

    Science.gov (United States)

    Maniscalco, W M; Finkelstein, J N; Parkhurst, A B

    1989-05-01

    De novo fatty acid synthesis may be an important source of saturated fatty acids for fetal lung disaturated phosphatidylcholine (DSPC) production. To investigate the roles of de novo fatty acid synthesis and exogenous fatty acids, we incubated dispersed fetal lung cells and freshly isolated adult type II cells with exogenous palmitate and oleate and measured DSPC synthesis. Unlike adult type II cells, fetal lung cells did not increase DSPC synthesis when exogenous palmitate was available; adult type II cells increased DSPC synthesis by 70% in the presence of palmitate. Exogenous oleate decreased DSPC synthesis by 48% in fetal cells but not in adult type II cells. Incubation of fetal lung cells with TOFA [2-furancarboxylate, 5-(tetradecyloxy)-sodium], a metabolic inhibitor of fatty acid synthesis, decreased fatty acid synthesis by 65%. There was a simultaneous 56% inhibition of DSPC production, but no effect on protein, DNA, or glyceride-glycerol production, measured by precursor incorporation. The inhibition of DSPC synthesis associated with TOFA was partially prevented by exogenous palmitate but not oleate. Fetal cells prepared from explants that had been cultured in dexamethasone also had TOFA-associated inhibition of DSPC synthesis that was similar to non-dexamethasone-exposed cells. These studies suggest that under baseline conditions of low fatty acid availability, such as in the fetus, de novo fatty acid synthesis in fetal cells, but not in adult type II cells, provides sufficient saturated fatty acids to support maximal DSPC production. Inhibition of de novo fatty acid synthesis resulting in decreased DSPC production in fetal lung cells in conditions of low fatty acid availability suggests that fatty acid synthesis may be central to maintain DSPC synthesis in the fetus.

  17. The cytomegalovirus homolog of interleukin-10 requires phosphatidylinositol 3-kinase activity for inhibition of cytokine synthesis in monocytes.

    Science.gov (United States)

    Spencer, Juliet V

    2007-02-01

    Human cytomegalovirus (CMV) has evolved numerous strategies for evading host immune defenses, including piracy of cellular cytokines. A viral homolog of interleukin-10, designated cmvIL-10, binds to the cellular IL-10 receptor and effects potent immune suppression. The signaling pathways employed by cmvIL-10 were investigated, and the classic IL-10R/JAK1/Stat3 pathway was found to be activated in monocytes. However, inhibition of JAK1 had little effect on cmvIL-10-mediated suppression of tumor necrosis factor alpha (TNF-alpha) production. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway had a more significant impact on TNF-alpha levels but did not completely relieve the immune suppression, demonstrating that cmvIL-10 stimulates multiple signaling pathways to modulate cell function.

  18. 3-(2-Alkylsulfanyl-6-benzothiazolylaminomethyl-2-benzoxazolethiones - Synthesis and Photosynthesis-Inhibiting Activity in Spinach Chloropasts

    Directory of Open Access Journals (Sweden)

    Dusan Loos

    1999-03-01

    Full Text Available The synthesis and photosynthesis-inhibiting activity of 14 new 3-(2-alkylsulfanyl-6-benzothiazolylaminomethyl-2-benzoxazolethiones are reported. The new compounds were prepared by the reaction of 2-alkylsulfanyl-6-aminobenzothiazoles with 3-hydroxymethyl-2-benzoxazolethione. The structures of the compounds were verified by 1H NMR spectra. The compounds inhibit photosynthetic electron transport in spinach chloroplasts. The photosynthetic activity was found to depend on the calculated lipophilicity of the new compounds. Some structure characteristics and quantum chemical parameters were calculated by AM1 method.

  19. Gamma-ray induced inhibition of DNA synthesis in ataxia telangiectasia fibroblasts is a function of excision repair capacity

    International Nuclear Information System (INIS)

    Smith, P.J.; Paterson, M.C.

    1980-01-01

    The extent of the deficiency in γ-ray induced DNA repair synthesis in an ataxia telangiectasia (AT) human fibroblast strain was found to show no oxygen enhancement, consistent with a defect in the repair of base damage. Repair deficiency, but not repair proficiency, in AT cells was accompanied by a lack of inhibition of DNA synthesis by either γ-rays or the radiomimetic drug bleomycin. Experiments with 4-nitroquinoline 1-oxide indicated that lack of inhibition was specific for radiogenic-type damage. Thus excision repair, perhaps by DNA strand incision or chromatin modification, appears to halt replicon initiation in irradiated repair proficient cells whereas in repair defective AT strains this putatively important biological function is inoperative

  20. Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Wang Qilin

    2012-12-01

    Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

  1. Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation

    International Nuclear Information System (INIS)

    Ventura, A.M.; Meneghini, R.

    1984-01-01

    Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nonsubstituted DNA, was noticed in the density-distribution profiles. The density distribution of DNA along the gradient can provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement. (Auth.)

  2. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

    Science.gov (United States)

    Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

    2014-01-01

    Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

  3. The chemopreventive effect of the dietary compound kaempferol on the MCF-7 human breast cancer cell line is dependent on inhibition of glucose cellular uptake.

    Science.gov (United States)

    Azevedo, Cláudia; Correia-Branco, Ana; Araújo, João R; Guimarães, João T; Keating, Elisa; Martel, Fátima

    2015-01-01

    Our aim was to investigate the effect of several dietary polyphenols on glucose uptake by breast cancer cells. Uptake of (3)H-deoxy-D-glucose ((3)H-DG) by MCF-7 cells was time-dependent, saturable, and inhibited by cytochalasin B plus phloridzin. In the short-term (26 min), myricetin, chrysin, genistein, resveratrol, kaempferol, and xanthohumol (10-100 µM) inhibited (3)H-DG uptake. Kaempferol was found to be the most potent inhibitor of (3)H-DG uptake [IC50 of 4 µM (1.6-9.8)], behaving as a mixed-type inhibitor. In the long-term (24 h), kaempferol (30 µM) was also able to inhibit (3)H-DG uptake, associated with a 40% decrease in GLUT1 mRNA levels. Interestingly enough, kaempferol (100 µM) revealed antiproliferative (sulforhodamine B and (3)H-thymidine incorporation assays) and cytotoxic (extracellular lactate dehydrogenase activity determination) properties, which were mimicked by low extracellular (1 mM) glucose conditions and reversed by high extracellular (20 mM) glucose conditions. Finally, exposure of cells to kaempferol (30 µM) induced an increase in extracellular lactate levels over time (to 731 ± 32% of control after a 24 h exposure), due to inhibition of MCT1-mediated lactate cellular uptake. In conclusion, kaempferol potently inhibits glucose uptake by MCF-7 cells, apparently by decreasing GLUT1-mediated glucose uptake. The antiproliferative and cytotoxic effect of kaempferol in these cells appears to be dependent on this effect.

  4. Inhibition of IGF-1-Mediated Cellular Migration and Invasion by Migracin A in Ovarian Clear Cell Carcinoma Cells.

    Science.gov (United States)

    Ukaji, Tamami; Lin, Yinzhi; Banno, Kouji; Okada, Shoshiro; Umezawa, Kazuo

    2015-01-01

    Previously we isolated migracin A from a Streptomyces culture filtrate as an inhibitor of cancer cell migration. In the present research, we found that migracin A inhibited migration and invasion of ovarian clear cell carcinoma ES-2 cells. In the course of our mechanistic study, migracin A was shown to enhance vasohibin-1 expression in an angiogenesis array. We also confirmed that it increased the mRNA expression of this protein. Moreover, overexpression of vasohibin-1 lowered the migration but not the invasion of ES-2 cells. Then, we looked for another target protein employing a motility array, and found that migracin A lowered the IGF-1 expression. Knockdown of IGF-1 by siRNA decreased the migration and invasion of ES-2 cells. Migracin A also decreased Akt phosphorylation involved in the downstream signaling. Crosstalk analysis indicated that overexpression of vasohibin-1 decreased the IGF-1 expression. On the other hand, it showed no direct anticancer activity in terms of the ES-2 growth in agar. Migracin A inhibited the migration and IGF-1 expression in not only ES-2 but also another ovarian clear cell carcinoma JHOC-5 cells. In addition, it also inhibited capillary tube formation of human umbilical vein endothelial cells. Since its cytotoxicity is very low, migracin A may be a candidate for an anti-metastasis agent not exhibiting prominent toxicity.

  5. Fibroblast growth factor 23 inhibits extrarenal synthesis of 1,25-dihydroxyvitamin D in human monocytes.

    Science.gov (United States)

    Bacchetta, Justine; Sea, Jessica L; Chun, Rene F; Lisse, Thomas S; Wesseling-Perry, Katherine; Gales, Barbara; Adams, John S; Salusky, Isidro B; Hewison, Martin

    2013-01-01

    Vitamin D is a potent stimulator of monocyte innate immunity, and this effect is mediated via intracrine conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)(2) D). In the kidney, synthesis of 1,25(OH)(2) D is suppressed by fibroblast growth factor 23 (FGF23), via transcriptional suppression of the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). We hypothesized that FGF23 also suppresses CYP27B1 in monocytes, with concomitant effects on intracrine responses to 1,25(OH)(2) D. Healthy donor peripheral blood mononuclear cell monocytes (PBMCm) and peritoneal dialysate monocyte (PDm) effluent from kidney disease patients were assessed at baseline to confirm the presence of mRNA for FGF23 receptors (FGFRs), with Klotho and FGFR1 being more strongly expressed than FGFR2/3/4 in both cell types. Immunohistochemistry showed coexpression of Klotho and FGFR1 in PBMCm and PDm, with this effect being enhanced following treatment with FGF23 in PBMCm but not PDm. Treatment with FGF23 activated mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) pathways in PBMCm, demonstrating functional FGFR signaling in these cells. FGF23 treatment of PBMCm and PDm decreased expression of mRNA for CYP27B1. In PBMCm this was associated with downregulation of 25OHD to 1,25(OH)(2) D metabolism, and concomitant suppression of intracrine induced 24-hydroxylase (CYP24A1) and antibacterial cathelicidin (LL37). FGF23 suppression of CYP27B1 was particularly pronounced in PBMCm treated with interleukin-15 to stimulate synthesis of 1,25(OH)(2) D. These data indicate that FGF23 can inhibit extra-renal expression of CYP27B1 and subsequent intracrine responses to 1,25(OH)(2) D in two different human monocyte models. Elevated expression of FGF23 may therefore play a crucial role in defining immune responses to vitamin D and this, in turn, may be a key determinant of infection in patients with chronic kidney disease (CKD). Copyright © 2013 American Society for

  6. FGF23 inhibits extra-renal synthesis of 1,25-dihydroxyvitamin D in human monocytes

    Science.gov (United States)

    Bacchetta, Justine; Sea, Jessica L; Chun, Rene F; Lisse, Thomas S; Wesseling-Perry, Katherine; Gales, Barbara; Adams, John S.; Salusky, Isidro B; Hewison, Martin

    2012-01-01

    Vitamin D is a potent stimulator of monocyte innate immunity, with this effect being mediated via intracrine conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)2D). In the kidney synthesis of 1,25(OH)2D is suppressed by fibroblast growth factor 23 (FGF23), via transcriptional suppression of the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). We hypothesized that FGF23 also suppresses CYP27B1 in monocytes, with concomitant effects on intracrine responses to 1,25(OH)2D. Monocytes from healthy donor peripheral blood mononuclear cells (PBMCm) and from peritoneal dialysate effluent from kidney disease patients (PDm) were assessed at baseline to confirm the presence of mRNA for FGF23 receptors (FGFRs), with Klotho and FGFR1 being more strongly expressed than FGFR2/3/4 in both cell types. Immunohistochemistry showed co-expression of Klotho and FGFR1 in PBMCm and PDm, with this effect being enhanced following treatment with FGF23 in PBMCm but not PDm. Treatment with FGF23 activated MAP kinase (MAPK) and Akt pathways in PBMCm, demonstrating functional FGFR signaling in these cells. FGF23 treatment of PBMCm and PDm decreased expression of mRNA for CYP27B1. In PBMCm this was associated with downregulation of 25OHD to 1,25(OH)2D metabolism, and concomitant suppression of intracrine induced 24-hydroxylase (CYP24A1) and antibacterial cathelicidin (LL37). FGF23 suppression of CYP27B1 was particularly pronounced in PBMCm treated with interleukin-15 to stimulate synthesis of 1,25(OH)2D. These data indicate that FGF23 can inhibit extra-renal expression of CYP27B1 and subsequent intracrine responses to 1,25(OH)2D in two different human monocyte models. Elevated expression of FGF23 may therefore play a crucial role in defining immune responses to vitamin D and this, in turn, may be a key determinant of infection in patients with CKD. PMID:22886720

  7. Alzheimer's Disease Brain-Derived Amyloid-{beta}-Mediated Inhibition of LTP In Vivo Is Prevented by Immunotargeting Cellular Prion Protein.

    LENUS (Irish Health Repository)

    Barry, Andrew E

    2011-05-18

    Synthetic amyloid-β protein (Aβ) oligomers bind with high affinity to cellular prion protein (PrP(C)), but the role of this interaction in mediating the disruption of synaptic plasticity by such soluble Aβ in vitro is controversial. Here we report that intracerebroventricular injection of Aβ-containing aqueous extracts of Alzheimer\\'s disease (AD) brain robustly inhibits long-term potentiation (LTP) without significantly affecting baseline excitatory synaptic transmission in the rat hippocampus in vivo. Moreover, the disruption of LTP was abrogated by immunodepletion of Aβ. Importantly, intracerebroventricular administration of antigen-binding antibody fragment D13, directed to a putative Aβ-binding site on PrP(C), prevented the inhibition of LTP by AD brain-derived Aβ. In contrast, R1, a Fab directed to the C terminus of PrP(C), a region not implicated in binding of Aβ, did not significantly affect the Aβ-mediated inhibition of LTP. These data support the pathophysiological significance of SDS-stable Aβ dimer and the role of PrP(C) in mediating synaptic plasticity disruption by soluble Aβ.

  8. Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

    OpenAIRE

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-01-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell...

  9. Inhibition of the cellular function of perforin by 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazoles.

    Science.gov (United States)

    Lyons, Dani M; Huttunen, Kristiina M; Browne, Kylie A; Ciccone, Annette; Trapani, Joseph A; Denny, William A; Spicer, Julie A

    2011-07-01

    A high throughput screen showed the ability of a 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazole analogue to directly inhibit the lytic activity of the pore-forming protein perforin. A series of analogues were prepared to study structure-activity relationships (SAR) for the this activity, either directly added to cells or released in situ by KHYG-1 NK cells, at non-toxic concentrations. These studies showed that the pyridobenzimidazole moiety was required for effective activity, with strongly basic centres disfavoured. This class of compounds was relatively unaffected by the addition of serum, which was not the case for a previous class of direct inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na+,K+-ATPase Blocks HIV-1 Infection

    Directory of Open Access Journals (Sweden)

    Silvia Agostini

    2017-07-01

    Full Text Available Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na+,K+-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.

  11. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Heyward, Scott; Moeller, Timothy [Bioreclamation In Vitro Technologies, Baltimore, MD 21227 (United States); Swaan, Peter W. [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Wang, Hongbing, E-mail: hwang@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States)

    2014-08-15

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  12. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    International Nuclear Information System (INIS)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao; Heyward, Scott; Moeller, Timothy; Swaan, Peter W.; Wang, Hongbing

    2014-01-01

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  13. Effect of inhibition of DNA synthesis on recovery of X-irradiated L5178Y-S cells. I

    International Nuclear Information System (INIS)

    Kapiszewska, M.; Lange, C.S.

    1989-01-01

    Irradiated L5178Y-S cells (LY-S) were characterized by an exponential survival curve and the potentiation effect of split -dose irradiation. Previously it was found that in LY-S cells the reduction of DNA replicative synthesis rate affected the balance between the fixation and repair of sublethal damage (SLD) and of potentially lethal damage (PLD) in favor of repair. It was found now that a block of DNA synthesis by aphidicolin (APC), an inhibitor of DNA polymerase alpha, was sufficient to protect LY-S cells from fixation of PLD and SLD induced by X-rays. Treatment with APC 0.5 μg/ml for 2 h, efficiently inhibited DNA replication (95%) with minimal effect on survival. Inhibition of DNA synthesis by combined irradiation and APC was not significantly different from APC treatment alone. The level of protection by APC was dependent on the length of time between irradiation and APC application. An opposite effect was observed when the drug treatment had preceded irradiation: The killing effect of X-ray increased. The effect of aphidicolin treatment remained even after removal of APC and was dependent on the drug concentration and time between drug removal and irradiaton. These results are interpreted as indicating that X-ray damage was fixed in LY-S cells, because of their lack of ability to maintain the nucleotide pool balance, and that fixation took place during progression through the cell cycle. (author). 6 figs., 22 refs

  14. Design, characterization, and in vitro cellular inhibition and uptake of optimized genistein-loaded NLC for the prevention of posterior capsular opacification using response surface methodology.

    Science.gov (United States)

    Zhang, Wenji; Li, Xuedong; Ye, Tiantian; Chen, Fen; Sun, Xiao; Kong, Jun; Yang, Xinggang; Pan, Weisan; Li, Sanming

    2013-09-15

    This study was to design an innovative nanostructured lipid carrier (NLC) for drug delivery of genistein applied after cataract surgery for the prevention of posterior capsular opacification. NLC loaded with genistein (GEN-NLC) was produced with Compritol 888 ATO, Gelucire 44/14 and Miglyol 812N, stabilized by Solutol(®) HS15 by melt emulsification method. A 2(4) central composite design of 4 independent variables was performed for optimization. Effects of drug concentration, Gelucire 44/14 concentration in total solid lipid, liquid lipid concentration, and surfactant concentration on the mean particle size, polydispersity index, zeta potential and encapsulation efficiency were investigated. Analysis of variance (ANOVA) statistical test was used to assess the optimization. The optimized GEN-NLC showed a homogeneous particle size of 90.16 nm (with PI=0.33) of negatively charged surface (-25.08 mv) and high encapsulation efficiency (91.14%). Particle morphology assessed by TEM revealed a spherical shape. DSC analyses confirmed that GEN was mostly entrapped in amorphous state. In vitro release experiments indicated a prolonged and controlled genistein release for 72 h. In vitro growth inhibition assay showed an effective growth inhibition of GEN-NLCs on human lens epithelial cells (HLECs). Preliminary cellular uptake test proved a enhanced penetration of genistein into HLECs when delivered in NLC. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Cellular and molecular insight into the inhibition of primary root growth of Arabidopsis induced by peptaibols, a class of linear peptide antibiotics mainly produced by Trichoderma spp.

    Science.gov (United States)

    Shi, Wei-Ling; Chen, Xiu-Lan; Wang, Li-Xia; Gong, Zhi-Ting; Li, Shuyu; Li, Chun-Long; Xie, Bin-Bin; Zhang, Wei; Shi, Mei; Li, Chuanyou; Zhang, Yu-Zhong; Song, Xiao-Yan

    2016-04-01

    Trichoderma spp. are well known biocontrol agents that produce a variety of antibiotics. Peptaibols are a class of linear peptide antibiotics mainly produced by Trichoderma Alamethicin, the most studied peptaibol, is reported as toxic to plants at certain concentrations, while the mechanisms involved are unclear. We illustrated the toxic mechanisms of peptaibols by studying the growth-inhibitory effect of Trichokonin VI (TK VI), a peptaibol from Trichoderma longibrachiatum SMF2, on Arabidopsis primary roots. TK VI inhibited root growth by suppressing cell division and cell elongation, and disrupting root stem cell niche maintenance. TK VI increased auxin content and disrupted auxin response gradients in root tips. Further, we screened the Arabidopsis TK VI-resistant mutant tkr1. tkr1 harbors a point mutation in GORK, which encodes gated outwardly rectifying K(+)channel proteins. This mutation alleviated TK VI-induced suppression of K(+)efflux in roots, thereby stabilizing the auxin gradient. The tkr1 mutant also resisted the phytotoxicity of alamethicin. Our results indicate that GORK channels play a key role in peptaibol-plant interaction and that there is an inter-relationship between GORK channels and maintenance of auxin homeostasis. The cellular and molecular insight into the peptaibol-induced inhibition of plant root growth advances our understanding of Trichoderma-plant interactions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  16. 1α,25-Dihydroxyvitamin D(3) inhibits vascular cellular adhesion molecule-1 expression and interleukin-8 production in human coronary arterial endothelial cells.

    Science.gov (United States)

    Kudo, Keiko; Hasegawa, Shunji; Suzuki, Yasuo; Hirano, Reiji; Wakiguchi, Hiroyuki; Kittaka, Setsuaki; Ichiyama, Takashi

    2012-11-01

    Kawasaki disease is an acute febrile vasculitis of childhood that is associated with elevated production of inflammatory cytokines, causing damage to the coronary arteries. The production of proinflammatory cytokines and expression of adhesion molecules in human coronary arterial endothelial cells (HCAECs) is regulated by nuclear transcription factor-κB (NF-κB) activation. We have previously reported that the active form of vitamin D, 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)), inhibits tumor necrosis factor-α (TNF-α)-induced NF-κB activation. In this study, we examined the anti-inflammatory effects of 1α,25-(OH)(2)D(3) on TNF-α-induced adhesion molecule expression (vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1)) and cytokine production (interleukin-6 (IL-6) and IL-8) in HCAECs. Pretreatment with 1α,25-(OH)(2)D(3) significantly inhibited TNF-α-induced VCAM-1 expression and IL-8 production in HCAECs. Our results suggest that adjunctive 1α,25-(OH)(2)D(3) therapy may modulate the inflammatory response during Kawasaki disease vasculitis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Dietary uptake of Cu sorbed to hydrous iron oxide is linked to cellular toxicity and feeding inhibition in a benthic grazer

    Science.gov (United States)

    Cain, Daniel J.; Croteau, Marie-Noele; Fuller, Christopher C.; Ringwood, Amy H.

    2016-01-01

    Whereas feeding inhibition caused by exposure to contaminants has been extensively documented, the underlying mechanism(s) are less well understood. For this study, the behavior of several key feeding processes, including ingestion rate and assimilation efficiency, that affect the dietary uptake of Cu were evaluated in the benthic grazer Lymnaea stagnalis following 4–5 h exposures to Cu adsorbed to synthetic hydrous ferric oxide (Cu–HFO). The particles were mixed with a cultured alga to create algal mats with Cu exposures spanning nearly 3 orders of magnitude at variable or constant Fe concentrations, thereby allowing first order and interactive effects of Cu and Fe to be evaluated. Results showed that Cu influx rates and ingestion rates decreased as Cu exposures of the algal mat mixture exceeded 104 nmol/g. Ingestion rate appeared to exert primary control on the Cu influx rate. Lysosomal destabilization rates increased directly with Cu influx rates. At the highest Cu exposure where the incidence of lysosomal membrane damage was greatest (51%), the ingestion rate was suppressed 80%. The findings suggested that feeding inhibition was a stress response emanating from excessive uptake of dietary Cu and cellular toxicity.

  18. The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways.

    Science.gov (United States)

    Youns, Mаhmoud; Abdel Halim Hegazy, Wael

    2017-01-01

    Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.

  19. The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Mаhmoud Youns

    Full Text Available Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2, colorectal (Caco-2 and pancreatic (Suit-2 cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.

  20. The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways

    Science.gov (United States)

    Youns, Mаhmoud; Abdel Halim Hegazy, Wael

    2017-01-01

    Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes. PMID:28052097

  1. Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

    Directory of Open Access Journals (Sweden)

    Kim CH

    2016-05-01

    Full Text Available Cy Hyun Kim,1,2,* Jin-Hong Shin,1,3,* Sung Jun Hwang,1,2 Yung Hyun Choi,4 Dae-Seong Kim,1,3 Cheol Min Kim2,51Research Institute of Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, 2Center for Anti-Aging Industry, Pusan National University, Busan, 3Department of Neurology, Pusan National University Yangsan Hospital, Yangsan, 4Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan, 5Department of Biomedical Informatics, Pusan National University School of Medicine, Yangsan, Republic of Korea*These authors contributed equally to this work Abstract: Schisandrae fructus (SF has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 µg/mL of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 µg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged

  2. Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis.

    Science.gov (United States)

    Rahmanzadeh, R; Hüttmann, G; Gerdes, J; Scholzen, T

    2007-06-01

    Expression of the nuclear Ki-67 protein (pKi-67) is strongly associated with cell proliferation. For this reason, antibodies against this protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi-67 is still missing. Recently, we proposed a function of pKi-67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi-67 in this process by photochemical inhibition using chromophore-assisted light inactivation (CALI). Anti-pKi-67 antibodies were labelled with the fluorochrome fluorescein 5(6)-isothiocyanate and were irradiated after binding to their target protein. Performing CALI in vitro on cell lysates led to specific cross-linking of pKi-67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross-link formation, indicating a close spatial proximity of UBF and pKi-67. CALI in living cells, using micro-injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi-67-CALI resulted in dramatic inhibition of RNA polymerase I-dependent nucleolar rRNA synthesis, whereas RNA polymerase II-dependent nucleoplasmic RNA synthesis remained almost unaltered. Our data presented here argue for a crucial role of pKi-67 in RNA polymerase I-dependent nucleolar rRNA synthesis.

  3. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  4. Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia.

    Science.gov (United States)

    Nishida, Takashi; Hara, Naho; Watanabe, Kenta; Shimizu, Takashi; Fujishima, Masahiro; Watarai, Masahisa

    2018-01-01

    Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires' disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena . The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila , but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC -deletion mutant. To assess the phagocytic activity of P. tetraurelia , Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella -containing vacuoles from P. tetraurelia . We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity

  5. Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia

    Directory of Open Access Journals (Sweden)

    Takashi Nishida

    2018-04-01

    Full Text Available Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308 and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show

  6. High Throughput Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

    National Research Council Canada - National Science Library

    Boger, Dale L

    2005-01-01

    .... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

  7. High Throughout Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

    National Research Council Canada - National Science Library

    Boger, Dale

    2003-01-01

    .... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

  8. High Throughput Synthesis and Screening for Agents Inhibiting Androgen Receptor Mediated Gene Transcription

    National Research Council Canada - National Science Library

    Boger, Dale

    2004-01-01

    .... This entails the high throughput synthesis of DNA binding agents related to distamycin, their screening for binding to androgen response elements using a new high throughput DNA binding screen...

  9. Inhibition of DNA virus: Herpes-1 (HSV-1 in cellular culture replication, through an antioxidant treatment extracted from rosemary spice

    Directory of Open Access Journals (Sweden)

    Dalva Assunção Portari Mancini

    2009-03-01

    Full Text Available This work aimed to evaluate antiviral properties in antioxidants from spices. Phenolic compounds extracted from rosemary (Rosmarinus officinallis, L by hot water, had their antioxidant activity determined by spectrophotometry using β carotene/linoleic acid system. The rosemary extract was evaluated by antiviral assay of Herpes Virus type-1 (HSV-1 replication in VERO cells, in the presence or absence of the spice. 10,000 TCID50/mL of the HSV-1 was kept for 3 h at 4º C, with 300 ppm of rosemary extract, and 100 ppm of butyl hydroxyl toluene (BHT. Then, these viruses were inoculated in VERO cells incubated at 37º C in CO2-5 %, for seven days. Daily, they were examined and the end point was based on 100% of CPE in virus control (without antioxidants. The HSV-1 replication inhibition percentage (IP measured the antiviral action from antioxidants, showing viral reductions of the 82.0, 82.5%, in the presence of rosemary and rosemary + BHT, respectively. As an extension, cell test corresponded to the similar viral decrease (IP = 85.0 and 86.3% in both aforementioned situations. Results lead to conclude that phenolic compounds from rosemary revealed an antiviral action on herpesvirus-1.Neste estudo foi avaliada a ação antiviral de antioxidantes de especiaria. Extrato aquoso de alecrim (Rosmarinus officinalis, L, que apresentou atividade antioxidante através de espectrofotometria usando o sistema β caroteno/ácido linoléico, foi avaliado em ensaios com vírus herpes-1 na replicação em células VERO. Nestes ensaios foram utilizados 10.000 TCID50%/mL do vírus HSV-1, mantidos em contato com 300 ppm do extrato de alecrim e com 100 ppm de butil hidroxi tolueno (BHT, durante 3h a 4°C. Esses vírus, em seguida, foram inoculados em células VERO incubadas a 37 °C/5% de CO2 por sete dias. Pelo efeito citopático (ECP e o "end point" de ECP do controle de vírus (sem antioxidante, foi possível observar que houve reduções na replicação viral de 82

  10. Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells

    NARCIS (Netherlands)

    Hanemaaijer, R.; Visser, H.; Koolwijk, P.; Sorsa, T.; Salo, T.; Golub, L.M.; Hinsbergh, V.W. van

    1998-01-01

    Doxycycline is a commonly used broad-spectrum antibiotic. Recently, it has been shown that it also inhibits the activity of mammalian collagenases and gelatinases, an activity unrelated to its antimicrobial efficacy. In this study, we show that doxycycline not only inhibits MMP-8 and MMP-9

  11. Cell growth state determines susceptibility of repair DNA synthesis to inhibition by hydroxyurea and 1-beta-D-arabinofuranosylcytosine

    International Nuclear Information System (INIS)

    Mullinger, A.M.; Collins, A.R.; Johnson, R.T.

    1983-01-01

    The effects of inhibitors of replicative DNA synthesis on repair DNA synthesis have been examined by autoradiography in several different cell types and in cells in different growth states. Hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), administered together, influence unscheduled DNA synthesis (UDS) in a manner which is independent of the status of the cell culture (normal or transformed) and of the species, but which is strongly affected by whether the cells are proliferating or quiescent. In proliferating human, Chinese hamster and Microtus cell cultures, UDS is not inhibited by HU and ara C, and may even appear to be stimulated. In quiescent cultures of these cells UDS is reduced by HU and ara C. In cells reseeded from a confluent culture and followed during proliferation and back to quiescence the effect of inhibitors parallels the growth pattern. The results are interpreted in terms of changes in the sizes of endogenous DNA precursor pools; they underline the potential problems associated with quantitating UDS in the presence of inhibitors

  12. Inhibition of protein synthesis but not β-adrenergic receptors blocks reconsolidation of a cocaine-associated cue memory.

    Science.gov (United States)

    Dunbar, Amber B; Taylor, Jane R

    2016-08-01

    Previously consolidated memories have the potential to enter a state of lability upon memory recall, during which time the memory can be altered before undergoing an additional consolidation-like process and being stored again as a long-term memory. Blocking reconsolidation of aberrant memories has been proposed as a potential treatment for psychiatric disorders including addiction. Here we investigated of the effect of systemically administering the protein synthesis inhibitor cycloheximide or the β-adrenergic antagonist propranolol on reconsolidation. Rats were trained to self-administer cocaine, during which each lever press resulted in the presentation of a cue paired with an intravenous infusion of cocaine. After undergoing lever press extinction to reduce operant responding, the cue memory was reactivated and rats were administered systemic injections of propranolol, cycloheximide, or vehicle. Post-reactivation cycloheximide, but not propranolol, resulted in a reactivation-dependent decrease in cue-induced reinstatement, indicative of reconsolidation blockade by protein synthesis inhibition. The present data indicate that systemically targeting protein synthesis as opposed to the β-adrenergic system may more effectively attenuate the reconsolidation of a drug-related memory and decrease drug-seeking behavior. © 2016 Dunbar and Taylor; Published by Cold Spring Harbor Laboratory Press.

  13. Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell

    International Nuclear Information System (INIS)

    Chiou, S.-H.; Chen, S.-J.; Peng, C-H.; Chang, Y.-L.; Ku, H.-H.; Hsu, W.-M.; Ho, Larry L.-T.; Lee, C.-H.

    2006-01-01

    Fluoxetine is a widely used antidepressant compound which inhibits the reuptake of serotonin in the central nervous system. Recent studies have shown that fluoxetine can promote neurogenesis and improve the survival rate of neurons. However, whether fluoxetine modulates the proliferation or neuroprotection effects of neural stem cells (NSCs) needs to be elucidated. In this study, we demonstrated that 20 μM fluoxetine can increase the cell proliferation of NSCs derived from the hippocampus of adult rats by MTT test. The up-regulated expression of Bcl-2, Bcl-xL and the cellular FLICE-inhibitory protein (c-FLIP) in fluoxetine-treated NSCs was detected by real-time RT-PCR. Our results further showed that fluoxetine protects the lipopolysaccharide-induced apoptosis in NSCs, in part, by activating the expression of c-FLIP. Moreover, c-FLIP induction by fluoxetine requires the activation of the c-FLIP promoter region spanning nucleotides -414 to -133, including CREB and SP1 sites. This effect appeared to involve the phosphatidylinositol-3-kinase-dependent pathway. Furthermore, fluoxetine treatment significantly inhibited the induction of proinflammatory factor IL-1β, IL-6, and TNF-α in the culture medium of LPS-treated NSCs (p < 0.01). The results of high performance liquid chromatography coupled to electrochemical detection further confirmed that fluoxentine increased the functional production of serotonin in NSCs. Together, these data demonstrate the specific activation of c-FLIP by fluoxetine and indicate the novel role of fluoxetine for neuroprotection in the treatment of depression

  14. Cellular corepressor TLE2 inhibits replication-and-transcription- activator-mediated transactivation and lytic reactivation of Kaposi's sarcoma-associated herpesvirus.

    Science.gov (United States)

    He, Zhiheng; Liu, Yunhua; Liang, Deguang; Wang, Zhuo; Robertson, Erle S; Lan, Ke

    2010-02-01

    Replication and transcription activator (RTA) encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential and sufficient to initiate lytic reactivation. RTA activates its target genes through direct binding with high affinity to its responsive elements or by interaction with cellular factors, such as RBP-Jkappa, Ap-1, C/EBP-alpha, and Oct-1. In this study, we identified transducin-like enhancer of split 2 (TLE2) as a novel RTA binding protein by using yeast two-hybrid screening of a human spleen cDNA library. The interaction between TLE2 and RTA was confirmed by glutathione S-transferase (GST) binding and coimmunoprecipitation assays. Immunofluorescence analysis showed that TLE2 and RTA were colocalized in the same nuclear compartment in KSHV-infected cells. This interaction recruited TLE2 to RTA bound to its recognition sites on DNA and repressed RTA auto-activation and transactivation activity. Moreover, TLE2 also inhibited the induction of lytic replication and virion production driven by RTA. We further showed that the Q (Gln-rich), SP (Ser-Pro-rich), and WDR (Trp-Asp repeat) domains of TLE2 and the Pro-rich domain of RTA were essential for this interaction. RBP-Jkappa has been shown previously to bind to the same Pro-rich domain of RTA, and this binding can be subject to competition by TLE2. In addition, TLE2 can form a complex with RTA to access the cognate DNA sequence of the RTA-responsive element at different promoters. Intriguingly, the transcription level of TLE2 could be upregulated by RTA during the lytic reactivation process. In conclusion, we identified a new RTA binding protein, TLE2, and demonstrated that TLE2 inhibited replication and transactivation mediated by RTA. This provides another potentially important mechanism for maintenance of KSHV viral latency through interaction with a host protein.

  15. Inhibition of putrescine synthesis blocks development of the polychete Ophryotrocha labronica at gastrulation.

    Science.gov (United States)

    Emanuelsson, H; Heby, O

    1978-01-01

    Development eggs of the polychete Ophryotrocha labronica were analyzed for polyamines during the first 6 days after fertilization. The spermine content dominated initially, but gradually decreased. It was surpassed by putrescine, which rapidly increased to a maximum on the 3rd day, i.e., at the inception of grastrulation. The spermidine content was low during the entire period. Treatment of eggs with the putrescine synthesis inhibitor alpha-methylornithine from the onset of development led to developmental arrest at gastrulation and to an abnormally low content of putrescine in the treated embryos. Methylglyoxal bis(guanylhydrazone), an inhibitor of spermine and spermidine synthesis, had no visible effect of development. Our observations strongly suggest that putrescine synthesis is indispensable in early embryonic development of Ophryotrocha. Images PMID:273215

  16. A Customizable Quantum-Dot Cellular Automata Building Block for the Synthesis of Classical and Reversible Circuits.

    Science.gov (United States)

    Moustafa, Ahmed; Younes, Ahmed; Hassan, Yasser F

    2015-01-01

    Quantum-dot cellular automata (QCA) are nanoscale digital logic constructs that use electrons in arrays of quantum dots to carry out binary operations. In this paper, a basic building block for QCA will be proposed. The proposed basic building block can be customized to implement classical gates, such as XOR and XNOR gates, and reversible gates, such as CNOT and Toffoli gates, with less cell count and/or better latency than other proposed designs.

  17. Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

    International Nuclear Information System (INIS)

    Wang, Yijun; Lu, Hongjuan; Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun; Taylor, Ethan Will; Zhang, Jinsong

    2012-01-01

    Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

  18. Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yijun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Lu, Hongjuan [Productivity Center of Jiangsu Province, Nanjing 210042, Jiangsu (China); Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Taylor, Ethan Will [Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Zhang, Jinsong, E-mail: zjs@ahau.edu.cn [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China)

    2012-12-15

    Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

  19. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

    Science.gov (United States)

    Taniguchi, Masayuki; Ochiai, Akihito; Kondo, Hiroshi; Fukuda, Shun; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-05-01

    Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Psoralen plus ultraviolet radiation-induced inhibition of DNA synthesis and viability in human lymphoid cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kraemer, K H; Waters, H L [National Cancer Inst., Bethesda, MD (USA); Ellingson, O L; Tarone, R E

    1979-08-01

    The present study investigated whether conditions of 8-methoxypsoralen (8-MOP) concentration and of exposure to high intensity long wavelength ultraviolet radiation (UV-A) during psoriasis and mycosis fungoides therapy might be sufficient to result directly in decreased lymphoid cell DNA synthesis and viability in vitro. Tritiated thymidine (/sup 3/HtdR) incorporation and cell growth following UV-A exposure alone or with 8-MOP was examined in peripheral blood lymphocytes and in Ebstein-Barr virus transformed human lymphoblastoid cell lines. UV-A exposure alone induced a dose-dependent inhibition of /sup 3/HTdR incorporation in both types of lymphoid cells. Pre-incubation with 0.1 ..mu..g/ml 8-MOP before UV-A exposure induced a significantly greater inhibition of /sup 3/HTdr incorporation. Further inhibition of /sup 3/HTdR incorporation was observed by preincubation of the lymphoblastoid cells with 1.0 ..mu..g/ml 8-MOP but not in the lymphocytes. The concentration of viable lymphoblastoid cells did not decrease below the original concentration after the highest dose of UV-A alone (29,00 J/m/sup 2/) but preincubation with 0.1 ..mu..g/ml 8-MOP resulted in 40% and 0.6% survival respectively after 3000 J/m/sup 2/. This study suggested that the low doses of 8-MOP and UV-A received by patients' lymphocytes may be sufficient to explain the decreased DNA synthesis found in their circulating leucocytes. (author).

  1. Smad3 induces atrogin-1, inhibits mTOR and protein synthesis, and promotes muscle atrophy in vivo.

    Science.gov (United States)

    Goodman, Craig A; McNally, Rachel M; Hoffmann, F Michael; Hornberger, Troy A

    2013-11-01

    Myostatin, a member of the TGF superfamily, is sufficient to induce skeletal muscle atrophy. Myostatin-induced atrophy is associated with increases in E3-ligase atrogin-1 expression and protein degradation and decreases in Akt/mechanistic target of rapamycin (mTOR) signaling and protein synthesis. Myostatin signaling activates the transcription factor Smad3 (Small Mothers Against Decapentaplegic), which has been shown to be necessary for myostatin-induced atrogin-1 expression and atrophy; however, it is not known whether Smad3 is sufficient to induce these events or whether Smad3 simply plays a permissive role. Thus, the aim of this study was to address these questions with an in vivo model. To accomplish this goal, in vivo transfection of plasmid DNA was used to create transient transgenic mouse skeletal muscles, and our results show for the first time that Smad3 expression is sufficient to stimulate atrogin-1 promoter activity, inhibit Akt/mTOR signaling and protein synthesis, and induce muscle fiber atrophy. Moreover, we propose that Akt/mTOR signaling is inhibited by a Smad3-induced decrease in microRNA-29 (miR-29) expression and a subsequent increase in the translation of phosphatase and tensin homolog (PTEN) mRNA. Smad3 is also sufficient to inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) promoter activity and to increase FoxO (Forkhead Box Protein, Subclass O)-mediated signaling and the promoter activity of plasminogen activator inhibitor 1 (PAI-1). Combined, this study provides the first evidence that Smad3 is sufficient to regulate many of the events associated with myostatin-induced atrophy and therefore suggests that Smad3 signaling may be a viable target for therapies aimed at preventing myostatin-induced muscle atrophy.

  2. Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization

    Directory of Open Access Journals (Sweden)

    Yujia Li

    2018-04-01

    Full Text Available The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5 incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC. PIV5 containing CD59 (PIV5-CD59 showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59.

  3. Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy

    NARCIS (Netherlands)

    Platt, F. M.; Jeyakumar, M.; Andersson, U.; Priestman, D. A.; Dwek, R. A.; Butters, T. D.; Cox, T. M.; Lachmann, R. H.; Hollak, C.; Aerts, J. M.; van Weely, S.; Hrebícek, M.; Moyses, C.; Gow, I.; Elstein, D.; Zimran, A.

    2001-01-01

    The glycosphingolipid (GSL) lysosomal storage diseases are caused by mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes. In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and

  4. Chemo-Enzymatic Synthesis of Branched N-Acetyllactosamine Glycan Oligomers for Galectin-3 Inhibition

    Czech Academy of Sciences Publication Activity Database

    Laaf, D.; Steffens, H.; Pelantová, Helena; Bojarová, Pavla; Křen, Vladimír; Elling, L.

    2017-01-01

    Roč. 359, č. 22 (2017), s. 4015-4024 ISSN 1615-4150 R&D Projects: GA ČR GC15-02578J; GA MŠk(CZ) LTC17005 Institutional support: RVO:61388971 Keywords : biomimetic synthesis * galectin-3 * glycosylation Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 5.646, year: 2016

  5. Inhibition of skeletal muscle protein synthesis in septic intra-abdominal abscess

    International Nuclear Information System (INIS)

    Vary, T.C.; Siegel, J.H.; Tall, B.D.; Morris, J.G.; Smith, J.A.

    1988-01-01

    Chronic sepsis is always associated with profound wasting leading to increased release of amino acids from skeletal muscle. Net protein catabolism may be due to decreased rate of synthesis, increased rate of degradation, or both. To determine whether protein synthesis is altered in chronic sepsis, the rate of protein synthesis in vivo was estimated by measuring the incorporation of [ 3 H]-phenylalanine in skeletal muscle protein in a chronic (5-day) septic rat model induced by creation of a stable intra-abdominal abscess using an E. coli + B. fragilis-infected sterile fecal-agar pellet as foreign body nidus. Septic rats failed to gain weight at rates similar to control animals, therefore control animals were weight matched to the septic animals. The skeletal muscle protein content in septic animals was significantly reduced relative to control animals (0.18 +/- 0.01 vs. 0.21 +/- 0.01 mg protein/gm wet wt; p less than 0.02). The rate of incorporation of [ 3 H]-phenylalanine into skeletal muscle protein from control animals was 39 +/- 4 nmole/gm wet wt/hr or a fractional synthetic rate of 5.2 +/- 0.5%/day. In contrast to control animals, the fractional synthetic rate in septic animals (2.6 +/- 0.2%/day) was reduced by 50% compared to control animals (p less than 0.005). The decreased rate of protein synthesis in sepsis was not due to an energy deficit, as high-energy phosphates and ATP/ADP ratio were not altered. This decrease in protein synthesis occurred even though septic animals consumed as much food as control animals

  6. Synthesis of magnetic resonance–, X-ray– and ultrasound-visible alginate microcapsules for immunoisolation and noninvasive imaging of cellular therapeutics

    Science.gov (United States)

    Barnett, Brad P; Arepally, Aravind; Stuber, Matthias; Arifin, Dian R; Kraitchman, Dara L; Bulte, Jeff W M

    2011-01-01

    Cell therapy has the potential to treat or cure a wide variety of diseases. Non-invasive cell tracking techniques are, however, necessary to translate this approach to the clinical setting. This protocol details methods to create microcapsules that are visible by X-ray, ultrasound (US ) or magnetic resonance (MR) for the encapsulation and immunoisolation of cellular therapeutics. Three steps are generally used to encapsulate cellular therapeutics in an alginate matrix: (i) droplets of cell-containing liquid alginate are extruded, using an electrostatic generator, through a needle tip into a solution containing a dissolved divalent cation salt to form a solid gel; (ii) the resulting gelled spheres are coated with polycations as a cross-linker; and (iii) these complexes are then incubated in a second solution of alginate to form a semipermeable membrane composed of an inner and an outer layer of alginate. The microcapsules can be rendered visible during the first step by adding contrast agents to the primary alginate layer. Such contrast agents include superparamagnetic iron oxide for detection by 1H MR imaging (MRI); the radiopaque agents barium or bismuth sulfate for detection by X-ray modalities; or perfluorocarbon emulsions for multimodal detection by 19F MRI, X-ray and US imaging. The entire synthesis can be completed within 2 h. PMID:21799484

  7. Synthesis, solubilization, and surface functionalization of highly fluorescent quantum dots for cellular targeting through a small molecule

    Science.gov (United States)

    Galloway, Justin F.

    To achieve long-term fluorescence imaging with quantum dots (QDs), a CdSe core/shell must first be synthesized. The synthesis of bright CdSe QDs is not trivial and as a consequence, the role of surfactant in nucleation and growth was investigated. It was found that the type of surfactant used, either phosphonic or fatty acid, played a pivotal role in the size of the CdSe core. The study of surfactant on CdSe synthesis, ultimately led to an electrical passivation method that utilized a short-chained phosphonic acid and highly reactive organometallic precursors to achieve high quantum yield (QY) as has been previously described. The synthesis of QDs using organometallic precursors and a phosphonic acid for passivation resulted in 4 out of 9 batches of QDs achieving QYs greater than 50% and 8 out of 9 batches with QYs greater than 35%. The synthesis of CdSe QDs was done in organic solutions rendering the surface of the particle hydrophobic. To perform cell-targeting experiments, QDs must be transferred to water. The transfer of QDs to water was successfully accomplished by using single acyl chain lipids. A systematic study of different lipid combinations and coatings demonstrated that 20-40 mol% single acyl chained lipids were able to transfer QDs to water resulting in monodispersed, stable QDs without adversely affecting the QY. The advantage to water solubilization using single acyl chain lipids is that the QD have a hydrodynamic radius less than 15 nm, QYs that can exceed 50% and additional surface functionalization can be down using the reactive sites incorporated into the lipid bilayer. QDs that are bright and stable in water were studied for the purpose of targeting G protein-coupled Receptors (GPCR). GPCRs are transmembrane receptors that internalize extracellular cues, and thus mediate signal transduction. The cyclic Adenosine Monophosphate Receptor 1 of the model organism Dictyostelium disodium was the receptor of interest. The Halo protein, a genetically

  8. Nitrogen and phosphorus co-doped graphene quantum dots: synthesis from adenosine triphosphate, optical properties, and cellular imaging.

    Science.gov (United States)

    Ananthanarayanan, Arundithi; Wang, Yue; Routh, Parimal; Sk, Mahasin Alam; Than, Aung; Lin, Ming; Zhang, Jie; Chen, Jie; Sun, Handong; Chen, Peng

    2015-05-07

    Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of applications, particularly, as superior fluorescent reporters for bio-imaging and optical sensing. Heteroatom doping can endow GQDs with new or improved photoluminescence properties. Here, we demonstrate a simple strategy for the synthesis of nitrogen and phosphorus co-doped GQDs from a single biomolecule precursor (adenosine triphosphate - ATP). Such ATP-GQDs exhibit high fluorescence quantum yield, strong two-photon upconversion, small molecular weight, high photostability, and good biocompatibility. Furthermore, transferrin conjugated ATP-GQDs have been used for imaging and real-time tracking of transferrin receptors in live cells.

  9. Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI).

    Science.gov (United States)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-03-01

    Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (Kinact) of 2.64 ± 0.15 min(-1) and half maximal rate of inactivation (KI) of 7.69 ± 2.48 μM with Kinact/KI ratio of 0.343 min(-1) μM(-1). Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Study on the Synthesis and Corrosion Inhibition Performance of Mannich-Modified Imidazoline

    Directory of Open Access Journals (Sweden)

    Xiangjun Kong

    2016-07-01

    Full Text Available A novel Mannich-modified imidazoline (MMI as cationic emulsifier was synthesised for corrosion harm reduction, through three steps — acylation, cyclization, and Mannich reaction. The surface activity was characterized by determination of surface tensions and critical micelle concentration (CMC. The corrosion inhibition performance of five types of steels in the simulated corrosion solution in the presence of the MMI was investigated by static weight loss tests. The results showed that the MMI had good surface activities, with CMC of 19.8 μg g−1 and surface tension of 36.4 mN m−1. The corrosion test results indicated that the corrosion rates of different materials were decreased significantly, and degrees of corrosion inhibition were always higher than 80.0 %. The main inhibition mechanism was most likely due to the adsorption of the corrosion inhibitor on the steel surface, leading to the prevention of corrosion medium from the metal surface.

  11. Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland.

    Science.gov (United States)

    Romano, M I; Machiavelli, G A; Pérez, R L; Carricarte, V; Burdman, J A

    1984-07-01

    The relationship between the release of LH and the synthesis of DNA was studied in the anterior pituitary gland of castrated rats. Cell types were characterized immunocytochemically. Castration significantly (P less than 0.01) increased the concentration of LH in serum (1326%) and the incorporation of [3H]thymidine into pituitary DNA (72%). This was accompanied by an increment in the activity of the enzyme DNA polymerase-alpha (58%) and in the number of mitoses (from 2 +/- 0.1/mm2 in intact rats to 21 +/- 0.8/mm2 15 days after castration). Only 20% of the mitoses found in the pituitary gland of castrated rats were positively stained with the antiserum against the beta-subunit of LH. The other 80% did not stain either with LH antiserum or with antisera against the other pituitary hormones. There was a significant (P less than 0.01) increase in the number of LH cells in castrated rats (48%). All the changes produced in the anterior pituitary gland after castration were prevented by the administration of dihydrotestosterone. The results demonstrate that a stimulation of LH release is followed by an increase of DNA synthesis and cell proliferation of gonadotrophs in the anterior pituitary gland.

  12. Synthesis of 2-acylated and sulfonated 4-hydroxycoumarins: In vitro urease inhibition and molecular docking studies.

    Science.gov (United States)

    Rashid, Umer; Rahim, Fazal; Taha, Muhammad; Arshad, Muhammad; Ullah, Hayat; Mahmood, Tariq; Ali, Muhammad

    2016-06-01

    Sixteen 4-hydroxycoumarin derivatives were synthesized, characterized through EI-MS and (1)H NMR and screened for urease inhibitory potential. Three compounds exhibited better urease inhibition than the standard inhibitor thiourea (IC50=21±0.11μM) while other four compounds exhibited good to moderate inhibition with IC50 values between 29.45±1.1μM and 69.53±0.9μM. Structure activity relationship was established on the basis of molecular docking studies, which helped to predict the binding interactions of the most active compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Effect of inhibition of protein synthesis on the development of thermotolerance

    International Nuclear Information System (INIS)

    Chang, P.Y.; Blakely, E.A.; Gonzalez-Flores, I.

    1986-01-01

    The authors have chosen to use a temperature-sensitive mutant line, CHO-TSH1, which shuts down protein synthesis at nonpermissive temperatures of 40 0 C and above by the inactivation of its cytoplasmic nonmitochondrial leucyl-transfer RNA (t-RNA) synthetase enzyme. The parent cell line, CHO-SC1, was used as the control for these experiments. Exponentially growing, asynchronous CHO-TSH1 and CHO-SC1 cell populations were treated for times up to 8 hours at 41.5 0 C, 42 0 C, and 42.5 0 C. The wild-type cells showed the development of tolerance to heat killing at 41.5 0 C, 42 0 C, and possibly at 42.5 0 C, although the survival level at which tolerance developed at 42.5 0 C was too low to be statistically significant. The CHO-TSH1 mutant cell showed no tolerance at any of those temperatures. The rate of total protein synthesis was measured in both cell lines in pulse-labeling experiments with 3 H-leucine under the conditions of the experiment. Results indicated that the rate of synthesis dropped precipitously within the initial hour of exposure to 42 0 C and remained low during the 3 hours of 42 0 C treatment. When each cell line was returned to 35 0 C after the 3-hour treatment at 42 0 C, protein synthesis immediately resumed and eventually returned to control levels after 7 hours at 35 0 C

  14. Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats.

    OpenAIRE

    Schubring-Giese Maximilian; Leemburg Susan; Luft Andreas Rüdiger; Hosp Jonas Aurel

    2016-01-01

    Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of ...

  15. Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

    International Nuclear Information System (INIS)

    Burkovics, Peter; Sebesta, Marek; Kolesar, Peter; Sisakova, Alexandra; Marini, Victoria; Plault, Nicolas; Szukacsov, Valeria; Pinter, Lajos; Haracska, Lajos; Robert, Thomas; Kolesar, Peter; Gangloff, Serge; Krejci, Lumir

    2013-01-01

    Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. (authors)

  16. Synthesis facilitates an understanding of the structural basis for translation inhibition by the lissoclimides

    Science.gov (United States)

    Könst, Zef A.; Szklarski, Anne R.; Pellegrino, Simone; Michalak, Sharon E.; Meyer, Mélanie; Zanette, Camila; Cencic, Regina; Nam, Sangkil; Voora, Vamsee K.; Horne, David A.; Pelletier, Jerry; Mobley, David L.; Yusupova, Gulnara; Yusupov, Marat; Vanderwal, Christopher D.

    2017-11-01

    The lissoclimides are unusual succinimide-containing labdane diterpenoids that were reported to be potent cytotoxins. Our short semisynthesis and analogue-oriented synthesis approaches provide a series of lissoclimide natural products and analogues that expand the structure-activity relationships (SARs) in this family. The semisynthesis approach yielded significant quantities of chlorolissoclimide (CL) to permit an evaluation against the National Cancer Institute's 60-cell line panel and allowed us to obtain an X-ray co-crystal structure of the synthetic secondary metabolite with the eukaryotic 80S ribosome. Although it shares a binding site with other imide-based natural product translation inhibitors, CL engages in a particularly interesting and novel face-on halogen-π interaction between the ligand's alkyl chloride and a guanine residue. Our analogue-oriented synthesis provides many more lissoclimide compounds, which were tested against aggressive human cancer cell lines and for protein synthesis inhibitory activity. Finally, computational modelling was used to explain the SARs of certain key compounds and set the stage for the structure-guided design of better translation inhibitors.

  17. Pyridine-substituted thiazolylphenol derivatives: Synthesis, modeling studies, aromatase inhibition, and antiproliferative activity evaluation.

    Science.gov (United States)

    Ertas, Merve; Sahin, Zafer; Berk, Barkin; Yurttas, Leyla; Biltekin, Sevde N; Demirayak, Seref

    2018-04-01

    Drugs used in breast cancer treatments target the suppression of estrogen biosynthesis. During this suppression, the main goal is to inhibit the aromatase enzyme that is responsible for the cyclization and structuring of estrogens either with steroid or non-steroidal-type inhibitors. Non-steroidal derivatives generally have a planar aromatic structure attached to the triazole ring system in their structures, which inhibits hydroxylation reactions during aromatization by coordinating the heme group. Bioisosteric replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase the selectivity for aromatase enzyme inhibition. In this study, pyridine-substituted thiazolylphenol derivatives, which are non-steroidal triazole bioisosteres, were synthesized using the Hantzsch method, and physical analysis and structural determination studies were performed. The IC 50 values of the compounds were determined by a fluorescence-based aromatase inhibition assay. Then, their antiproliferative activities on the MCF7 and HEK 293 cell lines were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the crystal structure of human placental aromatase was subjected to a series of docking experiments to identify the possible interactions between the most active structure and the active site. Lastly, an in silico technique was performed to analyze and predict the drug-likeness, molecular and ADME properties of the synthesized molecules. © 2018 Deutsche Pharmazeutische Gesellschaft.

  18. One-pot synthesis of FePt/CNTs nanocomposites for efficient cellular imaging and cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weihong; Zheng, Xiuwen, E-mail: xwzheng1976@163.com [Linyi University, School of Chemistry & Chemical Engineering, Shandong Provincial Key Laboratory of Detection Technology for Tumor Makers (China); Li, Shulian [Linyi Tumor Hospital (China); Zhang, Wei; Wen, Xin [Linyi University, School of Chemistry & Chemical Engineering, Shandong Provincial Key Laboratory of Detection Technology for Tumor Makers (China); Yue, Ludan [Shandong Normal University (China); Wang, Jinlong [Shandong University of Technology (China)

    2015-11-15

    Here, we developed a facile route to synthesize carbon nanotube-based FePt nanocomposites (FePt/CNTs) as a potential theranostic platform in the cancer treatment. FePt/CNTs were firstly synthesized via one-pot polyol route, and then functionalized with 6-arm-polyethylene glycol-amine polymer. The average size of FePt nanoparticles (NPs) is 3–4 nm, which is dispersed on the CNT surface (ca.50–150 nm). The as-prepared FePt NPs display high cytotoxicity by highly reactive oxygen species in cancer cells. Folic acid and fluorescein isothiocyanate are assembled onto the surface of FePt/CNTs for effective targeting of folate receptor-positive cancer cells and simultaneously for the visualization of cellular uptake. Therefore, the FePt/CNTs NPs capability of simultaneously performing diagnosis, therapy, and targeting is, therefore, promising for future potential widespread application in biomedicine.

  19. Cellular proliferation and infiltration following interstitial irradiation of normal dog brain is altered by an inhibitor of polyamine synthesis

    International Nuclear Information System (INIS)

    Fike, John R.; Gobbel, Glenn T.; Chou, Dean; Wijnhoven, Bas P. L.; Bellinzona, Mattia; Nakagawa, Minoru; Seilhan, Theresa M.

    1995-01-01

    Purpose: The objectives of this study were to quantitatively define proliferative and infiltrative cell responses after focal 125 I irradiation of normal brain, and to determine the effects of an intravenous infusion of α-difluoromethylornithine (DFMO) on those responses. Methods and Materials: Adult beagle dogs were irradiated using high activity 125 I sources. Saline (control) or DFMO (150 mg/kg/day) was infused for 18 days starting 2 days before irradiation. At varying times up to 8 weeks after irradiation, brain tissues were collected and the cell responses in and around the focal lesion were quantified. Immunohistochemical stains were used to label astrocytes (GFAP), vascular endothelial cells (Factor VIII), polymorphonuclear leukocytes (PMNs; MAC 387) and cells synthesizing deoxyribonucleic acid (DNA) (BrdU). Cellular responses were quantified using a histomorphometric analysis. Results: After radiation alone, cellular events included a substantial acute inflammatory response followed by increased BrdU labeling and progressive increases in numbers of capillaries and astrocytes. α-Difluoromethylornithine treatment significantly affected the measured cell responses. As in controls, an early inflammatory response was measured, but after 2 weeks there were more PMNs/unit area than in controls. The onset of measurable BrdU labeling was delayed in DFMO-treated animals, and the magnitude of labeling was significantly reduced. Increases in astrocyte and vessel numbers/mm 2 were observed after a 2-week delay. At the site of implant, astrocytes from DFMO-treated dogs were significantly smaller than those from controls. Conclusions: There is substantial cell proliferation and infiltration in response to interstitial irradiation of normal brain, and these responses are significantly altered by DFMO treatment. Although the precise mechanisms by which DFMO exerts its effects in this model are not known, the results from this study suggest that modification of radiation

  20. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    Science.gov (United States)

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Rocuronium Bromide Inhibits Inflammation and Pain by Suppressing Nitric Oxide Production and Enhancing Prostaglandin E2 Synthesis in Endothelial Cells.

    Science.gov (United States)

    Baek, Sang Bin; Shin, Mal Soon; Han, Jin Hee; Moon, Sang Woong; Chang, Boksoon; Jeon, Jung Won; Yi, Jae Woo; Chung, Jun Young

    2016-12-01

    Rocuronium bromide is a nondepolarizing neuromuscular blocking drug and has been used as an adjunct for relaxation or paralysis of the skeletal muscles, facilitation of endotracheal intubation, and improving surgical conditions during general anesthesia. However, intravenous injection of rocuronium bromide induces injection pain or withdrawal movement. The exact mechanism of rocuronium bromide-induced injection pain or withdrawal movement is not yet understood. We investigated whether rocuronium bromide treatment is involved in the induction of inflammation and pain in vascular endothelial cells. For this study, calf pulmonary artery endothelial (CPAE) cells were used, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Western blot, nitric oxide detection, and prostaglandin E 2 immunoassay were conducted. Rocuronium bromide treatment inhibited endothelial nitric oxide synthase and suppressed nitric oxide production in CPAE cells. Rocuronium bromide activated cyclooxygenase-2, inducible nitric oxide synthase and increased prostaglandin E 2 synthesis in CPAE cells. Rocuronium bromide induced inflammation and pain in CPAE cells. Suppressing nitric oxide production and enhancing prostaglandin E 2 synthesis might be associated with rocuronium bromide-induced injection pain or withdrawal movement.

  2. Histone deacetylase inhibition decreases cholesterol levels in neuronal cells by modulating key genes in cholesterol synthesis, uptake and efflux.

    Directory of Open Access Journals (Sweden)

    Maria João Nunes

    Full Text Available Cholesterol is an essential component of the central nervous system and increasing evidence suggests an association between brain cholesterol metabolism dysfunction and the onset of neurodegenerative disorders. Interestingly, histone deacetylase inhibitors (HDACi such as trichostatin A (TSA are emerging as promising therapeutic approaches in neurodegenerative diseases, but their effect on brain cholesterol metabolism is poorly understood. We have previously demonstrated that HDACi up-regulate CYP46A1 gene transcription, a key enzyme in neuronal cholesterol homeostasis. In this study, TSA was shown to modulate the transcription of other genes involved in cholesterol metabolism in human neuroblastoma cells, namely by up-regulating genes that control cholesterol efflux and down-regulating genes involved in cholesterol synthesis and uptake, thus leading to an overall decrease in total cholesterol content. Furthermore, co-treatment with the amphipathic drug U18666A that can mimic the intracellular cholesterol accumulation observed in cells of Niemman-Pick type C patients, revealed that TSA can ameliorate the phenotype induced by pathological cholesterol accumulation, by restoring the expression of key genes involved in cholesterol synthesis, uptake and efflux and promoting lysosomal cholesterol redistribution. These results clarify the role of TSA in the modulation of neuronal cholesterol metabolism at the transcriptional level, and emphasize the idea of HDAC inhibition as a promising therapeutic tool in neurodegenerative disorders with impaired cholesterol metabolism.

  3. Carvacrol Induces Heat Shock Protein 60 and Inhibits Synthesis of Flagellin in Escherichia coli O157:H7▿

    Science.gov (United States)

    Burt, Sara A.; van der Zee, Ruurd; Koets, Ad P.; de Graaff, Anko M.; van Knapen, Frans; Gaastra, Wim; Haagsman, Henk P.; Veldhuizen, Edwin J. A.

    2007-01-01

    The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7. PMID:17526792

  4. Low‑dose halofuginone inhibits the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes.

    Science.gov (United States)

    Li, Zeng; Fei, Hao; Wang, Zhen; Zhu, Tianyi

    2017-09-01

    Full‑thickness and large area defects of articular cartilage are unable to completely repair themselves and require surgical intervention, including microfracture, autologous or allogeneic osteochondral grafts, and autologous chondrocyte implantation. A large proportion of regenerative cartilage exists as fibrocartilage, which is unable to withstand impacts in the same way as native hyaline cartilage, owing to excess synthesis of type I collagen in the matrix. The present study demonstrated that low‑dose halofuginone (HF), a plant alkaloid isolated from Dichroa febrifuga, may inhibit the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. In addition, HF was revealed to inhibit the phosphorylation of mothers against decapentaplegic homolog (Smad)2/3 and promoted Smad7 expression, as well as decrease the synthesis of type I collagen synthesis. Results from the present study indicated that HF treatment suppressed the synthesis of type I collagen by inhibiting the transforming growth factor‑β signaling pathway in chondrocytes. These results may provide an alternative solution to the problems associated with fibrocartilage, and convert fibrocartilage into hyaline cartilage at the mid‑early stages of cartilage regeneration. HF may additionally be used to improve monolayer expansion or 3D cultures of seed cells for the tissue engineering of cartilage.

  5. Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec- mutant of Escherichia coli K12

    International Nuclear Information System (INIS)

    Pirsel, M.; Slezarikova, V.

    1977-01-01

    A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA - ) or by chloramphenicol (CAP) treatment prior to UV irradiation (2.5 J m -2 ) increased more than tenfold the resistance of the strain Escherichia coli K12 SR19 to UV radiation. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation, and a higher stability of both parental and newly synthesized DNAs could be demonstrated. (author)

  6. Design and Synthesis of Bifunctional Oxime Reactivators of OP- inhibited Cholinesterase

    Science.gov (United States)

    2013-08-01

    N O O OH N O O O O N OH O O N N O O N N N O N N N N N O N+ N N N OH MeOH/APS THP NaBH4 MsCl HN N p-Toluenesulfonic Acid Swern Oxidation H2N O S O O O...pendant general acid groups. Compound 8 has been delivered to ICD for testing (synthesis and characterization below). This strategy is in place to...as a THP ether and the trifluoromethylketone installed by coupling the organolithium with trifluoroacetylpiperidine. We again used our direct

  7. Inhibition of synthesis of heparan sulfate by selenate: Possible dependence on sulfation for chain polymerization

    International Nuclear Information System (INIS)

    Dietrich, C.P.; Nader, H.B.; Buonassisi, V.; Colburn, P.

    1988-01-01

    Selenate, a sulfation inhibitor, blocks the synthesis of heparan sulfate and chondroitin sulfate by cultured endothelial cells. In contrast, selenate does not affect the production of hyaluronic acid, a nonsulfated glycosaminoglycan. No differences in molecular weight, [ 3 H]glucosamine/[ 35 S]sulfuric acid ratios, or disaccharide composition were observed when the heparan sulfate synthesized by selenate-treated cells was compared with that of control cells. The absence of undersulfated chains in preparations from cultures exposed to selenate supports the concept that, in the intact cell, the polymerization of heparan sulfate might be dependent on the sulfation of the saccharide units added to the growing glycosaminoglycan chain

  8. Kaempferol inhibits fibroblast collagen synthesis, proliferation and activation in hypertrophic scar via targeting TGF-β receptor type I.

    Science.gov (United States)

    Li, Hongwei; Yang, Liu; Zhang, Yuebing; Gao, Zhigang

    2016-10-01

    Hypertrophic scar (HPS) formation is a debilitating condition that results in pain, esthetic symptom and loss of tissue function. So far, no satisfactory therapeutic approach has been available for HPS treatment. In this study, we discovered that a natural small molecule, kaempferol, could significantly inhibit HPS formation in a mechanical load-induced mouse model. Our results also demonstrated that kaempferol remarkably attenuated collagen synthesis, proliferation and activation of fibroblasts in vitro and in vivo. Western blot analysis further revealed that kaempferol significantly down-regulated Smad2 and Smad3 phosphorylation in a dose-dependent manner. At last, we found that such bioactivity of kaempferol which resulted from the inhibition of TGF-β1/Smads signaling was induced by the selective binding of kaempferol to TGF-β receptor type I (TGFβRI). These findings suggest that kaempferol could be developed into a promising agent for the treatment of HPS or other fibroproliferative disorders. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Synthesis, Antimycobacterial, Antifungal and Photosynthesis-Inhibiting Activity of Chlorinated N-phenylpyrazine-2-carboxamides †

    Directory of Open Access Journals (Sweden)

    Katarina Kralova

    2010-11-01

    Full Text Available A series of sixteen pyrazinamide analogues with the -CONH- linker connecting the pyrazine and benzene rings was synthesized by the condensation of chlorides of substituted pyrazinecarboxylic acids with ring-substituted (chlorine anilines. The prepared compounds were characterized and evaluated for their antimycobacterial and antifungal activity, and for their ability to inhibit photosynthetic electron transport (PET. 6-Chloro-N-(4-chlorophenylpyrazine-2-carboxamide manifested the highest activity against Mycobacterium tuberculosis strain H37Rv (65% inhibition at 6.25 μg/mL. The highest antifungal effect against Trichophyton mentagrophytes, the most susceptible fungal strain tested, was found for 6-chloro-5-tert-butyl-N-(3,4-dichlorophenylpyrazine-2-carboxamide (MIC = 62.5 μmol/L. 6-Chloro-5-tert-butyl-N-(4-chlorophenylpyrazine-2-carboxamide showed the highest PET inhibition in spinach chloroplasts (Spinacia oleracea L. chloroplasts (IC50 = 43.0 μmol/L. For all the compounds, the relationships between the lipophilicity and the chemical structure of the studied compounds as well as their structure-activity relationships are discussed.

  10. Synthesis, characterization and corrosion inhibition efficiency of N-(4-(Morpholinomethyl Carbamoyl Phenyl Furan-2-Carboxamide

    Directory of Open Access Journals (Sweden)

    N. Zulfareen

    2016-01-01

    Full Text Available A mannich base namely N-(4-(Morpholinomethyl Carbamoyl Phenyl Furan-2-Carboxamide (MFC was synthesized and characterized by FT-IR, 1H NMR, and 13C NMR. The molecular weight of MFC was confirmed by LC-MS. The inhibition effect of MFC on brass in 1 M HCl medium has been investigated by weight loss measurement, potentiodynamic polarization, electrochemical impedance spectroscopy (EIS and cyclic voltametry (CV. Thermodynamic parameters such as free energy, entropy and enthalpy were calculated to describe the mechanism of corrosion inhibitor. The inhibition efficiency of MFC increases with increase in concentration and temperature ranges from 30 °C to 60 °C. Polarization measurements indicated that MFC acts as a mixed type corrosion inhibitor. AC impedance indicates that Rct value increases with increase in the concentration of inhibitor. CV reveals that the oxidation of the copper is controlled by the addition of inhibitor on the brass metal. Surface analysis using scanning electron microscope (SEM shows a significant morphological improvement on the brass surface with the addition of the inhibitor. The adsorption of MFC on brass obeys Langmuir adsorption isotherm. The molecular structure of MFC was distorted to quantum chemical indices using density functional theory (DFT which indicates that the inhibition efficiency of MFC is closely related to quantum parameters.

  11. Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS.

    Science.gov (United States)

    Ott, Ingo; Koch, Thao; Shorafa, Hashem; Bai, Zhenlin; Poeckel, Daniel; Steinhilber, Dieter; Gust, Ronald

    2005-06-21

    Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.

  12. Inhibition of bovine viral diarrhea virus RNA synthesis by thiosemicarbazone derived from 5,6-dimethoxy-1-indanone.

    Science.gov (United States)

    Castro, Eliana F; Fabian, Lucas E; Caputto, María E; Gagey, Dolores; Finkielsztein, Liliana M; Moltrasio, Graciela Y; Moglioni, Albertina G; Campos, Rodolfo H; Cavallaro, Lucía V

    2011-06-01

    In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV).

  13. Inhibition of Bovine Viral Diarrhea Virus RNA Synthesis by Thiosemicarbazone Derived from 5,6-Dimethoxy-1-Indanone▿

    Science.gov (United States)

    Castro, Eliana F.; Fabian, Lucas E.; Caputto, María E.; Gagey, Dolores; Finkielsztein, Liliana M.; Moltrasio, Graciela Y.; Moglioni, Albertina G.; Campos, Rodolfo H.; Cavallaro, Lucía V.

    2011-01-01

    In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV). PMID:21430053

  14. Synthesis of a Neutral Mixed-Valence Diferrocenyl Carborane for Molecular Quantum-Dot Cellular Automata Applications.

    Science.gov (United States)

    Christie, John A; Forrest, Ryan P; Corcelli, Steven A; Wasio, Natalie A; Quardokus, Rebecca C; Brown, Ryan; Kandel, S Alex; Lu, Yuhui; Lent, Craig S; Henderson, Kenneth W

    2015-12-14

    The preparation of 7-Fc(+) -8-Fc-7,8-nido-[C2 B9 H10 ](-) (Fc(+) FcC2 B9 (-) ) demonstrates the successful incorporation of a carborane cage as an internal counteranion bridging between ferrocene and ferrocenium units. This neutral mixed-valence Fe(II) /Fe(III) complex overcomes the proximal electronic bias imposed by external counterions, a practical limitation in the use of molecular switches. A combination of UV/Vis-NIR spectroscopic and TD-DFT computational studies indicate that electron transfer within Fc(+) FcC2 B9 (-) is achieved through a bridge-mediated mechanism. This electronic framework therefore provides the possibility of an all-neutral null state, a key requirement for the implementation of quantum-dot cellular automata (QCA) molecular computing. The adhesion, ordering, and characterization of Fc(+) FcC2 B9 (-) on Au(111) has been observed by scanning tunneling microscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Sleep, Plasticity and the Pathophysiology of Neurodevelopmental Disorders: The Potential Roles of Protein Synthesis and Other Cellular Processes

    Directory of Open Access Journals (Sweden)

    Dante Picchioni

    2014-03-01

    Full Text Available Sleep is important for neural plasticity, and plasticity underlies sleep-dependent memory consolidation. It is widely appreciated that protein synthesis plays an essential role in neural plasticity. Studies of sleep-dependent memory and sleep-dependent plasticity have begun to examine alterations in these functions in populations with neurological and psychiatric disorders. Such an approach acknowledges that disordered sleep may have functional consequences during wakefulness. Although neurodevelopmental disorders are not considered to be sleep disorders per se, recent data has revealed that sleep abnormalities are among the most prevalent and common symptoms and may contribute to the progression of these disorders. The main goal of this review is to highlight the role of disordered sleep in the pathology of neurodevelopmental disorders and to examine some potential mechanisms by which sleep-dependent plasticity may be altered. We will also briefly attempt to extend the same logic to the other end of the developmental spectrum and describe a potential role of disordered sleep in the pathology of neurodegenerative diseases. We conclude by discussing ongoing studies that might provide a more integrative approach to the study of sleep, plasticity, and neurodevelopmental disorders.

  16. The cellular origin of the hepatic cholesterol synthesis (1961); Origine cellulaire du cholesterol hepatique de synthese (1961)

    Energy Technology Data Exchange (ETDEWEB)

    Chevallier, F [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

    1961-07-01

    If rats are sacrificed within minutes after an injection of acetate 1 - {sup 14}C the specific radioactivities of sterols precipitable with digitonine, extracted from liver parenchyma cells and from Kupffer cells are very close to each other, whatever the duration of the experiment may be. It follows that cholesterol synthesis probably occurs in both types of cells. A validation of this conclusion requires that the validity of certain assumptions be established. (authors) [French] Si l'on sacrifie des rats dans les minutes qui suivent une injection d'acetate 1- {sup 14}C, les valeurs des radioactivites specifiques des sterols, precipitables par la digitonine, extraits des cellules parenchymateuses du foie et des cellules de Kupffer sont tres proches l'une de l'autre quelle que soit la duree de l'experience. On en deduit que la synthese du cholesterol s'effectue probablement dans les deux types de cellules. Cette conclusion pour etre valable, exige que le bien fonde de certaines hypotheses soit verifie. (auteurs)

  17. Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response.

    Science.gov (United States)

    Bayry, Jagadeesh; Beaussart, Audrey; Dufrêne, Yves F; Sharma, Meenu; Bansal, Kushagra; Kniemeyer, Olaf; Aimanianda, Vishukumar; Brakhage, Axel A; Kaveri, Srini V; Kwon-Chung, Kyung J; Latgé, Jean-Paul; Beauvais, Anne

    2014-08-01

    In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  18. Luminescent cyclometalated iridium(III) polypyridine indole complexes--synthesis, photophysics, electrochemistry, protein-binding properties, cytotoxicity, and cellular uptake.

    Science.gov (United States)

    Lau, Jason Shing-Yip; Lee, Pui-Kei; Tsang, Keith Hing-Kit; Ng, Cyrus Ho-Cheong; Lam, Yun-Wah; Cheng, Shuk-Han; Lo, Kenneth Kam-Wing

    2009-01-19

    A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser

  19. Pharmacological inhibition of eicosanoid synthesis and hyperalgesia in yeast-injected rat paws

    International Nuclear Information System (INIS)

    Opas, E.E.; Dallob, A.; Herold, E.; Luell, S.; Humes, J.L.

    1986-01-01

    Brewer's yeast caused an inflammation characterized by edema and hyperalgesia when injected into the hindpaw of a rat. These events were temporally distinct and each was associated with increases of specific arachidonic and oxygenation products. As determined by radioimmunoassay (RIA) on whole paw lipid extracts, the 5-lipoxygenase (5-LO) products, leukotrienes C 4 and D 4 and 5-hydroxyeicosatetraendic acid (5-HETE) were synthesized concurrently with the onset of edema (maximal at 15 minutes after yeast injection). The hyperalgesic phase of the inflammation (3-4 hr after yeast injection) was associated with increased tissue levels of the cyclooxygenase (CO) products, prostaglandin E 2 and thromboxane B 2 (TXB 2 ) as well as increases in levels of the 5-LO products, leukotriene B 4 (LTB 4 ) and 5-HETE. Pharmacological agents modulated the synthesis of eicosanoids and suppressed the hyperalgesic response

  20. A novel multiple-stage antimalarial agent that inhibits protein synthesis

    Science.gov (United States)

    Baragaña, Beatriz; Hallyburton, Irene; Lee, Marcus C. S.; Norcross, Neil R.; Grimaldi, Raffaella; Otto, Thomas D.; Proto, William R.; Blagborough, Andrew M.; Meister, Stephan; Wirjanata, Grennady; Ruecker, Andrea; Upton, Leanna M.; Abraham, Tara S.; Almeida, Mariana J.; Pradhan, Anupam; Porzelle, Achim; Martínez, María Santos; Bolscher, Judith M.; Woodland, Andrew; Norval, Suzanne; Zuccotto, Fabio; Thomas, John; Simeons, Frederick; Stojanovski, Laste; Osuna-Cabello, Maria; Brock, Paddy M.; Churcher, Tom S.; Sala, Katarzyna A.; Zakutansky, Sara E.; Jiménez-Díaz, María Belén; Sanz, Laura Maria; Riley, Jennifer; Basak, Rajshekhar; Campbell, Michael; Avery, Vicky M.; Sauerwein, Robert W.; Dechering, Koen J.; Noviyanti, Rintis; Campo, Brice; Frearson, Julie A.; Angulo-Barturen, Iñigo; Ferrer-Bazaga, Santiago; Gamo, Francisco Javier; Wyatt, Paul G.; Leroy, Didier; Siegl, Peter; Delves, Michael J.; Kyle, Dennis E.; Wittlin, Sergio; Marfurt, Jutta; Price, Ric N.; Sinden, Robert E.; Winzeler, Elizabeth A.; Charman, Susan A.; Bebrevska, Lidiya; Gray, David W.; Campbell, Simon; Fairlamb, Alan H.; Willis, Paul A.; Rayner, Julian C.; Fidock, David A.; Read, Kevin D.; Gilbert, Ian H.

    2015-06-01

    There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

  1. Synthesis of Cyclic Antifreeze Glycopeptide and Glycopeptoids and Their Ice Recrystallization Inhibition Activity

    International Nuclear Information System (INIS)

    Ahn, Mija; Murugan, Ravichandran N.; Bang, Jeong Kyu; Kim, Hak Jun; Shin, Song Yub; Kim, Eunjung; Lee, Jun Hyuck

    2012-01-01

    Until now, few groups reported the antifreeze activity of cyclic glycopeptides; however, the tedious synthetic procedure is not amenable to study the intensive structure activity relationship. A series of N-linked cyclic glycopeptoids and glycopeptide have been prepared to evaluate antifreeze activity as a function of peptide backbone cyclization and methyl stereochemical effect on the rigid Thr position. This study has combined the cyclization protocol with solid phase peptide synthesis and obtained significant quantities of homogeneous cyclic glycopeptide and glycopeptoids. Analysis of antifreeze activity revealed that our cyclic peptide demonstrated RI activity while cyclic glycopeptoids showed no RI activity. These results suggest that the subtle changes in conformation and Thr orientation dramatically influence RI activity of N-linked glycopeptoids

  2. Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency.

    Science.gov (United States)

    Blanco, Beatriz; Sedes, Antía; Peón, Antonio; Lamb, Heather; Hawkins, Alastair R; Castedo, Luis; González-Bello, Concepción

    2012-05-14

    Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.

  3. Regulation of ribonucleic acid synthesis by polyamines. Reversal by spermine of inhibition by methylglyoxal bis(guanylhydrazone) of ribonucleic acid synthesis and histone acetylation in rabbit heart.

    Science.gov (United States)

    Caldarera, C M; Casti, A; Guarnier, C; Moruzzi, G

    1975-10-01

    The relationship between polyamines and RNA synthesis was studied by considering the action of spermine on histone acetylation in perfused heart. In addition, the effect of methylglyoxal bis(guanylhydrazone), inhibitor of putrescine-activated S-adenosylmethionine decarboxylase activity, on RNA and polyamine specific radioactivity and on acetylation of histone fractions was also investigated in perfused heart. Different concentrations of spermine and/or methylglyoxas bis(guanylhydrazone) were injected into the heart, 15 min after beginning the perfusion. The results demonstrate that spermine stimulates the specific radioactivity of RNA of subcellular fractions. Acetylation of the arginine-rich histone fractions, involved in the regulation of RNA transcription, is enhanced by spermine. The perfusion with methylglyoxal bis(guanylhydrazone) causes a decrease in the specific radioactivity of polyamines and RNA, and in acetylation of histone fractions. However, spermine is able to reverse the methylglyoxal bis(guanylhydrazone) inhibition when injected simultaneously. From these results we may assume a possible role for spermine in the regulation of RNA transcription.

  4. Synthesis of cytochrome c oxidase 1 (SCO1) inhibits insulin sensitivity by decreasing copper levels in adipocytes.

    Science.gov (United States)

    Wei, Xiang-Bo; Guo, Liang; Liu, Yang; Zhou, Shui-Rong; Liu, Yuan; Dou, Xin; Du, Shao-Yue; Ding, Meng; Peng, Wan-Qiu; Qian, Shu-Wen; Huang, Hai-Yan; Tang, Qi-Qun

    2017-09-23

    Dysregulation of insulin signaling leads to type 2 diabetes mellitus (T2DM) and other metabolic disorders. Obesity is an important contributor to insulin resistance, and although the understanding of this relationship has improved in recent years, the mechanism of obesity-induced insulin resistance is not completely understood. Disorders of copper metabolism tend to accompany the development of obesity, which increases the risk of insulin resistance. Synthesis of cytochrome c oxidase 1 (SCO1) functions in the assembly of cytochrome c oxidase (COX) and cellular copper homeostasis. However, the role of SCO1 in the regulation of metabolism remains unknown. Here, we found that obese mice had higher expression of SCO1 and lower levels of copper in white adipose tissue (WAT) than did the control mice. Overexpression of SCO1 in adipocytes was associated with copper deficiency. Copper increased insulin sensitivity by decreasing the level of phosphatase and tensin homolog (PTEN) protein. Ectopic expression of SCO1 led to insulin resistance and was accompanied by a decrease in intracellular copper level, and addition of copper abolished the inhibitory effect of SCO1 on insulin sensitivity. Our results demonstrated a novel role of SCO1 in modulating insulin sensitivity via the regulation of copper concentration in WAT and suggested a potential therapeutic target for T2DM. Copyright © 2017. Published by Elsevier Inc.

  5. δ- and γ-tocopherols inhibit phIP/DSS-induced colon carcinogenesis by protection against early cellular and DNA damages.

    Science.gov (United States)

    Chen, Jayson X; Liu, Anna; Lee, Mao-Jung; Wang, Hong; Yu, Siyuan; Chi, Eric; Reuhl, Kenneth; Suh, Nanjoo; Yang, Chung S

    2017-01-01

    Tocopherols, the major forms of vitamin E, are a family of fat-soluble compounds that exist in alpha (α-T), beta (β-T), gamma (γ-T), and delta (δ-T) variants. A cancer preventive effect of vitamin E is suggested by epidemiological studies. However, past animal studies and human intervention trials with α-T, the most active vitamin E form, have yielded disappointing results. A possible explanation is that the cancer preventive activity of α-T is weak compared to other tocopherol forms. In the present study, we investigated the effects of δ-T, γ-T, and α-T (0.2% in diet) in a novel colon cancer model induced by the meat-derived dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by dextran sodium sulfate (DSS)-induced colitis in CYP1A-humanized (hCYP1A) mice. PhIP/DSS treatments induced multiple polypoid tumors, mainly tubular adenocarcinomas, in the middle to distal colon of the hCYP1A mice after 10 wk. Dietary supplementation with δ-T and γ-T significantly reduced colon tumor formation and suppressed markers of oxidative and nitrosative stress (i.e., 8-oxo-dG and nitrotyrosine) as well as pro-inflammatory mediators (i.e., NF-κB p65 and p-STAT3) in tumors and adjacent tissues. By administering δ-T at different time periods, we obtained results suggesting that the inhibitory effect of δ-T against colon carcinogenesis is mainly due to protection against early cellular and DNA damages caused by PhIP. α-T was found to be ineffective in inhibiting colon tumors and less effective in attenuating the molecular changes. Altogether, we demonstrated strong cancer preventive effects of δ-T and γ-T in a physiologically relevant model of human colon cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

    DEFF Research Database (Denmark)

    Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer

    2015-01-01

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example......, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an 18F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[18F]fluorobenzoate, and the [18F]ASIS was purified on a PD-10 desalting...... column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [18F]ASIS to TF and to a specific anti-factor VII...

  7. Synthesis of water-soluble curcumin derivatives and their inhibition on lysozyme amyloid fibrillation

    Science.gov (United States)

    Wang, Sujuan; Peng, Xixi; Cui, Liangliang; Li, Tongtong; Yu, Bei; Ma, Gang; Ba, Xinwu

    2018-02-01

    The potential application of curcumin was heavily limited in biomedicine because of its poor solubility in pure water. To circumvent the detracting feature, two novel water-soluble amino acid modified curcumin derivatives (MLC and DLC) have been synthesized through the condensation reaction between curcumin and Nα-Fmoc-Nε-Boc-L-lysine. Benefiting from the enhanced solubility of 3.32 × 10- 2 g/mL for MLC and 4.66 × 10- 2 g/mL for DLC, the inhibition effects of the as-prepared derivatives on the amyloid fibrillation of lysozyme (HEWL) were investigated detaily in water solution. The obtained results showed that the amyloid fibrillation of HEWL was inhibited to a great extent when the concentrations of MLC and DLC reach to 20.139 mM and 49.622 mM, respectively. The fluorescence quenching upon the addition of curcumin to HEWL provide a support for static and dynamic recombination quenching process. The binding driving force was assigned to classical hydrophobic interaction between curcumin derivatives and HEWL. In addition, UV-Vis absorption and circular dichroism (CD) spectra confirmed the change of the conformation of HEWL.

  8. Synthesis and Characterization of (Z-5-Arylmethylidene-rhodanines with Photosynthesis-Inhibiting Properties

    Directory of Open Access Journals (Sweden)

    Josef Jampilek

    2011-06-01

    Full Text Available A series of rhodanine derivatives was prepared. The synthetic approach, analytical and spectroscopic data of all synthesized compounds are presented. Lipophilicity of all the discussed rhodanine derivatives was analyzed using the RP-HPLC method. The compounds were tested for their ability to inhibit photosynthetic electron transport (PET in spinach (Spinacia oleracea L. chloroplasts and reduce chlorophyll content in freshwater alga Chlorella vulgaris. Structure-activity relationships between the chemical structure, physical properties and biological activities of the evaluated compounds are discussed. For majority of the tested compounds the lipophilicity of the compound and not electronic properties of the R1 substituent were decisive for PET-inhibiting activity. The most potent PET inhibitor was (5Z-5-(4-bromobenzylidene-2-thioxo-1,3-thiazolidin-4-one (IC50 = 3.0 μmol/L and the highest antialgal activity was exhibited by (5Z-5-(4-chlorobenzylidene-2-thioxo-1,3-thiazolidin-4-one (IC50 = 1.3 μmol/L.

  9. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    Energy Technology Data Exchange (ETDEWEB)

    Forward, Nicholas A.; Conrad, David M. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Power Coombs, Melanie R.; Doucette, Carolyn D. [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Furlong, Suzanne J. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Lin, Tong-Jun [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

    2011-04-22

    Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  10. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    International Nuclear Information System (INIS)

    Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.; Doucette, Carolyn D.; Furlong, Suzanne J.; Lin, Tong-Jun; Hoskin, David W.

    2011-01-01

    Highlights: → Curcumin inhibits CD4 + T-lymphocyte proliferation. → Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4 + T-lymphocytes. → Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. → IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4 + T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca 2+ release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4 + CD25 + regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  11. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    International Nuclear Information System (INIS)

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-01-01

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  12. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  13. Inhibition of nitric oxide synthesis by systemic N(G)-monomethyl-L-arginine administration in humans

    DEFF Research Database (Denmark)

    Frandsen, U; Bangsbo, J; Langberg, Henning

    2000-01-01

    (controls) and with prior N(G)-nitro-L-arginine methyl ester (L-NAME) infusion (4 mg/kg, intravenously). Samples from the interstitial fluid were obtained at rest, during exercise and after exercise with the microdialysis technique. Interstitial adenosine in controls increased (p0.05) to controls. The 6......-keto-prostaglandin F1alpha concentration in controls was 1.17+/-0.20 ng/ml at rest and increased (p0.05) in L-NAME. The interstitial K(+) concentration in controls increased (p......We examined whether the formation or the release of the vasodilators adenosine, prostacyclin (PGI(2)) and potassium (K(+)) increase in skeletal muscle interstitium in response to nitric oxide synthase (NOS) inhibition. Five subjects performed one-legged knee extensor exercise at 30 W without...

  14. Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

    Science.gov (United States)

    Erlandsson, Maria; Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Jesper B; Petersen, Lars C; Madsen, Jacob; Kjaer, Andreas

    2015-05-15

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

    International Nuclear Information System (INIS)

    Deng, Yu; Cao, Hong; Cu, Fenglong; Xu, Dan; Lei, Youying; Tan, Yang; Magdalou, Jacques; Wang, Hui; Chen, Liaobin

    2013-01-01

    Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes

  16. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    Energy Technology Data Exchange (ETDEWEB)

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States); Rozengurt, Enrique, E-mail: erozengurt@mednet.ucla.edu [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Metformin inhibits cancer cell growth but the mechanism(s) are not understood. Black-Right-Pointing-Pointer We show that the potency of metformin is sharply dependent on glucose in the medium. Black-Right-Pointing-Pointer AMPK activation was enhanced in cancer cells incubated in physiological glucose. Black-Right-Pointing-Pointer Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. Black-Right-Pointing-Pointer Metformin, at low concentrations, inhibited DNA synthesis through AMPK. -- Abstract: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser{sup 79} and Raptor at Ser{sup 792}, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the {alpha}{sub 1} and {alpha}{sub 2} catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

  17. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    International Nuclear Information System (INIS)

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert; Rozengurt, Enrique

    2013-01-01

    Highlights: ► Metformin inhibits cancer cell growth but the mechanism(s) are not understood. ► We show that the potency of metformin is sharply dependent on glucose in the medium. ► AMPK activation was enhanced in cancer cells incubated in physiological glucose. ► Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. ► Metformin, at low concentrations, inhibited DNA synthesis through AMPK. -- Abstract: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser 79 and Raptor at Ser 792 , was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05–0.1 mM) that were 10–100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α 1 and α 2 catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

  18. Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Yu; Cao, Hong; Cu, Fenglong [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Xu, Dan [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Lei, Youying [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Tan, Yang [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Magdalou, Jacques [UMR 7561 CNRS-Nancy Université, Faculté de Médicine, Vandoeuvre-lès-Nancy (France); Wang, Hui [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Chen, Liaobin, E-mail: lbchen@whu.edu.cn [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China)

    2013-05-15

    Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes.

  19. Fluoride induces endoplasmic reticulum stress and inhibits protein synthesis and secretion.

    Science.gov (United States)

    Sharma, Ramaswamy; Tsuchiya, Masahiro; Bartlett, John D

    2008-09-01

    Exposure to excessive amounts of fluoride (F(-)) causes dental fluorosis in susceptible individuals; however, the mechanism of F(-)-induced toxicity is unclear. Previously, we have shown that high-dose F(-) activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells. In this study we determined if low-dose F(-) causes ER stress and activates the UPR, and we also determined whether F(-) interferes with the secretion of proteins from the ER. We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F(-)-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F(-) were examined for eucaryotic initiation factor-2, subunit alpha (eIF2alpha) phosphorylation. We found that F(-) decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F(-). These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2alpha. Importantly, F(-)-induced phosphorylation of eIF2alphawas confirmed in vivo. These data suggest that F(-) initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis.

  20. Synthesis, α-glucosidase inhibition and molecular docking study of coumarin based derivatives.

    Science.gov (United States)

    Taha, Muhammad; Shah, Syed Adnan Ali; Afifi, Muhammad; Imran, Syahrul; Sultan, Sadia; Rahim, Fazal; Khan, Khalid Mohammed

    2018-04-01

    We have synthesized seventeen Coumarin based derivatives (1-17), characterized by 1 HNMR, 13 CNMR and EI-MS and evaluated for α-glucosidase inhibitory potential. Among the series, all derivatives exhibited outstanding α-glucosidase inhibition with IC 50 values ranging between 1.10 ± 0.01 and 36.46 ± 0.70 μM when compared with the standard inhibitor acarbose having IC 50 value 39.45 ± 0.10 μM. The most potent derivative among the series is derivative 3 having IC 50 value 1.10 ± 0.01 μM, which are many folds better than the standard acarbose. The structure activity relationship (SAR) was mainly based upon by bring about difference of substituent's on phenyl part. Molecular docking studies were carried out to understand the binding interaction of the most active compounds. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Synthesis, β-glucuronidase inhibition and molecular docking studies of hybrid bisindole-thiosemicarbazides analogs.

    Science.gov (United States)

    Taha, Muhammad; Ismail, Nor Hadiani; Imran, Syahrul; Rahim, Fazal; Wadood, Abdul; Khan, Huma; Ullah, Hayat; Salar, Uzma; Khan, Khalid Mohammed

    2016-10-01

    Hybrid bisindole-thiosemicarbazides analogs (1-18) were synthesized and screened for β-glucuronidase activity. All compounds showed varied degree of β-glucuronidase inhibitory potential when compared with standard d-saccharic acid 1,4-lactone (IC50=48.4±1.25μM). Compounds 4, 7, 9, 6, 5, 12, 17 and 18 showed exceptional β-glucuronidase inhibition with IC50 values ranging from 0.1 to 5.7μM. Compounds 1, 3, 8, 16, 13, 2 and 14 also showed better activities than standard with IC50 values ranging from 7.12 to 15.0μM. The remaining compounds 10, 11, and 15 showed good inhibitory potential with IC50 values 33.2±0.75, 21.4±0.30 and 28.12±0.25μM respectively. Molecular docking studies were carried out to confirm the binding interaction of the compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Bacterial Peptide Deformylase Inhibition of Tetrazole-Substituted Biaryl Acid Analogs: Synthesis, Biological Evaluations, and Molecular Docking Study.

    Science.gov (United States)

    Khan, Firoz A Kalam; Patil, Rajendra H; Patil, Manjiri; Arote, Rohidas; Shinde, Devanand B; Sangshetti, Jaiprakash N

    2016-12-01

    The synthesis and screening of tetrazole-substituted biaryl acid analogs 7a-l as bacterial peptide deformylase (PDF) enzyme inhibitors is reported. The compounds 7e (IC 50 value = 5.50 μM) and 7g (IC 50 value = 7.25 μM) showed good PDF inhibition activity. The compounds 7e (MIC range = 10.75-11.66 μg/mL) and 7g (MIC range = 8.91-12.83 μg/mL) also showed potent antibacterial activity when compared with the standard ciprofloxacin (MIC range = 25-50 μg/mL). Thus, the active derivatives were not only potent PDF enzyme inhibitors but also efficient antibacterial agents. In order to gain more insight into the binding mode of the compounds with the PDF enzyme, the most active compounds 7e and 7g, the moderately active compound 7k, and the least active compound 7h were docked against the PDF enzyme of Escherichia coli. The docking study of the most active compounds 7e and 7g against the PDF enzyme exhibited good binding properties. Hence, we believe our synthesized compounds 7a-l could serve as reservoir for bacterial PDF inhibitor development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Electron beam assisted synthesis of silver nanoparticle in chitosan stabilizer: Preparation, stability and inhibition of building fungi studies

    Science.gov (United States)

    Jannoo, Kanokwan; Teerapatsakul, Churapa; Punyanut, Adisak; Pasanphan, Wanvimol

    2015-07-01

    Silver nanoparticles (AgNPs) in chitosan (CS) stabilizer were successfully synthesized using electron beam irradiation. The effects of irradiation dose, molecular weight (MW) of CS stabilizer, concentration of AgNO3 precursor and addition of tert-butanol on AgNPs production were studied. The stability of the AgNPs under different temperatures and storage times were also investigated. The AgNPs formation in CS was observed using UV-vis, FT-IR and XRD. The characteristic surface plasmon resonance (SPR) of the obtained AgNPs was around 418 nm. The CS stabilizer and its MW, AgNO3 precursor and irradiation doses are important parameters for the synthesis of AgNPs. The optimum addition of 20% v/v tert-butanol could assist the formation of AgNPs. The AgNPs in CS stabilizer were stable over a period of one year when the samples were kept at 5 °C. The AgNPs observed from TEM images were spherical with an average particle size in the range of 5-20 nm depending on the irradiation doses. The AgNPs in CS solution effectively inhibited the growth of several fungi, i.e., Curvularia lunata, Trichoderma sp., Penicillium sp. and Aspergillus niger, which commonly found on the building surface.

  4. Low Protein Diet Inhibits Uric Acid Synthesis and Attenuates Renal Damage in Streptozotocin-Induced Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Jianmin Ran

    2014-01-01

    Full Text Available Aim. Several studies indicated that hyperuricemia may link to the worsening of diabetic nephropathy (DN. Meanwhile, low protein diet (LPD retards exacerbation of renal damage in chronic kidney disease. We then assessed whether LPD influences uric acid metabolism and benefits the progression of DN in streptozotocin- (STZ- induced diabetic rats. Methods. STZ-induced and control rats were both fed with LPD (5% and normal protein diet (18%, respectively, for 12 weeks. Vital signs, blood and urinary samples for UA metabolism were taken and analyzed every 3 weeks. Kidneys were removed at the end of the experiment. Results. Diabetic rats developed into constantly high levels of serum UA (SUA, creatinine (SCr and 24 h amounts of urinary albumin excretion (UAE, creatintine (UCr, urea nitrogen (UUN, and uric acid (UUA. LPD significantly decreased SUA, UAE, and blood glucose, yet left SCr, UCr, and UUN unchanged. A stepwise regression showed that high UUA is an independent risk factor for DN. LPD remarkably ameliorated degrees of enlarged glomeruli, proliferated mesangial cells, and hyaline-degenerated tubular epithelial cells in diabetic rats. Expression of TNF-α in tubulointerstitium significantly decreased in LPD-fed diabetic rats. Conclusion. LPD inhibits endogenous uric acid synthesis and might accordingly attenuate renal damage in STZ-induced diabetic rats.

  5. The tragedy of TRIUMPH for nitric oxide synthesis inhibition in cardiogenic shock: where do we go from here?

    Science.gov (United States)

    Bailey, Alison; Pope, Theodore W; Moore, Scott A; Campbell, Charles L

    2007-01-01

    evaluated compounds with little selectivity for iNOS and their failure may have been due, in part, to the inhibition of the other NOS isoforms. In this review, we describe the biochemistry of NO synthesis, the regulation of NO production, and the clinical trials evaluating the efficacy of NOS inhibition with an eye to future trials with more selective inhibitors of iNOS.

  6. Normal inhibition of DNA synthesis following γ-irradiation of radiosensitive cell lines from patients with Down's syndrome and Alzheimer's disease

    International Nuclear Information System (INIS)

    Lavin, M.F.; Le poidevin, P.; Chen, P.C.; Bates, P.

    1989-01-01

    Inhibition of DNA synthesis was studied in γ-iradiated lymphoblastoid cells from patients with Alzheimer's disease and Down's syndrome. A normal biphasic pattern of inhibition was observed over a dose range of 0-4 krad of γ-rays in all of the cell lines 3 out of 4 Down's and all the Alzheimer's cell lines were shown to be hypersensitive to ionizing radiation based on induced chromosomal aberrations. Increased G2 phase delay, comparable to that occurring in ataxia-telangiectasia cells, was observed for some of the cell lines, after exposure to γ-rays. Contrary to other data in the literature these results demonstrate that radioresistand DNA synthesis is not an intrinsic feature of all disorders characterized by radiosensitivitey. (author).; 25 refs.; 2 figs.; 1 tab

  7. Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

    Directory of Open Access Journals (Sweden)

    Li Xing'an

    2010-08-01

    Full Text Available Abstract Background Cooperation of constituents of the ubiquitin proteasome system (UPS with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. Conclusions It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition.

  8. A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

    Science.gov (United States)

    Lai, Yu-Chiang; Liu, Yang; Jacobs, Roxane; Rider, Mark H

    2012-10-01

    PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

  9. Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor-dependent pathway

    Directory of Open Access Journals (Sweden)

    Barańska Sylwia

    2009-03-01

    Full Text Available Abstract Background Mucopolysaccharidoses (MPS are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs. Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome. Methods To learn on details of the molecular mechanism of genistein-mediated inhibition of GAG synthesis, efficiency of this process was studied by measuring of incorporation of labeled sulfate, storage of GAGs in lysosomes was estimated by using electron microscopic techniques, and efficiency of phosphorylation of epidermal growth factor (EGF receptor was determined by using an ELISA-based assay with fluorogenic substrates. Results Effects of genistein on inhibition of GAG synthesis and accumulation in fibroblasts from patients suffering from various MPS types were abolished in the presence of an excess of EGF, and were partially reversed by an increased concentration of genistein. No such effects were observed when an excess of 17β-estradiol was used instead of EGF. Moreover, EGF-mediated stimulation of phsophorylation of the EGF receptor was impaired in the presence of genistein in both wild-type and MPS fibroblasts. Conclusion The results presented in this report indicate that the mechanism of genistein-mediated inhibition of GAG synthesis operates through epidermal growth factor (EGF-dependent pathway.

  10. Comparison of specificity of inhibition of polyamine synthesis in bovine lymphocytes by ethylglyoxal bis(guanylhydrazone) and methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Igarashi, K; Porter, C W; Morris, D R

    1984-11-01

    Ethylglyoxal bis(guanylhydrazone) (EGBG) was compared as an inhibitor of polyamine biosynthesis with methylglyoxal bis(guanylhydrazone) (MGBG) in bovine small lymphocytes stimulated by concanavalin A. EGBG brought about a decrease in spermidine and spermine levels equal to that found with MGBG, but at a 5-fold lower intracellular drug concentration. Despite identical polyamine levels, the degree of inhibition of DNA and protein synthesis by EGBG was smaller than that observed with MGBG, in either the presence or absence of the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. It was found that in vitro protein synthesis and in vivo mitochondrial function were inhibited by concentrations of MGBG necessary to inhibit polyamine synthesis in cells (1 to 3 mM), but not by efficacious levels of EGBG (0.2 to 0.6 mM). These results suggest that EGBG is more suitable as a specific inhibitor of polyamine biosynthesis and that use of this drug, rather than MGBG, in combination with alpha-difluoromethylornithine may be useful for studying the physiological functions of polyamines in animal cells.

  11. Effect of chlorpromazine on lipid metabolism in aortas from cholesterol-fed rabbits and normal rats, in vitro: inhibition of sterol esterification and modification of phospholipid synthesis

    International Nuclear Information System (INIS)

    Bell, F.P.

    1983-01-01

    Chlorpromazine (CPZ), a major tranquilizer, was found to be a potent inhibitor of acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes and in intact arterial tissue from the rat and cholesterol-fed rabbit in vitro. In isolated rabbit arterial microsomes, CPZ resulted in a concentration-dependent inhibition of ACAT with 50% inhibition of [1-14C]oleoylCoA incorporation into [14C]cholesteryl esters occurring at 0.1 mM CPZ. CPZ also effectively inhibited the incorporation of [14C]oleate into triglycerides without affecting incorporation into diglycerides. Additionally, CPZ altered the pattern of arterial phospholipids synthesized from [1-14C]oleate. Incorporation into phosphatidylcholine was depressed while incorporation into phosphatidylinositol was increased. Since diglyceride synthesis appeared to be unaffected by CPZ, a redirection of phosphatidic acid into the CDP-diglyceride pathway of glycerolipid synthesis does not adequately account for the effect of CPZ on arterial phospholipid and triglyceride synthesis in these experiments

  12. Inhibition of DNA synthesis in cultured lymphocytes and tumor cells by extracts of betel nut, tobacco, and miang leaf, plant substances associated with cancer of the ororespiratory epithelium.

    Science.gov (United States)

    Yang, J A; Huber, S A; Lucas, Z J

    1979-12-01

    The high incidence of oropharyngeal, esophageal, and laryngeal cancers in certain parts of the world has been ascribed to conjugated tannins found in certain folk medicinal herbs. We extracted miang leaf and betel nut with phosphate-buffered saline (0.14 M NaCl, 0.15 M potassium phosphate buffer, pH 7.4) and found that the extracts inhibited [3H]thymidine incorporation by phytohemagglutinin-stimulated human lymphocytes and by rat mammary tumor and mouse L-cells in logarithmic growth. Pretreating the lymphocytes for 1 or 4 hr with the extracts inhibited phytohemagglutinin-induced thymidine incorporation 72 hr later. At concentrations of 2.5 volumes % or lower, miang and betel nut extracts inhibited thymidine incorporation by 40 to 98% without any apparent signs of toxicity as demonstrated by the 66Rb equilibrium assay. In addition, neither extract inhibited cytotoxicity of rat mammary tumor cells by immune syngeneic spleen cells. The molecular weights of the inhibitory factors were between 1,000 and 10,000 daltons as determined by ultrafiltration and were unaffected by boiling for 3 min or by treatment with alcohol and, therefore, are probably not proteins. This in vitro demonstration of inhibition of DNA synthesis by these plant extracts presumably enriched for conjugated tannins may relate to inhibition of growth of rats and chicks fed conjugated tanin-contaminated sorghum feed. The carcinogenic potential of either these extracts or conjugated tannins is not yet established.

  13. Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hua-Yu; Li, Chao; Zheng, Zhao; Zhou, Qin; Guan, Hao; Su, Lin-Lin; Han, Jun-Tao; Zhu, Xiong-Xiang; Wang, Shu-yue; Li, Jun, E-mail: lijunfmmu@163.com; Hu, Da-Hai, E-mail: hudahaifmmu@aliyun.com

    2015-03-27

    The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at the gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs. - Highlights: • PPAR-γ agonist inhibits collagen synthesis in HSFBs. • Smad3 and type I collagen expression are decreased by PPAR-γ agonist. • miR-145 expression is increased by PPAR-γ agonist in HSFBs. • Increased miR-145 inhibits collagen synthesis by targeting Smad3. • miR-145 regulates collagen synthesis.

  14. Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Ming Ming

    Full Text Available Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC cells (PANC-1, MiaPaCa-2 with the isoquinoline alkaloid berberine (0.3-6 µM inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70% the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244 and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

  15. Inhibition of microRNA-153 protects neurons against ischemia/reperfusion injury in an oxygen-glucose deprivation and reoxygenation cellular model by regulating Nrf2/HO-1 signaling.

    Science.gov (United States)

    Ji, Qiong; Gao, Jianbo; Zheng, Yan; Liu, Xueli; Zhou, Qiangqiang; Shi, Canxia; Yao, Meng; Chen, Xia

    2017-07-01

    MicroRNAs are emerging as critical regulators in cerebral ischemia/reperfusion injury; however, their exact roles remain poorly understood. miR-153 is reported to be a neuron-related miRNA involved in neuroprotection. In this study, we aimed to investigate the precise role of miR-153 in regulating neuron survival during cerebral ischemia/reperfusion injury using an oxygen-glucose deprivation and reoxygenation (OGD/R) cellular model. We found that miR-153 was significantly upregulated in neurons subjected to OGD/R treatment. Inhibition of miR-153 significantly attenuated OGD/R-induced injury and oxidative stress in neurons. Nuclear factor erythroid 2-related factor 2 (Nrf2) was identified as a target gene of miR-153. Inhibition of miR-153 significantly promoted the expression of Nrf2 and heme oxygenase-1 (HO-1). However, silencing of Nrf2 significantly blocked the protective effects of miR-153 inhibition. Our study indicates that the inhibition of miR-153 protects neurons against OGD/R-induced injury by regulating Nrf2/HO-1 signaling and suggests a potential therapeutic target for cerebral ischemia/reperfusion injury. © 2017 Wiley Periodicals, Inc.

  16. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    Science.gov (United States)

    Jänne, J; Morris, D R

    1984-03-15

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

  17. An antisense peptide nucleic acid against Pseudomonas aeruginosa inhibiting bacterial-induced inflammatory responses in the cystic fibrosis IB3-1 cellular model system

    DEFF Research Database (Denmark)

    Montagner, Giulia; Bezzerri, Valentino; Cabrini, Giulio

    2017-01-01

    of the essential acpP gene of P. aeruginosa, and previously shown to inhibit bacterial growth, concomitantly also strongly inhibits induced up-regulation of the pro-inflammatory markers IL-8, IL-6, G-CSF, IFN-γ, IP-10, MCP-1 and TNF-α in IB3-1 cystic fibrosis cells infected by P. aeruginosa PAO1. Remarkably...... are significant considering the key role of this protein in the cystic fibrosis inflammatory process exacerbated by P. aeruginosa infection....

  18. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    Energy Technology Data Exchange (ETDEWEB)

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-08-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: (/sup 3/H)thymidine incorporation decreased by 80% and (/sup 14/C)leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of (/sup 3/H)histidine/(/sup 14/C)leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine.

  19. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    International Nuclear Information System (INIS)

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-01-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: [ 3 H]thymidine incorporation decreased by 80% and [ 14 C]leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of [ 3 H]histidine/[ 14 C]leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine

  20. Epac is required for exogenous and endogenous stimulation of adenosine A2B receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation.

    Science.gov (United States)

    Phosri, Sarawuth; Bunrukchai, Kwanchai; Parichatikanond, Warisara; Sato, Vilasinee H; Mangmool, Supachoke

    2018-01-10

    Angiotensin II (Ang II) plays an important role on the pathogenesis of cardiac fibrosis. Prolong and overstimulation of angiotensin II type 1 receptor with Ang II-induced collagen synthesis and myofibroblast differentiation in cardiac fibroblasts, leading to cardiac fibrosis. Although adenosine and its analogues are known to have cardioprotective effects, the mechanistic by which adenosine A 2 receptors (A 2 Rs) inhibit Ang II-induced cardiac fibrosis is not clearly understood. In the present study, we examined the effects of exogenous adenosine and endogenous adenosine on Ang II-induced collagen and myofibroblast differentiation determined by α-smooth muscle action (α-SMA) overexpression and their underlying signal transduction. Elevation of endogenous adenosine levels resulted in the inhibition of Ang II-induced collagen type I and III and α-SMA synthesis in cardiac fibroblasts. Moreover, treatment with exogenous adenosine which selectively stimulated A 2 Rs also suppressed Ang II-induced collagen synthesis and α-SMA production. These antifibrotic effects of both endogenous and exogenous adenosines are mediated through the A 2B receptor (A 2B R) subtype. Stimulation of A 2B R exhibited antifibrotic effects via the cAMP-dependent and Epac-dependent pathways. Our results provide new mechanistic insights regarding the role for cAMP and Epac on A 2B R-mediated antifibrotic effects. Thus, A 2B R is one of the potential therapeutic targets against cardiac fibrosis.

  1. 14-3-3theta protects against neurotoxicity in a cellular Parkinson's disease model through inhibition of the apoptotic factor Bax.

    Directory of Open Access Journals (Sweden)

    Sunny R Slone

    Full Text Available Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP(+ in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

  2. Cellular gravity

    NARCIS (Netherlands)

    F.C. Gruau; J.T. Tromp (John)

    1999-01-01

    textabstractWe consider the problem of establishing gravity in cellular automata. In particular, when cellular automata states can be partitioned into empty, particle, and wall types, with the latter enclosing rectangular areas, we desire rules that will make the particles fall down and pile up on

  3. Metabotropic glutamate receptor I (mGluR1) antagonism impairs cocaine-induced conditioned place preference via inhibition of protein synthesis.

    Science.gov (United States)

    Yu, Fei; Zhong, Peng; Liu, Xiaojie; Sun, Dalong; Gao, Hai-Qing; Liu, Qing-Song

    2013-06-01

    Antagonism of group I metabotropic glutamate receptors (mGluR1 and mGluR5) reduces behavioral effects of drugs of abuse, including cocaine. However, the underlying mechanisms remain poorly understood. Activation of mGluR5 increases protein synthesis at synapses. Although mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome, it remains unknown whether group I mGluR-mediated protein synthesis is involved in any behavioral effects of drugs of abuse. We report that group I mGluR agonist DHPG induced more pronounced initial depression of inhibitory postsynaptic currents (IPSCs) followed by modest long-term depression (I-LTD) in dopamine neurons of rat ventral tegmental area (VTA) through the activation of mGluR1. The early component of DHPG-induced depression of IPSCs was mediated by the cannabinoid CB1 receptors, while DHPG-induced I-LTD was dependent on protein synthesis. Western blotting analysis indicates that mGluR1 was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. We also show that cocaine conditioning activated translation machinery in the VTA via an mGluR1-dependent mechanism. Furthermore, intra-VTA microinjections of mGluR1 antagonist JNJ16259685 and protein synthesis inhibitor cycloheximide significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP) and activation of translation elongation factors. Taken together, these results suggest that mGluR1 antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine.

  4. Hamowanie syntezy chlorofilu i RNA w izolowanych liścieniach ogórka przez N-hydroksymocznik [Inhibition of chlorophyll and RNA synthesis by N-hydroxyurea in detached cucumber cotyledons

    Directory of Open Access Journals (Sweden)

    A. Rennert

    2015-01-01

    Full Text Available N-hydroxyurea (HU at the concentration of 5 x 10-4 M decreased the content of chlorophyll in detached cucumber cotyledons; at this concentration it has no inhibitory effect on growth. Benzylaminopurine, gibberelic acid and KCl partially reversed the inhibitory effect of HU on chlorophyll synthesis. HU stimulated yellowing of barley first leaf sections. The compound had little effect on leucine-14C incorporation to protein, and markedly inhibited uracil-14C incorporation in to RNA of the greening cucumber cotyledons. It is suggested that the inhibition of RNA and chlorophyll synthesis in HU-treated cucumber cotyledons follows the HU-dependent inhibition of DNA replication.

  5. Inhibition of Glutathione Synthesis Induced by Exhaustive Running Exercise via the Decreased Influx Rate of L-Cysteine in Rat Erythrocytes.

    Science.gov (United States)

    Xiong, Yanlian; Xiong, Yanlei; Zhou, Shuai; Yu, Zhenhai; Zhao, Dongmei; Wang, Zhiqiang; Li, Yuling; Yan, Jingtong; Cai, Yu; Zhang, Wenqian

    2016-01-01

    The main purpose of this study was to investigate the effect of exhaustive exercise on L-cysteine uptake and its effect on erythrocyte glutathione (GSH) synthesis and metabolism. Rats were divided into three groups: sedentary control (C), exhaustive running exercise (ERE) and moderate running exercise (MRE) (n=12 rats/group). We determined the L-cysteine efflux and influx in vitro in rat erythrocytes and its relationship with GSH synthesis. Total anti-oxidant potential of plasma was measured in terms of the ferric reducing ability of plasma (FRAP) values for each exercise group. In addition, the glucose metabolism enzyme activity of erythrocytes was also measured under in vitro incubation conditions. Biochemical studies confirmed that exhaustive running exercise significantly increased oxidative damage parameters in thiobarbituric acid reactive substances (TBARS) and methemoglobin levels. Pearson correlation analysis suggested that L-cysteine influx was positively correlated with erythrocyte GSH synthesis and FRAP values in both the control and exercise groups. In vitro oxidation incubation significantly decreased the level of glucose metabolism enzyme activity in the control group. We presented evidence of the exhaustive exercise-induced inhibition of GSH synthesis due to a dysfunction in L-cysteine transport. In addition, oxidative stress-induced changes in glucose metabolism were the driving force underlying decreased L-cysteine uptake in the exhaustive exercise group. © 2016 The Author(s) Published by S. Karger AG, Basel.

  6. Partial reversal by beta-D-xyloside of salicylate-induced inhibition of glycosaminoglycan synthesis in articular cartilage

    International Nuclear Information System (INIS)

    Palmoski, M.J.; Brandt, K.D.

    1982-01-01

    While net 35 S-glycosaminoglycan synthesis in normal canine articular cartilage was suppressed by 10(-3)M sodium salicylate to about 70% of the control value, addition of xyloside (10(-6)M-10(-3)M) to the salicylate-treated cultures led to a concentration-dependent increase in glycosaminoglycan synthesis, which rose to 120-237% of controls. Similar results were obtained when 3 H-glucosamine was used to measure glycosaminoglycan synthesis, confirming that salicylate suppresses and xyloside stimulates net glycosaminoglycan synthesis, and not merely sulfation. Salicylate (10-3)M) did not affect the activity of xylosyl or galactosyl transferase prepared from canine knee cartilage, and net protein synthesis was unaltered by either salicylate or xyloside. The proportion of newly synthesized proteoglycans existing as aggregates when cartilage was cultured with xyloside was similar to that in controls, although the average hydrodynamic size of disaggregated proteoglycans and of sulfated glycosaminoglycans was diminished

  7. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    OpenAIRE

    Jänne, J; Morris, D R

    1984-01-01

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylm...

  8. Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

    Science.gov (United States)

    Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

    2017-11-01

    GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

  9. Rare ginsenoside Ia synthesized from F1 by cloning and overexpression of the UDP-glycosyltransferase gene from Bacillus subtilis: synthesis, characterization, and in vitro melanogenesis inhibition activity in BL6B16 cells.

    Science.gov (United States)

    Wang, Dan-Dan; Jin, Yan; Wang, Chao; Kim, Yeon-Ju; Perez, Zuly Elizabeth Jimenez; Baek, Nam In; Mathiyalagan, Ramya; Markus, Josua; Yang, Deok-Chun

    2018-01-01

    Ginsenoside F1 has been described to possess skin-whitening effects on humans. We aimed to synthesize a new ginsenoside derivative from F1 and investigate its cytotoxicity and melanogenesis inhibitory activity in B16BL6 cells using recombinant glycosyltransferase enzyme. Glycosylation has the advantage of synthesizing rare chemical compounds from common compounds with great ease. UDP-glycosyltransferase (BSGT1) gene from Bacillus subtilis was selected for cloning. The recombinant glycosyltransferase enzyme was purified, characterized, and utilized to enzymatically transform F1 into its derivative. The new product was characterized by NMR techniques and evaluated by MTT, melanin count, and tyrosinase inhibition assay. The new derivative was identified as (20 S )-3 β ,6 α ,12 β ,20-tetrahydroxydammar-24-ene-20- O - β -D-glucopyranosyl-3- O - β -D-glucopyranoside (ginsenoside Ia), which possesses an additional glucose linked into the C-3 position of substrate F1. Ia had been previously reported; however, no in vitro biological activity was further examined. This study focused on the mass production of arduous ginsenoside Ia from accessible F1 and its inhibitory effect of melanogenesis in B16BL6 cells. Ia showed greater inhibition of melanin and tyrosinase at 100 μmol/L than F1 and arbutin. These results suggested that Ia decreased cellular melanin synthesis in B16BL6 cells through downregulation of tyrosinase activity. To our knowledge, this is the first study to report on the mass production of rare ginsenoside Ia from F1 using recombinant UDP-glycosyltransferase isolated from B. subtillis and its superior melanogenesis inhibitory activity in B16BL6 cells as compared to its precursor. In brief, ginsenoside Ia can be applied for further study in cosmetics.

  10. Synthesis, Characterization and In Vitro Anticancer Activity of C-5 Curcumin Analogues with Potential to Inhibit TNF-α-Induced NF-κB Activation

    Directory of Open Access Journals (Sweden)

    Amit Anthwal

    2014-01-01

    Full Text Available In a search of new compounds active against cancer, synthesis of a series of C-5 curcumin analogues was carried out. The new compounds demonstrated good cytotoxicity against chronic myeloid leukemia (KBM5 and colon cancer (HCT116 cell lines. Further, these compounds were found to have better potential to inhibit TNF-α-induced NF-κB activation in comparison to curcumin, which show their potential to act as anti-inflammatory agents. Some compounds were found to show higher cytotoxicity against cancer cell lines in comparison to curcumin used as standard.

  11. Bistable switches control memory and plasticity in cellular differentiation

    Science.gov (United States)

    Wang, Lei; Walker, Brandon L.; Iannaccone, Stephen; Bhatt, Devang; Kennedy, Patrick J.; Tse, William T.

    2009-01-01

    Development of stem and progenitor cells into specialized tissues in multicellular organisms involves a series of cell fate decisions. Cellular differentiation in higher organisms is generally considered irreversible, and the idea of developmental plasticity in postnatal tissues is controversial. Here, we show that inhibition of mitogen-activated protein kinase (MAPK) in a human bone marrow stromal cell-derived myogenic subclone suppresses their myogenic ability and converts them into satellite cell-like precursors that respond to osteogenic stimulation. Clonal analysis of the induced osteogenic response reveals ultrasensitivity and an “all-or-none” behavior, hallmarks of a bistable switch mechanism with stochastic noise. The response demonstrates cellular memory, which is contingent on the accumulation of an intracellular factor and can be erased by factor dilution through cell divisions or inhibition of protein synthesis. The effect of MAPK inhibition also exhibits memory and appears to be controlled by another bistable switch further upstream that determines cell fate. Once the memory associated with osteogenic differentiation is erased, the cells regain their myogenic ability. These results support a model of cell fate decision in which a network of bistable switches controls inducible production of lineage-specific differentiation factors. A competitive balance between these factors determines cell fate. Our work underscores the dynamic nature of cellular differentiation and explains mechanistically the dual properties of stability and plasticity associated with the process. PMID:19366677

  12. A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

    1990-09-01

    A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

  13. Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

    Science.gov (United States)

    Montero-Villegas, Sandra; Polo, Mónica; Galle, Marianela; Rodenak-Kladniew, Boris; Castro, María; Ves-Losada, Ana; Crespo, Rosana; García de Bravo, Margarita

    2017-01-01

    The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [ 14 C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32 µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [ 14 C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.

  14. Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.

    Science.gov (United States)

    Beltz, Hervé; Clauss, Céline; Piémont, Etienne; Ficheux, Damien; Gorelick, Robert J; Roques, Bernard; Gabus, Caroline; Darlix, Jean-Luc; de Rocquigny, Hugues; Mély, Yves

    2005-05-20

    The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

  15. Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis

    International Nuclear Information System (INIS)

    Ernst, S.G.; Oleinick, N.L.; Rustad, R.C.; Greenblatt, R.M.

    1979-01-01

    Sublethal doses of γ radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrphoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena. (Auth.)

  16. Synthesis and characterization of an effective organic/inorganic hybrid green corrosion inhibitive complex based on zinc acetate/Urtica Dioica

    International Nuclear Information System (INIS)

    Salehi, E.; Naderi, Reza; Ramezanzadeh, B.

    2017-01-01

    Highlights: • An organic/inorganic hybrid green corrosion inhibitive pigment was synthesized and characterized. • Chemical structure and morphology of the hybrid complex were characterized. • Zinc acetate/Urtica Dioica showed effective inhibition action in saline solution on carbon steel. • The synergistic effect between Zn 2+ cations and inhibitive compounds existed in U.D resulted in protective film deposition on the steel surface. - Abstract: This study aims at synthesis and characterization of an effective corrosion inhibitive complex based on zinc acetate/Urtica Dioica (ZnA-U.D) for corrosion protection of mild steel in chloride solution. The chemical structure and morphology of the complex were characterized by Fourier transform infrared spectroscopy (FT-IR), UV–vis, thermal gravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The corrosion protection performance of the mild steel samples dipped in 3.5 wt.% NaCl solutions with and without ZnA-U.D extract was investigated by visual observations, open circuit potential (OCP) measurements, electrochemical impedance spectroscopy (EIS) and polarization test. Results revealed that the ZnA successfully chelated with organic inhibitive compounds (i.e Quercetin, Quinic acid, Caffeic acid, Hystamine and Serotonin) present in the U.D extract. The electrochemical measurements revealed the effective inhibition action of ZnA-U.D complex in the sodium chloride solution on the mild steel. The synergistic effect between Zn 2+ and organic compounds present in the U.D extract resulted in protective film deposition on the steel surface, which was proved by SEM and XPS analyses.

  17. Synthesis and characterization of an effective organic/inorganic hybrid green corrosion inhibitive complex based on zinc acetate/Urtica Dioica

    Energy Technology Data Exchange (ETDEWEB)

    Salehi, E.; Naderi, Reza [School of Metallurgy and Materials Engineering, College of Engineering, University of Tehran, P.O. Box 11155-4563, Tehran (Iran, Islamic Republic of); Ramezanzadeh, B., E-mail: ramezanzadeh@aut.ac.ir [Department of Surface Coatings and Corrosion, Institute for Color Science and Technology (ICST), PO 16765-654, Tehran (Iran, Islamic Republic of)

    2017-02-28

    Highlights: • An organic/inorganic hybrid green corrosion inhibitive pigment was synthesized and characterized. • Chemical structure and morphology of the hybrid complex were characterized. • Zinc acetate/Urtica Dioica showed effective inhibition action in saline solution on carbon steel. • The synergistic effect between Zn{sup 2+} cations and inhibitive compounds existed in U.D resulted in protective film deposition on the steel surface. - Abstract: This study aims at synthesis and characterization of an effective corrosion inhibitive complex based on zinc acetate/Urtica Dioica (ZnA-U.D) for corrosion protection of mild steel in chloride solution. The chemical structure and morphology of the complex were characterized by Fourier transform infrared spectroscopy (FT-IR), UV–vis, thermal gravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The corrosion protection performance of the mild steel samples dipped in 3.5 wt.% NaCl solutions with and without ZnA-U.D extract was investigated by visual observations, open circuit potential (OCP) measurements, electrochemical impedance spectroscopy (EIS) and polarization test. Results revealed that the ZnA successfully chelated with organic inhibitive compounds (i.e Quercetin, Quinic acid, Caffeic acid, Hystamine and Serotonin) present in the U.D extract. The electrochemical measurements revealed the effective inhibition action of ZnA-U.D complex in the sodium chloride solution on the mild steel. The synergistic effect between Zn{sup 2+} and organic compounds present in the U.D extract resulted in protective film deposition on the steel surface, which was proved by SEM and XPS analyses.

  18. The Fruits of Wampee Inhibit H2O2-Induced Apoptosis in PC12 Cells via the NF-κB Pathway and Regulation of Cellular Redox Status

    Directory of Open Access Journals (Sweden)

    Xiaobin Zeng

    2014-06-01

    Full Text Available Wampee (Clausena lansium fruits (CLS, whose pulp can be used to prepare fruit cups, desserts, jam, or jelly, can be eaten along with the peel. In this study, a PC12 cell model was built to observe the protective effect of CLS against H2O2-induced oxidative stress. We found that pretreatment with CLS increased cell viability and inhibited cytotoxicity, caspase-3 activity and DNA condensation. CLS also attenuated the increase in ROS production and MMP reduction. Moreover, we attempted to determine whether CLS suppressed the expression and phosphorylation of NF-κB. Western blot and immunostaining assay revealed that CLS inhibited H2O2-induced up-regulation of NF-κB p65 and pNF-κB p65. And CLS significantly suppressed the translocation of NF-κB p65 and pNF-κB p65 from cytoplasm to nuclear. Also, seven major compounds including a new flavanoid, luteolin-4'-O-β-d-gluco-pyranoside (3 and six known compounds 1,2, 4–7 were isolated and identified from CLS. Their antioxidative and H2O2-induced PC12 cell apoptosis-reversing activity were determined. These findings suggest that CLS and its major constituents (flavanoids may be potential antioxidant agents and should encourage further research into their use as a functional food for neurodegenerative diseases.

  19. Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status.

    Science.gov (United States)

    Michaelis, Martin; Rothweiler, Florian; Löschmann, Nadine; Sharifi, Mohsen; Ghafourian, Taravat; Cinatl, Jindrich

    2015-07-10

    The PKCβ inhibitor enzastaurin was tested in parental neuroblastoma and rhabdomyosarcoma cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53-depleted cells. Enzastaurin IC50s ranged from 3.3 to 9.5 μM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125 μM interfered with ABCB1-mediated drug transport. PKCα and PKCβ may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations ≥ 1.25 μM. The investigated cell lines did not express PKCβ. PKCα depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. These findings are important for the further development of therapies combining enzastaurin with ABC transporter substrates.

  20. Delphinidin prevents high glucose-induced cell proliferation and collagen synthesis by inhibition of NOX-1 and mitochondrial superoxide in mesangial cells

    Directory of Open Access Journals (Sweden)

    Seung Eun Song

    2016-04-01

    Full Text Available This study examined the effect of delphinidin on high glucose-induced cell proliferation and collagen synthesis in mesangial cells. Glucose dose-dependently (5.6–25 mM increased cell proliferation and collagen I and IV mRNA levels, whereas pretreatment with delphinidin (50 μM prevented cell proliferation and the increased collagen mRNA levels induced by high glucose (25 mM. High glucose increased reactive oxygen species (ROS generation, and this was suppressed by pretreating delphinidin or the antioxidant N-acetyl cysteine. NADPH oxidase (NOX 1 was upregulated by high glucose, but pretreatment with delphinidin abrogated this upregulation. Increased mitochondrial superoxide by 25 mM glucose was also suppressed by delphinidin. The NOX inhibitor apocynin and mitochondria-targeted antioxidant Mito TEMPO inhibited ROS generation and cell proliferation induced by high glucose. Phosphorylation of extracellular signal regulated kinase (ERK1/2 was increased by high glucose, which was suppressed by delphinidin, apocynin or Mito TEMPO. Furthermore, PD98059 (an ERK1/2 inhibitor prevented the high glucose-induced cell proliferation and increased collagen mRNA levels. Transforming growth factor (TGF-β protein levels were elevated by high glucose, and pretreatment with delphinidin or PD98059 prevented this augmentation. These results suggest that delphinidin prevents high glucose-induced cell proliferation and collagen synthesis by inhibition of NOX-1 and mitochondrial superoxide in mesangial cells.

  1. MCK1 is a novel regulator of myo-inositol phosphate synthase (MIPS that is required for inhibition of inositol synthesis by the mood stabilizer valproate.

    Directory of Open Access Journals (Sweden)

    Wenxi Yu

    Full Text Available Myo-inositol, the precursor of all inositol compounds, is essential for the viability of eukaryotes. Identifying the factors that regulate inositol homeostasis is of obvious importance to understanding cell function and the pathologies underlying neurological and metabolic resulting from perturbation of inositol metabolism. The current study identifies Mck1, a GSK3 homolog, as a novel positive regulator of inositol de novo synthesis in yeast. Mck1 was required for normal activity of myo-inositol phosphate synthase (MIPS, which catalyzes the rate-limiting step of inositol synthesis. mck1Δ cells exhibited a 50% decrease in MIPS activity and a decreased rate of incorporation of [13C6]glucose into [13C6]-inositol-3-phosphate and [13C6]-inositol compared to WT cells. mck1Δ cells also exhibited decreased growth in the presence of the inositol depleting drug valproate (VPA, which was rescued by supplementation of inositol. However, in contrast to wild type cells, which exhibited more than a 40% decrease in MIPS activity in the presence of VPA, the drug did not significantly decrease MIPS activity in mck1Δ cells. These findings indicate that VPA-induced MIPS inhibition is Mck1-dependent, and suggest a model that unifies two current hypotheses of the mechanism of action of VPA-inositol depletion and GSK3 inhibition.

  2. MCK1 is a novel regulator of myo-inositol phosphate synthase (MIPS) that is required for inhibition of inositol synthesis by the mood stabilizer valproate.

    Science.gov (United States)

    Yu, Wenxi; Daniel, Joshua; Mehta, Dhara; Maddipati, Krishna Rao; Greenberg, Miriam L

    2017-01-01

    Myo-inositol, the precursor of all inositol compounds, is essential for the viability of eukaryotes. Identifying the factors that regulate inositol homeostasis is of obvious importance to understanding cell function and the pathologies underlying neurological and metabolic resulting from perturbation of inositol metabolism. The current study identifies Mck1, a GSK3 homolog, as a novel positive regulator of inositol de novo synthesis in yeast. Mck1 was required for normal activity of myo-inositol phosphate synthase (MIPS), which catalyzes the rate-limiting step of inositol synthesis. mck1Δ cells exhibited a 50% decrease in MIPS activity and a decreased rate of incorporation of [13C6]glucose into [13C6]-inositol-3-phosphate and [13C6]-inositol compared to WT cells. mck1Δ cells also exhibited decreased growth in the presence of the inositol depleting drug valproate (VPA), which was rescued by supplementation of inositol. However, in contrast to wild type cells, which exhibited more than a 40% decrease in MIPS activity in the presence of VPA, the drug did not significantly decrease MIPS activity in mck1Δ cells. These findings indicate that VPA-induced MIPS inhibition is Mck1-dependent, and suggest a model that unifies two current hypotheses of the mechanism of action of VPA-inositol depletion and GSK3 inhibition.

  3. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    International Nuclear Information System (INIS)

    Wang Lei; Sasai, Ken; Akagi, Tsuyoshi; Tanaka, Shinya

    2008-01-01

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development

  4. Inhibition of microtubules and dynein rescues human immunodeficiency virus type 1 from owl monkey TRIMCyp-mediated restriction in a cellular context-specific fashion.

    Science.gov (United States)

    Pawlica, Paulina; Dufour, Caroline; Berthoux, Lionel

    2015-04-01

    IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5α (tripartite motif protein 5, isoform α) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5α maps to the N-terminal domain of the retroviral capsid proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA)-coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits human immunodeficiency virus type 1 (HIV-1). Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5α. Here, we showed that efficient restriction of HIV-1 by omkTRIMCyp was similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by small interfering RNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp. © 2015 The Authors.

  5. Reduced antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells of salivary polymorphonuclear leukocytes and inhibition of peripheral blood polymorphonuclear leukocyte cytotoxicity by saliva.

    Science.gov (United States)

    Ashkenazi, M; Kohl, S

    1990-06-15

    Blood polymorphonuclear leukocytes (BPMN) have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-infected cells. Although HSV infections are frequently found in the oral cavity, the ADCC capacity of salivary PMN (SPMN) has not been studied, mainly because methods to isolate SPMN were not available. We have recently developed a method to isolate SPMN, and in this study have evaluated their ADCC activity against HSV-infected cells. SPMN were obtained by repeated washings of the oral cavity, and separated from epithelial cells by nylon mesh filtration. ADCC was quantitatively determined by 51Cr release from HSV-infected Chang liver cells. SPMN in the presence of antibody were able to destroy HSV-infected cells, but SPMN were much less effective in mediating ADCC than BPMN (3.4% vs 40.7%, p less than 0.0001). In the presence of antiviral antibody, SPMN were able to adhere to HSV-infected cells, but less so than BPMN (34% vs 67%), and specific antibody-induced adherence was significantly lower in SPMN (p less than 0.04). The spontaneous adherence to HSV-infected cells was higher for SPMN than BPMN. SPMN demonstrated up-regulation of the adhesion glycoprotein CD18, but down-regulation of the FcRIII receptor. Incubation with saliva decreased ADCC capacity of BPMN, up-regulated CD18 expression, and down-regulated FcRIII expression.

  6. L-Cysteine in vitro can restore cellular glutathione and inhibits the expression of cell adhesion molecules in G6PD-deficient monocytes.

    Science.gov (United States)

    Parsanathan, Rajesh; Jain, Sushil K

    2018-04-06

    L-Cysteine is a precursor of glutathione (GSH), a potent physiological antioxidant. Excess glucose-6-phosphate dehydrogenase (G6PD) deficiency in African Americans and low levels of L-cysteine diet in Hispanics can contributes to GSH deficiency and oxidative stress. Oxidative stress and monocyte adhesion was considered to be an initial event in the progression of vascular dysfunction and atherosclerosis. However, no previous study has investigated the contribution of GSH/G6PD deficiency to the expression of monocyte adhesion molecules. Using human U937 monocytes, this study examined the effect of GSH/G6PD deficiency and L-cysteine supplementation on monocyte adhesion molecules. G6PD/GSH deficiency induced by either siRNA or inhibitors (6AN/BSO, respectively) significantly (p adhesion molecules (ICAM-1, VCAM-1, SELL, ITGB1 and 2); NADPH oxidase (NOX), reactive oxygen species (ROS) and MCP-1 were upregulated, and decreases in levels of GSH, and nitric oxide were observed. The expression of ICAM-1 and VCAM-1 mRNA levels increased in high glucose, MCP-1 or TNF-α-treated G6PD-deficient compared to G6PD-normal cells. L-Cysteine treatment significantly (p adhesion molecules. Thus, GSH/G6PD deficiency increases susceptibility to monocyte adhesion processes, whereas L-cysteine supplementation can restore cellular GSH/G6PD and attenuates NOX activity and expression of cell adhesion molecules.

  7. Inhibition of central de novo ceramide synthesis restores insulin signaling in hypothalamus and enhances β-cell function of obese Zucker rats

    Directory of Open Access Journals (Sweden)

    Mélanie Campana

    2018-02-01

    Full Text Available Objectives: Hypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity. Methods: Hypothalamic GT1-7 neuronal cells were treated with palmitate. De novo ceramide synthesis was inhibited either by pharmacological (myriocin or molecular (si-Serine Palmitoyl Transferase 2, siSPT2 approaches. Obese Zucker rats (OZR were intracerebroventricularly infused with myriocin to inhibit de novo ceramide synthesis. Insulin resistance was determined by quantification of Akt phosphorylation. Ceramide levels were quantified either by a radioactive kinase assay or by mass spectrometry analysis. Glucose homeostasis were evaluated in myriocin-treated OZR. Basal and glucose-stimulated parasympathetic tonus was recorded in OZR. Insulin secretion from islets and β-cell mass was also determined. Results: We show that palmitate impaired insulin signaling and increased ceramide levels in hypothalamic neuronal GT1-7 cells. In addition, the use of deuterated palmitic acid demonstrated that palmitate activated several enzymes of the de novo ceramide synthesis pathway in hypothalamic cells. Importantly, myriocin and siSPT2 treatment restored insulin signaling in palmitate-treated GT1-7 cells. Protein kinase C (PKC inhibitor or a dominant-negative PKCζ also counteracted palmitate-induced insulin resistance. Interestingly, attenuating the increase in levels of hypothalamic ceramides with intracerebroventricular infusion of myriocin in OZR improved their hypothalamic insulin-sensitivity. Importantly, central myriocin treatment partially restored glucose tolerance in OZR. This latter effect is related to the restoration of glucose-stimulated insulin

  8. Some Anilides of 2-Alkylthio- and 2-Chloro-6-Alkylthio-4-Pyridinecarboxylic Acids: Synthesis and Photosynthesis-Inhibiting Activity

    Directory of Open Access Journals (Sweden)

    Miloš Macháček

    2001-06-01

    Full Text Available Many compounds containing a -CONH- group display photosynthesis inhibiting activity. Based on this structural feature, a group of anilides of 2-alkylthio-(1b-4f or 2-chloro-6-alkylthio-4-pyridinecarboxylic acids (5a-6c was synthesised. The prepared compounds were tested for their inhibition of the oxygen evolution rate (OER in spinach chloroplasts. A quasi-parabolic dependence between photosynthesis-inhibiting activity and the lipophilicity of the compounds was determined for 1b-4f as well as for 5a-6c. The inhibitory activity of compounds 1b-4f was higher than that of 5a-6c for comparable lipophilicity values.

  9. Synthesis and characterization of an effective organic/inorganic hybrid green corrosion inhibitive complex based on zinc acetate/Urtica Dioica

    Science.gov (United States)

    Salehi, E.; Naderi, Reza; Ramezanzadeh, B.

    2017-02-01

    This study aims at synthesis and characterization of an effective corrosion inhibitive complex based on zinc acetate/Urtica Dioica (ZnA-U.D) for corrosion protection of mild steel in chloride solution. The chemical structure and morphology of the complex were characterized by Fourier transform infrared spectroscopy (FT-IR), UV-vis, thermal gravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The corrosion protection performance of the mild steel samples dipped in 3.5 wt.% NaCl solutions with and without ZnA-U.D extract was investigated by visual observations, open circuit potential (OCP) measurements, electrochemical impedance spectroscopy (EIS) and polarization test. Results revealed that the ZnA successfully chelated with organic inhibitive compounds (i.e Quercetin, Quinic acid, Caffeic acid, Hystamine and Serotonin) present in the U.D extract. The electrochemical measurements revealed the effective inhibition action of ZnA-U.D complex in the sodium chloride solution on the mild steel. The synergistic effect between Zn2+ and organic compounds present in the U.D extract resulted in protective film deposition on the steel surface, which was proved by SEM and XPS analyses.

  10. Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

    International Nuclear Information System (INIS)

    Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.; Kuo, M.-L.; Ho, Y.-S.; Lee, W.-S.

    2008-01-01

    Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest

  11. Activated Macrophages as a Novel Determinant of Tumor Cell Radioresponse: The Role of Nitric Oxide-Mediated Inhibition of Cellular Respiration and Oxygen Sparing

    International Nuclear Information System (INIS)

    Jiang Heng; De Ridder, Mark; Verovski, Valeri N.; Sonveaux, Pierre; Jordan, Benedicte F.; Law, Kalun; Monsaert, Christinne; Van den Berge, Dirk L.; Verellen, Dirk; Feron, Olivier; Gallez, Bernard; Storme, Guy A.

    2010-01-01

    Purpose: Nitric oxide (NO), synthesized by the inducible nitric oxide synthase (iNOS), is known to inhibit metabolic oxygen consumption because of interference with mitochondrial respiratory activity. This study examined whether activation of iNOS (a) directly in tumor cells or (b) in bystander macrophages may improve radioresponse through sparing of oxygen. Methods and Materials: EMT-6 tumor cells and RAW 264.7 macrophages were exposed to bacterial lipopolysaccharide plus interferon-γ, and examined for iNOS expression by reverse transcription polymerase chain reaction, Western blotting and enzymatic activity. Tumor cells alone, or combined with macrophages were subjected to metabolic hypoxia and analyzed for radiosensitivity by clonogenic assay, and for oxygen consumption by electron paramagnetic resonance and a Clark-type electrode. Results: Both tumor cells and macrophages displayed a coherent picture of iNOS induction at transcriptional/translational levels and NO/nitrite production, whereas macrophages showed also co-induction of the inducible heme oxygenase-1, which is associated with carbon monoxide (CO) and bilirubin production. Activation of iNOS in tumor cells resulted in a profound oxygen sparing and a 2.3-fold radiosensitization. Bystander NO-producing, but not CO-producing, macrophages were able to block oxygen consumption by 1.9-fold and to radiosensitize tumor cells by 2.2-fold. Both effects could be neutralized by aminoguanidine, a metabolic iNOS inhibitor. An improved radioresponse was clearly observed at macrophages to tumor cells ratios ranging between 1:16 to 1:1. Conclusions: Our study is the first, as far as we are aware, to provide evidence that iNOS may induce radiosensitization through oxygen sparing, and illuminates NO-producing macrophages as a novel determinant of tumor cell radioresponse within the hypoxic tumor microenvironment.

  12. microRNA-494 is a potential prognostic marker and inhibits cellular proliferation, migration and invasion by targeting SIRT1 in epithelial ovarian cancer.

    Science.gov (United States)

    Yang, Aijun; Wang, Xuenan; Yu, Chunna; Jin, Zhenzhen; Wei, Lingxia; Cao, Jinghe; Wang, Qin; Zhang, Min; Zhang, Lin; Zhang, Lei; Hao, Cuifang

    2017-09-01

    Ovarian cancer is one of the most common types of gynecological malignancy worldwide, and is the fourth leading cause of cancer-associated mortality among women. Despite improvements in therapeutic treatments, the prognosis for epithelial ovarian cancer (EOC) remains poor, mainly due to the rapid growth and metastasis of ovarian cancer tumors. An increasing number of studies have indicated that microRNAs (miRNAs) are involved in the carcinogenesis and progression of human cancer, suggesting that miRNAs may be used in clinical prognosis and as a therapeutic target in EOC. The aim of the present study was to investigate the expression levels of miRNA-494 in EOC tissues and cell lines. The clinical significance of miRNA-494 in patients with EOC was also evaluated. The results demonstrated that miRNA-494 was significantly downregulated in EOC tissues and cell lines. Low expression levels of miRNA-494 were associated with poor prognostic features, including International Federation of Gynecology and Obstetrics stage, tumor size and lymph node metastasis. In vitro functional studies demonstrated that overexpression of miRNA-494 inhibited proliferation, migration and invasion in EOC cells. By contrast, knockdown of miRNA-494 enhanced cell growth, migration and invasion in EOC cells. Notably, sirtuin 1 (SIRT1) was identified as a direct target of miRNA-494 in EOC. Furthermore, MTT, cell migration and invasion assays verified that EOC cell proliferation, migration and invasion were completely restored with forced miRNA-494 expression and SIRT1 restoration. Together, these findings suggest that miRNA-494 is a potential prognostic marker, and may provide novel therapeutic regimens of targeted therapy for EOC.

  13. Cellular metabolism

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Walters, R.A.

    1977-01-01

    Progress is reported on the following research projects: chromatin structure; the use of circular synthetic polydeoxynucleotides as substrates for the study of DNA repair enzymes; human cellular kinetic response following exposure to DNA-interactive compounds; histone phosphorylation and chromatin structure in cell proliferation; photoaddition products induced in chromatin by uv light; pollutants and genetic information transfer; altered RNA metabolism as a function of cadmium accumulation and intracellular distribution in cultured cells; and thymidylate chromophore destruction by water free radicals

  14. Compounds isolated from the aerial part of Crataegus azarolus inhibit growth of B16F10 melanoma cells and exert a potent inhibition of the melanin synthesis.

    Science.gov (United States)

    Mustapha, Nadia; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2015-02-01

    Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study, we have investigated inhibitory capacity of vitexin-2''-O-rhamnoside as well as the extract from which it was isolated, i.e. the ethyl acetate extract obtained from the leaves of Crataegus azarolus, on mouse melanoma (B16F10) proliferation. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475nm. Ethyl acetate extract and vitexin-2''-O-rhamnoside exhibited significant anti-proliferative activity against B16F10 melanoma cells after incubation for 48hours with IC50s of 50μg/mL and 20μM, respectively. Furthermore, these two compounds have the ability to reduce the melanin content by inhibiting the tyrosinase activity of B16F10 cells. Thus, further investigations are merited to ascertain their potential application in treating hyperpigmentation disorders. Copyright © 2014. Published by Elsevier Masson SAS.

  15. Synthesis and corrosion inhibition application of NATN on mild steel surface in acidic media complemented with DFT studies

    Science.gov (United States)

    Al-Baghdadi, Shaimaa B.; Hashim, Fanar G.; Salam, Ahmed Q.; Abed, Talib K.; Gaaz, Tayser Sumer; Al-Amiery, Ahmed A.; Kadhum, Abdul Amir H.; Reda, Khalid S.; Ahmed, Wahab K.

    2018-03-01

    The corrosion inhibition effectiveness of thiosemicarbazide compound, namely 3-nitro-5-(2-amino-1,3,4-thiadiazolyl)nitrobenzene (NATN), on mild steel in 1 M hydrochloric acid media has been investigated by weight loss technique. The results exhibit that the corrosion ratio of mild steel was reduced regarding to adding NATN. The corrosion inhibition rate for the NATN was 92.3% at the highest investigated NATN concentration. From the weight loss results it could be concluded that NATN with sulfur, nitrogen and oxygen atoms has clarified best corrosion inhibition achievement comparing to 3,5-dinitrobenzoic acid. Regarding to theoretical studies, DFT was employee to figured geometrical structure and electronic characteristics on NATN. The investigation have been extensive to the HOMO and LUMO analysis to evaluate the energy gap, Ionization potential, Electron Affinity, Global Hardness, Chemical Potential, Electrophilicity, Electronegativity and Polarizability.

  16. Synthesis and corrosion inhibition application of NATN on mild steel surface in acidic media complemented with DFT studies

    Directory of Open Access Journals (Sweden)

    Shaimaa B. Al-Baghdadi

    2018-03-01

    Full Text Available The corrosion inhibition effectiveness of thiosemicarbazide compound, namely 3-nitro-5-(2-amino-1,3,4-thiadiazolylnitrobenzene (NATN, on mild steel in 1 M hydrochloric acid media has been investigated by weight loss technique. The results exhibit that the corrosion ratio of mild steel was reduced regarding to adding NATN. The corrosion inhibition rate for the NATN was 92.3% at the highest investigated NATN concentration. From the weight loss results it could be concluded that NATN with sulfur, nitrogen and oxygen atoms has clarified best corrosion inhibition achievement comparing to 3,5-dinitrobenzoic acid. Regarding to theoretical studies, DFT was employee to figured geometrical structure and electronic characteristics on NATN. The investigation have been extensive to the HOMO and LUMO analysis to evaluate the energy gap, Ionization potential, Electron Affinity, Global Hardness, Chemical Potential, Electrophilicity, Electronegativity and Polarizability. Keywords: NATN, Mild steel, Weight loss, Dinitrobenzoic acid

  17. Synthesis, Characterization, Antimicrobial Studies and Corrosion Inhibition Potential of 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane: Experimental and Quantum Chemical Studies

    Directory of Open Access Journals (Sweden)

    Henry U. Nwankwo

    2016-02-01

    Full Text Available The macrocylic ligand, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (MHACD was synthesized by the demetallation of its freshly synthesized Ni(II complex (NiMHACD. Successful synthesis of NiMHACD and the free ligand (MHACD was confirmed by various characterization techniques, including Fourier transform infra-red (FT-IR, proton nuclear magnetic resonance (1H-NMR, carbon-13 nuclear magnetic resonance (13C-NMR, ultraviolet-visible (UV-vis, and energy dispersive x-ray (EDX spectroscopic techniques. The anti-bacteria activities of MHACD were investigated against Staphylococcus aureus and Enterococcus species and the results showed that MHACD possesses a spectrum of activity against the two bacteria. The electrochemical cyclic voltammetry study on MHACD revealed that it is a redox active compound with promising catalytic properties in electrochemical applications. The inhibition potential of MHACD for mild steel corrosion in 1 M HCl was investigated using potentiodynamic polarization method. The results showed that MHACD inhibits steel corrosion as a mixed-type inhibitor, and the inhibition efficiency increases with increasing concentration of MHACD. The adsorption of MHACD obeys the Langmuir adsorption isotherm; it is spontaneous and involves competitive physisorption and chemisorption mechanisms. Quantum chemical calculations revealed that the energy of the highest occupied molecular orbital (HOMO of MHACD is high enough to favor forward donation of charges to the metal during adsorption and corrosion inhibition. Natural bond orbital (NBO analysis revealed the presence of various orbitals in the MHACD that are capable of donating or accepting electrons under favorable conditions.

  18. Dose- and time-dependent radiation inhibition of RNA and glycosaminoglycan synthesis in embryonic cartilage: an in vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Cornelissen, M.; Thierens, H.; De Ridder, L. (Ghent Rijksuniversiteit (Belgium))

    1990-05-01

    Radiation effects on the RNA and glycosaminoglycan (GAG) synthesis of embryonic cartilaginous tibiae were studied in vitro during a 4- or 7-day culture period. Before culture, tibiae received single radiation doses of 20, 50 or 100 Gy. A limited, dose-dependent immediate effect on RNA and GAG synthesis was found. This effect was unchanged for 2 days. After this period a time-dependent delayed effect was observed. For each radiation dose, and for each precursor, the same time-related pattern was found. At the end of the culture period acid phosphatase activity, an early indicator of apoptosis, was higher in irradiated tibiae than in controls. No other morphological ultrastructural differences were observed at this time. The authors conclude that metabolic alterations are probably due to stimulation of initial stages of the apoptotic process in the irradiated cartilage cells. (author).

  19. Inhibition of protein synthesis but not β-adrenergic receptors blocks reconsolidation of a cocaine-associated cue memory

    OpenAIRE

    Dunbar, Amber B.; Taylor, Jane R.

    2016-01-01

    Previously consolidated memories have the potential to enter a state of lability upon memory recall, during which time the memory can be altered before undergoing an additional consolidation-like process and being stored again as a long-term memory. Blocking reconsolidation of aberrant memories has been proposed as a potential treatment for psychiatric disorders including addiction. Here we investigated of the effect of systemically administering the protein synthesis inhibitor cycloheximide ...

  20. Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells

    International Nuclear Information System (INIS)

    Hill, T.M.; Sinden, R.R.; Sadler, J.R.

    1983-01-01

    Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff

  1. The relationship between DNA synthesis and incorporation of (14C) lysine into different histone fractions in Ehrlich ascites tumour cells

    International Nuclear Information System (INIS)

    Malec, J.; Kornacka, L.; Wojnarowska, M.; Moscicka, M.

    1974-01-01

    The effect of inhibition of DNA synthesis by hydroxyurea on ( 14 C) lysine incorporation into the main four histone fractions in Ehrlich ascites tumor cells, was examined in vitro. The radioactivity of lysine-rich histones, especially of histone f1, was preferentially decreased. The smallest decrease was observed for histone f3. The incorporation into other cellular proteins was but slightly affected. (author)

  2. Synthesis, Biological Evaluation, and Docking Studies of Novel Bisquaternary Aldoxime Reactivators on Acetylcholinesterase and Butyrylcholinesterase Inhibited by Paraoxon

    Directory of Open Access Journals (Sweden)

    Kamil Kuca

    2018-05-01

    Full Text Available Nerve agents and oxon forms of organophosphorus pesticides act as strong irreversible inhibitors of two cholinesterases in the human body: acetylcholinesterase (AChE; EC 3.1.1.7 and butyrylcholinesterase (BChE; EC 3.1.1.8, and are therefore highly toxic compounds. For the recovery of inhibited AChE, antidotes from the group of pyridinium or bispyridinium aldoxime reactivators (pralidoxime, obidoxime, HI-6 are used in combination with anticholinergics and anticonvulsives. Therapeutic efficacy of reactivators (called “oximes” depends on their chemical structure and also the type of organophosphorus inhibitor. Three novel oximes (K131, K142, K153 with an oxime group in position four of the pyridinium ring were designed and then tested for their potency to reactivate human (Homo sapiens sapiens AChE (HssACHE and BChE (HssBChE inhibited by the pesticide paraoxon (diethyl 4-nitrophenyl phosphate. According to the obtained results, none of the prepared oximes were able to satisfactorily reactivate paraoxon-inhibited cholinesterases. On the contrary, extraordinary activity of obidoxime in the case of paraoxon-inhibited HssAChE reactivation was confirmed. Additional docking studies pointed to possible explanations for these results.

  3. Maternal obesity reduces milk lipid production in lactating mice by inhibiting acetyl-CoA carboxylase and impairing fatty acid synthesis.

    Science.gov (United States)

    Saben, Jessica L; Bales, Elise S; Jackman, Matthew R; Orlicky, David; MacLean, Paul S; McManaman, James L

    2014-01-01

    Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.

  4. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

    Science.gov (United States)

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

  5. Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide

    Energy Technology Data Exchange (ETDEWEB)

    Jobson, Andrew G.; Lountos, George T.; Lorenzi, Philip L.; Llamas, Jenny; Connelly, John; Cerna, David; Tropea, Joseph E.; Onda, Akikazu; Zoppoli, Gabriele; Kondapaka, Sudhir; Zhang, Guangtao; Caplen, Natasha J.; Cardellina, II, John H.; Yoo, Stephen S.; Monks, Anne; Self, Christopher; Waugh, David S.; Shoemaker, Robert H.; Pommier, Yves; (NIH)

    2010-04-05

    Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {l_brace}4-[1-(guanidinohydrazone)-ethyl]-phenyl{r_brace}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

  6. Identification of ergosterol and inhibition of sterol synthesis by Δ5-sterols in GL7, an auxotrophic mutant of yeast

    International Nuclear Information System (INIS)

    Dhanuka, I.C.

    1988-01-01

    Synthesis of ergosterol was demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the exogenous sterol was 5α-cholestan-3β-ol, 5α-cholest-8(14)-en-3β-ol, or 24β-methyl-5α-cholest-8(14)-en-3β-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol. Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labelling from [ 3 H]acetate. Except for some cholest-5-en-3β-ol (cholesterol) which was derived from the 5α-cholestan-3β-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the exogenous sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5α-cholest-8(9)-en-3β-ol and 5α-cholest-7-en-3β-ol (lathosterol) were converted to cholesta-5,7-dien-3β-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol

  7. Metabonomics Indicates Inhibition of Fatty Acid Synthesis, β-Oxidation, and Tricarboxylic Acid Cycle in Triclocarban-Induced Cardiac Metabolic Alterations in Male Mice.

    Science.gov (United States)

    Xie, Wenping; Zhang, Wenpeng; Ren, Juan; Li, Wentao; Zhou, Lili; Cui, Yuan; Chen, Huiming; Yu, Wenlian; Zhuang, Xiaomei; Zhang, Zhenqing; Shen, Guolin; Li, Haishan

    2018-02-14

    Triclocarban (TCC) has been identified as a new environmental pollutant that is potentially hazardous to human health; however, the effects of short-term TCC exposure on cardiac function are not known. The aim of this study was to use metabonomics and molecular biology techniques to systematically elucidate the molecular mechanisms of TCC-induced effects on cardiac function in mice. Our results show that TCC inhibited the uptake, synthesis, and oxidation of fatty acids, suppressed the tricarboxylic acid (TCA) cycle, and increased aerobic glycolysis levels in heart tissue after short-term TCC exposure. TCC also inhibited the nuclear peroxisome proliferator-activated receptor α (PPARα), confirming its inhibitory effects on fatty acid uptake and oxidation. Histopathology and other analyses further confirm that TCC altered mouse cardiac physiology and pathology, ultimately affecting normal cardiac metabolic function. We elucidate the molecular mechanisms of TCC-induced harmful effects on mouse cardiac metabolism and function from a new perspective, using metabonomics and bioinformatics analysis data.

  8. An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

    International Nuclear Information System (INIS)

    Reigadas, Sandrine; Ventura, Michel; Andreola, Marie-Line; Michel, Justine; Gryaznov, Sergei; Tarrago-Litvak, Laura; Litvak, Simon; Astier-Gin, Therese

    2003-01-01

    We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC 50 values lower than 1 μM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2'-O-methyl modification increased the inhibitor activity 11-fold (IC 50 = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3'-end of the minus-strand RNA

  9. Synthesis and Antimycobacterial and Photosynthesis-Inhibiting Evaluation of 2-[(E-2-Substituted-ethenyl]-1,3-benzoxazoles

    Directory of Open Access Journals (Sweden)

    Ales Imramovsky

    2014-01-01

    Full Text Available A series of twelve 2-[(E-2-substituted-ethenyl]-1,3-benzoxazoles was designed. All the synthesized compounds were tested against three mycobacterial strains. The compounds were also evaluated for their ability to inhibit photosynthetic electron transport (PET in spinach (Spinacia oleracea L. chloroplasts. 2-[(E-2-(4-Methoxyphenylethenyl]-1,3-benzoxazole, 2-[(E-2-(2,3-dihydro-1-benzofuran-5-ylethenyl]-1,3-benzoxazole and 2-{(E-2-[4-(methylsulfanylphenyl]ethenyl}-1,3-benzoxazole showed the highest activity against M. tuberculosis, M. kansasii, and M. avium, and they demonstrated significantly higher activity against M. avium and M. kansasii than isoniazid. The PET-inhibiting activity of the most active ortho-substituted compound 2-[(E-2-(2-methoxyphenylethenyl]-1,3-benzoxazole was IC50 = 76.3 μmol/L, while the PET-inhibiting activity of para-substituted compounds was significantly lower. The site of inhibitory action of tested compounds is situated on the donor side of photosystem II. The structure-activity relationships are discussed.

  10. Rocaglamide overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells by attenuating the inhibition of caspase-8 through cellular FLICE-like-inhibitory protein downregulation.

    Science.gov (United States)

    Luan, Zhou; He, Ying; He, Fan; Chen, Zhishui

    2015-01-01

    The enhancement of apoptosis is a therapeutic strategy used in the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, hepatocellular carcinoma (HCC) cells exhibit marked resistance to the induction of cell death by TRAIL. The present study investigated whether rocaglamide, a naturally occurring product isolated from the genus Aglaia, is able to sensitize resistant HCC cells to TRAIL-mediated apoptosis. Two HCC cell lines, HepG2 and Huh-7, were treated with rocaglamide and/or TRAIL and the induction of apoptosis and effects on the TRAIL signaling pathway were investigated. The in vivo efficacy of rocaglamide was determined in TRAIL-resistant Huh-7-derived tumor xenografts. Rocaglamide significantly sensitized the TRAIL-resistant HCC cells to apoptosis by TRAIL, which resulted from the rocaglamide-mediated downregulation of cellular FLICE-like inhibitory protein and subsequent caspase-8 activation. Furthermore, rocaglamide markedly inhibited tumor growth from Huh-7 cells propagated in severe combined immunodeficient mice, suggesting that chemosentization also occurred in vivo. These data suggest that rocaglamide acted synergistically with TRAIL against the TRAIL-resistant HCC cells. Thus, it is concluded that rocaglamide as an adjuvant to TRAIL-based therapy may present a promising therapeutic approach for the treatment of HCC.

  11. Synthesis, characterization and corrosion inhibition properties of benzamide-2-chloro-4-nitrobenzoic acid and anthranilic acid-2-chloro-4-nitrobenzoic acid for mild steel corrosion in acidic medium

    Science.gov (United States)

    Pandey, Archana; Verma, Chandrabhan; Singh, B.; Ebenso, Eno E.

    2018-03-01

    The present study deals with the synthesis of two new compounds namely, benzamide - 2-chloro-4-nitrobenzoic acid (BENCNBA) and anthranilic acid-2-chloro-4-nitrobenzoic acid (AACNBA) using solid phase reactions. The phase diagram studies revealed that formation of the investigated compounds occurs in 1:1 molar ratio. The synthesized compounds were characterized using several spectral techniques such as FT-IR, 1H and 13C NMR, UV-Vis, powder X-ray diffraction (PXRD). Single crystal XRD (SCXRD) study showed that both BENCNBA and AACNBA compounds crystallize in triclinic crystal system with P-1 space group. Further, the presence of intermolecular hydrogen bonding between the constituent components was also supported by single crystal X-ray diffraction (SCXRD) method. Heat of mixing, entropy of fusion, roughness parameter, interfacial energy and excess thermodynamic functions have also been computed using the enthalpy of fusion values derived from differential scanning calorimeter (DSC) study. The inhibition effect of BENCNBA and AACNBA on the mild steel corrosion in hydrochloric acid solution was tested using electrochemical methods. Electrochemical impedance spectroscopy (EIS) study revealed that both BENCNBA and AACNBA behaved as interface corrosion inhibitors and showed maximum inhibition efficiencies of 95.71% and 96.42%, respectively at 400 ppm (1.23 × 10-3 M) concentration. Potentiodynamic polarization (PDP) measurements suggested that BENCNBA and AACNBA acted as mixed type corrosion inhibitors. EIS and PDP results showed that BENCNBA and AACNBA act as efficient corrosion inhibitors for mild steel and their inhibition efficiencies enhances on increasing their concentrations.

  12. mTORC1 Balances Cellular Amino Acid Supply with Demand for Protein Synthesis through Post-transcriptional Control of ATF4

    Directory of Open Access Journals (Sweden)

    Yeonwoo Park

    2017-05-01

    Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 is a master regulator of cell growth that is commonly deregulated in human diseases. Here we find that mTORC1 controls a transcriptional program encoding amino acid transporters and metabolic enzymes through a mechanism also used to regulate protein synthesis. Bioinformatic analysis of mTORC1-responsive mRNAs identified a promoter element recognized by activating transcription factor 4 (ATF4, a key effector of the integrated stress response. ATF4 translation is normally induced by the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α through a mechanism that requires upstream open reading frames (uORFs in the ATF4 5′ UTR. mTORC1 also controls ATF4 translation through uORFs, but independently of changes in eIF2α phosphorylation. mTORC1 instead employs the 4E-binding protein (4E-BP family of translation repressors. These results link mTORC1-regulated demand for protein synthesis with an ATF4-regulated transcriptional program that controls the supply of amino acids to the translation machinery.

  13. Synthesis, Density Functional Theory (DFT), Urease Inhibition and Antimicrobial Activities of 5-Aryl Thiophenes Bearing Sulphonylacetamide Moieties.

    Science.gov (United States)

    Noreen, Mnaza; Rasool, Nasir; Gull, Yasmeen; Zubair, Muhammad; Mahmood, Tariq; Ayub, Khurshid; Nasim, Faiz-Ul-Hassan; Yaqoob, Asma; Zia-Ul-Haq, Muhammad; de Feo, Vincenzo

    2015-11-05

    A variety of novel 5-aryl thiophenes 4a-g containing sulphonylacetamide (sulfacetamide) groups were synthesized in appreciable yields via Pd[0] Suzuki cross coupling reactions. The structures of these newly synthesized compounds were determined using spectral data and elemental analysis. Density functional theory (DFT) studies were performed using the B3LYP/6-31G (d, p) basis set to gain insight into their structural properties. Frontier molecular orbital (FMOs) analysis of all compounds 4a-g was computed at the same level of theory to get an idea about their kinetic stability. The molecular electrostatic potential (MEP) mapping over the entire stabilized geometries of the molecules indicated the reactive sites. First hyperpolarizability analysis (nonlinear optical response) were simulated at the B3LYP/6-31G (d, p) level of theory as well. The compounds were further evaluated for their promising antibacterial and anti-urease activities. In this case, the antibacterial activities were estimated by the agar well diffusion method, whereas the anti-urease activities of these compounds were determined using the indophenol method by quantifying the evolved ammonia produced. The results revealed that all the sulfacetamide derivatives displayed antibacterial activity against Bacillus subtiles, Escherichia coli, Staphylococcus aureus, Shigella dysenteriae, Salmonella typhae, Pseudomonas aeruginosa at various concentrations. Furthermore, the compound 4g N-((5-(4-chlorophenyl)thiophen-2-yl)sulfonyl) acetamide showed excellent urease inhibition with percentage inhibition activity ~46.23 ± 0.11 at 15 µg/mL with IC50 17.1 µg/mL. Moreover, some other compounds 4a-f also exhibited very good inhibition against urease enzyme.

  14. Prostaglandin synthesis can be inhibited locally by infusion of NSAIDS through microdialysis catheters in human skeletal muscle

    DEFF Research Database (Denmark)

    Mikkelsen, Ulla Ramer; Helmark, Ida Carøe; Kjaer, Michael

    2008-01-01

    of nonsteroidal anti-inflammatory drugs (NSAIDs). However, to study the local role of prostaglandins, the formation of prostaglandins within the tissue must be controlled. Microdialysis enables determination of local concentrations of water-soluble substances within the tissue. In the present study......, the microdialysis method was used to infuse NSAIDs locally into human skeletal muscles producing a local block of prostaglandin formation. In addition, the graded blockade at various distances from the infusion site within the muscle during rest, exercise and recovery was determined. Microdialysis was performed...... in thigh muscles (vastus lateralis muscle) in six healthy men. One of the microdialysis catheters was used to block prostaglandin synthesis by infusion of the NSAID indomethacin. Additional catheters were placed 1 and 4 cm away from the infusion and in the contralateral leg (working control). Following 2 h...

  15. Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-01-01

    Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inh......Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation...... of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism...

  16. RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

    Science.gov (United States)

    Yu, W; Sanders, B G; Kline, K

    1997-01-01

    RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

  17. Facile, Large-Quantity Synthesis of Stable, Tunable-Color Silicon Nanoparticles and Their Application for Long-Term Cellular Imaging.

    Science.gov (United States)

    Zhong, Yiling; Sun, Xiaotian; Wang, Siyi; Peng, Fei; Bao, Feng; Su, Yuanyuan; Li, Youyong; Lee, Shuit-Tong; He, Yao

    2015-06-23

    We herein introduce a facile, low-cost photochemical method capable of rapid (nanoparticles (SiNPs) of tunable optical properties (peak emission wavelength in the range of 470-560 nm) under ambient air conditions, by introducing 1,8-naphthalimide as a reducing agent and surface ligands. The as-prepared SiNPs feature robust storage stability and photostability preserving strong and stable fluorescent during long-term (>3 h) high-power UV irradiation, in contrast to the rapid fluorescence quenching within 2 h of conventional organic dyes and II-VI quantum dots under the same conditions. The as-prepared SiNPs serving as photostable nanoprobes are workable for cellular imaging in long-term manners. Our findings provide a powerful method for mild-condition and low-cost, large-quantity production of highly fluorescent and photostable SiNPs for various promising applications.

  18. Inhibition of β-galactosidase and α-glucosidase synthesis in petroleum refinery effluent bacteria by phenolic compounds

    Directory of Open Access Journals (Sweden)

    Gideon C. Okpokwasili

    2011-04-01

    Full Text Available Inhibition of α-glucosidase (EC 3.2.1.20 and β-galactosidase (EC 3.2.1.23 biosynthesis by phenolic compounds (phenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol in Escherichia coli, Bacillus and Pseudomonas species isolated from petroleum refinery wastewater was assessed. At sufficient concentrations, phenols inhibited the induction of α-glucosidase and β-galactosidase. The patterns of these toxic effects can be mathematically described with logistic and sigmoid dose-response models. The median inhibitory concentrations (IC50 varied among the phenols, the bacteria and enzymes. Quantitative structure–activity relationship (QSAR models based on the logarithm of the octanol–water partition coefficient (log10Kow were developed for each bacterium. The correlation coefficients varied between 0.84and 0.99 for the enzymes. The test results indicated α-glucosidase and β-galactosidase biosynthesis as important microbial indices for evaluation of toxicity of phenolic compounds.

  19. Synthesis of fluorescent carbon dots via microwave carbonization of citric acid in presence of tetraoctylammonium ion, and their application to cellular bioimaging

    International Nuclear Information System (INIS)

    Bhaisare, Mukesh Lavkush; Talib, Abou; Khan, M. Shahnawaz; Pandey, Sunil; Wu, Hui-Fen

    2015-01-01

    A jelly-like form of carbon dots (C-dots) was prepared by microwave-assisted synthesis from citric acid in the presence of tetraoctylammonium bromide. The effect of the concentration of tetraoctylammonium bromide was examined. The synthesized carbon dots were characterized by UV–vis, XRD, FTIR, fluorescence and HR-TEM. Fluorescence extends from 350 to 600 nm, and the corresponding excitation wavelengths range from 300 to 460 nm. Quantum yields are at around 0.11. A cytotoxicity study showed carbon dots to be cell permeable and biocompatible which renders them appropriate for imaging applications. The dots were used to image HeLa cell lines via the blue fluorescence of the dots. (author)

  20. Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

    International Nuclear Information System (INIS)

    Hunting, D.J.; Dresler, S.L.

    1985-01-01

    We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

  1. HET0016, a selective inhibitor of 20-HETE synthesis, decreases pro-angiogenic factors and inhibits growth of triple negative breast cancer in mice.

    Directory of Open Access Journals (Sweden)

    Thaiz Ferraz Borin

    Full Text Available A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and in vitro cell line. MDA-MB-231 tumor cells were implanted in animals' right flank and randomly assigned to early (1 and 2, starting treatments on day 0, or delayed groups (3 and 4 on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p<0.05. Tumor growth was completely inhibited on three of ten animals on early treatment group. Treatment groups showed significantly lower expression of pro-angiogenic factors compared to control at 21 days; however, there was no significant difference in HIF-1α expression after treatments. Similar results were found in vitro at 24 hrs of HET0016 treatment. After 28 days, significant increase of angiogenin, angiopoietin-1/2, EGF-R and IGF-1 pro-angiogenic factors were found (p<0.05 compared to control, as well as an higher intensity of all factors were found when compared to that of 21 day's data, suggesting a treatment resistance. HET0016 inhibited tumor growth by reducing expression of different set of pro-angiogenic factors; however, a resistance to treatment seemed to happen after 21 days.

  2. Increased renal sodium absorption by inhibition of prostaglandin synthesis during fasting in healthy man. A possible role of the epithelial sodium channels

    Directory of Open Access Journals (Sweden)

    Graffe Carolina C

    2010-10-01

    Full Text Available Abstract Background Treatment with prostaglandin inhibitors can reduce renal function and impair renal water and sodium excretion. We tested the hypotheses that a reduction in prostaglandin synthesis by ibuprofen treatment during fasting decreased renal water and sodium excretion by increased absorption of water and sodium via the aquaporin2 water channels and the epithelial sodium channels. Methods The effect of ibuprofen, 600 mg thrice daily, was measured during fasting in a randomized, placebo-controlled, double-blinded crossover study of 17 healthy humans. The subjects received a standardized diet on day 1, fasted at day 2, and received an IV infusion of 3% NaCl on day 3. The effect variables were urinary excretions of aquaporin2 (u-AQP2, the beta-fraction of the epithelial sodium channel (u-ENaCbeta, cyclic-AMP (u-cAMP, prostaglandin E2 (u-PGE2. Free water clearance (CH2O, fractional excretion of sodium (FENa, and plasma concentrations of vasopressin, angiotensin II, aldosterone, atrial-, and brain natriuretic peptide. Results Ibuprofen decreased u-AQP2, u-PGE2, and FENa at all parts of the study. During the same time, ibuprofen significantly increased u-ENaCbeta. Ibuprofen did not change the response in p-AVP, u-c-AMP, urinary output, and free water clearance during any of these periods. Atrial-and brain natriuretic peptide were higher. Conclusion During inhibition of prostaglandin synthesis, urinary sodium excretion decreased in parallel with an increase in sodium absorption and increase in u-ENaCbeta. U-AQP2 decreased indicating that water transport via AQP2 fell. The vasopressin-c-AMP-axis did not mediate this effect, but it may be a consequence of the changes in the natriuretic peptide system and/or the angiotensin-aldosterone system Trial Registration Clinical Trials Identifier: NCT00281762

  3. N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

    Science.gov (United States)

    Yi, Dan; Hou, Yongqing; Wang, Lei; Long, Minhui; Hu, Shengdi; Mei, Huimin; Yan, Liqiong; Hu, Chien-An Andy; Wu, Guoyao

    2016-02-01

    Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

  4. Amino acid containing thapsigargin analogues deplete androgen receptor protein via synthesis inhibition and induce the death of prostate cancer cells

    DEFF Research Database (Denmark)

    Griend, Donald J Vander; Antony, Lizamma; Dalrymple, Susan L

    2009-01-01

    There are quantitative and/or qualitative mechanisms allowing androgen receptor (AR) growth signaling in androgen ablation refractory prostate cancer cells. Regardless of the mechanism, agents that deplete AR protein expression prevent such AR growth signaling. Thapsigargin (TG) is a highly cell......-penetrant sequiterpene-lactone that once inside cells inhibits (IC(50), approximately 10 nmol/L) critically important housekeeping SERCA 2b calcium pumps in the endoplasmic reticulum. Using a series of five genetically diverse androgen ablation refractory human prostate cancer lines (LNCaP, LAPC-4, VCaP, MDA-PCa-2b......-specific proteases, such as prostate-specific antigen and prostate-specific membrane antigen, or cancer-specific proteases, such as fibroblast activation protein, so that toxicity of these prodrugs is selectively targeted to metastatic sites of prostate cancer. Based on these results, these prodrugs are undergoing...

  5. Synthesis, DNA Cleavage Activity, Cytotoxicity, Acetylcholinesterase Inhibition, and Acute Murine Toxicity of Redox-Active Ruthenium(II) Polypyridyl Complexes.

    Science.gov (United States)

    Alatrash, Nagham; Narh, Eugenia S; Yadav, Abhishek; Kim, Mahn-Jong; Janaratne, Thamara; Gabriel, James; MacDonnell, Frederick M

    2017-07-06

    Four mononuclear [(L-L) 2 Ru(tatpp)] 2+ and two dinuclear [(L-L) 2 Ru(tatpp)Ru(L-L) 2 ] 4+ ruthenium(II) polypyridyl complexes (RPCs) containing the 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) ligand were synthesized, in which L-L is a chelating diamine ligand such as 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me 4 phen) or 4,7-diphenyl-1,10-phenanthroline (Ph 2 phen). These Ru-tatpp analogues all undergo reduction reactions with modest reducing agents, such as glutathione (GSH), at pH 7. These, plus several structurally related but non-redox-active RPCs, were screened for DNA cleavage activity, cytotoxicity, acetylcholinesterase (AChE) inhibition, and acute mouse toxicity, and their activities were examined with respect to redox activity and lipophilicity. All of the redox-active RPCs show single-strand DNA cleavage in the presence of GSH, whereas none of the non-redox-active RPCs do. Low-micromolar cytotoxicity (IC 50 ) against malignant H358, CCL228, and MCF7 cultured cell lines was mainly restricted to the redox-active RPCs; however, they were substantially less toxic toward nonmalignant MCF10 cells. The IC 50 values for AChE inhibition in cell-free assays and the acute toxicity of RPCs in mice revealed that whereas most RPCs show potent inhibitory action against AChE (IC 50 values <15 μm), Ru-tatpp complexes as a class are surprisingly well tolerated in animals relative to other RPCs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

    Science.gov (United States)

    Domarkas, Juozas; Dudouit, Fabienne; Williams, Christopher; Qiyu, Qiu; Banerjee, Ranjita; Brahimi, Fouad; Jean-Claude, Bertrand Jacques

    2006-06-15

    According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

  7. Acute inhibition of estradiol synthesis impacts vestibulo-ocular reflex adaptation and cerebellar long-term potentiation in male rats.

    Science.gov (United States)

    Dieni, Cristina V; Ferraresi, Aldo; Sullivan, Jacqueline A; Grassi, Sivarosa; Pettorossi, Vito E; Panichi, Roberto

    2018-03-01

    The vestibulo-ocular reflex (VOR) adaptation is an ideal model for investigating how the neurosteroid 17 beta-estradiol (E2) contributes to the modification of behavior by regulating synaptic activities. We hypothesized that E2 impacts VOR adaptation by affecting cerebellar synaptic plasticity at the parallel fiber-Purkinje cell (PF) synapse. To verify this hypothesis, we investigated the acute effect of blocking E2 synthesis on gain increases and decreases in adaptation of the VOR in male rats using an oral dose (2.5 mg/kg) of the aromatase inhibitor letrozole. We also assessed the effect of letrozole on synaptic plasticity at the PF synapse in vitro, using cerebellar slices from male rats. We found that letrozole acutely impaired both gain increases and decreases adaptation of the VOR without altering basal ocular-motor performance. Moreover, letrozole prevented long-term potentiation at the PF synapse (PF-LTP) without affecting long-term depression (PF-LTD). Thus, in male rats neurosteroid E2 has a relevant impact on VOR adaptation and affects exclusively PF-LTP. These findings suggest that E2 might regulate changes in VOR adaptation by acting locally on cerebellar and extra-cerebellar synaptic plasticity sites.

  8. Synthesis, spectroscopic, computational (DFT/B3LYP), AChE inhibition and antioxidant studies of imidazole derivative

    Science.gov (United States)

    Ahmad, Faheem; Alam, Mohammad Jane; Alam, Mahboob; Azaz, Shaista; Parveen, Mehtab; Park, Soonheum; Ahmad, Shabbir

    2018-01-01

    The present study reports the synthesis and evaluation of biological properties of 3a,8a-dihydroxy-8-oxo-1,3,3a,8a-tetrahydroindeno[1,2-d]imidazol-2(1H)-iminium chloride (3). The structure was confirmed by the FTIR, NMR, MS, CHN microanalysis and X-ray crystallographic analysis. Quantum chemical calculations have been performed at B3LYP-D3/6-311++G(d,p) level of theory to study the molecular geometry, IR, (1H and 13C) NMR, UV/Vis spectra and other molecular parameters of the asymmetric unit of crystal of imidazole compound (3). An empirical dispersion correction to hybrid functional (B3LYP-D3) has been incorporated in the present calculations due to presence of non-covalent interaction, Cl⋯H-O, in the present compound. The remarkable agreement has been observed between theoretical data and those measured experimentally. Moreover, the Hirshfeld analysis was carried out to ascertain the secondary interactions and associated 2D fingerprint plots. The synthesized imidazole derivative showed promising antioxidant property and inhibitory activity against acetylcholinesterase (AChE). Molecular docking was also performed in order to explain in silico antioxidant studies and to ascertain the probable binding mode of compound with the amino acid residues of protein.

  9. Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

    Science.gov (United States)

    Hussain, Tahir; Yogavel, Manickam; Sharma, Amit

    2015-04-01

    Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. AmyI-1-18, a cationic α-helical antimicrobial octadecapeptide derived from α-amylase in rice, inhibits the translation and folding processes in a protein synthesis system.

    Science.gov (United States)

    Taniguchi, Masayuki; Ochiai, Akihito; Fukuda, Shun; Sato, Teppei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-10-01

    In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; AmyI-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1-18 strongly bound to RNA with a KD value of 1.4 × 10(-8) M but not to DNA and that AmyI-1-18 specifically bound to DnaK with a KD value of 4.4 × 10(-6) M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis.

    Science.gov (United States)

    Millard, E E; Srivastava, K; Traub, L M; Schaffer, J E; Ory, D S

    2000-12-08

    The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoprotein cholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function of NPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein (CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to the endoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1 cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increase in PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterol synthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role for NPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulated gene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, these studies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol.

  12. Enterobacter sp. I-3, a bio-herbicide inhibits gibberellins biosynthetic pathway and regulates abscisic acid and amino acids synthesis to control plant growth.

    Science.gov (United States)

    Radhakrishnan, Ramalingam; Park, Jae-Man; Lee, In-Jung

    2016-12-01

    Very few bacterial species were identified as bio-herbicides for weed control. The present research was focused to elucidate the plant growth retardant properties of Enterobacter sp. I-3 during their interaction by determining the changes in endogenous photosynthetic pigments, plant hormones and amino acids. The two bacterial isolates I-4-5 and I-3 were used to select the superior bacterium for controlling weed seeds (Echinochloa crus-galli L. and Portulaca oleracea L.) germination. The post-inoculation of I-3 (Enterobacter sp. I-3) significantly inhibited the weeds seed germination than their controls. The mechanism of bacterium induced plant growth reduction was identified in lettuce treated with I-3 bacterium and compared their effects with known chemical herbicide, trinexapac-ethyl (TE). The treatment of I-3 and TE showed a significant inhibitory effect on shoot length, leaf number, leaf length, leaf width, shoot weight, root weight and chlorophyll content in lettuce seedlings. The endogenous gibberellins (GAs) and abscisic acid (ABA) analysis showed that Enterobacter sp. I-3 treated plants had lower levels of GAs (GA 12 , GA 19 , GA 20 and GA 8 ) and GAs/ABA ratio and then, the higher level of ABA when compared to their controls. Indeed, the individual amino acids ie., aspartic acid, glutamic acid, glycine, threonine, alanine, serine, leucine, isoleucine and tyrosine were declined in TE and I-3 exposed plants. Our results suggest that the utilization of Enterobacter sp. I-3 inhibits the GAs pathway and amino acids synthesis in weeds to control their growth can be an alternative to chemical herbicides. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. Low Concentrations of o,p’-DDT Inhibit Gene Expression and Prostaglandin Synthesis by Estrogen Receptor-Independent Mechanism in Rat Ovarian Cells

    Science.gov (United States)

    Liu, Jing; Zhao, Meirong; Zhuang, Shulin; Yang, Yan; Yang, Ye; Liu, Weiping

    2012-01-01

    o,p’-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p’-DDT at range of 0.3–500 ng/g (8.46×10−10 M−1.41×10−6 M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p’-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p’-DDT (10−12−10−8 M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p’-DDT at 0.5–1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p’-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p’-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p’-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p’-DDT observed in the population likely poses a health risk to female reproduction. PMID:23209616

  14. Synthesis, characterization and applications of carboxylated and polyethylene-glycolated bifunctionalized InP/ZnS quantum dots in cellular internalization mediated by cell-penetrating peptides.

    Science.gov (United States)

    Liu, Betty R; Winiarz, Jeffrey G; Moon, Jong-Sik; Lo, Shih-Yen; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2013-11-01

    Semiconductor nanoparticles, also known as quantum dots (QDs), are widely used in biomedical imaging studies and pharmaceutical research. Cell-penetrating peptides (CPPs) are a group of small peptides that are able to traverse cell membrane and deliver a variety of cargoes into living cells. CPPs deliver QDs into cells with minimal nonspecific absorption and toxic effect. In this study, water-soluble, monodisperse, carboxyl-functionalized indium phosphide (InP)/zinc sulfide (ZnS) QDs coated with polyethylene glycol lipids (designated QInP) were synthesized for the first time. The physicochemical properties (optical absorption, fluorescence and charging state) and cellular internalization of QInP and CPP/QInP complexes were characterized. CPPs noncovalently interact with QInP in vitro to form stable CPP/QInP complexes, which can then efficiently deliver QInP into human A549 cells. The introduction of 500nM of CPP/QInP complexes and QInP at concentrations of less than 1μM did not reduce cell viability. These results indicate that carboxylated and polyethylene-glycolylated (PEGylated) bifunctionalized QInP are biocompatible nanoparticles with potential for use in biomedical imaging studies and drug delivery applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Synthesis of an excellent electrocatalyst for oxygen reduction reaction with supercritical fluid: Graphene cellular monolith with ultrafine and highly dispersive multimetallic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Yazhou; Cheng, Xiaonong; Yen, Clive H.; Wai, Chien M.; Wang, Chongmin; Yang, Juan; Lin, Yuehe

    2017-04-01

    Graphene cellular monolith (GCM) can be used as an excellent support for nanoparticles in widespread applications. However, it's still a great challenge to deposit the desirable nanoparticles in GCM that have small size, controllable structure, composition, and high dispersion using the current methods. Here we demonstrate a green, efficient and large-scale method to address this challenge using supercritical fluid (SCF). By this superior method, graphene hydrogel can be transferred into GCM while being deposited with ultrafine and highly dispersive nanoparticles. Specifically, the bimetallic PtFe/GCM and the trimetallic PtFeCo/GCM catalysts are successfully synthesized, and their electrocatalytic performances toward oxygen reduction reaction (ORR) are also studied. The resultant PtFe/GCM shows the significant enhancement in ORR activity, including a factor of 8.47 enhancement in mass activity (0.72 A mgPt-1), and a factor of 7.67 enhancement in specific activity (0.92 mA cm-2), comparing with those of the commercial Pt/C catalyst (0.085 A mgPt-1, 0.12 mA cm-2). Importantly, by introducing the Co, the trimetallic PtFeCo/GCM exhibits the further improved ORR activities (1.28 A mgPt-1, 1.80 mA cm-2). The high ORR activity is probably attributed to the alloying structure, ultrafine size, highly dispersive, well-defined, and a better interface with 3D porous graphene support.

  16. The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

    International Nuclear Information System (INIS)

    Mu, J.-J.; Tsay, Y.-G.; Juan, L.-J.; Fu, T.-F.; Huang, W.-H.; Chen, D.-S.; Chen, P.-J.

    2004-01-01

    Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [ 3 H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication

  17. DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography.

    Science.gov (United States)

    Prabhakaran, R; Kalaivani, P; Huang, R; Poornima, P; Vijaya Padma, V; Dallemer, F; Natarajan, K

    2013-02-01

    Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.

  18. Cellular dosimetry

    International Nuclear Information System (INIS)

    Humm, J.L.; Chin, L.M.

    1989-01-01

    Radiation dose is a useful predictive parameter for describing radiation toxicity in conventional radiotherapy. Traditionally, in vitro radiation biology dose-effect relations are expressed in the form of cell survival curves, a semilog plot of cell survival versus dose. However, the characteristic linear or linear quadratic survival curve shape, for high- and low-LET radiations respectively, is only strictly valid when the radiation dose is uniform across the entire target population. With an external beam of 60 Co gamma rays or x-rays, a uniform field may be readily achievable. When radionuclides are incorporated into a cell milieu, several new problems emerge which can result in a departure from uniformity in energy deposition throughout a cell population. This nonuniformity can have very important consequences for the shape of the survival curve. Cases in which perturbations of source uniformity may arise include: 1. Elemental sources may equilibrate in the cell medium with partition coefficients between the extracellular, cytosol, and nuclear compartments. The effect of preferential cell internalization or binding to cell membrane of some radionuclides can increase or decrease the slope of the survival curve. 2. Radionuclides bound to antibodies, hormones, metabolite precursors, etc., may result in a source localization pattern characteristic of the carrier agent, i.e., the sources may bind to cell surface receptors or antigens, be internalized, bind to secreted antigen concentrated around a fraction of the cell population, or become directly incorporated into the cell DNA. We propose to relate the distribution of energy deposition in cell nuclei to biological correlates of cellular inactivation. The probability of each cell's survival is weighted by its individual radiation burden, and the summation of these probabilities for the cell population can be used to predict the number or fraction of cell survivors

  19. Synthesis, characterization and histomorphometric analysis of cellular response to a new elastic DegraPol® polymer for rabbit Achilles tendon rupture repair.

    Science.gov (United States)

    Buschmann, Johanna; Calcagni, Maurizio; Bürgisser, Gabriella Meier; Bonavoglia, Eliana; Neuenschwander, Peter; Milleret, Vincent; Giovanoli, Pietro

    2015-05-01

    Tendon rupture repair is a surgical field where improvements are still required due to problems such as repeat ruptures, adhesion formation and joint stiffness. In the current study, a reversibly expandable and contractible electrospun tube based on a biocompatible and biodegradable polymer was implanted around a transected and conventionally sutured rabbit Achilles tendon. The material used was DegraPol® (DP), a polyester urethane. To make DP softer, more elastic and surgeon-friendly, the synthesis protocol was slightly modified. Material properties of conventional and new DP film electrospun meshes are presented. At 12 weeks post-surgery, tenocyte and tenoblast density, nuclei and width, collagen fibre structure and inflammation levels were analyzed histomorphometrically. Additionally, a comprehensive histological scoring system by Stoll et al. (2011) was used to compare healing outcomes. Results showed that there were no adverse reactions of the tendon tissue following the implant. No differences were found whether the DP tube was applied or not for both traditional and new DP materials. As a result, the new DP material was shown to be an excellent carrier for delivery of growth factors, stem cells and other agents responsible for tendon healing. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Microwave-assisted synthesis of BSA-modified silver nanoparticles as a selective fluorescent probe for detection and cellular imaging of cadmium(II)

    International Nuclear Information System (INIS)

    Gu, Yu; Li, Nan; Gao, Mengmeng; Wang, Zilu; Xiao, Deli; Li, Yun; Jia, Huning; He, Hua

    2015-01-01

    We have developed a microwave-assisted method for the synthesis of silver nanoparticles (AgNPs) whose surface is modified with bovine serum albumin (BSA). The reaction involves reduction of the BSA-Ag(I) complex by tyrosine in strongly alkaline solution to form BSA-AgNPs. The reaction takes a few minutes only owing to rapid and uniform microwave heating. The modified AgNPs were characterized by UV–vis and fluorescence spectroscopy, transmission electron microscopy and X- ray photoelectron spectroscopy. The BSA-AgNPs are yellow and display luminescence with a maximum at 521 nm if excited at 465 nm. They have a hydrodynamic diameter of 3–5 nm and possess good colloidal stability in the pH 4.6 to 12.0 range. The fluorescence of the BSA-AgNPs is enhanced by Cd(II) ion due to the formation of a stable hybrid conjugate referred to as Cd-BSA-AgNPs. The effect was exploited to quantify Cd(II) in spiked real water samples with a 4.7 nM detection limit, and also to fluorescently image Cd(II) in Hepatoma cells. (author)

  1. Novel piplartine-containing ruthenium complexes: synthesis, cell growth inhibition, apoptosis induction and ROS production on HCT116 cells.

    Science.gov (United States)

    D'Sousa Costa, Cinara O; Araujo Neto, João H; Baliza, Ingrid R S; Dias, Rosane B; Valverde, Ludmila de F; Vidal, Manuela T A; Sales, Caroline B S; Rocha, Clarissa A G; Moreira, Diogo R M; Soares, Milena B P; Batista, Alzir A; Bezerra, Daniel P

    2017-11-28

    Piplartine (piperlongumine) is a plant-derived molecule that has been receiving intense interest due to its anticancer characteristics that target the oxidative stress. In the present paper, two novel piplartine-containing ruthenium complexes [Ru(piplartine)(dppf)(bipy)](PF 6 ) 2 (1) and [Ru(piplartine)(dppb)(bipy)](PF 6 ) 2 (2) were synthesized and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes are more potent than metal-free piplartine in a panel of cancer cell lines on monolayer cultures, as well in 3D model of cancer multicellular spheroids formed from human colon carcinoma HCT116 cells. Mechanistic studies uncovered that the complexes reduced the cell growth and caused phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase-3 activation and loss of the mitochondrial transmembrane potential on HCT116 cells. Moreover, the pre-treatment with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, reduced the complexes-induced apoptosis, indicating cell death by apoptosis through caspase-dependent and mitochondrial intrinsic pathways. Treatment with the complexes also caused a marked increase in the production of reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and nitric oxide, and decreased reduced glutathione levels. Application of N-acetyl-cysteine, an antioxidant, reduced the ROS levels and apoptosis induced by the complexes, indicating activation of ROS-mediated apoptosis pathway. RNA transcripts of several genes, including gene related to the cell cycle, apoptosis and oxidative stress, were regulated under treatment. However, the complexes failed to induce DNA intercalation. In conclusion, the complexes are more potent than piplartine against different cancer cell lines and are able to induce caspase-dependent and mitochondrial intrinsic apoptosis on HCT116 cells by ROS-mediated pathway.

  2. Feeding Unprotected CLA Methyl Esters Compared to Sunflower Seeds Increased Milk CLA Level but Inhibited Milk Fat Synthesis in Cows

    Directory of Open Access Journals (Sweden)

    F. Dohme-Meier

    2012-01-01

    Full Text Available An experiment was conducted to compare the effect of the same amount of 18:2 offered either as 18:2n-6 or as a mixture of unprotected 18:2c9t11 and 18:2t10c12 on feed intake, milk components as well as plasma and milk fatty acid profile. Fifteen cows were blocked by milk yield and milk fat percentage and within block assigned randomly to 1 of 3 treatments (n = 5. Each cow passed a 12-d adjustment period (AP on a basal diet. After the AP cows received 1 of 3 supplements during an 18-d experimental period (EP. The supplements contained either 1.0 kg ground sunflower seeds (S, 0.5 kg conjugated linoleic acid (CLA-oil (C or 0.75 kg of a mixture of ground sunflower seeds and CLA-oil (2:1; SC. All 3 supplements contained the same amount of 18:2 either as CLA (∑18:2c9t11+18:2t10c12, 1:1 or as 18:2c9c12. During the last 2 d of AP and the last 4 d of EP feed intake and milk yield were recorded daily and milk samples were collected at each milking. Blood samples were collected from the jugular vein on d 11 of AP and d 15 and 18 of EP. The 18:2 intake increased in all treatments from AP to EP. Regardless of the amount of supplemented CLA, the milk fat percentage decreased by 2.35 and 2.10%-units in treatment C and SC, respectively, whereas in the treatment S the decrease was with 0.99%-unit less pronounced. Thus, C and SC cows excreted daily a lower amount of milk fat than S cows. The concentration of trans 18:1 in the plasma and the milk increased from AP to EP and increased with increasing dietary CLA supply. While the concentration of 18:2c9t11 and 18:2t10c12 in the plasma and that of 18:2t10c12 in the milk paralleled dietary supply, the level of 18:2c9t11 in the milk was similar in C and CS but still lower in S. Although the dietary concentration of CLA was highest in treatment C, the partial replacement of CLA by sunflower seeds had a similar inhibitory effect on milk fat synthesis. Comparable 18:2c9t11 levels in the milk in both CLA treatments

  3. A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

    Science.gov (United States)

    Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

    1996-09-01

    The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

  4. Cellular pharmacodynamics of the cytotoxic guanidino-containing drug CHS 828. Comparison with methylglyoxal-bis(guanylhydrazone).

    Science.gov (United States)

    Ekelund, S; Sjöholm, A; Nygren, P; Binderup, L; Larsson, R

    2001-04-20

    N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828) is a new guanidino-containing compound with antitumoral activity both in vitro and in vivo. Its activity profile differs from those of standard cytotoxic drugs but the mechanism of action is not yet fully understood. CHS 828 is presently in early phase I and II clinical trials. In the present study, the pharmacodynamic effects at the cellular level of CHS 828 was compared to another compound containing two guanidino groups, methylglyoxal-bis(guanylhydrazone) (MGBG). MGBG is known to inhibit the synthesis of polyamines, which are important in, e.g., proliferation and macromolecular synthesis. The concentration-response relationship of CHS 828 closely resembled that of MGBG and the drugs were similar with respect to inhibition of DNA and protein synthesis. On the other hand, CHS 828 induced a significant increase in cellular metabolism while MGBG did not. The cytotoxic effect of MGBG was reversed by the addition of exogenous polyamines, while that of CHS 828 was unaffected. Unlike MGBG, there was also no effect of CHS 828 on the levels of decarboxylating enzymes in the polyamine biosynthesis. In conclusion, CHS 828 does not appear to share any major mechanisms of action with the polyamine synthesis inhibitor MGBG. Further studies will be required to define the exact mechanism of action of CHS 828.

  5. Synthesis and characterization of fluorinated magnetic core-shell nanoparticles for inhibition of insulin amyloid fibril formation

    International Nuclear Information System (INIS)

    Skaat, Hadas; Margel, Shlomo; Belfort, Georges

    2009-01-01

    Maghemite (γ-Fe 2 O 3 ) magnetic nanoparticles of 15.0 ± 2.1 nm are formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Uniform magnetic γ-Fe 2 O 3 /poly (2,2,3,3,4,4,4-heptafluorobutyl acrylate) (γ-Fe 2 O 3 /PHFBA) core-shell nanoparticles are prepared by emulsion polymerization of the fluorinated monomer 2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) in the presence of the maghemite nanoparticles. The kinetics of the insulin fibrillation process in the absence and in the presence of the γ-Fe 2 O 3 /PHFBA core-shell nanoparticles are elucidated. A significant direct slow transition from α-helix to β-sheets during insulin fibril formation is observed in the presence of the γ-Fe 2 O 3 /PHFBA nanoparticles. This is in contradiction to our previous manuscript, which illustrated that the γ-Fe 2 O 3 core nanoparticles do not affect the kinetics of the formation of the insulin fibrils, and to other previous publications that describe acceleration of the fibrillation process by using various types of nanoparticles. These core-shell nanoparticles may therefore be also useful for the inhibition of conformational changes of other amyloidogenic proteins that lead to neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, mad cow and prion diseases.

  6. Novel piperonal 1,3,4-thiadiazolium-2-phenylamines mesoionic derivatives: Synthesis, tyrosinase inhibition evaluation and HSA binding study.

    Science.gov (United States)

    Lopes, Natália Drumond; Chaves, Otávio Augusto; de Oliveira, Márcia C C; Sant'Anna, Carlos Mauricio R; Sousa-Pereira, Danilo; Netto-Ferreira, José Carlos; Echevarria, Aurea

    2018-06-01

    A novel series of piperonal mesoionic derivatives (PMI 1-6) was synthesized. Tyrosinase inhibition in the presence of PMI-1, -2, -3, -4, -5 and -6 as well as human serum albumin (HSA) binding studies with PMI-5 and PMI-6 were done by spectroscopic and theoretical methods. The mesoionic compound PMI-5 is the most promising tyrosinase inhibitor with a noncompetitive inhibitory mechanism and an IC 50 =124μmolL -1 . In accordance with the kinetic profile, molecular docking results show that PMI-5 is able to interact favorably with the tyrosinase active site containing the substrate molecule, L-DOPA, interacting with Val-247, Phe-263 and Val-282 residues. The spectroscopic results for the interaction HSA:PMI-5 and HSA:PMI-6 indicated that these mesoionic compounds can associate with HSA in the ground state and energy transfer can occur with high probability. The binding was moderate, spontaneous and can perturb significantly the secondary structure of the albumin. The molecular docking results suggest that PMI-5 and PMI-6 are able to be accommodated inside the Sudlow's site I in HSA, interacting with hydrophobic and hydrophilic amino acid residues. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Bacterial Peptide deformylase inhibition of cyano substituted biaryl analogs: Synthesis, in vitro biological evaluation, molecular docking study and in silico ADME prediction.

    Science.gov (United States)

    Khan, Firoz A Kalam; Patil, Rajendra H; Shinde, Devanand B; Sangshetti, Jaiprakash N

    2016-08-15

    Herein, we report the synthesis and screening of cyano substituted biaryl analogs 5(a-m) as Peptide deformylase (PDF) enzyme inhibitors. The compounds 5a (IC50 value=13.16μM), 5d (IC50 value=15.66μM) and 5j (IC50 value=19.16μM) had shown good PDF inhibition activity. The compounds 5a (MIC range=11.00-15.83μg/mL), 5b (MIC range=23.75-28.50μg/mL) and 5j (MIC range=7.66-16.91μg/mL) had also shown potent antibacterial activity when compared with ciprofloxacin (MIC range=25-50μg/mL). Thus, the active derivatives were not only potent PDF inhibitors but also efficient antibacterial agents. In order to gain more insight on the binding mode of the compounds with PDF, the synthesized compounds 5(a-m) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. In silico ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Understanding the sub-cellular dynamics of silicon transportation and synthesis in diatoms using population-level data and computational optimization.

    Directory of Open Access Journals (Sweden)

    Narjes Javaheri

    2014-06-01

    Full Text Available Controlled synthesis of silicon is a major challenge in nanotechnology and material science. Diatoms, the unicellular algae, are an inspiring example of silica biosynthesis, producing complex and delicate nano-structures. This happens in several cell compartments, including cytoplasm and silica deposition vesicle (SDV. Considering the low concentration of silicic acid in oceans, cells have developed silicon transporter proteins (SIT. Moreover, cells change the level of active SITs during one cell cycle, likely as a response to the level of external nutrients and internal deposition rates. Despite this topic being of fundamental interest, the intracellular dynamics of nutrients and cell regulation strategies remain poorly understood. One reason is the difficulties in measurements and manipulation of these mechanisms at such small scales, and even when possible, data often contain large errors. Therefore, using computational techniques seems inevitable. We have constructed a mathematical model for silicon dynamics in the diatom Thalassiosira pseudonana in four compartments: external environment, cytoplasm, SDV and deposited silica. The model builds on mass conservation and Michaelis-Menten kinetics as mass transport equations. In order to find the free parameters of the model from sparse, noisy experimental data, an optimization technique (global and local search, together with enzyme related penalty terms, has been applied. We have connected population-level data to individual-cell-level quantities including the effect of early division of non-synchronized cells. Our model is robust, proven by sensitivity and perturbation analysis, and predicts dynamics of intracellular nutrients and enzymes in different compartments. The model produces different uptake regimes, previously recognized as surge, externally-controlled and internally-controlled uptakes. Finally, we imposed a flux of SITs to the model and compared it with previous classical kinetics

  9. MPEG-CS/Bmi-1RNAi Nanoparticles Synthesis and Its Targeted Inhibition Effect on CD133+ Laryngeal Stem Cells.

    Science.gov (United States)

    Wei, Xudong; He, Jian; Wang, Jingyu; Wang, Wei

    2018-03-01

    Previous studies have confirmed that CD133+ cells in laryngeal tumor tissue have the characteristics of cancer stem cells. Bmi-1 gene expression is central to the tumorigenicity of CD133+ cells. In this study, we tried to develop a new siRNA carrier system using chitosan-methoxypolyethylene nanoparticles (CS-mPEG-NPs) that exhibit higher tumor-targeting ability and enhanced gene silencing efficacy in CD133+ tumor stem cells. It is hoped to block the self-renewal and kill the stem cells of laryngeal carcinoma. The mPEG-CS-Bmi-1RNAi-NPs were synthesized and their characters were checked. The changes in invasion ability and sensitivity to radiotherapy and chemotherapy of CD133+Hep-2 tumor cells were observed after Bmi-1 gene silencing. The mPEG-CS-Bmi-1RNAi-NPs synthesized in this experiment have a regular spherical form, a mean size of 139.70 ±6.40 nm, an encapsulation efficiency of 85.21 ± 1.94%, with drug loading capacity of 18.47 ± 1.83%, as well as low cytotoxicity, providing good protection to the loaded gene, strong resistance to nuclease degradation and high gene transfection efficiency. After Bmi-1 gene silencing, the invasion ability of CD133+ cells was weakened. Co-cultured with paclitaxel, the survival rates of CD133+Bmi-1RNAi cells were lower. After radiotherapy, the mean growth inhibition rate of CD133+/Bmi-1RNAi cells was significantly lower than CD133+ cells. In conclusion, the mPEG-CS nano-carrier is an ideal vector in gene therapy, while silencing the Bmi-1 gene can enhance the sensitivity of CD133+ tumor stem cells to chemoradiotherapy and abate their invasion ability.

  10. Nicotinamide starvation and inhibition of poly(ADP-Ribose) synthesis enhance the induced mutation in Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Okada, Gensaku; Kaneko, Ichiro; Mitsui, Hideki.

    1987-01-01

    The effects of nicotinamide (NA) deficiency and added NA and 3-aminobenzamide (3AB) on the cytotoxicity and the induction of mutations in Chinese hamster V79-14 cells were investigated. In NA deficiency the addition of NA (up to 4 mM) and 3AB (up to 7.5 mM) was not cytotoxic. The presence of NA prior to exposure to mitomycin C (MMC) or γ-rays produced a dose-dependent increase in the relative cloning ability of DNA-damaged cells. The lethality of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was significantly potentiated by pre-treatment with 5 mM 3AB, but no potentiation by 3AB was observed for MMC, ultraviolet (UV)-B light, or γ-rays. Among cells pre-cultured in NA-free medium there were increased frequencies of mutations at both the hypoxanthineguanine phosphoribosyltransferase (HGPRT) and the adenine phosphoribosyltransferase (APRT) loci following DNA damage. The enhancing effect by NA deficiency was time-dependent. Incubation with NA prior to DNA damage produced a significant reduction in the frequency of mutations. The addition of 3AB to the nicotinamide adenine dinucleotide (NAD + )-depleted cell cultures before or after the DNA damage also strongly increased the frequency of induced mutations, with increasing concentrations of 3AB up to 5 mM, but the frequency was reduced at higher concentrations. The interaction between NA deficiency and the addition of 3AB appears to act synergistically on mutation induction. A correlation was observed between the potential of inhibiting poly (ADP-ribose) polymerase and the enhancement of mutation frequency. (author)

  11. Synthesis and characterization of fluorinated magnetic core-shell nanoparticles for inhibition of insulin amyloid fibril formation

    Energy Technology Data Exchange (ETDEWEB)

    Skaat, Hadas; Margel, Shlomo [Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900 (Israel); Belfort, Georges [Howard P Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 (United States)], E-mail: ch348@mail.biu.ac.il, E-mail: belfog@rpi.edu, E-mail: Shlomo.margel@mail.biu.ac.il

    2009-06-03

    Maghemite ({gamma}-Fe{sub 2}O{sub 3}) magnetic nanoparticles of 15.0 {+-} 2.1 nm are formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Uniform magnetic {gamma}-Fe{sub 2}O{sub 3}/poly (2,2,3,3,4,4,4-heptafluorobutyl acrylate) ({gamma}-Fe{sub 2}O{sub 3}/PHFBA) core-shell nanoparticles are prepared by emulsion polymerization of the fluorinated monomer 2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) in the presence of the maghemite nanoparticles. The kinetics of the insulin fibrillation process in the absence and in the presence of the {gamma}-Fe{sub 2}O{sub 3}/PHFBA core-shell nanoparticles are elucidated. A significant direct slow transition from {alpha}-helix to {beta}-sheets during insulin fibril formation is observed in the presence of the {gamma}-Fe{sub 2}O{sub 3}/PHFBA nanoparticles. This is in contradiction to our previous manuscript, which illustrated that the {gamma}-Fe{sub 2}O{sub 3} core nanoparticles do not affect the kinetics of the formation of the insulin fibrils, and to other previous publications that describe acceleration of the fibrillation process by using various types of nanoparticles. These core-shell nanoparticles may therefore be also useful for the inhibition of conformational changes of other amyloidogenic proteins that lead to neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, mad cow and prion diseases.

  12. Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

    Science.gov (United States)

    Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

    2016-06-01

    Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc

  13. Inhibition and recovery of semiconservative DNA synthesis in normal and solar UV sensitive ICR 2A frog cell lines following the induction of non-dimer DNA damage by sunlamp UV > 315 nm

    Energy Technology Data Exchange (ETDEWEB)

    Rosenstein, B.S. (Brown Univ., Providence, RI (USA). Dept. of Radiation Medicine)

    1989-08-01

    Cultures of solar UV-sensitive cell lines DRP 36 and DRP 153, and of the parental ICR 2A cell line, were exposed to 150 kJ/m{sup 2} of sunlamp UV>315nm plus photoreactivating light, resulting in the induction primarily of non-dimer DNA damage. Following either 0, 3, 6, 12 or 24 h incubation, cultures were pulse-labelled with ({sup 3}H) thymidine, and the synthesis of different size classes of replicon intermediates measured using alkaline step elution assay. For all three cell lines tested, an immediate depression of low molecular weight DNA synthesis was observed, followed by inhibition of all size classes of replicon intermediates. Within 12 h following irradiation, recovery of DNA synthesis was observed, generally most apparent for low molecular weight DNA. The ICR 2A cells exhibited a nearly full recovery in all size classes of DNA synthesized by 24 h. A much smaller recovery of continued inhibition was primarily in the synthesis of full replicon size DNA, and most pronounced for DRP 36 cells. (author).

  14. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by β-Cl-alanine

    International Nuclear Information System (INIS)

    Medlock, K.A.; Merrill, A.H. Jr.

    1987-01-01

    Serine palmitoyltransferase (SPT) is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the first committed step of long-chain base (LCB) synthesis. Inhibition of SPT activity and de novo biosynthesis of sphinganine and sphingosine was observed in vitro and in intact Chinese hamster ovary cells (CHO). In vitro studies revealed that inhibition was irreversible and concentration- and time-dependent, which are characteristics of suicide inhibition. Incubation of intact CHO cells with 5 mM β-Cl-alanine for 15 min completely inhibited SPT activity and LCB synthesis from [ 14 C]serine. The concentration dependences of inhibition of SPT activity and LCB formation were identical. There was no loss of viability of recovery of SPT activity over the 2 hour time course of these experiments. The synthesis of several other lipids was not affected by the same treatment. These results establish the association between the activity of SPT and the cellular rate of LCB formation and indicate that β-Cl-alanine can be used to study alterations in cellular LCB synthesis

  15. Inhibition of p38 MAPK during cellular activation modulate gene expression of head kidney leukocytes isolated from Atlantic salmon (Salmo salar) fed soy bean oil or fish oil based diets.

    Science.gov (United States)

    Holen, E; Winterthun, S; Du, Z-Y; Krøvel, A V

    2011-01-01

    Head kidney leukocytes isolated from Atlantic salmon fed either a diet based on fish oil (FO) or soy bean oil (VO) were used in order to evaluate if different lipid sources could contribute to cellular activation of the salmon innate immune system. A specific inhibitor of p38 MAPK, SB202190, was used to investigate the effect of lipopolysaccharide (LPS) signalling in the head kidney leukocytes. The results show that LPS up regulate IL-1β, TNF-α, Cox2 expression in leukocytes isolated from fish fed either diet. The p38 MAPK inhibitor, SB202190, reduced the LPS induced expression of these genes in both dietary groups. In LPS stimulated leukocytes isolated from VO fed fish, SB202190 showed a clear dose dependent inhibitory effect on IL-1β, TNF-α and Cox2 expression. This effect was also observed for Cox2 in leukocytes isolated from FO fed fish. Furthermore, there was a stronger mean induction of Cox2 in LPS stimulated leucocytes isolated from the VO-group compared to LPS stimulated leukocytes isolated from the FO-group. In both dietary groups, LPS stimulation of salmon head kidney leukocytes increased the induction of CD83, a dendrite cell marker, while the inhibitor reduced CD83 expression in the VO fed fish only. The inhibitor also clearly reduced hsp27 expression in VO fed fish. Indicating a p38 MAPK feedback loop, LPS significantly inhibited the expression of p38MAPK itself in both diets, while SB202190 increased p38MAPK expression especially in the VO diet group. hsp70 expression was not affected by any treatment or feed composition. There were also differences in p38MAPK protein phosphorylation comparing treatment groups but no obvious difference comparing the two dietary groups. The results indicate that dietary fatty acids have the ability to modify signalling through p38 MAPK which may have consequences for the fish's ability to handle infections and stress. Signalling through p38MAPK is ligand dependent and affects gene and protein expression differently

  16. Iron induction of ferritin synthesis in soybean cell suspensions.

    Science.gov (United States)

    Proudhon,